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REPRESENTATIVE ABRSTRACTS


 

Published in the International Journal of Neuropsychopharmacology(2013) 

Four weeks of Lithium Treatment Alters Neuronal Dendrites in the Rat Hippocampus.

SS Shim1, MD Hammonds2, and RF Mervis3,4
 

1Department of Psychiatry, Case Western Reserve University School of Medicine, Cleveland, OH, USA.,
2Cleveland State University School of Health Sciences, Cleveland, OH
3Department of Neurosurgery and Brain Repair, Center for Aging and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL
4Neurostructural Research Labs, Inc., Tampa, F 

Abstract
A large body of evidence from molecular, cellular and human studies suggests that lithium may enhance synaptic plasticity, which may be associated with its therapeutic efficacy. However, only a small number of studies have directly assessed this. To determine whether lithium treatment alters structural synaptic plasticity, this study examined the effect of 4 wk lithium treatment on the amount and distribution of dendrites in the dentate gyrus (DG) and hippocampal area CA1 of young adult rats. Following 4 wk lithium or control chow feeding, animals were decapitated, the hippocampi were prepared and stained using a rapid Golgi staining technique and the amount and distribution of the dendritic branching was evaluated using Sholl analyses (method of concentric circles). In the DG, lithium treatment increased the amount and distribution of dendritic branches in the proximal half of dendritic trees of the granule cells and reduced branching in the distal half. In area CA1, the same treatment also increased the number of dendritic branches in the proximal half of apical dendritic trees of CA1 pyramidal cells and reduced branching in the distal half of apical dendritic trees but had no effect on basilar dendritic trees. The lithium treatment altered the total density of dendritic trees in neither the DG nor area CA1. These findings suggest that, in the DG and apical CA1, chronic lithium treatment rearranges neuronal morphology to increase dendritic branching and distribution to where major afferent input is received.


 

To be presented at the Society for Neuroscience, San Diego, CA (2013)

Hippocampal CA1 Dendritic Neuroplasticity in Mild Cognitive Impairment during the Progression of Alzheimer’s disease


R. F. MERVIS1,2, S. K. FOLEY1,3, C. J. PATEL3, S. ZAJD3, M. ARADI3,  G. S. WALHA3, P. SOLANKI3, S. W. SCHEFF4, E. J. MUFSON5;

1Neurostructural Research. Labs, Tampa, FL;
2Ctr. for Aging and Brain Repair, Univ. of South Florida Morsani College of Medicine., Tampa, FL;
3The Honors College., Univ. of South Florida, Tampa, FL;
4Sanders-Brown Ctr. on Aging, Univ. of Kentucky, Lexington, KY;
5Dept of Neurological. Sciences., Rush Univ. Med. Ctr., Chicago, IL

 
Abstract
Mild Cognitive Impairment (MCI) is a prodromal stage of Alzheimer’s disease (AD) and as many as thirty percent of these individuals transition to AD. The CA1 projection neurons of the hippocampus, a hub of the medial temporal lobe memory circuit, undergo degeneration early in the disease process. However, there is very little information on the structural integrity of CA1 neurons during the onset of AD. The present study characterized changes in the dendritic branching of the basilar tree of hippocampal CA1 pyramidal neurons using formalin-fixed tissue for Golgi impregnation and analyzed for dendritic branching in cases harvested from the University of Kentucky Alzheimer’s Center who died with a clinical diagnosis of no cognitive impairment (NCI), MCI, and AD. Camera lucida drawings were prepared of the basilar tree of randomly selected CA1 neurons. Analysis of the amount, distribution and complexity of the arbor showed a significant increase in these parameters (length, + 18%; complexity, +23%) in MCI CA1 neurons compared to NCI. Conversely, there was a significant reduction in branch length (-39%) and arbor complexity (-25%) in the progression from MCI to AD.. These findings suggest that the observed increase in CA1 dendritic parameters is a neuroplastic compensatory response to a loss of cortical or subcortical input early in the course of the disease. CA1 neuroplasticity represents a viable window for potential therapeutic strategies aimed at restoring or maintaining hippocampal function during MCI.


To be presented at the Society for Neuroscience, San Diego, CA (2013)

Neurotoxic Effects of Pre-weaning Benzyl Butyl Phthalate (BBP) Exposure on Dendritic Parameters in the Rat

S. K. FOLEY1,2, R. F. MERVIS3,1, K. SHAKFEH2,
P. HANNA2, S. BHASKAR2, D. PHAM2, D. DEBARTOLO4, A. BETZ4

1Neurostructural Research. Labs, Tampa, FL;
2The Honors College., University of South Florida, Tampa, FL;
3Dept of Neurosurgery & Brain Repair, Center for Aging and Brain Repair, Univ. of South Florida Morsani College of Medicine., Tampa, FL;
4Dept of Psychology, Quinnipiac University., Hamden, CT
 
 
Abstract
Benzyl butyl phthalate (BBP) is a widely used industrial plasticizer which, if ingested, appears to have endocrine disrupting properties and may have deleterious consequences on the developing nervous system. In the present study, pregnant Sprague-Dawley rats were fed sweetened food pellets containing 10ug/ml of BBP. This dose was continued up until post-natal day 23 at which time the rat pups were sacrificed. Pregnant control rats received sweetened food pellets with no BBP. All brains were fixed in 10% neutral buffered formalin. Coronal blocks incorporating the parietal cortex and underlying hippocampus of 4 BBP-treated and 4 control 23 day-old male rats were Golgi stained. From coded slides camera lucida drawings of randomly selected granule cells of the dentate gyrus were prepared and evaluated for extent, complexity, and distribution of the dendritic arbor. Granule cell morphology was largely unaffected by this pre-weaning BBP exposure. Layer V pyramids, conversely, were significantly impacted by BBP. Analysis of the basilar dendritic tree showed that the BBP-exposed pups had significantly larger and more complex dendritic trees of the cortical pyramids. Dendritic spine density on the layer V pyramids was not significantly altered, but greater branching indicates that there was an overall increased synaptic presence in the layer V basilar tree. The present findings suggest that pre-weaning BBP exposure - e.g., during the period of the rodent brain growth spurt - may contribute to either additional cortical dendritic growth or diminished dendritic pruning in the course of the maturation of cortical circuits. Additional studies will determine if this is a transient or long-lasting phenomena, and if additional cell populations are also at risk from BBP exposure. Regardless, there is now evidence that environmental BBP may have deleterious consequences on neuronal development which could influence brain circuitry and, ultimately, behavior.


To be presented at the Society for Neuroscience, San Diego, CA (2013)

Treatment of Mild Traumatic Brain injury in Mice with a Nrf2 Activator Affects
Dendritic Complexity
 
Bruce A. Citron1, Jessica N. Chang1, Ronald F. Mervis2,4,
Stephanie K. Foley2, Peter Hanna2, Lital Rachmany3, Anat Shaer3
Vardit Rubovitch3, and Chaim G. Pick3
 

1Laboratory of Molecular Biology, Research & Development 151, Bay Pines VA Healthcare System, Bay Pines, FL  and Department of Molecular Medicine, University of South Florida Morsani College of Medicine, Tampa, FL
2NeuroStructural Research Labs, Inc, Tampa, FL.
3Department of Anatomy and Anthropology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
4Center for Aging and Brain Repair, Dept of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL
  
Abstract
Worldwide, the incidence of TBI is approximately 0.5% per year and the vast majority of TBIs are mild yet they frequently do result in significant negative effects on brain function.  Our lab has uncovered molecular mechanisms, in particular certain inflammatory responsive regulatory factors involved in the health of neurons.  We have previously presented cognitive improvements produced by treatment activating the transcription factor, Nrf2, by administration of tBHQ after injury and have identified pathway changes involved in the neuroprotection.  Here we describe the changes seen in dendritic complexity after tBHQ treatment and TBI in mice.  We employed a closed head system involving a weight drop induced impact and rotational injury and have measured behavioral, mRNA, and protein changes.  To determine alterations in connectivity, brain samples were stained with the Golgi method to quantitatively assess dendritic branching and spine morphologies and densities.  Dendritic complexity, determined by several different measures including branch point analysis, was higher in the untreated TBI brains compared to sham injured or to brains from mice exposed to TBI plus treatment.  This may indicate a preferential loss of the least connected neurons in the injured, untreated mice.  Through these experiments we seek to develop effective treatment strategies to combat the secondary effects of traumatic brain injury.


Published in Neuroscience (2013)

Dendritic Spine Alterations in the Hippocampus and Parietal Cortex of Alpha7 Nicotinic Acetylcholine Receptor Knockout Mice

BJ Morley1 and RF Mervis2,3

1Boys Town National Research Hospital, Omaha, NE
2Neurostructual Research Labs, Inc., Tampa, FL
3Center for Aging and Brain Repair, Dept of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL
 
Abstract
The α7 nicotinic acetylcholine receptor (nAChR) is involved in higher cognitive and memory functions, and is associated with the etiology of neurological diseases involving cognitive decline, including Alzheimer's disease (AD). We hypothesized that spine changes in the α7 knockout might help to explain the behavioral deficits observed in α7 knockout mice and prodromal hippocampal changes in AD. We quantified several measures of dendritic morphology in the CA1 region of the mouse hippocampus in Golgi-stained material from wildtype and α7 knockout mice at P24. The most significant difference was a 64% increase in thin (L-type) dendritic spines on the CA1 basilar tree in knockout mice (p<.05). There were small decreases in the number of in N-type (-15%), M-type (-14%) and D-type (-4%) spine densities. The CA1 basilar dendritic tree of knockout mice had significantly less branching in the regions near the soma in comparison with wildtype animals (p<.01), but not in the more distal branching. Changes in the configuration of CA1 basilar dendritic spines have been observed in a number of experimental paradigms, suggesting that basilar dendritic spines are highly plastic. One component of cognitive dysfunction may be through α7-modulated GABAergic interneurons synapsing on CA1 basal dendrites


To be presented at the Radiation Research Society, New Orleans, September, 2013

Effect of Fractionated Whole Brain Irradiation on Neuronal Morphology of rat cortex and hippocampus: Impact of late delayed brain injury

Dana M. Greene-Schloesser, PhD1; A Mehta2; S K. Foley2,4
 S Rye-Buckingham2; T Stalas2; Ann M. Peiffer, PhD1; Linda Metheny-Barlow, PhD1; Mike E. Robbins1; and Ronald F. Mervis, PhD3,4

1Wake Forest University Baptist Medical Center, Winston-Salem, NC;
 2The Honors College, University of South Florida, Tampa, FL;
3University of South Florida Morsani College of Medicine, Tampa, FL
4Neurostructural Research Labs, Inc., Tampa, FL 

 
Abstract
Fractionated partial or whole-brain irradiation (fWBI) is routinely used for the treatment of primary and metastatic brain cancer. Presently, ~200,000 patients/year are treated with fWBI, and ~ 100,000/year survive long enough (>6 months) to develop radiation-induced brain injury, including cognitive impairment, which can adversely impact their quality of life. Using a well-established fWBI rat model, the effects of fWBI on brain microcircuitry were evaluated 6 months following irradiation.
Methods: Six 10-12 week old F344xBN male rats were lightly anesthetized and received either: (1) 40 Gy fWBI or, (2) sham-irradiation. Coronal tissue blocks encompassing parietal cortex and hippocampus were Golgi stained. From coded slides, granule cells of the dentate gyrus (DG; 9 neurons/brain) and layer V pyramids (6 neurons/brain) were randomly selected for analysis of amount, distribution and complexity of the dendritic arbors. Dendritic spines were also evaluated on DG granule cells (5 neurons/brain).
Results: DG Granule Cell branching: Total branch length was not affected by fWBI however; the distribution of the arbor was shifted in the fWBI subjects with more dendritic material in the inner 2/3rds and less arbor in the outer 3rd of the tree. This was accompanied by a similar redistribution of dendritic complexity. Dendritic spines on the granule cells showed no significant change; however, Mushroom (M)-type spines in the neurons of fWBI subjects showed a 16% loss. Layer V Pyramidal Cell branching: A significant loss of dendritic arbor in the neurons of fWBI subjects was seen restricted to the inner half of the tree. This loss of arbor was mirrored by a reduction of dendritic arbor complexity.
Conclusions: Six months following 40 Gy fWBI, rats that received fWBI had significant alterations in dendritic morphology. Hippocampal and cortical neurons were significantly affected by distortion (granule cells) or reduction (layer V pyramids) of the arbor. Additionally, fWBI led to a significant decrease in the number of M-type spines in granular cells of the DG. These spines are thought to be related to memory function. In turn, these dendritic changes could negatively impact communication between neurons and represent a neurostructural basis for late-delayed radiation-induced cognitive impairment

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Published in Behavioral Brain Research 2012
 
Dendritic morphology of amygdala and hippocampal neurons in more and less predator stress responsive rats and more and less spontaneously anxious handled controls

*R. ADAMECa, M. HEBERTa, J. BLUNDELLa, R. F. MERVISb,c

a
Memorial University, St. John's, Newfoundland, Canada, A1B 3X9;
bCenter of Excellence for Aging & Brain Repair, Dept of Neurosurgery & Brain Repair, Univ South Florida College of Medicine, Tampa, FL;
cNeurostructural Research Labs, Inc., 12409 Telecom Drive, Tampa, FL 33637, USA
 

Abstract
We investigated the neurobiological bases of variation in response to predator stress (PS). Sixteen days after treatment (PS or handling), rats were grouped according to anxiety in the elevated plus maze (EPM).  Acoustic startle was also measured. We examined the structure of dendritic trees of basolateral amygdala (BLA) output neurons (stellate and pyramidal cells) and of dorsal hippocampal (DHC) dentate granule cells of less anxious (LA) and more (extremely) anxious (MA) stressed animals (PSLA and PSMA).  Handled controls (HC) which were less anxious (HCLA) and spontaneously more anxious (HCMA) equivalently to predator stressed subgroups were also studied. Golgi analysis revealed BLA output neurons of HCMA rats exhibited longer, more branched dendrites with higher spine density than the other groups of rats, which did not differ. Finally, spine density of DHC granule cells was equally depressed in HCMA and PSMA rats relative to HCLA and PSLA rats. Total dendritic length of BLA pyramidal and stellate cells (positive predictor) and DHC spine density (negative predictor) together accounted for 96% of the variance of anxiety of handled rats. DHC spine density was a negative predictor of PSMA and PSLA anxiety, accounting for 70% of the variance. Data are discussed in the context of morphological differences as phenotypic markers of a genetic predisposition to anxiety in handled controls, and a possible genetic vulnerability to predator stress expressed as reduced spine density in the DHC. Significance of findings for animal models of anxiety and hyperarousal comorbidities of PTSD are discussed.

 
Presented at the Society for Neuroscience, New Orleans, LA. (2012)
Dendritic Alterations in Transthyretin (TTR) Knockout Mice

R. F. MERVIS1,2, S. K. FOLEY3,2, P. PANTCHEVA2, I. PASSIOURA3, M. KONSTANTINOVIC3, S. RYE-BUCKINGAM3, M. ARADI3, P. SAWANGKUM3, R. CROUSE3, N. ARRUDA3.,J. N. BUXBAUM4

1Department of Neurosurgery. & Brain Repair, Univ. of South Florida Coll Med., TAMPA, FL; 2Neurostructural Res. Labs, Inc., Tampa, FL; 3The Honors College, Univ.of South Florida, Tampa, FL; 4Dept of Molecular  & Exptl. Medicine., The Scripps Research.Institute., La Jolla, CA

Abstract
Transthyretin (TTR) (formerly, thyroxine binding prealbumin) is an evolutionarily conserved serum and cerebrospinal fluid protein that transports holo-retinol-binding protein (RBP) and thyroxine. TTR can suppress the Alzheimer's disease phenotype in transgenic animal models of cerebral Abeta deposition. In the current study, we sought to evaluate dendritic morphology of cortical, hippocampal, and striatal neurons in TTR knockout mice. There were five 6 month-old controls and 4-5 age-matched TTR knockouts . Fixed brain tissue blocks were stained using a Rapid Golgi procedure and all slides were coded. Camera lucida drawings were made of randomly selected neurons of 3 populations: In the hippocampus, from granule cells of the dentate gyrus; in the neocortex, layer II-III pyramids of the parietal cortex; and, in the striatum, medium spiny neurons. Branching analyses of the KO mice showed no differences from controls for granule cells and only subtle loss in the cortical neurons. Striatal neurons, however, were significantly impacted in the TTR KO mice with atrophy of branching (-18%) and loss of dendritic complexity (-22%). The basis for the differential sensitivity of the various neuronal populations to the KO genotype is unknown but may be related to reduced levels of RBP or of thyroxine which is known to influence brain development.

Presented at the Society for Neuroscience Washington DC 2011 
 
Anxiety and Dendritic Spine Density in the Granule Cells of the Rat Hippocampal Dentate Gyrus: Implications for PTSD  

*N. SHAKFEH1, N. MEYLAN2, R. F. MERVIS2,3, R. ADAMEC4

1
The Honors College, Univ. of South Florida, Tampa, FL;
2Neurostructural Res. Labs, Inc., Tampa, FL;
3Center of Excellence for Aging & Brain Repair, Dept of Neurosurgery & Brain Repair, Univ South Florida College of Medicine, Tampa, FL
4Department of Psychology, Memorial University, St. John’s, Newfoundland, Canada
 
Abstract
Post-Traumatic Stress Disorder  (PTSD) is an anxiety response to severe stress.  Not all individuals exposed to the same stressor show a long-lasting fear response.  Using a rat model, the goal of this study was to ascertain the relationship between innate anxiety levels and a morphological indicator of hippocampal brain circuitry: the spine density on granule cells of the dentate gyrus of the dorsal hippocampus.

Synaptic density may be altered in the presence of various stressors.In a rat, predator stress (e.g, exposure to a cat).is an intense stressor The response to predator stress is also contingent upon whether the subject’s innate level of anxiety is low (e.g, subject is well adapted) or high (e.g., mal-adapted).  Anxiety was previously determined by the rat’s response in an elevated plus maze. In the present study, we investigated if less anxious (LA) and more anxious (MA) stressed and unstressed animals differed in the dendritic spine density of granule cells of the dentate gyrus of the dorsal hippocampus from right and left hemispheres.

Two weeks after exposure to predator stress (or non-stress “handled” conditions), the rats were sacrificed.  The fixed brain tissue was Golgi stained and coded slides prepared. Dendritic spine density was assessed on randomly selected granule cells of the dorsal hippocampal dentate gyrus from both hemispheres.

Analysis showed that spine density of the granule cells was bilaterally and equally depressed in both high anxious groups (stressed and unstressed) relative to the low anxious groups. Thus, regardless of exposure to a stressor, the granule cells from “high anxious”  (= mal-adapted) rats had significantly  less spine density  (- 10-14%) than from the “less anxious”  (= well-adapted) rats.

These results suggest that altered hippocampal circuitry (partially a function of dendritic spine density) in the mal-adapted (high anxiety) rats may represent the basis for a possible genetic vulnerability to stress anxiety, a co-morbid symptom of PTSD.

 

Presented at the Society for Neuroscience Washington DC 2011 
 
Cortical Astrocyte Morphologic Alterations During the Progression of Alzheimer’s  Disease

*S. K. FOLEY1, R. LCROUSE1, N. MEYLAN2,
S. SCHEFF
3, E. MUFSON4, R. F. MERVIS2,5

1The Honors College, Univ. of South Florida, Tampa, FL;
2Neurostructural Res. Labs, Inc., Tampa, FL;
3Sanders-Brown Ctr. for Aging, Univ. of Kentucky, Lexington, KY;
4Dept. Neurological Sciences, Rush Medical University, Chicago, IL;
5Center of Excellence for Aging & Brain Repair, Dept of Neurosurgery & Brain Repair, Univ South Florida College of Medicine, Tampa, FL

Abstract
Astrocytes are the most numerous cells in the brain and play a critical role in maintaining various homeostatic responses within the CNS. Protoplasmic astrocytes, which are primarily located  in cortical grey matter rcrouse@mail.usf.edu play an important but enigmatic role in the pathophysiology of many neurodegenerative diseases.  The objective of this study was to evaluate changes in cortical protoplasmic morphology and whether these structural changes correlated with the progression of Alzheimer’s Disease (AD) -- from individuals who died with a clinical diagnosis of no cognitive impaired (NCI), mild Cognitive Impairment (MCI) and AD.

Tissue harvested from the inferior temporal cortex (Brodmann area 21) from each clinical group was Golgi impregnated and coded slides prepared for analysis of stained protoplasmic astrocytes located in superficial layers II-III.  Camera lucida drawings of areal domains from randomly selected astrocytes were evaluated and the numbers of branch tips of astrocytic processes were quantified.

Layer II-III astrocytes from NCI and MCI temporal cortex showed no difference in the extent of their areal domains.  However, astrocytes were significantly smaller (20%) in AD compared to NCI or MCI.  In all three groups there was no difference in the number of astrocytic branch processes.  These data suggest that in AD, there is a dying-back phenomenon of the astrocytic processes such that glia are smaller but without a reduction in their complexity. These findings indicate that the homeostatic capabilities in astrocytes may be compromised, thus contributing to the downward spiral of neuronal and synaptic damage in AD.

Supported by The USF Honors College and AG14449.

 

Presented at The 4th International Conference on Stress and Behavior, Tulane University School of Medicine, New Orleans, LA 2011
 
Dendritic Alterations and Neurocircuitry Abnormalities in Stress and Neuropsychiatric-Related Disorders.

Ronald F. Mervis, PhD.,

Abstract
The dendritic arbor of the typical neuron of the mammalian brain comprises over 95% of the volume of the neuron.  In addition, the dendrites of almost all neurons are embellished with thousands of dendritic spines which serve as the post-synaptic loci for the transfer of information between neurons.  Morphometric assessment of dendritic branching and spines of a neuronal population therefore represents a critical index of the microcircuitry of those neurons.  Dysmorphic alterations in these dendritic parameters include both atrophic and neuroplastic changes which can influence transfer of information -- disruption of which ultimately may be reflected by changes in learning, memory, or behavior.  Examples of these scenarios are presented which will demonstrate how neuroplastic and/or neurodegenerative changes in dendritic branching and spines may impact on brain circuitry and behavior. 
 

 

Presented at the Society for Neuroscience, San Diego, Nov., 2010

Distinctive patterns of alterations in dendritic branching are seen in different cortical regions in mild cognitive impairment and Alzheimer’s disease.


*V. LOZANO1,2, A. R. LOZANO2,3, J. J. MILLER4,5, R. A. LONG2,3, J. D. KOTICK3,6,
M. L. SHAH
3,7, A. WINKLER8, S. SCHEFF10, E. J. MUFSON11, R. F. MERVIS9;

1Palm Harbor, FL;
2The Honors College, Univ. of South Florida, Tampa, FL;
3Neurostructural Res. Labs, Inc., Tampa, FL;
4Neurostructural Res. Labs, Inc, Tampa, FL;
5Program in Mol. Med., Univ. of South Florida, Tampa, FL;
6Univ. of Miami Miller Sch. of Med., Miami, FL;
7Univ. of Florida Col. of Med., Gainesville, FL;
8Univ. of South Florida Col. of Med., Tampa, FL;
9Ctr. for Aging and Brain Repair, Dept. Neurosurg.,
10Sanders-Brown Ctr. for Aging, Univ. of Kentucky, Lexington, KY;
11Neurolog. Sci., Rush Univ. Med. Ctr., Chicago, IL

Abstract
Alzheimer’s Disease (AD) is the major cause of dementia in the elderly. The extensive cognitive dysfunction associated with frank AD is often initially apparent as a subtle form of memory loss referred to as Mild Cognitive Impairment (MCI). The dendritic branching arbor of cortical neurons comprises approximately 95% of the volume of the neuron; therefore, it is reasonable that loss of dendritic branching network (and the concomitant loss of synapses on those branches) would be reflected by diminished cognitive function. Using postmortem tissue harvested from 3 different cortical regions, e.g., superior frontal cortex (SFC, Brodmann area 9), inferior parietal cortex (IPC, areas 39,40), and inferior temporal cortex (ITC, area 21), the purpose of the present study was to assess changes in dendritic branching in individuals diagnosed as Non-Cognitively Impaired (NCI), MCI, or AD. The tissue was stained by the rapid Golgi method. All slides were coded, and layer II-III pyramidal neurons were randomly selected for analysis by investigators blinded to clinical status. Quantitative changes in the amount and complexity of dendritic branching of the basilar tree were evaluated. Results showed that each cortical region had a distinctive pattern of dendritic change in the progression from normal aging (NCI) to MCI and thence to AD. The ITC showed an initial 20% loss of branching in MCI, with a subsequent additional 5% loss of branching in AD for a total loss of 25%. In the IPC, in MCI, II-III pyramids initially showed a mild (-4%) loss of branching in MCI, with a subsequent additional 10% loss in AD. In contrast, in the SFC, the pyramidal cells showed an initial 16.5% increase in dendritic branching in MCI, with a subsequent 36% loss of branching in AD. Compared to the NCI group, the total loss of branching in the frontal cortex in AD amounted to 20% of the dendritic arbor. This initial neuroplastic increase in dendritic branching in MCI may reflect a compensatory response by the damaged neurons of the frontal lobe to maintain cortical function.

 

Presented at the Society for Neuroscience, San Diego, Nov., 2010

Alterations of cortical microcircuitry in Alzheimer’s disease and mild cognitive impairment: Changes in dendritic spines in layer II-III Pyramids


*S. ARADI1,2, J. D. KOTICK3,4, M. L. SHAH5,4, A. WINKLER6, A. R. LOZANO2,4, K. KASIMOS2, S. SCHEFF8, E. J. MUFSON9, R. F. MERVIS7,4;

1Largo, FL;
2The Honors College, Univ. of South Florida, Tampa, FL;
3Univ. of Miami Miller Sch. of Med., Miami, FL;
4Neurostructural Res. Labs, Inc., Tampa, FL;
5Univ. of Florida Col. of Med., Gainesville, FL;
6Univ. of South Florida Col. of Med., Tampa;
7Ctr. of Aging and Brain Repair, Dept Neurosurg., FL;
8Sanders-Brown Ctr. on Aging, Univ. of Kentucky Col. of Med., Lexington, KY;
9Dept of Neurolog. Sci., Rush Univ. Med. Ctr., Chicago, IL

Abstract
Dendritic spines are the primary loci for synaptic transfer of information between neurons. Loss of dendritic spines would likely be associated with cognitive impairment in Alzheimer’s disease (AD), the major cause of dementia in the elderly. The initial manifestation of subtle memory loss is Mild Cognitive Impairment (MCI) and this is associated with a higher risk for the subsequent development of Alzheimer’s disease. In this study we wanted to evaluate how dendritic spines were being affected in the progression from normal aging -- e.g., non-cognitive impairment (NCI) ? MCI ? AD. Postmortem tissue was harvested from different cortical regions: superior frontal cortex (SFC, Brodmann area 9), inferior parietal cortex (IPC, area 39,40), and superior temporal cortex (STC, area 21). Fixed tissue blocks were stained using the Golgi method. All slides were coded. Dendritic spines were quantified on randomly selected layer II-III pyramids from the frontal, parietal, and temporal cortices. In MCI, the SFC showed a non-significant spine loss of 3.5%; however, in AD there was an additional 15% spine reduction (p < 0.05). In the ITC, in MCI, layer II-III pyramids showed a 10% reduction of spines compared to the NCI group. In AD, there was also an additional 15% loss of spines, for a total loss of 25% (p < 0.05 compared to the NCI group). In the IPC, MCI was associated with a significant 22% loss of spines (p < 0.05). There was no additional significant loss of spines in AD. In sum, in AD all cortical regions examined showed a significant loss of dendritic spines - reflecting a breakdown in cortical microcircuitry. However, in MCI, the initial spine loss in the SFC was minimal (-4%), with greater spine loss in the ITC, and significantly more MCI-associated spine loss in the parietal cortex. This data suggests that dendritic spines from different cortical regions have various degrees of vulnerability to the initial manifestations of MCI, but that AD is associated with significant spine loss in all cortical regions evaluated.

 

Presented at the Society for Neuroscience, San Diego, Nov., 2010

Structural neuroplasticity and neurodegeneration within the cortical connectome in mild cognitive impairment and Alzheimer's disease

*R. F. MERVIS1,2, S. ARADI3, V. LOZANO3, A. R. LOZANO3,2, R. A. LONG3, J. D. KOTICK4,2,
A. WINKLER
5, M. SHAH2,6, K. KASIMOS3, J. J. MILLER3,2, S. SCHEFF7, E. J. MUFSON8;

1Neurosurg. & Brain Repair, Univ. South Florida Coll Med., TAMPA, FL;
2Neurostructural Res. Labs, Inc., Tampa, FL;
3Honors College, Univ. South Florida, Tampa, FL;
4Univ. of Miami Miller Sch. of Med., Miami, FL;
5USF Col. of Med., Tampa, FL;
6Univ. of Florida Col. of Med., Gainesville, FL;
7Sanders-Brown Ctr. on Aging, Univ. of Kentucky, Lexington, KY;
8Dept of Neurolog. Sci., Rush Univ. Med. Ctr., Chicago, IL

Abstract
People with mild cognitive impairment (MCI), who present with memory loss, are at a higher risk for the development Alzheimer’s disease (AD). The disruption of the neuronal “connectome”, which represents the network of elements and connections underlying the neurostructural substrate of cognition and memory, plays a key role in the onset of dementia. Alterations in brain circuitry can be evaluated utilizing a morphometric assessment of dendritic branching and spines. Here, we characterized the earliest alterations in cortical circuitry associated with MCI; and, subsequently, any additional disruption of the connectome associated with onset of frank AD. We compared changes in layer II-III pyramidal cell morphology from 3 cortical regions: the inferior parietal cortex (IPC, Brodmann areas 39, 40), the inferior temporal cortex (ITC, area 21), and the superior frontal cortex (SFC, area 9). Formalin-fixed cortical tissue was harvested from individuals diagnosed antimortem with a clinical diagnosis of No Cognitive Impairment (NCI), MCI, or AD. Cortical tissue blocks were Golgi stained, all slides coded, and layer II-III pyramids were randomly selected for dendritic branching and spine analysis of the basilar dendritic arbor. A “Global Circuitry Index” (GCI) was devised to integrate changes of both dendritic parameters into a unitary component. Overall analysis revealed different patterns of alterations of brain circuitry with increasing loss of cognitive function depending upon the cortical region examined. In the MCI IPC, there was an initial 28% reduction of circuitry compared to NCI with essentially no additional loss in AD (total reduction of 29%). In MCI ITC, there was an initial 35.5% decrease in the connectome GCI followed by a additional 14.3% decrease in AD (total loss relative to NCI = 44.8%). The change in the GCI in the frontal cortex was uniquely different: In MCI, there was a striking 37% increase in the GCI frontal cortex layer II-III pyramids compared to NCI. This was followed by a 55.7% reduction of the index from MCI to AD. Overall, this left the AD SFC with an 18.7% loss in circuitry compared to normal NCI brain tissue. Thus, as opposed to the initial diminution of the circuitry index associated with MCI seen in IPC and ITC, frontal cortex pyramidal neurons display a remarkable compensatory neuroplastic response in MCI, which may be an attempt to rescue the cortical connectome and ,in turn, to maintain cognitive function prior to additional disruption of cortical circuitry in frank AD.

 

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Published in the Journal of Neuroscience 2010
 
SIRT1 Is Essential for Normal Cognitive Function and sSynaptic Plasticity
 
Shaday Michán1,2*, Ying Li3,4*, Maggie Meng-Hsiu Chou3, Edoardo Parrella4, Huanying Ge5, Jeffrey M. Long6, Joanne S. Allard6, Kaitlyn Lewis6, Marshall Miller6, Wei Xu3,
Ronald F. Mervis7, Jing Chen8, Karen I. Guerin8, Lois E. H. Smith8, Michael W. McBurney9, David A. Sinclair1, Michel Baudry3, Rafael de Cabo
6, Valter D. Longo3,4.

Author addresses:
1Paul F. Glenn Laboratories, Department of Pathology,
Harvard Medical School, Boston, MA, USA.
2Instituto de Geriatría, Institutos Nacionales de Salud, México D.F., México.
3Neuroscience Program, University of Southern California, Los Angeles, CA, USA.
4Andrus Gerontology Center and Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA.
5Molecular and Computational Biology, Department of Biology Sciences, University of Southern California, Los Angeles, CA, USA.
6Laboratory of Experimental Gerontology, National Institute on Aging, National Institutes of Health, Bethesda, Maryland, USA.
7Center of Excellence in Aging and Brain Repair, University of South Florida & NeuroStructural Research Labs Inc. FL, USA.
8Department of Ophthalmology, Harvard Medical School, Children’s Hospital Boston, MA, USA. 9Ottawa Health Research Institute, Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada.

Abstract
Conservation of normal cognitive functions relies on the proper performance of the nervous system at the cellular and molecular level. The mammalian NAD+-dependent deacetylase, SIRT1, impacts different processes potentially involved in the maintenance of brain integrity such as chromatin remodeling, DNA repair, cell survival and neurogenesis. Here we show that SIRT1 is expressed in neurons of the hippocampus, a key structure in learning and memory. Using a combination of behavioral and electrophysiological paradigms we analyzed the effects of SIRT1 deficiency and overexpression on mouse learning and memory as well as on synaptic plasticity. We demonstrated that the absence of SIRT1 impaired cognitive abilities, including immediate memory, classical conditioning and spatial learning. In addition, we found that the cognitive deficits in SIRT1 knockout mice were associated with defects in synaptic plasticity without alterations in basal synaptic transmission or NMDA receptor function. Brains of SIRT1-KO mice exhibited normal morphology and dendritic spine structure but display a decrease in dendritic branching, branch length and complexity of neuronal dendritic arbors. Also, a decrease in ERK1/2 phosphorylation and altered expression of hippocampal genes involved in synaptic function, lipid metabolism and myelination were detected in SIRT1-KO mice. In contrast, mice with high levels of SIRT1 expression in brain exhibited regular synaptic plasticity and memory. We conclude that SIRT1 is indispensable for normal learning, memory and synaptic plasticity in mice.

 

Presented at the Meeting of the American Society for Neural Transplantation and Repair, Clearwater Beach, Florida, 2009

MINIMAL TRAUMATIC BRAIN INJURY IN A MOUSE MODEL OF MULTIPLE CONCUSSIONS: EFFECTS ON CORTICAL DENDRITIC SPINES.
Preliminary Findings.

Scott Moradian1, Chiam G. Pick2, Jared Kaminsky3, and Ronald F. Mervis3,4

1The Honors College, The University of South Florida, Tampa, FL 33612;
2
Dept of Anatomy and Anthropology, Tel-Aviv University,
Sackler School of Medicine, Tel-Aviv 69978, Israel;
3
Neurostructural Research Labs, Inc. Tampa, FL 33637;
4
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida College of Medicine, Tampa, FL 33612.

Abstract
Single and multiple mild concussions are a frequent occurrence in sports. Clear morphological deficits are difficult to discern but there may be long lasting behavioral and learning disturbances, the basis of which is poorly understood. The Golgi impregnation method randomly stains the dendritic branches and spines of about 6-8% of all neurons. The vast majority of synapses occur on the dendritic spines. In this study, using analysis of Golgi-impregnated neurons, we evaluated these dendritic parameters in a closed-head model of minimal traumatic brain injury (mTBI) in mice exposed to a weight drop of 10g (to the temporal right hemisphere) either 1 time, or for 2 or 3 times (with a week interval between mTBI episodes). An additional group was used to compare the effects a single 30g weight drop. Animals were sacrificed 72 hours after the last traumatic event. All slides were coded and layer V pyramids (apical and basilar trees) from the injured hemisphere were randomly selected for spine analysis. Most notable in these early findings are that: (1) a single mTBI episode appeared not to significantly affect dendritic spines; (2) two mTBI episodes appeared to reduce spines in both apical and basilar trees; and (3) surprisingly, three 10mg mTBI episodes or a single 30g mTBI episode was not accompanied by spine loss, but by an increase in spines. Although the time frames for these 2 groups are different, these findings may be attributed to a compensatory dendritic hypertrophy mechanism associated with neuronal dropout. Additional assessment of dendritic branching and stereological evaluation of neuronal density will further clarify the roles of these parameters in modifying cortical circuitry following multiple concussion-related traumatic brain injury.

 

Published in Epilepsia. 2009 Apr;50(4):629-45. Epub 2008 Jul 24.

Decreased number of interneurons and increased seizures in neuropilin 2 deficient mice: implications for autism and epilepsy.
Gant JC, Thibault O, Blalock EM, Yang J, Bachstetter A, Kotick J, Schauwecker PE, Hauser KF, Smith GM, Mervis R, Li Y, Barnes GN.

Departments of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, Lexington, KY, USA.

Abstract
PURPOSE: Clinically, perturbations in the semaphorin signaling system have been associated with autism and epilepsy. The semaphorins have been implicated in guidance, migration, differentiation, and synaptic plasticity of neurons. The semaphorin 3F (Sema3F) ligand and its receptor, neuropilin 2 (NPN2) are highly expressed within limbic areas. NPN2 signaling may intimately direct the apposition of presynaptic and postsynaptic locations, facilitating the development and maturity of hippocampal synaptic function. To further understand the role of NPN2 signaling in central nevous system (CNS) plasticity, structural and functional alterations were assessed in NPN2 deficient mice. METHODS: In NPN2 deficient mice, we measured seizure susceptibility after kainic acid or pentylenetetrazol, neuronal excitability and synaptic throughput in slice preparations, principal and interneuron cell counts with immunocytochemical protocols, synaptosomal protein levels with immunoblots, and dendritic morphology with Golgi-staining. RESULTS: NPN2 deficient mice had shorter seizure latencies, increased vulnerability to seizure-related death, were more likely to develop spontaneous recurrent seizure activity after chemical challenge, and had an increased slope on input/output curves. Principal cell counts were unchanged, but GABA, parvalbumin, and neuropeptide Y interneuron cell counts were significantly reduced. Synaptosomal NPN2 protein levels and total number of GABAergic synapses were decreased in a gene dose-dependent fashion. CA1 pyramidal cells showed reduced dendritic length and complexity, as well as an increased number of dendritic spines. DISCUSSION: These data suggest the novel hypothesis that the Sema 3F signaling system's role in appropriate placement of subsets of hippocampal interneurons has critical downstream consequences for hippocampal function, resulting in a more seizure susceptible phenotype.

 

Published in Environmental and Health Perspectives (2009) Mar;117(3):426-35.

Developmental exposure to polychlorinated biphenyls interferes with experience-dependent dendritic plasticity
and ryanodine receptor expression in weanling rats.

Yang D, Kim KH, Phimister A, Bachstetter AD, Ward TR, Stackman RW, Mervis RF, Wisniewski AB, Klein SL, Kodavanti PR, Anderson KA, Wayman G, Pessah IN, Lein PJ.

Center for Research on Occupational and Environmental Toxicology, Oregon Health & Science University, Portland, Oregon, USA.

Abstract
BACKGROUND: Neurodevelopmental disorders are associated with altered patterns of neuronal connectivity. A critical determinant of neuronal connectivity is the dendritic morphology of individual neurons, which is shaped by experience. The identification of environmental exposures that interfere with dendritic growth and plasticity may, therefore, provide insight into environmental risk factors for neurodevelopmental disorders. OBJECTIVE: We tested the hypothesis that polychlorinated biphenyls (PCBs) alter dendritic growth and/or plasticity by promoting the activity of ryanodine receptors (RyRs). METHODS AND RESULTS: The Morris water maze was used to induce experience-dependent neural plasticity in weanling rats exposed to either vehicle or Aroclor 1254 (A1254) in the maternal diet throughout gestation and lactation. Developmental A1254 exposure promoted dendritic growth in cerebellar Purkinje cells and neocortical pyramidal neurons among untrained animals but attenuated or reversed experience-dependent dendritic growth among maze-trained littermates. These structural changes coincided with subtle deficits in spatial learning and memory, increased [3H]-ryanodine binding sites and RyR expression in the cerebellum of untrained animals, and inhibition of training-induced RyR upregulation. A congener with potent RyR activity, PCB95, but not a congener with negligible RyR activity, PCB66, promoted dendritic growth in primary cortical neuron cultures and this effect was blocked by pharmacologic antagonism of RyR activity. CONCLUSIONS: Developmental exposure to PCBs interferes with normal patterns of dendritic growth and plasticity, and these effects may be linked to changes in RyR expression and function. These findings identify PCBs as candidate environmental risk factors for neurodevelopmental disorders, especially in children with heritable deficits in calcium signaling.

 

Published in Journal of Neuroimmune Pharmacol. 2008 Dec;3(4):241-5.

Chronic methamphetamine induces structural changes in frontal cortex neurons and upregulates type I interferons.
Coutinho A, Flynn C, Burdo TH, Mervis RF, Fox HS.

Molecular and Integrative Neurosciences Department, The Scripps Research Institute, 10550 N. Torrey Pines Road, SP30-2030 La Jolla, CA 92037, USA.

Abstract
While methamphetamine-induced changes in brain neurotransmitters, their receptors, and transporters are well studied, the means by which methamphetamine abuse results in cognitive and behavioral abnormalities is unknown. Here, we administered methamphetamine chronically, in doses relevant to recreational usage patterns, to nonhuman primates. Neurostructural analysis revealed decreased dendritic material and loss of spines in frontal lobe neurons. Molecular examination demonstrated that type I interferons (interferon-alpha and interferon-beta) increased in the frontal lobe in response to chronic methamphetamine treatment, in correlation with the neuronal changes. Chronic methamphetamine thus results in significant changes in the primate brain, inducing cytokines and altering neuronal structure, both of which can contribute to functional abnormalities.

 

 Published in Pediatric Diabetes. 2008 Dec;9(6):531-9.

Hyperglycemia not hypoglycemia alters neuronal dendrites
and impairs spatial memory.
Malone JI, Hanna S, Saporta S, Mervis RF, Park CR, Chong L, Diamond DM.

Department of Pediatrics, University of South Florida College of Medicine, Tampa, FL, USA. jmalone@hsc.usf.edu

Comment in:
Pediatr Diabetes. 2008 Dec;9(6):527-30.

Abstract
BACKGROUND/OBJECTIVE: We previously reported that chronic hyperglycemia, but not hypoglycemia, was associated with the reduction of neuronal size in the rat brain. We hypothesized that hyperglycemia-induced changes in neuronal structure would have negative consequences, such as impaired learning and memory. We therefore assessed the effects of hyperglycemia and hypoglycemia on neuronal dendritic structure and cognitive functioning in young rats. DESIGN/METHODS: Experimental manipulations were conducted on male Wistar rats for 8 wk, beginning at 4 wk of age. At the completion of the treatments, all rats were trained in the radial-arm water maze, a spatial (hippocampus-dependent) learning and memory task. Three groups of rats were tested: an untreated control group, a streptozotocin-induced diabetic (STZ-D) group, and an intermittent hypoglycemic group. Following behavioral training, the brains of all animals were examined with histologic and biochemical measurements. RESULTS: Peripheral hyperglycemia was associated with significant increases in brain sorbitol (7.5 +/- 1.6 vs. 5.84 +/- 1.0 microM/mg) and inositol (9.6 +/- 1.4 vs. 7.1 +/- 1.1 microM/mg) and reduced taurine (0.65 +/- 0.1 vs. 1.3 +/- 0.1 mg/mg). Histologic evaluation revealed neurons with reduced dendritic branching and spine density in STZ-D rats but not in control or hypoglycemic animals. In addition, the STZ-D group exhibited impaired performance on the water maze memory test. CONCLUSIONS: Hyperglycemia, but not hypoglycemia, was associated with adverse effects on the brain polyol pathway activity, neuronal structural changes, and impaired long-term spatial memory. This finding suggests that the hyperglycemic component of diabetes mellitus has a greater adverse effect on brain functioning than does intermittent hypoglycemia.

 

Presented at the Meeting of the Society for Neuroscience 2008

Hippocampal dendritic branching and spine changes precede ß-amyloid plaque formation in the young ArcAß transgenic mouse model of Alzheimer’s Disease

*R. F. Mervis1,2, M. Knobloch3, N. Jani4, S. Moradian4, J. Yesudas4, B. A. Thomas4, L. Nattkemper5, U. Konietzko6 and R. M. Nitsch6

1Ctr. for Aging & Brain Repair, Univ. South Florida Coll Med., Tampa, FL
2Neurostructural Res. Labs, Inc., Tampa, FL
3Inst. of Cell Biol., ETH Zurich, Zurich, Switzerland
4The Honors College, Univ. of South Florida, Tampa, FL
5 Program in Mol. Med., The Grad. School, Univ. of South Florida, Tampa, FL
6Div. of Psychiatry Res., Univ. of Zurich, Zurich, Switzerland

Abstract:
The arcAß transgenic mouse expresses human APP695 with the combined Swedish and Arctic mutations. This results in a transgenic mouse model of brain ß-amyloid pathology caused by both increased production of Aß and enhanced formation of oligomeric Aß species. Small intraneuronal Aß aggregates occur as early as 3 months of age and precede ß-amyloid plaque formation in the neuropil by several months. Furthermore, severe behavioral deficits and impaired long-term potentiation also manifest before plaque deposition is detectable, suggesting a toxic role of small oligomeric Aß species in this mouse model. The purpose of this study was to determine if hippocampal dysfunction in these young mice (e.g., LTP deficits) correlates with alterations in dendritic branching and spines in hippocampus neurons. Methods: Formalin fixed brain blocks from 3.5 month-old transgenic mice (N= 5) and age-matched non-transgenic controls (N = 5) were stained using the rapid Golgi method. This technique shows the soma, dendritic branching and spines of a small randomly-stained population of neurons. All slides were coded. Dendritic branching and spine analyses of randomly selected hippocampal CA1s and granule cells of the dentate gyrus were carried out. Results: Sholl analysis of the basilar tree of CA1 pyramids in the 3.5 month-old ArcAß mice had significantly less dendritic branching (-35%, p < 0.0002) and complexity (-30%, p < 0.009) than age-matched non-transgenic littermate controls. There was also a significant loss of dendritic spines on the apical (-15%, p < 0.02) and basilar (-20%, p < 0.004) trees of the transgenic mice. For the granule cells of the dentate gyrus, the 3.5 month-old arcAß tg mouse again showed a significant loss of spines (12%, p < 0.01). Conclusions: The reduction in hippocampal dendritic branching and spines in the arcAß transgenic mice occurs before the deposition of extracellular ß-amyloid plaques, but coincides with the occurance of small intraneronal Aß aggregates. This present data therefore suggests that small Aß species might damage neuronal dendritic and synaptic circuitry. This disruption of neuronal networks may, in turn, underlie the subsequent functional impairment seen in these arcAß tg mice.

 

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Published in Nature Neuroscience 2008

Activation of Estrogen Receptor Beta Regulates Hippocampal Synaptic Plasticity and Improves Memory

1Feng Liu, 2Mark Day, 1Luis C. Muñiz, 1Robert Arias, 3Daniel Bitran,
4Raquel Revilla-Sanchez, 1Steve Grauer, 1Guoming Zhang, 1Virginia Pulito, 1Amy Sung, 5,6 Ronald Mervis, 1Rachel Navarra, 1Warren Hirst, 1Peter Reinhart, 1Karen L. Marquis, 4Stephen J. Moss, 1Menelas N. Pangalos and 1Nicholas J. Brandon

1Wyeth Research, Discovery Neuroscience, Princeton, NJ
2Wyeth Research, Translational Medicine, Collegeville, PA
3Department of Psychology, College of the Holy Cross, Worcester, MA
4Department of Neuroscience, University of Pennsylvania, Philadelphia, PA
5Neurostructural Research Labs, Inc., Tampa, FL 33637
6Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, Tampa, FL.

Estrogens have long been implicated in influencing cognitive processes, yet the molecular mechanisms underlying these effects and the relative roles of the estrogen receptors alpha (ERa) and beta (ERß) remain unclear. Utilizing pharmacological, biochemical and behavioral techniques, we demonstrate for the first time that the effects of estrogen on hippocampal synaptic plasticity and memory in rodents are mediated through ERß. Selective ERb agonists increased levels of key synaptic proteins in vivo including PSD-95, synaptophysin and the AMPA-receptor subunit GluR1. These effects were absent in ERb knockout mice. In hippocampal slices ERb activation enhanced long-term potentiation (LTP), an effect that was absent in slices from ERb knockout mice. ERb activation induced morphological changes in hippocampal neurons in vivo including increased dendritic branching and density of mushroom-type spines. An ERb agonist, but not an ERa agonist, also improved performance in a variety of hippocampal-dependent memory tasks. Taken together, our data suggest that activation of ERb can regulate hippocampal synaptic plasticity and improve hippocampal-dependent cognition.

 

Presented at the American Society for Neural Transplantation and Repair, Clearwater, FL 2008

NEUROSTRUCTURAL CONSEQUENCES OF DEVELOPMENTAL EXPOSURE TO MODERATE DOSES OF CANNABINOIDS IN A RAT MODEL

Ronald F. Mervis1,2, Nikki Vyas3 , Scott Moradian3, Jeremy Yesudas3, Bryan Thomas3, Leigh Nattkemper4, Silvana Gaetani5, Maria Tattoli5, Addolorata Coluccia2, and Vincenzo Cuomo1

1Ctr of Excellence for Aging & Brain Repair,
Univ. South Florida College of Medicine, Tampa, FL, USA
4NeuroStructural Research Labs, Inc., Tampa, FL, US
5The Honors College, USF, Tampa, FL, USA
1Dept of Human Physiology and Pharmacology, Univ. of Rome “La Sapienza”, Italy
2Dept of Pharmacology and Human Physiology, Univ. Of Bari;

Even though marijuana is the most widely used illegal drug among women of reproductive age, reports dealing with the effects of prenatal exposure to this substance of abuse are still controversial. More complex and less understood is the scenario concerning the possible long-term consequences of in utero exposure to cannabis derivatives on cognitive functions.

In this study the synthetic CB1 agonist WIN 55,212-2 (WIN) was administered daily to pregnant rats from gestational day 5 to 20 (0.5mg/kg). This dose is equivalent to a moderate or low exposure to marijuana in humans and has no overt toxic effects. The treatment with WIN did not affect gestational and reproduction parameters and WIN-exposed pups did not show any sign of malformations or malnutrition. However, a deeper investigation revealed that prenatal treatment with WIN altered pup performance in homing behavior and produced a decrease in the rate of separation-induced ultrasonic vocalizations. Behavioral deficits that resulted were long-lasting, since prenatal WIN exposure caused a disruption of memory retention in young and adult offspring subjected either to a passive or an active avoidance task. Moreover, an altered dendritic morphology of hippocampal CA1 pyramids was detected in young (40 day-old) rats. In particular, in the prenatally WIN-exposed rats, there was a significant 12% increase in estimated total dendritic length and a highly significant increase in branching complexity in the middle-third of the dendritic tree. These findings suggest that moderate exposure to cannabinoids during crucial periods of brain development can cause dysmorphic maturation of the hippocampus. Such subtle morphological alterations and commensurate changes in brain circuitry would be, in turn, a factor underlying the behavioral deficits observed both in early and late postnatal life.

 


Presented at the Society for Neuroscience, San Diego, November, 2007

A Chronic Stress Regimen with Unpredictable Stressor Presentation Decreases the Density of Spines on the CA3 Pyramidal Neurons of the Hippocampus in the Adult C57BL/6J Male Mouse


D.B. Miller
1, A.D. Bachstetter2, R.F Mervis2,3 , S. A. Benkovic1

1Centers for Disease Control and Prevention, Morgantown, WV 26505
2Center of Excellence for Aging and Brain Repair,
University of South Florida College of Medicine, Tampa, FL 33612
3Neurostructural Research Labs, Inc., Tampa, FL 33637

The neural changes associated with chronic stress are of great interest to those interested in brain health as they may be linked to the cognitive impairments and neurodegeneration observed in many brain diseases. To examine how chronic stress impacts brain structural elements adult C57BL/6J male mice (N=10) were exposed to 21 consecutive days of the following stressors in random order: (1) 8 hrs restraint; (2) 3 mins forced swim in ice water; (3) 8 hrs of light-cycle disruption; (4) 8 hrs at 15o; (5) social reorganization or remained in their home-cage. Immediately following the last stressor mice were anesthetized, perfused with paraformaldehyde, the brains removed and maintained in fixative until sections were prepared and examined for alterations in neuronal plasticity. This chronic stress regimen, designed to minimize habituation by presenting the stressors in an unpredictable random order, produced a measurable but non-significant change in body weight. It also caused a 27% decrease in thymus weight indicating a sustained activation of the HPA axis throughout the stress period although this decrease is minimal compared to the 93% reduction observed in animals exposed to supra-physiological levels of corticosterone through pellet (200 mg) implant for the 21 days. Dendritic spines were quantified for spine density along terminal tip and internal branch segments of the apical tree of the CA3 pyramidal neurons of the hippocampus. Spines were also categorized into “L-“ (lollipop), “N-“ ( nubby) , and “D-type” (dimple) configurations. Five to six randomly selected Golgi-impregnated CA3 neurons were evaluated from each subject and spines quantified along 30 um Golgi-impregnated sections. Findings revealed a ~ 15.5% decrease in L-and N- but not D-type spines on the apical dendrites. A lesser but still significant decrease in all spine types was also observed on the internal segments of the apical dendrites. A less dramatic loss of spines was observed in mice exposed continuously to supra-physiological levels of corticosterone. Reduced spine density may be indicative of reduced neuronal plasticity. Thus, our data provides evidence that the CRS regimen alters plasticity in the hippocampal area of the mouse as it does in the rat and provides a chronic stress model that can be used in future studies utilizing this rodent species to examine the signaling pathways suspected to mediate the effects of stress on plasticity.

 

Presented at the Society for Neuroscience, San Diego, November, 2007

Neurotoxic Consequences of Chlorpyrifos Exposure on Dendritic Circuitry in the Adult Rat

S. M. ANTOINE
1, A. COLE1, J. D. KOTICK1, M. L. SHAH1,
A. DECAMILLO1, A. V. TERRY2, R. F. MERVIS3,4

1Honors College, Univ. South Florida, Tampa, FL
2Pharmacol. & Toxicology, Med. College Georgia, Augusta, GA
3NeuroStructural Research Labs, Inc., Tampa, FL
4Ctr. Aging & Brain Repair, Dept Neurosurgery, Univ. South Florida Col. Med., Tampa, FL

Chlorpyrifos (CPF) is a common organophosphate (OP) insecticide. It has extensive use in agriculture as a pesticide. The primary mechanism of action is inhibition of acetylcholinesterase (AchE). Exposure to OPs may have deleterious neurobehavioral consequences. This has been well documented in neonatal and developing animals; however, the impact on the adult brain is more poorly understood. Analysis of Golgi stained neurons was chosen for morphological assay which allows for complete visualization of entire dendritic branching arbor and dendritic spines and quantification of the morphometric changes. Adult CPF-treated rats were given 18mg/kg for 14 days. There were six CPF and six control subjects in each group. Five randomly-seclected neurons were evaluated from each region of each subject: the cingulate gyrus of the frontal cortex, CA1s in the hippocampus, and granule cells of the dentate gyrus. Dendritic branching was assessed by preparation of camera lucida drawings and subsequent Sholl analysis which profiles the amount and distribution of the arbor. Dendritic spines were counted along 30um long dendritic segments.
Results: Whereas the cingulate and CA1 neurons showed no effects of the CPF-exposure on dendritic branching or spine density, in the dentate gyrus, analysis of the dendritic arbor of the granule cells revealed a significant reduction of branching across the entire tree in the chlorpyrifos exposed animals (p<0.0001, Wilcoxon Test). This corresponded to a ~16% decrease in dendritic material. Branch point analysis of these animals revealed a trend toward less complex dendritic arbors in the CPF treatment group. Dendritic spine analysis of the granule cells showed a significant ~7% (p<0.01, unpaired T-test) decrease of spine density in the middle third of the dendritic tree of the granule cells in the CPF treatment group.
These results show that a 14 day exposure to CPF causes a significant reduction in dendritic branching, along with spine loss in the dentate gyrus of the hippocampus. Relative to the cortical neurons and the hippocampal CA1s where no neurotoxic effects were observed, the granule cells appear to be selectively sensitive to CPF-exposure. Damage to granule cells -- and to the related hippocampal circuitry -- may be the underlying neuroanatomical basis for cognitive dysfunction observed in OP-exposed subjects.

 

Presented at the Society for Neuroscience, San Diego, November, 2007

Heavy Particle Irradiation and Neuronal Damage: A Potential Risk to Future Interplanetary Exploration?


Quasem I1, Kasimos K1, Kalmbach K5, Rabin BM2, Shukitt-Hale B3, Joseph JA3, R. F. Mervis4,5

1The Honors College, University of South Florida, Tampa, FL
2Dept. Psychology, Univ. Maryland Baltimore County, Baltimore, MD
3USDA Human Nutrition Lab, Tufts University, Boston, MA
4Dept Neurosurgery, Ctr for Aging and Brain Repair, Univ South Florida Med Ctr, Tampa, FL
5Neurostructural Research Labs, Inc., Tampa, FL

In future deep space missions, astronauts will be exposed to heavy particle radiation over lengthy periods. Potentially, this exposure could result in neuronal damage, impair brain circuitry, and compromise the astronaut’s cognitive and behavioral capabilities which would threaten mission success. At this time, relatively little is known of the extent of this exposure on neuronal circuitry. The purpose of this study was to evaluate the effects of high-energy (56)Fe particles on dendritic branching and spine parameters in layer II-III cortical neurons in the adult rat. These parameters are critical to the integrity of brain circuitry: dendritic branching comprises about 95% of total neuronal volume and the vast majority of synapses occur on the dendritic spines.
4 month-old Sprague-Dawley rats were exposed to a single exposure of (56)Fe radiation (1.5Gy of 1GeV/n). 28 days later the animals were sacrificed. Formalin fixed coronal tissue blocks were stained using the rapid Golgi method and, from coded slides, randomly selected layer II-III pyramids of the frontal cortex were evaluated for dendritic branching and spines. Camera lucida drawings of the basilar tree were assessed using Sholl analysis. Results showed that there was a statistically significant loss of dendritic material (~10%) in the dendritic arbor (p =0.008, Wilcoxon test) of the neurons exposed to the heavy particle irradiation.
These results suggest that exposure of the rat brain to heavy particle irradiation equivalent to that which astronauts might encounter on deep space missions has a deleterious effect on neuronal morphology and hence, on brain circuitry. Neuroprotective strategies may need to be devised to minimize brain damage and assure mission success.

 

Presented at the Society for Neuroscience, San Diego, November, 2007

The Effect of Chronic Lithium Chloride on Dendritic Branching in the Adult Rat Neocortex and Hippocampus


Halwani G
1, Quasem I2, Nehaul K1, Kotick JD1, Kalmbach K3, Shim S
5, R. F. Mervis3,4,6

1College of Arts & Sciences, USF
2The Honors College, USF
3NeuroStructural Research Labs, Inc., Tampa, FL
4Center of Excellence for Aging and Brain RepairCollege of Medicine, Tampa, FL.
5Dept. of Psychiatry, Case Western Reserve Medical Center, Cleveland, OH
6Department of Neurosurgery, USF College of Medicine, Tampa, FL

Lithium may serve as a treatment for bipolar disorders and other psychiatric conditions by counteracting both mania and depression. Lithium can affect neurotransmitter activity by increasing serotonin levels and decreasing noradrenaline discharge. Lithium may also influence neuroplasticity and the dendritic parameters that are the underlying neurostructural basis for the cognitive and behavioral manifestations.
We evaluated the effects of chronic administration of lithium chloride to adult rats on the morphology of the dendritic arbor of Golgi stained granule cells of the dentate gyrus and layer II-III pyramids of the prefrontal cortex. The dendritic arbor comprises about 95% of the volume of the typical neuron. Adult rats were injected with lithium chloride (1mEq/kg/day IP injection for 14 days) or saline. Formalin fixed blocks of tissue were Golgi stained and camera lucida drawings were prepared from randomly selected neurons. Sholl analysis was used to assess amount and distribution of the dendritic arbor.
Lithium treatment resulted in a neuroplastic remodeling of the dendritic arbor of the granule cells: relative to the controls, there was significantly increased branching in the proximal region (p=0.0028, Wilcoxon test) and decreased branching in the distal portion of the arbor (p = 0.0024, Wilcoxon). There was also a significant increase in branching complexity in the medial portion of the tree.(Previous studies have indicated that neurophysiological changes in dentate gyrus occur in lithium treated rats.)
In the prefrontal layer II-III pyramids, the lithium treatment also resulted in a significant neuroplastic increase of the basilar dendritic arbor which was most pronounced in the medial portion of the tree. Conversely, in the apical dendritic tree, the lithium resulted in a decrease in the amount of dendritic material in the proximal region of the arbor.
Although the specific mechanisms for these neuroplastic dendritic alterations are not currently known, the changes in the dendritic arbor due to the chronic lithium can clearly modify the transfer of information in cortical and hippocampal brain circuits and this can, in turn, influence behavior.

 

Presented at the Nutrisciences and Health Conference, Charlottetown, PEI, Canada, July, 2007

Dietary Supplementation in Aged Rats with Blueberry Extract, a Nutritional Antioxidant, Enhances Dendritic Neuroplasticity in Cortical Neurons

Patricia Hwang1, Steve Antoine1, James Kotick, Mrunal Shah1, Keri Kalmbach2,
Barbara Shukitt-Hale3, James Joseph3 and Ronald F. Mervis2,4

1The Honors College, University of South Florida, Tampa, FL
2Neurostructural Research Labs, Inc. , Tampa, FL
3US Dept Agriculture, Human Nutrition Research Center at Tufts University Medical Center, Boston, MA
4Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida College of Medicine, Tampa, FL

Aged-related oxidative stress results in the formation of reactive oxygen species (ROS) which can damage neurons and may an important component associated with disruption of brain circuits and concomitant age-related cognitive impairment. This study attempts to characterize the potential beneficial effects of the nutritional antioxidants (e.g., polyphenols) found in blueberries (BB), on morphological indices of brain circuitry in old rats. Nineteen month old male Fischer 344 rats were given either a standard NIH-31 rat chow (controls) or an NIH chow enriched with 2% BB extract for 2.5 months. Following behavioral testing, the 21.5 mon-old rats were killed and their brains stained using the Golgi method (N = 3 subjects/group).. Golgi staining permits microscopic visualization and quantitative analysis of the dendritic parameters of the impregnated neurons. From coded slides, randomly selected layer II-III cortical neurons of frontal cortex in the 21.5 month old rats (7 neurons/subject) were evaluated for the extent of their dendritic branching. Two and half months of the BB-enriched diet resulted in a significant increase in dendritic branching, primarily in the proximal half of the basilar dendritic arbor of the layer II-III neurons (a 14% increase). This suggests that even in old subjects, BB dietary supplementation appears to have a neuroplastic impact on neuronal morphology: it can mitigate normal age-related dendritic atrophy. This would suggest that the BB-extract can exert anti-aging neuroprotection in maintaining or enhancing the integrity of brain circuitry in the old rats.

 

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Published in Environmental Health Perspectives 2007

Ontogenetic Alterations in Molecular and Structural Correlates of Dendritic
Growth After Developmental Exposure to Polychlorinated Biphenyls.

Lein PJ1, Yang D1, Bachstetter AD2,3, Tilson HA4, Harry GJ5, Mervis RF2,3, Kodavanti PR4

1Center for Research on Occupational and Environmental Toxicology, Oregon Health &
Science University, Portland, OR
2Neurostructural Research Labs, Inc. Tampa, FL,3Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, Tampa, FL
4Cellular and Molecular Toxicology Branch, Neurotoxicology Division, National Health and Environmental Effects Research Lab, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC
5National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC

OBJECTIVE: Perinatal exposure to polychlorinated biphenyls (PCBs) is associated with decreased IQ scores, impaired learning and memory, psychomotor difficulties, and attentional deficits in children. It is postulated that these neuropsychological deficits reflect altered patterns of neuronal connectivity. To test this hypothesis, we examined the effects of developmental PCB exposure on dendritic growth. METHODS: Rat dams were gavaged from gestational day 6 through postnatal day (PND) 21 with vehicle (corn oil) or the commercial PCB mixture Aroclor 1254 (6 mg/kg/day). Dendritic growth and molecular markers were examined in pups during development. RESULTS: Golgi analyses of CA1 hippocampal pyramidal neurons and cerebellar Purkinje cells indicated that developmental exposure to PCBs caused a pronounced age-related increase in dendritic growth. Thus, even though dendritic lengths were significantly attenuated in PCB-treated animals at PND22, the rate of growth was accelerated at later ages such that by PND60, dendritic growth was comparable to or even exceeded that observed in vehicle controls. Quantitative reverse transcriptase polymerase chain reaction analyses demonstrated that from PND4 through PND21, PCBs generally increased expression of both spinophilin and RC3/neurogranin mRNA in the hippocampus, cerebellum, and cortex with the most significant increases observed in the cortex. CONCLUSIONS: This study demonstrates that developmental PCB exposure alters the ontogenetic profile of dendritogenesis in critical brain regions, supporting the hypothesis that disruption of neuronal connectivity contributes to neuropsychological
deficits seen in exposed children.

 

Presented at the 2006 Annual Meeting of the Society for Neuroscience. Atlanta, GA

Dendritic atrophy and spine plasticity in frontal cortex neurons in Mild Cognitive Impairment: A quantitative Golgi analysis.

Ronald F. Mervis
1,2,3, James Kotick3,4 , Mrunal Shah3,4 , Steven Scheff5, Elliott J. Mufson,6

1Ctr of Excellence for Aging & Brain Repair, Univ South Florida College of Medicine, Tampa, FL
2Dept Neurosurgery, USF, Tampa, FL
3NeuroStructural Research Labs, Inc., Tampa, FL
4Honors College, USF, Tampa, FL
5Sanders-Brown Ctr for Aging, Univ Kentucky, Lexington, KY
6Dept Neurol Sciences, Rush Univ Med Center, Chicago, IL

Disruption of dendritic branching and/or spine components likely play a role in the neuropathology underlying the onset of cognitive impairment seen in Alzheimer disease (AD). Whether disruption in these components of brain circuitry is altered in elderly people with a clinical diagnosis of mild cognitive impairment (MCI) without frank AD remain unknown. To evaluate these dendritic alterations, formalin fixed frontal (area 9) cortical tissue blocks were Golgi stained from individuals who died with a clinical diagnosis of either No Cognitive Impairment (NCI), MCI, or AD obtained from the University of Kentucky. Layer II-III pyramidal neurons (6/subject) were randomly selected from coded slides. This data represents the initial phase of a larger on-going Golgi investigation of neo- and limbic brain circuitry in the progression of AD. Sholl analysis revealed significant dendritic atrophy in the MCI compared to AD and controls. However, there was a significant increase in pyramidal neuron spine density in MCI as compared to controls and AD (which did not differ from each other). The underlying molecular mechanisms are unknown: mutations in amyloid beta (Aß) (and/or tau) genes may enhance dendritic pruning (microtubule depolymerization in the shafts of dendrites) by promoting enhanced calcium influx from the endoplasmic reticulum, or Aß could promote spine formation via actin polymerization. Similar dichotomous findings were seen in the 3xtg triple AD transgenic mouse model of AD, further implicating genetic mutation in APP and tau genes in this process. These findings may represent compensatory neuroplastic responses, which may assist in maintaining cortical circuitry and delay severe cognitive dysfunction in MCI.

 

Presented at the 2006 Annual Meeting of the Society for Neuroscience. Atlanta, GA

Dichotomous Dendritic Changes in the Aging Triple Transgenic Mouse Model of Alzheimer’s Disease: Dendritic Spines Increase and Branching Decreases in Layer V Pyramidal Cells.

Anthony R. Dicamillo1, Mrunal L. Shah1,4, James Kotick1,4, Mohamed R. Mughal5,
Mark P. Mattson5, and Ronald F. Mervis2,3,4

1The Honors College, Univ South Florida, Tampa, FL
2Center of Excellence for Aging and Brain Repair, USF, Tampa, FL
3Dept Neurosurgery, USF, Tampa, FL
4NeuroStructural Research Labs, Inc. Tampa, FL
5Labratory of Neurosciences, NIA IRP, Baltimore, MD

The triple transgenic mouse model of Alzheimer’s disease (3xTgAD) was generated by expressing three different mutant genes (APP, PS1 and tau) linked to inherited forms of dementia. 3xTgAD mice exhibit age-related amyloid and tau pathology that is associated with synaptic dysfunction and memory impairment. From randomly selected Golgi stained neurons, we evaluated neocortical layer V dendritic spine density (basilar tree and main apical branch) and basilar dendritic branching in aging (15 mon-old) 3xTgAD male and female mice and age-matched controls. Branching analysis of the basilar dendritic arbor of both 3xTg males and females showed significant mild-to-moderate branching atrophy in the distal 2/3rds of the tree (~15%). Conversely, for both aging males and females, there was an overall significant average increase in spines (~ 50%) for these layer V cortical neurons. A possible mechanism for the contradictory dendritic findings is that Abeta, the PS1 mutation (and tau mutations as well) promote enhanced calcium influx and release from the ER; therefore, this would tend to cause dendritic pruning (microtubule depolymerization in the shafts of dendrites), but could also promote spine formation (actin polymerization). We have also seen a similar dichotomous dendritic response in cortical neurons from autopsied humans diagnosed with mild cognitive impairment (a prodromal stage of AD); this lends additional strength to the 3xtg mouse as a valid model of Alzheimer’s. These dendritic findings would suggest that alterations in cortical circuitry are contributing to the cognitive dysfunction associated with the progression of the disease.

 

Presented at the 2006 Annual Meeting of the Society for Neuroscience. Atlanta, GA

Human Umbilical Cord Blood Cell Treatment Mitigates Loss of Dendritic Branching and Spines in the Aged Rat Brain.


N. Rajadhyaksha1, S. Khan2, K. Shah2, J. Kotick2,6, M. L. Shah2,6, R.F. Mervis3,4,6,
P. Bickford3,4,5, P. R. Sanberg3,4, P. Walczak5, R. Shamek3,4, N. Chen3,4, and A.Willing3,4

1College of Liberal Arts & Sciences, Univ South Florida, Tampa, FL
2The Honors College, USF, Tampa, FL
3Ctr of Excellence for Aging & Brain Repair, Tampa, FL
4Dept Neurosurgery, USF, Tampa, FL
5J.A. Haley VA Hospital, Tampa, FL
6Neurostructural Research Labs, Inc. Tampa, FL

Human Umbilical Cord Blood (HUCB) cells are enriched for stem cells that have the potential to initiate and maintain tissue repair. The potential neuroprotective role of HUCB on the circuitry of the aging brain is unknown. However, HUCB may release certain neurotrophic factors that protect the dendritic and synaptic circuitry from age-related deterioration. This study was designed to provide new insight into the neuroprotective role of HUCB in aging rats. The brains from 22 month-old aging rats and 4 month-old young controls were evaluated for dendritic branching and spine morphology from several cell populations. The old rats were administered a single treatment of HUCB either i.v. or directly into the hippocampus. Cortical and hippocampal neurons were assessed using Golgi impregnated preparations which can reveal the amount of dendritic material and the numbers of dendritic spines of randomly stained neurons. In the neocortex and hippocampus, layer II-III pyramids and granule cells, respectively, showed age-related spine loss which was largely attenuated by the single HUCB cell treatment. Also, age-related granule cell dendritic branch atrophy was reversed by HUCB. These results will have significant impact on future applications of umbilical cord therapies.

 

Presented at the 2006 Annual Meeting of the Society for Neuroscience. Atlanta, GA

Disruption of Normal Cortical Dendritic Growth in the Akt3 KnockOut Mouse.

Elizabeth Brachowicz1, Rachael M. Easton2, Morris J. Birnbaum2,3,
and Ronald F. Mervis3,4,6

1The Honors College, University of South Florida, Tampa, FL
2Department of Medicine
3The Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
4Center of Excellence for Aging & Brain Repair, USF College of Medicine, Tampa, Fl, Dept Neurosurgery, USF College of Medicine, Tampa, FL
5NeuroStructural Research Labs, Inc., Tampa, Florida


In the CNS, the insulin signal transduction pathway regulates many diverse processes ranging from metabolism to memory formation. Binding of insulin to the insulin receptor results in activation of phosphatidylinositol 3’-kinase (PI3K), generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3), and activation of the serine/ threonine kinase Akt / protein kinase B. In mammals, three highly conserved proteins, Akt1, Akt2, and Akt3, comprise the Akt family. Akt3 is the predominant isoform, representing about half of total Akt protein and is the most prevalent isoform in the cortex and hippocampus. Akt3 is required for normal brain growth and mice deficient in Akt3 demonstrate a selective 20% decrease in overall brain size. Signaling through the PI3K-Akt pathway can modulate dendritic branching. Here, we demonstrate the in vivo requirement of Akt3 for regulation of dendrite growth. Using Golgi-stained neurons, compared to the wild-type controls, analysis of the basilar tree of parietal layer V pyramids of Akt3-deficient brains showed a 20% reduction in the amount and distribution of dendrite branching (p< 0.0001, Wilcoxon test). There was also a trend toward reduction of dendritic spine density on the terminal tips of these neurons in the Akt3 deficient mice (-9%, p=0.07, unpaired t-test). Thus, the serine/ threonine kinase Akt3 influences normal brain neuronal branch growth and development of normal brain circuitry.

 

Presented at the 2006 Annual Meeting of the Society for Neuroscience. Atlanta, GA

Effects of Hyper- and Hypoglycemia on Dendritic Branching and Spines in the Young Adult Diabetic Rat.

Jaimie Waite1, Corinne Althauser2, Ronald F. Mervis3,4,5, Suzanne Hanna6,
Samuel Saporta3,4,7, John I. Malone6

1Honors College, Univ South Florida, Tampa, FL
2Coll Arts & Sciences, USF, Tampa, FL
3Ctr of Excellence Aging & Brain Repair, USF, Tampa, FL
4Dept Neurosurgery, USF, Tampa, FL
5NeuroStructural Research Labs, Inc., Tampa, FL
6Dept Pediatrics, USF, Tampa, FL
7Dept Anatomy, USF, Tampa, FL

Childhood diabetes and related medical issues are a growing health problem. Children with diabetes onset before 5 years old may have reduced neurocognitive function. Typically, this problem has been attributed to hypoglycemia, a complication of insulin therapy. However, hyperglycemia is much more common than intermittent hypoglycemia during early childhood diabetes. Relatively little is known about the effects of hyperglycemia on development of neural circuitry. The purpose of this study was to evaluate the effects of chronic hyperglycemia and intermittent hypoglycemia on dendritic branching and spines in the young rat neocortex. Starting at four weeks of age, experimental rats were exposed to 4 weeks of either chronic hyperglycemia or intermittent (3 hours, 3x/week) hypoglycemia. Tissue was stained using Golgi impregnation methods to assess dendritic parameters. Compared to age-matched controls, evaluation of the basilar tree of layer II-III pyramids in the parietal cortex showed that although the intermittent hypoglycemia did not affect dendritic branching, the hyperglycemic paradigm resulted in significantly smaller (-16%, p < 0.05) and less complex dendritic arbors relative to the controls. There was also a 9% loss of dendritic spines on the internal branch segments of the basilar tree (p < 0.004). These findings indicate that chronic hyperglycemia in the developing brain – which is associated with early childhood diabetes – may result in damage to cortical neurons, compromise brain circuitry and could be the neuroanatomical basis for the neurocognitive impairment found in this disorder. Other studies on hippocampal neurons and using additional dosages are currently in progress. (Supported by a grant from the Juvenile Diabetes Research Foundation)

 

Presented at the Annual Meeting of the Society of Toxicology, San Diego, March, 2006

Stress and Depleted Uranium Exposure Alter Dendritic Morphology in the Rat

R.F Mervis1,4, D. S. Barber2, M. Ehrich3, J. Hinckley3, J. Kotick1,5,
M. Shah1,5, T. Amato5, B.S. Jortner3

1Neurostructural Research Laboratories, Tampa, FL
4Center for Aging & Brain Repair, University of South Florida College of Med, Tampa, FL 2Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL
3Laboratory for Neurotoxicity Studies, Virginia Tech, Blacksburg, VA
5The Honors College, USF, Tampa, FL

Studies of rats with intramuscular implantation of depleted uranium (DU) reveal that in addition to the well-described accumulation of this metal in kidney and bone, it is also increased in brain (Pellmar et al, 1999). In addition, Gulf War I veterans with long-term residual DU shrapnel had lowered performance efficiency in selected neurocognitive tests (McDairmond et al, 2000). This study was undertaken to additionally assess the neurotoxicologic effects of implanted DU in rats, and to see if stress altered this response, focusing on changes to the hippocampal pyramidal cells. Adult male Sprague-Dawley rats each had 20 DU or tantalum (control) pellets (1mm x 2mm) implanted in the gastrocnemius muscles, for a six month period. Stress was applied 5 days/week for the entire exposure period in a random pattern of handling, restraint, and swimming to minimize habituation. At terminal sacrifice, cerebral regions from animals exposed to the stress, DU, the combination of stress and DU and the tantalum controls were removed and stained by the Golgi impregnation procedure. The basilar dendritic arbor of hippocampal CA1 pyramidal cells was assessed using Sholl analysis. Preliminary data shows that relative to the negative controls, there was an increase in dendritic material in the inner 1/3 of the arbor in rats administered DU alone. This diminished in the outer 2/3 of the arbor, a region where the combination of stress and DU elicited increase in dendritic material. There also was decrease in dendritic material in animals only exposed to stress in this outer region. Thus, 24 weeks exposure to a high dose of implanted DU pellets alters the dendritic tree of CA1 pyramidal neurons which may influence cognitive behavior. (Supported by DAMD17-01-1-0775, US Army Medical Research and Materiel Command)

 

Presented at the annual meeting of the Society for Neuroscience, Washington, DC, November, 2005

The Increase in CA1 Spines Produced by Spatial Learning is Blocked by Pre-Training, but Not Pre-Retrieval, Predator Stress.

Diamond, D.M.1,2,3, Campbell, A.M.1,2, Woodson, J.C.4, Park, C.R.1,2, Bachstetter, A.D.5, Mervis, R.F.5

1Medical Research, VA Hospital, Tampa, FL, USA
2Psychology,
3Pharmacology, University of South Florida, Tampa, FL, USA
4Psychology, Univ of Tampa, Tampa, FL
5Neurostructural Research Labs, Tampa, FL USA


Rats exhibit strong long-term spatial memory when they are trained in the radial arm water maze (RAWM). However, when rats were exposed to a cat after learning the location of the hidden escape platform, their spatial memory was impaired (Hippocampus, 9:542-552, 1999; Learning & Memory, 10:326-336, 2003). Here, we investigated the influence of spatial learning and predator stress on long-term (24 hr) memory and dendritic spines in CA1. In addition, we have tested the hypothesis that there will be a differential expression of learning and stress-induced amnesia on well-developed, lollypop-shaped (L-type) spines, compared to stubby, nubbin-shaped (N-type) spines.
Adult male SD rats were trained to learn the location of a hidden platform in the water maze, and then their spatial memory was tested 1 and 24 hr later. Rats were exposed to a cat for 30 min either before they learned the platform location (Stress Day 1), before the 24 hr memory test trial (Stress Day 2) or not at all (No Stress), with brain extraction immediately after the 24 hr memory test trial. The three groups showed equivalent learning and 1 hr memory on Day 1, but both stress groups showed impaired performance on the 24 hr memory test. Thus, the Stress Day 1 group exhibited impaired storage of the spatial information, and the Stress Day 2 group exhibited impaired retrieval of stored information. The No Stress and Stress Day 2 groups both exhibited a significant increase in N-type spines, with no change in the number of L-Type spines. There was no increase in either type of spine in the Stress Day 1 Group.
These findings indicate that rats that had 24 hr to consolidate spatial information (the “No stress” and “Stress Day 2” groups) exhibited increase in spines. Rats that were stressed before learning (Stress Day 1 group) had impaired consolidation and lacked an increase in spines. Moreover, the spine changes were specific to N-type spines, which are known to be more plastic than L-type spines. Overall, these findings support the idea that increases in N-type spines are involved in the storage of information. We have also provided the novel observation that morphological evidence of memory storge (spine changes) remain when acute stress interferes with the retrieval of a stored memory.

Supported by the VA

 

Presented at the Annual Meeting of the Society for Neuroscience, Washington DC, November, 2005

Hyperglycemia Induced Alteration of Cortex and Hippocampus in the Rat

Samuel Saporta1,2,4, Suzan Hanna3, Ronald F. Mervis2,4,5,
Christopher P. Phelps1 and John I. Malone3

Departments of 1Anatomy, 3Pediatrics and 2Neurosurgery,
University of South Florida College of Medicine, Tampa, FL 33612
4Center of Excellence for Aging and Brain Repair,
Univ South Florida College of Medicine, Tampa, FL 33612
5Neurostructural Research Labs, Inc., Tampa, FL 33612

Children with diabetes onset before 5 years of age have reduced neurocognitive function. This problem has been attributed to hypoglycemia, a complication of insulin therapy. The eye, kidney and nerve complications of diabetes have been reduced by intensified Insulin therapy which is associated with a 3 fold increase in severe hypoglycemia and therefore was not recommended for children less than13 years of age. Since hyperglycemia is much more common than intermittent hypoglycemia during early childhood diabetes, it is important to determine if hyperglycemia affects brain growth and development. Rats were exposed to 4 weeks of either chronic hyperglycemia or intermittent (3 hours, 3 times/week) hypoglycemia from 4 to 8 weeks of age. The brains of these animals were compared to those of similarly aged normal control animals. Overall cell density, and the density of Map-2-positive neurons and S-100b-positive astrocytes in the entorhinal cortex and hippocampus of these animals were compared between groups. The number of cells was increased and the cell size reduced in the cortex of diabetic animals as assessed by DNA/ wet weight of brain and protein/DNA content. Reduced amounts of protein, fatty acids, and cholesterol/ µgram DNA also indicate smaller cells with reduced myelin content in the cortex of the diabetic animals. Histologic evaluation of these brains confirmed that there was an increase in the number of small neurons in these areas with a slight decrease in the number of astrocytes. These observations require further confirmation and evaluation, but indicate that chronic hyperglycemia may be more damaging than intermittent hypoglycemia to the developing brain.

Supported by a grant 1-2004-751 from the Juvenile Diabetes Research Foundation

 

Presented at the Society for Neuroscience NIDA Satellite Symposium “Frontiers in Addiction Research”, Washington, DC, November, 2005

Neurofunctional Consequences of Developmental Exposure to Moderate Doses of Cannabinoids in a Rat Model.

Silvana Gaetani1, Maria Tattoli2, Addolorata Coluccia2, Ronald F Mervis3,4,
Adam Bachstetter4, Nisida Berberi5, and Vincenzo Cuomo1

1Dept of Human Physiology and Pharmacology, Univ. of Rome “La Sapienza”, Italy
2Dept of Pharmacology and Human Physiology, Univ. Of Bari
3Ctr of Excellence for Aging & Brain Repair,
Univ. South Florida College of Medicine, Tampa, FL, USA
4NeuroStructural Research Labs, Inc., Tampa, FL, USA
5Honors College, USF, Tampa, FL, USA.

Even though marijuana is the most widely used illegal drug among women of reproductive age, reports dealing with the effects of prenatal exposure to this substance of abuse are still controversial. More complex and less understood is the scenario concerning the possible long-term consequences of in utero exposure to cannabis derivatives on cognitive functions.
In this study the synthetic CB1 agonist WIN 55,212-2 (WIN) was administered daily to pregnant rats from gestational day 5 to 20 (0.5mg/kg). This dose is equivalent to a moderate or low exposure to marijuana in humans and has no overt toxic effects. The treatment with WIN did not affect gestational and reproduction parameters and WIN-exposed pups did not show any sign of malformations or malnutrition. However, a deeper investigation revealed that prenatal treatment with WIN altered pup performance in homing behavior and produced a decrease in the rate of separation-induced ultrasonic vocalizations. Behavioral deficits that resulted were long-lasting, since prenatal WIN exposure caused a disruption of memory retention in young and adult offspring subjected either to a passive or an active avoidance task. Moreover, an altered dendritic morphology of hippocampal CA1 pyramids was detected in young (40 day-old) rats. In particular, in the prenatally WIN-exposed rats, there was a significant 12% increase in estimated total dendritic length and a highly significant increase in branching complexity in the middle-third of the dendritic tree. These findings suggest that moderate exposure to cannabinoids during crucial periods of brain development can cause dysmorphic maturation of the hippocampus. Such subtle morphological alterations and commensurate changes in brain circuitry would be, in turn, a factor underlying the behavioral deficits observed both in early and late postnatal life.

 

Presented at the meeting of the Neurobehavioral Society, St. Pete Beach, FL, June, 2005

This Is Your (Child's) Brain on Drugs: in utero Exposure to a Cannabinoid Agonist Affects Dendritic Morphology of Hippocampal CA1 Neurons in the Young Rat.


Ronald F. Mervis1,2, N. Berberi3*, Adam Bachstetter,2*,T. Cassanno4*,
M.G. Morgese4 *, S. Gaetani
5* and V. Cuomo5*.

1Ctr for Aging & Brain Repair, Dept Neurosurgery.,
Univ. South Florida College of Medicine, Tampa, FL
2NeuroStructural Research Labs, Tampa, FL
3Honors College, Univ of South Florida, Tampa, FL
4Univ Foggia, Italy
5Univ La Sapienza Rome, Italy.

Among women of reproductive age marijuana is the most widely used illegal drug. The impact of cannabis exposure on the developing brain is poorly understood. A specific cannab-inoid receptor (CB1) is highly expressed in many brain regions, including the hippocampus. The synthetic CB1 agonist WIN 55,212-2 was administered daily to pregnant rats from gestational day 5 to 20 (0.5mg/kg). This dose is equivalent to a moderate or low exposure of marijuana in humans and has no overt toxic effects. The behavioral consequences in 40 day-old (do) rats included hyperactive behavior and memory impairment. To assess the effects of this prenatal exposure on dendritic morphology of CA1 pyramids of the hippocampus, fixed brain tissue from WIN-exposed 40 do offspring (N= 6) and age-matched controls (N=5) was Golgi stained. The basilar dendritic arbors of CA1s were quantified. In the prenatally WIN-exposed 40 do rats, there was a significant 12% increase in estimated total dendritic length and a highly significant increase in branching in the middle-third of the dendritic tree. This finding suggests that hippocampal circuitry was affected by the cannabinoid agonist; perhaps due to failure of normal developmental pruning back of the dendritic tree. Dysmorphic maturation of the hippocampus would be a factor underlying behavioral and cognitive changes seen in the young 40 day-old rats.

 

Presented at the 7th International Conference on Alzheimer's and Parkinson's Diseases, Sorrento, Italy, March, 2005

Cortical and Hippocampal Dendritic Spine Alterations in the Triple Transgenic Mouse Model of Alzheimer's Disease.

Ronald F. Mervis1,2, Adam Bachstetter2, Mohamed Mughal3,
Peter Mouton
3,4 and Mark P. Mattson3

1Center for Aging and Brain Repair, Department of Neurosurgery,
University of South Florida College of Medicine, Tampa, FL
2NeuroStructural Research Labs, Tampa, FL
3Laboratory of Neurosciences, National Institute on Aging IRP, Baltimore, MD
4Stereology Resource Center, Chester, MD

The triple transgenic mouse model of Alzheimer's disease (3xTgAD) was generated by expressing three different mutant genes (APP, PS1 and tau) linked to inherited forms of dementia. 3xTgAD mice exhibit age-related amyloid and tau pathology which is associated with synaptic dysfunction and memory impairment. Memory impairment in AD correlates strongly with synaptic loss. In the present study we evaluated the density and configuration of dendritic spines in neocortex and hippocampus of male 3xTgAD mice. From coded slides, dendritic spines of Golgi stained neurons were evaluated on randomly selected layer II/III pyramids of the parietal neocortex and on the basilar tree of hippocampal CA1 pyramids. For 3xTgAD mice (12 months old, n=4) and nontransgenic controls (11.5 months old, n=5) spines were assessed for density and configuration, properties that are known to influence synaptic efficacy. In CA1 neurons, spines on the terminal tip segments of the basilar tree were quantified. Total spine density was reduced in the aged 3xTgAD mice. Most strongly affected were spines lacking well-defined spine heads. On neocortical layer II/III pyramids, spines were assessed throughout the dendritic arbor. Compared to controls, there was no significant total spine loss in the 3xTgAD mice. However, there was a significant increase in small-headed spines, a form of spine believed to be dysfunctional. These initial findings suggest that both hippocampal and cortical circuitry in the aged 3xTgAD mice are altered, but in different ways. In the hippocampus spine loss occurs, whereas in the parietal neocortex an increase in dysfunctional spines may occur.

 

Presented at the meeting of the Society for Neuroscience, San Diego,
November, 2004

Alterations in the Morphology of Dendrites in the Nucleus Accumbens and Frontal Cortex Following Repeated Neonatal Isolation Stress.

W.J. Shoemaker1, J. Costill1, A. Bachstetter2, A. Cupples3,
J. Kotick4 and R.F. Mervis2,5.

1University of Connecticut Health Center., Farmington, CT,
2
NeuroStructural Research Labs, Tampa, FL
3
College of Arts & Sciences, University of South Florida, Tampa, FL
4
Honors College, University of South Florida, Tampa, FL
5
Center of Excellence in Aging & Brain Repair, Dept. Neurosurgery,
University of South Florida College of Medicine, Tampa, FL

Newborn rat pups isolated from the dam and littermates for 1 hr. per day (PN 2-9) display behavioral sensitization when tested as juveniles and adults and also have a hyper-responsive mesolimbic dopamine system to either a drug or stress challenge. We have analyzed fixed brain tissue from adult male rats using the rapid Golgi method in order to quantify changes in dendrites and synaptic spines. In addition, some animals were given memantine, an NMDA receptor antagonist, at the time of isolation to test whether blockade of NMDA receptors will attenuate stress-induced sensitized behavior and morphological changes. Memantine (10 mg/kg) was given by oral gavage at each animal’s 1-hr isolation on each day from PN 2-9. Isolation-only (ISO), non-isolation only (NON-ISO) and memantine only (MEM) groups were included in the design. The drug was tolerated well and had no effect on body weight over time for any treatment group. Examination of Golgi-stained sections using the Sholl analysis reveals that, compared to NON-ISO controls, ISO animals had a 23% increase in the branching of dendrites in medium spiny neurons of the nucleus accumbens shell. There were no significant alterations in spine number or type, or in cell soma size. ISO-MEM and MEM animals had dendritic branching levels intermediate between the ISO and NON-ISO levels, consistent with behavioral data that indicated memantine could attenuate the effects of isolation, but had other effects of its own. In the layer II/III pyramidal cells of the frontal cortex, there were no differences between ISO and NON-ISO animals in dendritic branching, but both groups receiving memantine were significantly lower than controls. There was a trend to increase in spine number (14%) but it was not significant. However, soma size of pyramidal cells was smaller in isolated groups, regardless of memantine treatment.

 

Presented at the meeting of the International Society for
Developmental Neuroscience, Edinburgh, Scotland, August, 2004

Cortical and Hippocampal Dendritic Alterations in the ASMKO Mouse –
A Knockout Model of Niemann-Pick Disease, Type A.

R.F. Mervis1, 2*, A. Bachstetter2 , J. Kotick3 , G.R. Stewart4

1University of South Florida College of Medicine, Dept. Neurosurgery,
Center of Excellence for Aging & Brain Repair, Tampa, FL 33612, USA;
2
NeuroStructural Research Labs, Tampa, FL 33612, USA;
3
Honors College, University of South Florida, Tampa, FL 33612;
4
Genzyme Corporation, Framingham, MA 01701, USA.
Contact: RonMervis@NeuroStructural.org

Type A Niemann-Pick Disease (NP-A) is caused by deficiency of acid sphingomyelinase (ASM) which results in sphingomyelin accumulation. NP-A is a severe neurologic disease that begins in the first few months of life, is accompanied by progressive psychomotor retardation, and leads to death by 2 to 3 years of age. The NP-A knockout mouse model was created by targeted disruption of the gene encoding acid sphingomyelinase.

Formalin-fixed brains of 18 week-old ASMKO(-/-) mice (N=8) and normal littermate controls (N=8) were stained using the Rapid Golgi method. This reveals the soma, dendritic arbor, and spines of randomly stained neurons.

From coded slides dendritic branching, spines and soma size were quantified on 6 randomly selected neurons from parietal neocortex layer V pyramids , CA1 pyramids, and granule cells of the dentate gyrus of each brain. Grossly, nearly all CA1 pyramids of the hippocampus of the ASMKO mice were distinguished by the widespread appearance of "meganeurites" – neuritic structures near the soma swollen by accumulation of sphingomyelin.

Results. Parietal Layer V Pyramids: In KO mice, dendritic length was significantly reduced (-19% ; p = 0.01) but without significant dendritic spine loss (-7%, NS); soma size was significantly increased (+15%, p=0.02). Granule cells of the Dentate Gyrus: branching was reduced by 14% (p=0.03), spines were reduced by 9% (p=0.02), soma size was unaffected. CA1 Pyramids: the distribution of dendritic branching was not significantly reduced; there was a significant increase in spines (+10%, p=0.02), and a 25% increase in soma size (p=0.0001).

Conclusion: In ASMKO mice Layer V pyramids and granule cells showed evidence of damage; however, despite the widespread presence of large dysmorphic meganeurites on all CA1 pyramids, these neurons showed no loss of dendritic arbor and, indeed, an increase in spines. This suggests that meganeurite formation in ASMKO mice may sequester accumulated sphingomyelin in a less toxic format and, therefore, meganeurites may have some neuroprotective function.

 

Presented at the meeting of the International Behavioral Neuroscience Society, Key West, Florida, June, 2004

Abnormally Excessive Cortical Dendritic Branching in a Knockout Mouse Model of the Fragile X Mental Retardation Syndrome.

Mervis, R.F.1 2, Bachstetter, A.D.2, Ather,T.3, Maloney,T. 3, Cupples,A. 3, Toth, M.4

1Center of Excellence for Aging and Brain Repair, Dept. Neurosurgery,
University of South Florida College of Medicine, Tampa, FL 33612,
2
NeuroStructural Research Labs, Tampa, FL 33612;
3
College of Arts & Sciences, USF, Tampa, FL 33512;
4
Dept. Pharmacology, Weill Medical College of Cornell University, NY, NY 10021.

Fragile X syndrome is the most common cause of inherited mental retardation and is caused by a mutation in the FMR1 gene leading to absence of the fragile X mental retardation protein (FMRP). Knockout of the gene associated with Fragile X, Fmr1, results in mice with abnormalities analogous to human symptoms. Neuroanatomical findings include an excess of abnormally thin, immature dendritic spines. FMRP may play a role in the normal process of dendritic spine growth and pruning of excess immature synapses. The impact of Fmr1 deletion on development of dendritic branching is poorly understood and is the focus of this study. Using Golgi impregnated neurons, we evaluated dendritic arborization of layer II/III pyramids of the parietal cortex of 10 week-old fragile X mice. Coronal blocks of the parietal cortex from fmr-1 knockout mice and WT controls (FVB/NJ) were stained using the Rapid Golgi method (N=5 per group). From coded slides, 5-6 layer II/III pyramidal neurons were randomly selected. Camera lucida drawings of the basilar tree were made of each neuron. Sholl analysis showed that the Fragile X mice had significantly more dendritic material throughout the extent of the basilar tree (p = 0.0002, Wilcoxon test). Estimated total dendritic length was 28% longer (p=0.001, unpaired T test) and branching complexity was greater. These findings suggest that Fragile X mice may have abnormalities of developmental pruning of both branching and spines which would result in anomalous processing of information in cortical circuits. This would contribute to the cognitive dysfunction and behavioral problems associated with Fragile X syndrome.

 

Presented at the annual meeting of the Society for Neuroscience, New Orleans, November, 2003

ANTEROGRADE AMNESIA IN CARDIAC ARREST SURVIVORS:
A RODENT MODEL

E.R. Glasper1; G.N. Neigh2; R.F. Mervis3,4; J. Kofler5; R.J. Traystman5;
A. Bachstetter3; A.C. DeVries1,2

1Dept. Psychology, Ohio State University, Columbus , OH 43210 USA;
2
Dept Neuroscience, Ohio State University, Columbus, OH 43210 USA;
3
NeuroStructural Research Labs, Tampa, FL 33612 USA;
4
Center for Excellence in Aging and Brain Repair, Dept Neurosurgery, University of South Florida College of Medicine, Tampa, FL 33612 USA;
5
Dept of Anesthesiology and Perioperative Medicine, Oregon Health and Sciences University, Portland, OR USA

Progress in medical technology has led to an increased number of cardiac arrest survivors resulting in a population of patients with behavioral and physiological impairments. The hippocampus is particularly sensitive to arrest-induced global hypoxia and clinical evidence suggests that cardiac arrest and cardiopulmonary resuscitation (CA/CPR) survivors report memory deficits. The current study used the Morris water maze to characterize the learning and memory deficits elicited by 8 minutes of cardiac arrest in a mouse model. The Morris water maze has been widely used as a test of spatial discrimination and place navigation in rodents, and is sensitive to alterations in the function of the hippocampal formation. Over the 8 days of pre-surgical water maze training, all animals showed a reduction in the mean distance traveled and latency to reach the hidden platform, indicating a mean improvement in performance. Following CA/CPR, mice were allowed to recover for 7 days. CA/CPR and SHAM animals performed the same on probe trials, indicating that the animals were able to identify the spatial location that previously contained the hidden platform. There also were no post-surgical differences observed in the visible platform trials, indicating both groups were motivated to find the platform. CA/CPR animals exhibited a performance decrement during the reversal training (when platform is moved to a new location), suggesting that CA/CPR may have impaired their ability to learn new spatial tasks. Ultimately, a better understanding of the behavioral deficits that result from CA/CPR and the dendritic alterations that underlie these changes may provide insight into the memory deficits reported by CA/CPR survivors. Support Contributed By: NIH NS40267

 

Published in Journal of Neuroscience Research 73: 1-9 (2003)

Insulin-like Growth Factor 1 Is Essential for Normal Dendritic Growth

C.M. Cheng1, R.F. Mervis 2,3, S-L Niu4 , N. Salem, Jr. 4,
L.A. Witters 5, V. Tseng1, R. Reinhardt1, and C.A. Bondy1

1 Developmental Endocrinology Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD; 2 NeuroStructural Research Labs, Inc. Tampa, FL
3 Dept of Neurosurgery, Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, Tampa, FL
4 Laboratory of Membrane Biophysics and Biochemistry, National Institute of Alcohol Abuse and Alcoholism, NIH, Bethesda, MD
5 Dept of Medicine and Biochemistry, Dartmouth Medical School, Hanover, NH

This study evaluated somatic and dendritic growth of neurons in the frontoparietal cortex of Igf1-/- brains. Pyramidal neuron density was increased by approximately 25% (P =.005) and soma size reduced by approximately 10% (P <.001). Golgi staining revealed that cortical layer II-III neurons exhibited a significant reduction in dendritic length and complexity in Igf1 null mice. Dendritic spine density and presumably synaptic contacts were reduced by 16% (P =.002). Similar findings were obtained for cortical layer V and piriform cortex pyramids. Supporting a reduction in synapses, synaptotagmin levels were reduced by 30% (P <.02) in the Igf1 null brain. Investigation of factors critically involved in dendritic growth and synaptogenesis showed an approximately 50% reduction in cortical CDC42 protein expression (P <.001) and an approximately 10% reduction in brain cholesterol levels (P <.01) in Igf1 null mice. Evidence is presented that Igf1 deletion causes disruptions in lipid and microtubule metabolism, leading to impaired neuronal somatic and dendritic growth. Published 2003 Wiley-Liss, Inc.

 

Presented at the meeting of the Society for Neuroscience
New Orleans, November, 2003

EFFECT OF PILOCARPINE-INDUCED TEMPORAL LOBE EPILEPSY ON DENDRITIC MORPHOLOGY IN THE ENTORHINAL CORTEX OF ADULT RATS: A QUANTITATIVE GOLGI ANALYSIS.

R.F. Mervis1,2 A. Bachstetter1, C. Leroy3, A. Nehlig3.

1NeuroStructural Research Labs, Tampa, FL 33612;
 
2Dept. of Neurosurgery, Center of Excellence for Aging & Brain Repair, University of South Florida College of Medicine, Tampa, FL 33612;
3
INSERM U405, Universite Louis Pasteur, Faculté de Médecine, 67085 Strasbourg, France


Many clinical and neuropathological features of human temporal lobe epilepsy (TLE) are reproduced using the lithium-pilocarpine (Li-Pilo) rat model. Status epilepitus is followed by a "silent" seizure free stage (14-25 days) and then by life-long spontaneous recurrent seizures (SRS) (2-3/weeks). This TLE model results in necrosis-related neuronal damage to the hippocampus, neocortex, and piriform and entorhinal cortices. We evaluated the effects of TLE on dendritic morphology of stellate cells in the entorhinal cortex. METHODS: Adult S-D male rats were administered lithium and pilocarpine or saline (controls). Subjects were sacrificed about 2 months after the appearance of the SRS. Coronal and horizontal blocks encompassing the entorhinal cortex were stained using the Rapid Golgi method. From coded slides, camera lucida drawings of dendritic arbors of randomly selected layer II stellate cells were drawn and Sholl analysis was used to assess the amount and distribution of dendritic material. Dendritic spine number and configuration were also evaluated on terminal tip segments. RESULTS: Sholl analysis showed that the stellate cells from the Li-Pilo rats had significantly less dendritic material (p<0.0001, Wilcoxon test). Estimated total dendritic length was reduced by 18%. TLE also significantly reduced dendritic spine density by 29% (p = 0.0002, Mann-Whitney test). All spine types were compromised. CONCLUSIONS: These dendritic changes would compromise entorhinal circuitry, alter normal signalling patterns to the dentate gyrus, and could contribute to epileptogenesis. TLE damage to stellate cells may thus be intimately involved in the widespread epileptic activity seen in this model. (Supported in part by INSERM)

 

Presented at the 6th International Conference on Neuroprotective Agents
Hilton Head, September, 2002

The Ultra Low Weight Glycosaminoglycan, C3, Protects Against Loss of Dendritic Branching and Spines of Pyramidal Cells Following Cortical Damage in the Rat

Ronald F. Mervis1, Adam Bachstetter1, Bertalan Dudas2, Umberto Cornelli2, and Israel Hanin2.

1NeuroStructural Research Laboratories, Columbus, Ohio 43212, USA
2
Department of Pharmacology and Experimental Therapeutics,
Loyola University Medical Center, Maywood, IL 60153, USA

Introduction and Methods
The group of glycosaminoglycans (or GAGs) is
composed of heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate, and their analogs, including a novel GAG, C3. C3 is an ultra-low molecular weight analog of heparin produced by fractionation of heparin via g-irradiation, followed by gel filtration. It is comprised mainly of 6-10 oligosaccharides, with a molecular weight of 2.1 kD, and a USP value of 12 U/mg. C3 can cross the blood brain barrier, and in previous studies has been shown to have neurotrophic properties resulting in an increase in dendritic branching and spines in young adult rats. Other investigations have indicated that it may beneficial in neurodegenerative disorders such as Alzheimer’s disease.

As part of a larger series of studies investigating C3, one group of adult rats had previously been subjected to saline injections into their lateral ventricles (ICV), with the needle cannulae inserted bilaterally through the frontal cortices (saline group, N = 5). Another group of rats, which also had an ICV saline injection, had been given C3 orally (once daily, by gavage, 25mg/kg) beginning one week prior to the ICV saline and continuing for one week following saline administration (C3 group, N = 4) . Total treatment time for C3 was thus 14 days.

Initial observations of Golgi-stained neurons from the region adjacent to the needle track showed damaged cortical neurons as assessed by dendritic atrophy and spine loss. In view of findings from earlier studies suggesting the neurotrophic potential of C3, we decided to assess whether oral C3 treatment may have a neuroprotective role against the cortical stab wound-like damage caused by the needle inserted into the ventricle.

Dendritic branching and spines were quantitatively evaluated from Golgi stained tissue. As dendritic material of the cortical neuron comprises over 95% of its volume and the vast majority of synapses occur on spines, these dendritic parameters mirror the integrity and sophistication of cortical circuitry as well as the general health of the neurons.

Following 14 days of C3 treatment or saline, the rats were euthanized, their brains removed and immersion fixed in 4% paraformaldehyde for subsequent Golgi staining. 3mm coronal blocks of frontal cortex encompassing the needle cannula track were stained using the Rapid Golgi method and all slides were coded for neuronal analysis by an observer blind to treatment. In addition to the layer V pyramidal neurons selected adjacent to the needle track from the C3 treated or untreated animals, a third group of layer V pyramids were evaluated from saline treated subjects (N = 5): randomly selected neurons located in the same region of frontal cortex – but distal from the needle track. (and hence, undamaged). These neurons represented a baseline control by which to compare with the damaged neurons.

For assessment of dendritic branching, camera lucida drawings were made of the basilar tree of randomly selected, well-stained layer V pyramidal cells (n = 5 per subject). The basilar trees were evaluated for extent and distribution of their arbors by the Sholl method of concentric circles. This also provides an estimate of total dendritic length. Dendritic spines were counted along 30 micron segments from approximately 4 terminal tips of the basilar dendritic branches of each neuron (5 neurons per subject).

Results and Conclusions
Compared to the (undamaged) layer V pyramids distal from the needle track, e.g., baseline controls, neurons adjacent to the needle track from the untreated (saline) subjects showed a significant loss of both dendritic material (-15%, Sholl analysis: p < 0.001, Wilcoxon) and dendritic spines (–21.4%, p < 0.0001, Mann-Whitney). This indicated that the needle cortical stab wound resulted in significant damage to these neurons. By contrast, in comparison to the damaged neurons from the saline-treated subjects, the neurons from rats given C3 orally (for 7 days prior to, and 7 days after, the cortical stab wound) had significantly more dendritic material (p < 0.001, Wilcoxon) and 26% more spines (p < 0.0001, Mann-Whitney). Combining branching and spine data together (a so-called Circuitry Index reflecting estimated total synaptic connections), the cortical stab wound-induced neurotrauma resulted in a 33% reduction in circuitry in nearby neurons compared to neurons distal from the wound (p = 0.0079, Mann-Whitney). The C3 treatment protected neurons adjacent to the stab wound from this damage, the Circuitry Index now being the same as the baseline neuronal values (p = 0.9, N.S.).

In conclusion, C3 treatment has clearly exhibited a strong neuroprotective role maintaining normal cortical circuitry for neurons that would otherwise show significant neurotrauma-related injury. Further studies will be required to determine if C3 achieved this result primarily by means of minimizing initial damage and/or by enhancing neuroplastic recovery of the neurons from the cortical stab wound injury.

(Supported in part by NIA STTR Grant # 1-R41-AG15740-2 awarded to RFM and IH)

 

Presented at the meeting of the Society for Neuroscience,
Orlando, FL, November, 2002

Rapid Nonhuman Primate Model for NeuroAIDS: Evidence for Neuronal Damage.

R.F. Mervis1, A. Bachstetter1, H. Singleton1, S. Henriksen2, L. Madden2, E. Roberts2, M. Burudi2, C. Marcondes2, M. Taffe2, and H. Fox2.

1
NeuroStructural Research Labs, Columbus, OH 43212;
2
Dept. Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037

CNS dysfunction induced by HIV infection, known as neuroAIDS, continues to be a significant problem in the AIDS pandemic. We have obtained a reproducible model of neuroAIDS in rhesus macaques, utilizing a CD8-depletion regimen at the time of SIV inoculation. Most of the monkeys show cognitive, motor, neurophysiological, and neuropathological abnormalities within four months after infection. To more fully characterize the neuronal changes accompanying the CNS disease, we utilized Golgi-impregnation staining to evaluate pathological alterations in dendritic branching and spines from multiple brain regions. Randomly selected pyramidal neurons located in layers II/III and V in the Anterior Cingulate Gyrus (ACG), the Middle Frontal Gyrus (MFG), the Superior Temporal Gyrus (STG) and in CA1 hippocampus were evaluated (3 subjects/group; 6 neurons/region/subject). From coded slides, camera lucida drawings were made of the basilar pyramidal trees. Sholl analysis was used to quantify the extent and distribution of neuronal dendritic arbors. Dendritic spines were counted along 30 micra lengths of terminal dendritic tip segments. In the SIV-infected monkeys the presence of damaged neurons was often in spotty patches and was characterized by dendritic varicosities, spine loss, and dendritic branch atrophy. Most areas showed significant loss of branching and/or spines with an average reduction of neural circuitry throughout each of these brain regions of almost 17%. These findings validate this reproducible nonhuman primate model for neuroAIDS. (Supported by NIH grants MH59468, MH62261, MH61692 and DA12444)

 

Presented at the meeting of the Society for Neuroscience,
Orlando, FL, November, 2002

IGF1 IS ESSNTIAL FOR NORMAL DENDRITIC GROWTH

Clara M Cheng1, Ronald F Mervis2, Shui-Lin Niu3, and Carolyn A Bondy1.

1Developmental Endocrinology Branch, National Institute Child Health & Human Development, NIH, Bethesda, MD 20892
2
NeuroStructural Research Laboratories, Columbus, Ohio 43212
3
Laboratory of Membrane Biophysics and Biochemistry, National Institute Alcohol Abuse and Alcoholism, NIH, Rockville, MD

  IGF1 gene deletion results in reduced brain growth and mental retardation. We have previously shown that brain glucose utilization is profoundly impaired during early postnatal development in the IGF1 null mouse. Given that IGF1 is normally selectively expressed by projection neurons that grow the most extensive dendritic arbors, we hypothesized that absence of IGF1 would result in attenuation of dendritic growth and synaptogenesis. Sholl analysis of the basilar trees of Golgi stained fronto-parietal cortical layer II-III pyramids in IGF1-/- and wild-type (WT) 50 day-old mice shows that IGF1-/- neurons have significant deficits in both dendritic length and complexity. IGF1-/- neurons have fewer dendritic intersections, particularly at increasing distances away from the soma (P=0.0002) and fewer branch points at each branch order measured indicating reduced branching complexity. IGF1-/- dendrites are ~ 25% shorter than WT (P<0.001). Dendritic spines are reduced by 16% (P = 0.002) and synaptotagmin levels are reduced by 30% (P = 0.01) in the IGF1-/- brain, suggesting decreased synaptogenesis. Similar findings were obtained in cortical layer V and in the piriform cortex. Cholesterol and phospholipid are each reduced by ~10% in IGF1-/- brains (P<0.01 and P<0.05 respectively), suggesting impaired lipogenesis in the IGF1-/- brain. These data show that IGF1 is essential for normal dendritic growth and synaptogenesis, probably through its insulin-like, anabolic effects on glucose utilization and lipogenesis.

 

Presented at the meeting of the Society for Neuroscience, Orlando, FL, November, 2002

Neuropathology of the Niemann Pick A (ASMKO) Mouse

S.L. Macauley1, L. Shihabuddin*1, E.H. Schuchman2,
R.F. Mervis3, T. Taksir1, and G.R. Stewart1

1Genzyme Corporation, Framingham, MA 01701;
2
Mount Sinai School of Medicine, New York, NY, 10029;
3
NeuroStructural Research Laboratories, Columbus, OH, 43212

    This study describes the nature, distribution and progression of neuropathology in the Niemann Pick Type A (NP-A) mouse that was created by targeted disruption of the gene encoding acid sphingomyelinase (ASM). ASMKO (-/-) and normal littermate (+/+) animals from 4 to 28 weeks of age were prepared by fixation perfusion and in most cases the brains was processed for paraffin histology. In H& E stained specimens, lysosomal accumulation was readily apparent in the cytoplasm of ASMKO animals, particularly in larger neurons throughout the brain and spinal cord. Sensory neurons within dorsal root ganglion were also severely affected with massive lysosomal accumulation giving the cells a foamy appearance classically associated with visceral NP-A pathology. Mild lysosomal accumulation was present as early as six weeks and progressively worsened with age. Brain regions most severely affected included pyramidal cells in the cortex (layers II, III, V) and hippocampus (CA1), most subnuclei of the thalamus, and magnocellular brainstem structures such as the trigeminal nucleus. Despite advanced disease in older animals, the only region observed to undergo neurodegeneration was an almost complete depletion of Purkinje cells from the cerebellum (see poster by Stewart et al). Silver impregnation staining did reveal evidence for more widespread degenerating structures (see poster by Switzer). However, the use of retrograde tracers demonstrated that diseased neurons were able to efficiently transport dyes and that distant brain structures retained normal, intact connections (e.g. striatum to nigra). Further structural analysis using Golgi staining revealed relatively preserved fine neuronal morphology, although there was evidence for stripping of dendritic spines as well as meganeurite formation within some CA1 and cortical pyramidal neurons.

 

Presented at the meeting of the Society for Neuroscience,
Orlando, Fl, November, 2002

TETRACHLOROAZOBENZENE (TCAB)-INDUCED DEVELOPMENTAL NEUROTOXICITY IN CORTEX AND CEREBELLUM IN THE RAT:
A Quantitative Morphometric Golgi Analysis.

A. Bachstetter1, R.F. Mervis1, H. Singleton1, G. Wolfe2, J. Bishop3, and G. J. Harry3,

1NeuroStructural Research Labs, Columbus, OH 43212;
2
Therimmune Research Corp., Gaithersberg, MD 20878;
3
NIH, National Institute for Environmental Health Sciences,
Research Triangle Park, NC 27709

   3,3’,4,4’-tetrachloroazobenzene (TCAB) is a dioxin-like organochlorine compound formed as a byproduct in the manufacture of herbicides. It impairs thyroid function, and thus controls critical aspects of brain development. The potential neurotoxic consequences of TCAB exposure need to be determined. TCAB was administered by gavage to SD female rats starting two weeks before pregnancy until the pups were weaned. TCAB dose levels were 0.1 mg/kg/day (low dose) or 3.0 mg/kg/day (high dose). Controls received corn oil. Male pups were sacrificed when 22 days old. Neocortex was stained using the Rapid Golgi method and cerebella, using the Golgi-Cox variant. Using coded slides, from each subject 6 randomly selected layer V pyramids and 6 Purkinje cells (PCs) were evaluated. Analysis of dendritic arbor of the V pyramids by Sholl analysis showed that control arbor > low dose TCAB > hi dose TCAB. Significant dendritic spine loss was seen only in the high dose TCAB. The Neuronal Circuitry Index (branching/spine index) showed that low dose TCAB reduced pyramidal cell circuitry by 18%, and high dose TCAB exposure, by 32%. In cerebellum, PC dendritic area was significantly reduced only in the high dose TCAB group. However, both low dose and high dose groups had small ectopic dysmorphic PC-like neurons located in the molecular layer. The results suggest that the impaired development of neocortical and cerebellar neurons may be mediated by thyroid dysfunction due to the TCAB exposure.

 

Presented at the meeting of the Society for Neuroscience,
Orlando, FL, November, 2002

DEVELOPMENTAL EXPOSURE TO HEXACHLOROBENZENE (HCB)
IN THE RAT RESULTS IN ECTOPIC AND DYSMORPHIC
CEREBELLAR PURKINJE CELLS

A. Bachstetter1, R.F. Mervis1, H. Singleton1, G. Wolfe2, J. Bishop3, and G. J. Harry3.

1NeuroStructural Research Labs, Columbus, OH 43212;
2
Therimmune Research Corp., Gaithersberg, MD 20878;
3
NIH, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709

Hexachlorobenzene (HCB) is a dioxin-like organochlorine compound that binds to the aryl hydrocarbon receptor (Ah). HCB may impair thyroid metabolism. HCB is widespread in the environment and accumulates in biological systems. It is transplacentally transferred from mother to fetus during pregnancy and through breast milk. Neurotoxic consequences of HCB exposure on the developing brain are, however, poorly documented. HCB (2.5 mg/kg/day) was administered by gavage to Sprague-Dawley female rats starting two weeks before pregnancy until the pups were weaned. Controls received corn oil. Pups were sacrificed when 22 days-old and cerebella were stained using the Golgi-Cox method. There were seven male subjects per group. Using coded slides, from each subject seven randomly selected Purkinje cells (PCs) were evaluated for soma size and areal extent of their dendritic arbor. There were no differences between groups for PCs that had correctly migrated and were located in the Purkinje cell layer. However, there were also a significant number of neurons with Purkinje cell-like characteristics that had improperly migrated into the molecular cell layer. These highly dysmorphic PCs had dendritic areas 90% smaller than normal, and somas which were 75% smaller than normal. Since thyroid function influences critical aspects of cerebellar development, the abnormal migration of PCs and their dysmorphic development may reflect the neurotoxic impact of HCB exposure on thyroid function.

 

Presented at the meeting of the Endocrinology Society
San Francisco, CA, June, 2002

Endogenous Brain IGF1 is Essential for Normal Dendritic Growth.

Clara M Cheng, Ronald F Mervis, Shui-Lin Niu, and Carolyn A Bondy

Developmental Endocrinology Branch, NICHD, NIH, Bethesda, MD 20892
NeuroStructural Research Laboratories, Columbus, Ohio 43212
Laboratory of Membrane Biophysics and Biochemistry, NIAAA, NIH

IGF1 deficiency results in reduced postnatal brain development in both human and animal models. Our previous studies on IGF1 gene deleted mouse brain (IGF1-/-) have shown that despite the reduction in brain size, the IGF1-/- brain anatomy and cell numbers are normal for the most part. Given that the dendritic neuropil occupies a very large percentage of the brain parenchyma, the attenuation in IGF1-/-brain size may result from the deficit in the growth of dendritic processes in the absence of endogenous brain IGF1. To test this hypothesis, we used Golgi histological staining and biochemical analysis to evaluate the dendritic growth and complexity of cortical neurons in the IGF1-/- and wild-type (WT) brains of 50 day-old mice. Morphometric analysis of fronto-parietal cortical pyramidal layer II-III neurons shows that the soma size of IGF1-/- neuron is reduced by 10% (P<0.001), and that the cell density is significantly higher by 25% compared to that of WT (P=0.005). Analysis of camera lucida drawing of the basilar trees of the Golgi stained II/III pyramids shows that IGF1-/- neurons have less dendritic material than WT’s as reflected by deficits in both dendritic length and complexity. Sholl analysis of the basilar trees reveals that IGF1-/- neurons have fewer dendritic intersections with the measuring concentric circles, particularly at the increasing distances away from the soma (P=0.0002). Branch point analysis shows that IGF1-/- neurons have fewer branch points at each branch order measured indicating less branching complexity. IGF1-/- neurons have an average of 25% shorter dendrites than WT’s (P<0.001). Supporting this finding, brain total lipid, the major substance for constructing dendrites, is decreased by 29% in the IGF1-/- brain homogenates (P=0.059). Cholesterol and phospholipid, the two primary components of the brain lipid, are reduced by 9% in IGF1-/- brains individually (P<0.01 for cholesterol and P<0.05 for phospholipid). Further investigation of the enzymes involved in neuronal morphogenesis demonstrates that the cdc42, a member belonging to the Rho GTPase family, is reduced in IGF1-/- brains by 30% compared to WT’s (P<0.005). Rac1 and RhoA, the other two members in the Rho family, show no change in IGF1-/- brains. Our results suggest that the normal growth of dendritic processes is significantly impaired in the absence of endogenous brain IGF1 and that the cdc 42 is one of the major modulators for the action IGF1 in regulating brain dendritic development.

 

Presented at the forthcoming meeting of the International
Society for the Study of Fatty Acids & Lipids, Montreal, May, 2002

ALTERATIONS IN BEHAVIOR AND NEUROANATOMY IN DHA DEFICIENT RATS

Norman Salem, Jr.1, Ronald F. Mervis2, Aneeq Ahmad1, Toru Moriguchi1, Janice Nicklay1, Rebecca Greiner1, Sunyoung Lim1, Adam Bachstetter2, and Burton Slotnick3.

1Section of Nutritional Neuroscience, Lab of Membrane Biochemistry & Biophysics, NIAAA, NIH;
2NeuroStructural Research Laboratories, Columbus, Ohio;
3Department of Psychology, American University, Washington, D.C.

If docosahexaenoic acid (DHA) is essential for optimal nervous system function, then diets that lead to a loss in brain DHA would be expected to result in functional deficits. Brain function is usually assessed using behavioral tasks of varying complexity. In this work, rats were given diets with only safflower oil as their source of essential fats (n-3 Def group) or safflower oil plus sources of alpha-linolenate and DHA (n-3 Adq group) for two generations. Male pups (behavior) and female pups (anatomy) were weaned to the same diets that their mothers consumed and then tested at various ages. In spatial tasks, animals in the n-3 Def group acquired the Morris water maze more slowly and performed poorly on the retention trial. In a go, no-go olfactory discrimination task, n-3 Def rats again acquired the task more slowly and failed to acquire a learning set. Anatomical studies indicated smaller neuronal areas of cell bodies in the hippocampus as well as other brain areas. Golgi staining and Sholl analysis indicated that granule cells in the dentate gyrus had significantly less dendritic arborization in the n-3 Def animals at both 21 and 68 days of age than the n-3 Adq group. A similar loss of dendritic arborization was also observed in cortical pyramidal neurons in 21-day old n-3 Def rats, however, this difference was no longer found in 68 d old animals apparently due to greater dendritic pruning in the n-3 Adq brains. Persistent alterations in hippocampal circuitry in the n-3 Def animals may be the neurostructural basis for the observed behavioral deficits.

 

Presented at the Seventh International Geneva/Springfield
Symposium on Advances in Alzheimer Therapy, April, 2002

GLYCOSAMINOGLYCANS AS A POTENTIAL THERAPY FOR ALZHEIMER’S DISEASE

I. Hanin, B. Dudas, Q. Ma, M. Rose, J. Fareed, S.Lorens, M. Hejna,
J. Lee, M. Walzer, *R. Mervis, and U. Cornelli,

Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, and *NeuroStructural Research Laboratories, Columbus, Ohio, USA.

Over the past decade a mixture of glycosaminoglycans (GAGs) has been shown to improve cognition in aged rats and to reverse cognitive and neurochemical deficits in patients with Alzheimer’s disease (AD). This mixture of GAGs (called GAP or GAG-polysulfate), which is composed of heparin (10-20%), heparan sulfate (40-60%), dermatan sulfate (20-35%) and chondroitin sulfate (2-8%), has earned a use-patent in several European countries. The effects mentioned above may be attributable to the ability of GAGs to compete with endogenous proteoglycans (PG), such as heparan sulfate PG, and to prevent their tendency to: a) induce amyloid formation via an increase in aggregation rate of b amyloid (Ab) into b-pleated fibrils and b) increase polymerization of tau into paired helical proteins, which make up the neurofibrillary tangles of AD. During the past three years we have been working to identify the active component(s) of the GAP mixture, by fractionating each of the components of GAP into smaller molecular weight fractions, and testing their respective protective activity on several animal models of brain neuron degeneration.

C3 is an ultra-low molecular weight analog of heparin produced by fractionation of heparin via g-irradiation, followed by gel filtration. It is comprised mainly of 4-10 oligosaccharides, with an apparent molecular weight of 2.1 kD, and a USP value of 12 U/mg. The fractionation procedure has been standardized; thus we can manufacture a reproducible product in large quantities (kgs). This substance has been found to be the most active of several products obtained via fractionation of the above-mentioned components of GAP).

To date we have observed, using C3, that: a) it can cross the blood brain barrier (BBB); b) it prevents tau-2 immunoreactivity and reactive astrocytosis in both the AF64A, and the Ab(25-35) treated rat; c) its protective effect is obtained following either oral or subcutaneous administration of C3; d) it has neurotrophic effects in the brain, in vivo, as measured by Golgi impregnation technology; e) it is devoid of significant anticoagulant activity; and f) it is safe at high doses.

Thus, C3 is a promising candidate for further development and clinical testing in patients with AD and other central neurodegenerative disease states. It will soon be employed in Phase I clinical studies.

(Supported in part by NIA STTR Grant # 1-R41-AG15740)

 

Presented at the meeting of the Society of Toxicology,
Nashville, April, 2002

LONG-LASTING NEUROSTRUCTURAL CONSEQUENCES IN THE RAT HIPPOCAMPUS BY DEVELOPMENTAL EXPOSURE TO A MIXTURE OF POLYCHLORINATED BIPHENYLS (PCBs).

R F Mervis1, A D Bachstetter1, G J Harry2, H A Tilson3, ,and P R S Kodavanti3.

1NeuroStructural Research Labs, Columbus, OH, USA;
2
NIH, NIEHS, RTP, NC, USA, and
3
Neurotox Div., USEPA, NHEERL/ORD, Research Triangle Park, NC, USA.

The objective of the study was to assess the effects of developmental exposure to a commercial mixture of PCBs (Aroclor 1254) on neuronal dendritic morphology of hippocampal CA1 pyramidal neurons in postnatal day (PND) 22 and PND 60 male Long-Evans rats. Rat pups were born to mothers who were exposed to Aroclor 1254 (AccuStandard Inc., Lot # 124-191; 0 and 6 mg/kg/day) from gestational day 6 through PND 21. Thus, pups were exposed to PCBs in utero and through weaning. Male rats (N = 5-6 per group) were sacrificed on PND 22 and PND 60. Brains were formalin-fixed for Rapid Golgi staining of tissue blocks; coded slides of hippocampus were prepared. For branching and spine analysis, 6 CA1 pyramids were randomly selected from each brain. Camera lucida drawings of the basilar dendritic tree were analyzed using the Sholl method of concentric circles. For spine analysis, counts were made along internal and terminal tip segments of 6-7 neurons from each brain. Results of the branching analysis for the PND 22 PCB-exposed rats showed that, compared to controls, there was significantly less dendritic branching in the outer 2/3rds of the dendritic tree (p = 0.002, Wilcoxon test). Spine analysis also showed a reduction in spines on the terminal tips segments of 22 day old PCB-exposed rats (p=0.005, T-test). These results suggest that perinatal exposure to Aroclor 1254 resulted in morphometric changes in hippocampus. By PND 60, spine density on terminal tip segments had returned to normal levels. However, branching analysis now showed that compared to controls there was an excessive amount of dendritic material in the distal 2/3rds of the tree (p=0.001, Wilcoxon test). This suggested a possible structural "hyperplasticity" in neurons damaged by PCB exposure during the developmental period with a residual long-term dysmorphic impact on hippocampal circuitry. (This abstract does not necessarily reflect USEPA policy).

 

Presented at the 2nd Annual Neurobiology of Aging Conference
San Diego, November, 2001

Global Dendritic Neuroplasticity in the Young Adult Rat Brain FollowingAdministration of C3, an Ultra Low Molecular Weight Glycosaminoglycan:  Implications for Treatment of Alzheimer’s Disease.

R.F. Mervis1, H. Waters1, A. Bachstetter1, H. Singleton1,2,
B. Dudas3, U. Cornelli3, J.M. Lee3, and I. Hanin3.

1NeuroStructural Research Laboratories, Columbus, OH 43212;
2
Capital University, Columbus, OH
3
Loyola University Medical Center, Maywood, IL 60153

     Glycosaminoglycans (GAGs) are linear polysaccharides consisting of a hexosamine, dissacchride units, and sulfate substituents. Proteoglycans (PG), such as heparin sulfate PG, have been shown to facilitate the occurrence of classical neuropathological hallmarks of Alzheimer’s disease (AD) such as amyloid formation and increased polymerization of tau protein into neurofibrillary tangles. GAGs compete with endogenous PGs to inhibit these occurrences. Therefore, it was thought that treatment with GAGs might attenuate the neuropathological changes associated with AD and would be a useful treatment strategy. We now show that C3 also exerts widespread neuroplastic effects on dendritic branching in young adult rats.
     F344 rats (N=5) were administered C3 subcutaneously (s.c.) for 32 days (2.5 mg/kg s.c., b.i.d.). Controls were administered saline. Formalin fixed tissue blocks were Golgi-stained and coded slides prepared
. Neurons were randomly selected from three areas for quantitative analysis of dendritic branching: layer V pyramids of frontoparietal cortex; CA1 pyramids of the hippocampus, and granule cells of the dentate gyrus. Camera lucida drawings were made from 6 neurons from each area and Sholl analysis (Method of Concentric Circles) was carried out to assess the amount of dendritic material and its distribution.
     In all three populations there was evidence of dendritic neuroplasticity. This was predominately seen by increased arborization which was significant for CA1s (p=0.001), for layer V pyramids (p=0.004), and for the outer half of the arbor of granule cells of the dentate gyrus (p =0.001). Since the vast majority of synapses are on dendritic branches, it is feasible that GAG-mediated structural plasticity would also result in additional synaptic connections. Since Alzheimer’s dementia is associated with synaptic loss, C3 treatment may be efficacious in maintaining brain circuitry and minimizing cognitive loss.

(Supported by NIH grant #1 R4 AG15740-01 to RFM, IH, and JML)

 

Presented at the meeting of the Society for Neuroscience, San Diego,
November, 2001

DIETARY DEFICIENCY OF DOCOSAHEXAENOIC ACID (DHA) IMPAIRS NORMAL DENDRITIC DEVELOPMENT IN RAT NEOCORTEX.

R.F. Mervis1, A. Bachstetter1,2, H. Waters1, A. Ahmad3,
T. Moriguchi3, and N. Salem3.

1NeuroStructural Research Laboratories, Columbus, OH 43212,
2
Capital University, Columbus, OH 43209,
3
Laboratory of Membrane Biochemistry & Biophysics, NIAAA, NIH, Rockville, MD 20852.

     Docosahexaenoic acid (DHA), an n-3 fatty acid, is a critical structural component of neuronal and synaptic membranes. Since DHA is rapidly deposited during the period of normal brain development, reduced dietary intake of n-3 fats leads to lowered brain DHA that, in turn, may cause losses in brain function. Currently, infant formula in North America is devoid of DHA. Rats were made deficient in brain DHA (DEF group) by limiting n-3 dietary fats through 3 generations. Controls received an n-3 adequate diet (ADQ group) that contained flaxseed oil and DHASCO. Rats were sacrificed at 21 and 68 days of age (N=8/group) and fronto-parietal cortex was Golgi-stained. From coded slides, 40 layer V pyramids were randomly selected from each group and camera lucida drawings were made of the basilar dendritic trees. Statistical analysis (Sholl Analysis, method of concentric circles) showed that in the 21 d group, there was 15% less dendritic material in the DHA DEF group compared to the ADQ group (p = 0.0001, Wilcoxin test). Between 21 to 68 days, dendritic pruning reduced the arbor of the ADQ control group by 25%, and of the DEF group by 13%. Thus, by 68 days the extent of dendritic branching in pyramid neurons from both groups was equivalent. Since DHA deficiency can result in long-term cognitive deficits, our findings suggest possible remaining neurostructural consequences that may result from n-3 deficiency.

 

Presented at the meeting of the Society for Neuroscience, San Diego
November, 2001

DENDRITIC AND SOMA SIZE ALTERATIONS IN HIPPOCAMPUS AND CORTEX OF RATS FOLLOWING NEONATAL BORNA VIRUS INFECTION

M. Hornig1,2, R.F. Mervis4, H. Waters4; A. Bachstetter4,5, H. Singleton4,5, D. Chian2,
M. Sindelar2, W.I. Lipkin1,2,3*

Departments of Neurology1, Microbiology & Molecular Genetics2,
Anatomy & Neurobiology3, UC Irvine, Irvine, CA, USA
4
NeuroStructural Research Labs, Columbus, OH, USA
5
Capital University, Columbus, OH, USA

     Altered neuronal morphology and cell losses are reported in postmortem brain of subjects with autism, including small neurons, decreased dendritic complexity in hippocampus, cerebellar Purkinje cell loss, and at least one report of cortical involvement. Neonatal infection of Lewis rats with Bornavirus, a neurotropic RNA virus, results in neuropathologic, behavioral, and neurochemical abnormalities resembling features of autism. Understanding the processes influencing synaptic dysfunction and selective neuronal losses by apoptosis in this animal model may reveal mechanisms important in neurodevelopmental disorders, including autism. Using Golgi-stained tissue, we evaluated changes in neuronal morphology in dentate gyrus and layer V of fronto-parietal cortex in neonatally infected rats. At 3 weeks postinfection, dendritic branching was decreased in dentate gyrus granule cells (Sholl analysis, p=0.0004) and in layer V pyramidal cells (p=0.0006). Distal dendrites were especially affected. The soma of the granule cells (but not of cortical neurons) were significantly smaller (p=0.0043). Other changes included branch atrophy, spine loss, and varicosities. These findings suggest a morphologic substrate underlying processes that disrupt synaptic plasticity or increase vulnerability to apoptosis during brain development, and support the use of this animal model for uncovering fundamental aspects of neurodevelopmental disorder pathogenesis.
Supported by: NIH grants K08 MH01608 and RO1 HD37546

 

Presented at the Yale University School of Medicine Symposium on
Cognitive Enhancers, Anti-Alzheimer and Neuroprotective Drugs
New Haven, October, 2000

Neurotrophic Effects of the Glycosaminoglycan C3 on Dendritic Arborization and Spines in the Adult Rat Hippocampus: A Quantitative Golgi Study.

R. F. Mervis1, J. McKean1, S. Zats1,2, A. Gum1,2, R. Reinhart1, B. Dudas3, U. Cornelli3,
J.M. Lee3, S.A. Lorens3, J. Fareed3, and I. Hanin3.

1NeuroStructural Research Laboratories, Columbus, Ohio 43212.
2
Ohio Dominican College, Columbus, Ohio
3
Dept. of Pharmacology and Experimental Therapeutics,
Loyola University Chicago Stritch School of Medicine, Maywood, Illinois 60153.

Glycosaminoglycans (GAGs) may be effective as a therapeutic strategy in the treatment of Alzheimer’s Disease (AD). Previous work from this group using a rat model showed that a single intra-amygdaloid injection of A beta (25-35) could induce abnormal tau-immunoreactive perikarya in the hippocampus. Furthermore, administration of the GAG C3, an ultra low molecular weight heparin mixture of 4-10 oligosaccharides (MW~2.1) could decrease Abeta25-35-induced tau-2 immunoreactivity in this model of AD.

In this study, we evaluated the effects of stereotaxic intra-amygdaloid injection of A Beta (25-35) 5nml/3ul)and of C3-treatment (administered subcutaneously, s.c.) on dendritic morphology of Golgi-impregnated CA1 pyramids of the hippocampus. Subjects were young-adult, male F344 rats.

There were four groups:

  • the controls were rats with intra-amygdaloid (i-a) injection of vehicle (trifluoroacetic acid, TFA, 3ul) followed by saline (32 days, s.c., 1ml/kg, b.i.d.)(N=10);
  • treatment groups consisted of:
  • i-a injection of Abeta unmollfollowed by 32 days of saline (s.c., 1ml/kg, b.i.d.)(N=5); i-a injection of Abetanmollfollowed by GAG-treatment (32 days of C3, 2.5 mg/kg s.c., b.i.d.)(N=5); andi-a injection of vehicle (TFA, 3ul) followed by the C3 GAG (32 days, 2.5 mg/kg s.c. , b.i.d.)(N=5).

Formalin-fixed coronal blocks of rat frontoparietal cortex, which included the underlying hippocampus, were stained using the Rapid Golgi method. Slides were sectioned at 120 m. All brains were coded, and 6 well-impregnated CA1 neurons were randomly chosen from each subject from the hemisphere ipsilateral to the intra-amygdaloid injection of the A25-35or the vehicle. For analysis of dendritic branching, camera lucida drawings were made of the basilar tree. The drawings were then quantified by the Sholl analysis (method of concentric circles) which provides a profile of the extent and distribution of the dendritic material. Dendritic spines were quantified on 4-5 randomly selected terminal tip segments from each neuron.

Results. Compared to the controls (TFA vehicle), dendritic branching of the CA1s was not diminished by A however, dendritic spine density was significantly attenuated (p<0.001). GAG (C3) treatment in the Atreated animals had only a minor effect on increasing dendritic branching (p=0.039, Wilcoxon signed-rank test) and had no impact on spines. However, C3 GAG-treatment alone (e.g., in the TFA vehicle-injected controls) had a remarkable neurotrophic impact on both dendritic branching and spines. C3 treatment increased total dendritic length by 36% (p<0.001) while increasing branching complexity (p<0.05). Simultaneously, dendritic spine density on the terminal tips was increased by 17%. Combining the C3-induced neurotrophic changes in both branching and spines, it was determined that 32 days of C3-treatment resulted in an overall 61% increase in total synaptic contacts on the basilar tree of the CA1 pyramids in these young-adult rats (p<0.001).

These results indicate that the GAG C3 appears to have a remarkable neurotrophic impact on the hippocampal circuitry of the brain of the normal young-adult rat. The apparent inability of the GAG treatment to produce the same effect in the A-injected AD model may suggest that a more extended time frame (and/or a different dosage) is required for the damaged neurons to respond to C3 treatment. These and other related studies (including the assessment of the C3 treatment on neurons in aged rats) are in progress. In addition, while it possible that C3 treatment could enhance learning or memory, it is also feasible that the extensive neurotrophic response in the young rat may be deleterious to learning (due to the "noise" induced by the presence of inappropriate synapses). This, too, is the subject of future study.

The present findings demonstrate a clear and dramatic effect of the GAG C3 on inducing an extensive neurotrophic effect on both dendritic branching and spines in the undamaged hippocampus of the adult rat. While awaiting the results of future and more targeted studies, they also suggest that such treatment may be useful in the treatment of AD or in attenuating age-related dendritic atrophy. The study also demonstrates the value of analysis of dendritic branching and spines from Golgi-impregnated tissue to demonstrate and quantify this structural neuroplasticity.

(Supported by HHS STTR grant # AG15740-01awarded to RFM and IH)

 

Presented at the Society for Neuroscience, October, 2000, New Orleans

ATROPHIC DENDRITIC BRANCHING OF CORTICAL PYRAMIDAL NEURONS RESULTS FROM INSULIN-LIKE GROWTH FACTOR 1 (IGF1) GENE DELETION:

A QUANTITATIVE GOLGI ASSESSMENT.

R.F. Mervis1, J. McKean1, S. Zats1,2, C. Cheng3, A. Gum1,2 and Bondy,C.A3

1NeuroStructural Research Laboratories, Columbus, Ohio 43212, USA;
2
Ohio Dominican College, Columbus, Ohio, USA;
3
Developmental Endocrinology Branch, NICHD, NIH, Bethesda, MD 20892, USA;

Based on its high level expression in the developing brain and on the finding of mental retardation in the case of human IGF1 gene deletions, IGF1 is thought to play an important role in brain development. The murine Igf1 (-/-) brain demonstrates preserved cell numbers in most structures, but a generalized reduction in neuropil— suggesting a decrease in growth of neuronal processes and compromised circuitry. In this study, we quantified the extent of dendritic branching in cortical layer II-III pyramidal neurons in adult Igf1 targeted gene deletion mice. Formalin-fixed blocks of frontoparietal cortex of 50 day-old Igf1 (-/-) mice (N=7) and wild-type littermate controls (N=6) were stained using the Rapid Golgi method. From coded slides, five layer II/III pyramids were randomly selected from each subject. Camera lucida drawings were made of the basilar dendritic tree of each neuron and the dendritic arbor was assessed using the Sholl analysis and other quantitative means. Sholl analysis showed that the dendritic arbor of II/III pyramids from the Igf1 (-/-) mice had significantly less dendritic material (p=0.0002). The estimated total dendritic length of the basilar tree was reduced by 24% in the knockout mice. Branch point analysis confirmed that these neurons also had a less complex dendritic arbor. Conclusions: This study conclusively shows significant dendritic atrophy of layer II/III pyramids in 50 day-old Igf1 targeted gene deletion mice. Similar, albeit more moderate, dendritic atrophy was also seen in layer V pyramids of these knockout mice. These results support a critical role for IGF1 in the normal maturation of cortical neurons. (Supported by NICHD)

 

Presented at the Meeting of the Society for Neuroscience, 2000

STRIKING NEUROTROPHIC EFFECT OF CHRONIC GLYCOSAMINOGLYCAN (GAG) TREATMENT ON THE DENDRITIC ARBOR OF HIPPOCAMPAL CA1 PYRAMIDAL NEURONS IN ADULT F344 RATS: A QUANTITATIVE GOLGI ANALYSIS

R.F. Mervis1, J. McKean1, T. Pindell1, R. Rinehart1, S. Zats2, A. Gum2, J.M Lee,3,4,
M.Hejna3, B. Dudas4, and I. Hanin4

1NeuroStructural Research Labs, Columbus, OH 43212;
2
Ohio Dominican College, Columbus, OH,
3
Dept. of Pathology and 4Dept of Pharmacology & Exp. Therapeutics,
Loyola Univ. Chicago Medical Center, Maywood, IL 60153.

In order to to test the efficacy of a novel therapeutic strategy in a animal model of Alzheimer’s disease (AD), neurostructural assessment of the extent of the dendritic arbor was made of CA1 pyramids in adult rats in which amyloid- (25-35) (or vehicle) had been stereotaxically injected directly into the amygdala. This was followed by chronic sub-cutaneous administration of the GAG compound. Previously, amyloid- was foundto result in trans-synaptic cytoskeletal pathology including tau2 immunoreactivity distally in cingulate cortex and hippocampus (Sigurdsson et al, 1996). In this study there were four groups of subjects: (1) Young-adult (3 mon-old) F344 rats (N=8) were given a single intra-amygdala injection of A (25-35) (5nm/3ul) and killed 32 days post-operatively; (2) Vehicle-injected Controls (N=12); (3) A+ GAG-treated Ss (N=6), and (4) the s.c. GAG-treatment alone (N=5). Formalin-fixed tissue blocks were Golgi-stained and CA1 pyramids (6/brain) were randomly selected from coded slides. The basilar dendritic trees were analyzed using Sholl analysis. While intra-amydgaloid A+ chronic GAG treatment (32 days) resulted in a small increase (+6%) in dendritic arbor above control levels, chronic GAG treatment alone resulted in a and highly significant increase (+32%) in the amount of dendritic branching in the hippocampal neurons in the young adult rats. These findings lend support the neurotrophic potential of of GAGs and suggests that it may be useful as a therapeutic strategy in AD. (Supported by NIH/SBIR Grant #1-R41-AG15740-01)

 

Published in the Journal of Neuroscience (2000)

HIPPOCAMPAL ABNORMALITIES AND ENHANCED EXCITABILITY IN A MURINE MODEL OF HUMAN LISSENCEPHALY

Mark Fleck1,4, Shinji Hirtsune2,5, Michael J. Gambello2,6, Emily Phillips-Tansey1,
Gregory Suares1, Ronald F. Mervis3, Anthony Wynshaw-Boris2,6, and Chris J. McBain1

1Laboratory of Cellular & Molecular Neurophysiology, NICHD, NIH, Bethesda, MD 20892; 2Laboratory of Genetic Disease Research, National Human Genome Research Institute, NIH, Bethesda, MD 20892;
3
NeuroStructural Research Laboratories, Columbus, OH 43212;
4
Dept of Pharmacology and Neuroscience, Albany Medical College, Albany, NY 12208;
5
The Institute for Animal Genetics, Odakura, Fukushima 961, Japan;
6
Depts of Pediatrics and Medicine, UCSD School of Medicine, La Jolla, CA 92093

Abstract
     Human cortical heterotopia and neuronal migration disorders result in epilepsy; however, the precise mechanisms remain elusive. Here we demonstrate severe neuronal dysplasia and heterotopia throughout the granule cell and pyramidal cell layers of mice containing a heterozygous deletion of Lis1, a mouse model of human 17p13.3-linked lissencephaly. Birth-dating analysis using bromodeoxyuridine revealed that neurons are born at the appropriate time but fail in migration to form a defined cell layer. Heterotopic pyramidal neurons in Lis1+/- mice were stunted and possessed fewer dendritic branches, whereas dentate granule cells were hypertrophic and formed spiney basilar dendrites from which the principal axon emerged. Both somatostatin- and parvalumin-containing-containing inhibitory neurons were heterotopic and displaced into both stratum radiatum and stratum lacunosum-moleculare. Mechanisms of synaptic transmission were severely disrupted, revealing hyperexcitability at Schaffer collateral-CA1 synapses and depression of mossy fiber-CA3 transmission. In addition, the dynamic range of frequency-dependent facilitation of Lis1+/- mossy fiber transmission was less than that of wild type. Consequently, Lis1+/- hippocampi are prone to interictal electrographic seizure activity in an elevated [K+](o) model of epilepsy. In Lis1+/- hippocampus, intense interictal bursting was observed on elevation of extracellular potassium to 6.5mM, a condition that resulted in only minimal bursting in wild type. These anatomical and physiological hippocampal defects may provide a neuronal basis for seizures associated with lissencephaly.

 

Published in CNS Drug Reviews, 5, suppl 1 (1999)

QUANTITATIVE ASSESSMENT OF NEURONAL DAMAGE IN A TRANSGENIC MURINE MODEL OF ALZHEIMER’S DISEASE

R.F. Mervis1, D. Campbell1, T. Pindell1, J. McKean1,
I. Lieberburg2, D. Schenk2, K. Kahn2, and D. Games2.


1
NeuroStructural Research Labs, Columbus, OH 43212
2
Elan Pharmaceuticals, South San Francisco, CA 94080

Abstract
     Golgi-stained preparations reveal the dendritic arbor and spines of randomly impregnated neurons in exquisite detail. This permits highly quantitative assessments of dendritic parameters (e.g., branching and spines) which reflect the health of the neurons and the extent of circuitry in that brain region.
     The PD-APP transgenic (tg) mouse overexpresses human amyloid precursor protein (APP717Và F) and is an animal model of Alzheimer’s Disease (AD). These mice develop A-beta plaques in an age-related manner, and show aberrant axonal sprouting. Although increased amyloid deposition has not been demonstrated to result in neuronal loss in these mice, injured neurons should show dendritic atrophy amd related alterations. Using Golgi preparations we characterized dendritic changes in the hippocampus of aged tg PDAPP mice and non-transgenic (non-tg) wild-type controls.
     Coronal blocks of formalin-fixed tissue encompassing the hippocampal formation from 26 month-old PD-APP mice and age-matched controls were stained using the Rapid Golgi method. From coded slides, CA1 pyramids (n=66) and granule cells (n=67) in the dentate gyrus were randomly selected for dendritic branching analysis. Camera lucida drawings were analyzed using the Sholl Method of Concentric Circles. This analysis produces a profile delineating the extent and distribution of the dendritic field. Dendritic spines along selected branch segments were quantified directly from the microscope at 1,008x.

Results:

  • CA1 Pyramids – (basilar tree) Branching: The basilar tree of the CA1 pyramids from the PD-APP mice showed significant widespread atrophic changes throughout the entire arbor characterized by both loss of dendritic material and simplification of branching. Spines: There was a 30% loss of total estimated spines on the basilar tree.
  • Granule Cells -- Branching: The dendritic arbor of the granule cells showed significant atrophic changes specifically restricted only to the outer 2/3rds of the molecular layer. This portion of the dendritic tree receives afferent input from the entorhinal cortex, an area prominently affected in AD. Spines: There was a 45% loss of total estimated spines on the distal outer 1/3rd of the granule cells in the molecular layer. Middle and inner molecular layer segments showed no spine loss.

Summary:
     Our findings are in general agreement with previous studies of hippocampal dendritic atrophy in AD brains. Moreover, since these hippocampal neurons receive afferent innervation from the entorhinal cortex and this latter region is widely damaged in AD, our results suggest that amyloid deposition and partial deafferentiation of entorhinal input may both contribute to hippocampal damage with concomitant disruption of brain circuitry. These findings support the use of these PD-APP mice which overexpress amyloid as an animal model of AD. It also serves as validation to the efficacy and sensitivity of quantitative Golgi studies for assessing the extent of neuronal damage and, subsequently, for testing the efficacy of various treatment strategies.

 

Presented at the Society for Neuroscience, Miami Beach, October, 1999

HIPPOCAMPAL DENDRITIC ATROPHY IN AGING PD-APP TRANSGENIC MICE OVEREXPRESSING HUMAN AMYLOID PRECURSOR PROTEIN:
A quantitative Golgi analysis of ca1 pyramids and dentate granule cells.

R.F. Mervis1, D. Campbell1, T. Pindell1, J. McKean1,
I. Lieberburg2, D. Schenk2, K. Kahn2, and D. Games2.

1NeuroStructural Research Labs, Columbus, OH 43212
2
Elan Pharmaceuticals, South San Francisco, CA 94080

Abstract
     PD-APP transgenic (tg) mice overexpress human amyloid precursor protein (hAPP) V717F, develop A-beta plaques, and mirror other Alzheimer-like pathology. Here we further characterize neurostructural changes in the hippocampus of these mice. Coronal blocks of formalin-fixed tissue encompassing the hippocampal formation from aged (26 mons-old) PD-APP mice and age-matched controls were stained using the Rapid Golgi method. CA1 pyramids (n=66) and granule cells (n=66) in the dentate gyrus were randomly selected from coded slides. Camera lucida drawings were made of their dendritic arbors and analyzed using the Sholl Method. The basilar tree of the CA1 pyramids from the PD-APP mice showed significant atrophic changes including loss of dendritic material and simplification of branching. By contrast, the dendritic arbor of the granule cells showed atrophic changes only in the outer 2/3rds of the molecular layer, e.g., that dendritic region receiving input from the entorhinal cortex, an area severely affected in AD. These findings support the validity of this animal model of AD and provide an anatomical substrate for the cognitive impairment described in these mice. (Supported by Elan Pharmaceuticals)

 

Presented at the Society for Neuroscience, Miami Beach, October, 1999

GLYCOSAMINOGLYCANS REVERSE THE DENDRITIC ATROPHY OF CINGULATE PYRAMIDS FOLLOWING INTRA-AMYDGALOID INJECTION OF AMYLOID-b IN ADULT F344 RATS: A Quantitative Golgi Analysis

J. McKean1, T. Pindell1, J.M Lee2,3, M.Hejna2, I. Hanin3, and R.F. Mervis1

1NeuroStructural Research Labs, Columbus, OH 43212;
2
Department of Pathology and 3Department of Pharmacology & Experimental Therapeutics,
Loyola University,Chicago Medical Center, Maywood, IL 60153

Abstract
     A potential animal model of Alzheimer’s disease (AD) was assessed, based on the neurotoxic effects of amyloid-
b (25-35) stereotaxically injected directly into the amygdala. Previously, this was found (Sigurdsson et al, 1996) to result in progressive trans-synaptic cytoskeletal and astroglial reactions, including tau2 immunoreactivity distally in cingulate cortex and hippocampus. The present study reveals additional information regarding neurostructural damage of cingulate neurons in this model. Young-adult (3 mon-old) F344 rats (N=8) were given a single intra-amygdala injection of Ab (25-35) (5nm/3l) and killed 32 days post-operatively. Controls (N=7) were vehicle-injected. Formalin-fixed fronto-parietal tissue blocks were Golgi-stained. Layer V cingulate neurons (6/brain) were randomly selected from coded slides. Camera lucida drawings of the basilar tree were analyzed using Sholl analysis. Results revealed small, but consistent and statistically significant dendritic atrophy in Ab injected rats (Wilcoxin test, p<.05). The findings lend additional validity to this animal model of AD and suggest that it may be useful in the evaluation of therapeutic strategies.
(Supported by NIH/SBIR Grant #1-R41-AG15740-01)

 

Presented at the Meeting of the Society for Neuroscience, Miami Beach, October,1999

HIPPOCAMPAL DISORGANIZATION AND DENDRITIC ATROPHY FOLLOWING ABNORMAL NEURONAL MIGRATION IN LIS1-DEFICIENT MICE:
a quantitative analysis of golgi-stained tissue.

T. Pindell1, R.F. Mervis1, J. McKean1, M.W. Fleck2, S. Hirotsune3, M. Gambello3,
A. Wynshaw-Boris3, and C. J. McBain4.

1NeuroStructural Research Labs, Columbus, OH 43212;
2
Dept. Pharmacology & Neuroscience, Albany Medical College, Albany, NY 12208;
3
Lab of Genetic Disease Research, National Human Genome Research Institute, NIH, Bethesda, MD

4
Lab. of Cellular & Molecular Physiology, NICHD, NIH, Bethesda, MD 20892

Abstract
     Human Type I lissencephaly is a severe neuronal migration disorder that results from mutation or deletion of the gene encoding the
b subunit of platelet-activating factor acetylhydrolase (Lis1). Mice with a heterozygous deletion of Lis1 show abnormal neuronal migration. In the hippocampus of Lis1+/- mice both normally located, and a "stranded" subgroup of CA1 pyramidal neurons are found. We characterized dendritic parameters of both populations of CA1 neurons. Formalin-fixed coronal blocks from mutants (N=4) and wild type (WT) controls (N=7) were Golgi stained. From coded slides, normally located CA1 pyramids (in the str. pyramidale) and stranded CA1s were randomly selected for analysis. Camera lucida drawings were quantified. Dendritic analysis showed that normally located CA1s in both the KO mice and WT controls were not structurally different. However, stranded CA1s were significantly atrophic and had far more simplistic dendritic arbors. Nevertheless, spine density on these stranded CA1 neurons was normal. Such structural abnormalities may explain in part the reduced electrographic seizure threshold observed in Lis1+/- mice. (Supported by NIH.)

 

Presented at the meeting of the Neurotrauma Society, Miami Beach, October, 1999

NEOCORTICAL NEUROPROTECTION BY BONE MORPHOGENIC PROTEIN-7 FOLLOWING MIDDLE CEREBRAL ARTERY OCCLUSION IN THE RAT:
A Novel and Sensitive Histopathological Assessment using Golgi-Stained Tissue.

Ronald F. Mervis*1, J.M. McKean1, T.P. Pindell1, P.L. Kaplan2J-M Ren3, S.P. Finklestein3

1NeuroStructural Research Laboratories, Columbus, OH 43212,
2
Creative BioMolecules, Inc., Boston, MA 02199,
3
Dept. of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114

Abstract
     Bone Morphogenic Protein-7 (BMP-7) a.k.a Osteogenic protein-1 (OP-1) has previously been shown to enhance recovery of function when administered 24 hours after unilateral middle cerebral artery occlusion (MCAO) in the rat. Using the microscopic appearance of Golgi-stained neuropil as a highly sensitive histopathological technique for assessing tissue damage, we now show that BMP-7 reduced the extent of tissue damage surrounding the infarct.
     Adult S-D rats were subjected to permanent right MCAO. Approximately twenty-four and seventy-two hours after infarction the animals were administered BMP-7 (~0.4nmol) or vehicle by injection into the cisterna magna. All subjects were sacrificed 21 days post-MCAO. Brains were immersion-fixed in formalin and 3mm-thick coronal blocks within the infarcted region were removed and stained using the rapid Golgi method.
     Neocortex from the infarcted hemisphere was categorized as: (1) missing; (2) heavily damaged (severely necrotic with no Golgi-impregnated neurons); (3) moderate-to-mildly damaged (with well-impregnated neurons and a grainy neuropil background); and, (4) undamaged (impregnated neurons with a clear neuropil background). From coded slides, neocortical areas ascribed to the different categories were quantified.
     BMP-7 treated rats had less tissue missing (p=0.0012) and more lightly-to-moderately damaged (p=0.0098) and undamaged tissue (p=0.001) than control rats. Since Golgi-stained neurons in the moderately-to-lightly damaged penumbral region had less dendritic material (p=0.002) this population of neurons may be characterized by injured cells which were rescued from subsequent neuronal death by BMP-7.
(Funded by Creative BioMolecules, Inc.)

 

Presented at the IBC Conference on Alzheimer’s Disease, Boston, MA 1999

AGING PDAPP TRANSGENIC MICE WITH A-beta DEPOSITION SHOW DIFFERENTIAL PATTERNS DENDRITIC ATROPHY OF HIPPOCAMPAL CA1 PYRAMIDS
& DENTATE GRANULE CELLS.
A quantitative Golgi study of an animal model of Alzheimer’s disease

Ronald F. Mervis1, Deborah Campbell1, Timothy Pindell1, Jody McKean1,
Ivan Lieberburg2, Dale Schenk2, Karen Kahn2, and Dora Games2

1NeuroStructural Research Labs, Columbus, OH 43212
2
Elan Pharmaceuticals, South San Francisco, CA 94080

Abstract
     PD-APP transgenic (tg) mice overexpress human amyloid precursor protein (hAPP) V717F, develop A-beta plaques, and mirror other Alzheimer-like pathology. Here we further characterize neurostructural changes in the hippocampus of these mice. Coronal blocks of formalin-fixed tissue encompassing the hippocampal formation from aged (26 mons-old) PD-APP mice and age-matched controls were stained using the Rapid Golgi method. CA1 pyramids (n=66) and granule cells (n=66) in the dentate gyrus were randomly selected from coded slides. Camera lucida drawings were made of their dendritic arbors and analyzed using the Sholl Method. The basilar tree of the CA1 pyramids from the PD-APP mice showed significant atrophic changes including loss of dendritic material and simplification of branching. By contrast, the dendritic arbor of the granule cells showed atrophic changes only in the outer 2/3rds of the molecular layer, e.g., that dendritic region receiving input from the entorhinal cortex, an area severely affected in AD. These findings support the validity of this animal model of AD and provide an anatomical substrate for the cognitive impairment described in these mice. (Supported by Elan Pharmaceuticals)


 

Presented at The American Association of Veterinary Anatomists
July, 1999

ANALYSIS OF SELECTED GOLGI-STAINED BRAIN REGIONS OF TOTTERING, LEANER AND COMPOUND HETEROZYGOUS, TOTTERING/LEANER MICE.

L. C. Abbott*, J. McKean, T. Pindell and R. F. Mervis.

*Dept. of Veterinary Anatomy & Public Health, Texas A&M University, College Station, TX 77843 NeuroStructural Research Laboratories, 2109 West Fifth Ave., Columbus,OH 43212.

Abstract
     Tottering (tg/tg), leaner (tgla/tgla), and compound heterozygous tottering/leaner (tg/tgla), mutant mice exhibit, to different degrees: ataxia, petit-mal-like epilepsy and an intermittent, myoclonus-like movement disorder. The tottering (tg) and leaner (tgla) mutations occur in the alpha1A calcium channel subunit which is highly expressed by many CNS neurons. This suggests a correlation between calcium channel dysfunction and occurrence of the neurological disorders exhibited by these mice, but the pathogenesis is far from clear. We initiated a morphologic study of the CNS to gain information concerning the influence these mutations have on CNS neurons. We have carried out a quantitative morphometric analysis of tg/tgla cerebellar Purkinje cells, as 90+ % of calcium channels expressed by these cells contain alpha1A subunits. We also examined the cerebral cortex to study neurons in another brain region. Brains from adult mutant and control mice were prepared using the Golgi-Cox method. Decreased branching area and spine density were observed for tg/tgla cerebellar Purkinje cells. However, tg/tgla cerebral layer II/III pyramidal neurons showed significantly larger dendritic arbors compared to controls, but no significant difference in spine density was observed. Cerebral pyramidal neurons may undergo compensatory neuroplastic changes, possibly in response to impaired cerebellar circuitry. Supported by a NIH grant (K08NS01681) to L.C.A.

 

Published in Brain Research, 1998

DENDRITIC ALTERATIONS IN CORTICAL PYRAMIDAL CELLS
IN THE SPARCE FUR MOUSE

K.J. Hopkins1, J. McKean 2, R.F. Mervis 2, M-L. Oster-Granite3

1
Dept of Environmental Toxicology, University of California, Riverside, CA 92521;
2
NeuroStructural Research Labs, Columbus, OH 43212;
3
Division of Biomedical Sciences, University of California, Riverside, CA 92521

Abstract
     Ornithine carbamoyltransferase deficiency, an X-linked trait, leads to toxic hyperammonemia in sparce fur (spf/Y) mice. Quantitative analysis of the basilar dendritic tree of layer V pyramidal cell in frontoparietal cortex stained by the Golgi Kopsch method revealed a significant decrease in both the complexity of the dendritic arbor and in dendritic terminal spine density (60%) in spy/Y mice compared with controls. Such reductions may contribute to behavioral dysfunction observed in spy/Y mice.

 

Presented at the Annual Meeting of the Neurotrauma Society, Los Angeles, November, 1998

QUANTITATIVE ASSESSMENT OF CORTICAL DENDRITIC ALTERATIONS FOLLOWING TRAUMATIC BRAIN INJURY IN THE RAT: A GOLGI-IMPREGNATION STUDY.

Ronald F. Mervis*1, Timothy Pindell1, Jody McKean1, Joseph S. Soblosky2, Michael E. Carey2

1NeuroStructural Research Laboratories, 2109 West Fifth Avenue, Columbus, OH 43212 USA, 2Department of Neurosurgery, LSU Medical Center, New Orleans, LA 70112 USA.

Abstract
    
Traumatic brain injury (TBI) was produced by a controlled pneumatic impactor striking the entire right sensorimotor cortex of the anesthetized rat. Neurologic testing reveals initial deficits with a biphasic pattern of functional behavioral recovery. Golgi-impregnation staining – which permits detailed microscopic assessment of degenerative and regenerative dendritic changes – was used to quantitatively evaluate the basilar trees in superficial (layers II-IV) and deep (layers V-VI) pyramidal cell populations in the impact zone ("damaged region") and adjacent cortical ("undamaged") region. Animals were sacrificed at 6 days (n=3) , 3 months (n=3), and 4.5 months (n=1) after the TBI. There were 5 sham controls. A total of 219 neurons were evaluated. Slides were coded and assessments made blind with respect to the post-trauma times. Parameters quantified included Sholl analyses, measurement of total dendritic lengths, and changes in branch segments. The most salient findings were: Six days following TBI, neurons in both damaged and undamaged cortex revealed widespread dendritic atrophy. Deep pyramidal neurons appeared to be more affected than superficial pyramids. Indices of damage included loss of terminal branches, reduction in dendritic length, and significant reductions in Sholl dendritic arbor profiles. At 3 months post-injury, most dendritic parameters still were suppressed in both damaged and undamaged regions. However, there was an increase in terminal tip segments, possibly predicting new neuronal growth. By 4.5 months we find preliminary but compelling evidence of neuroplasticity in both damaged and undamaged cortex, the undamaged region showing the more robust effect. Golgi-staining thus appears to be a valuable tool to help ascertain the extent of neuronal damage and dendritic plasticity and could be useful in evaluating the efficacy of neuroprotective agents . (Supported by U.S. Army contract DAMD-17-93-C-3008)

 

Presented at the Annual Meeting of the Society for Neuroscience, Los Angeles, November, 1998

COMPENSATORY CORTICAL NEUROPLASTICITY IN THE COMPOUND, HETEROZYGOUS, TOTTERER-LEANER MUTANT MOUSE.

Ronald F. Mervis*, Timothy Pindell*, Jody McKean*, and Louise C. Abbott1

*NeuroStructural Research Laboratories, Columbus, OH 43212,
1
Dept. Veterinary Anatomy & Public Health, Texas A&M University, College Station, TX 77843

Abstract
     Compound heterozygous, tottering/leaner mice (tg/tgla) exhibit ataxia, intermittent myoclonus and absence seizures. These mice carry one allele each of two autosomal recessive mutations: tottering (tg) and leaner (tgla). Golgi impregnation studies of the cerebellum showed Purkinje cells (PC) from adult tg/tgla mice have reduced branching area and fewer dendritic spines (Abbott et al, Soc. Neurosci. Abst., 1998). Broad interaction between cortex and cerebellum with respect to motor and memory functions infers that reduced cerebellar circuitry in these mutant mice may influence cortical circuitry. Blocks of fronto-parietal cortex from tg/tgla (N=5) and control (+/+) (N=4) mice were stained using the Golgi-Cox method. Sections were cut at 120um. From coded slides, five layer II/III pyramidal neurons were randomly selected from each subject. Camera lucida drawings were made of the basilar tree of each neuron. Sholl analysis showed that the dendritic arbors from tg/tgla mutant mice were significantly larger than controls (Wilcoxin test, p=0.001). There was no significant difference in the spine density along 30um terminal tip segments between tg/tgla mutants and controls. Overall, the increased amount of dendritic branching in the mutant suggests that cortical synaptic circuitry is enhanced in this group. Previously, it was found that Lurcher mutants (with postnatal loss of PCs) showed enhanced dendritic arbor of cortical pyramids (Goss et al, Soc. Neurosci. Abst. 22:1132, 1996). The tg/tgla mutant mouse reveals an apparently comparable compensatory neuroplastic response of cortical neurons -- possibly to subserve functions associated with impaired cerebellar circuitry. [Supported by a NIH grant (K08NS01681) to L.C.A.]

 

Published in the Journal of Neurochemistry, 69, 1997

ENHANCED HEMICHOLINIUM BINDING AND ATTENUATED DENDRITE BRANCHING IN COGNITIVELY IMPAIRED ACETYLCHOLINESTERASE-TRANSGENIC MICE

R. Beeri (1), N. LeNovere (2), R.F. Mervis (3), T. Huberman (1), E. Grauer (4), J.P. Changeux (2),
and M. Soreq (1)

(1)Department of Biological Chemistry, Hebrew University of Jerusalem, Jerusalem, Israel;
(2)
Molecular Neurobiology Laboratory, Biotechnologies Department,
Pasteur Institute, Paris, France
(3)
NeuroStructural Research Laboratories, Columbus, Ohio 43212 USA
(4)
Department of Pharmacology, Israel Institute for Biological Research, Ness-Ziona, Israel

Abstract
     In a search for behavioral, neuroanatomical, and metabolic characteristics of Alzheimer’s disease that may result from cholinergic malfunction, we used transgenic mice overexpressing acetylcholinesterase (AChE) mRNA and active enzyme in brain neurons. Mapping by in situ hybridization revealed that transgenic and host AChE mRNAs were distributed similarly. In a Morris water maze working memory paradigm, adult transgenic mice did not display the characteristic improvement found control mice either between or within test days and spent less time than control mice in the platform zone. In 5-week-old transgenic mice, the basilar dendritic trees of layer V pyramidal neurons from the frontoparietal cortex were essentially as developed as in age-matched controls. However, branching totally ceased after this age, whereas in control adults it continued up to at least 7 months. Therefore, dendritic arbors became smaller in adult transgenic mice than those of controls. Furthermore, the average number of spines was significantly lower on dendritic branches of 7-month-old but not 5 week-old transgenics compared to controls. Binding of tritiated hemicholinium-3, a blocker of the high affinity uptake characteristic of active cholinergic terminals, was over two-fold enhanced in the brain of transgenic mice. In contrast, no differences were observed in the mRNA and ligand binding levels of several different subtypes of nicotinic and muscarinic acetylcholine receptors. These findings suggest that three different hallmarks associated with Alzheimer’s disease – namely, progressive cognitive failure, cessation of dendrite branching and spine formation, and enhanced high-affinity choline uptake – are outcomes of cholinergic malfunction.




 
Ronald F. Mervis, Ph.D.
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