Abstract List for 2011 meeting

#123: Transport of Parkinson’s Disease Proteins

Anderson, Eric ; Gunawardena, Shermali ;

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

Parkinson’s disease (PD) is a common neurodegenerative disease that is characterized by loss of dopaminergic (DA) neurons in the substantia nigra par compacta and lewy body formation. Thus far, more than eight genes with different functions have been implicated in PD. However, little is known about how defects in these genes contribute to neuronal death or lewy body formation. Using drosophila as our model system, we tested whether proteins implicated in PD (α-synucelin, PINK1, and DJ1a) can cause axonal transport defects. We found that expression of α-syn induces axonal blockages in larval neurons and Familial PD mutations in α-syn (Ala30Pro and Ala53Thr) increases blockages. To further investigate how these proteins may affect transport, we initiated a genetic interaction analysis with kinesin (KHC) and dynein (Rob1k) motor proteins. We find that reduction of kinesin and dynein enhances blockages in α-syn expressing larval neurons. Preliminary biochemical analysis suggests that α-syn is membrane associated. Further, although expression of PINK1 and DJ1a does not induce blockages, expression of these with reduction of kinesin and dynein causes blockages indicating a possible role in axonal transport. Together, our results show that three proteins involved in PD have some role in axonal transport. Thus, disruption of the axonal transport pathway could contribute to early neuropathology observed in Parkinson’s disease.

#137: The Urotensin II Receptor is Expressed Presynaptically by Cholinergic Mesopontine Neurons

Clark, Stewart D ;

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

In the last decade there has been the discovery of a number of neuropeptides which have greatly increased our understanding of brain function. We have previously shown that the mRNA of the receptor for the novel neuropeptide urotensin II (UII) is expressed by mesopontine cholinergic neurons (LDTg and PPTg). It was also found that areas known to be axonal projection sites of these mesopontine tegmental neurons, which had no detectable UII receptor (UIIR) mRNA, express the UIIR protein (in situ binding of [¹²⁵I]-UII; lateral septal nucleus (LSD), dorsal, medial habenular nucleus (MHb), ventral tegmental area (VTA)). Subsequently, we have shown that UII microinjection into the VTA produces a dose-dependent, sustained dopamine efflux in the nucleus accumbens (NAc). Both drugs of abuse and 'rewarding' stimuli also increase extracellular levels of dopamine in the NAc. Therefore, it is thought that UII may have a role in regulating goal-oriented behaviors. Our working hypothesis is that UII-R is expressed presynaptically by cholinergic mesopontine neurons, and that UIIR activation facilitates release of neurotransmitter(s) from these terminals that subsequently excite VTA dopaminergic neurons.
To demonstrate that UIIR expressed in the VTA, and other areas, are due to presynaptic expression by the LDTg and PPTg neurons, we have used a combination of [¹²⁵I]-UII in situ binding and a novel fusion toxin (UII-diphtheria). Injection of the toxin into the mesopontine selectively kills the UIIR expressing neurons. Bilateral stereotaxic injections into both the LDTg and PPTg were performed. Removal of the UIIR expressing neurons of the mesopontine abolished [¹²⁵I]-UII binding in the VTA suggesting that the UIIR there are presynaptic. However, [¹²⁵I]-UII binding in the LSD and MHb remained intact, and so, UIIR expressed in these areas are not due to mesopontine innervation.
Funding: NIDA R00DA024754

#136: The Essential Role of the Small GTPase Rac1 in Cocaine-Induced Plasticity

Dietz, David ;

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

Addiction is marked by long-lasting changes in behavior that persist despite prolonged abstinence. Increasing evidence suggests that morphological changes in neurons that comprise the brain's reward circuitry contribute to these lasting behavioral abnormalities. For example, repeated administration of psychostimulants induces a persistent increase in dendritic spine density on medium spiny neurons of the nucleus accumbens (NAc), a key brain reward region. However, the molecular mechanisms mediating these changes are poorly understood. While there have been several reports directly linking transcriptional mechanisms to cocaine-induced NAc dendritic plasticity, the events more proximal to spine growth and actin remodeling remain unknown. The data presented here examines how repeated cocaine exposure primes NAc neurons for transient drug-induced downregulation of the small GTPase, Rac1, and that such repression of Rac1 activity, via signaling to the actin filament-severing protein cofilin, is responsible for the expansion of dendritic spines on NAc neurons and for enhanced cocaine reward.

#139: Cell-cell and cell-matrix interactions during Schwann cell development

Feltri, Laura ;

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

Myelin is a multilamellar extension of glial cells that enwrap axons. The interaction between glia and neurons and the cytoarchitecture of myelin are among the most fascinating and spectacular examples of cell-cell communication and cell polarization. Myelin is required for proper neuronal development and function including rapid conduction of nerve impulses and neuroprotection. Several neurological diseases are due to failure to synthesize myelin or myelin breakdown. In my talk I will present cell biological studies that address how adhesion, cytoskeleton and cell signaling molecules that are involved in human diseases effect glial cells development in the peripheral nervous system.

#126: The MT₁ and MT₂ Melatonin Receptors as Essential Mediators of Methamphetamine-Induced Sensitization

Hutchinson, Anthony J ¹; Stepien, Iwona ¹; Hudson, Randall L ²; Dubocovich, Margarita L ¹;

¹Department of Pharmacology and Toxicology, ²Department of Physiology and Biophysics, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

Studies showing dosing-time dependence of psychostimulant-induced responses suggest that the circadian timekeeping system may regulate these responses (Manev et al., Int Rev Neurobiology 2009;88:25-41). Diurnal variations in locomotor sensitization have been reported and may require endogenous melatonin, as melatonin-proficient C3H/HeJ mice undergo a larger magnitude of sensitization during the day compared to night whereas melatonin-deficient C57BL/6J mice exhibit no variation (Uz et al. Life Sci 2002;70:3069-75). The goal of this study was to determine the roles of the MT₁ and MT₂ melatonin receptors in dosing time-dependent effects of sensitization. C3H/HeN mice lacking the MT₁ (MT₁KO), MT₂ (MT₂KO) or both receptors (MT₁/MT₂KO) were given 6 daily pretreatments of methamphetamine (MTA; 1.2 mg/kg, i.p.) or vehicle (VEH). Following 4 days of withdrawal, sensitization was assessed by measuring the increase in distance traveled by MTA-pretreated mice compared to VEH-pretreated controls for 1h following MTA challenge (1.2 mg/kg, i.p.). At ZT 5-7 (lights on at ZT 0, 12h:12h light-dark cycle), locomotor sensitization was observed in wild-type (WT; 71.4±15.1m, n=8), MT₁KO (61.9±12.3m, n=16), and MT₂KO (73.26±12.1m, n=9); but sensitization was absent in MT₁/MT₂KO mice (15.2±11.9m, n=12, p<0.05) compared to the other genotypes. WT mice were tested at ZT 19-21 and sensitization was not observed (0.4±15.4m, n=11). This loss of sensitization at night was due to increased activity expressed by the VEH-pretreatment group (132.3±8.9m, n=11), which was greater than the corresponding value at ZT 5-7 (74.9±16.8m, n=8, p<0.05) and may indicate a form of sensitization induced by the novelty of the experimental procedure. Our findings indicate that melatonin acts through the MT₁ and MT₂ receptors to potentiate MTA-induced sensitization. The MT₁ and MT₂ melatonin receptors appear to be targets for the development of treatments for MTA addiction.
Support by DA021870 to MLD.

#106: Characterizing kinetics of calcium influx through the NMDA receptor

Maki, Bruce A ¹; Aman, Teresa K ²; Popescu, Gabriela K ²;

¹Center for Neuroscience, ²Department of Biochemistry, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

NMDA receptors (NMDARs) are a unique class of ionotropic glutamate receptors in that they are selectively permeable to Ca²⁺, which accounts for ~10% of the channel’s total charge. This Ca²⁺ influx is requisite for neurophysiological processes such as synaptic plasticity and excitotoxicity. To understand mechanisms by which NMDAR-mediated Ca²⁺ influx can be modulated, we investigated the kinetic mechanism by which Ca²⁺ permeates single NMDARs. In order to characterize the stationary gating kinetics of NMDARs Ca²⁺ flux, we performed cell-attached single channel recordings of wild type receptors containing GluN1-1a and GluN2A subunits in the presence of high levels of Ca²⁺ (75 mM) and no other permeating ion. Under these conditions, two distinct conductance levels can be resolved: a main level (1.9 ± 0.04 pA) and a sub level (0.94 ± 0.03 pA), having channel occupancies of 0.35 ± 0.06 and 0.05 ± 0.01, respectively. The decreased occupancy in the sub-level can be accounted for by decreased entry into the sub-level state (both from closed and main-level states), as well as a significantly lower mean open time (MOT) compared to the main level (main-level: 4.9 ± 0.6 ms; sub-level: 3.0 ± 0.5 ms). This kinetic model proposes the possibility that two or more channel structures with distinct Ca²⁺ binding affinities exist. Further investigation is required to understand the relative Ca²⁺ permeabilities of these distinct kinetic states, and how each state can be allosterically modulated.

#108: How Schwann cells control the clustering of sodium channels at nodes of Ranvier

Peles, Elior ;

Department of Molecular Cell Biology, Weizmann Institute of Science;

Although myelin is an excellent insulator that allows axons to conduct at faster speed than achievable without it, action potentials would decay over a few millimeters and thus must be regenerated at regular intervals. Nodes of Ranvier are gaps in the myelin that are exquisitely designed for just this purpose, equipped with high density of voltage-gated sodium channels. In the peripheral nervous system, initial clustering of sodium channels in heminodes that border each myelin segment requires gliomedin, NrCAM and neurofascin 186 (NF186), three cell adhesion molecules (CAMs) that mediate the interaction between Schwann cells and the axon. Heminodal clustering coincides with a second, paranodal junction (PNJ)-dependent mechanism that allows Na⁺ channels to accumulate in mature nodes by restricting their distribution between two growing myelin segments. Thus, Schwann cells govern the assembly of nodes in the PNS by two independent adhesion systems that provide reciprocal backup capacities.

#129: Multiple sweet sensing mechanism in the retina

Yang, Jae Young ; Myers, Jason; Slaughter, Malcolm M ;

Center for Neuroscience, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

Purpose: Sweet taste receptors (STRs) are expressed in various tissues including gut, pancreas, and hypothalamus. We determined the molecular and physiological characteristics of sweet sensing mechanisms in rat retina. Methods: Acutely dissociated or cultured retinal cells from 10 - 16 day Sprague-Dawley rats were treated with various superfused drugs. Internal calcium was monitored using fluo3-based imaging while calcium currents were measured using whole cell patch clamp. Single cell RT-PCR and RACE (rapid amplification of cDNA Ends) were used to determine STR expression. Gene knockdown of T1R2 was done in a retinal cell line, R28. Results: We found mRNA of T1R1 (umami TR) and T1R2 (sweet TR) expressed in rat retina. In fluo3 calcium imaging, various sweeteners including aspartame, saccharin, D-amino acids, and glucose raised intracellular calcium in isolated retinal neurons. The full sequences of T1R1 and T1R2 from rat retina were identical to T1R1 and T1R2 of tongue. Yet rat T1R2 in the tongue does not respond to aspartame. In single cell RT-PCR analysis, aspartame responding neurons have both T1R1 and T1R2. But when T1R1 and T1R2 were transfected into HEK293 cells with GIRK and Galpha15, these cells did not respond to sweeteners. In gene knockdown experiments with siRNA against T1R2 in R28 cells, the response to 1mM saccharin was reduced 45% compared to controls, but the response to 1mM aspartame remained. This suggests T1R2 in rat retina is functionally similar to tongue. Part of the aspartame response is mediated by calcium permeable membrane channels because it could be eliminated by removing extracellular calcium. TRPV1 blockers (10µM SB366791) blocked aspartame’s action and part of the glucose response in rat retina. Aspartame also altered the ERG b-wave amplitude. Glucose was also found to activate the TRPV1 channel, and this effect of glucose could be blocked with insulin. Conclusions: There are multiple sweet sensing pathways in retina, including the canonical T1R2 system found in tongue and a TRPV1 system. Saccharin is detected by the former, aspartame by the latter. TRPV1 transduces glucose levels in neurons.

#105: Phenotypic Array Analysis Reveals Differential Behaviors in C57WT and MT1KO mice along the 24h day

Adamah-Biassi, Ekue B ¹; Stepien, Iwona ¹; Hudson, Randall L ²; Dubocovich, Margarita L ¹;

¹Department of Pharmacology and Toxicology, ²Department of physiology and Biophysics, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

The goal of this study was to investigate the role of the MT1 melatonin receptor in a behavior array using C57 wild type (WT) (n=8) and C57MT1 knockout (MT1KO) (n=8) mice kept in a 14/10 L/D cycle by visualizing and recognizing sixteen distinct behaviors for 24 hours using the HomeCageScan system (Cleversys Inc., Reston, VA). Behaviors assessed were activity related behaviors (i.e. travel distance, walk, hang, jump), anxiety related behaviors (i.e. dig, groom, rear up, sniff, stretch), rest related behaviors (i.e. awake, remain low, rest, twitch) and metabolism related behaviors (i.e. drink, eat). Phenotypic array analysis revealed distinct behavioral rhythms in WT and MT1KO mice, including anticipation to light and dark. Time spent in rest related behaviors, such as rest, twitch and awake were decreased while the time spent in activity related behaviors such as hang and jump behaviors were significantly increased in both male and female MT1KO compared to WT, indicating hyperactivity and decrease resting behaviors in these mice. The time spent in anxiety related behaviors were increased in male (i.e. groom, rear up) and female (i.e. dig). Using the marble burying test we confirmed that male MT1KO mice buried less marble than the WT indicating positive effects of the MT1 melatonin receptor deletion on trait anxiety behavior. The time spent in most behaviors was differentially affected by sex (i.e. travel distance, rear up, awake, stretch, drink and eat) and time of day (i.e. dig, groom, rear up, stretch and twitch) in both WT and MT1KO. Together, these results suggest that removal of the MT1 melatonin receptor negatively affects resting behaviors, increases general activities and have a positive effect on anxiety related behaviors in C57/BL6 mice.
Support: NS061068

#124: Protein kinase A decreases the stability of microscopic NMDA receptor desensitization

Aman, Teresa K ; Ruffino, Thomas J ; Popescu, Gabriela K ;

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

Calcium influx through glutamatergic NMDA receptors is required for many processes, including long-term potentiation (LTP), the cellular basis of memory, and excitotoxicity, the cause of neuronal death in many diseases. Understanding mechanisms that modulate NMDA receptors, therefore, is necessary to determine how plasticity and excitotoxicity can be regulated. Protein kinase A (PKA) has been shown to increase current amplitude and calcium influx through NMDA receptors. Further, PKA can alter LTP size and threshold. The mechanism by which NMDA receptors are modulated by PKA, however, is unknown. We have investigated the biophysical and kinetic mechanisms by which PKA modulates single NMDA receptors by making cell-attached recordings of single GluN1-1a/GluN2B receptors in HEK293 cells with a PKA inhibitor, PKI. Within minutes of PKI (1 mM) application, channel activity was reduced by half (N=8). Channel mean open time was unaltered, whereas mean closed time doubled. Kinetic modeling of channel gating demonstrated that longer closures result from slowed recovery rates from desensitized states and reaction rates towards opening. In contrast, water application did not change any gating parameter (N=6). Next, we tested the involvement of serine 1166 on GluN2B, known to be modulated by PKA. Consistent with the pharmacological results, the phosphomimic mutation (S1166D, N=11) had an increased P_o and decreased MCT compared to the phosphodeficient mutation (S1166A, N=8), suggesting that this residue influences gating, potentially through PKA modulation. Finally, simulations of macroscopic currents indicate that blocking PKA may reduce calcium influx during theta burst stimulations. Therefore, PKA phosphorylation increases channel activity by decreasing the stability of desensitized states. These details about NMDA receptor regulation give a better understanding of the processes underlying memory and may lead to novel pharmacological approaches to address excitotoxicity.