Abstract List for 2017 meeting

#432: Tet1 catalytic domain knocking-out impairs neural differentiation of human embryonic stem cells

Li, Hanqin ; Feng, Jian ;

Department of Physiology and Biophysics, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

Background DNA methylation is one of the major epigenetic mechanism regulating gene expression. Tet family encode 5-methylcytosine (5mC) demethylases, and play critical roles in a variety of developmental, physiological and pathological processes. Tet1 is the highest expressed tet in both mouse and human embryonic stem cells (ESC). So far, functions of tet1 in maintaining pluripotency of mESC are controversial, and the functions of tet1 in human ESCs haven’t been elucidated. Results In this study, we successfully generated tet1 catalytic domain knock-out (tet1-CDKO) hESCs using CRISPR/Cas9 system. Tet1-CDKO hESCs express classical pluripotency markers and are able to differentiate to all three germ layers, even though their 5hmC level decreasing to as low as 20% comparing to wild type hESCs. When differentiating to neuroectoderm in morphogen-free condition, tet1 CDKO expressed much lower level of critical neuroectoderm marker genes, like PAX6, SOX1 and FOXG1, than wild type at embryoid body (EB) stage. And the final percentage of cells committing to neuroectoderm (SOX1+PAX6+Nestin+) were also significantly less than the number observed in wild type. These evidence suggest tet1 catalytic domain knocking-out impair in vitro neural differentiation of hESCs. Our next step is trying to figure out how loss of tet1 catalytic activity causes this developmental defect.

#429: Drebrin regulates opiate-induced behavioral and structural plasticity in the NAc

Martin, Jennifer A ¹; Werner, Craig T ¹; Wang, Zi-Jun ²; Siemian, Justin N ¹; Zhong, Ping ²; Hagarty, Devin ³;

¹Department of Pharmacology and Toxicology, ²Department of Physiology and Biophysics, SUNY University at Buffalo, School of Medicine and Biomedical Sciences; ³Department of Psychology, California State University, Bakersfield;

Opiate addiction, a chronic relapsing disease, places a large societal and financial burden on our country. In particular, the fundamental issue for the therapeutic treatment of opiate addiction is the persistent drug craving, seeking, and high rates of relapse following drug abstinence. These behaviors are characterized by persistent behavioral and cellular plasticity in key regions of the mesolimbic dopamine system such as the nucleus accumbens (NAc), including structure and density of dendritic spines on Medium Spiny Neurons (MSNs). To date the cellular and molecular mechanism governing opiate-induced structural plasticity remains undetermined. Following heroin self-administration we find decreased expression of the essential actin-binding protein drebrin in the NAc, an effect that is transcriptionally regulated through HDAC2 binding on the drebrin promoter. Using viral mediated gene transfer, overexpression of drebrin attenuated, while CRISPR-Cas9 deletion exacerbated, heroin-primed, but not sucrose primed, relapse-like behaviors. In addition, drebrin overexpression produced a downward, while CRISPR-Cas9 produced an upward, vertical shift in a within-session dose-response curve; demonstrating drebrin regulates the reinforcing properties of heroin. Restoration of drebrin levels following heroin self-administration reversed the heroin-induced decreases in spine density and reduction in AMPA and NMDA receptor conductance. Taken together, these data demonstrate an essential role for drebrin in mediating the molecular mechanisms underlying opiate-induced behavioral and structural plasticity. More importantly, understanding these cellular responses will help lead to the therapeutic intervention for the prevention of relapse.

#446: Magnetothermal genetic deep brain stimulation of motor behaviors in awake, freely moving mice

Munshi, Rahul ; Qadri, Shahnaz M ; Pralle, Arnd ;

Department of Physics, SUNY University at Buffalo, College of Arts and Sciences;

Magnetothermal stimulation is a minimally invasive technique to modulate deep brain neurons, with high specificity and repeatability. Here, we show how magnetothermal modulation can be used to evoke motor behaviors in awake mice. We used alternating magnetic fields to heat magnetic nanoparticles attached to the membrane of neurons, heat sensitized by TRPV1 overexpression. Magnetothermal genetic stimulation in the motor cortex evoked ambulation, deep brain stimulation in the striatum caused rotation around the body-axis, and stimulation near the ridge between ventral and dorsal striatum caused freezing-of-gait. The duration of the behavior correlated tightly with field application. Being implant-free and highly precise, the technique serves as a unique toolkit for establishing causality between neuronal activities and ensuing behaviors, and has the potential for therapeutic usage.

#445: Magi1 scaffolds Na_V1.8 channels in Dorsal Root Ganglion neurons regulating excitability and pain

Pryce , Kerri D ;

Department of Pharmacology and Toxicology, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

The voltage-gated sodium channel isoform NaV1.8 (Scn10A) is exclusively expressed in pain-sensing Dorsal Root Ganglion (DRG) neurons. Due to their unique expression in nociceptive neurons and their role in action potential (AP) firing, NaV1.8 channels represent ideal targets for analgesic development. Moreover, during injury, these channels traffic to the membrane and cause the repetitive AP firing associated with nociceptor sensitization. The underlying molecular machinery responsible for the trafficking and membrane expression of NaV1.8 in DRGs however, remains to be fully elucidated. Here, we have identified Magi-1 as a major ion channel scaffold and regulator of sodium signaling in neurons. We describe for the first time that both the membrane and total expression of NaV channels, especially NaV1.8 channels, are largely dependent upon scaffolding by Magi-1. To study the physiological roles of Magi-1 in cultured DRG neurons, we used small interfering RNAs (siRNA) targeted against Magi-1 and observed a dramatic decrease in the ability for DRG neurons to fire action potentials with a concomitant decrease in both tetrodotoxin-sensitive and -resistant sodium currents. To further investigate a putative role of Magi-1 in pain signaling we unilaterally injected plasmid shRNAs targeted against Magi-1 directly into the sciatic nerve of naïve mice. This spinal nerve injection technique is novel and we have modified it for mice, achieving long-lasting Magi-1 knockdown in DRG neurons in vivo. After Magi-1 knockdown, we observed a significant reduction in thermal nociception and inflammatory pain behavior compared to control mice. More importantly, because of the absence of Magi-1, we observed an almost complete loss of NaV1.8 protein, indicating that Magi-1 is an obligate protein partner for this channel. Together, these data suggest that Magi-1 is a critical scaffold for sodium channels in DRG neurons and targeting it could lead to the development of novel analgesic drugs.

#436: Understanding the role of calcineurin in a mouse model of Charcot Marie Tooth 1B neuropathy

Reed, Chelsey B ¹; Sidoli, Mariapaola ²; Feltri, M. Laura ³; Wrabetz, Lawrence ⁴;

¹Neuroscience, ³Department of Biochemistry, ⁴Department of Neurology, SUNY University at Buffalo, School of Medicine and Biomedical Sciences; ²Department of Developmental Biology, Stanford University;

Charcot Marie Tooth disease (CMT) is the most common inherited neuromuscular disorder. Mutations in Myelin Protein Zero (MPZ, P0) cause the dominant CMT1B demyelination neuropathy. In the transgenic CMT1B-S63del mouse model, the folding of P0 is disrupted and causes a toxic gain of funtion in Schwann cells. The accumulation of P0 in the endoplasmic reticulum (ER) leads to an unfolded protein response (UPR) and demyelination. The UPR sensor, PERK kinase, usually acts to relieve ER stress by phosphorylating eIF2alpha and attenuating protein translation. Surprisingly, Schwann cell-specific ablation of Perk in S63del mice, instead improves myeliantion, indicating a possible pathogenic role for PERK. Calcineurin, an important promyelinating signal, has recently been identified as a novel PERK substrate in other cells types. Here we present evidence for a physical interaction between PERK and calcineurin and increased calcineurin phosphatase activity in S63del nerves. Moreover, ablation of calcineurin b specifically in Schwann cells produces hypomyelination, which is synergystic with the hypomyelination of S63del nerves. These data suggest that calcineurin plays a role in the pathogenesis of CMT1B-S63del mice through an interaction with PERK.

#428: Excessive central auditory gain enhancement and disrupted loudness perception following hearing loss

Auerbach, Benjamin D ; Radziwon, Kelly ; Chen, Guang-Di ; Salvi, Richard ;

Center for Hearing and Deafness, SUNY University at Buffalo, College of Arts and Sciences;

The central auditory system displays a remarkable ability to adapt its response properties to changes in sound level. This includes compensatory increases in neuronal gain that can partially restore sound detection following long-term hearing loss. A price of this plasticity, however, is the potential for maladaptive sound encoding in response to abrupt or extreme changes in auditory input. To explore this issue, we examined the relationship between central auditory gain changes and behavioral measures of loudness following acoustic trauma. Simultaneous recordings from multiple levels of the central auditory system demonstrated that noise-induced gain changes decoupled cortical intensity coding from subcortical auditory responses, resulting in excessive sound-evoked activity in the auditory cortex. In chronic recordings from behaviorally-trained animals, we found this cortical hyperactivity to be strikingly correlated with changes in loudness perception. These results indicate that loss of afferent drive to the auditory cortex leads to maladaptive gain increases that directly affect sound perception, highlighting the role of central auditory plasticity in the perceptual sequela of hearing loss. This is particularly relevant to auditory perceptual disorders associated with hearing loss like hyperacusis, a debilitating disorder where everyday sounds are perceived as intolerably loud or painful.

#456: The loop region of Presenilin is essential for Glycogen synthase kinase-3β mediated axonal transport

Banerjee, Rupkatha ; Rudloff, Zoe ; Naylor, Crystal ; Gunawardena, Shermali ;

Department of Biological Sciences, SUNY University at Buffalo;

Neurons require intracellular transport of essential components for function and viability. Defects in axonal transport have been implicated in many neurodegenerative diseases. Although multiple levels of regulation of motor protein function must exist for proper transport of components within axons, little is known about these mechanisms. One possible mechanism by which transport defects can occur is by improper regulation of molecular motors. Previous work has shown that reduction of Presenilin (PS) or Glycogen synthase kinase-3β (GSK-3β) stimulated APP vesicle motility. Excess GSK-3β causes axonal transport defects and increased motor binding to membranes, while reduction of PS decreased active GSK-3β and motor binding to membranes, suggesting that PS and GSK-3β may function together during axonal transport. Since PS and GSK3β are known to interact in the β-Catenin pathway, we hypothesize that PS influences GSK3β activity for motor regulation. Using Drosophila genetics, we found that excess PS rescued GSK-3β mediated axonal blockages. Intriguingly, the catalytic region of PS, (PS loop), which is known to bind to GSK3β and β-Catenin, is essential for this rescue. Disruption of PS loop (PSΔE9) exacerbated GSK3β-mediated axonal blocks, while excess of PS loop suppressed it. Together, our observations suggest that functional PS with an intact PS loop region is required to modulate GSK-3β-mediated roles during axonal transport. Perhaps, PS and GSK-3β physically interact to regulate motor activity during axonal transport similar to GSK-3β-mediated mechanisms in the β-Catenin pathway<

#449: Patterns of correlated activity driven by structural connectivity in human brain networks

Bansal, Kanika ¹; Lieberman, Gregory ²; Verstynen, Timothy D ³; Vettel, Jean M ²; Muldoon, Sarah F ¹;

¹Department of Mathematics, SUNY University at Buffalo; ²Human Research and Engineering, Army Research Lab, APG, MD; ³Department of Psychology, Carnegie Mellon University, Pittsburgh, PA;

Dynamic patterns of coordinated activity in human brain networks capture and predict human behavior. Fundamentally, this activity originates as interacting neural populations form domains of synchronization, a state referred to as ‘chimera’ in a complex systems framework. The appearance of chimera states is due to an interplay between the characteristic dynamics of the interacting network elements, topology, and coupling functions. Here, we analyze the emergence of chimera states using brain data across a cohort of thirty individuals. Using diffusion weighted imaging, anatomical white matter connectivity matrices are derived for each individual and used in a data-driven computational model of individual brain dynamics. We analyze spatial structures of emergent chimera and other dynamical states across functionally defined cognitive brain sub-networks. We observe that the organization of the human brain is divided into groups that show drastically different levels of variability across subjects. Sub-networks defining the default mode and subcortical systems show patterns of chimeras that are similar across subjects, which could indicate a limited role of these regions in driving individual variability. On the other hand, sub-networks defining the auditory, cingulo-opercular and frontoparietal systems show a high level of variability, suggesting that these systems drive variability across individuals.

#472: Heterogeneous dopamine regulation in the nucleus accumbens induced by methamphetamine

Bhimani, Rohan V ¹; Park, Jinwoo ²;

¹Neuroscience, ²Biotechnical and Clinical Laboratory Sciences, SUNY University at Buffalo;

Psychostimulants such as methamphetamine (METH) increase extracellular concentrations of dopamine (DA) in the nucleus accumbens shell (NAc), a brain structure critically involved in the reinforcing and behavioral effects of psychostimulants. It is known that at high doses (≥ 5.0 mg/kg), METH exerts its effects on the DA system by displacing vesicular stores, inhibiting DA transporters and inducing non-vesicular release through reverse transport. However, at lower doses (<5.0 mg/kg), the mechanisms by which METH modulates extracellular DA regulation (release and clearance) are not clear. In this study, we used fast-scan cyclic voltammetry (FSCV) to monitor rapid, sub-second changes of DA in discrete brain regions to determine how acute exposure to increasing doses of METH (0.5, 2.0 and 5.0 mg/kg) modulates NAc-DA regulation in anesthetized and behaving rats. We found that METH dose-dependently increased the frequency and concentration of naturally occurring DA transients (phasic release) in behaving rats. However, at high doses, increases in basal DA levels were also observed with these transients. Rats exhibited greater hyperactivity at doses ≤ 2.0 mg/kg whereas at higher doses, locomotion decreased and rats engaged in repetitive, in-place stereotypies. Using an anesthetized rat preparation, we characterized how METH dose-dependently modulated DA regulation free from environmental confounds and changes in affective state. Uptake continued to decrease with increasing doses while electrically evoked release reached a plateau at doses (≥ 2.0 mg/kg). Lastly, we show that METH dose-dependently activates DA D2-autoreceptors while at higher doses of METH, extracellular DA concentrations increase in a D2-autoreceptor independent manner. These results demonstrate that METH augments DA release via different mechanisms depending on the concentration administered.  These findings will set the stage for future research on how METH repeated exposure disrupts DA regulation.

#457: Brain and Body Temperature Effects of Heroin: State Dependency and Environmental Modulation

Bola, R. Aaron ¹; Kiyatkin, MD, PhD, Eugene A ²;

¹Jacobs School of Medicine and Biomedical Sciences, SUNY University at Buffalo; ²Behavioral Neuroscience Research Branch, National Institute on Drug Abuse;

We examined how intravenous heroin at a dose that maintains self-administration (0.1 mg/kg) affects brain temperature homeostasis in freely moving rats under conditions that seek to mimic some aspects of human drug use. When administered under standard laboratory conditions (quiet rest at 22 °C ambient temperature), heroin induced moderate temperature increases (1.0-1.5 °C) in the nucleus accumbens (NAc), a critical structure of the brain motivation-reinforcement circuit. By simultaneously recording temperatures in the temporal muscle and skin, we demonstrate that the hyperthermic effects of heroin results primarily from inhibition of heat loss due to strong and prolonged skin vasoconstriction. Heroin-induced brain temperature increases were enhanced during behavioral activation (i.e., social interaction) and in a moderately warm environment (29 °C). By calculating the "net" effects of the drug in these two conditions, we found that this enhancement results from the summation of the hyperthermic effects of heroin with similar effects induced by either social interaction or a warmer environment. When the dose of heroin was increased (to 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/kg), brain temperature showed a biphasic down-up response. The initial temperature decrease was dose-dependent and resulted from a transient inhibition of intra-brain heat production coupled with increased heat loss via skin surfaces-the effects typically induced by general anesthetics. These initial inhibitory effects induced by large-dose heroin injections could be related to profound CNS depression-the most serious health complications typical of heroin overdose in humans.