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To be 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 S5, 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. Morgese
4 *, S. Gaetani5* 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 Mouton3,4
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