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6th ANNUAL NEUROSCIENCE, BEHAVIOR AND HEALTH RESEARCH FORUM The University of Vermont Dudley H. Davis Center Livak Ballroom / Mansfield Room January 22 - 23, 2016 Platform Talks and Poster Abstracts Sponsored by: Society for Neuroscience Society for Neuroscience Vermont Chapter UVM Neuroscience, Behavior and Health Initiative UVM Neuroscience Graduate Program Neuroscience COBRE MBF Bioscience Med Associates / Catamount Research NBH RESEARCH FORUM SCHEDULE OF EVENTS FRIDAY, JANUARY 22 4:00 pm Registration: White Pine Lounge, Davis Center 4th Floor 4:20 pm Welcome and Introduction: Victor May, PhD, President, Vermont Chapter for the Society of Neuroscience; Livak Ballroom, Davis Center 4:30 pm Keynote Lecture: Ronald Emeson, PhD, Professor of Pharmacology, Molecular Physiology and Biophysics and Psychiatry, Vanderbilt University School of Medicine “RNA Editing in Nervous System Function and Dysfunction” Livak Ballroom, Davis Center 5:30 pm Reception: White Pine Lounge, Davis Center 4th Floor SATURDAY, JANUARY 23 8:00 am Registration, Handy Room, Davis Center 4th Floor Light Breakfast - coffee/tea: White Pine Lounge Poster Setup, Mansfield Room, Davis Center 2nd Floor 8:30 am Introductory Remarks: Mark Bouton,, Director, Neuroscience, Behavior and Health Research Initiative, Livak Ballroom, Davis Center Platform Talks Session I Chairs: Marion Weir and Gregory Johnson Departments of Biology and Neuroscience Graduate Program, University of Vermont 8:40 am Dawei Li, PhD, Department of Microbiology and Molecular Genetics, UVM “Viral DNA integration in the human genome and brain disease” 9:00 am Estelle Spear, NGP, Department of Neurological Sciences, UVM “Altered gastrointestinal motility in multiple sclerosis” 9:20 am Chenyi Liao, Department of Chemistry, UVM “Microsecond MD simulations to reveal dynamics and mechanisms of a Class B GPCR” 9:40 am Megan Shipman, NGP, Department of Psychological Science, UVM “Inactivation of the prelimbic cortex attenuates context-dependent excitatory operant responding” 10:00 am S. Bradley King, Department of Psychological Science, UVM “Chronic stress potentiates the response to intra-BNST PACAP infusion” 10:20 am Coffee Break Session II Chairs: Roman Popov and Bradley King Neuroscience Graduate Program and Department of Psychological Science University of Vermont 10:40 am James Bishop, NGP, Department of Neurological Sciences, UVM “Porcine Model to Define Pain Structural Neuroplasticity” 11:00 am Riley St. Clair, NGP, Department of Biology, UVM “Putting the [P] in [P]lexin: characterizing the phosphorylation of PlexinA2 by tyrosine kinase Fyn” 11:20 am Arielle Baker, Department of Physiology and Neurobiology, Dartmouth “Selective cholinergic modulation of layer 5 projection neurons in the mouse prefrontral cortex” 11:40 am Max Mehlman, Department of Psychological & Brain Sciences, Dartmouth “Functional and anatomical relationships between the dorsal striatum, medial precentral cortex and head direction circuit” 12:00 pm Sydney Trask, Department of Psychological Science, UVM “Learning to suppress behavior during operant extinction: implications for cue exposure therapy” 12:20 pm Olivia Miles, Department of Psychological Science, UVM “Intra-bed nucleus of the stria terminalis (BNST) pituitary adenylate cyclase activating polypeptide (PACAP) infusion reinstates cocaine seeking in rats” 12:40 pm Lunch Break / Poster Viewing and Judging Mansfield Room, Davis Center 2:00 pm Awards and Closing NEUROSCIENCE, BEHAVIOR AND HEALTH RESEARCH FORUM POSTER SESSION Poster 1 Regulation of Microtubule dynamics by Tau Rehan Ali and Christropher Berger Department of Molecular Physiology, University of Vermont College of Medicine, Burlington, VT 05405 Poster 2 Temporal coordination of hippocampal place cells reflects cognitive outcome post febrile status epilepticus Jeremy Barry Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, Vermont 05405 Poster 3 Optogentic manipulation of hippocampal theta rhythm can obstruct performance in the active avoidance task Benjamin Blumberg, eremy Barry, Sean Flynn and Gregory L. Holmes Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VT 05405 Poster 4 Parabrachial PACAP activation of amygdala endosomal ERK signaling regulates the emotional component of pain Galen Missig1 , Linda Mei1 , Margaret A. Vizzard1 , James A. Waschek2 , Sayamwong E. Hammack3, Victor May1 and Karen M. Braas1 Departments of Neurological Sciences1 and Psychological Science3 , University of Vermont College of Medicine, Burlington, VT 05405, Department of Psychiatry and Behavioral Sciences2 , University of California Los Angeles, Los Angeles, CA 90095 Poster 5 PKM-. is involved in cerebellar-dependent learning and memory Kutibh Chihabi, John T. Green1 and Anthony D. Morielli2 Departments of Psychological Science1 and Pharmacology2 ,University of Vermont, Burlington, VT 05405 Poster 6 The COBRE Imaging and Physiology Core: Two New Confocal Setups Fully Operational Todd A. Clason, Cindy J. Forehand, and Rodney L. Parsons Department of Neurological Sciences, University of Vermont, College of Medicine, Burlington, VT 05405 Poster 7 Hypotensive actions of A2 noradrenergic neurons are abolished by increased BDNF expression in the PVN Nick C. Cruickshank, Chris L. Schaich, Zachary D. Einwag, Theresa L. Wellman and Benedek Erdos Department of Pharmacology, University of Vermont College of Medicine, Burlington, VT 05405 Poster 8 Does the adaptive algorithm in the stop signal task introduce a confound in neuroimaging studies? Nicholas D’Alberto1 , Bader Chaarani1 , Philip Spechler1 , Kelsey Hudson1 , Scott Mackay1 , Nick Allgaier1 , Matthew Albaugh1 , Catherine Orr1 , Mitchell Snowe1 , Robert Althoff1, Alexandra Potter1, Hugh Garavan1, and the IMAGEN consortium. Department of Psychiatry, University of Vermont College of Medicine, Burlington, VT 05405 Poster 9 WNK-1 regulation of Kv1.2: A novel hypothesis for cognitive impairment in schizophrenia Adrian Dutkiewicz, MA; Anthony Morielli, PhD Neuroscience Graduate Program, Department of Pharmacology,University of Vermont College of Medicine, Burlington, VT 05405 Poster 10 The Role of the Plexin A Family in Eye Development. Emerson, S.E.1 , Light, S.E.1 , St. Clair, R.M.1,2 , Ballif, B.A.1,2 and Ebert, A.M.1,2 1 Department of Biology, 2 Neuroscience Graduate Program, University of Vermont, Burlington VT 05405 Poster 11 Contributions of pituitary adenylate cyclase-activating polypeptide (PACAP)/receptor signaling to increased voiding frequency and somatic sensitivity in mice with urothelium-specific overexpression (OE) of nerve growth factor (NGF) in the urinary bladder. Beatrice M. Girard, Susan Malley, Morgan E. Mathews, Margaret A. Vizzard Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VT 05405 Poster 12 The effect of cyclophosphamide on salt preference and detection threshold Michael Gomella, Evan Lowry, Ben Jewkes, Joy Benner, Eugene Delay Department of Biology, University of Vermont, Burlington, VT 05405 Poster 13 Mechanism(s) of transforming growth factor-beta (TGF-$) mediated bladder afferent nerve hyperexcitability E.J. Gonzalez, M.A. Vizzard Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VT 05405 Poster 14 Does axon initial segment plasticity regulate neuron excitability? Allan T. Gulledge1 and Jaime J. Bravo2 1 Department of Physiology and Neurobiology, Geisel School of Medicine, 2 Thayer School of Engineering, Dartmouth College, Hanover, NH 03755 Poster 15 ACTH Prevents Deficits in Fear Extinction Associated with Early Life Seizures Andrew T. Massey1,2, David K. Lerner3, Gregory L. Holmes1, Rod C. Scott1,4, Amanda E. Hernan1 1 Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VT, USA 05405, 2University of Bath, Department of Biological Sciences, Bath, UK, 3Dartmouth College, College of Arts and Sciences, Hanover, NH, USA 03756, 4 University College London, Institute of Child Health, London WC1N 1EH, UK Poster 16 Characterization of mPFC layer 5/6 neurons following sub-anesthetic ketamine Gregory Johnson and Sayamwong ‘Jom’ Hammack Department of Psychological ScienceUniversity of Vermont, Burlington, VT 05405 Poster 17 Preliminary QTL Mapping Suggests Candidate Regions for EthanolInduced Phenotypes in the Diversity Outbred Mouse Population Steven Kasparek1 , Troy Wilcox2 , Dan Gatti2 , Eric Busch3 , Drew Kreuzman1 , Benjamin Mansky1 , Sophie Masneuf3 , Erica Sagalyn3 , Kayvon Sharif1 , Dominik Taterra1 , Walter Taylor1 , MaryThomas1 , Elissa J. Chesler2 , Andrew Holmes3 , and Clarissa C Parker1 1 Department of Psychology and Program in Neuroscience, Middlebury College, VT 05753, 2 Center for Genome Dynamics, The Jackson Laboratory, Bar Harbor, ME 04609, 3 Laboratory of Behavioral and Genomic Neuroscience, NIAAA, NIH, Rockville MD 20852 Poster 18 Identifying genes associated with conditioned fear in the Diversity Outbred mouse population using a forward genetic, genome-wide approach Andrew Kreuzman1 , Troy Wilcox2 , Dan Gatti2 , Eric Busch3 , Steven Kasparek1 , Drew Kreuzman1, Benjamin Mansky1, Sophie Masneuf3, Erica Sagalyn3, Kayvon Sharif1 , Dominik Taterra1 , Walter Taylor1 , Mary Thomas1 , Elissa J. Chesler2 , Andrew Holmes3 , and Clarissa C. Parker1,4 1Program in Neuroscience, Middlebury College, VT 05673, 2Center for Genome Dynamics, The Jackson Laboratory, Bar Harbor, ME 04609, 3Laboratory of Behavioral and Genomic Neuroscience, NIAAA, NIH, Rockville MD 20852 4Department of Psychology, Middlebury College, VT 05673 Poster 19 Abundant 5-HT release from EC cells disrupts colonic motility Brigitte Lavoie, Dmitri Tchitchkan, Gary M. Mawe Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VT 05405 Poster 20 Kv1.2 potassium channel role in Cerebellar learning and memory. S. C. Madasu1 , M. L. Shipman2 , J. T. Green2 , A. D. Morielli1 1 Departments of Pharmacology and Psychological Science, University of Vermont, Burlington, VT 05405 Poster 21 TRPV1-mediated Ca2+ Influx and Constriction of Middle Meningeal Arteries Inessa Manuelyan1, Masayo Koide1, Arsalan U. Syed1, Bo Shui2, Swapnil Sonkusare1 , Michael I. Kotlikoff2 , Mark T. Nelson1 and George C. Wellman1 Department of Pharmacology, University of Vermont College of Medicine, Burlington, VT1, College of Veterinary Medicine Cornell University, Ithaca, NY2 Poster 22 Pituitary adenylate cyclase-activating polypeptide (PACAP) expression in lower urinary tract pathways (LUT) with cyclophosphamide (CYP)-induced cystitis in PACAP promoter-dependent EGFP BAC transgenic mice Morgan E. Mathews1, Susan Malley1, Beatrice M. Girard1, Karen M. Braas1, James A. Waschek2 , Victor May1 and Margaret A. Vizzard1 Dept. of Neurological Sciences1 , University of Vermont College of Medicine, Burlington, VT 05405 Dept. of Psychiatry and Behavioral Sciences2, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA Poster 23 High resolution mapping of PACAP neurocircuits using a PACAP-EGFP transgenic mouse model. Michael C. Condro1 , Anna Matynia2,3 , Nicholas N. Foster4 , Yukio Ago5 , Abha K.Rajbhandari1 , Bhavaani Jayaram1 , Sachin Parikh2,3 , Eileen Nguyen2,3 , Victor May6, Hong-Wei Dong4 and James A. Waschek1 1 Department of Psychiatry and Psychology, 2 Department of Ophthalmology, and 3 Brain Research Institute, University of California - Los Angeles, USA 90095; 4 Institute of Neuro Imaging and Informatics, University of Southern California, Los Angeles, CA USA 90089, 5 Laboratory of Medicinal Pharmacology, Osaka University, Osaka, Japan; 6 Department of Neurological Sciences, University of Vermont, USA 05405 Poster 24 Loss of mTOR complex 1 function impairs synaptic transmission and blocks the effects of Pten deletion. Matthew McCabe and Matthew Weston Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VT 05405 Poster 25 Sequential reprogramming and re-differentiation of reactive astrocytes to identify downstream targets of Jagged1/Notch1 signaling in the peri-infarct area after stroke Matthew D. LeComte, Issei S. Shimada, Andrea L. Bibeau, and Jeffrey L. Spees Department of Medicine, University of Vermont College of Medicine, Burlington, VT 05405 Poster 26 Origin of Locally-Derived Neural Spheres from the Peri-Infarct Area Following Stroke Issei S. Shimada and Jeffrey L. Spees Department of Medicine, University of Vermont College of Medicine, Burlington, VT 05405 Poster 27 Minimally Complex Robotic Model of Human Step Initiation with Anticipatory Postural Adjustments Roman E. Popov1,2 , Jesse V. Jacobs1 and Josh C. Bongard2 Departments of Rehabilitation and Motion Science1, and Computer Science2, University of Vermont, Burlington, VT 05405 Poster 28 Food-Seeking Inhibited While Hungry Can Renew in the Context of Satiation Scott T. Schepers and Mark E. Bouton Department of Psychological Science, University of Vermont Poster 29 Mechanisms of serotonergic 2A-receptor-mediated excitation in callosal projection neurons in the mouse prefrontal cortex Emily K. Stephens and Allan T. Gulledge Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth, Lebanon, NH Poster 30 It’s a HARS Knock Life for Fish: Characterizing Histidyl tRNA Synthetase in the Zebrafish Ashley Waldron1, Susan Robey-Bond2, Christopher Francklyn2, and Alicia Ebert1 1 Department of Biology and 2 Biochemistry, University of Vermont, Burlington, VT 05405 Poster 31 Disciplined self-observation of aphasic disturbances: recovery of a left hemisphere injured brain, and the insights learned from primary optic aphasia, and how my changed brain works now. Sarah Robinson Burlington, VT 05405 Poster 32 Renewal of extinguished behavior in the context of the preceding response Jeremey M. Trott, Eric A. Thrailkill and Mark E. Bouton Department of Psychological Science, University of Vermont, Burlington, VT 05405 Platform Talk 1 Viral DNA integration in the human genome and brain disease Dawei Li Department of Microbiology and Molecular Genetics and Computer Science University of Vermont College of Medicine, Burlington, VT 05405 Viral DNA integration into the host genome is required for retroviral replication and also occurs with many non-retroviruses. Converging evidence implies that viral integration may trigger numerous diseases, such as the herpes simplex virus-1 with Alzheimer’s disease. Bornavirus can integrate into the genome of brain cells, which might cause mutations that interfere with brain function, leading to development of psychiatric disorders. Due to the lack of high-throughput analytic tool, our knowledge about virome-wide viral integrations is very limited. We are developing a novel approach based on the next-generation deep sequencing to identify and characterize variations caused by viral integrations in the human genomes. We applied our method to the human genomes containing known viral integrations and achieved 100% sensitivity. Platform Talk 2 Altered Gastrointestinal Motility in Multiple Sclerosis Spear, E.T., Lavoie, B., Haag, M.M., Applebee, A., Teuscher, C.,and Mawe, G.M. Department of Neurological Sciences University of Vermont College of Medicine, Burlington, VT 05405 Multiple sclerosis (MS) patients often experience constipation, but the etiology of this symptom is unknown. To gain a better understanding of this condition, we tested whether gastrointestinal (GI) function is altered in mice with experimental autoimmune encephalomyelitis (EAE), and whether their blood and/or blood from MS patients contains antibodies directed against targets in enteric ganglia. EAE was induced in C57BL/6J and SJL/J mice by injection of complete Freund’s adjuvant (CFA) with myelin oligodendrocyte glycoprotein (C57 mice) and proteolipid protein 1 or mouse spinal cord homogenate (SJL mice). After somatic motor symptoms developed, small intestinal transit was measured by calculating the leading edge (LE) and geometric center (GC) 20 minutes after oral gavage of rhodamine dextran. Whole GI transit time was determined by oral gavage of carmine red and calculating the latency for dye to appear in fecal pellets. Fecal water content was measured by weighing fecal pellets before and after dehydration. Colonic transit was measured by the time to expulsion of a glass bead inserted 2 cm into the colon. Targets of antisera were evaluated by immunohistochemical staining of guinea pig intestine whole mount preparations with EAE, MS and control plasma. Small intestinal transit was significantly slower in EAE mice than in CFA controls (C57 MOG:GC, p<0.05; SJL PLP:GC, p<0.05; LE, p<0.05; SJL MSCH n.s.). The rate of whole GI transit was significantly longer in EAE mice versus controls (All models: p<0.05). Fecal water content was significantly lower in all EAE models compared to controls (C57 MOG: p<0.05 SJL PLP: p<0.0001 SJL MSCH: p<0.0002). Plasma from MS patients yielded more intense immunoreactivity in myenteric ganglia than plasma from control subjects (p<0.01), with immunostained structures including neurons, nerve processes, and enteric glia. In summary, EAE causes delayed small intestinal, whole GI, and colonic transit time compared to control mice, which could be representative of the bowel dysmotility exhibited by multiple sclerosis patients. The symptomology may be explained by the presence of GI-targeted antibodies in the blood of EAE mice or human MS patients. Platform Talk 3 Microsecond MD simulations to reveal the dynamics and mechanisms of a Class B GPCR Chenyi Liao and Jianing Li Department of Chemistry, University of Vermont, Burlington, VT 05405 We have studied a class B G-protein coupled receptor (GPCR), pituitary adenylate cyclaseactivating polypeptide receptor (PAC1R), which is crucial for transducing signal in nervous system and thus considered a potential drug target for psychiatric treatments. Given the lack of knowledge about the three-dimensional structure, it is a challenging task to understand the activation pathway and the regulation mechanism of this GPCR. Although its antagonism is believed to provide promising treatment to chronic stress, rational design of small-molecule antagonists has not been successfully achieved. To gain structural and mechanistic insight at the molecular level, we created reliable PAC1R homology models, and carried out microsecondlong simulations. We have observed high stability of the transmembrane domain and N-terminal extracellular domain in our simulations, while the linker connecting these domains is fairly flexible. In particular, two major states, for the first time, have been identified, which are likely related to ligand binding and unbinding behaviors. Further, a careful analysis based on the community network has revealed critical information about the protein dynamics, providing hints for us to propose the probable activation mechanism. In summary, our long simulation studies reveal the detailed dynamics to shed light on rational antagonist design to modulate the targeted class-B GPCR receptor. Platform Talk 4 Inactivation of the prelimbic cortex attenuates context-dependent excitatory operant responding Megan L. Shipman, Sydney Trask, John T. Green, and Mark E. Bouton Department of Psychological Science University of Vermont, Burlington, VT 05405 The renewal effect following operant extinction in rodents is used as a model of drug relapse and has been demonstrated using many different reinforcers including cocaine (Fuchs et al., 2007), alcohol (Willcocks & McNally, 2013) and heroin (Bossert et al., 2011). In operant renewal, extinguished operant behavior can recover when tested outside the context in which it was extinguished. In our lab, Eddy et al. (2016) utilized a simple sucrose reinforcer to show that inactivation of the prelimbic (PL) region of the medial prefrontal cortex (mPFC) by baclofen/muscimol (B/M) during testing attenuates renewal of an extinguished response when testing occurs in Context A (e.g., ABA renewal). One explanation for this attenuated renewal is that the PL may play a role in context-dependent excitatory responding (e.g., Thrailkill & Bouton, 2015). Two experiments tested this prediction. In Experiment 1, rats learned to lever press for a sucrose-pellet reward. Once the behavior was acquired, animals received an infusion of either B/M or saline vehicle into the PL and were tested in the acquisition context, Context A, or a different context, Context B. Although both groups showed a decrement in responding in Context B (a typical context-switch effect), inactivation of the PL decreased responding in Context A relative to the vehicle controls. Given that PL inactivation decreased behavioral control by the acquisition context, other types of renewal in which testing occurs outside the acquisition context should not be affected by PL inactivation. Therefore, in Experiment 2, the same rats again responded for the sucrose reinforcer in Context A. Responding was then extinguished in a new context, Context C. Animals then received an infusion of either B/M or saline into the PL before being tested in the extinction context, Context C, or another context, Context D. As predicted, both groups showed ACD renewal that was unaffected by PL inactivation. A final test of ABA renewal verified that the cannulae were still functional and replicated Eddy et al. (2016). Rather than affecting renewal generally, inactivation of the prelimbic cortex attenuates ABA renewal by reducing context-dependent excitatory responding in the conditioning context. These results may have implications for better understanding the neurobiology of context-related relapse. Platform Talk 5 Chronic Stress Potentiates the Response to Intra-BNST PACAP Infusion S. Bradley King1, Kim R. Lezak1, Victor May2 and Sayamwong E. Hammack1 Departments of Psychological Science1 and Neurological Sciences2 University of Vermont, Burlington, VT 05405 Chronic or repeated exposure to stressful stimuli can result in several maladaptive consequences, including increased anxiety-like behaviors and altered peptide expression in brain structures involved in emotion. Among these structures, the bed nucleus of the stria terminalis (BNST) has been implicated in emotional behaviors as well as regulation of hypothalamic-pituitary-adrenal (HPA) axis activity. In rodents, chronic variate stress (CVS) has been shown to increase BNST pituitary adenylate cyclase activating polypeptide (PACAP) and its cognate PAC1 receptor transcript, and BNST PACAP signaling may mediate the maladaptive changes associated with chronic stress. In order to determine whether chronic stress would potentiate the behavioral and/or endocrine response to subthreshold BNST PACAP infusion, rats were exposed to a 7 day CVS paradigm previously shown to upregulate BNST PAC1 receptor transcripts; control rats were not stressed. 24 hr following the last stressor, stressed and control rats were bilaterally infused into the BNST with 0.5 g PACAP. Startle response to intra-BNST PACAP infusion was assessed post-infusion in Experiment 1. In Experiments 2 and 3, blood was sampled via a tail nick 30 min following PACAP infusion to assess the corticosterone response to PACAP following CVS. We found an increase in startle amplitude and an increase in plasma corticosterone levels 30 minutes following BNST PACAP infusion only in rats that had been previously exposed to CVS. These results were likely mediated via PAC1 receptors, as equimolar infusion of the VPAC1/2 receptor ligand vasoactive intestinal polypeptide (VIP) had no effect on plasma corticosterone levels. These results suggest that repeated exposure to stressors sensitizes the neural circuits underlying the behavioral and endocrine responses to BNST PACAP infusion and BNST PACAP/PAC1 receptor signaling likely plays a critical role in mediating stress responses. Platform Talk 6 A Porcine Model to Define Pain Structural Neuroplasticity James Bishop and Magdalena Naylor Department of Psychiatry, University of Vermont College of Medicine Burlington, VT 05405 Chronic pain is associated with altered CNS structure and function. Specifically, it is speculated that changes in movement patterns and behavior have profound effects on thoracolumbar fascia dynamics as well as in both grey and white matter microstructure. Advances in magnetic resonance imaging acquisitions and analyses have been pivotal at defining CNS alterations, however, the mechanisms that contribute to these brain changes remain largely unknown. The objective of this work is to develop an animal model of low back pain that mimics the thoracolumbar fascia pathophysiology observed in clinical low back pain patients. Using this model, we first define a novel neuroimaging methodology to elucidate structural changes post mortem. Subsequently, this will be followed up by immunohistochemistry and histological evaluation to explore the cellular underpinnings of chronic pain neuroplasticity. The results of this translational work will contribute significantly to the interpretation of clinical MRI findings across chronic pain conditions. Platform Talk 7 Putting the [P] in [P]lexin: Characterizing the Phosphorylation of PlexinA2 by the Tyrosine Kinase Fyn Riley M. St. Clair 1,2, Marion E. Weir1, Alicia M. Ebert 1, 2, and Bryan A. Ballif1, 2* 1 Department of Biology, 2Neuroscience Graduate Program University of Vermont, Burlington, VT 05405, USA Cellular responses to extracellular cues are necessary for nervous system development. We have shown that one such signal transduction pathway, Semaphorin6A-PlexinA2, is critical for vertebrate eye development. However, the mechanisms underlying this pathway are not yet fully understood. Semaphorin6A is a guidance molecule that, upon binding to the receptor PlexinA2, regulates neuronal migration and retinal precursor cell position. PlexinA2 is hypothesized to interact with the Src-family tyrosine kinase Fyn to initiate downstream signaling, ultimately governing cytoskeletal dynamics. We and others have previously shown that Fyn can phosphorylate PlexinA2. However, the mechanisms of this phosphorylation event are not well characterized, including which PlexinA2 residues are phosphorylated by Fyn. Using bioinformatics and mass spectrometry data, we propose specific PlexinA2 amino acids as candidate sites for Fyn-induced phosphorylation. We validated that Fyn can phosphorylate these sites using PlexinA2 mutant constructs in a cell culture system. Future studies will determine the functional relevance of these sites in the development of the vertebrate visual system. Platform Talk 8 Selective cholinergic modulation of layer 5 projection neurons in the mouse prefrontal cortex Arielle L. Baker, Ryan J. O’Toole and Allan T. Gulledge Department of Physiology and Neurobiology, Geisel School of Medicine Dartmouth College, Hanover, NH 03755 Pyramidal neurons in layer 5 of the mouse prefrontal cortex comprise two broad classes of projection neurons: commissural/callosal (COM) neurons, and corticopontine (CPn) neurons. These two neuron subtypes have distinct morphological and physiological characteristics, including divergent responses to neuromodulators such as serotonin, acetylcholine (ACh), and norepinephrine. To further characterize the role of ACh in regulating cortical circuits, we compared phasic responses to exogenous and endogenous ACh in labeled COM and CPn neurons in the medial prefrontal cortex (mPFC) of wild-type mice and mice expressing channelrhodopsin-2 in cholinergic neurons (ChAT-ChR2 mice; Jackson Labs). When paired with suprathreshold depolarization, exogenous ACh (100 µM for 100 ms) generated a brief inhibitory response followed by an excitatory response in COM and CPn neurons (data pooled from wildtype and ChAT-ChR2 mice). Apamin-sensitive inhibitory responses were qualitatively similar in COM (n = 26) and CPn (n = 23) neurons, but were of substantially longer duration in COM neurons (1.838 ± 0.159 s vs. 1.052 ± 0.101 s in COM and CPn neurons, respectively; p < 0.05). The excitatory responses of CPn neurons to exogenous ACh, quantified as the peak increase in firing rate (% over baseline), were larger in magnitude (162 ± 15% vs. 79 ± 28% in CPn and COM neurons, respectively; p < 0.05) and had shorter latencies to peak response (3.370 ± 0.206 vs. 4.439 ± 0.282 s in CPn and COM neurons, respectively; p < 0.05) than in COM neurons. Endogenous release of ACh triggered by flashes of blue light (100 pulses, 5 ms each, at 59 Hz) had no effect in COM (n = 10) or CPn (n = 6) neurons from wild-type animals, and did not induce inhibitory responses in neurons from ChAT-ChR2 mice, but increased firing rates (by 18 ± 5% and 47 ± 7%, respectively) in both COM (n = 18) and CPn (n = 22) neurons (p < 0.05) from ChAT-ChR2 mice. Remarkably, single flashes of light (5 ms duration) enhanced action potential generation selectively in CPn neurons. In baseline conditions, periodic current steps (1.5 s duration) generated 8.2 ± 0.2 and 8.7 ± 0.4 action potentials in COM (n = 24) and CPn (n = 20) neurons, respectively. Single flashes of blue light increased the number of action potentials generated in both cell types, but preferentially so in CPn neurons (9 ± 1% vs. 30 ± 1% in COM vs. CPn neurons, respectively; p < 0.05). Together, our results demonstrate cell-type selectivity in phasic cholinergic signaling in projection neurons in the mPFC, with ACh preferentially promoting excitation in CPn neurons. Platform Talk 9 Functional and anatomical relationships between the dorsal striatum, medial precentral cortex and head direction circuit. Max L. Mehlman, Shawn S. Winter, Stephane Valerio & Jeffrey S. Taube Department of Psychological and Brain Sciences Dartmouth College, Hanover, NH Animals must maintain a sense of direction to efficiently navigate within their environment. At the neural level, direction is represented by the activity of head direction (HD) cells. These neurons fire as a function of the animal’s allocentric directional heading, operating much like a compass. While most rodent HD cells are located within limbic system structures that form the canonical limbic HD circuit, small proportions are found elsewhere in the brain, including the dorsal striatum (DS) and medial precentral cortex (PrCM). Is the HD signal in these structures derived from limbic HD circuit output or is it generated independently? To examine this issue we recorded single unit activity in the DS and PrCM of freely moving rats and compared HD cell activity observed in control animals to that observed in animals with neurotoxic lesions of the anterodorsal thalamic nuclei (ADN), a manipulation known to disrupt the limbic HD circuit. Large ADN lesions (>75%) completely abolished the HD signal in both the DS and PrCM. Animals with smaller lesions exhibited degraded HD cell activity in the DS; these HD cells fired over a significantly wider directional range compared to HD cells from control animals. We conclude that the HD signal is first generated by the limbic HD circuit and then projected to the DS and PrCM. To determine if HD cell activity in the limbic HD circuit is influenced by feedback from the DS and PrCM, we recorded HD cells in the ADN of animals with neurotoxic lesions of the DS and PrCM. We found no difference between control and lesioned animals in regards to HD cell activity observed in the ADN. This suggests a unidirectional functional relationship between the limbic HD circuit and the DS and PrCM. Finally, to examine the anatomical projections that could convey the HD signal to the DS and PrCM, we injected fluorescent retrograde neuronal tracers into these structures and examined labeling throughout the limbic HD circuit. We found that the DS receives input from almost every structure in the limbic HD circuit: the lateral mammillary nuclei (LMN), ADN, postsubiculum, medial entorhinal cortex (MEC) and retrosplenial cortex (RSP). Interestingly, the PrCM has less robust connectivity with the limbic HD circuit, receiving input from only the LMN, MEC and RSP. Given that the DS receives input from the PrCM, two possibilities exist; either the HD signal is projected serially from the MEC or RSP to the PrCM, which in turn conveys the HD signal to the DS, or the HD signal is projected in parallel from one or more nodes in the limbic HD circuit directly to both the DS and PrCM. Platform Talk 10 Learning to suppress behavior during operant extinction: Implications for cue exposure therapy Sydney Trask1, Rodrigo Carranza-Jasso2, and Mark E. Bouton1 1 The Department of Psychological Science, University of Vermont, Burlington, VT 05405 2 Department of Psychology, Autonomous University of Aguascalientes, Mexico Many negative health outcomes in humans can be linked directly to voluntary behavior. These include obesity, drug-dependence, and smoking. Operant conditioning in animals, in which actions are associated with reinforcers in the environment, is an important model of voluntary behavior that allows the study of its basic underlying processes. Importantly, in both humans and animals, operant or voluntary behavior can be reduced through a process known as extinction in which the reinforcing outcome is no longer produced by the response. While extinction is easily achieved in a laboratory setting, it is not typical for humans to perform a response (such as cigarette-smoking or drug-taking) without the reinforcing outcome present. Instead, human extinction treatments (“cue-exposure therapy”) typically omit the response and instead present cues (or discriminative stimuli) that have been associated with the response-outcome pairing in an attempt to reduce the motivational power of those cues to elicit responding. However, treatments of this type often have little to no effect on suppressing behavior (see Conklin & Tiffany, 2002). Here we suggest that this may be true because the organism needs to learn to suppress (or inhibit) its behavior more directly. In a first experiment, rats were trained to respond for a food outcome only in the presence of a brief discriminative stimulus, or S. Responding was not reinforced when S was not present. During an extinction phase, the rats then received either simple exposure to S (as in “cueexposure therapy”), or were allowed to make the response (and learn that it had no consequence) during S. A control received neither treatment. The results indicated that only the group that was allowed to make the response showed weakened responding when the rats were later tested for their responding during S. In a second experiment, responding on two different responses was reinforced in the presence of the same S. Extinction was conducted as before: one group received simple exposure to S, one group was allowed to make one response (R1) during S, and one group received neither treatment. The group allowed to make the R1 response was suppressed on only that response during S and not the second response, R2. Neither other group showed any effect of the extinction treatment. A third experiment training the same response with two separate stimuli (S1 and S2), each signaling the response would be followed by a distinct outcome (e.g., O1 or O2). Only rats that were allowed to perform the response during extinction showed suppressed responding during a test; that suppression was evident in both S1 and S2, suggesting that they had generally learned to inhibit the response regardless of its setting stimulus or associated outcome. Together, these results suggest that making the response in the presence of a discriminative stimulus is an important part of learning to inhibit that response. Further, while this type of training does not transfer to other responses, it does transfer well between stimuli that signal the same response. These results provide a potential mechanism to account for the failure of cue-exposure treatments in humans. Platform Talk 11 Intra-bed nucleus of the stria terminalis (BNST) pituitary adenylate cyclase activating polypeptide (PACAP) infusion reinstates cocaine seeking in rats Olivia W. Miles1, Eric A. Thrailkill1, Victor May2, Mark E. Bouton1, Sayamwong E. Hammack1 1 Department of Psychological Science and 2Neurological Sciences University of Vermont, Burlington, VT 05405 The tendency of users to relapse severely hinders adequate treatment of addiction. Physical and psychological stressors often contribute to difficulties in maintaining behavior change, and may play a significant role in relapse. We have previously shown that the activation of pituitary adenylate cyclase activating peptide (PACAP) systems in the bed nucleus of the stria terminalis (BNST) mediate many consequences of chronic stressor exposure. Hence, chronic stress substantially increased BNST PACAP levels, intra-BNST PACAP infusions produced the behavioral and endocrine consequences of stressor exposure, and BNST PACAP antagonism blocked many of the consequences of chronic stress. In the present set of studies, we investigated the role of BNST PACAP in stress-induced reinstatement of cocaine seeking. All rats self-administered cocaine (3mg/ml; 0.5mg/ig/infusion, i.v.) for 1hr daily over 10 days followed by extinction training in which lever pressing no longer resulted in cocaine delivery. In the first experiment we showed that intra-BNST PACAP infusion (1 μg; 0.5 μl per side) could reinstate previously extinguished cocaine seeking behavior. In the second experiment we found that intra-BNST infusions of the PAC1/VPAC2 antagonist, PACAP 6-38 (1 μg; 0.5 μl per side) blocked reinstatement following stressor exposure (5 sec 2mA footshock). Overall, these data suggest that BNST PACAP systems mediate stress-induced reinstatement to drug seeking. Understanding the neuropharmacology of BNST PACAP in stress-induced reinstatement and the role of PACAP systems may lead to viable targets for relapse prevention. Poster 1 Regulation of Microtubule dynamics by Tau Rehan Ali and Christropher Berger Department of Molecular Physiology University of Vermont College of Medicine, Burlington, VT 05405 The Microtubule associated protein, Tau, is implicated in a class of neurodegenerative diseases known as “Tauopathies”. One of the common feature of these disease states is misregulation of axonal transport. Thus to better understand the pathology of these diseases it is important to understand the role that Tau plays in regulation of axonal transport. Tau is known to regulate microtubule dynamics in vitro, which is thought to be an important function in stabilizing the microtubule tracks required for efficient axonal transport; however the mechanisms by which Tau regulates microtubule dynamics are not well understood. Moreover, six isoforms of Tau are expressed in adult human brain, and the isoform specificity of Tau’s function is also not completely clear. To address these issues, we use total internal reflection fluorescence (TIRF) microscopy to examine the dynamics of individual microtubules in the absence and presence of different isoforms of Tau. We are currently working with 3RS- and 3RLisoforms of Tau, which both contain three microtubule-binding repeats in the C-terminal microtubule binding domain, but differ in the number of N-terminal acidic inserts that they contain (0 and 2, respectively) in the N-terminal projection domain. Consistent with previous studies, we confirm that both of these isoforms reduce microtubule catastrophe frequencies. Interestingly, our findings also show that only the isoform in which the N-terminal acidic inserts are present (3RL-Tau) increases microtubule rescue frequency. We further demonstrate that Tau alters the rate at which microtubule tip structure evolves while it is growing in an isoform specific manner. 3RL-Tau holds tip taper at a steady value independent of the microtubule length and in the presence of 3RS-Tau there is an initial increase in microtbule tip taper followed by a steady decrease as microtubule length increases.This is a previously unknown mechanism by which Tau can alter microtubule catastrophe frequency. Currently, work is on-going with other isoforms which differ in their number of microtubule binding repeats (three or four), as well as high resolution electron microscopy imaging of microtubule tips in the presence and absence of Tau. In summary, we are elucidating in detail the role of Tau in regulation of microtubule dynamics in an isoform specific manner, leading to new insight as to how misregulation of Tau’s function affects microtubule dynamics and in turn axonal transport during the disease process. Poster 2 Temporal coordination of hippocampal place cells reflects cognitive outcome post febrile status epilepticus Jeremy Barry Department of Neurological Sciences, University of Vermont College of Medicine Burlington, Vermont 05405 The coordination of dynamic neural activity within and between neural networks is believed to underlie normal cognitive processes. Conversely, cognitive deficits that occur following neurological insults may result from network discoordination. We hypothesized that cognitive outcome following febrile status epilepticus (FSE) depends on network efficacy within and between layers CA1 and CA3 to dynamically organize cell activity by theta phase. Control and FSE rats were trained to forage or perform an active avoidance spatial task. FSE rats were sorted by those that were able to reach task criterion (FSE-L) and those that could not (FSE-NL). FSE-NL CA1 place cells did not exhibit phase preference in either context and exhibited poor cross-theta interaction between CA1 and CA3. FSE-L and control CA1 place cells exhibited phase preference at peak theta that shifted during active avoidance to the same static phase preference observed in CA3. Temporal coordination of neuronal activity by theta phase may therefore explain variability in cognitive outcome following neurological insults in early development. Poster 3 Optogentic manipulation of hippocampal theta rhythm can obstruct performance in the active avoidance task Benjamin Blumberg, Jeremy Barry, Sean Flynn and Gregory L. Holmes Department of Neurological Sciences University of Vermont College of Medicine Burlington, VT 05405 Recent theories suggest that temporal coordination of hippocampal neurons within and between components of the hippocampal circuit by local theta rhythm are necessary for learning and memory. We are testing this theory by optogenetically manipulating the hippocampal theta pacemaker, the medial septum, to influence the frequency and power of hippocampal theta oscillations. We hypothesized that by causing temporal discoordination of hippocampal neurons during learning, we can cause a cognitive impairment in a complex spatial task. Preliminary data shows that optically maintaining a 6 Hz oscillation in the hippocampus can prevent learning of the active avoidance task. Moreover, the stimulations can also induce memory deficits the spatial task after it has already been learned. This data supports the theory that the temporal coordination is important for spatial cognition. Poster 4 Parabrachial PACAP activation of amygdala endosomal ERK signaling regulates the emotional component of pain Galen Missig1, Linda Mei1, Margaret A. Vizzard1,, James A. Waschek2, Sayamwong E. Hammack3, Victor May1 and Karen M. Braas1 Departments of Neurological Sciences1 and Psychological Science3, University of Vermont College of Medicine, Burlington, VT 05405, USA; Department of Psychiatry and Behavioral Sciences2, University of California Los Angeles, Los Angeles, CA 90095, USA Chronic pain and stress-related psychopathologies, such as depression and anxiety-associated abnormalities, are mutually reinforcing; however, the neuronal circuits and mechanisms that underlie this reinforcement are still not well understood. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) and its cognate PAC1 receptor (Adcyap1r1) are expressed in peripheral nociceptive pathways, participate in anxiety-related responses and have been have been linked to posttraumatic stress disorder (PTSD) and other mental health afflictions. In a partial sciatic nerve ligation chronic constriction injury (CCI) model, we show that chronic neuropathic pain increases PACAP expression at multiple tiers along the spinoparabrachioamygdaloid tract, and bilaterally augments nociceptive amygdala (CeA) PACAP immunoreactivity, ERK phosphorylation and c-Fos activation, in parallel with heightened anxiety-like behavior and nociceptive hypersensitivity. Acute CeA infusions with the PACAP receptor antagonist PACAP(6-38) blocked CCI-induced behavioral responses; further, pretreatments with MEK or endocytosis inhibitors to block endosomal PACAP receptor ERK signaling attenuated PACAP-induced CeA neuronal activation and nociceptive responses. Accordingly, chronic pain-induced PACAP neuroplasticity and signaling in spinoparabrachioamygdaloid projections can impact CeA stress- and nociception-associated maladaptive responses which can be ameliorated upon receptor antagonism even during injury progression. Poster 5 PKM- is involved in cerebellar-dependent learning and memory Kutibh Chihabi, John T. Green1 and Anthony D. Morielli2 Departments of Psychological Science1 and Pharmacology2 University of Vermont, Burlington, VT 05405 PKM- has long been implicated in a hippocampal cellular correlate of learning, long-term potentiation (LTP), through its regulation of hippocampal AMPA receptors (Ling et al., 2002; Yao et al., 2008). Disruption of PKM- with Zeta-inhibitory peptide (ZIP) can irreversibly disrupt hippocampal memory that has been maintained for many weeks (Hernandez et al., 2003). Here, we show that PKC- and PKM- are highly expressed in the cerebellar cortex, with highest expression found in Purkinje cell (PC) nuclei. Despite being highly expressed in the cerebellum (Oster et al., 2004), no studies have examined how regulation of cerebellar PKM- may affect cerebellar-dependent learning and memory. We have shown for the first time that infusion of ZIP in the lobulus simplex of the cerebellum can significantly disrupt delay eye-blink conditioning (EBC) in rats, a form of cerebellar-dependent learning. Infusion of 0.50 l 20 mM ZIP or 0.50 l PBS vehicle occurred 2 hours prior to the first acquisition session of EBC, ipsilateral to the conditioned eye; rats underwent a total of 6 daily sessions of 350-ms delay EBC. We hypothesized that PKM- may have regulatory effects on voltage-gated potassium channel alpha-subunit 1.2 (Kv1.2). Several studies have shown that PKC- can coimmunoprecipitate with and phosphorylate a subunit that associates with cerebellar Kv1.2 (Gong et al., 1999; Croci et al., 2003). Kv1.2 is highly expressed in cerebellar basket cell axon terminals and Purkinje cell dendrites and our lab has shown that Kv1.2 is important for cerebellar EBC in rats (Williams et al., 2012). Here we demonstrate that PKM- can significantly reduce Kv1.2 surface expression in HEK 293 cells. Poster 6 The COBRE Imaging and Physiology Core: Two New Confocal Setups Fully Operational Todd A. Clason, Cindy J. Forehand, and Rodney L. Parsons Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VT 05405 The University of Vermont COBRE Imaging and Physiology Core is a central resource which offers a wide range of imaging hardware for fixed and live-cell immunofluorescence studies. We now have three confocal systems and two multiphoton systems in routine operation. Our newest confocal, the Nikon C2 is a point-scanning system with three fluorescent detectors as well as a transmitted detector. The C2 system has some very useful features, including a motorized stage with software control, which allows the collection of large tiled images in Z-series. The lasers and detectors can also be variably controlled through a Z-series, so that higher gain and laser power can be used at depth to maintain consistent image dynamic range throughout a volume. We have used this feature to great effect imaging cleared brain tissue courtesy of Felix Eckenstein. Our Nikon/Andor CSU-W1 Yokogawa Spinning Disk confocal microscope is now in routine use and is a significant improvement to our high speed imaging capabilities. This spinning disk confocal system can simultaneously record two low light signals with dual 512x512 EM CCD cameras at 52 fps (frames per second), a rate well above our other instrumentation. Higher resolution (2560X2160) imaging for brighter samples can be achieved with the Zyla sCMOS camera. These cameras allow great flexibility for the multiple sample types encountered in this facility. For data analysis, we have recently acquired the Bitplane Imaris software package (Oxford Instruments), which complements our other image processing software, including MetaMorph (Universal Imaging), Volocity (Perkin Elmer) and ImageJ (NIH) for general analysis and rendering. Supported by NIH Grant Numbers 5 P30 RR032135 from the COBRE Program of the National Center for Research Resources and 8 P30 GM 103498 from the National Institute of General Medical Sciences. Poster 7 Hypotensive actions of A2 noradrenergic neurons are abolished by increased BDNF expression in the PVN Nick C. Cruickshank, Chris L. Schaich, Zachary D. Einwag, Theresa L. Wellman and Benedek Erdos Department of Pharmacology University of Vermont College of Medicine, Burlington, VT 05405 The nucleus of the solitary tract (NTS) and the paraventricular nucleus of the hypothalamus (PVN) play important roles in central regulation of cardiovascular function both during baseline conditions and during stress. Sympathoregulatory PVN neurons and NTS noradrenergic (NAergic) A2 neurons are connected by reciprocal projections. However, the importance of this PVN – NTS interaction is not fully understood. Brain derived neurotrophic factor (BDNF) expression in the PVN increases in response to hypertensive stimuli including stress, and we have recently shown that BDNF is a significant pro-hypertensive mediator in the PVN. BDNF has also been shown to modulate NA-ergic neuronal function elsewhere in the central nervous system, but the effect of BDNF released from PVN neurons on PVN-projecting NTS A2 neurons has never been investigated. In this study, we tested the hypothesis that cardiovascular actions of BDNF within the PVN are mediated in part by altered function of PVN-projecting A2 NA-ergic neurons. To test this hypothesis, Sprague-Dawley rats received bilateral PVN injections of viral vectors (AAV2) expressing either green fluorescent protein (GFP) or BDNF and bilateral NTS injections of PBS or the anti-dopamine--hydroxylase-conjugated saporin (DSAP), a neurotoxin selective to NA-ergic neurons. Radiotelemetry was used to monitor blood pressure during baseline conditions and during acute restraint stress experiments. Our results show that BDNF overexpression in the PVN results in significantly elevated tyrosine hydroxylase and dopamine-hydroxylase (DBH) expression in the NTS and elevated DBH immunoreactivity in the PVN. BDNF overexpression also significantly increased baseline blood pressure, but reduced acute stress-induced blood pressure elevations. Lesioning NTS NA-ergic neurons with DSAP led to a significant rise in baseline blood pressure in GFP-treated rats, whereas it had no additional hypertensive effects in BDNF-treated rats. DSAP treatment also led to an increased blood pressure response during the second half of restraint stress in GFP rats, while it had no effect in the BDNF group. In summary, our findings indicate that elevated BDNF expression in the PVN abolishes hypotensive effects of A2 neurons despite a significant upregulation of catecholamine biosynthesizing enzymes in these neurons. Poster 8 Does the adaptive algorithm in the stop signal task introduce a confound in neuroimaging studies? Nicholas D’Alberto1, Bader Chaarani1, Philip Spechler1, Kelsey Hudson1, Scott Mackay1, Nick Allgaier1, Matthew Albaugh1, Catherine Orr1, Mitchell Snowe1, Robert Althoff1, Alexandra Potter1, Hugh Garavan1, and the IMAGEN consortium. Department of Psychiatry, University of Vermont College of Medicine Burlington, VT 05405 The Stop Signal Task (SST) is widely used in research examining inhibitory control (1). The SST measures an individual’s ability to inhibit a response that has already been initiated. There exist two main versions of the task. In one version participants are tested on a defined set of stop signal delays (SSD), such that each participant in the study performs an identical task. In the second version, participants are tested with an adaptive algorithm where SSD length typically varies so that each participant is able to successfully inhibit on 50% of stop trials. Although the tracking algorithm results in the same inhibitory success rate across participants, this is achieved by varying the set of SSDs across participants. Thus, a good inhibitor compared to a poor inhibitor will receive, on average, longer SSDs to reach 50% accuracy on stop trials thereby creating a potential confound (2). When interpreting fMRI activation differences, it is difficult to disseminate between inhibitory ability and objective task difficulty given that successfully withholding a response is more difficult as the SSD lengthens. The goal of the present study is to explore neural activation associated with SSD length and to determine how it relates to activation differences associated with SSRT ability. Poster 9 WNK-1 regulation of Kv1.2: A novel hypothesis for cognitive impairment in schizophrenia Adrian Dutkiewicz, MA; Anthony Morielli, PhD Neuroscience Graduate Program, Department of Pharmacology University of Vermont College of Medicine, Burlington, VT 05405 Our lab studies the voltage-gated potassium channel Kv1.2 and the chloride-modulated regulatory kinases WNK-1 and WNK-3. These proteins collectively exert a strong influence on neuronal excitability by maintaining resting membrane potential. We are attempting to apply our research on membrane excitability to understand the cognitive symptoms of schizophrenia. Over the course of the disease, pyramidal cell firing in the dorsolateral prefrontal cortex (dlPFC) becomes dysregulated due to hypoactive GABAergic input that they receive from chandelier cells. A reduction of inward chloride currents through GABAA channels – predicted from the hypoactivity of chandelier cells – could result in greater activation of the chloride-inhibited kinase WNK-1. Preliminary studies reveal that WNK-1 inhibits Kv1.2. We therefore predict that reduced GABAergic stimulation through GABAA receptors will result in increased WNK-1 mediated inhibition of Kv1.2, which in turn should increase membrane excitability. In addition, compensatory responses to normalize chloride concentrations that are mediated through WNK-3 may also contribute to potential anomalies in Kv1.2 deactivation. The dysregulation in pyramidal cell firing in the dlPFC and the consequent discoordination of gamma band oscillations have been described extensively but have been commonly attributed to lack of IPSPs from the impaired chandelier cells and other abnormalities ? rather than the type of aberrant ion channel trafficking that we predict. Therefore, this study could reveal new mechanisms involved in schizophrenia and identify new potential molecular targets for rational drug design. Poster 10 The Role of the Plexin A Family in Eye Development. Emerson, S.E.1, Light, S.E.1, St. Clair, R.M.1,2, Ballif, B.A.1,2 and Ebert, A.M.1,2 1 Department of Biology, 2Neuroscience Graduate Program, University of Vermont, Burlington VT 05405, USA During development, migrating neurons navigate to their correct synaptic targets by using a variety of transmembrane and secreted guidance cues in their environment. Plexins and Semaphorins are a family of signaling factors that were initially discovered to act as repulsive signals to migrating neurons by influencing actin dynamics in axonal growth cones. It is becoming widely appreciated that the Plexin/Semaphorin family has a much broader role in development than axon guidance. Using a combination of Morpholino knockdowns, in situ hybridization and immunohistochemistry in zebrafish, we have uncovered a novel early role for PlexinA2 in maintaining proper cohesion and proliferation of migrating optic vesicles. Using a microarray we determined a set of downstream genes that are differentially regulated by PlexinA2 and Semaphorin6A involved in migration and proliferation. We have also shown that PlexinA1 has a compensatory role for PlexinA2 in this system. Further work will investigate the expression patterns and roles of the remaining Plexin A family members in eye development. Poster 11 Contributions of pituitary adenylate cyclase-activating polypeptide (PACAP)/receptor signaling to increased voiding frequency and somatic sensitivity in mice with urothelium-specific overexpression (OE) of nerve growth factor (NGF) in the urinary bladder. Beatrice M. Girard, Susan Malley, Morgan E. Mathews, Margaret A. Vizzard Department of Neurological Sciences, University of Vermont College of Medicine Burlington, VT 05405 NGF-OE in the urothelium stimulates neuronal sprouting or proliferation in the urinary bladder, produces increased voiding frequency and non-voiding contractions, and results in increased referred somatic sensitivity. Additional NGF-mediated changes might contribute to the urinary bladder hyperreflexia and pelvic hypersensitivity observed in these transgenic mice such as upregulation of neuropeptide/receptor systems. NGF-OE in the urothelium was achieved through the use of a highly urothelium-specific, uroplakin II promoter. We examined PACAP, vasoactive intestinal polypeptide (VIP), and associated receptor (PAC1, VPAC1, VPAC2) transcripts or protein expression in urothelium and detrusor smooth muscle and lumbosacral dorsal root ganglia in NGF-OE and littermate wildtype (WT) mice using real-time quantitative reverse transcription-polymerase chain reaction and immunohistochemical approaches. Results demonstrate upregulation of PAC1 receptor transcript and PAC1-immunoreactivity (IR) in urothelium of NGF-OE mice whereas PACAP transcript and IR were decreased in urothelium. In contrast, VPAC1 receptor transcript was decreased in both urothelium and detrusor smooth muscle of NGF-OE mice. VIP transcript expression and IR was not altered in urinary bladder of NGF-OE mice. Given the presence of PAC1-IR fibers, the expression of PAC1 receptor expression in bladder tissues, and the abilities of PACAP to facilitate detrusor contractility, whether PACAP/receptor signaling contributes to bladder hyperreflexia and somatic sensitivity was evaluated. Intravesical administration of PACAP6-38 (300 nM) significantly (p ≤ 0.01) increased bladder capacity (2.0-fold), intercontraction interval and void volume in NGF-OE mice. Intravesical instillation of PACAP6-38 also decreased filling pressure and peak micturition pressure in NGF-OE mice. PACAP6-38 had no effects on WT mice. Intravesical administration of PACAP6-38 (300 nM) significantly (p ≤ 0.01) reduced pelvic sensitivity in NGF-OE mice but was without effect in WT mice. These studies demonstrate that additional NGF-mediated pleiotropic changes, such as modulation of neuropeptide/receptor systems, contribute to the increased voiding frequency and pelvic sensitivity observed in NGF-OE mice. Support: NIH-NIDDK 2R01DK051369, 2R01DK060481 Poster 12 The effect of cyclophosphamide on salt preference and detection threshold Michael Gomella, Evan Lowry, Ben Jewkes, Joy Benner, Eugene Delay Department of Biology, University of Vermont Burlington, VT 05405 Chemotherapy is one of the most commonly used cancer treatments, yet it has many reported side effects including altered taste. The mammalian taste bud is comprised of four distinct types of cells, and type I cells are thought to detect salt through amiloride sensitive pathways. However, other receptors may be involved in the detection of salt, including amiloride insensitive channels. The goal of this ongoing study is to elucidate the effect that cyclophosphamide, a common chemotherapeutic agent, has on salt taste of mice. Conditioned taste aversion (CTA) methodology is being used to test if salt preference changes within several weeks following a single dose of cyclophosphamide. Concurrently, changes in detection threshold are being used tested to determine if salt sensitivity changed following treatment. We hypothesized that cyclophosphamide treatment would kill type I taste receptor cells and reduce the salt aversion, as well as raise the threshold for detecting salt. Preliminary data suggest that CTA mice injected with cyclophosphamide show diminished aversion around day 5 and day 19 post-injection while threshold mice showed significance differences between cyclophosphamide injected mice and control mice from day 16 to day 32 post-injection. These results could suggest patterns in receptor cell loss and regeneration following cyclophosphamide treatment. Poster 13 Mechanism(s) of transforming growth factor-beta (TGF-β) mediated bladder afferent nerve hyperexcitability E.J. Gonzalez, M.A. Vizzard Department of Neurological Sciences, University of Vermont College of Medicine Burlington, VT 05405 The neural circuitry underlying the micturition reflex is often compromised following neural diseases, injuries and inflammatory conditions. We have previously demonstrated that TGF-β1 contributes to afferent nerve hyperexcitability in an experimental cystitis model of Bladder Pain Syndrome (BPS)/Interstitial Cystitis (IC). We hypothesize that afferent hyperexcitability may result from urothelial cells secreting neuroactive molecules, including adenosine triphosphate (ATP), that signal to the suburothelial nervous network. We used whole bladder preparations (n=4-8) isolated from C57BL6 mice (3-6 month old, male) to determine the role of TGF-β1 in urothelial ATP release. Intravesical instillation of recombinant TGF-β1 (10 ng/ml) significantly (p ≤ 0.01) increased ATP release. The release of ATP was attenuated with the co-administration of a TβR-1 inhibitor, SB505124 (5 µM), suggesting the response was specific to TGF-β1 instillation. TGF-β-mediated ATP release was also reduced with brefeldin A (10 µM) but not 10Panx (50 µM) suggesting TGF-β1 stimulates ATP release via vesicular exocytosis with minimal contribution from pannexin-1 channels. Taken together, these results demonstrate a role for TGF-β in urothelial signaling that may contribute to afferent nerve hyperexcitability and underlie peripheral and central sensitization. Targeting TGF-β within the sensory components of the micturition reflex may be a therapeutic approach to improve bladder function. NIH-NIDDK 2R01DK051369-16S1, 2R01DK060481 Poster 14 Does axon initial segment plasticity regulate neuron excitability? Allan T. Gulledge1 and Jaime J. Bravo2 1 Department of Physiology and Neurobiology, Geisel School of Medicine Thayer School of Engineering, Dartmouth College, Hanover, NH 03755 2 In many neurons, action potentials (APs) are initiated in the axon initial segment (AIS), a specialized region of proximal axon having a high density of voltage-gated sodium channels. Previous studies have reported both diversity and activity-dependent plasticity of AIS morphology, and have speculated that neurons may reduce their excitability by moving the AIS away from the soma (see Grubb et al., J. Neurosci. 31:16049-55, 2011). Alternatively, the capacitive load of somatodendritic compartments may reduce excitability at proximal axonal locations. Using computational models of simplified and morphologically realistic neurons, we tested the impact of AIS location on neuron excitability. In most simulations, a 30 m-long AIS attached to an extended myelinated axon was shifted from 0 to 70 m from the soma. Using somatic or dendritic current injections, current thresholds (rheobase) for AP initiation were measured across a range of AIS locations and dendritic morphologies. In smaller neurons, rheobase was lowest (most “optimal”) when the AIS was adjacent to the soma. On the other hand, increasing the number of dendrites, or their size, shifted the optimal AIS location toward more distal locations, up to the maximal location of 70 m from the soma. For any given dendritic morphology, increasing dendritic membrane capacitance or conductance (i.e., decreasing membrane resistivity) moved the optimal AIS position to more distal axonal locations. Reduced rheobase for distal AIS locations did not depend on changes in whole-cell time constant, active conductances in dendrites, or the presence of myelinated axons. Optimal AIS location in morphologically realistic models was also dependent on morphology, with distal AIS locations favored in large neurons (e.g., pyramidal neurons), and proximal AIS locations favored in smaller neurons (e.g., medium spiny neurons). Finally, in models having a uniform unmyelinated axon (without an AIS), increasing dendritic complexity, membrane capacitance, or membrane conductance, shifted the site of AP initiation to more distal axonal locations. Our results demonstrate that the impact of dynamic changes in AIS location will depend on somatodendritic morphology, and that in many neurons distal AIS locations may increase, rather than decrease, neuronal excitability. Poster 15 ACTH Prevents Deficits in Fear Extinction Associated with Early Life Seizures Andrew T. Massey1,2 *, David K. Lerner BA3*, Gregory L. Holmes MD1, Rod C. Scott MD PhD1,4, Amanda E. Hernan PhD1 1 Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VT, USA 05405 2 University of Bath, Department of Biological Sciences, Bath, UK 3 Dartmouth College, College of Arts and Sciences, Hanover, NH, USA 03756 4 University College London, Institute of Child Health, London WC1N 1EH, UK *These authors contributed equally to this work Abstract Early life seizures are often associated with cognitive and psychiatric comorbidities that are detrimental to quality of life. Using a rat model of early life seizures, we explored long-term cognitive outcomes in adult rats. Using ACTH, an endogeneous HPA-axis hormone given to children with severe epilepsy, we sought to prevent cognitive deficits. While rats with a history of ELS were able to acquire a conditioned fear learning paradigm as well as controls, these rats had significant deficits in their ability to extinguish fearful memories and ACTH treatment was able to significantly improve this fear extinction. This ACTH effect was specific for fear extinction deficits and not for spatial learning deficits in a water maze. Additionally, ACTH did not alter seizure latency or duration suggesting that cognitive and seizure outcomes may be dissociable. Expression levels of melanocortin receptors, which bind ACTH, were found to be significantly lower in animals that had experienced ELS than in control animals, potentially implicating central melanocortin receptor dysregulation in the effects of ELS and suggesting a mechanism of action for ACTH. Taken together, these data suggest that early treatment with ACTH can have significant longterm consequences for cognition in animals with a history of ELS independently of seizure cessation, and may act in part through a CNS melanocortin receptor pathway. Poster 16 Characterization of mPFC layer 5/6 neurons following sub-anesthetic ketamine Gregory Johnson and Sayamwong ‘Jom’ Hammack Department of Psychological Science University of Vermont, Burlington, VT 05405 In recent years the NMDA receptor antagonist ketamine has been shown to have rapid-acting, long lasting anti-depressant effects at sub-anesthetic doses in humans and animals. More recently it has also been shown that prior exposure of ketamine can induce stress resistance in several animal models associated with anxiety- and depression-like behavioral changes. For example, ketamine pre-treatment has recently been shown to prevent the constellation of depression-like behavioral changes that typically follow inescapable stress when injected either systemically or directly into the medial prefrontal cortex (mPFC). These and other data have suggested that the mPFC is an important site of action for ketamine in promoting stress-resistance; however, the changes in cellular physiology of mPFC neurons following ketamine exposure remain unclear. In the current set of studies we are using whole cell patch clamp electrophysiology to characterize the effects of prior ketamine on the intrinsic excitability of neuronal subpopulations in the mPFC, as well as the responses of mPFC neurons to serotonin and norepinephrine. Poster 17 Preliminary QTL Mapping Suggests Candidate Regions for Ethanol-Induced Phenotypes in the Diversity Outbred Mouse Population Steven Kasparek1, Troy Wilcox2, Dan Gatti2, Eric Busch3, Drew Kreuzman1, Benjamin Mansky1, Sophie Masneuf3, Erica Sagalyn3, Kayvon Sharif1, Dominik Taterra1, Walter Taylor1, MaryThomas1, Elissa J. Chesler2, Andrew Holmes3, Clarissa C Parker1 1 Department of Psychology and Program in Neuroscience, Middlebury College, VT 05753 2 Center for Genome Dynamics, The Jackson Laboratory, Bar Harbor, ME 04609, 3 Laboratory of Behavioral and Genomic Neuroscience, NIAAA, NIH, Rockville MD 20852 Alcohol Use Disorders (AUD) are widespread in the United States and often have devastating behavioral and emotional consequences. Altered sensitivity to alcohol is a strong predictor of the development of AUDs. Thus, identifying the genes responsible for AUD susceptibility is a key step for treatment and prevention. Traditionally, mouse mapping populations have only been able to identify large chromosome regions, rather than individual genes that contribute to ethanolrelated traits. This is largely due to a lack of genetic diversity and low recombination in the populations used. In a departure from tradition, the present study utilized the newly developed Diversity Outbred (DO) population (n ~ 608) to study three ethanol-induced phenotypes associated with AUDs: ataxia, hypothermia, and loss of the righting response (LORR). The DO population is highly recombinant, and more closely approximates the genetic variation found in human populations. A paired samples t-test indicated that DO mice exhibited ethanol-induced ataxia, remaining on the rotarod for significantly shorter lengths of time following ethanol injections (t(600) = 25.53, p < 0.0001, d = 1.04. Similarly, DO mice exhibited ethanol-induced hypothermia, with the body temperature of the mice significantly decreasing over time following ethanol injections F(2.956, 1747.087) = 797.788, p < 0.0001, 2p = 0.574. Finally, DO mice exhibited ethanol-induced LORR, following ethanol injection, with the majority of subjects (85.5%) losing and regaining the righting response during the testing period. We genotyped a subset of these mice (N = 288) at ~140k markers across the genome and performed high precision QTL mapping using the R program DOQTL. We identified numerous suggestive and significant QTLs associated with ethanol sensitivity. This information can in turn be used to identify alleles that contribute to AUDs in humans, elucidate causative biological mechanisms, or assist in the development of putative treatment strategies. Poster 18 Identifying genes associated with conditioned fear in the Diversity Outbred mouse population using a forward genetic, genome-wide approach Andrew Kreuzman1, Troy Wilcox2, Dan Gatti2, Eric Busch3, Steven Kasparek1, Drew Kreuzman1, Benjamin Mansky1, Sophie Masneuf3, Erica Sagalyn3, Kayvon Sharif1, Dominik Taterra1, Walter Taylor1, Mary Thomas1, Elissa J. Chesler2, Andrew Holmes3 Clarissa C. Parker1,4 1 Program in Neuroscience, Middlebury College, VT 05673 Center for Genome Dynamics, The Jackson Laboratory, Bar Harbor, ME 04609, 3 Laboratory of Behavioral and Genomic Neuroscience, NIAAA, NIH, Rockville MD 20852 4 Department of Psychology, Middlebury College, VT 05673 2 While genome-wide association studies (GWAS) have been largely successful in polygenic human traits, their adoption in mouse genetics has faced several large barriers. This has led to increased interest in the development of genetically diverse, highly recombinant mouse populations. Such populations allow for a greater range of phenotypic variation and improved mapping resolution, thus increasing potential for efficient identification of genes correlated with a trait of interest. We tested 509 male JAX Diversity Outbred mice (DO) using a three-day conditioned fear (CF) paradigm to fine-map quantitative trait loci (QTLs) associated with acquisition, extinction, and renewal of CF. A one-way repeated measures ANOVA found a significant increase in freezing following each tone-shock pairing during acquisition, (F 2.1, 1069.4 = 692.8, p < 0.001; ??2p = 0.57), demonstrating the ability to learn to associate the tone and foot-shock. Freezing behavior in response to the tone significantly decreased across trialblocks during extinction training (F 5.9, 2983.5 = 177.9, p < 0.001; ??2p = 0.26) suggesting mice were able to successfully extinguish the fearful association over time. On the renewal test, mice displayed less freezing relative to the first trial-block of extinction training (t(508) = 19.79, p < 0.001). Importantly, DO mice demonstrated promising variation for all these traits, which is crucial for later identifying correlated genetic variation. Promisingly, QTL analyses on a small subset of mice (N = 288) identified numerous suggestive and significant QTLs associated with conditioned fear. As we increase our sample size, mapping power and resolution will increase. Additionally, we are collecting hippocampal tissue from a subset of mice for future RNA-Seq experiments that will explore the network of correlations between CF, DNA sequence, and gene expression. Poster 19 Abundant 5-HT release from EC cells disrupts colonic motility Brigitte Lavoie, Dmitri Tchitchkan, Gary M. Mawe Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VT 05405 Serotonin (5-HT) that is synthesized by enterochromaffin (EC) cells in the intestinal mucosa can activate the ascending contractile and descending relaxant limbs of the peristaltic reflex. However, recent studies have demonstrated that peristalsis persists in the absence of mucosal 5HT, raising the question of whether mucosal 5-HT actually influences propulsive motility. In this study we evaluated the actions of emetic drugs (ipecac, emetine and cisplatin), which are known to induce massive release of 5-HT from EC cells, on propulsive motility in isolated segments of guinea pig distal colon. A Gastrointestinal Motility Monitor (GIMM; Catamount Research) system was used to record and analyze the rate of fecal pellet propulsive motility. Following a 30 minute equilibration period, pellet velocity was evaluated over three trials separated by 5 minute rest periods, and under control conditions was typically in the range of 1.8-2.4 mm/sec. Drugs were then added to the solution for 10 min, and an additional 3 trials were recorded. Intraluminal infusion of ipecac (2-10%), emetine (1-10mM) or cisplatin (3µM-1mM) to the bath slowed motility in a concentration-dependent manner, and halted transit at high concentrations. Bath application of the 5HT3/5HT4 antagonist SDZ-205-557 hydrochloride (10µM) inhibited the effects of the emetic compounds, but intraluminal application of the antagonist had no effect. In summary, extensive activation of 5-HT release from EC cells over an entire segment of the colon has a negative impact on propulsive motility. These findings support the concept that release of mucosal 5-HT can influence propulsive motility in the intestines. Supported by DK62267 Poster 20 Kv1.2 potassium channel role in Cerebellar learning and memory. S. C. Madasu1, M. L. Shipman2, J. T. Green2, A. D. Morielli1 1Departments of Pharmacology and Psychological Science University of Vermont, Burlington, VT 05405 Learning is regulated by synaptic and intrinsic plasticity. Synaptic plasticity in turn is regulated by ion channel function. Major contributors of neuronal excitability and intrinsic plasticity are voltage-gated ion channels. The regulation and surface expression of ion channels such as voltage-gated potassium channel Kv1.2 has been shown not only to govern Purkinje cell (PC) excitability in the cerebellum(1) but also cerebellum-dependent associative learning and memory such as eye blink conditioning (EBC). Our lab has previously shown that inhibition of Kv1.2 in cerebellar cortex via infusion of the potent and selective Kv1.2 blocker tityustoxin-K facilitates EBC in rats (2). AMPAR endocytosis during mGluR1 stimulated long term depression (LTD) at parallel fiber-PC synapses has been proposed as a mechanism for EBC. mGluR1 knock out mice show impaired EBC and impaired LTD (3, 4). However, mutation studies in mouse that block AMPAR endocytosis and prevent LTD have no effect on EBC. (5). Thus, mGluR1, but possibly not AMPAR endocytosis, is important for cerebellar dependent learning. Here we show that mGluR1 stimulation via DHPG decreases surface expression of both Kv1.2 and AMPAR*. We propose an alternative mechanism for mGluR1 involvement in EBC that involves modulation of surface expression of Kv1.2 in cerebellar cortex. Poster 21 TRPV1-mediated Ca2+ Influx and Constriction of Middle Meningeal Arteries Inessa Manuelyan1, Masayo Koide1, Arsalan U. Syed1, Bo Shui2, Swapnil Sonkusare1, Michael I. Kotlikoff2, Mark T. Nelson1 and George C. Wellman1 Department of Pharmacology, University of Vermont College of Medicine, Burlington, VT1, College of Veterinary Medicine Cornell University, Ithaca, NY2 Transient receptor potential cation channel subfamily V member 1 (TRPV1) is widely studied in sensory nerves and has been implicated as an important component in the pain pathway. The role of this non-selective cation channel in vascular smooth muscle cells, however, remains largely unexplored. We used several ex-vivo methods to test the hypothesis that capsaicin (CAP), a TRPV1 agonist, constricts middle meningeal artery (MMA) by activation of smooth muscle TRPV1 channels. Diameter measurements of isolated, pressurized rat and mouse MMA revealed that CAP induced a concentration-dependent constriction of MMA (EC50 100 nM). CAPinduced MMA constriction was blocked by the TRPV1-selective antagonists, capsazepine (1 M), SB366791 (1 M) and was absent in arterioles isolated from TRPV1 knock-out mice. TRPV1-tdTomato reporter mouse demonstrated expression of TRPV1 channels in MMA, intercostal arteries, nerves, skin as well as skeletal muscle arteries. CAP also induced similar constriction in intercostal and skeletal muscle arteries. Interestingly, td-Tomato fluorescence was absent in brain arteries of TRPV1-tdTomato reporter mice and diameter measurements revealed that CAP (10 M) did not constrict cerebral arteries. To explore the effect of CAP on Ca2+ influx in smooth muscle directly, MMA myocytes were dissociated from acta2-GCaMP5mCherry transgenic mice expressing the ratiometric Ca2+ indicator protein, GCaMP5-mCherry, driven by the smooth muscle promoter acta2. In freshly isolated MMA myocytes from acta2GCaMP5-mCherry mice, 1 M CAP caused a 4-fold increase in the ratio of Ca2+-sensitive GCaMP5 to Ca2+-independent mCherry fluorescence. Imaging of Ca2+ influx events (sparklets) in slit-open MMA using the Ca2+ indicator dye, Fluo4-AM, revealed elementary CAP-induced signals with a quantal fractional fluorescence change of 0.19 F/F0, consistent with the single channel conductance of TRPV1 channels. Further, CAP-induced MMA constriction was reduced by approximately 30% by the voltage-dependent Ca2+ channel (VDCC) blocker diltiazem (100 M). Together, these findings support our hypothesis that CAPinduced MMA constriction results from activation of TRPV1 on vascular smooth muscle cells. The reduction of MMA constriction in the presence of diltiazem suggests that the CAP-induced Ca2+ influx in MMA smooth muscle is likely the combination of direct Ca2+ entry via TRPV1 channels and VDCC activation via TRPV1-mediated Na+ influx. Poster 22 Pituitary adenylate cyclase-activating polypeptide (PACAP) expression in lower urinary tract pathways (LUT) with cyclophosphamide (CYP)-induced cystitis in PACAP promoter-dependent EGFP BAC transgenic mice Morgan E. Mathews1, Susan Malley1, Beatrice M. Girard1, Karen M. Braas1, James A. Waschek2, Victor May1 and Margaret A. Vizzard1 Dept. of Neurological Sciences1, University of Vermont College of Medicine, Burlington, VT 05405 Dept. of Psychiatry and Behavioral Sciences2, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA The PACAP/receptors system exists in rodent lower urinary tract (LUT); the majority derived from sensory neurons. PACAP expression in sensory neurons following nerve injury is changed; however, few studies have examined PACAP expression following inflammation. We have previously demonstrated an upregulation of PACAP expression in rodent micturition pathways following CYPinduced cystitis. We now examined the effects of CYP-induced cystitis (4 h, 48 h, chronic) in PACAP promoter-dependent EGFP BAC transgenic mice. We induced bladder inflammation in adult mice by injecting CYP intraperitoneally to produce acute (150 mg/kg; 4 h), intermediate (150 mg/kg; 48 h), and chronic (75 mg/kg; every third day for 10 days) cystitis. In control (no inflammation) animals, low basal expression of PACAP-EGFP+ fibers was present in the superficial DH at all segmental levels examined (L1, L2, L4-S1). Dorsal root ganglia (DRG; L1, L2, L6, S1) from control animals also exhibited PACAP-EGFP+ cells. After CYP-induced cystitis, PACAP-EGFP+ cells increased dramatically in spinal segments and DRG (L1, L2, L6, and S1) involved in micturition reflexes. Small diameter, PACAP-EGFP+ DRG cells co-localized with TRPV1- and TRPV4-IR. The density of PACAP-EGFP+ nerve fibers was increased in the superficial laminae (I-II) of the L1, L2, L6, and S1 DH. No changes in PACAP-EGFP+ nerve fibers were observed in the L4-L5 segments. PACAP-EGFP+ nerve fibers also increased in the lateral collateral pathway in L6-S1 spinal cord. Following CYPinduced cystitis, PACAP-EGFP+ urothelial cells were observed and the number of PACAP-EGFP+ urothelial cells increased with duration of cystitis. PACAP-EGFP+ urothelial cells were co-localized with TRPV4-IR. Changes in PACAP expression in LUT pathways after cystitis may play a role in altered visceral sensation (allodynia) and/or increased voiding frequency in the chronic inflammatory pain syndrome, interstitial cystitis/bladder pain syndrome. Support: NIH-NIDDK 2R01DK051369, 2R01DK060481 Poster 23 High resolution mapping of PACAP neurocircuits using a PACAP-EGFP transgenic mouse model. Michael C. Condro1, Anna Matynia2,3, Nicholas N. Foster4, Yukio Ago5, Abha K.Rajbhandari1, Bhavaani Jayaram1, Sachin Parikh2,3, Eileen Nguyen2,3, Victor May6, Hong-Wei Dong4 and James A. Waschek1 1 Department of Psychiatry and Psychology, 2 Department of Ophthalmology, and 3Brain Research Institute, University of California - Los Angeles, USA 90095; 4 Institute of Neuro Imaging and Informatics, University of Southern California, Los Angeles, CA USA 90089, 5Laboratory of Medicinal Pharmacology, Osaka University, Osaka, Japan; 6 Department of Neurological Sciences, University of Vermont, USA 05405 Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) expression in the central and peripheral nervous systems is typically identifed and mapped using immunocytochemical and in situ hybridization techniques. We introduce here a transgenic mouse model that harbors in its genome a bacterial artificial chromosome that containing an EGFP expression cassette inserted upstream of the PACAP ATG translation initiation codon. Analysis of EGFP expression in brain sections of these mice revealed EGFP in distinct neuronal perikarya and nerve fibers in major brain regions; the PACAP-expressing cell bodies and fiber processes were easy to detect, allowing morphological characterization and anatomical circuits to be better resolved. The PACAP-EGFP expression patterns closely mimicked those described by other techniques; notably, several immunocytochemically identified PACAP expression sites were not recapitulated. The PACAP-EGFP expression was found to be strongly upregulated in motor neurons after peripheral axotomy in the ipsilateral facial motor nucleus in the brainstem, mimicking reported changes in PACAP gene expression reported in the same injury model. The high resolution maps of PACAP expression using these mice will be useful in delineating the anatomical expression and plasticity of PACAP in the nervous system. Poster 24 Loss of mTOR complex 1 function impairs synaptic transmission and blocks the effects of Pten deletion. Matthew McCabe and Matthew Weston Department of Neurological Sciences University of Vermont College of Medicine, Burlington, VT 05405 The mammalian target of rapamycin (mTOR) signaling cascade is a ubiquitous pathway that regulates essential functions such as cellular growth, proliferation and protein synthesis. Hyperactivation of mTOR in both humans and animal models can lead to cortical malformations, cognitive disabilities, autistic behavior and epilepsy. In addition, the mTOR pathway may be abnormally activated by acquired injuries that produce epilepsy and trigger downstream processes of epileptgenesis. Therefore, a more complete understanding of how the mTOR pathway contributes to epilepsy and epileptogenesis may provide new targets for therapeutic intervention early on or even prior to the onset of epilepsy and its accompanying neurocognitive defects. Pten is a negative regulator of the PI3K-mTOR signaling pathway. Pten null mice die during embryogenesis, but deletion of Pten in subsets of neurons leads to macro-encephaly, autism-like behavior and seizures. Previous studies of synaptic transmission after genetic or pharmacological manipulation of the mTOR signaling cascade, and Pten in particular, have reported alterations in synaptic transmission and plasticity. Specifically, Pten loss leads to enhanced synaptic currents in both glutamatergic and GABAergic neurons, and increases in both the number of synaptic vesicles in the readily releasable pool and the postsynaptic response to single vesicle fusion are underlying mechanisms. 72 hour treatment with rapamycin completely blocked these changes and caused an additional increase in the probability of synaptic vesicle fusion. The goal of the present research was to explore the role of mTOR complex 1 (mTORC1) in mediating this effect. We selectively impaired mTORC1 in cultured hippocampal neurons by genetic deletion of Raptor, an association protein necessary for the formation of mTORC1. We observed a reduction in peak evoked EPSC (eEPSC) amplitude as well as a reduction in the size of the synaptic vesicle readily releasable pool (RRP) following Raptor knockout. When both Raptor and Pten were deleted, peak eEPSC amplitude and RRP size resembled wild-type cells. Taken together, this indicates that mTORC1 is involved in mediating the effect of Pten knockout, but also suggests additional unknown mechanisms are also involved. Poster 25 Sequential reprogramming and re-differentiation of reactive astrocytes to identify downstream targets of Jagged1/Notch1 signaling in the peri-infarct area after stroke Matthew D. LeComte, Issei S. Shimada, Andrea L. Bibeau, and Jeffrey L. Spees Department of Medicine, University of Vermont College of Medicine Burlington, VT 05405 Stroke is the fourth leading cause of mortality and the principle cause of disability in the United States. Reactive astrocytes surround ischemic and necrotic brain tissue and help to reduce inflammation, protect neurons, and maintain and repair the blood brain barrier. Notch signaling is required for the proliferation of reactive astrocytes that most closely border the infarct area. Conditional knockout of Notch1 from reactive astrocytes after stroke increases immune cell invasion into the brain parenchyma. The role of Notch signaling in regulating the protective and reparative functions of reactive astrocytes is unknown. Poster 26 Origin of Locally-Derived Neural Spheres from the Peri-Infarct Area Following Stroke Issei S. Shimada and Jeffrey L. Spees Department of Medicine, University of Vermont College of Medicine Burlington, VT 05405 Neural stem/progenitor cells (NSCs) are multipotent cells that differentiate into neurons, astrocytes and oligodendrocytes and are found in neurogenic niches such as the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus. NSCs reside as astrocyte-like cells in neurogenic niches and some NSCs express the astrocyte marker glial fibrillary acidic protein (GFAP). The existence of stem/progenitors in non-neurogenic niches, such as the cortex, remains under debate. Astrocytes constitute the most abundant cell type in the brain. Although both NSCs and astrocytes express many proteins in common, such as GFAP and Sox2, adult cortical astrocytes are typically not considered as stem/progenitor cells. Astrocytes present in the vicinity of brain injury or within the peri-infarct region after stroke are activated and called “reactive astrocytes”. Recent studies in brain stab injury indicate that reactive astrocytes possess greater plasticity than previously understood, and may share several properties with NSCs. Poster 27 Minimally Complex Robotic Model of Human Step Initiation with Anticipatory Postural Adjustments Roman E. Popov1,2, Jesse V. Jacobs1 and Josh C. Bongard2 Departments of Rehabilitation and Motion Science1, and Computer Science2 University of Vermont, Burlington, VT 05405 A wide range of neurological diseases results in gait impairments, including Parkinson's disease (PD). PD specifically impairs step initiation; this impairment is best illustrated by altered parameters of anticipatory postural adjustments (APAs) (e.g., delayed and diminished amplitudes of backward and lateral displacements of the center of pressure during the APA). These alterations are in turn associated with decreased stability, step length, and step velocity. Inability to initiate locomotion properly has a large negative impact on everyday life of patients with PD. Due to limitations of contemporary human subject research and animal models, a computational model of human step initiation is necessary to provide additional insights and potentially generate new hypotheses about neural mechanisms of motor impairments. In this study, a minimally complex model of human step initiation with APAs was created. Iterative search for a model with target motor behavior (i.e. stepping forward to a target located on the floor) was directed from the simplest models to more complex in order to obtain a model with the lowest complexity necessary. This approach provided knowledge about the contribution of each new additional model feature towards achieving target behavior. Models were created and tested in a simulated environment that replicates real physics. The final model’s body consisted of a pelvis, two hips, two lower legs, and two feet. There were no explicit kinematic instructions given; models learned through iterative optimization, which was achieved by an evolutionary algorithm named “differential evolution” (DE). Default DE parameters were used. The neural controller was implemented based on a continuous-time recurrent neural network (CTRNN). CTRNNs did not have recurrent connections; time constant was kept identical for all neurons. The CTRNN had two input neurons, two hidden neurons, and 12 output neurons. The size of input and output layers was dictated by the model’s sensory and motor systems: it had two touch sensors located in its feet; and there were 12 joint actuators (six in each leg, two per joint). Only the model described above was able to perform stable step initiation without falling and elicited APAs similar to those produced by humans. All simpler models failed to produce the target behavior. The model’s joint angle trajectories in phase space were compared to those recorded in the experiment with human participants performing the same task and were found to lie within the 95% confidence intervals of human-derived trajectories. The model found in this study can be used for future experiments analyzing impact of PD on motor behavior. Specifically, the model can be altered to have a wide range of “lesions” (i.e. changes that negatively affect the target motor behavior) in its neural controller, morphology, as well as in learning environments. These alterations can represent impaired central sensorimotor processing, improper function of proprioceptive or motor system, or risk aversion acquired due to the disease. Comparing these altered models with “healthy” ones may provide insights into the neural mechanisms of pathology and compensation in human subjects with PD as well as into other degenerative neurological diseases with a motor component. Knowledge gained from these simulations can be used to generate new testable hypotheses or to narrow down the scope of hypotheses currently used in human studies. Poster 28 Food-Seeking Inhibited While Hungry Can Renew in the Context of Satiation Scott T. Schepers and Mark E. Bouton Department of Psychological Science, University of Vermont Recent research suggests that pathologies related to being overweight or obese are associated with as many as 4 million deaths in a single year. Obesity is an especially salient issue in the United States, as approximately 1/3 of US adults are considered obese. Accordingly, research aimed to identify conditions that promote problem-eating behaviors is especially important. Further, like other behaviors, dieting behavior that produces weight loss is often susceptible to relapse. Several studies with rat subjects therefore examined whether food-seeking behavior established while the rat is satiated can be renewed by satiation after it has been inhibited (extinguished) in the context of hunger. In two initial experiments, rats received unlimited access to food in their homecages (i.e., they were sated) while they received 12 daily sessions in which lever pressing was reinforced with highly palatable food pellets (i.e., sweet/fatty or sucrose pellets). Then, over the next 4 days, all rats received restricted access to food in the home cage and a daily session of extinction training (lever pressing no longer produced food pellets). Two test sessions then examined lever responding when rats remained on food restriction and when they were again given unlimited food access in the home cage. Counter-intuitively, the rats exhibited more food-seeking behavior when they were sated than when they were food restricted. Apparently, satiation and hunger can function as contexts (A and B, respectively) and support ABA renewal effects. A third experiment found that the interoceptive states connected with food deprivation and its absence (e.g., the feeling of hunger or satiation) may be more salient as cues for controlling learned behavior than are the absence or presence of food in the home cage. Thus, when individuals learn to eat junk food when they are not hungry and then inhibit the behavior when they are hungry (e.g., while dieting), they may further lapse when the diet is broken and they are in the state of satiation again. Poster 29 Mechanisms of serotonergic 2A-receptor-mediated excitation in callosal projection neurons in the mouse prefrontal cortex Emily K. Stephens and Allan T. Gulledge Department of Physiology and Neurobiology Geisel School of Medicine at Dartmouth, Lebanon, NH Serotonin (5-HT) selectively excites subpopulations of pyramidal neurons in the neocortex via activation of 5HT2A (2A) receptors. Classically, 2A and other Gq coupled excitatory responses are thought to result from turning off potassium conductances, including those mediated by KCNQ channels. However, the precise mechanism underlying the 2A-dependent excitation in the cortex has not yet been established. We tested several potential mechanisms of serotonergic excitation in callosal projection neurons (COM neurons) in layer 5 of the mouse medial prefrontal cortex. Under baseline conditions, focally applied 5-HT increased the rate of action potential generation during suprathreshold DC somatic current injections. To confirm that potassium conductances are involved in serotonergic excitation, we measured 2A-excitation in control conditions and after reducing extracellular potassium from 3 mM to 0.5 mM. Surprisingly, increasing the driving force for potassium failed to enhance 2A-mediated excitatory responses (mean percent change in serotonergic excitation from baseline levels was -28 22 %; n = 11, p = 0.24), even as the KCNQ channel blocker XE 991 (10 or 20 M) reduced serotonergic excitation (by 32 9%; n = 10, p < 0.05). These data suggest that serotonergic excitation is mediated by combined suppression of KCNQ channels and activation of nonspecific cation channels permeable to potassium, similar to the Gq-mediated cholinergic excitation observed in these same neurons (see, for instance, Haj-Dahmane and Andrade, J. Neurosci., 1996). Indeed, we found that activation of muscarinic receptors with bath-applied carbachol (50 M) occluded 2A-mediated excitation, confirming that both cholinergic and serotonergic excitation utilize the same Gq pathway and effector mechanisms in COM neurons. Finally, we found that 2A mediated excitation was not dependent on intracellular calcium, as responses were enhanced above control levels (by 176%), rather than reduced, when internal calcium was chelated with BAPTA (10 mM in patch pipette; n = 12, p < 0.05). Our results point to a role for multiple ionic effectors, including a nonspecific cation conductance and KCNQ-mediated Mcurrent, working together to mediate serotonergic and cholinergic excitation in prefrontal pyramidal neurons. Poster 30 It’s a HARS Knock Life for Fish: Characterizing Histidyl tRNA Synthetase in the Zebrafish Ashley Waldron1, Susan Robey-Bond2, Christopher Francklyn2, and Alicia Ebert1 1 Department of Biology and 2Biochemistry, University of Vermont, Burlington, VT 05405 Histidyl tRNA Synthetase (HARS) is a member of the family of enzymes responsible for attaching amino acids to their appropriate tRNA molecules. Without the proper function of these enzymes, translation of mRNA into protein would be impossible. Recently, a recessive, missense mutation in HARS was associated with a human deafness-blindness disorder (Usher Syndrome IIIB). Why a mutation in a ubiquitously expressed protein such as HARS would cause such a tissue specific disorder is not well understood and could imply that HARS has some important role in the development and maintenance of the auditory and visual systems. We are using the zebrafish to help better understand what this role may be. HARS is highly conserved between humans and zebrafish. However, while humans encode cytoplasmic and mitochondrial versions of the enzyme in separate genes, zebrafish utilize alternative splicing of a single gene to generate each protein. We have begun characterizing the genomic and functional aspects of zebrafish HARS. Using morpholino antisense oligonucleotides that specifically target one splice variant or the other, we have found that knock-down of either variant results in retinal and hair cell phenotypes. These results suggest that both cytoplasmic and mitochondrial HARS are particularly important in these sensory systems. Poster 31 Disciplined self-observation of aphasic disturbances: recovery of a left hemisphere injured brain, and the insights learned from primary optic aphasia, and how my changed brain works now. Sarah Robinson Burlington, VT 05405 Recommendations for a focus on syntax as well as semantics are necessary to understand how an injured brain is able to create and recall long term memory. Aphasic disturbances of several types are examined and discussed in terms of how I have learned to work around and with them over the last two and a half years — the first year with 7 months of bottom-up rehabilitation therapy (4 months in, and 3 months out patient), and engaged in self-directed top down therapy for the last 22 months. What is referred to as semantic learning in Neuroscience f-MRI studies of injured brains, refer to semantic learning for creating and recalling memory. Directly observed of my efforts with memory recovery has taught me that this process also involves syntactic learning, (e.g, that is to say it takes meaning in context), in order to create and recover new long term memory. I discuss this in terms of vision and language, and connect the syntactic processes in terms of the importance of historical and cultural context for the importance of deep, (as defined in Greek Mythos), meaning in memory creation in recovery.Illustrations, from both the visual arts and linguistics, specific to my personal experience with aphasic disturbances, are employed to communicate the key points and themes of this research project and conclusions and recommendations. An overview of my trial and error repetitive exercises that were incrementally changed with recovery, as well as learned acceptance of impairments that are permanent and must be worked around, are presented to share for further research and application in language and visual aphasic disturbances. It is hoped that this is taken as an invitation for more multidisciplinary research in social and neuroscience on the living injured human brain Poster 32 Renewal of extinguished behavior in the context of the preceding response Jeremy M. Trott, Eric A. Thrailkill and Mark E. Bouton Department of Psychological Science, University of Vermont Burlington, VT 05405 Behavior often occurs in the form of sequences, or chains, of responses that may include procurement behavior leading to a final consumption behavior. For example, a drug user or overeater must first buy the drug or food before he or she can use or eat it. Treatments to reduce drug use or overeating thus need to reduce behaviors that are a part of a chain. One of the key facts from research on extinction, the process in which organisms learn to stop doing a behavior, is that extinction learning is particularly context-dependent. Another point is that contexts are defined broadly, and can take many forms. In the case of a behavior chain, in which procurement and consumption responses must occur in sequence in order to be reinforced, consumption responses take place in the context of having performed the procurement response. Therefore, extinction of consumption alone might result in inhibition of the response that is specific to the context of having not performed the procurement response. These experiments asked whether separately extinguished consumption behavior renews upon return to the heterogeneous behavior chain (procurement responding preceding consumption responding). Rats learned to make a procurement response (e.g., a chain pull) in the presence of a procurement discriminative stimulus (SD), which led to the presentation of a consumption SD and the opportunity to perform a consumption response (e.g., lever press). In Experiment 1, consumption behavior that was extinguished outside the chain renewed when it was returned to the context of the chain. In Experiment 2, rats learned to make two separate behavior chains before extinction of each consumption response. Only the specific procurement response associated with the consumption response was able to cause renewal. In each case, renewal of consumption depended on the animal having made the procurement response. Therefore, in a behavior chain, the procurement response can serve as the context for consumption. Implications for increasing the durability of extinction will be discussed, particularly as it relates to behaviors that often occur as part of a chained sequence of behaviors.
