Abstract List for 2012 meeting

#186: Protein kinase A potentiates NMDA receptor currents by accelerating activation and resensitization

Aman, Teresa K ; 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 brain processes, including long-term potentiation (LTP) and excitotoxicity. Understanding the mechanisms that modulate NMDA receptors, therefore, is necessary to determine how these processes can be regulated. Protein kinase A (PKA) increases macroscopic current amplitude and calcium influx through NMDA receptors; however, the mechanism of this regulation is unknown. We therefore studied how PKA affects the biophysical and kinetic properties of single NMDA receptors using cell-attached recordings of receptors expressed in HEK293 cells. After measuring baseline activity, a PKA inhibitor (PKI, 1 μM) was added to the bath and within ~5 min, the activity of GluN1/GluN2B receptors decreased more than 2-fold (Po = 0.16 ± 0.06 vs. 0.06 ± 0.03, n = 8, p < 0.05). The decrease in activity was entirely due to increased mean closed time. Kinetic modeling indicated that longer closures resulted from slower channel activation and slower recovery from desensitized states. The gating effect was: i) specific to PKI, since water application to GluN1/GluN2B-expressing cells had no effect on gating (Po = 0.17 ± 0.05 vs. 0.14 ± 0.04, n = 14, p = 0.4); ii) was isoform specific, since PKI application to cells expressing GluN1/GluN2A receptors was ineffective (Po = 0.48 ± 0.1 vs. 0.47 ± 0.1, n = 4; p = 0.9); and iii) was mediated by the C-terminal domain (CTD) of GluN2B, since switching CTDs between GluN2A and GluN2B receptors conferred PKI sensitivity to receptors that contained the GluN2B CTD (Po = 0.47 ± 0.1 vs. Po = 0.33 ± 0.1, n = 5, p < 0.05). Based on these results, we conclude that constitutive PKA activity maintains elevated levels of GluN2B receptor activity via the CTD by increasing channel activation and recovery from desensitization. These new insights into the mechanism of NMDA receptor regulation by PKA provide a more thorough understanding of the processes underlying LTP and excitotoxicity.

#151: Proximal Astrocytes Alter the Iron Efflux Mechanism of Human Brain Microvascular Endothelial Cells

McCarthy, Ryan C ; Kosman, Daniel J ;

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

The role of astrocytes in the regulation of iron flux from the brain microvasculature endothelial cells (BMVEC) of the blood-brain barrier (BBB) remains unknown. Here, we utilize a human BMVEC line (hBMVEC) to understand their mechanism(s) of iron efflux which are, in part, regulated by spatially adjacent astrocytes. Not surprisingly, we determined that iron efflux from hBMVEC requires an exocytoplasmic ferroxidase and Ferroportin (Fpn). We found that astrocytes, exclusively, stimulate iron efflux from hBMVEC via a contact-independent mechanism. Upon further investigation we observed that upon contact, astrocytes induce the depletion of endogenous hBMVEC Hephaestin (Hp). This is an entirely novel observation and suggests that astrocytes play a direct regulatory role in the transport of iron by hBMVEC. Taken together, our findings demonstrate that proximal astrocytes regulate iron flux through hBMVEC in vitro.

#161: NAcc and VP neurons preferentially encode the incentive value of a food-associated cue

Meyer, Paul J ;

Department of Psychology, SUNY University at Buffalo;

Cues predicting food or drug rewards cues can acquire incentive salience, that is, the power to motivate, attract, and reinforce behaviors. However, the degree of incentive salience attributed to these cues varies among individuals. We hypothesized that the incentive salience of cues is preferentially encoded by two areas of the ventral basal ganglia: the nucleus accumbens (NAcc) and the ventral pallidum (VP). To achieve this, we measured individual differences in the propensity to attribute incentive salience to a reward cue during a Pavlovian conditioned approach paradigm. During training, rats were given presentations of a retractable lever for eight seconds, followed by the immediate delivery of a sucrose pellet. Approach to the lever (sign-tracking) and food-receptacle (goal- tracking) were used to categorize rats that attributed relatively high or low levels of incentive value to the cue, respectively. Rats were then implanted with moveable recording electrodes into the NAcc and VP, and single unit activity was recorded during additional testing sessions. Neurons from both brain areas reacted to cue presentations and encoded the behaviors evoked by the cue. A greater percentage of neurons from sign-tracking rats responded to some aspect of the cue, and only neurons from sign-trackers encoded the cue onset. This suggests that nucleus accumbens and ventral pallidum neurons preferentially process the incentive value of a reward-associated stimulus. Understanding the precise nature of cue-processing in these two parts of the brain will help us understand the nature of cue-controlled compulsions, such as drug addiction, in humans.

#150: Activation of membrane-associated estrogen receptors decreases ingestive behavior in female rats

Santollo, Jessica ; Marshall, Aniko ; Daniels, Derek ;

Department of Psychology, SUNY University at Buffalo;

Estradiol (E2) decreases food and water intake in a variety of species, including rats. Available evidence suggests that this is mediated by genomic mechanisms that are most often attributed to nuclear estrogen receptors (ERs). More recent studies indicate that membrane-associated estrogen receptors (mERs) also can influence gene expression through the activation of transcription factors; yet it is unclear if mERs are involved in mediating the hypophagic and anti-dipsetic effects of E2. In the present experiments, we injected E2 or a membrane-impermeable form of E2 (E17BSA) into the lateral cerebral ventricle of ovariectomized female rats and evaluated the effect on 23 h food and water intake. First, we found that higher doses of E2 were necessary to reduce water intake than were sufficient to reduce food intake. Analysis of drinking microstructure revealed that the decrease in water intake following E2 treatment was mediated by both a decrease in burst number and burst size. Next, activation of mERs with E17BSA decreased both overnight food and water intake and analysis of drinking microstructure indicated that the decreased water intake resulted from a decrease in burst number. Finally, E17BSA did not condition a taste aversion, suggesting that the inhibitory effects on food and water intake were not secondary to malaise. Together, these findings suggest that activation of mERs is sufficient to decrease food and water intake in female rats.

#181: Agmatine attenuates methamphetamine-induced hyperthermia in rats

Thorn, David A ; Winter, Jerrold C ; Li, Jun-Xu ;

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

Methamphetamine (MA) abuse and addiction is a significant health care problem but currently there is no effective pharmacotherapy available. MA induces robust rewarding (measured with a conditioned place preference [CPP] paradigm) and neurotoxic (hyperthermia) effects in laboratory animals. In our previous studies, the cationic polyamine agmatine attenuates MA-induced CPP. However, the effects of agmatine on the neurotoxic effects of MA are currently unknown. To address this issue, we examined the effects of agmatine on MA-induced hyperthermia by measuring rectal temperature in rats. A large dose of MA (10 mg/kg) induced a significant hyperthermia. Although agmatine (10-32 mg/kg) alone had no effect on body temperature in rats, it markedly decreased MA-induced hyperthermia. To investigate the receptor mechanism mediating this effect, the α2 adrenoreceptor antagonist/ imidazoline I2 receptor antagonist idazoxan was studied in combination with agmatine and MA. Idazoxan (3 mg/kg) not only reversed the effects of agmatine on MA-induced hyperthermia, but it potentiated MA-induced hyperthermia, suggesting the involvement of an α2-independent mechanism. Due to the potentiation of the hyperthermic effects of MA by idazoxan, the interaction between idazoxan and MA was then investigated. Idazoxan (3 mg/kg) significantly enhanced the body temperature changes induced by MA (3.2-10 mg/kg). In summary, MA produced robust hyperthermia in rats, which was attenuated by agmatine. In addition, there appears to be an interaction between compounds acting as antagonists on the α2 adrenoreceptor and MA. Given that agmatine has been shown to attenuate the rewarding effects of MA, combined with the finding that it also attenuates MA-induced neurotoxic effects, suggests agmatine may have some value against MA abuse.

#152: SNAREs are essential in the potentiation of NMDARs by Group II Metabotropic Glutamate Receptors

Cheng, Jia ; Liu, Wenhua ; Yan, Zhen ;

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

The group II metabotropic glutamate receptor (mGluRII) has emerged as a new drug target for schizophrenia treatment. To understand the potential molecular mechanisms underlying the antipsychotic effects of mGluRII, we examined its impact on NMDA receptors, since NMDAR hypofunction has been implicated in schizophrenia. We previously found that application of APDC, a highly selective mGluRII agonist, caused a potent enhancement of NMDAR-mediated currents in cortical pyramidal neurons. Here we examined whether this effect of mGluRII involves the exocytosis of NMDA receptors mediated by SNARE proteins, such as SNAP-25 (synaptosomal-associated protein of 25 kDa) and Syntaxin 4. We found that the enhancing effect of APDC on NMDAR currents was abolished when SNARE complex was disrupted by delivering Botulinum toxin or SNAP-25 C-terminal blocking peptide into the neurons. Moreover, knockdown of Syntaxin 4 blocked mGluRII potentiation of NMDAR currents. Syntaxin 4 is a postsynaptic component interacting with Rab4, a small Rab GTPase mediating fast recycling from early endosome to the plasma membrane. The effect of APDC on NMDAR currents was abolished by dominant negative Rab4, and occluded by constitutively active Rab4, suggesting the involvement of Rab4-mediated NMDAR exocytosis to the cell membrane. Taken together, these results have revealed the key molecules involved in mGluRII enhancement of NMDA receptor trafficking and function.

#171: Corticosterone stress hormone increases significantly during salicylate induced tinnitus

Decker, Brandon ; Manohar, Senthilvelan ; Sheppard, Adam ; Salvi, Richard ;

Center for Hearing and Deafness, SUNY University at Buffalo;

Subjective tinnitus refers to the phantom ringing sensation that occurs in the absence of sound. Anecdotal reports suggest that periods of high stress can trigger the onset or exacerbate tinnitus in patients with hearing loss. Since high levels of stress could conceivably play a critical role in the development of tinnitus, we hypothesized that the onset of tinnitus might be linked to an increase in the circulating stress hormones. To test this hypothesis, we induced tinnitus in adult, male Sprague-Dawley rats with a treatment of 250 mg/kg of sodium salicylate (active ingredient in aspirin), a drug dose that reliably induces hearing loss and tinnitus. Blood was sampled at different time points after salicylate treatment and corticosterone levels were measured using a sensitive ELISA assay. The 250 mg/kg dose of salicylate produced a massive increase in corticosterone levels 2 h after salicylate treatment compared to saline treated rats (600 vs 25 ng corticosterone/ml serum). Subsequent corticosterone measures were obtained from rats treated with different doses of salicylate (250, 150, 50 mg salicylate/kg weight). The 50 mg/kg dose had no effect on corticosterone level whereas the 150 mg/kg dose produced a modest increase in only a third of the rats. This results support the hypothesis that high levels of stress may contribute to the induction of tinnitus.

#165: Shank3 Deficiency Induces NMDA Receptor Hypofunction via an Actin-Dependent Mechanism

Duffney, Lara J ; Yan, Zhen ;

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

Shank3, which encodes a scaffolding protein at glutamatergic synapses, is a genetic risk factor of autism. It has been found that Shank3 binds to the NMDA-type glutamate receptor, a key player in cognition and mental illnesses. In this study, we examined the physiological impact of Shank3 deficiency on NMDAR function and the underlying mechanisms. We found that knockdown of Shank3 caused a significant reduction of NMDAR-mediated current in cortical cultures, while AMPAR-mediated current was unchanged. The surface expression of NMDAR subunits was also decreased by Shank3 knockdown. The effect of Shank3 siRNA on NMDAR currents was blocked in neurons exposed to the actin stabilizer phalloidin, and was occluded by the actin destabilizer latrunculin B, but was unaffected by compounds that perturb microtubule networks, suggesting the involvement of actin dynamics. Since actin cytoskeleton is regulated by the GTPase Rac1 and down-stream effector PAK, we further examined Shank3 regulation of NMDARs when Rac1 or PAK was inhibited. We found that the reducing effect of Shank3 siRNA on NMDAR currents was mimicked and occluded by treatment with EHT (specific Rac1 inhibitor), IPA or PAK18 (specific PAK inhibitors), as well as by transfection with a dominant-negative PAK. Taken together, these results suggest that Shank3 knockdown induces NMDAR hypofunction by interfering with the Rac1/PAK/actin signaling, leading to the loss of NMDAR membrane delivery and stability. It provides a potential mechanism for the role of Shank3 in cognitive deficit in autism.

#166: Withdrawal from cocaine self-administration alters Transforming Growth Factor-Beta (TGF-β) signaling

Gancarz, Amy M ¹; Schroeder, Gabrielle L ²; Panganiban, Clarisse ²; Kausch, Michael A ²; Clark, Stewart D ²; Dietz, David M ²;

¹Research Institute on Addictions, ²Department of Pharmacology and Toxicology, SUNY University at Buffalo;

The addicted phenotype is characterized as a long-lasting, chronically relapsing disorder that persists following long periods of abstinence, suggesting that the underlying molecular changes are stable and persist for long periods. There is ample evidence that indicates repeated exposure to psychomotor stimulants results in an increase in dendritic spine density in the nucleus accumbens (NAc) medium spiny neurons. Here, we investigated key regulators of morphological plasticity in animals that were tested for cue-induced reinstatement following periods of withdrawal from cocaine self-administration (SA). Rats were trained to self-administer cocaine or saline and underwent either 0 or 7 days of withdrawal. When compared to saline, cocaine SA resulted in a transient down-regulation in gene expression of LIMK, an enzyme involved in regulating actin filament dynamics, in the NAc following early drug cessation, which returned to baseline following 7 days of withdrawal. In contrast, expression of cofilin, an actin binding protein that promotes filament disassembly, showed the opposite pattern of regulation. Furthermore, we investigated changes in the transforming growth factor-beta (TGF-β) family, an intracellular signaling cascade that has both direct and indirect (transcriptional) interactions with actin cycling. When compared to saline, we show that cocaine SA results in an increase in the type 1 receptor (TGF-βR1) in the NAc. A period of withdrawal following cocaine SA induced expression of SMAD3, a key component TGF-β signaling pathway that transmits TGF-β signals from the cell surface into the nucleus, allowing for transcriptional control. In addition, there was an increased proportion of the active protein form, p-SMAD3 suggesting cocaine increase the transcriptional activity of SMAD3. Taken together, these data indicate that TGF-β signaling is regulated in a time-dependent manner following cocaine self-administration.

#179: The MT1 Melatonin Receptor Regulates Methamphetamine-induced Locomotor Activity at Night

Hutchinson, Anthony J ¹; Ma, Jason 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;

Locomotor sensitization refers to the enhancement of locomotor responses by successive psychostimulant treatments and reflects drug-induced neuroadaptations [Robinson & Berridge, Addiction 2000;95 Suppl 2:S91-117]. We have shown that genetic deletion of the MT1 and MT2 receptors, which mediate the functions of melatonin in an array of physiological processes, attenuates sensitization in C3H/HeN mice [Hutchinson et al., J Pineal Res 2012; in press]. Here we describe the function of the MT1 receptor in diurnal variations of methamphetamine (METH)-induced sensitization. C3H/HeN mice were pretreated in novel test arenas with 6 daily doses of saline (VEH) or METH (1.2 mg/kg, i.p.) at Zeitgeber time (ZT) 5-7 or ZT 19-21 (ZT 0=lights on, 12h:12h light-dark cycle). Five days after the last pretreatment at ZT5-7, both WT and MT1KO mice pretreated with METH expressed sensitization upon METH challenge (WT: 152.5±11.37 m traveled, n=11, p<0.05; MT1KO: 114.9±12.3 m, n=16, p<0.05), whereas VEH-pretreated mice did not (WT: 72.5±11.35 m, n=12; MT1KO: 53.0±8.0 m, n=16). At ZT 19-21, VEH-pretreated WT mice (236.3±15.7 m, n=11), but not MT1KO mice (109.9±20.8 m, n=8), expressed the same locomotor response to the challenge as their METH-pretreated counterparts (WT: 207.9±28.4 m, n=10, n.s.; MT1KO: 182.2±21.7 m, n=9, p<0.05). This nocturnal sensitization in the absence of METH pretreatment is attributed to environmental novelty, because repeated exposure to the arenas induced sensitization to METH challenge at ZT19-21 but not at ZT 5-7. We also tested C57BL/6 mice for locomotor sensitization, and found that a single METH pretreatment elicited sensitization at both ZT 5-7 and ZT 19-21 in WT mice, but not MT1KO at either time. Novelty had no effect on the nocturnal expression of sensitization in C57BL/6 mice. We conclude that the MT1 receptor may mediate the effects of both prior drug experience and environmental features such as novelty on responses to METH in mice.