Abstract List for 2014 meeting

#249: Methylphenidate Exerts Dose-Dependent Effects on Glutamate Receptors and Behaviors

Cheng, Jia ; Xiong, Zhe ; Duffney, Lara J ; Wei, Jing ; Liu, Aiyi ; Yan, Zhen ;

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

Methylphenidate (MPH) is a psychostimulant drug approved for the treatment of attention-deficit hyperactivity disorder (ADHD). While MPH produces the effects of increasing alertness and improving attention at therapeutic dose, its misuse has been associated with an increased risk of aggression and psychosis. In this study, we sought to determine the molecular mechanism underlying the complex actions of MPH at different doses. We firstly found that a single administration of low-dose MPH (0.5 mg/kg) facilitated the temporal order recognition memory (TORM) and attentional-set shifting without remarkably change in locomotor activity in adolescent (4-week-old) rats. In contrast, animals injected with high-dose (10mg/kg) MPH exhibited significantly impaired cognitive functions and elevated locomotor activity. At neuronal level, we found that low-dose MPH selectively potentiated NMDAR-mediated excitatory synaptic currents (EPSC), while high-dose MPH suppressed both NMDAR- and AMPAR-EPSC in the prefrontal cortex (PFC) of adolescent rats. The dual effects of MPH on EPSCs were associated with bi-directional changes in the surface level of glutamate receptor subunits. Moreover, we found that the enhancing effect of low-dose MPH on NMDAR-EPSC was attributed to norepinephrine reuptake inhibition and adrenergic receptor activation. Inhibiting the function of SNAP-25, a key SNARE proteins involved in the exocytosis of NMDARs, blocked the increase of NMDAR-EPSC by low-dose MPH. In animals exposed to repeated stress, administration of low-dose MPH effectively restored NMDAR function and recognition memory via a mechanism dependent on SNAP-25.Taken together, our results have provided a potential mechanism underlying the cognitive enhancing effects of low-dose MPH, as well as the psychosis inducing effects of high-dose MPH.

#253: Protons Potentiate GluN1/GluN3A Glycinergic NMDA Receptor Currents

Cummings, Kirstie A ; Popescu, Gabriela K ;

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

Glycinergic N-methyl-D-aspartate receptors (NMDARs) are tetramers of GluN1 and GluN3 subunits that are distinct from traditional NMDARs in that that they are activated by glycine alone and are insensitive to glutamate. While very little is known about their function, it is hypothesized that GluN1/GluN3A receptors are tonically activated by basal levels of glycine in the brain and therefore serve to keep cells in a more depolarized/excitable state. GluN3A is normally present during development where it controls maturation of dendritic spines however its aberrant expression into adulthood has been shown in schizophrenia, Huntington’s disease, and cocaine addiction. More recently, it was demonstrated that following ischemia, GluN1/GluN3A receptors are selectively upregulated while ‘classical’ GluN1/GluN2A and GluN1/GluN2B NMDA receptors are downregulated. Here, we investigated the effects of protons on GluN1/GluN3A receptor currents. In stark contrast to GluN1/GluN2 receptors, we discovered that protons profoundly potentiated GluN1/GluN3A receptor currents and significantly decreased the population of desensitized receptors. We also observed that protons modulated pharmacological properties of these receptors by decreasing sensitivities to both glycine and zinc. Importantly, we found that when protons were applied to steady-state currents, which mimick tonic GluN1/GluN3A physiological activity, a large influx of current occurred which significantly depolarized the membrane. Taken together, these results indicate that both physiological and pathological fluxes in proton concentration potently modulate GluN1/GluN3A receptor currents and that protons may serve as an atypical neurotransmitter for GluN1/GluN3A receptors in vivo.

#250: Activin/Smad3 induction in the nucleus accumbens mediates cocaine relapse

Gancarz-Kausch, Amy M ¹; Schroeder, Gabrielle L ¹; Adank, Danielle N ¹; Humby, Monica S ¹; Thorn, David ¹; Li, Jun-Xu ²; Dietz, David M ¹;

¹Department of Pharmacology and Toxicology, SUNY University at Buffalo; ²Department of Pharmacology and Toxicology, SUNY University at Buffalo, Buffalo, NY;

The addicted phenotype is characterized by a chronic, relapsing disorder that persists despite long periods of abstinence, suggesting that the underlying molecular changes are stable and enduring. Many of the long-term effects of cocaine have been shown to be dependent on alterations in gene expression that lead to prolonged adaptations, such as structural changes of medium spiny neurons in the reward circuitry of the brain. We have previously shown that withdrawal from cocaine self-administration (SA) activates TGF-Beta signaling in the nucleus accumbens (NAc). Here, we investigate Activin receptor-mediated signaling via downstream Smad3 protein following withdrawal from cocaine SA. The Activin type II receptor was increased in the NAc at both the mRNA and protein levels following 7 days of withdrawal from cocaine SA. Direct pharmacologic antagonism of the Activin receptor in the NAc resulted in decreased SA and attenuated drug-induced reinstatement behaviors without effecting locomotor activity or food-maintained responding. Pharmacologic activation of the Activin receptor via microinjections of Activin A into the NAc potentiated cocaine-primed reinstatement, without effecting locomotor activity. Withdrawal from cocaine SA also increased the expression of phosphorylated-Smad3, the downstream intracellular mediator of Activin signaling. Using viral-mediated gene transfer, we found that overexpression of Smad3 in the NAc potentiated cocaine-primed reinstatement. Importantly, blockade of Smad3 signaling via overexpression of a dominant negative Smad3 attenuated cocaine SA. Taken together, these data indicate that Activin/Smad3 signaling is induced following withdrawal from cocaine SA and such regulation may be a key molecular mechanism underlying behavioral plasticity. Our ongoing studies examine how these molecular pathways regulate downstream transcriptional events and structural plasticity following withdrawal from SA.

#258: WT1 regulates the development of the posterior taste field

Gao, Yankun ; Toska, Eneda ; Denmon, Dane ; Roberts, Stefan G E; Medler, Kathryn F ;

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

Despite the importance of taste in determining nutrient intake, our understanding of the processes that control the development of the peripheral taste system is lacking. Several early regulators of taste development have been identified including Sonic-Hedgehog (Shh), Bone Morphogenetic Protein 4 (BMP4) and multiple members of the Wnt/beta-catenin signaling pathway. However, the regulation of these factors, including their induction, is still poorly understood. In this study, we identify a critical role for the Wilms’ tumor 1 protein (WT1) in circumvallate papillae (CV) development. WT1 is a transcription factor which is important in the normal development of multiple tissues, including both the olfactory and visual systems. In mice, WT1 expression is detectable by day E12.5 when the circumvallate taste placode begins to visually form. In mice lacking WT1, CV fails to develop normally and markers of early taste development are dysregulated compared to wild type. We demonstrate that expression of the WT1 target genes Lef1, Ptch1 and BMP4 are significantly reduced in developing tongue tissue derived from WT1 knock out (KO) mice and in normal tongues, WT1 is bound to the promoter regions of these genes. Our data identify WT1 as a critical transcription factor in the development of the CV through the regulation of multiple signaling pathways that have established roles in the formation and patterning of taste placodes.

#272: Defining the Relationship between Diet-Induced Obesity and Peripheral Taste Responses

Ahart, Zachary C ; Medler, Kathryn F ;

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

As rates of obesity continue to rise, many health-related complications associated with obesity are also increasing. While the causes of obesity are complex, it is primarily due to over-consumption coupled with a sedentary lifestyle. Dietary consumption is regulated by appetite which is in turn controlled by multiple systems, including the taste system. However, our understanding of the relationship between taste and obesity is still quite poor. Multiple studies have shown that taste perception in the brain is altered in obese animals and humans, and we recently reported that responses in the taste receptor cells are altered in diet-induced obesity (DIO) mice (Maliphol et al., 2013). This is important because taste receptor cells are the initiation site for the detection and perception of taste stimuli. In this earlier study, we used C57Bl/6 mice which readily become obese when placed on a high fat diet. We measured how taste-evoked calcium signals were affected in the obese mice and found that significantly fewer taste cells were responsive to some taste stimuli. In the current study, we are asking if the high fat diet or the weight gain is responsible for the impairment of the peripheral taste cells. Our results suggest that both diet and weight can impact the taste system. Using calcium imaging on isolated taste receptor cells, we found a decrease in the number of responsive cells due to diet alone for certain stimuli while an increase in weight was required for other stimuli. Behavioral experiments revealed a decrease in preference for certain tastants as a result of diet while other stimuli were affected by weight increases. Taken together, this data supports that diet alone can affect the peripheral taste system but that weight gains may be responsible for some effects on this system.

#261: Interaction of NRG1-III & Laminin 211 Signaling Within Schwann Cells

Ameroso, Dominique ¹; Ghidinelli, Monica ²; Hurley, Edward ²; Nave, Klaus-Armin ³; Wrabetz, Lawrence ²; Feltri, Laura ²;

¹Neuroscience Program, ²Department of Biochemistry, SUNY University at Buffalo, School of Medicine and Biomedical Sciences; ³Department of Neurogenetics, Max Planck Institute for Experimental Medicine;

Proper ensheathment and myelination of axons within the Peripheral Nervous System require Schwann Cell (SC) interaction with both axons and the extracellular matrix (ECM). SCs rely on these signals in order to recognize and ensheath axons of a large enough caliber, a process called radial sorting. Once sorted, SCs use these signals to form myelin wraps of appropriate thickness around the axons. This peripheral nerve myelination serves to increase action potential propagation speed, while also providing support to axons. Improper myelin formation and function lead to a variety of detrimental diseases. The neuronal protein crucial for these SC processes is neuregulin 1 type III (NRG1-III), while the crucial ECM components are laminins. Previous findings with genetically modified mice have suggested that laminins and NRG1-III interact to regulate both radial sorting and myelination. Specifically, it appears that laminin 211 (Lm211) works to inhibit NRG1-III pathways during radial sorting. After radial sorting, Lm211 appears to activate parallel pathways that act as both positive and negative regulators of the NRG1-III pathway to affect myelination. The current study focuses on investigation of the mechanism underlying NRG1-III and Lm211 interaction in both radial sorting and myelination. Current findings suggest that in radial sorting Lm211 limits activation of cAMP and PKA to work as a break pedal on the NRG1-III pathway, thus allowing radial sorting to occur properly prior to the onset of myelination. It appears that Lm211’s both positive and negative effects on myelination are due to differential activation of laminin receptors. Investigation is currently focused on integrin a6b4 as the potential laminin receptor expressed on SC’s which Lm211 uses to limit myelination via inhibition of the NRG1-III pathway.

#281: Abnormal α-synuclein disrupts Rab3 movement in Drosophila larval axons

Anderson, Eric ; Gunawardena, Shermali ;

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

α-Synuclein (α-syn), a predominantly presynaptic protein of unknown function, is found in lewy bodies in Parkinson’s disease (PD). Evidence suggests that α-syn is involved in activity-dependent plasticity and membrane dynamics at synaptic terminals. Rab3 is a presynaptic GTPase that is involved in synaptic vesicle exocytosis and trafficking. Work has shown that abnormal α-syn is associated with Rab3 in PD patients and in mouse models of PD. Further, α-syn and Rab3 can exist on the same vesicle at the synapse. However, it is unknown whether α-syn and Rab3 are associated during axonal transport and whether abnormal α-syn disrupts the motility of Rab3 vesicles. To test this hypothesis we imaged Rab3 motility live in Drosophila larval axons. We found that Rab3-YFP moves bi-directionally, and that reduction in kinesin and dynein motors impairs the motility of Rab3, indicating that these motors are required for the movement of Rab3 within axons. Strikingly, expression of normal or diseased forms of α-syn (familial Parkinson’s disease (FPD) mutations, A30P and A53T) dramatically perturbs the motility of Rab3. This disruption was specific to Rab3 as excess of α-syn had no effect on the motility of Rab5 or a membrane bound red fluorescence protein (mCD8-mRFP). Furthermore, Rab3 and α-syn were observed in axonal blocks. Taken together these results suggest that Rab3 and α-syn interact during axonal transport and maybe part of the same vesicular complex. Perhaps disruption of Rab3 motility mediated by abnormal α-syn could contribute to neuropathology observed in PD.

#295: The role of GLP-1 in motivation to emit an appetitive response for a highly palatable food reward

Balba, Nadir M ¹; Montagne, Laurelle ¹; Shatz, Kelcie C ¹; Strand, Lisa M ¹; King, Chris P ¹; McKay, Naomi J ¹; Thompson, Alexis C ²; Daniels, Derek ¹;

¹Department of Psychology, ²Research Institute on Addictions, SUNY University at Buffalo;

Activation of GLP-1 receptors (GLP-1R) induces significant decreases in feeding and drinking behavior, as well as alcohol and cocaine self-administration. The latter observations may suggest that GLP-1R agonists could serve as a treatment for substance use disorders. Using an operant conditioning paradigm in a runway maze with Sprague Dawley rats and dilute sweetened condensed milk (SCM) as a reinforcer, we tested the effects of GLP-1R activation on a non-homeostatically regulated feeding behavior. The goals were to model self-administration behaviors and evaluate the effect of a GLP-1R agonist on motivation and reinforcer strength. After training and extinction, rats were re-exposed to 5 consecutive trials in which SCM was again available and the effect of GLP-1R activation, tested by injecting the GLP-1R agonist exendin-4 (EX4, 2.4 µg/kg, or saline, IP), was measured before the 1^st and 5^th re-exposure trial. The change in run time on trials 1 and 5 tested the effect of EX4 in two different motivational states (extinction test [trial 1]- low motivation, after re-training [trial 5]- high motivation). The run time in trial 2, the day after the first re-exposure to SCM, measured the reinforcing strength of SCM on trial 1. EX4 significantly increased run time during trial 1, but not during trial 5. Ex4 did not affect the reinforcing value of SCM. To test if tolerance could explain the change in EX4 effect between trials 1 and 5, another group of rats treated with saline on trial 1 and EX4, or saline, on trial 5. Again EX4 had no effect on run time in trial 5, indicating tolerance did not explain the change and supporting a conclusion that the effects of EX4 varies by motivational state. A final study was conducted that verified that EX4 decreases ad lib intake of SCM as previously published. These results suggest that GLP-1R activation does not modify the reinforcing value of a highly palatable reward but does reduce appetitive motivation under some circumstances.

#298: Transcriptomic analysis of Neuregulin-1 modulation in normal or neuropathic myelination

Belin, Sophie ;

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

NRG1 type III is a key protein in peripheral nervous system development. Schwann cell survival and the ensheathment fate of axons depends on the amount of NRG1 type III present on axons. NRG1 type III is known to regulate peripheral myelination through the transcriptional regulation of myelin genes (MPZ, PMP22, MAG) by Krox20. Also myelin genes require precise transcriptional regulation or pathologic myelin development occurs. We engineered a mouse line of Congenital Hypomyelination Neuropathy (CHN) due to a non-sense mutation in Myelin Protein Zero (MPZ), called P0Q215X. The mouse phenotype reproduces the human pathology, developing hypomyelination in the peripheral nervous system. We hypothesized that Nrg1t3 modulation could be a therapeutic strategy for the CHN in Q215X mice. Therefore, we crossed Q215X/+ mice with transgenic mice that overexpress Nrg1t3 by 2-3 fold. We conducted morphological and RNA deep sequencing analyses to identify the downstream molecular mechanisms depending on NRG1 type III in a normal and pathologic context. Surprisingly, while myelin thickness is restored in Nrg1t3;Q215X mice, the global transcription is unchanged. Using a transcriptional correlation analysis approach with available published data on the known NRG1 type III signal pathways, we observed strong evidence of activation of MAPK/ERK target genes, which was further confirmed at the biochemical level. Instead, Krox20 target genes were not found to be correlated, corroborating the increase in myelin thickness independent from the canonical myelin gene regulation. We hypothesized that NRG1 type III overexpression triggers an unbalanced activation of its downstream pathways, where MAPK/ERK might induce myelination in either normal or pathological contexts. Further transcriptomic analyses are necessary to identify MAPK/ERK target genes that promote myelination after NRG1 type III gain of function.

#276: Altered GABA profile in mouse CNS due to neuroinflammation initiated by Toxoplasma gondii infection

Brooks, Justin M ¹; Fox, Michael A ²; Blader, Ira J ¹;

¹Department of Microbiology and Immunology, SUNY University at Buffalo, School of Medicine and Biomedical Sciences; ²Department of Biological Sciences, VTCRI;

Understanding the role of immunology in the central nervous system (CNS) is an important emerging field.  Various neuropathologies have recently been linked to the upregulation of immunological regulators within the brain that demonstrate an ability to modulate “normal” CNS function. To better understand the effects of neuroinflammation on neuronal operation, our lab aims to understand the consequences of Toxoplasma gondii (T. gondii) infections on cellular function in immune privileged tissues. T. gondii infiltration in the CNS changes fear response in rodents and causes neurological deficits in immunocompromised humans. We employed immunohistochemical (IHC) staining on cortical and thalamic tissues in T. gondii infected mice, and we discovered a disruption in the intensity and localization of GAD67, an enzyme responsible for synthesis of the inhibitory GABA neurotransmitter. These observations are important because perturbation of the inhibitory signaling in the CNS potentially leads to the development of epilepsy, mood disorders, and schizophrenia. Interestingly, we have also observed that excitatory glutamatergic neuron terminals in cortical and thalamic areas appear “normal” in infected mice, which leads us to believe that the presence of T. gondii elicits a selective neuropathic effect in inhibitory interneuron populations. Further IHC revealed concurrent upregulation in the appearance of reactive glial cells throughout the brain, including astrocytes and microglia, in response to CNS infection. Previous studies have focused on T. gondii mediated effects on excitatory dopaminergic signaling; however, we hypothesize a new mechanism by which T. gondii infection and/or induced inflammatory responses selectively alter inhibitory GABAergic signaling in the brain, causing aberrant excitatory (i.e. dopaminergic) signaling.