Jeffrey G. Edwards

Myosin Vb Mobilizes Recycling Endosomes and AMPA Receptors for Postsynaptic Plasticity

Learning-related plasticity at excitatory synapses in the mammalian brain requires the trafficking of AMPA receptors and the growth of dendritic spines. However, the mechanisms that couple plasticity stimuli to the trafficking of postsynaptic cargo are poorly understood. Here we demonstrate that myosin Vb (MyoVb), a Ca2+-sensitive motor, conducts spine trafficking during long-term potentiation (LTP) of synaptic strength. Upon activation of NMDA receptors and corresponding Ca2+ influx, MyoVb associates with recycling endosomes (REs), triggering rapid spine recruitment of endosomes and local exocytosis in spines. Disruption of MyoVb or its interaction with the RE adaptor Rab11-FIP2 abolishes LTP-induced exocytosis from REs and prevents both AMPA receptor insertion and spine growth. Furthermore, induction of tight binding of MyoVb to actin using an acute chemical genetic strategy eradicates LTP in hippocampal slices. Thus, Ca2+-activated MyoVb captures and mobilizes REs for AMPA receptor insertion and spine growth, providing a mechanistic link between the induction and expression of postsynaptic plasticity.

TRPV1 Channels Mediate Long-Term Depression at Synapses on Hippocampal Interneurons

TRPV1 receptors have classically been defined as heat-sensitive, ligand-gated, nonselective cation channels that integrate nociceptive stimuli in sensory neurons. TRPV1 receptors have also been identified in the brain, but their physiological role is poorly understood. Here we report that TRPV1 channel activation is necessary and sufficient to trigger long-term synaptic depression (LTD). Excitatory synapses onto hippocampal interneurons were depressed by either capsaicin, a potent TRPV1 channel activator, or the endogenously released eicosanoid, 12-(S)-HPETE, whereas neighboring excitatory synapses onto CA1 pyramidal cells were unaffected. TRPV1 receptor antagonists also prevented interneuron LTD. In brain slices from TRPV1-/- mice, LTD was absent, and neither capsaicin nor 12-(S)-HPETE elicited synaptic depression. Our results suggest that, in the hippocampus, TRPV1 receptor activation selectively modifies synapses onto interneurons. Like other forms of hippocampal synaptic plasticity, TRPV1-mediated LTD may have a role in long-term changes in physiological and pathological circuit behavior during learning and epileptic activity.

Transient receptor potential vanilloid 1 agonists modulate hippocampal CA1 LTP via the GABAergic system.

Transient receptor potential vanilloid 1 (TRPV1) was shown to modulate hippocampal CA1 pyramidal cell synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Synaptic plasticity is the cellular mechanism thought to mediate declarative learning and memory in the hippocampus. Although TRPV1 is involved in modulating hippocampal plasticity, it has yet to be determined how TRPV1 mediates its effects. Using field electrophysiology in hippocampal CA1 stratum radiatum we investigated how TRPV1 agonists modulate LTP, low frequency stimulation-induced LTD, and (RS)-3,5-dihydroxyphenylglycine (DHPG)-induced LTD. First we confirmed that TRPV1 agonists induce enhancement of CA1 pyramidal cell LTP in the absence the GABA(A) receptor antagonist picrotoxin. Because it was recently determined that TRPV1 mediates a novel form of LTD in CA1 inhibitory GABAergic interneurons, which can disinhibit CA1 pyramidal cells, we used picrotoxin to block the effect of the GABAergic circuitry on CA1 LTP. When using picrotoxin, the TRPV1 agonist-induced enhancement of CA1 LTP was eliminated suggesting that the GABAergic circuitry is required for TRPV1 agonist mediated increases. Regarding LTD, in contrast to previously reported data, we did not see TRPV1 agonist-mediated effect on low frequency-induced stimulus LTD. However, during DHPG-induced LTD, TRPV1 was involved in the acute, but not the long-term depression phase of this plasticity. In summary, our findings support TRPV1 agonist involvement in hippocampal synaptic plasticity, including its enhancement of CA1 LTP. We demonstrate that the enhancement mediated by TRPV1 agonists requires GABA input to pyramidal cells thus providing a mechanism for how TRPV1 agonists modulate hippocampal synaptic plasticity.

The role of connexin-36 gap junctions in alcohol intoxication and consumption.

Ventral tegmental area (VTA) GABA neurons appear to be critical substrates underlying the acute and chronic effects of ethanol on dopamine (DA) neurotransmission in the mesocorticolimbic system implicated in alcohol reward. The aim of this study was to examine the role of midbrain connexin‐36 (Cx36) gap junctions (GJs) in ethanol intoxication and consumption. Using behavioral, molecular, and electrophysiological methods, we compared the effects of ethanol in mature Cx36 knockout (KO) mice and age‐matched wild‐type (WT) controls. Compared to WT mice, Cx36 KO mice exhibited significantly more ethanol‐induced motor impairment in the open field test, but less disruption in motor coordination in the rotarod paradigm. Cx36 KO mice, and WT mice treated with the Cx36 antagonist mefloquine (MFQ), consumed significantly less ethanol than their WT controls in the drink‐in‐the‐dark procedure. The firing rate of VTA GABA neurons in WT mice was inhibited by ethanol with an IC50 of 0.25 g/kg, while VTA GABA neurons in KO mice were significantly less sensitive to ethanol. Dopamine neuron GABA‐mediated sIPSC frequency was reduced by ethanol (30 mM) in WT mice, but not affected in KO mice. Cx36 KO mice evinced a significant up‐regulation in DAT and D2 receptors in the VTA, as assessed by quantitative RT‐PCR. These findings demonstrate the behavioral relevance of Cx36 GJ‐mediated electrical coupling between GABA neurons in mature animals, and suggest that loss of coupling between VTA GABA neurons results in disinhibition of DA neurons, a hyper‐DAergic state and lowered hedonic valence for ethanol consumption. Synapse, 2011.

TRPV1 in the Central Nervous System: Synaptic Plasticity, Function, and Pharmacological Implications

The function of TRPV1 in the peripheral nervous system is increasingly being investigated for its anti-inflammatory and antinociceptive properties in an effort to find a novel target to fight pain that is nonaddictive. However, in recent years, it was discovered that TRPV1 is also associated with a wide array of functions and behaviors in the central nervous system, such as fear, anxiety, stress, thermoregulation, pain, and, more recently, synaptic plasticity, the cellular mechanism that allows the brain to adapt to its environment. This suggests a new role for brain TRPV1 in areas such as learning and memory, reward and addiction, and development. This wide array of functional aspects of TRPV1 in the central nervous system (CNS) is in part due to its multimodal form of activation and highlights the potential pharmacological implications of TRPV1 in the brain. As humans also express a TRPV1 homologue, it is likely that animal research will be translational to humans and therefore worthy of exploration. This review outlines the basic expression patterns of TRPV1 in the CNS along with what is known regarding its signaling mechanisms and its role in the aforementioned brain functions. As TRPV1 involvement in synaptic plasticity has never been fully reviewed elsewhere, it will be a focus of this review. The chapter concludes with some of the potential pharmaceutical implications of further TRPV1 research.

A novel non-CB1/TRPV1 endocannabinoid-mediated mechanism depresses excitatory synapses on hippocampal CA1 interneurons.

Endocannabinoids (eCBs) mediate various forms of synaptic plasticity at excitatory and inhibitory synapses in the brain. The eCB anandamide binds to several receptors including the transient receptor potential vanilloid 1 (TRPV1) and cannabinoid receptor 1 (CB1). We recently identified that TRPV1 is required for long‐term depression at excitatory synapses on CA1 hippocampal stratum radiatum interneurons. Here we performed whole‐cell patch clamp recordings from CA1 stratum radiatum interneurons in rat brain slices to investigate the effect of the eCB anandamide on excitatory synapses as well as the involvement of Group I metabotropic glutamate receptors (mGluRs), which have been reported to produce eCBs endogenously. Application of the nonhydrolysable anandamide analog R‐methanandamide depressed excitatory transmission to CA1 stratum radiatum interneurons by ∼50%. The Group I mGluR agonist DHPG also depressed excitatory glutamatergic transmission onto interneurons to a similar degree, and this depression was blocked by the mGluR5 antagonist MPEP (10 μM) but not by the mGluR1 antagonist CPCCOEt (50 μM). Interestingly, however, neither DHPG‐mediated nor R‐methanandamide‐mediated depression was blocked by the TRPV1 antagonist capsazepine (10 μM), the CB1 antagonist AM‐251 (2 μM) or a combination of both, suggesting the presence of a novel eCB receptor or anandamide target at excitatory hippocampal synapses. DHPG also occluded R‐methanandamide depression, suggesting the possibility that the two drugs elicit synaptic depression via a shared signaling mechanism. Collectively, this study illustrates a novel CB1/TRPV1‐independent eCB pathway present in the hippocampus that mediates depression at excitatory synapses on CA1 stratum radiatum interneurons.

Calcineurin is required for TRPV1-induced long-term depression of hippocampal interneurons.

Transient receptor potential vanilloid 1 (TRPV1) mediates a novel form of presynaptic long-term depression (LTD) in hippocampal interneurons. To date, while TRPV1 is currently being heavily studied in the PNS for its anti-nociceptive and anti-inflammatory properties, much less is known regarding TRPV1 signaling and function in the CNS, including the mechanism mediating hippocampal interneuron LTD. Here we performed whole-cell voltage clamp electrophysiology experiments on CA1 hippocampal interneurons from Sprague-Dawley male rats to identify this signaling mechanism. Because calcineurin is linked to multiple synaptic plasticity types, we investigated whether TRPV1 activates presynaptic calcineurin, which in turn induces LTD. To do so we employed calcineurin inhibitors cyclosporin A or FK-506. To determine the location of the calcineurin involved we either bath applied calcineurin antagonists, blocking calcineurin activity ubiquitously in the slice, presynaptically and postsynaptically, or applied antagonists to the internal solution to block calcineurin postsynaptically. Both calcineurin antagonists applied to the bath blocked TRPV1-dependent LTD, indicating calcineurin involvement in LTD. Because calcineurin antagonist applied to the internal solution did not block TRPV1-LTD, it suggests presynaptic calcineurin is required for LTD. Finally, because high frequency stimulus used to induce LTD could potentially activate receptors besides TRPV1, we confirmed that bath, but not intracellularly applied cyclosporin A, also blocked TRPV1 agonist-induced depression of CA1 interneurons. In conclusion, these data illustrate that presynaptic calcineurin activity is required for both TRPV1-induced LTD and TRPV1 agonist-induced depression. This finding is the first to demonstrate the TRPV1-induced signaling mechanism in CA1 hippocampus.

Ventral tegmental area dopamine and GABA neurons: Physiological properties and expression of mRNA for endocannabinoid biosynthetic elements

The ventral tegmental area (VTA) is involved in adaptive reward and motivation processing and is composed of dopamine (DA) and GABA neurons. Defining the elements regulating activity and synaptic plasticity of these cells is critical to understanding mechanisms of reward and addiction. While endocannabinoids (eCBs) that potentially contribute to addiction are known to be involved in synaptic plasticity mechanisms in the VTA, where they are produced is poorly understood. In this study, DA and GABAergic cells were identified using electrophysiology, cellular markers, and a transgenic mouse model that specifically labels GABA cells. Using single-cell RT-qPCR and immunohistochemistry, we investigated mRNA and proteins involved in eCB signaling such as diacylglycerol lipase α, N-acyl-phosphatidylethanolamine-specific phospholipase D, and 12-lipoxygenase, as well as type I metabotropic glutamate receptors (mGluRs). Our results demonstrate the first molecular evidence of colocalization of eCB biosynthetic enzyme and type I mGluR mRNA in VTA neurons. Further, these data reveal higher expression of mGluR1 in DA neurons, suggesting potential differences in eCB synthesis between DA and GABA neurons. These data collectively suggest that VTA GABAergic and DAergic cells have the potential to produce various eCBs implicated in altering neuronal activity or plasticity in adaptive motivational reward or addiction.

Identification of mRNA for endocannabinoid biosynthetic enzymes within hippocampal pyramidal cells and CA1 stratum radiatum interneuron subtypes using quantitative real-time polymerase chain reaction.

The hippocampus is required for short-term memory and contains both excitatory pyramidal cells and inhibitory interneurons. These cells exhibit various forms of synaptic plasticity, the mechanism underlying learning and memory. More recently, endocannabinoids were identified to be involved in synaptic plasticity. Our goal was to describe the distribution of endocannabinoid biosynthetic enzymes within CA1 stratum radiatum interneurons and CA3/CA1 pyramidal cells. We extracted mRNA from single interneurons and pyramidal cells and used real-time quantitative polymerase chain reaction (RT-PCR) to detect the presence of 12-lipoxygenase, N-acyl-phosphatidylethanolamine-specific phospholipase D, diacylglycerol lipase α, and type I metabotropic glutamate receptors, all known to be involved in endocannabinoid production and plasticity. We observed that the expression of endocannabinoid biosynthetic enzyme mRNA does occur within interneurons and that it is coexpressed with type I metabotropic glutamate receptors, suggesting interneurons have the potential to produce endocannabinoids. We also identified that CA3 and CA1 pyramidal cells express endocannabinoid biosynthetic enzyme mRNA. Our data provide the first molecular biological evidence for putative endocannabinoid production in interneurons, suggesting their potential ability to regulate endocannabinoid-mediated processes, such as synaptic plasticity.

Nicotine Enhances the Excitability of Gaba Neurons in the Ventral Tegmental Area via Activation of Alpha 7 Nicotinic Receptors on Glutamate Terminals

Ventral tegmental area dopamine (DA) and GABA neurons express nicotinic acetylcholine receptor (nAChR) subtypes, whose net activation results in enhancement of DA release in the nucleus accumbens (NAc). This effect decreases after repeated nicotine (NIC) treatment via desensitization. We evaluated the effects of acute NIC on glutamate decarboxylase (GAD67)-positive GABA neurons in the VTA of green fluorescent protein (GFP) knockin (GAD-GFP) mice, and determined the expression of selected nAChR subunits in VTA GABA neurons. In vivo, tachylphylaxis accrued to repeated systemic, but not local administration of NIC. Microelectrophoretic application of NIC and the α7 nAChR partial agonist JN403 markedly enhanced the firing rate of VTA GABA neurons. This activation was suppressed by intraperitoneal administration of the α7 nAChR antagonist methyllycaconitine (MLA, 1 mg/kg), or the glutamate (GLU) NMDA receptor antagonist APV (1 mg/kg), but not by the non-selective non-competitive antagonist mecamylamine (MEC, 1 mg/kg). In patch clamp studies in the slice preparation, the α7 nAChR agonist choline (1-10 mM), in the presence of the muscarinic cholinergic antagonist atropine (50 μM), enhanced sEPSC and mini-EPSC frequency, but not amplitude, which was blocked by MLA (0.5 μM). JN403 (0.1-1 μM) enhanced evoked EPSCs, without affecting membrane currents of VTA GABA neurons. In patch clamp studies in dissociated VTA GABA neurons, choline+atropine failed to induce whole-cell current responses in most cells tested. Single cell RT-qPCR revealed that most GABA neurons did not express α7 nAChRs. Together, this indicates that NIC excites VTA GABA neurons via α7 nAChRs located on GLUergic terminals.