Neuroscience 2015 - Presentations from Winnipeg scientists

Abstracts are below the schedule.

  Session Start Time
Presentation Time
Location
Pres #
& Type
Poster Board # Authors & Institutions Abstract Title
Session # & Title
  Sat 10/17 1:00 PM
1:00 PM - 5:00 PM

Hall A
29.17
Poster
A31 J. L. C. CADIEUX, S. SELAMAT, *S. S. HANNILA; Human Anat. and Cell Sci., Univ. of Manitoba, Winnipeg, MB, Canada Molecular mechanisms of MAG and myelin-induced Smad2 phosphorylation

29.Axon Growth and Guidance: Extrinsic Mechanisms
  Sat 10/17 1:00 PM
1:00 PM - 5:00 PM

Hall A
39.08
Poster
B95 *S. ZHU1, J.-F. WANG2, X.-M. LI3; 1Dept. of Pharmacol. and Therapeut., 2Pharmacol. and Therapeut., Univ. of Manitoba, Winnipeg, MB, Canada; 3Psychiatry, Univ. of Alberta, Edmonton, AB, Canada Changes in amyloid deposition and neuroinflammation with age and its relationship with learning deficits in a mouse model of Alzheimerīs disease

39.Neuroinflammation and Alzheimer's Disease
  Sat 10/17 1:00 PM
1:00 PM - 5:00 PM

Hall A
74.18
Poster
R19 B. VUONG1,2, G. ODERO1,2, M. STEVENSON1,2, S. M. KERELIUK2,3, T. J. PEREIRA2,3, V. W. DOLINSKY2,3, *T. M. KAUPPINEN1,2,3; 1Kleysen Inst. for Advanced Med., 2Univ. of Manitoba, Winnipeg, MB, Canada; 3the Children's Hosp. Res. Inst. of Manitoba, Winnipeg, MB, Canada Gestational diabetes mellitus in pregnant rats induces chronic neuroinflammation, synaptic degradation and behavioral changes in the offspring

74.Neuroinflammation: Endogenous and Exogenous Modulation
  Sat 10/17 1:00 PM
1:00 PM - 5:00 PM

Hall A
77.11
Poster
U23 *W. SNOW1,2, S. K. ROY CHOWDHURY1, J. DJORDJEVIC1, D. MCCALLISTER1, C. CADONIC1,3, P. FERNYHOUGH1,2, B. C. ALBENSI1,2,3; 1Div. of Neurodegenerative Disorders, St. Boniface Hosp. Res. Ctr., Winnipeg, MB, Canada; 2Pharmacol. & Therapeut., 3Biomed. Engin., Univ. of Manitoba, Winnipeg, MB, Canada NF-gulates neuronal bioenergetics In vitro

77.Thermoregulation and Energy Metabolism
  Sat 10/17 1:00 PM
1:00 PM - 5:00 PM

Hall A
93.06
Poster
BB31 *C. CADONIC1,6,2, E. THOMSON1,6, W. SNOW6, S. ROY CHOWDHURY6, D. MCALLISTER6, J. FIEGE3, P. FERNYHOUGH4,6, S. PORTET5,2, B. C. ALBENSI4,6,2; 2Grad. Program in Biomed. Engin., 3Physics and Astronomy, 4Pharmacol. and Therapeut., 5Mathematics, 1Univ. of Manitoba, Winnipeg, MB, Canada; 6Div. of Neurodegenerative Disorders, St. Boniface Hosp. Res., Winnipeg, MB, Canada Development of a mathematical model of mitochondrial bioenergetics in cortical neurons

93.Computation
  Sun 10/18 8:00 AM
8:00 AM - 8:15 AM

S405
112.01
Nanosymposium
  K. D. PHILIBERT1, M. WUNGJIRANIRUN1,2, P. LEW3, T. M. MIZUNO3, *M. J. GLUCKSMAN4; 1Biochem. & Mol. Biol., RFUMS/Chicago Med. Sch., North Chicago, IL; 2Beth Israel Deaconess Med. Ctr., Boston, MA; 3Dept. of Physiol. and Pathophysiology, Univ. of Manitoba, Winnipeg, MB, Canada; 4Biochem. & Mol. Biol., Rosalind Franklin Univ. of Med. & Science/Chicago Med. Sch., North Chicago, IL The neuropeptide processing enzyme EP24.15 is a regulator of xenin signaling

112.Food Intake and Energy Regulation
  Sun 10/18 8:00 AM
8:00 AM - 12:00 PM

Hall A
131.16
Poster
D24 *J. DJORDJEVIC1, S. ROY CHOWDHURY2, W. M. SNOW2,3, D. MCALLISTER2, C. CADONIC2,4, E. THOMSON2, P. FERNYHOUGH2,3, B. C. ALBENSI2,3,4; 2Div. of Neurodegenerative Disorders, 1St. Boniface Hosp. Res., Winnipeg, MB, Canada; 3Pharmacol. & Therapeut., 4Biomed. Engin., Univ. of Manitoba, Winnipeg, MB, Canada Mitochondrial dysfunction in 3xTg mouse brain is accompanied by deficits in Complex I

131.Alzheimer's Disease: Synaptic and Neuronal Dysfunction
  Sun 10/18 1:00 PM
1:00 PM - 5:00 PM

Hall A
203.07
Poster
A7 *S. CHUNG1, S. BISWAS2, J. SOHN3, P. JIANG2, C. CHEN2, F. CHMILEWSKY1, W. AYAZ1, H. MARZBAN4, W. DENG2; 1Oral Biol., Univ. of Illinois At Chicago, Chicago, IL; 2Biochem. and Mol. Med., Univ. of California, Davis, Sacramento, CA; 3Inst. for Pediatric Regenerative Medicine, Shriners Hosp. for Children, Sacramento, CA; 4Univ. of Manitoba, Winnipeg, AB, Canada In vivo role of p38alpha mitogen activated protein kinase in oligodendrocyte development and myelination

203.Oligodendrocyte Differentiation
  Sun 10/18 1:00 PM
1:00 PM - 5:00 PM

Hall A
210.08
Poster
B41 *M. E. RUBIO1, J. I. NAGY2; 1Otolaryngology, Univ. of Pittsburgh Med. Sch., Pittsburgh, PA; 2Dept. of Physiol. and Pathophysiology, Fac. of Med., Univ. of Manitoba, Winnipeg, MB, Canada Connexin36 expression in major centers of the auditory system in mouse and rat: evidence for neurons forming purely electrical synapses and morphologically mixed synapses

210.Synaptic transmission: Modulation
  Mon 10/19 8:00 AM
8:00 AM - 12:00 PM

Hall A
310.22
Poster
H21 *C. HART, S. M. DYCK, S. KARIMI-ABDOLREZAEE; Physiol. and Pathophysiology, Univ. of Manitoba, Winnipeg, MB, Canada Activated astrocytes modulate survival and fate specifications of adult spinal cord neural precursor cells

310.Spinal Cord Injury: Animal Models and Human Studies
  Mon 10/19 1:00 PM
1:00 PM - 5:00 PM

Hall A
421.11
Poster
P25 *K. STECINA; Physiol., Univ. of Manitoba, Winnipeg, MB, Canada Examination of functional connectivity between brain and spinal neural networks in adult mice, In vivo with the use of fictive motor output

421.Rhythmic Motor Pattern Connectivity
  Mon 10/19 1:00 PM
1:00 PM - 5:00 PM

Hall A
422.06
Poster
Q7 *X. CHEN1, C. MACDONELL1, K. STECINA1, S. ZHAO2, E. COUTO-ROLDAN1, U. SLAWINSKA3, K. GARDINER1, P. GARDINER1, L. JORDAN1; 1Physiol., 2Biol., Univ. of Manitoba, Winnipeg, MB, Canada; 3Nencki Inst. of Exptl. Biol., Warsaw, Poland Distribution and function of the 5HT7 receptors in the lumbar spinal cord of the adult rat

422.Rhythmic Motor Patterns: Neuromodulation
  Tue 10/20 8:00 AM
8:00 AM - 12:00 PM

Hall A
478.08
Poster
B4 *R. SHI1,2, Q. SHAO3, K. LINDSTROM4, J. KELLY3, A. SCHROEDER5, J. JUUSOLA6, K. LEVINE6, J. L. ESSELTINE3, S. PENUELA3, M. F. JACKSON1,2, D. W. LAIRD3; 1Kleysen Inst. for Advanced Med., Winnipeg, MB, Canada; 2Dept. Pharmacol. & Therapeut., Univ. of Manitoba, Winnipeg, MB, Canada; 3Dept. Anat. & Cell Biol., Univ. of Western Ontario, London, ON, Canada; 4Div. of Genet. and Metabolism, Phoenix Childrenīs Hosp., Phoenix, AZ; 5Div. of Genet., Univ. of Rochester Med. Ctr., Rochester, NY; 6GeneDx, Gaithersburg, MD A PANX1 loss-of-function mutation identified from a patient with intellectual disability, hearing loss and endocrine disorders

478.Ion Channels
  Tue 10/20 8:00 AM
8:00 AM - 12:00 PM

Hall A
501.02
Poster
J11 V. BHARTI, H. TAN, Z. ZHOU, Y. WANG, *J.-F. WANG; Dept. of Pharmacol. & Therapeutics, Fac. of Medicine, Univ. of Manitoba, Winnipeg, MB, Canada Role of oxidative cysteine protein modification in Amphetamine induced neurotoxicity

501.Cell Death Mechanisms: Oxidative Stress
  Tue 10/20 1:00 PM
1:00 PM - 5:00 PM

Hall A
591.08
Poster
H31 *N. PANDIAN1, M. IQBAL2, E. EFTEKHARPOUR3; 1Physiol., Dept. of Physiology, Univ. of Manitoba, Winnipeg, MB, Canada; 2Physiol., Dept. of Physiol. and Pathophysiology, Univ. of Manitoba, Winnipeg, MB, Canada; 3Physiol. and Pathophysiology, Univ. of Manitoba, Winnipeg, MB, Canada Pro-survival role of thioredoxin reductase under metabolic stress condition: Interplay between autophagy and apoptosis

591.Cell Death Mechanisms: Apoptosis and Mitochondria
  Tue 10/20 1:00 PM
1:00 PM - 5:00 PM

Hall A
594.21
Poster
J2 *M. RASTEGAR, V. R. B. LIYANAGE, R. M. ZACHARIAH; Biochem. and Med. Genet., Univ. of Manitoba, Winnipeg, MB, Canada DNA methylation, neural stem cells and Fetal Alcohol Spectrum Disorders; implicating the role of MeCP2 regulatory network

594.Alcohol: Effects of Prenatal Exposure
  Tue 10/20 1:00 PM
1:00 PM - 5:00 PM

Hall A
635.02
Poster
CC59 *H. WANG1, S. LI1, G. J. KIROUAC1,2; 1Oral Biol., 2Psychiatry, Univ. of Manitoba, Winnipeg, MB, Canada Effect of a single episode of footshock on anxiety and neuropeptide precursors in the striatum and extended amygdala

635.Fear and Anxiety: Molecular and Cellular Mechanisms
  Wed 10/21 8:00 AM
8:00 AM - 12:00 PM

Hall A
716.08
Poster
X37 A. R. FREEMAN1, J. F. HARE2, G. ANDERSON2, *H. K. CALDWELL1; 1Dept. of Biol. Sci., Kent State Univ., Kent, OH; 2Biol. Sci., Univ. of Manitoba, Manitoba, MB, Canada Arginine vasopressin influences the social behavior of free-living Richardsonīs ground squirrels (Urocitellus richardsonii)

716.Social Behavior: Oxytocin and Vasopressin


ABSTRACTS

29.17/A31. Molecular mechanisms of MAG and myelin-induced Smad2 phosphorylation
J. L. C. CADIEUX, S. SELAMAT, *S. S. HANNILA;
Human Anat. and Cell Sci., Univ. of Manitoba, Winnipeg, MB, Canada
Inhibitory proteins in myelin such as myelin-associated glycoprotein (MAG), Nogo-A, and oligodendrocyte myelin glycoprotein pose a significant barrier to axonal regeneration in the central nervous system (CNS) after injury. Previous studies have shown that all three proteins bind to Nogo receptor 1 (NgR1) and paired immunoglobulin receptor B (PirB) with high affinity, and MAG also binds to low-density lipoprotein receptor-related protein-1 (LRP1). In addition to these receptors, we now show that myelin-associated inhibitors can also activate the transforming growth factor Fceptor and Smad2/3 signaling pathway. For P6 rat cerebellar granule neurons (CGN), P1 cortical neurons, and P1 hippocampal neurons, treatment with 20 ĩg/ml MAG-Fc, Nogo-AP, or CNS myelin for 30 minutes strongly induced phosphorylation of Smad2. In our recent work, we have also shown that myelin basic protein (MBP), one of the most abundant proteins in myelin, does not induce Smad2 phosphorylation, which indicates that this effect is specific to myelin-associated inhibitors. To elucidate the role of the TGFeptor in this process, P6 CGN were treated with the TGFceptor inhibitors SB431542 and SB505124 for 1 hour, followed by the addition of 20 ĩg/ml MAG-Fc for 30 minutes. In samples from neurons that received SB431542 or SB505124, MAG-induced Smad2 phosphorylation was completely blocked, which suggests that MAG activates the TGFeptor. We are now characterizing the onset and duration of Smad2 activation by myelin-associated inhibitors and investigating whether the TGFceptor is being transactivated by the known MAG receptors NgR1, PirB, and LRP1. Elucidating the pathways involved in mediating inhibition of axonal growth is the first step towards developing therapies for enhancing functional recovery after spinal cord injury, and ultimately, we plan to test whether TGFeptor inhibitors are capable of overcoming inhibition by myelin and promoting axonal regeneration in vivo.



39.08/B95. Changes in amyloid deposition and neuroinflammation with age and its relationship with learning deficits in a mouse model of Alzheimerīs disease
*S. ZHU1, J.-F. WANG2, X.-M. LI3;
1Dept. of Pharmacol. and Therapeut., 2Pharmacol. and Therapeut., Univ. of Manitoba, Winnipeg, MB, Canada; 3Psychiatry, Univ. of Alberta, Edmonton, AB, Canada
The pathology of Alzheimerīs disease (AD) includes amyloid plaques and neurofibrillary tangles as well as chronic neuroinflammation characterized by frank microglial/astroglial activation and subsequent upregulation of proinflammatory cytokines. Both amyloid deposition and neuroinflammation appear in the early course of AD and become notably conspicuous as disease progresses. However, the progression of neuroinflammation and its relationship with amyloid deposition and behavioural changes have not been characterized as many underlying mechanisms rarely occur in isolation. The present study will thoroughly characterize the behaviour of the APP/PS1 mouse model of AD, using a comprehensive test battery designed to assess a variety of behaviours including: working memory, reference memory, long-term memory, anxiety, and motor ability. Using a cross-sectional design, these behaviours will be assessed in mice aged 2-3 months, 6-7 months, 9-10 months, 12-13 months, and 15-16 months. Brain pathology measures for amyloid deposition and neuroinflammation are done post-mortem. APP/PS1 mice exhibited significant learning deficits from the age of 6 month, which were aggravated at the later stages of life. However, the degree of memory impairment plateaus after 12 months. Histological analyses showed that an early appearance of amyloid plaques at 3 months of age with a linear progressive increase up to 22 months. This pronounced amyloid deposition was accompanied by a steady increase of the glial fibrillary acidic protein (GFAP) positive astrocytes and CD11b positive microglia up to the age of 9-12 months. Interestingly the expression levels of GFAP rose steeply from the age of 5 months to the age of 9 months and then stabilized at the age of 12 months which coincided with the observed pattern of learning deficits in the APP/PS1 mice. This work aims to provide advances in the knowledge of the neuroinflammatory changes and its relationship with amyloid deposition and behavioural changes that occur in AD patients.



74.18/R19. Gestational diabetes mellitus in pregnant rats induces chronic neuroinflammation, synaptic degradation and behavioral changes in the offspring
B. VUONG1,2, G. ODERO1,2, M. STEVENSON1,2, S. M. KERELIUK2,3, T. J. PEREIRA2,3, V. W. DOLINSKY2,3, *T. M. KAUPPINEN1,2,3;
1Kleysen Inst. for Advanced Med., 2Univ. of Manitoba, Winnipeg, MB, Canada; 3the Children's Hosp. Res. Inst. of Manitoba, Winnipeg, MB, Canada
Gestational diabetes mellitus (GDM) is the most common complication of pregnancy and population health studies have linked it to impaired cognitive performance in the offspring. GDM and diets containing excess fats and sugars promote inflammatory responses. Prolonged inflammation can impair the neuronal circuitry development in the fetus resulting in lifelong effects on cognitive functions. We hypothesized that GDM causes adverse inflammatory responses in the fetus. This inflammatory environment could disturb the fine-tuning of developing neuronal networks impairing the neurocognitive abilities of the offspring. We induced GDM by feeding female rats a "junk food" diet high in sucrose and fatty acids 6 weeks prior to mating and throughout their pregnancy. Fetal (18.5E) and 15 week-old (young adult) offspring from GDM and lean dams were examined. The neurocognitive abilities of 15 week-old offspring were evaluated in Open field, Morris Water Maze and with Novel Object Recognition test, and the brains from both age groups were analyzed by immunohistochemistry. Complementary in vitro experiments involved analyzing microglial responses to elevated levels of glucose and/or fatty acids. Offspring from GDM dams showed atypical explorative behaviour in open field test. The reduced neurocognitive performance directly correlated to maternal glucose imbalance (fasting blood glucose) during gestation. Analysis of brain tissues derived from the fetal and 15 week-old offspring of GDM dams showed increased astroglial GFAP expression, increased microglial morphological activation, and reduced expression of synaptic vesicle protein. Consumption of a post-weaning high fat and sucrose diet by the GDM offspring further promoted GDM-induced abnormalities. Cultured microglia exposed to high glucose and/or fatty acids transformed into activated, amoeboid morphology, significantly increased nitric oxide production, and changed cytokine release profile. Both in vitro and in vivo data demonstrate that GDM induces chronic inflammatory responses in the brain of the offspring that persist into young adulthood. Microglia culture experiments confirmed that excess glucose and/or fatty acids induce pro-inflammatory responses. Detrimental pro-inflammatory responses combined with impaired microglial neurotropic functions could explain synaptic degradation that contributes to behavior changes, and memory and learning impairments in the offspring.



77.11/U23. NF-gulates neuronal bioenergetics In vitro
*W. SNOW1,2, S. K. ROY CHOWDHURY1, J. DJORDJEVIC1, D. MCCALLISTER1, C. CADONIC1,3, P. FERNYHOUGH1,2, B. C. ALBENSI1,2,3;
1Div. of Neurodegenerative Disorders, St. Boniface Hosp. Res. Ctr., Winnipeg, MB, Canada; 2Pharmacol. & Therapeut., 3Biomed. Engin., Univ. of Manitoba, Winnipeg, MB, Canada
Objective: Although considered important for immunity, the transcription factor nuclear factor kappa B (NF-s also implicated in learning and memory. Mice lacking the p50 subunit of NF-splay learning and memory deficits. NF-vels are altered after the induction of long-term potentiation (LTP), an experimental model of cellular learning and memory. Conversely, LTP is altered after blockade of NF- hippocampal brain slices. In addition to synaptic plasticity, the regulation of neuronal bioenergetics is also central to learning and memory. NF-kB modulates cellular energy regulation (i.e., mitochondrial dynamics, glycolysis) in cancer biology. Whether NF-erts similar influences on energy production in neurons, however, is not well established. NF- present in the mitochondrial matrix in neurons. Further, the antioxidant manganese superoxide dismutase (MnSOD), localized to mitochondria, is a downstream target of NF-his study investigated the effects of NF-ockade on cellular respiration and glycolysis in neuronal cultures as a means of investigating its role in neuronal energy homeostasis. Methods: Cortical neurons from embryonic CD1 mice (E15-16) were cultured in 24-well Seahorse (Seahorse Biosciences) culture plates (300,000/well). After 24-hr treatment with NF-hibitors sulfasalazine and SN50 at DIV 8-9, oxygen consumption rates (OCR) and glycolysis (extracellular acidification rates; ECAR) were measured in real time using the XF24 Analyzer (Seahorse Biosciences) and compared to untreated controls. Western blots were used to detect levels of NF-d MnSOD after NF-ockade. Results: Treatment (24 hr) with 1 mM sulfasalazine decreased maximal respiration (p<0.05) in neurons. SN50 decreased maximal respiration rates in a dose-dependent fashion (p<0.05). Sulfasalazine had no effect on ECAR at baseline. Glycolysis, however, was diminished in neurons treated with varying doses (300 uM-1 mM) of sulfasalazine (p<0.001), as was glycolytic capacity (p<0.01). Western blot experiments are ongoing. Conclusions: These data establish a novel role for NF- neuronal bioenergetics. Such results have important implications for the treatment of disorders in which brain energy regulation and memory are compromised, including Alzheimerīs disease. Acknowledgement: Funding from Natural Sciences and Engineering Research Council (NSERC, BA), Research Manitoba (WS and BA), St. Boniface Research (BA), and the Everett Endowment Fund (BA).



93.06/BB31. Development of a mathematical model of mitochondrial bioenergetics in cortical neurons
*C. CADONIC1,6,2, E. THOMSON1,6, W. SNOW6, S. ROY CHOWDHURY6, D. MCALLISTER6, J. FIEGE3, P. FERNYHOUGH4,6, S. PORTET5,2, B. C. ALBENSI4,6,2;
2Grad. Program in Biomed. Engin., 3Physics and Astronomy, 4Pharmacol. and Therapeut., 5Mathematics, 1Univ. of Manitoba, Winnipeg, MB, Canada; 6Div. of Neurodegenerative Disorders, St. Boniface Hosp. Res., Winnipeg, MB, Canada
In this project, a mathematical model for mitochondrial function has been developed from oxygen consumption rate (OCR) and oxygen concentration data measured in the Seahorse XF24 Analyzer (Seahorse Biosciences). Measurements in the XF24 Analyzer were conducted on embryonic-cultured cortical neurons from CD1 mice. Based on the biological mechanism of mitochondrial activity, a deterministic model was developed using biochemical kinetic modelling, and a stochastic validation model was developed using the stochastic simulation algorithm. The deterministic model was calibrated using the optimization genetic algorithm Ferret by fitting real-time OCR data. The model was then coded in MATLAB R2014a (Mathworks) for simulating mitochondrial bioenergetics in silico. To modulate the activity of the mitochondria, specific dysfunctions were introduced by injecting the inhibiting reagents oligomycin, rotenone, and antimycin A; and the uncoupling reagent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), during OCR measurements. To appropriately incorporate these changes, the model equations were adapted and then re-calibrated to the data for each biological condition. This indicated the flexibility of the model in addressing changes in the biological environment and the appropriate mitochondrial response. The model developed thus maintains the capacity to be expanded upon when calibrated to additional data sets, affording the ability to determine the mechanism of the kinetic changes required to represent biological results.



112.01. The neuropeptide processing enzyme EP24.15 is a regulator of xenin signaling
K. D. PHILIBERT1, M. WUNGJIRANIRUN1,2, P. LEW3, T. M. MIZUNO3, *M. J. GLUCKSMAN4;
1Biochem. & Mol. Biol., RFUMS/Chicago Med. Sch., North Chicago, IL; 2Beth Israel Deaconess Med. Ctr., Boston, MA; 3Dept. of Physiol. and Pathophysiology, Univ. of Manitoba, Winnipeg, MB, Canada; 4Biochem. & Mol. Biol., Rosalind Franklin Univ. of Med. & Science/Chicago Med. Sch., North Chicago, IL
Xenin is a 25 amino acid neuropeptide produced by a subgroup of chromogranin A+ cells in the duodenum with sequence homology and actions similar to hypothalamic and ileal neurotensin (a satiety factor) as well as binding to the neurotensin receptor. This neurohormone is involved in glucose homeostasis and increases the response to glucose-dependent insulinotropic peptide (GIP). Upon release after meal ingestion Xenin inhibits secretion of acid and pancreatic exocrine peptides in the gastrointestinal tract. The mode of regulation of Xenin remains elusive. The metalloendopeptidase EC 3.4.24.15 (thimet oligopeptidase, EP24.15) has been demonstrated to play a key role in the cleavage and subsequent regulation of several neuropeptides that also exist in the gut such as neurotensin and somatostatin. These studies began from structural analyses and in silico molecular modeling and implicated Xenin as a substrate of EP24.15 and thus, this enzyme as a possible regulator of Xenin signaling. To substantiate a potential regulatory mechanism for the functioning of Xenin, we first identified the cleavage site and measured enzymatic parameters, and then determined if EP24.15 and Xenin are co-expressed in gut regions relevant to glucose homeostasis function. EP24.15 and Xenin were co-incubated and subjected to matrix-assisted laser desorption ionization time-of-flight (MADLI-TOF) mass spectrometry to confirm that EP24.15 can cleave Xenin in vitro and enzyme kinetics performed with standards via high performance liquid chromatography yielded results consistent with known substrates. Notably, Xenin is cleaved in a homologous manner as neurotensin. Double-label immunohistochemistry demonstrated Xenin and EP24.15 immunoreactivity within the mouse small intestine and specifically co-expression within intestinal mucosa and submucosa. Furthermore, there is co-expression of EP24.15 and Xenin in cells of both human and mouse stomach in the gastric mucosa. Taken together this data suggests that EP24.15 may act to cleave Xenin in vivo and represents an additional facet of the control mechanism of glucose homeostasis. As a potential pharmacological therapeutic target, understanding regulation of Xenin by the neuropeptide processing enzyme, EP24.15, may provide insight into an alternative strategy for glucose regulation and diseases such as diabetes and obesity. Supported by NIH OD010662 (MJG) and CIHR MOP123208 (TMM).



131.16/D24. Mitochondrial dysfunction in 3xTg mouse brain is accompanied by deficits in Complex I
*J. DJORDJEVIC1, S. ROY CHOWDHURY2, W. M. SNOW2,3, D. MCALLISTER2, C. CADONIC2,4, E. THOMSON2, P. FERNYHOUGH2,3, B. C. ALBENSI2,3,4;
2Div. of Neurodegenerative Disorders, 1St. Boniface Hosp. Res., Winnipeg, MB, Canada; 3Pharmacol. & Therapeut., 4Biomed. Engin., Univ. of Manitoba, Winnipeg, MB, Canada
Objective: Alzheimerīs disease (AD), the most common late onset neurodegenerative dementing disorder, affects almost 800 000 elderly in Canada. AD disproportionately affects women in both prevalence and severity; 72% of AD sufferers in Canada are women. AD is characterized by progressive neuronal loss, especially in the hippocampus and cortex. While plaques and tangles are hallmarks of the disease, emerging evidence strongly support the idea that mitochondrial dysfunction is an early event in the onset and progression of AD. Although mitochondrial dysfunction was reported in AD cases, the origin(s) of the mitochondrial dysfunction, its causal relationship to oxidative stress and the mechanisms of downstream effects to yield synaptic dysfunction and neuronal death are not clear. These experiments characterize dysfunctional processes in brain mitochondria that contribute to AD at different points in disease progression in 3xTg AD mice. Methods: Hippocampus and cortex of 3xTg and control male and female mice (2, 6 and 14 month-old) were analyzed for mitochondrial function by measuring oxygen consumption rates (OCR) on the XF24 Analyzer, (Seahorse Bioscience) and Oxygraph 2K (Oroboros) instruments. Western blot experiments were used to determine protein levels of selected Complex I-V subunits in cortical and hippocampal mitochondria. Results: Our data showed significantly decreased OCR, maximal respiration, spare respiratory capacity, basal and coupled respiration in cortical mitochondria from both male and female 3xTg mice (*p < 0.05, n=4) compared to their age-matched controls (14 month old). However, hippocampal mitochondria showed decreased maximal respiration and spare respiratory capacity only in 3xTg females. Western blot results of Complex I-V subunits revealed significant decrease in Complex I protein level (almost 50%) in both brain structures in female 3xTg (*p < 0.05). Experiments to determine mitochondrial function and Complex I-V protein levels at younger ages are ongoing. Conclusion: Our preliminary data confirm compromised mitochondrial function in hippocampus and cortex of 14 month-old 3xTg mice and suggest that deficits in Complex I are mitigating factors. Acknowledgement: Funding from Natural Sciences and Engineering Research Council (NSERC), the St. Boniface Research, and the Everett Endowment Fund.



203.07/A7. In vivo role of p38alpha mitogen activated protein kinase in oligodendrocyte development and myelination
*S. CHUNG1, S. BISWAS2, J. SOHN3, P. JIANG2, C. CHEN2, F. CHMILEWSKY1, W. AYAZ1, H. MARZBAN4, W. DENG2;
1Oral Biol., Univ. of Illinois At Chicago, Chicago, IL; 2Biochem. and Mol. Med., Univ. of California, Davis, Sacramento, CA; 3Inst. for Pediatric Regenerative Medicine, Shriners Hosp. for Children, Sacramento, CA; 4Univ. of Manitoba, Winnipeg, AB, Canada
The mitogen-activated protein kinases (MAPK) belong to the family of serine/threonine protein kinases that allow cells to respond to stimuli received from their extracellular environment including mitogens as well as to intracellular stress. Previous in vitro studies using p38 inhibitors suggested that p38K is required for myelination and oligodendrocyte (OL) differentiation. The proper development of OL and myelination is essential for maintaining the efficiency and speed of electrical nerve impulse. In an effort to identify the specific roles of p38myelination during early postnatal development, we have bred p38xed mice with NG2 or PLP-cre mice to generate homozygous conditional NG2/Plp-specific p38ditional knockout (CKO) mice for the first time. Our main finding was that, although a myelination phenotype was not evident at a gross level, there were several myelination defects at the ultra-structural level. Specifically, myelin bundles in the corpus callosum failed to develop normally, and there was a delayed onset of myelination in the corpus callosum. These defects could be partly due to a delay in OL differentiation during postnatal development since OL progenitor cell (OPC) proliferation remained normal in these knockout mice. This was supported by our observation that gene expression levels of several critical transcription factors of OPC maturation such as Olig1, Zfp488, and the OPC marker NG2 were significantly downregulated during early neonatal development in these knockout mice. Additionally, similar to previous reports, an inherent myelination defect was apparent in the primary OPCs isolated from p38O mouse brains. These OPCs failed to synthesize MBP when differentiated in vitro. These data indicate that p38K is as an important regulator of positive effectors of OL differentiation and myelination progression. Taken together, present study suggests p38PK as a key regulator in OL development and myelination process in the CNS.



210.08/B41. Connexin36 expression in major centers of the auditory system in mouse and rat: evidence for neurons forming purely electrical synapses and morphologically mixed synapses
*M. E. RUBIO1, J. I. NAGY2;
1Otolaryngology, Univ. of Pittsburgh Med. Sch., Pittsburgh, PA; 2Dept. of Physiol. and Pathophysiology, Fac. of Med., Univ. of Manitoba, Winnipeg, MB, Canada
Electrical synapses formed by gap junctions composed of connexin36 (Cx36) are widely distributed in the mammalian central nervous system. Here, we used immunofluorescence methods to document the expression of Cx36 in the cochlear nucleus and along upstream nuclei of the auditory pathway of rat and mouse. Labelling of Cx36 visualized exclusively as Cx36-puncta was densely distributed primarily on the cell body and initial dendrites of neuronal populations in the ventral cochlear nucleus, and was abundant in superficial layers of the dorsal cochlear nucleus. Other auditory centers displaying Cx36-puncta included the medial nucleus of the trapezoid body (MNTB), regions surrounding the lateral superior olivary nucleus, the dorsal nucleus of the medial lemniscus, the nucleus sagulum, all subnuclei of the inferior colliculus, and the auditory cerebral cortex. In EGFP-Cx36 transgenic mice, EGFP reporter was detected in neurons located in each of auditory center that harboured Cx36-puncta. In the ventral cochlear nuclei and the MNTB, many neuronal somata were heavily innervated by nerve terminals containing vesicular glutamate transporter-1 (vglut1) and Cx36 was frequently localized at these terminals. Cochlear ablation caused a near total depletion of vglut1-positive terminals in the ventral cochlear nuclei, with a commensurate loss of labelling for Cx36 around most neuronal cell bodies, but preserved Cx36-puncta at somatic neuronal appositions. The results suggest that electrical synapses formed by Cx36-containing gap junctions occur in most of the widely distributed centers of the auditory system. Further, it appears that morphologically mixed chemical/electrical synapses formed by nerve terminals are abundant in the ventral cochlear nucleus, including those at endbulbs of Held formed by cochlear primary afferent fibers, and those at calyx of Held synapses on MNTB neurons.



310.22/H21. Activated astrocytes modulate survival and fate specifications of adult spinal cord neural precursor cells
*C. HART, S. M. DYCK, S. KARIMI-ABDOLREZAEE;
Physiol. and Pathophysiology, Univ. of Manitoba, Winnipeg, MB, Canada
Neural stem/progenitor cells (NPCs) that reside in the adult spinal cord become activated following spinal cord injury (SCI) and contribute to generation of new glial cells. Despite their intrinsic ability to generate both oligodendrocyte and astrocytes, adult NPCs predominantly differentiate into astrocytes in the post-SCI milieu, with only a limited number giving rise to oligodendrocytes. This evidence implicates a role for the injury microenvironment in influencing the regenerative response of spinal cord NPCs. Astrocytes are a key modulator of their microenvironment both in the normal and injury conditions. Following SCI, resident astrocytes undergo drastic changes that result in their transition into an activated inhibitory phenotype. Here, we investigated how reactive astrocytes influence the cellular properties of spinal cord derived NPCs in their post-injury environment using an in vitro model of reactive astrogliosis. Rat primary astrocyte cultures were activated by lipopolysaccharide (LPS) and transforming growth factor beta (TGF-strocyte reactivity was confirmed by increased expression of chondroitin sulfate proteoglycans (CSPGs) and proinflammatory cytokines in the conditioned media of astrocytes treated by TGF- LPS, respectively. Primary NPCs were harvested from the spinal cord of adult mice and propagated through passaging. Dissociated NPCs were subject to astrocyte conditioned media (ACM) of normal, LPS and TGF-eated astrocytes. Using various cellular and molecular in vitro assays, we found that exposure to LPS-treated ACM markedly decreased NPCs survival and reduced their capacity for oligodendrocyte differentiation compared to the control normal ACM or serum free media. Importantly, LPS-treated ACM favored astrocyte differentiation of NPCs. We found the same but smaller effect in spinal cord NPCs subject to TGF-ated ACM. Interestingly, our analysis showed that increased levels of CSPGs in LPS and TGF-ated astrocytes was in part responsible for these inhibitory effects since removal of CSPGs with chondroitinase ABC was able to reverse some of these effects. Our data suggests the impact of reactive astrocytes in regulating the properties of NPCs in their post-injury niche. Identification of astrocyte-derived factors involved in NPCs modulation can potentially improve the regenerative response of NPCs in injury condition. Supported by Natural Sciences and Engineering Council of Canada (NSERC).



421.11/P25. Examination of functional connectivity between brain and spinal neural networks in adult mice, In vivo with the use of fictive motor output
*K. STECINA;
Physiol., Univ. of Manitoba, Winnipeg, MB, Canada
The advantages offered by genetically modified mouse models to study neural networks have lead to this species being an increasingly common model of choice for neuroscience research. The experimental model presented here describes a preparation in which functional connectivity of central neural networks can be assessed in mature mice, in vivo by a variety of electrophysiological methods. Stereotaxically defined stimulation of regions in the intact mouse brain can evoke motor responses (while the animals are under neuromuscular blockade) as well as in decerebrate preparations fictive motor output can be evoked by focused electrical brain stimulation and without pharmacological manipulations of neural excitability. The motor behaviours expressed in this preparation include descending fibre-activation induced motor output, fictive rhythmic locomotor-like activity and fictive crossed extension. This model has been used to identify midbrain-lumbar spinal connectivity that is involved in the initiation of fictive locomotion. Analysis of spinal cord surface potentials and electroneurograms evoked in response to stimulation in the Cuneiform and pre-Cuneiform region in the adult nervous system has found that direct descending fibres from this region are projecting to lumbar spinal targets. These projections have been described anatomically, but this is the first report of their functional role in motor control. These direct descending projections may represent important species differences between the mice and larger mammals as cats, for example, do not seem to utilize direct connections from this region for motor output initiation.



422.06/Q7. Distribution and function of the 5HT7 receptors in the lumbar spinal cord of the adult rat
*X. CHEN1, C. MACDONELL1, K. STECINA1, S. ZHAO2, E. COUTO-ROLDAN1, U. SLAWINSKA3, K. GARDINER1, P. GARDINER1, L. JORDAN1;
1Physiol., 2Biol., Univ. of Manitoba, Winnipeg, MB, Canada; 3Nencki Inst. of Exptl. Biol., Warsaw, Poland
Monoamines are important neuromodulators of motor function. Specific monoamine receptors, also known as 5-hydroxy-tryptophan (5HT) receptors contribute to rhythmic, locomotor-like activity. Recent work has shown that activation of 5-HT7/1A receptors facilitates the activity of the locomotor central pattern generator (CPG) without direct actions on the output components of the locomotor system, including motoneurons in rats. The strong influence of the 5HT7 receptors on the CPG was observed in rats that underwent complete spinal transection and grafting of embryonic serotonergic neurons to aid hindlimb locomotor function recovery. As there is limited information on the distribution and on the actions of 5HT7 receptors in mature rats with intact spinal cord, we performed two studies to determine both the distribution and the function of the 5HT7 receptors. One study used in situ hybridization of 5HT7 receptors in the adult rat spinal cord, with specific focus on the thoraco-lumbar segments. An oligonucleotide probe complementary to bases 1341-1385 of rat 5HT7R mRNA, was labeled at the 3ī-end with digoxigenin-dUTP. We have verified the specificity of these methods in 5HT7 receptor knock out mice. The preliminary results show widespread distribution in the thoracic and lumbar spinal cord including lamina VII, VIII and X of the ventral horn where key neurons for patterned motor output have been previously identified. The other study utilized the adult, decerebrate rat model to investigate the role of 5HT7 receptors during locomotion evoked by the electrical stimulation in the mesencephalic locomotor region (MLR). In this model, fictive locomotor (i.e. similar to overground locomotion but in animals under neuromuscular blockade) activity was evoked and a highly specific 5HT7 receptor antagonist, SB269970, was delivered via an intrathecal cannula over the lumbar spinal segments. The role of 5HT7 receptors during locomotion was evaluated using peripheral electroneurogram activity from hindlimb nerves, peak mean arterial blood pressure (BP) and time-to-peak increase of the BP following 60 s of MLR stimulation. The application of the 5HT7 antagonist was effective in diminishing the fictive motor output and it also reduced the systemic blood pressure response that is evoked by the stimulation of the MLR. The results indicate that the effects on the blood pressure and on the locomotor activity have a similar time course This implies that lumbar spinal interneurons with 5HT7 receptors may represent shared components of CPG networks and those networks that control cardiac output.



478.08/B4. A PANX1 loss-of-function mutation identified from a patient with intellectual disability, hearing loss and endocrine disorders
*R. SHI1,2, Q. SHAO3, K. LINDSTROM4, J. KELLY3, A. SCHROEDER5, J. JUUSOLA6, K. LEVINE6, J. L. ESSELTINE3, S. PENUELA3, M. F. JACKSON1,2, D. W. LAIRD3;
1Kleysen Inst. for Advanced Med., Winnipeg, MB, Canada; 2Dept. Pharmacol. & Therapeut., Univ. of Manitoba, Winnipeg, MB, Canada; 3Dept. Anat. & Cell Biol., Univ. of Western Ontario, London, ON, Canada; 4Div. of Genet. and Metabolism, Phoenix Childrenīs Hosp., Phoenix, AZ; 5Div. of Genet., Univ. of Rochester Med. Ctr., Rochester, NY; 6GeneDx, Gaithersburg, MD
Aberrant activation of pannexin channels is associated with CNS disorders including ischemic injury, epileptiform bursting and cortical spreading depression underlying migraine aura. We now report the identification of a disease-linked PANX1 mutation in a 17-year-old female patient with intellectual disability, sensorineural hearing loss and other systemic dysfunctions, including skeletal defects (kyphoscoliosis) and primary ovarian failure. Whole exome sequencing identified a homozygous PANX1 gene mutation (c.650G>A) resulting in an arginine to histidine substitution at position 217 (R217H). Using a multidisciplinary approach the consequence of the R217H mutation on the expression and function of Panx1 channels was assessed. When expressed in N2A, NRK, HEK293T and Ad293 cells, the glycosylation and trafficking to the cell surface of R217H was unaltered when compared to wild type Panx1, suggesting that the surface expression of R217H is not impacted. To assess the functional consequence of the R217H mutation, we performed tight-seal whole-cell recordings from HEK293T cells expressing R217H or Panx1. In cells voltage-clamped to -60 mV, pannexin current amplitude and reversal potential was assessed from applied voltage-ramps (ą100 mV, 500 ms). Current amplitude at +100 mV in cells expressing R217H was reduced by 50% compared to wild type Panx1. In contrast no change in reversal potential was noted suggesting that reduced currents in R217H expressing cells could not be attributed to a change in ionic permeability. Both R217H- and Panx1-mediated currents were augmented by high extracellular potassium treatment (50 mM KGluconate), most notably at negative holding potentials. Nevertheless, high potassium augmented ramp currents in R217H expressing cells were reduced by 50% when compared to wild type Panx1. Confirming the specific contribution of Panx1-based channels, ramp currents from R217H and Panx1 expressing cells were suppressed by the pannexin blocker carbenoxolone. As Panx1 is permeable to solutes of up to 1 kDa, the functional consequence of the R217H mutation on dye uptake and ATP release was determined. Uptake of ethidium bromide and release of ATP in response to high potassium treatment was impaired in cells expressing R217H. Collectively, our functional studies monitoring ion flux, dye uptake, and ATP release demonstrate that R217H, associated with developmental abnormalities in a human patient, represents a loss-of-function mutation. The first three authors contributed equally to this work.



501.02/J11. Role of oxidative cysteine protein modification in Amphetamine induced neurotoxicity
V. BHARTI, H. TAN, Z. ZHOU, Y. WANG, *J.-F. WANG;
Dept. of Pharmacol. & Therapeutics, Fac. of Medicine, Univ. of Manitoba, Winnipeg, MB, Canada
Amphetamine is a highly abused psycho stimulant that can lead to dopaminergic neuron degeneration and increased the risk for neurodegenerative diseases with advanced age upon long term consumption. Many studies have shown that oxidative/nitrosative stress contributes significantly to AMPH-induced toxicity. Thiols of cysteine residues in many proteins are very susceptible to attack by reactive oxgen/nitrogen species H2O2 and NOo, subsequently inducing sulfenylation and nitrosylation. In this study, we analyzed the effects of amphetamine on H2O2 induced sulfenylation and NOo induced nitrosylation in rat brain using biotin switch method following by immunoblotting analysis. We found that repeated amphetamine treatment increased total sulfenylation and nitrosylation of proteins in rat frontal cortex. Vesicular monoamine transporter 2 (VMAT2) is mainly responsible for packaging of monoamine neurotransmitters and play an important role in neurotransmission. We found that VMAT2 can be sulfenylated and nitrosylated, and that repeated amphetamine treatment increased sulfenylation of VMAT2. Our finding suggested that nitrosylation and sulfenylation of proteins including VMAT2 may interrupt normal regulation of neurotransmission and lead to neurotoxicity



591.08/H31. Pro-survival role of thioredoxin reductase under metabolic stress condition: Interplay between autophagy and apoptosis
*N. PANDIAN1, M. IQBAL2, E. EFTEKHARPOUR3;
1Physiol., Dept. of Physiology, Univ. of Manitoba, Winnipeg, MB, Canada; 2Physiol., Dept. of Physiol. and Pathophysiology, Univ. of Manitoba, Winnipeg, MB, Canada; 3Physiol. and Pathophysiology, Univ. of Manitoba, Winnipeg, MB, Canada
Autophagy is a cellular physiologic phenomenon for normal turnover of long-term proteins and organelles. Temporary shortage of nutrients enhances autophagy through inhibition of new protein synthesis which prompts the cells to use their organelles as energy source. Upon resumption of growth material autophagy will be scaled back and cells will continue their normal activities. While short term autophagy is necessary to ensure cell survival during the stress conditions, prolonged autophagy will result in cell death. Although apoptotic cell death is the most common studied form of cell death after neurotrauma and ischemia, increasing evidence indicates the involvement of autophagy as an undergoing but less investigated form of cell damage. Rationale for studying autophagy comes from reports of functional improvements after application of autophagy inhibitors or inducers in neurotrauma or ischemia. This warrants the need for understanding the molecular mechanisms of autophagy that may lead to identification of novel therapeutic tools for CNS treatment. Oxidative stress is a major inducer of cell death. Upregulation of oxidizing agents such as oxygen and nitrogen reactive species results in oxidation of many signaling molecules and induction of autophagy or apoptosis. Thioredoxin (Trx) system is a major regulator of protein oxidation and modulator of oxidative damage. Trx is responsible for reduction of oxidized proteins but is oxidized in this process. Thioredoxin reductase (TrxR) reduces Trx and therefore maintains the reducing capacity of the cell. TrxR is upregulated under stressful conditions and its inhibition results in cell death. The exact role of TrxR in induction of autophagy or apoptosis has not been investigated. In this study we used neuroblastoma cell line (SH-SY5Y) to study autophagy and apoptosis under normal (10% serum) and metabolic stress (0% serum) conditions. TrxR was inhibited using pharmacological inhibitors, and gene-delivery approaches. We observed that inhibition of TrxR in normal growth conditions promotes apoptosis as indicated by upregulation of caspases. This was associated with upregulation of endoplasmic reticulum (ER) stress markers IRE1, and ATF6. Serum deprivation resulted in 40% cell death that was associated with upregulation of autophagy and apoptosis markers. Serum deprived SH-SY5Y cells were several folds more sensitive to TrxR inhibition and after inhibition of TrxR, apoptosis was the prevalent form of cell death. This is the first study to investigate the role of TrxR in normal and stressed conditions. Our observations further confirm the potential therapeutic capacity of Trx system for neuroprotection.



594.21/J2. DNA methylation, neural stem cells and Fetal Alcohol Spectrum Disorders; implicating the role of MeCP2 regulatory network
*M. RASTEGAR, V. R. B. LIYANAGE, R. M. ZACHARIAH;
Biochem. and Med. Genet., Univ. of Manitoba, Winnipeg, MB, Canada
Fetal alcohol spectrum disorders (FASD) are intellectual disabilities with facial-growth abnormalities and compromised brain function due to maternal alcohol exposure. FASD are common neurodevelopmental disorders that affect 1-3% of live-born children. Currently, FASD has no cure and the molecular mechanisms by which alcohol deregulates the gene expression program of differentiating neurons are poorly understood. Characterizing the cellular and molecular deficiencies of the affected neurons and identifying clinically relevant drugs to rescue these neurons are the major focus of this study. FASD etiology is thought to be via alcohol-gene interactions in differentiating embryonic neural stem cells during brain development. This process is tightly regulated by epigenetic mechanisms and epigenetic factors. One of the best-studied epigenetic mechanisms that control brain development is DNA methylation. The main protein that binds to the methylated DNA in brain is MeCP2 with key roles in brain development and function, recently linked to FASD. Accordingly, altered MeCP2 levels or loss-of-function mutations cause Rett Syndrome, autism, and X-linked mental retardation. Our initial studies in MeCP2-deficient mouse were proof-of-principle for the role of MeCP2 isoforms in neuronal maturation. We reported the developmental and cell type-specific expression of Mecp2/MeCP2 isoforms in brain. We identified and characterized Mecp2 regulatory elements that are controlled by DNA methylation. We now show that these sequences are involved in Mecp2/MeCP2 deregulation by ethanol, associated with altered inactive (5mC: 5-methylcytosine) and active (5hmC: 5-hydroxymethylcytosine) DNA marks. We find that different modes of ethanol exposure [continuous, single time exposure, or withdrawal] either induce or decrease Mecp2/MeCP2. In response to ethanol, we show globally altered 5mC and 5hmC levels associated with increased neuronal neurite branching that might be biologically important. MeCP2 is the main 5mC- and 5hmC- binding protein in brain and we show selective deregulation of MeCP2 regulatory network by ethanol. Further, we show altered Mecp2/MeCP2 levels by an epigenetic drug (DNA methylation inhibitor) that may have future applications for MeCP2-associated neurodevelopmental disorders. DNA methylation is reversible, thus uncovering deregulation of MeCP2 regulatory network and DNA methylation-mediated effects of ethanol in neurons is critical for possible drug therapy strategies. Our results indicate that targeting MeCP2 network might be a possible intervention-therapeutic avenue to rescue impaired neuronal function caused by ethanol.



635.02/CC59. Effect of a single episode of footshock on anxiety and neuropeptide precursors in the striatum and extended amygdala
*H. WANG1, S. LI1, G. J. KIROUAC1,2;
1Oral Biol., 2Psychiatry, Univ. of Manitoba, Winnipeg, MB, Canada
Corticotropin releasing factor (CRF) and dynorphin are neuropeptides that have been associated with negative emotional states. Experimental evidence indicates that dynorphin neurons located in the nucleus accumbens (NAc) and CRF neurons in the bed nucleus of the stria terminalis (BST) and the amygdala mediate the behavioral effects produced 24-48 hrs after exposure to a stressful episode. The present study was done to evaluate if changes in the levels of the mRNA for these peptides in the striatum, BST, and amygdala were associated with the long-lasting anxiety induced in a subset of rats exposed to moderately intense footshocks. On day 1, shocked rats were exposed to footshocks (5 Ũ 2 s of 1.5 mA presented randomly over 3 min), whereas nonshocked (NS) rats were placed in the shock chamber for 5 min. On day 2, rats were tested in a novel chamber and according to the amount of time spent immobile, shocked rats were grouped as high responders (HR; immobility > 60%) or low responders (LR; immobility < 40%). On day 3, fear to the shock context was assessed by measuring immobility to the shock chamber for 5 min. On days 10 & 11, the elevated T-maze (ETM) was used to assess avoidance (a measure of anxiety) and escape (a measure of helplessness). On day 14, the rats were anesthetized and the amygdala, BST, NAc and caudate putamen were removed for analysis using real-time RT-PCR. HR displayed long-lasting anxiety in the elevated T-maze whereas LR had low levels of anxiety similar to NS rats. An enhanced level of CRF mRNA was detected in the amygdala of the HR compared to LR and NS rats. In contrast, CRF, prodynorphin and proenkephalin mRNA levels in the striatum, BST and amygdala were not different between HR, LR and NS rats. This study provides evidence that CRF neurons in the amygdala play a role in the long-lasting anxiety produced in a subset of rats exposed to footshocks.



716.08/X37. Arginine vasopressin influences the social behavior of free-living Richardsonīs ground squirrels (Urocitellus richardsonii)
A. R. FREEMAN1, J. F. HARE2, G. ANDERSON2, *H. K. CALDWELL1;
1Dept. of Biol. Sci., Kent State Univ., Kent, OH; 2Biol. Sci., Univ. of Manitoba, Manitoba, MB, Canada
In many vertebrate taxa arginine vasopressin (Avp) and its homologues are important to the neural regulation of social behaviors. In rodents, Avp is best known for its modulation of affiliative behaviors such as grooming, sniffing, and the formation of social bonds and memories. Avp is also important for species-specific vocalizations, as demonstrated through work on birds, fish, and amphibians. More recently, Avp has been found to influence social communication in laboratory rodents (i.e. rat and mouse) by altering pup ultrasonic vocalizations. However, this work has not been extended to other rodent species or to free-living individuals of any species. Richardson's ground squirrels (Urocitellus richardsonii) are free-living social rodents, in which alarm calling serves as a proximate manifestation of sociality. In order to determine how Avp effects social communication in the wild, we implanted osmotic minipumps into Richardson's ground squirrels and administered Avp or saline intracerebroventricularly. We then examined behavior before and after Avp or saline administration using three assays: 1) a general behavior survey, 2) a predator model presentation, and 3) a social challenge experiment. While saline had no effect, Avp reduced aggression and increased antipredator vigilance and escape behavior in males, but had no effect on the propensity to emit alarm calls in response to a predator model. However, during the social challenge, Avp-treated males increased chirp-type vocalizations during social interactions. These context-specific effects (i.e. predator versus conspecific) on communication are interesting and mirror effects seen in olfaction experiments with laboratory rodents. This work is significant as it is the first of its kind to examine the effects of Avp on social communication in the wild. Further, our discovery of Avpīs effects on vigilance in a social setting is particularly exciting and highlights Avpīs extensive influence on social behavior.