GBCSFN2015 Schedule_withpics

P31 Li, Tuo

PROTEIN CROWDING WITHIN THE POSTSYNAPTIC DENSITY CAN IMPEDE THE ESCAPE OF TRANSMEMBRANE
PROTEINS
Tuo P. Li (1,2), Yu Song (3), Thomas A. Blanpied (2), and Sri Raghavachari (4)
(1) Medical Scientist Training Program; (2) Dept. of Physiology and Program in Neuroscience, University of
Maryland School of Medicine, Baltimore, MD; (3) Dept. of Physics, Duke University, Durham, NC, USA; (4) Dept.
of Biology, University of Maryland, College Park, MD.

Mechanisms that regulate the movement and positioning of AMPA-type glutamate receptors (AMPAR) within
the postsynaptic density (PSD) determine the strength of neurotransmission at excitatory synapses. Two key
processes that control synaptic AMPAR number are lateral diffusion on the plasma membrane and binding of
receptors to PSD scaffold proteins. Electron microscopy suggests that the PSD is highly crowded, potentially
restricting the diffusion of receptors even in cases of negligible receptor-scaffold binding. However, the
contribution of macromolecular crowding to receptor retention remains poorly understood. Here, we combine
experimental and computational approaches to test the effect of synaptic crowding on receptor mobility and
enrichment. To examine the distinct contributions of crowding and receptor-scaffold binding, we developed a
computational model for AMPAR diffusion in the extrasynaptic and synaptic spaces, containing immobile
obstacles representing scaffolding, receptor, and adhesion molecules in the PSD. The spatial distribution of
scaffold proteins was determined directly from photoactivated localization microscopy that mapped molecular
positions. AMPAR‒scaffold association (Kon) and dissociation (Koff) rates were adjusted by computer
simulations to fit single-particle tracking and fluorescence recovery after photobleaching (FRAP) measurements.
The model predicted that variation of receptor size strongly influences the fraction of synaptic area that the
receptor may traverse, and the proportion of receptors that may quickly exchange in and out of the synapse.
Strikingly, variation of Koff did not significantly alter receptor residence time or mobility. In order to test the
model experimentally, we used single-molecule tracking and FRAP to quantify the synaptic diffusion dynamics of
uniquely designed transmembrane (TM) proteins that mimic receptors. We find that a single-pass TM protein
with one binding motif is delivered to the synapse, yet the smaller TM protein was more mobile than the much
larger AMPAR within the synapse. Furthermore, we find that either a single binding motif or an increase in bulk
by adding only one GFP on the cytoplasmic domain slowed the synaptic movement of the single-pass TM
protein. These results strongly suggest that both crowding and binding can play important roles in impeding the
escape of AMPARs from the synapse. This is likely a general principle by which tight protein packing within the
PSD can modulate the synaptic dwell time of other TM proteins such as ion channels and adhesion molecules
that are critically important for synaptic function.

51

P32 Matous, Allison
DELAYED RECRUITMENT OF RIGHT INSULA IN SMOKERS DURING PARAMETRIC MODULATION OF RESPONSE
CONFLICT
Allison L. Matous (1), John R. Fedota (1), Thomas J. Ross (1), Elliot A. Stein (1)
(1) NIDA-IRP
An influential theory of addiction posits that substance abusers have deficits in a range of cognitive control
processes that contribute to ongoing abuse. Compelling empirical evidence supports this theory, but
inconsistencies across paradigms assessing different aspects of cognitive control remain problematic to its
interpretation. While smokers exhibit clear deficits in evaluative processes, including outcome monitoring,
differences in regulative processes, including conflict detection and initial response selection, have been less
consistently characterized. The present study assessed regulative cognitive control processes through
performance on a modified Eriksen flanker task during MRI scanning. Participants (25 smokers, 14 non-smoking
controls) responded to flanker trials parametrically manipulated to instantiate high, medium, and low levels
conflict. Conflict trials were intermixed with trials of no conflict. Compared to the traditional flanker paradigm,
parametrically modulating conflict was predicted to allow for better characterization of regulative control,
including when these processes are recruited. Results show that across groups, reaction times increased and
accuracy decreased with increased conflict. Yet, smokers’ behavioral responses to parametrically manipulated
conflict were indistinguishable from nonsmokers’. In both groups, neuroimaging results show monotonic
increases in activation with increasing conflict in brain areas sensitive to conflict. However, smokers process
intermediate conflict similarly to low or no conflict, exhibiting reduced activation in the right insula, while
nonsmokers show a dose-response increase in insula activation with increased conflict. Because this pattern was
only seen only for medium but not high conflict trials, conflict processing is not absent but rather delayed in
smokers. This pattern of activation may put smokers at risk for committing actions that are in conflict with
ultimate goals and may contribute to the perpetuation of maladaptive behavior.

52

P33 Metzbower, Sarah

THE ROLE OF SYNAPTIC NANOSTRUCTURE IN REGULATING NMDA RECEPTOR ACTIVATION
*S. Ransom Metzbower(1,2), S. Raghavachari (3), T. A. Blanpied ( 1,2)
(1) Physiol., (2) Program In Neurosci., Univ. of Maryland, Baltimore, Baltimore, MD; (3) Dept. of Biol., Univ. of
Maryland, College Park, MD;

Within one synapse, many factors may influence NMDA-type glutamate receptor (NMDAR) activation, including
the number and subtype of NMDARs, their subsynaptic distribution, and their relationship to sites of vesicle
release. To examine regulation of NMDAR activation in single synapses, we utilized the Ca2+ indicator GCaMP6f.
Spinning disk confocal time lapse imaging of cultured hippocampal neurons in the presence of TTX, 0 Mg2+, and
3 mM Ca2+ revealed miniature spontaneous NMDAR activation in individual dendritic spines. The GCaMP6f
response to spontaneous transmitter release appeared sensitive to the total amount of NMDAR channel
opening, because slight elevation of extracellular Mg from 0 to 30 µM decreased.

Immunocytochemistry and focal glutamate delivery have suggested that tens of NMDARs are present at a single
synapse. However, when we applied a low concentration of the high affinity NMDAR antagonist CPP, there was
a significant decrease in mSCTs frequency with minimal changes in amplitude. This suggests that very few
NMDARs open following spontaneous neurotransmitter release. The low open probability of NMDARs after
agonist binding likely contributes to this phenomenon, but, in addition, subsynaptic position of NMDARs away
from glutamate release sites may minimize their activation. Previously, super-resolution imaging using
photoactivated localization microscopy (PALM) has revealed nanodomains within the PSD that contain a
relatively high density of scaffold proteins and the NMDAR subunit GluN2B. To examine the functional impact of
subsynaptic NMDAR position, we first used Monte Carlo simulations to predict the influence of vesicle fusion
site on NMDAR activation. Model synapses were created using measured super-resolution maps of the NMDAR
distribution and PSDs. Simulating release over the model PSD revealed that the open probability of GluN2B
containing NMDARs sharply decreased as a function of distance from the site of release (~50% in 80 nm). To
study the relationship between PSD nanoscale organization and NMDAR activation, we used PALM to map the
subsynaptic distribution of PSD-95 in spines imaged with GCaMP6f. This revealed a positive correlation between
NMDAR activation at single spines and the fractional area of the PSD that was incorporated into a nanodomain.
Additionally, spines with nanodomains showed a wider range of average peak amplitudes than spines that
lacked nanodomains. Together, these data suggest PSD nanostructure is a novel mechanism for regulation of
NMDAR activation.

53

P34 Myslinski, N. R.

BRAIN BEE
Norbert Myslinski
UMB Dental School, Dept of Neural and Pain Sciences

Future neuroscientists from around the world met in Cairns, Australia to compete in the 17th International Brain
Bee (IBB) Championship coordinated by Prof. Linda Richards. The Brain Bee is the preeminent neuroscience
competition for teenage high school students. The event was hosted and sponsored by the International Society
for Neurochemistry and the Australasian Neuroscience Society at their convention with the Asian-Pacific Society
for Neurochemistry in August. Additional major sponsors included James Cook University, the University of
Queensland and the Queensland Brain Institute as well as donations from individuals to support competitors
from specific countries. Worldwide there are 150 chapter competitions, each one involving many schools. The
Chapter winners then compete in their respective regional championships to earn the right to compete in the
World Championship. They are tested on their knowledge of the human brain with oral and written tests, a
neuroanatomy exam using human brains, a patient diagnosis component, and a neurohistology exam. The
regions competing were not known at press time, but the regions that sent their champions to the IBB
Championship last year (and their coordinators) were Australia (Linda Richards), Brazil (Alfred Sholl-Franco),
Canada (Judy Shedden), China (Jiangjie Yu), Germany (Julianne R McCall), India (Seema Raghunathan), Italy (P.
Paolo Battaglini), Japan (Tetsu Okumura), Kenya (Nchafatso Gikenyi), Korea South (Seong-Whan Lee), Macau
(Thomas Lao), Malaysia (Jafri Malin Abdullah), Nepal (Sarun Koirala), New Zealand (S Louise Nicholson), Nigeria
(Polycarp Nwoha), Poland (Elzbieta Malgorzata Pyza), Romania (Cristian Gurzu), Tanzania (Rashidi Mussa),
United Arab Emirates (Sathy Parvathy), United States (Norbert Myslinski) and Wales (Vanessa Davies). The IBB’s
purpose is to motivate young men and women to learn about the human brain, and to inspire them to enter
careers in the basic and clinical brain sciences.

Dr. Norbert Myslinski founded the IBB in 1998 with 12 local
chapters in North America. An estimated 20,000 students compete annually. More than 100 newspapers, radio
and television stations cover the IBB and the student competitors at each stage of the competition, and about
50 web sites are devoted to the Brain Bee. Presidents, Ambassadors and other public officials have recognized
the IBB. Many former competitors are now working in neuroscience, neurology, psychology and related fields.
The Brain Bee is building better brains to fight brain disorders. The 2016 IBB championship will be hosted by the
Federation of European Neuroscience Societies at their convention in Copenhagen, Denmark.

54

P35 Myslinski, Norbert

BRAIN BEE INTERNATIONAL
Norbert Myslinski
University of Maryland Dental School, DNPS

After two days of intense competition, the 2015 USA Regional Brain Bee Champion is Soren Christensen. The
Brain Bee is a neuroscience competition for high school students. A record 52 Chapter winners from 33 states
competed at the University of Maryland, Baltimore. Soren goes to Thomas Jefferson High School. He won a
scholarship, a summer internship at a Georgetown University’s Department of Pharmacology & Physiology, and
the right to represent the USA at the seventeenth International Brain Bee (IBB) Championship in Australia where
he will compete against the regional champions from approximately 20 countries such as Australia, Brazil,
Canada, China, France, Germany, India, Italy, Japan, Kenya, Korea, Malaysia, New Zealand, Romania, Singapore,
Turkey, Ukraine, and others. The IBB Championship will be hosted by the Australasian Neuroscience Society, the
International Society for Neurochemistry and the Asian-Pacific Society for Neurochemistry at their combined
convention in Cairns, Australia (See IBB Poster). The Brain Bee tests a student’s knowledge of the human brain,
including such topics as intelligence, emotions, memory, vision, Alzheimer’s disease, Parkinson’s disease, and
many others. The USA Championship competition involves a neuroanatomy laboratory exam with human brains,
patient diagnosis involving face-to-face interactions with patient actors, brain histology with microscopes, and a
final question-and-answer component. To advance to the USA Championship, Soren had to win the Washington,
DC Chapter Brain Bee Competition coordinated by Emily Dilger of the Society for Neuroscience and hosted by
the AAAS. Soren’s Patient Partner was Himal Bikma. “Patient Partners” is a new component of the USA Brain Bee
where student competitors are matched with patients. If the student wins, then the partner also wins a financial
prize. It focuses on the real purpose of the Brain Bee, helping the people with brain disorders. The USA Regional
Brain Bee was founded by Dr. Norbert Myslinski, Department of Neural and Pain Sciences, University of
Maryland Dental School, and is one of 30 World-Wide Regional Brain Bees. It utilizes Limbic Learning to motivate
young men and women to learn about the brain, and inspire them to consider careers in the basic and clinical
neurosciences. Limbic Learning is a new term referring to emotion-enhanced learning. The enhancement is
dependent upon the limbic system, or emotion circuits, of the brain, especially the amygdala (See Limbic
Learning Poster). Dr. Myslinski says, “We need future clinicians and researchers to treat and find cures for
neurological and psychological disorders. We build better brains to fight brain disorders.”

55

P36 Nakamura, Mai
INVESTIGATING THE ROLE OF GDE2 AND GDE3 IN ALZHEIMER’S DISEASE PATHOGENESIS
Mai Nakamura (1), Clinton Cave (1), and Shanthini Sockanathan (1)
(1) The Solomon Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore,
MD
Alzheimer’s Disease (AD) pathogenesis is characterized by the overproduction of amyloid-β (Aβ) peptides. These
Aβ peptides are formed by sequential cleavage of Amyloid Precursor Protein (APP) by the proteases β- and γ-
secretase. Additionally, recent work has shown that APP can be cleaved by the α-secretase ADAM10 in a
pathway that confers neuroprotection by production of the secreted fragment sAPPα. In AD, however, the
homeostasis between α- and β-cleavage is perturbed, resulting in accumulation of Aβ peptides through
increased β-cleavage. While pathways regulating the production of Aβ have been extensively studied, very few
have focused on the biogenesis of neuroprotective sAPPα. Elucidating the molecular mechanisms regulating α-
cleavage could therefore significantly advance our knowledge of the pathogenesis underlying AD.
In neurodevelopment, the glycophosphatidylinositol- (GPI-) anchored protein RECK antagonizes the
metalloprotease ADAM10, which in turn modulates Notch signaling. Previously, our lab has found that the six-
transmembrane protein Glycerophosphodiester Phosphodiesterase 2 (GDE2), and its close paralog
Glycerophosphodiester Phosphodiesterase 3 (GDE3), regulates this process by cleaving the GPI anchor of RECK,
thus inactivating RECK and promoting ADAM10 enzymatic activity.
We have recently found evidence of neurodegenerative pathology in mice lacking GDE2 or GDE3 (Gde2-/-,
Gde3-/-). These results point to the exciting possibility that the GDE/RECK/ADAM10 signaling axis may not only
play a role in neurodevelopment but may also be involved in APP processing and therefore neurodegeneration.
My thesis project seeks to further characterize the neurodegenerative effects caused by loss of Gde2 and Gde3
and to investigate whether RECK disinhibition of ADAM10 by the GDE proteins is neuroprotective in AD.

56

P37 Nemeth Mertz, Christina
NANOPARTICLE THERAPY FOR TARGETED DRUG DELIVERY TO INJURED CELLS FOLLOWING NEONATAL HYPOXIC-
ISCHEMIC BRAIN INJURY
Nemeth CL(1,2), Drummond G(1), Mishra MK(4), Zhang F(4), Kannan RM(4), Kannan S(2), Wilson MA (1,2,3)
(1)Hugo W. Moser Research Institute at Kennedy Krieger, (2)Department of Neurology, (3)Department of
Neuroscience, (4)Center for Nanomedicine at the Wilmer Eye Institute, The Johns Hopkins Medical Institutions,
Baltimore, Maryland
Perinatal hypoxic-ischemic encephalopathy (HIE) is a major cause of neurodevelopmental disorders such as
cerebral palsy (CP) and related disabilities. Therapeutic hypothermia is the first intervention to reduce brain
injury in term infants after HIE, but protection is incomplete, thus driving the need for combination therapies.
Our research uses a postnatal day 7 (P7) mouse model of neonatal hypoxic ischemia (HI) to evaluate the
neuroprotective efficacy of hypothermia combined with targeted drug delivery using dendrimer nanoparticles.
The anti-inflammatory drug N-acetyl-l-cysteine (NAC) has poor bioavailability that limits efficacy for brain injury.
In contrast, NAC conjugated to hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimer nanoparticles (D-
NAC) can cross the impaired blood brain barrier in some models of brain injury, allowing for the selective
accumulation of nanoparticle-conjugated NAC within activated microglia, astrocytes, and injured neurons. To
evaluate the cellular distribution of D-NAC after neonatal HI and to determine if therapeutic hypothermia alters
the biodistribution of D-NAC, male and female P7 mice were exposed to HI (unilateral carotid ligation and
subsequent hypoxia (10% O2 for 15 min)) and were treated with hypothermia or normothermia in combination
with i.p. Cy5-labeled D-NAC given 0, 6, or 24 hours after HI. Mice survived 24 hours after D-NAC treatment and
the bio-distribution of D-NAC-Cy5 in microglia, astrocytes, and neurons was analyzed in both brain hemispheres.
D-NAC was localized in injured neurons and activated microglia, indicating increased permeability of the blood-
brain barrier to dendrimer following hypoxic ischemic injury and a potential for increased efficacy of NAC when
conjugated to dendrimer. Accumulation of D-NAC-Cy5 correlated with severity of injury and dendrimer was not
detected outside vessel walls in the undamaged hemisphere or in control mice. Future work will assess the
neuroprotective and functional benefit of this combination therapy to limit brain injury in the HIE model, as well
as effects of sex. Targeted anti-inflammatory therapeutics may have important implications for the treatment of
brain injury and inflammatory-related diseases.

57

P38 Panicker, Matthew
CORTICAL HIERARCHY GOVERNS CLAUSTROCORTICAL CIRCUIT ORGANIZATION
Matthew G. Panicker(1), Michael G. White(1), Brian N. Mathur(1)
(1)Department of Pharmacology, University of Maryland School of Medicine Baltimore, MD 21201
The claustrum is a telencephalic gray matter structure with various proposed functions including sensory
integration and attentional allocation. Underlying these concepts is the reciprocal connectivity of the claustrum
with most, if not all, areas of the cortex. What remains to be elucidated to further inform functional hypotheses
is whether a pattern exists in the strength of connectivity between a given cortical area and the claustrum. To
this end, we performed a series of retrograde neuronal tract tracer injections into rat cortical areas along the
cortical processing hierarchy, from primary sensory to frontal cortices. We observed that the number of
claustrocortical projections increased as a function of processing hierarchy; claustrum neurons projecting to
primary sensory cortices were scant and restricted in distribution across the claustrum while neurons projecting
to the cingulate cortex were densely packed and evenly distributed throughout the claustrum. This connectivity
pattern suggests that the claustrum may subserve executive functions orchestrated by the cingulate cortex.

58

P39 Patton, Mary
ETHANOL DISINHIBITS DORSOLATERAL STRIATAL MEDIUM SPINY NEURONS THROUGH ACTIVATION OF A
PRESYNAPTIC DELTA OPIOID RECEPTOR
Mary H. Patton (1,2), Brian N. Mathur (2)
(1) Program in Neuroscience, (2) Department of Pharmacology, University of Maryland School of Medicine, 655
W. Baltimore St. BRB 4-011, Baltimore, MD, 21201, USA
Habit formation is mediated by the dorsolateral striatum (DLS) and is expedited by exposure to alcohol. Across
species, alcohol exposure disinhibits the DLS by dampening GABAergic transmission onto this structure’s
principal medium spiny projection neurons (MSNs), providing a potential mechanistic basis for habitual alcohol
drinking. However, the molecular and circuit components underlying this disinhibition remain unknown. To
examine this, we used a combination of whole-cell patch-clamp recordings and optogenetics to demonstrate
that ethanol potently disinhibits the DLS by persistently depressing both MSN- and fast-spiking interneuron
(FSI)-MSN GABAergic synaptic transmission. Concentrating on the powerfully inhibitory FSI-MSN synapse, we
further show that acute exposure of ethanol (50 mM) to striatal slices activates delta opioid receptors that
reside on FSI axon terminals and negatively couple to adenylyl cyclase to induce a long-term depression of GABA
release onto both direct and indirect pathway MSNs. These findings elucidate a mechanism through which
ethanol globally disinhibits the DLS and point to the delta opioid receptor as a potential node of intervention
targeting habitual drinking.

59

P40 Payano Sosa, Janell

HYPOTHALAMIC FUNCTIONAL CONNECTIVITY IN ONGOING PAIN IN HEALTHY SUBJECTS AND SPONTANEOUS
PAIN IN BURNING MOUTH SYNDROME PATIENTS
(1,2), J. S. PAYANO SOSA, (2) S. A. KHAN, (1,2) T. J. MEEKER, (3) T. F. MEILLER, (1,2) D. SEMINOWICZ
1. Program in Neuroscience, School of Medicine, 2. Neural and Pain Sciences, 3. Oncology and Diagnostic
Sciences, School of Dentistry, University of Maryland, Baltimore

Burning mouth syndrome (BMS) is a chronic pain syndrome characterized by burning in the superficial oral
mucosa. About 12-18% percent of postmenopausal women experience a type of BMS. While the mechanisms of
BMS are unclear, several lines of evidence implicate dysfunction of the hypothalamic-pituitary-adrenal (HPA)
axis. Because the hypothalamus is known to link the endocrine and nervous systems and is involved in
thermoregulation and circadian rhythm, we examined hypothalamus connectivity as a potential target for
understanding BMS symptoms. We investigated resting-state fMRI functional connectivity in a homogeneous
sample of 9 female (age range 40 to 61) peri- or post-menopausal BMS Type I patients and 9 matched healthy
control subjects. Patients underwent two scanning sessions on the same day: in the morning when the burning
is typically absent or minimal, and in the afternoon when the burning is usually most intense. To control for
changes in functional connectivity that could be attributed to ongoing pain not specific to BMS, we included 14
healthy male and female controls (age range 23 to 61), with resting-state scans during pain-free and ongoing
pain conditions. Ongoing pain was induced by applying capsaicin cream on the leg and placing a warm thermode
on the site of application during the entire time of the scan. We used Conn Toolbox and SPM12 to assess seed-
to-voxel connectivity with four seed regions, bilateral medial (MH) and lateral (LH) hypothalamus. Here we focus
on the connectivity of the right MH. We hypothesized that compared to healthy controls, BMS patients have
increased connectivity of the MH to regions involved in thermoregulation, pain processing, and emotional
circuitry.

Patients had increased functional connectivity between MH and the stria terminalis, ventral striatum, midbrain,
anterior (aMCC) and posterior (pMCC) midcingulate cortex, posterior insula, and orbitofrontal cortex in the
afternoon scans when compared to healthy controls. The increased connectivity in the majority of these regions
was explained by the presence of ongoing pain in BMS patients (i.e. determined by BMS morning vs. afternoon).
In the ongoing pain healthy control group, increased aMCC overlapping with that in the BMS analyses was
related to ongoing pain.

We therefore showed abnormal connectivity of the MH in BMS, which was mostly explained by the presence of
ongoing pain. The MH-aMCC connectivity in particular appears to be related to ongoing pain, even in the
absence of a disease state."

60

P41 Peter, Julia

THE PARK10 GENE USP24 IS A NEGATIVE REGULATOR OF AUTOPHAGY
Julia Peter (1), Nivedita Hegdekar(1), Chinmoy Sarkar(1), Ola Awad(2), Ricardo Feldman(2) and Marta M.
Lipinski(1)
(1) Anesthesiology, (2) Microbiology and Immunology

Autophagy, a lysosome-dependent catabolic pathway involved in turnover of intracellular proteins, protein

aggregates and organelles plays an essential neuroprotective function during brain aging. Defects in autophagy

are causatively linked to neurodegenerative diseases, including Parkinson’s disease (PD), and its up-regulation

has been proposed as a potential prevention and treatment strategy. To reach this goal, it will be important to

understand how autophagy is dysregulated in both familiar and idiopathic PD and identify molecular targets for

its safe modulation. In a high-throughput functional screen of a human genome-wide siRNA library we identified

ubiquitin specific peptidase 24 (USP24) as a negative regulator of autophagy. USP24 is a poorly-characterized

gene located on chromosome 1 in the PARK10 locus associated with late-onset PD. Consistent with a specific

function for USP24 in PD, non-synonymous single nucleotide polymorphisms (SNPs) in the coding region of this

gene affect predisposition to PD in diverse populations. Our data demonstrate that USP24 protein levels are

increased in the substantia nigra of a subpopulation of non-familial PD patients, suggesting potential

involvement also in idiopathic PD. In human cell lines and in human iPS cell derived neurons USP24 knock-down

led to up-regulation in levels of cellular autophagy, as assessed by translocation of the GFP-LC3 autophagy

reporter from cytosolic to autophagosomal localization and by increase in the autophagosome associated LC3-II.

Levels of autophagy induced by USP24 knock-down were further enhanced by lysosomal inhibitors, indicating

that USP24 inhibition increased autophagy flux upstream of the lysosomes. Knock-down of USP24 also caused

accumulation of the type III PI3 kinase product, PtdIns3P, as assessed by quantification of the FYVE-dsRed

reporter. Consistently, induction of autophagy by loss of USP24 function was attenuated in the presence of the

type III PI3 kinase inhibitors, spautin 1 or 3MA. On the other hand, knock-down of USP24 failed to affect mTOR

activity, as indicated by lack of change in the phosphorylation of mTORC1 targets, ribosomal protein S6 and S6

kinase. Importantly, induction of autophagy following USP24 knock-down was not associated with loss of cell

viability. -synuclein A53T. Together, our data indicate

that USP24 is a novel negative regulator of autophagy flux in PD, acting upstream of the type III PI3 kinase but

downstream or independent of mTOR.

61

P42 Pickett, Lindsay

THE ROLE OF MAST CELLS AND MICROGLIA IN SCULPTING THE SEXUALLY DIMORPHIC NUCLEUS (SDN) OF THE
POA.
Lindsay A. Pickett (1,2), Kathryn M. Lenz (3) and Margaret M. McCarthy (1,2)
(1) Department of Pharmacology and (2) Program in Neuroscience, University of Maryland School of Medicine,
Baltimore, MD; (3) Department of Psychology, The Ohio State University, Columbus, OH.

The first neuroanatomical sex difference detected in the mammalian brain was reported in 1978 and named the
sexually dimorphic nucleus (SDN) due to its larger size in males compared to females (Brain Res. 148:333, 1978).
The SDN is a dense collection of calbindin-expressing neurons (J. Neurobiol. 42:315, 2000) located within the
central division of the medial preoptic nucleus (MPNc) of the preoptic area (POA), a critical brain region for the
control of partner preference and maternal behaviors (Horm. Behav. 55:611, 2009; Neurosci. Bull. 30:863,
2014). Previous studies have established that both sexes generate the same number of neurons in the SDN and
they selectively die off early in development in females due to a lack of the endogenous survival factor, estradiol
(Brain Res. 353:7, 1985; Brain Res. Dev. Brain Res. 52:17, 1990). This system is an excellent model for naturally
occurring cell death versus neuroprotection in the developing brain but the involvement of non-neuronal cells in
this model system has been largely ignored. We have previously established that innate immune cells of the
brain, microglia, and inflammatory mediators such as prostaglandins direct the development of sex-specific
synaptic patterns in the neonatal POA that correlate with sexual behavior in the adult rat (Nat. Neurosci. 7:643,
2004; J. Neurosci. 33:2761, 2013). We now turn our attention to an additional inflammatory cell type, mast cells,
which like microglia are of myeloid cell lineage with origins outside the nervous system. We determined there
are more mast cells in the POA of neonatal males than females and this sex difference was mediated by
estradiol. We have also found that pharmacological activation of mast cells in newborn females induces a male-
typical microglial morphology, with higher numbers of ameboid microglia and lower numbers of ramified,
phagocytic microglia. We are currently investigating whether mast cells modulate microglial primary
phagocytosis (phagoptosis) and whether this contributes to the sexual differentiation of SDN volume by
phagoptosis of neurons in the female SDN. Collectively these results indicate non-neuronal cells are crucial and
unappreciated factors shaping brain development and sex-specific physiological and behavioral outcomes.
Understanding the role of these cells in apoptotic and neuroprotective cascades during normal brain
development will allow for further studies of novel therapeutic strategies and potential sex differences in
efficacy of estradiol and/or immune cell inhibitors as neuroprotective agents.

62

P43 Ramirez-Framco, Jose Jorge
PHOGRIN, A LARGE DENSE CORE VESICLE-ASSOCIATED PROTEIN, IS A NOVEL MARKER OF INTERNEURONS IN THE
HIPPOCAMPUS
Ramirez-Franco J (1), Muñoz-Cuevas FJ (1), Jurado S (1).
(1) Department of Pharmacology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Large Dense Core Vesicles (LDCVs) store, transport and secrete a myriad of neuromodulators such as
endogenous neuropeptides, catecholamines, and neurotrophic factors. Despite their crucial role in modulating
synaptic transmission and plasticity, very little is known about the molecular mechanisms underlying LDCVs
release in neuronal tissue. Here, we have characterized the expression of phogrin, a receptor-type protein-
tyrosin phosphatases which expression is confined to the membrane of secretory vesicles in neuroendocrine
tissues Interestingly, phogrin has been proposed to regulate the exo and endocytosis of LDCVs, as well as to
participate in the their biogenesis and cargo sorting. While the expression of phogrin in the CNS has been
previously demonstrated by Western blot, a thorough characterization of its expression in the mammalian brain
is currently lacking. Therefore, we have characterized the expression of phogrin in the adult mouse
hippocampus using a combination of Immunohistochemistry and transgenic mice. In contrast to previous
reports, we have found that phogrin is primarily expressed in the somato-dendritic compartment of
hippocampal interneurons. More importantly, phogrin expression appears to be restricted to Parvalbumin (PV)
positive interneurons, as approximately 70% of phogrin positive cells are PV positive interneurons.
Unexpectedly, we were not able to find co-localization of phogrin with Chromogranin B, a canonical marker of
LDCVs which is abundant in Proopiomelanocortin (POMC) positive neurons. These results suggest the existence
of different populations of LDCVs that are restricted to particular hippocampal interneurons. Moreover, these
distinct LDCVs populations may be distinguished not only by their cargo but also by their molecular composition.

63

P44 Rodriguez, Gabriela
CELL AUTONOMOUS AND NETWORK EFFECTS OF NMDA RECEPTORS IN EXPERIENCE-DEPENDENT HOMEOSTATIC
SYNAPTIC SCALING
Gabriela Rodriguez(1), Ming Gao (3) and Hey-Kyoung Lee (1,2)
(1)Biol., (2)Dept. of Neurosci., Johns Hopkins Univ., Baltimore, MD; (3)Div. of Neurol., Barrow Neurolog. Inst.,
Pheonix, AZ
Homeostatic changes in synaptic connections are observed in the primary visual cortex (V1) after loss of vision.
It has previously been demonstrated that AMPA receptor mediated miniature excitatory postsynaptic currents
(AMPAR-mEPSCs) in layer 2/3 of V1 show larger amplitudes after visual deprivation for two days. This result is
consistent with the idea of a homeostatic adaptation to vision loss that allows restoration of neuronal firing rate
within its target dynamic range. A subsequent reexposure to light for two hours reverses these effects,
decreasing AMPAR-mEPSC amplitudes back to baseline levels. While the role of NMDA receptors (NMDARs) in
Hebbian plasticity is well established, whether they play a role in homeostatic plasticity in vivo is unclear. In this
study we take advantage of an NR1 flox mouse line, which allows us to selectively knockout NMDA receptors in
vivo. NMDAR knockout was achieved by injecting layer 2/3 of V1 with a Cre-GFP adeno-associated virus during
the critical period. Injected mice were either normal reared for 3 weeks, visually deprived in a dark room for 2
days after 3 weeks of normal vision or visually deprived and reexposed to light for two hours. AMPARs mEPSCs
were recorded from GFP positive NMDAR KO cells as well as GFP negative neighbor cells from all groups and
compared. We show that NMDAR knockout neurons fail to undergo scaling, indicating that NMDAR activation is
required for homeostatic plasticity. We also observe non-cell autonomous effects that hinder the ability of the
system to achieve homeostatic experience-dependent scaling.

64

P45 Rudzinskas, Sarah
EXPLORING THE ROLES OF PROGESTERONE RECEPTOR AND OXYTOCIN SIGNALING IN THE MECHANISM
MEDIATING METHAMPHETAMINE-FACILITATED SEX BEHAVIOR
Sarah A. Rudzinskas (1,2), Jessica A. Mong (1,2)
(1) Program in Neuroscience, University of Maryland, Baltimore, MD, 21201, (2) Pharmacology, University of
Maryland, Baltimore, MD, USA, 21201
Methamphetamine (MA) is a psychomotor stimulant which increases motivational drive for sexual activity. This
increased sex drive results in ‘high risk’ sex behaviors, and in women, these behaviors pose significant health
concerns such as unplanned pregnancies and STD transmission. Despite this, most MA studies use male subjects,
which limit our understanding of the mechanisms mediating MA-induced sexual arousal in women. Our previous
work with a rodent model demonstrated that the medial amygdala posterior dorsal (MePD) is a crucial mediator
of this MA and sex interaction. These studies resulted in the novel finding that MA, in the absence of hormones,
increases progesterone receptor (PR) in the MePD. This is surprising, as PRs are typically under tight regulation
by estrogen receptors. Our follow up studies now suggest that this increase may be directly mediated by the
D1Rs in the MePD. icv infusion of a D1R agonist SKF38393 increased the number of PR-ir cells in the MePD,
though not significantly (p<0.10). We further hypothesized that PRs may work in tandem with oxytocin
receptors (OXTRs), to increase MA-facilitated sex behavior, as OXTRs have long been understood to play critical
roles in mediating natural reward and partner preference in rats. Presently, our preliminary data show no
significant changes in OXTRs or overall oxytocin mRNA levels following MA administration, nor any effect of an
OXTR antagonist on MA-induced motivated sex behavior. This suggests oxytocin signaling does not play a role in
this drug-sex interaction. Future studies may more closely explore the role of PR in the MePD in sexual
motivation.

65

P46 Scaglione, Alessandro
OPTOGENETIC DISSECTION OF MOTIVATIONAL SALIENCE NEURONAL CIRCUITS IN THE BASAL FOREBRAIN
Alessandro Scaglione (1), Rhett Greenfield (1), Shih-Chieh Lin (1).
(1) NIA-NIH-IRP, Baltimore, MD
The survival of animals depends critically on prioritizing responses to motivationally salient stimuli. Recent
studies have identified a group of neurons in the basal forebrain (BF) that encodes motivational salience of
attended stimuli using robust bursting responses (Lin & Nicolelis, 2008). Such salience-encoding BF responses
generate an event-related potential response in the frontal cortex (Nguyen & Lin, 2014) and lead to faster
decision speed (Avila & Lin, 2014). However, the neurochemical identity of salience- encoding BF neurons
remains unknown. Determining their neurochemical identity is a key step toward understanding their circuit-
level functions especially because BF is an anatomically complex and heterogeneous region, comprised of
multiple spatially overlapping macrosystems and several distinct neuronal populations. The goal of this study is
to determine the neurochemical identity of salience-encoding BF neurons by optogenetically tagging and
recording the three major cortically-projecting BF neuronal populations in freely behaving mice. To achieve this
goal, we first established an experimental platform in three strains of transgenic Cre mice (PV-, ChAT- and
VGluT2-Cre) that combines operant behavior, electrophysiology and optogenetics. Mice were trained to
maintain fixation in a nosepoke port and then respond quickly to a reward-predicting tone to collect reward in
an adjacent port. Mice were able to achieve high hit rates (>90%) and high trial numbers in a session (>150
rewarded trials) with fast reaction times (<300ms).
Neuronal recording in combination with photo-stimulation of the BF show that: 1) The majority of recorded BF
neurons in mice showed robust bursting responses to the motivationally salient tone similar to salience-
encoding BF neurons in rats and 2) photo-stimulation of the three major cortically- projecting BF neuronal
populations can directly or indirectly modulate (excite or inhibit) bursting responses to the salient tone. Taken
together, these results suggest that the three main populations of neurons participate in concert, directly or
indirectly, in the circuit underlying motivational salience in the BF.

66

P47 Schenk, Lieven
DIFFERENTIAL NEURAL PROCESSING OF TREATMENT AND STIMULUS-EXPECTANCY
Lieven A. Schenk (1, 2), Selim Onat (2), Christian Sprenger (2), Christian Büchel (2)
(1) Department of Pain and Translational Symptom Science, University of Maryland, Baltimore, Baltimore, MD
21201, USA, (2) Department of Systems Neuroscience, University Medical Center Hamburg-Eppendorf, 20246
Hamburg, Germany
Background: The influence of contextual information on pain perception is a crucial part of placebo analgesia. In
two context groups, we compared neural pain responses associated with treatment expectation against
expectation regarding pain stimulus intensity.
Methods: A total of 48 healthy volunteers received heat pain stimuli along with two distinct cues anticipating
either treatment and/or pain (group 1: treatment/no treatment cue versus group 2: low/normal pain cue). fMRI
measurements were used to explore neural responses during conditioning and test sessions while volunteers
had to rate expected and experienced pain. We used standard univariate analysis and multivariate
Representational Similarity Analysis (RSA) to investigate the neural differences between the two groups.
Results: We observed lower expectancy and pain ratings with the treatment/low pain cue in both groups.
Importantly, this reduction was stronger in the treatment context compared to the stimulus intensity context
during the test phase. At the neural level, we observed univariate BOLD response differences between the
groups in the rostral ACC and the hippocampus during the conditioning phase and in the lateral PFC and the
amygdala during the test phase. In the multivariate RSA, we observed group differences in the bilateral ventral
striatum during the cue presentation and in the postcentral gyrus and the parietal operculum during the pain
stimulation.
Conclusion: Taken together, our data shows that a treatment context leads to larger expectancy effects on pain.
Both imaging analysis methods show significant differences in neural processing between both contexts.

67

P48 Schmid, Anne-Christine

BRUSH ALLODYNIA SUSCEPTIBILITY IS RELATED TO BASELINE HEAT PAIN SENSITIVITY; REGIONAL BLOOD FLOW
DIFFERENCES; AND SENSITIZATION-INDUCED HEAT ALLODYNIA AND MECHANICAL HYPERALGESIA
Schmid, A.-C. (1, 2, 3), Meeker, T.J. (1, 3, 4), Keaser, M. (1), Zhu, S. (5), Seminowicz, D.A. (1, 3, 4), Dorsey, S.G. (3,
4, 6), Greenspan, J.D. (1,4)
(1) Department of Neural and Pain Sciences, University of Maryland, Dental SChool, Baltimore, MD, (2)
Department of Neurosurgery, Johns Hopkins School of Medicine, Baltimore, MD, (3) Center to Advance Chronic
Pain Research, University of Maryland, Baltimore, MD, (4) Program in Neuroscience, University of Maryland,
Baltimore, MD, (5) Organizational Systems and Adult Health, University of Maryland, School of Nursing,
Baltimore, MD, (6) Pain and Translational Symptom Sciences, University of Maryland School of Nursing,
Baltimore, MD

Introduction: Brush allodynia (BA) often, but variably occurs in many chronic neuropathic pain conditions. It is
not known what factors determine allodynic development in any given individual, or to what extent BA is related
to hyperalgesia expression. The goal of this study was to determine 1) whether baseline pain sensitivity can
predict BA susceptibility, and 2) the extent to which a sensitizing provocation engages separate mechanisms for
hyperalgesia and allodynia.

Methods: 65 healthy participants (37f) between 18-43 years (27.4±6.2) were evaluated before and after
exposure to a sensitizing capsaicin-heat pain model (C-HP). The model consisted of topical application of 10%
capsaicin cream, accompanied by a heat stimulus for 35 minutes provoking a moderate level of pain. Sensory
testing included Heat Pain Thresholds (HPT), and Mechanical Pain Ratings (MPR) of sharp probes (512mN,
256mN, 128mN, 64mN). Further we assessed BA at multiple time points after C-HP exposure. Following these
sensory test sessions, a subset of subjects (N=23) participated in a replicate arterial spin label (ASL) session.

Results: Half of the participants (26) developed BA within 1 hr of capsaicin-heat exposure. The BA group had a
significantly lower HPT both pre- and post C-HP exposure compared with the no-BA group. Additionally,
mechanically evoked hyperalgesia (based on MPR) was significantly greater for the BA group post C-HP
exposure, at all forces tested: 512mN, 256mN, 128mN, 64mN.This group difference in MPR as well as HPT was
seen at all time points tested post C-HP. The ASL results revealed that the allodynic group showed greater CBF
than the non- allodynic group pre C-HP in multiple areas including Caudate, Putamen, Orbital Frontal Gyrus,
Postcentral Gyrus, Anterior Cingulate Cortex, Posterior Cingulate Cortex and Inferior Parietal Lobule.

Discussion: In this preliminary analysis we show that seisitization-induced brush allodynia is related to greater
baseline heat pain sensitivity. Further, BA is associated with greater heat allodynia and mechanical hyperalgesia,
although the time courses of these two phenomena are distinctly different, implysing separate underlying
mechanisms at work. Finally, the group difference in CBF at baseline could predict the susceptibility to BA.

68

P49 Sharma, Tanu
RESILIENT PROPERTIES OF OLFACTORY ENSHEATHING CELLS AFTER NEURONAL LOSS
Tanu Sharma (1,2,3) and Randall R. Reed (1,2,3)
(1) Center for Sensory Biology; (2) Department of Molecular Biology and Genetics; (3) Department of
Neuroscience
Olfactory ensheathing cells (OECs) are specialized glial cells found exclusively in the olfactory system. The
remarkable ability of the sensory neurons (OSN) to continually regenerate makes this tissue an excellent model
for studying neurogenesis and neuron-glial interactions. In our study, we focused on the interaction between
OSNs and the OECs that ensheath the OSN axon bundles projecting from the olfactory epithelium to the bulb.
Due to their close association, we hypothesized that OECs play a major role in OSN regeneration. The response
of OECs to OSN loss and regeneration in vivo remains largely unexplored. Therefore, the main objective of this
study was to elucidate the molecular and cellular characteristics of OECs in adult mice following acute OSN
degeneration and during regeneration. We examined OECs in their normal state and ascertained their
morphology, proliferative state and changes in gene expression in normal epithelium and at several points
during sensory neuron regeneration and axon extension. RNA-seq was performed on nasal OECs isolated directly
from mice at various time points during methyl bromide-induced OSN regeneration. Our RNA-seq results
showed that surprisingly few genes expressed in OECs were significantly up-/down-regulated during the lesion
time-course. We also examined OEC morphology at various time points during¬¬ neuronal regeneration. The
OEC morphology in vivo was previously unknown; therefore, we first developed a unique strategy to image
individual OECs. OECs were sparsely-labeled using cre-mediated recombination and subsequently, imaged in
whole olfactory mucosal tissue by employing SeeDB, a tissue clearing technique, combined with conventional
confocal microscopy. Several parameters, including length and surface area-to-volume ratios were examined for
each OEC to determine any morphological changes. Our analysis revealed a remarkable stability in
morphological parameters post-neuronal injury. Finally, methyl bromide-induced lesions did not induce
significant turnover of OECs. Overall, our results indicate that OECs remain largely unaffected by the loss and
regeneration of OSN. We conclude that the role for OECs in adult mice is to maintain an overall stable
environment for continuous regeneration of olfactory neurons. Currently, we are focusing on the effects of OEC
ablation on surrounding OSN.

69

P50 Shuo, Liu
DISRUPTED AUTOPHAGY AFTER SPINAL CORD INJURY IS ASSOCIATED WITH ER STRESS AND NEURONAL CELL
DEATH
Shuo Liu (1), Chinmoy Sarkar (2), Michael Dinizo (2), Alan I. Faden (2,3), Eugene Y. Koh (1), Junfang Wu (2), and
Marta M. Lipinski (2,3)
(1)Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, MD, 21201;
(2)Department of Anesthesiology, Shock, Trauma and Anesthesiology Research (STAR) Center, University of
Maryland School of Medicine, Baltimore, MD, 21201; (3)Department of Anatomy and Neurobiology, University
of Maryland School of Medicine, Baltimore, MD, 21201
Autophagy is a catabolic mechanism facilitating degradation of cytoplasmic proteins and organelles in a
lysosome-dependent manner. Autophagy flux is necessary for normal neuronal homeostasis and its dysfunction
contributes to neuronal cell death in several neurodegenerative diseases. Elevated autophagy has been
reported after spinal cord injury (SCI); however, its mechanism, cell type specificity, and relationship to cell
death are unknown. Using a rat model of contusive SCI, we observed accumulation of LC3-II positive
autophagosomes starting at post-trauma day 1. This was accompanied by a pronounced accumulation of
autophagy substrate protein p62, indicating that early elevation of autophagy markers reflected disrupted
autophagosome degradation. Levels of lysosomal protease cathepsin D and numbers of cathepsin D positive
lysosomes were also decreased at this time, suggesting that lysosomal damage may contribute to the observed
defect in autophagy flux. Normalization of p62 levels started by day 7 after SCI, and was associated with
increased cathepsin D levels. At day 1 after SCI accumulation of autophagosomes was pronounced in ventral
horn motor neurons and dorsal column oligodendrocytes and microglia. In motor neurons disruption of
autophagy strongly correlated with evidence of endoplasmic reticulum (ER) stress in these cells. As autophagy is
thought to protect against ER stress, its disruption after SCI may contribute to ER stress-induced neuronal
apoptosis. Consistently, motor neurons showing disrupted autophagy co-expressed ER-stress associated initiator
caspase 12 and cleaved executioner caspase 3. Together these findings indicate that SCI causes lysosomal
dysfunction that contributes to autophagy disruption and associated ER stress-induced neuronal apoptosis.

70

P51 Silva, Joyce
DOES ANTI-NGT REVERSE SYMPTOMS OF CHRRONIC NEUROPATHIC PAIN?
Silva, Joyce.T.(1,2), Evangelista, Bianca.G. (1), Seminowicz, David.A. (2), Chacur, Marucia. (1),*
(1) Laboratory of Functional Neuroanatomy of Pain, Anatomy department, ICB III, USP, São Paulo, Brazil. (2)
University of Maryland School of Dentistry, Department of Neural and Pain Sciences
Introduction: Nerve growth factor (NGF) has been widely studied by the scientific community for its pro-
nociceptive role in chronic pain conditions, and it is characterized as a chemical mediator responsible for the
induction and maintenance of these pathologies. Chronic neuropathic pain is characterized by spontaneous
burning pain accompanied by allodynia and hyperalgesia. Anti-NGF drugs have been used to reduce these
symptoms in cancer pain, irritable bowel pain and osteoarthritis in both animals and human models. However,
in chronic neuropathic pain its multiple actions are not fully understood.
Methods: Male Wistar rats (200-220g, 2 months old) underwent induction of neuropathic pain by chronic
constriction injury of the sciatic nerve (CCI). Control groups included sham-operated animals (Sham), which
underwent the same incision, but without nerve ligation, and Naive animals, which underwent no surgical
procedures. We performed Western blot to detect NGF in the sensory ganglia (DRG L4-6) of CCI, Sham and Naive
animals. In addition, anti-NGF was injected (1 and 3ug, ipl.) in the hindpaw 14 days after surgery and a dose-
response curve was performed. Sensory testing included mechanical nociceptive thresholds, thermal
hyperalgesia and cold allodynia.
Results: We observed an increase in NGF synthesis in the CCI group compared with the control groups. The CCI
animals demonstrated a reduction of the nociceptive threshold and increased thermal hyperalgesia and cold
allodynia compared to control groups. After pharmacological treatment with anti-NGF (CCI + anti-NGF), we
observed a reduction of hyperalgesia and allodynia in these animals.
Conclusions: Our results suggest that NGF is an important factor in the induction and maintenance of
neuropathic pain, since increased NGF levels were observed in the DRG after injury. We also demonstrated the
relevance of this mediator as a therapeutic target, since anti-NGF was able to reverse the often difficult to treat
symptoms of this pathology. Studies for the characterization of anti-NGF as well as magnetic resonance imaging
are underway to further elucidate these findings.

71

P52 Spita, Nathalie
COCHLEAR INNERVATION MEDIATED BY SEMAPHORIN SIGNALING
Nathalie Spita (1), Kaidi Zhang (1), and Thomas Coate (1)
(1) Georgetown University
Auditory function is dependent on the formation of specific innervation patterns between mechanosensory hair
cells and afferent spiral ganglion neurons (SGNs). In particular, type I SGNs precisely connect with inner hair cells
(IHCs), whereas type II SGNs only connect with nearby outer hair cells (OHCs). The factors that mediate these
patterning events are largely unknown. We have recently demonstrated that expression of Semaphorin-3F
(Sema-3F) in the OHC region inhibits type I SGN process extension by activating Neuropilin-2 (Nrp2) receptors
expressed on SGNs. The effect of Sema3F and Nrp2 must be mediated by a Plexin-A transmembrane receptor,
but the identity and function of this receptor remains to be determined. Immunostaining studies using Plexin-A-
specific antibodies have revealed the following expression patterns: Plexin-A1 is expressed by the SGNs and also
in the OHC stereocilia bundles, and Plexin-A3 is strongly expressed in the SGN processes adjacent to IHCs, but
reduced in the processes that cross into the OHC region. We are currently investigating whether the conditional
removal of either of these Plexins alters cochlear hair cell innervation patterns.
Previous studies have suggested also morphological differences within the type I SGN population. Approximately
half of these fibers are wide in diameter, exhibit a high spontaneous discharge rate (SR) and bind to the lateral
side of the IHC, whereas the remaining half are relatively thin, display a low SR, and target the medial side of the
IHC. Using genetic SGN sparse labeling mice, confocal imaging, and morphometric analyses using Imaris
software, we have begun to characterize the developmental time course of type I SGN development.
Preliminarily, we detect an 8-12% average difference in medially- and laterally-projecting type I SGNs. But, our
data also suggest that IHCs do not have a strictly bimodal pattern of innervation, as SGNs axons with diameters
of a wide range appear to innervate both sides of the IHC. Additionally, the molecular mechanism controlling
their differentiation and guidance to distinct sides of IHC are completely unknown and we are currently
determining the extent to which Sema3a and Nrp1 are involved in this process."

72

P53 Srinath, Ramanujan

CHARACTERIZING GCAMP6F NONLINEARITY USING TWO-PHOTON IMAGING OF FERRET V1
Ramanujan Srinath (1,2), Andrew Daniels (1), Augusto A. Lempel (1,2), Kristina J. Nielsen (1,2)
(1) Zanvyl Krieger Mind/Brain Institute; (2) Solomon H. Snyder Department of Neuroscience, Johns Hopkins
University

Genetically encoded calcium indicators (GECIs) are promising tools for measuring neural activity in vivo using
two-photon microscopy. Particularly the newest generation of GECIs, the GCaMP6 family, is receiving increased
attention for this reason. One important use of these indicators is the determination of neural tuning functions.
These experiments are based on the assumption that changes in indicator fluorescence linearly track underlying
changes in spike rate. Nonlinearities in the transformation from spike rate to GECI fluorescence would violate
this assumption, and would distort tuning curves measured using two-photon microscopy. Here, we investigate
possible nonlinearities in the spike-to-fluorescence transformation for GCaMP6f. To this end, we make use of
the established contrast invariance of orientation tuning in ferret primary visual cortex: Based on spike rate
measurements, changes in stimulus contrast do not impact the width of orientation tuning curves. Violations of
contrast invariance in two-photon data are thus indicative of nonlinearities in the fluorescence response, such as
saturation at high firing rates or clipping at low firing rates. This approach has previously been used to
demonstrate a saturating nonlinearity in the fluorescence response of the calcium indicator Oregon Green
BAPTA-1AM. Here, we used a viral vector (AAV1-syn-GCaMP6f-WPRE-SV40; UPenn vector core) to express
GCaMP6f in ferret primary visual cortex. We then used GCaMP6f responses to measure orientation tuning
functions at low (5 - 15%), medium (25 - 50%) and high (80 - 100%) contrast. Our results demonstrate that for a
large range of contrasts, orientation tuning curves measured with GCaMP6f are indeed contrast invariant.
However, at the lowest contrast (5%), we observe a violation of contrast invariance. At this contrast, tuning
widths are on average 36% narrower than at other contrasts. This difference is largely due to the fact that at low
contrasts, non-optimal stimulus orientations evoke a smaller than expected change in GCaMP6f fluorescence.
These are the stimulus conditions with the lowest firing rates. Our results thus suggest that the relationship
between spike rate and GCaMP6 fluorescence changes slope at low firing rates. Nonlinearities in GCaMP6f
responses will distort tuning curve measurements, and thus need to be taken into account when interpreting
two-photon data. Our data suggest that the relationship between firing rates and fluorescence is more linear for
GCaMP6f than for previous calcium indicators, but that responses to stimuli evoking low firing rates may be
underestimated.

73

P54 Steele, Vaughn
NEURAL MEASURES OF INCORRECT RESPONSES PREDICT COMPLETION OF SUBSTANCE ABUSE TREATMENT
Vaughn R. Steele(1, 2, 3), Eric D. Claus(2), Brandi C. Fink(3), J. Michael Maurer(2, 3), Mohammad R.
Arbabshirani(2), Vikram Rao(2), Vince D. Calhoun(2, 3), & Kent A. Kiehl(2, 3)
1 National Institute on Drug Abuse, 2 The Mind Research Network , 3 University of New Mexico
Illicit drug use is reported in 25 million Americans with a substantial economic annual cost of nearly $200 billion.
Considering as an individual receives more treatment the likelihood of positive outcomes increases, identifying
risk factors for discontinuing treatment at the individual level is imperative. Deficiencies in specific cognitive
functions could be used to identify those individuals at greatest risk of discontinuing treatment and poor future
outcomes such as drug abuse relapse. Two studies are presented using event-related potentials (ERPs; N = 89) or
functional magnetic resonance imaging (fMRI; N = 139) measures of error-processing elicited during a Go/NoGo
task. Neural measures of response errors were used to predict which treatment-seeking individuals would or
would not complete a 12-week cognitive behavioral substance abuse treatment program. Principal component
analysis (PCA) and independent component analysis (ICA) were used to reduce the ERP and fMRI data,
respectively, before calculating support vector machine (SVM) models. In each modality, neural measures of
error-processing differentiated groups and identified which individuals would discontinue treatment
prematurely with high accuracy in both ERP and fMRI; 83.33% and 78.00% respectively. Specifically, neural
deficiencies in post-error adjustments were highlighted to be most useful in predicting poor treatment
outcomes. The amplitude of the error-positivity (Pe), an ERP component, was increased for individuals who
prematurely discontinued treatment. Increased functional network connectivity between the caudal anterior
cingulate cortex (cACC) and insula and temporal regions also predicted premature treatment discontinuation.
Cross-modal similarities suggest future treatments could be refined by targeting post-error response adjustment
in at-risk individuals, which could lead to more favorable long-term outcomes. Also, baseline screening to
identify error-processing deficiencies may facilitate specialized and individualized treatment assignment thus
increasing the likelihood of positive outcomes.

74

P55 Stins, Monique

BRAIN ENDOTHELIAL ACTIVATION AND NEURONAL DAMAGE
Abhai Tripathi (2), Mary Motari (1), David Sullivan (2), and Monique F Stins (1,2)
(1) Department of Neurology, Johns Hopkins School of Medicine and (2) Malaria Research Institute, Bloomberg
School of Public Health, Baltimore MD, U.S.A.

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum infection, especially in children,
exposed non immune travelers and military personnel. Clinically, CM includes seizures, reversible coma and
often death. Upon clearance of the infection, patients are often left with neurologic sequelae, such as seizures,
learning and behavioral disorders. Recent data also show that ADHD can be linked to CM. Post malaria
syndrome can also include psychotic or acute confusional episodes and tremor.

CM pathology is characterized by sequestration of Plasmodium infected erythrocytes (PRBC) to human blood
brain barrier (BBB) endothelium without invasion into the brain. It is unclear as to how these PRBC that are
confined to the brain vasculature induce neurological dysfunction. In CM, the BBB lies at the interface of the
events occurring in blood and brain. The BBB is part of the neurovascular unit (NVU), a concept that emphasizes
homeostatic interactions between its components to ensure optimal functioning of the central nervous system.
It is hypothesized that activation of the BBB endothelium disturbs the homeostasis between the astro-glial and
neuronal components of the NVU leading to neurological dysfunction..

Using an in vitro model for the human BBB, PRBC exposure increased ICAM-1 expression on the cells and
decreased the integrity (barrier function) of monolayers in a dose dependent manner. Microarray and Gene
Ontology (GO) analysis indicated a predominance of the NFkappaB mediated proinflammatory responses among
the host signaling pathways. RT-PCR and protein analysis confirmed the increase in transcripts and release of
cytokines and chemokines.

To assess whether BBB secretions could affect the brain media was collected from the basal side of BBB models.
These basal secretions caused dose-dependent abnormal astroneuronal morphology and cell death. Blockade of
NFkappaB was able to rescue the effect of the basal media on astroneuronal cultures. Anti cytokines and
chemokines antibodies were also able to reduce the detrimental effects.

It is concluded that endothelial NFkappaB and cyto-chemokines released to the basolateral site of the BBB are
involved in conferring neurological dysfunction. Determination of the underlying pathogenesis of observed BBB
activation and astroneuronal effects may lead to development of adjunctive neurotherapeutics to ameliorate
neurologic sequelae."

75

P56 Stockman, Sara
SEX DIFFERENCE IN NEONATAL HIPPOCAMPAL NEUROGENESIS (MAY) IMPACT EARLY LIFE FORGETTING
Sara L. Stockman (1,2), J. Michael Bowers (2), Margaret M. McCarthy (1,2)
( 1)Deptartment of Pharmacology and (2) Program in Neuroscience, University of Maryland School of Medicine,
Baltimore, MD
Sexual differentiation of the brain is an important process in normal development and has implications on later
behavior and disease susceptibility. Our laboratory has identified a sex difference in neonatal hippocampal
neurogenesis in which male rats exhibit increased cellular proliferation in multiple subregions of the
hippocampus compared to females and more of these cells differentiate into neurons (Bowers et al., Biol. Sex
Diff., 2010). While sex differences are traditionally accomplished through elevation of testosterone in neonatal
males that is locally aromatized to estradiol in the brain, there is no sex difference in hippocampal estradiol
content at this time (Konkle et. al, Endocrinology, 2011). Therefore, we examined alternative sources for this sex
difference and have discovered contributions of epigenetic regulation. Canonical modes of epigenetic regulation
include DNA methylation and histone acetylation. Pharmacological inhibition of methylation through bilateral
intracerebroventricularinjections of Zebularine on the day of birth and one day after reduced cell proliferation
(measured by BrdU incorporation) in males to that of females without effecting proliferation in females.
Conversely, increased histone acetylation via administration of the HDAC inhibitor, Trichostatin A,
intraperitoneally to newborns elevated proliferation in females to levels comparable to males, but had no effect
on male proliferation. These opposing results suggest neonatal hippocampal cell proliferation is regulated
epigenetically in a sexually dimorphic manner whereby in females reduced acetylation silences pro-proliferation
genes and in males methylation silences anti-proliferation genes. Currently, we are exploring the functional
significance of this sex difference. A recent study has implicated a role for neurogenesis in forgetting. We
propose that increased neonatal hippocampal neurogenesis in males promotes forgetting and may contribute to
sex differences in susceptibility to the effects of adverse early life events.

76

P57 Straka, Malgorzata
INHIBITION AND STIMULUS COMPETITION WITHIN A GABAERGIC NUCLEUS IN THE MIDBRAIN ATTENTION
NETWORK
Malgorzata M. Straka (1), Shreesh P. Mysore (1,2)
(1); Psychological and Brain Science, Johns Hopkins University; (2) Neuroscience, Johns Hopkins University
To manage numerous stimuli competing for attention, the brain flexibly selects the most important stimulus and
processes it preferentially to guide behavior. A key circuit implicated such flexible competitive selection is the
midbrain attention network, which includes the optic tectum (OT) and midbrain tegmentum. Recent work has
suggested that the intermediate and deeper layers of the OT (OTid) is capable of performing flexible competitive
selection due to the long-range inhibition from a group of GABAergic neurons in the midbrain tegmentum (Imc),
given that focal blockade of activity in the Imc abolishes all competitive inhibition of OTid responses. However,
the neural mechanisms for flexibility of competitive selection are unknown. By blocking inhibition of Imc
neurons via iontophoresing a GABA receptor blocker, we show preliminary data showing that this flexibility is
due to inhibitory inputs to the Imc. First, we found that inhibitory inputs to the Imc are essential for an Imc
neuron to competitively switch from a high to low activity state with increasing competitor strength. This
demonstrates that information regarding competing stimuli is exclusively communicated via lateral inhibition
within the Imc, and not from the OT. Secondly, we found that the inhibitory inputs to the Imc neurons are
essential for OTid neurons to achieve flexibility during competitive selection. These results uncover, for the first
time, a specific neural circuit for dynamic, flexible selection among neural representations.

77

P58 Talishinsky, Aleksandr
PSYCHOSTIMULANT DRUG ADMINISTRATION MARKEDLY INCREASES VISUAL STIMULUS SEEKING BEHAVIOR IN
CALORIE-RESTRICTED RATS; UNDERLYING NEURAL MECHANISMS
Aleksandr Talishinsky (1), Satoshi Ikemoto (1)
(1) National Institute on Drug Abuse Intramural Research Program, National Institutes of Health
Administration of psychostimulant drugs augments goal-directed responses, especially in calorie-restricted
animals. Our lab has previously demonstrated that chronic calorie-restriction interacts synergistically with D-
amphetamine to potentiate visual stimulus-seeking behavior in rats. Recent work has shown that calorie-
restriction affects the sensitivity and responsiveness of the mesolimbic dopamine pathways. Continuing with
this work, we found that chronic calorie restriction increased visual-stimulus seeking potentiated by the
psychostimulant methylphenidate. To further characterize the neural circuitry underlying the interaction of
calorie-restricted physiological state and psychostimulant drug action, we have begun using DREADD-mediated
neuronal inhibition of brain regions upstream of the mesolimbic dopamine system, including the medial
prefrontal cortex and the basolateral amygdala. Our results suggest that chronic calorie restriction alters
behavioral responsiveness to both D-amphetamine and methylphenidate. Potentiated visual stimulus seeking
behavior in calorie-restricted rats may be dependent upon input from the basolateral amygdala. Future work
will aim to characterize the involvement of specific BLA efferents in potentiating visual stimulus seeking
behavior.

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P59 Tan, Han
GRIP1 IS REQUIRED FOR HOMEOSTATIC REGULATION OF AMPAR TRAFFICKING
Han L. Tan, Bridget N. Queenan, and Richard L. Huganir
Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD
21205
Homeostatic plasticity is a negative feedback mechanism that stabilizes neurons during periods of perturbed
activity. The best-studied form of homeostatic plasticity in the central nervous system is the scaling of excitatory
synapses. Postsynaptic AMPA-type glutamate receptors (AMPARs) can be inserted into synapses to compensate
for neuronal inactivity or removed to compensate for hyperactivity. However, the molecular mechanisms
underlying the homeostatic regulation of AMPARs remain elusive.
In this study, we found that the expression and subcellular distribution of GRIP1, a multi-PDZ (postsynaptic
density 95/discs large/zona occludens) domain AMPAR-binding protein, are regulated during synaptic scaling.
Hyperactivity increases total GRIP1, primarily in the cytoplasmic pool, whereas synaptic GRIP1 is reduced.
Conversely, inactivity decreases GRIP1 expression, reflecting a loss of cytoplasmic GRIP1, and the remaining
GRIP1 protein becomes enriched at synapses. Further, these changes result in altered interaction between
GRIP1 and GluA2 at specific subcellular locations. Further GRIP1 is essential for synapse strengthening during
homeostatic scaling up. GRIP1 knockout neurons have reduced surface AMPARs under basal conditions and are
unable to up-regulate surface AMPARs or synaptic strength during chronic activity suppression.
Our results support the model that GRIP1 regulates AMPAR trafficking by shuttling receptors between the two
distinct pools. Inactivity increases the pool of synaptic GRIP1 and strengthens the association between GRIP1
and synaptic AMPARs, stabilizing surface AMPARs at synapses. Simultaneously, cytoplasmic GRIP1–GluA2
interactions are disrupted to relieve intracellular retention, promoting additional trafficking and accumulation of
AMPARs at the plasma membrane. During elevated activity, GRIP1 is removed from synapses and accumulates
within the cytoplasm, consequently leading to reduced surface AMPARs.
Collectively, our data point to an essential role of GRIP1-mediated AMPAR trafficking during inactivity-induced
synaptic scaling.

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P60 Tang, Aihui

A TRANSSYNAPTIC NANOCOLUMN ORGANIZES SYNAPTIC PROTEIN DISTRIBUTION TO ALIGN
NEUROTRANSMITTER RELEASE WITH RECEPTORS
Ai-Hui Tang, Haiwen Chen and Thomas A. Blanpied
Department of Physiology, Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD
21201

Scaffold proteins provide the foundation of excitatory synaptic transmission by establishing the architecture of
the active zone (AZ) and PSD. Indeed, their deletion or mutation causes severe neuropsychiatric disorders
including autism, mental retardation, and schizophrenia. Previously, we found that major postsynaptic scaffold
proteins including PSD-95 are organized into distinctive nanodomains (NDs) within the PSD and enriched with
both GluA2-containing AMPARs and GluN2B-containing NMDARs. Numerical simulations have predicted that
overall synapse strength is strongly dependent on whether vesicle release points in the AZ are aligned with
these postsynaptic NDs. Strikingly, this central question has not been addressed experimentally, in part because
of the limited resolution of traditional light microscopy.

Here, using 3-dimensional dSTORM imaging, we compared the distribution of PSD-95 over the face of individual
synapses to the corresponding distributions of three key presynaptic AZ proteins involved in vesicle release:
RIM1/2, Munc13 and Bassoon. By developing a 3D cross-correlation algorithm, we found that distributions of
RIM1/2 and PSD-95 were highly correlated to one another. This arose mainly because NDs of the two proteins
were aligned across the cleft. In fact, enrichment of RIM1/2 was predicted by the presence of a PSD-95 ND on
the other side of a synapse, and vice versa. The alignment of PSD-95 with Munc13 was weaker, and Bassoon
intermediate, suggesting a hierarchical distribution of these three proteins. Other PSD scaffold proteins co-
enriched with PSD-95 postsynaptically. Critically, clustered GluA2 receptors were also significantly aligned with
RIM1/2 NDs. This ~80 nm diameter, vertically oriented compact molecular ensemble within the bounds of the
synapse suggests a “transsynaptic nanocolumn” organization.

Following acute NMDA exposure known to trigger a reduction in synaptic strength (LTD?), nanocolumn
organization was dramatically reorganized. Immediately after NMDA application, postsynaptic structure was
severely disrupted. However, the progression of plasticity following the acute stimulus involved a retrograde
and coordinated remodeling of both pre- and post-synaptic elements of nanoscale alignment.

Taken together, these data suggest the nanocolumn architecture may be a critical determinant of synaptic
strength at CNS synapses, particularly at the majority of synapses that have low release probability. This simple
organizational principle suggests an especially sensitive point whereby disease-associated pathways may disrupt
synapse function.

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P61 Tennekoon, Michael
NEURAL CORRELATES OF WORKING MEMORY PREDICT ACADEMIC SKILLS
Michael S Tennekoon (1), Gillian E. Cook (2), and James R. Booth (3)
(1) National Institute for Drug Abuse, Baltimore., (2) University of Illinois, Urbana., (3) University of Texas,
Austin.
Working memory is important for the development of academic skills. Neural correlates of verbal working
memory are implicated in the development of reading skill, while neural correlates of spatial working memory
are implicated in the development of calculation skill. However, the selectivity of working memory neural
correlates to predict current and future academic skill has not been investigated. This is important as it
delineates the role of verbal and spatial working memory in the development of academic skills. Here we
investigated these relationships by examining longitudinal data from seventeen typically developing boys (aged
9-11). Although the neural correlates of spatial working memory were not associated with current academic
skills, activation in the left middle frontal gyrus predicted a change in calculation skill over a 2-year period;
critically this relationship was selective to spatial, but not verbal activation as well as to calculation, but not
reading skill. Results also showed that neural correlates of verbal working memory did not predict change in
academic skills, but prefrontal activation was associated with both current reading and calculation skill. These
findings help elucidate the precise contributions of working memory to the development of academic skills and
should be considered when designing interventions to improve academic skills.

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P62 Tim, Jarvela
THE NEURAL CHAPERONE PROSAAS POTENTLY BLOCKS ALPHA-SYNUCLEIN FIBRILLATION AND NEUROTOXICITY
Timothy S. Jarvela (1), Hua A. Lam (2), Michael Helwig (1), Akina Hoshino (1), Nikolai Lorenzen (3), Daniel E.
Otzen (3), Nigel Maidement (2), and Iris Lindberg (1)
(1) Anatomy and Neurobiology, University of Maryland Baltimore, Baltimore, MD; (2) University of California Los
Angeles, Los Angeles, CA; (3) Department of Molecular Biology and Genetics, Aarhus University, Aarhus,
Denmark
Emerging evidence strongly suggests that chaperone proteins are cytoprotective in neurodegenerative
proteinopathies involving protein aggregation, for example the accumulation of aggregated alpha-synuclein into
the Lewy bodies present in Parkinson’s disease. Of the various chaperones that have been explored in
neurodegenerative disease, the small secretory chaperone known as proSAAS has many attractive properties.
ProSAAS, widely expressed in neurons throughout the brain, is capable of blocking both the aggregation of beta
amyloid into fibrils as well as blocking beta amyloid neurotoxicity. Here we investigate whether addition of
proSAAS can inhibit α-synuclein fibrillation and cytotoxicity. Recombinant proSAAS potently inhibits the
fibrillation of alpha-synuclein in an in vitro assay. In SH-SY5Y cells overexpressing α-synuclein, addition of
purified proSAAS to the culture media rescues synuclein mediated toxicity. Further, proSAAS-encoding lentivirus
blocks alpha-synuclein-induced cytotoxicity in primary cultures of nigral dopaminergic neurons. Lastly, we found
immunoreactive proSAAS to be associated with aggregated α-synuclein proteins in the substantia nigra of
Parkinson’s disease patients. Interestingly, four separate proteomics studies have identified proSAAS as a
potential biomarker in various neurodegenerative diseases, including Parkinson’s disease. Collectively, our
studies support the idea that neuronal proSAAS represents a potential therapeutic target in neurodegenerative
disease.

82

P63 Tran, Tammy
THE ROLE OF APOE-4 IN HIPPOCAMPAL HYPERACTIVITY IN AMNESTIC MILD COGNITIVE IMPAIRMENT
Tammy Tran (1), Caroline Speck (2), Aparna Pisupati (2), Michela Gallagher (1), Arnold Bakker (1,2)
(1) Psychological and Brain Sciences, Johns Hopkins University, (2) Psychiatric Neuorimaging Division, Johns
Hopkins University, School of Medicine
Increased fMRI activation in the hippocampus is recognized as a signature characteristic in the aMCI stage of
Alzheimer’s disease (AD) and a predictor of cognitive decline and progression to AD dementia. Recent studies
have localized this increased activation to the dentate gyrus / CA3 subregion of the hippocampus and showed a
correlation with memory impairments in those patients, as predicted by a rodent model of aging. Increased
hippocampal activation has also been reported in carriers of the ApoE-4 allelic variation independently of mild
cognitive impairment although these findings were not localized to a hippocampal sub region. Animal models of
ApoE-4 predict that this increased activation would be similarly localized to the dentate gyrus / CA3 region. To
assess whether ApoE-4 contributes to increased hippocampal fMRI activation, 62 patients with aMCI genotyped
for ApoE-4 status and 30 healthy age-matched control participants completed a high-resolution fMRI scan while
performing a memory task designed to tax hippocampal sub region specific functions as well a
neuropsychological assessment. Consistent with previous reports, patients with aMCI showed increased
hippocampal activation in the left dentate gyrus / CA3 sub region of the hippocampus as well as memory task
errors attributable to this subregion of the hippocampus. However, this increased fMRI activation in the
hippocampus did not differ between ApoE-4 carriers (N = 25) and ApoE-4 non-carriers (N = 37) and the
proportion of memory errors attributable to dentate gyrus / CA3 function did not differ between ApoE-4 carrier
and ApoE-4 non-carriers. These results indicate that increased fMRI activation of the hippocampus observed in
patients with aMCI is independent of ApoE-4 status and that ApoE-4 does not contribute to the dysfunctional
hippocampal activation or the memory errors attributable to this sub region in these patients.

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P64 Van Dyke, Adam

CO-ACTIVATION OF MULTIPLE SEROTONIN RECEPTORS UNDERLIES SUSTAINED POTENTIATION OF SYNAPTIC
TRANSMISSION BY FLUOXETINE IN THE HIPPOCAMPUS
Adam M. Van Dyke (1), Angy J. Kallarackal (1), Xiang Cai (1), and Scott M. Thompson (1)
(1) Department of Physiology, Program in Neuroscience, University of Maryland, School of Medicine, Baltimore,
MD, USA.

Serotonin (5-HT) and its receptors have long been major targets in the pharmacological treatment of depression.
Despite their widespread use, the mechanism of action underlying the therapeutic efficacy of SSRIs like
fluoxetine remains poorly understood. Our laboratory has shown that raising endogenous 5-HT can potentiate
certain excitatory synapses, including those that are weakened in depression models, and that this potentiation
is necessary for the therapeutic behavioral action of antidepressants. In the hippocampus, activation of Gi/Go-
coupled 5-HT1B receptors potentiates glutamatergic signaling in temporoammonic (TA) to CA1 pyramidal cell
synapses in the stratum lacunosum-moleculare (SLM). This occurs due to the phosphorylation of the AMPA
receptor subunit GluA1 at serine 831 via recruitment of the Phospholipase C/Ca2+/Calmodulin-dependent
Protein Kinase II (PLC/Ca2+/CaMKII) signaling cascade. Selective activation of 5HT1BRs produces a transient
potentiation, whereas elevation of endogenous 5-HT with the SSRI fluoxetine persistent potentiates TA-CA1
synapses in a 5HT1BR-dependent manner. We hypothesized that fluoxetine’s sustained potentiation of
hippocampal TA-CA1 synapses is due to the co-activation of 5-HT1BR and a GS-coupled serotonin receptor
causing activation of PKA signaling. We have addressed this hypothesis by using various pharmacological tools to
disrupt/activate specific receptors and their respective signaling pathways. We have observed that antagonists
of 5-HT1BRs prevents the potentiation of TA-CA1 field potentials by both fluoxetine and anpirtoline. Disruption
of PKA signaling had no effect on 1B-mediated potentiation but inhibition of PKA did prevent the sustained
potentiation by fluoxetine. Antagonists of the various Gs-coupled 5-HTRs revealed that 5-HT6R was underlying
this activation of PKA by fluoxetine, whereas 5-HT4R or 5-HT7R antagonists had no effect. Conversely, co-
activation of 5-HT1BRs and 5-HT6Rs, with their respective agonists, produced a sustained increase in synaptic
transmission. Western blotting is in progress to determine the time course of kinase activation and AMPA
receptor phosphorylation under different conditions. Taken together these data suggests that the SSRI
fluoxetine raises endogenous 5-HT resulting in the co-activation of multiple 5-HTRs to produce an increase in
glutamatergic transmission. 5-HT1BR activation initially induces the increase in synaptic strength through
PLC/Ca2+/CaMKII dependent phosphorylation of AMPA receptors and 5-HT6R activation maintains this increase
through the recruitment of PKA signaling.

84

P65 VanRyzin, Jonathan
ROLE OF MICROGLIA IN SEXUAL DIFFERENTIATION OF THE AMYGDALA
Jonathan W. VanRyzin (1), Kathryn J. Argue (2), and Margaret M. McCarthy (2).
(1) Program in Neuroscience; (2) Departments of Pharmacology and Physiology, University of Maryland,
Baltimore, Baltimore, MD, United States
Microglia are the dominant resident immune cells of the brain and function in multiple ways outside their
traditional capacity of responding to insult. During development microglia regulate tissue homeostasis,
neuronal precursor populations, and synaptic circuitry. We recently implicated microglia as an integral
component of sexual differentiation of the preoptic area and control of male copulatory behavior, suggesting
these immune cells also function to organize sex-specific brain structure and function (Lenz et al. J Neurosci
33(7), 2013). The amygdala is also a sexually dimorphic brain region that regulates social behaviors known to
differ in males and females. We reported a sex difference in the number of newly born cells in the developing
rat medial amygdala (MeA) that is mediated by endocannabinoids, with females having higher numbers of newly
born cells than males and a lower endocannabinoid tone. These differences in newly born cells correlated to
behavioral changes, as newborn females treated with the CB1/2 agonist WIN55,212-2 exhibited masculinized
juvenile social play behavior and reduced cell genesis in the developing amygdala (Krebs-Kraft et al. PNAS
107(47), 2010). Further investigation suggests microglia may be central in establishing the observed sex
difference in newly born cells, as males have more Iba1+ microglia exhibiting phagocytic morphology compared
to females in the medial amygdala during the sensitive period for sexual differentiation. Treatment with
minocycline, a tetracycline derivative and inhibitor of microglial activation, increased male BrdU+ cell counts to
female levels, but did not alter the number of female BrdU+ cells in the MeA. We are now deciphering the
relative contributions of microglia phagocytosis and trophic signaling in the regulation of newly born cells using
methods that selectively deplete microglia or prevent phagocytosis. Ultimately, these studies will provide
valuable insight into new facets of immune regulation of brain sexual differentiation and development.

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