The Role of Ryanodine Receptor 2 in Drug-Associated Learning.

Type-2 ryanodine receptor (RyR2) ion channels facilitate the release of Ca 2+ from stores and serve an important function in neuroplasticity. The role for RyR2 in hippocampal-dependent learning and memory is well established and chronic hyperphosphorylation of RyR2 (RyR2P) is associated with pathological calcium leakage and cognitive disorders, including Alzheimer's disease. By comparison, little is known about the role of RyR2 in the ventral medial prefrontal cortex (vmPFC) circuitry important for working memory, decision making, and reward seeking. Here, we evaluated the basal expression and localization of RyR2 and RyR2P in the vmPFC. Next, we employed an operant model of sucrose, cocaine, or morphine self-administration (SA) followed by a (reward-free) recall test, to reengage vmPFC neurons and reactivate reward-seeking and re-evaluated the expression and localization of RyR2 and RyR2P in vmPFC. Under basal conditions, RyR2 was expressed in pyramidal cells but not regularly detected in PV/SST interneurons. On the contrary, RyR2P was rarely observed in PFC somata and was restricted to a different subcompartment of the same neuron - the apical dendrites of layer-5 pyramidal cells. Chronic SA of drug (cocaine or morphine) and nondrug (sucrose) rewards produced comparable increases in RyR2 protein expression. However, recalling either drug reward impaired the usual localization of RyR2P in dendrites and markedly increased its expression in somata immunoreactive for Fos, a marker of highly activated neurons. These effects could not be explained by chronic stress or drug withdrawal and instead appeared to require a recall experience associated with prior drug SA. In addition to showing the differential distribution of RyR2/RyR2P and affirming the general role of vmPFC in reward learning, this study provides information on the propensity of addictive drugs to redistribute RyR2P ion channels in a neuronal population engaged in drug-seeking. Hence, focusing on the early impact of addictive drugs on RyR2 function may serve as a promising approach to finding a treatment for substance use disorders.

K v 7 Channel Opener Retigabine Reduces Self-Administration of Cocaine but Not Sucrose in Rats.

The increasing rates of drug misuse highlight the urgency of identifying improved therapeutics for treatment. Most drug-seeking behaviors that can be modeled in rodents utilize the repeated intravenous self-administration (SA) of drugs. Recent studies examining the mesolimbic pathway suggest that K v 7/KCNQ channels may contribute in the transition from recreational to chronic drug use. However, to date, all such studies used noncontingent, experimenter-delivered drug model systems, and the extent to which this effect generalizes to rats trained to self-administer drug is not known. Here, we tested the ability of retigabine (ezogabine), a K v 7 channel opener, to regulate instrumental behavior in male Sprague Dawley rats. We first validated the ability of retigabine to target experimenter-delivered cocaine in a CPP assay and found that retigabine reduced the acquisition of place preference. Next, we trained rats for cocaine-SA under a fixed-ratio or progressive-ratio reinforcement schedule and found that retigabine-pretreatment attenuated the self-administration of low to moderate doses of cocaine. This was not observed in parallel experiments, with rats self-administering sucrose, a natural reward. Compared to sucrose-SA, cocaine-SA was associated with reductions in the expression of the K v 7.5 subunit in the nucleus accumbens, without alterations in K v 7.2 and K v 7.3. Therefore, these studies reveal a reward specific reduction in SA behavior considered relevant for the study of long-term compulsive-like behavior and supports the notion that K v 7 is a potential therapeutic target for human psychiatric diseases with dysfunctional reward circuitry.

Targeting 5-HT2A receptors and Kv7 channels in PFC to attenuate chronic neuropathic pain in rats using a spared nerve injury model.

Chronic pain remains a disabling disease with limited therapeutic options. Pyramidal neurons in the prefrontal cortex (PFC) express excitatory Gq-coupled 5-HT2A receptors (5-HT2AR) and their effector system, the inhibitory Kv7 ion channel. While recent publications show these cells innervate brainstem regions important for regulating pain, the cellular mechanisms underlying the transition to chronic pain are not well understood. The present study examined whether local blockade of 5-HT2AR or enhanced Kv7 ion channel activity in the PFC would attenuate mechanical allodynia associated with spared nerve injury (SNI) in rats. Following SNI, we show that inhibition of PFC 5-HT2ARs with M100907 or opening of PFC Kv7 channels with retigabine reduced mechanical allodynia. Parallel proteomic and RNAScope experiments evaluated 5-HT2AR/Kv7 channel protein and mRNA. Our results support the role of 5-HT2ARs and Kv7 channels in the PFC in the maintenance of chronic pain.

Drosophila model of anti-retroviral therapy induced peripheral neuropathy and nociceptive hypersensitivity.

The success of antiretroviral therapy (ART) has improved the survival of HIV-infected patients significantly. However, significant numbers of patients on ART whose HIV disease is well controlled show peripheral sensory neuropathy (PSN), suggesting that ART may cause PSN. Although the nucleoside reverse transcriptase inhibitors (NRTIs), one of the vital components of ART, are thought to contribute to PSN, the mechanisms underlying the PSN induced by NRTIs are unclear. In this study, we developed a Drosophila model of NRTI-induced PSN that recapitulates the salient features observed in patients undergoing ART: PSN and nociceptive hypersensitivity. Furthermore, our data demonstrate that pathways known to suppress PSN induced by chemotherapeutic drugs are ineffective in suppressing the PSN or nociception induced by NRTIs. Instead, we found that increased dynamics of a peripheral sensory neuron may possibly underlie NRTI-induced PSN and nociception. Our model provides a solid platform in which to investigate further mechanisms of ART-induced PSN and nociceptive hypersensitivity.This article has an associated First Person interview with the first author of the paper.

Levels of Par-1 kinase determine the localization of Bruchpilot at the Drosophila neuromuscular junction synapses.

Functional synaptic networks are compromised in many neurodevelopmental and neurodegenerative diseases. While the mechanisms of axonal transport and localization of synaptic vesicles and mitochondria are relatively well studied, little is known about the mechanisms that regulate the localization of proteins that localize to active zones. Recent finding suggests that mechanisms involved in transporting proteins destined to active zones are distinct from those that transport synaptic vesicles or mitochondria. Here we report that localization of BRP-an essential active zone scaffolding protein in Drosophila, depends on the precise balance of neuronal Par-1 kinase. Disruption of Par-1 levels leads to excess accumulation of BRP in axons at the expense of BRP at active zones. Temporal analyses demonstrate that accumulation of BRP within axons precedes the loss of synaptic function and its depletion from the active zones. Mechanistically, we find that Par-1 co-localizes with BRP and is present in the same molecular complex, raising the possibility of a novel mechanism for selective localization of BRP-like active zone scaffolding proteins. Taken together, these data suggest an intriguing possibility that mislocalization of active zone proteins like BRP might be one of the earliest signs of synapse perturbation and perhaps, synaptic networks that precede many neurological disorders.

Correction: Active zone proteins are transported via distinct mechanisms regulated by Par-1 kinase.

[This corrects the article DOI: 10.1371/journal.pgen.1006621.].

Active zone proteins are transported via distinct mechanisms regulated by Par-1 kinase.

Disruption of synapses underlies a plethora of neurodevelopmental and neurodegenerative disease. Presynaptic specialization called the active zone plays a critical role in the communication with postsynaptic neuron. While the role of many proteins at the active zones in synaptic communication is relatively well studied, very little is known about how these proteins are transported to the synapses. For example, are there distinct mechanisms for the transport of active zone components or are they all transported in the same transport vesicle? Is active zone protein transport regulated? In this report we show that overexpression of Par-1/MARK kinase, a protein whose misregulation has been implicated in Autism spectrum disorders (ASDs) and neurodegenerative disorders, lead to a specific block in the transport of an active zone protein component- Bruchpilot at Drosophila neuromuscular junctions. Consistent with a block in axonal transport, we find a decrease in number of active zones and reduced neurotransmission in flies overexpressing Par-1 kinase. Interestingly, we find that Par-1 acts independently of Tau-one of the most well studied substrates of Par-1, revealing a presynaptic function for Par-1 that is independent of Tau. Thus, our study strongly suggests that there are distinct mechanisms that transport components of active zones and that they are tightly regulated.

Leptin upregulates the expression of plasminogen activator inhibitor-1 in human vascular endothelial cells.

A prothrombotic state in obesity may be partially responsible for the higher incidence of atherosclerotic complications. However the factors responsible for this prothrombotic state, linked with high levels of plasminogen activator inhibitor-1 (PAI-1), are not fully known. Leptin is elevated in obesity and studies have shown a positive correlation between leptin and PAI-1 levels in human subjects, along with a negative correlation with tissue-type plasminogen activator (tPA). We tested the hypothesis that leptin induces PAI-1 and inhibits tPA expression using human coronary artery endothelial cells (HCAEC) in culture as these cells play an important role in atherosclerosis. We demonstrate that leptin induces the transcription and translation of PAI-1 in HCAEC. The leptin dependent upregulation of PAI-1 mRNA and protein was comparable to insulin-induced PAI-1 expression. We show leptin concentration (0-150 ng/ml) dependent increases in PAI-1 mRNA and protein after 6 and 12h of leptin administration, respectively. Increased intracellular PAI-1 expression correlates with increased PAI-1 activity in conditioned media and inhibition of specific ERK1/2 pathway by treatment with PD98059 (20-40 microM) inhibits leptin dependent PAI-1 expression. However no changes in tPA expression were seen with time or increasing concentrations of leptin. Also leptin treatment did not alter total tPA concentration or tPA activity in conditioned media. In conclusion, our study shows that leptin upregulates the expression of PAI-1 in vascular endothelial cells via activation of ERK1/2 but does not regulate tPA expression. These studies demonstrate a novel mechanism for the prothrombotic role of leptin in development of atherosclerosis.