Effects of acid-sensing ion channel-1A (ASIC1A) on cocaine-induced synaptic adaptations.

Chronic drug abuse is thought to induce synaptic changes in nucleus accumbens medium spiny neurons (MSNs) that promote subsequent craving and drug-seeking behavior. Accumulating data suggest acid-sensing ion channels (ASICs) may play a critical role. In drug naïve mice, disrupting the ASIC1A subunit produced a variety of synaptic changes reminiscent of wild-type mice following cocaine withdrawal, including increased AMPAR/NMDAR ratio, increased AMPAR rectification, and increased dendrite spine density. Importantly, these changes in Asic1a -/- mice were normalized by a single dose of cocaine. Here we sought to understand the temporal effects of cocaine exposure in Asic1a -/- mice and the cellular site of ASIC1A action. Six hours after cocaine exposure, there was no effect. However, 15 h, 24 h and 4 days after cocaine exposure there was a significant reduction in AMPAR/NMDAR ratio in Asic1a -/- mice. Within 7 days the AMPAR/NMDAR ratio had returned to baseline levels. Cocaine-evoked changes in AMPAR rectification and dendritic spine density followed a similar time course with significant reductions in rectification and dendritic spines 24 h after cocaine exposure in Asic1a -/- mice. To test the cellular site of ASIC1A action on these responses, we disrupted ASIC1A specifically in a subpopulation of MSNs. We found that effects of ASIC1A disruption were cell autonomous and restricted to neurons in which the channels are disrupted. We further tested whether ASIC1A disruption differentially affects MSNs subtypes and found AMPAR/NMDAR ratio was elevated in dopamine receptor 1-expressing MSNs, suggesting a preferential effect for these cells. Finally, we tested if protein synthesis was involved in synaptic adaptations that occurred after ASIC1A disruption, and found the protein synthesis inhibitor anisomycin normalized AMPAR-rectification and AMPAR/NMDAR ratio in drug-naïve Asic1a -/- mice to control levels, observed in wild-type mice. Together, these results provide valuable mechanistic insight into the effects of ASICs on synaptic plasticity and drug-induced effects and raise the possibility that ASIC1A might be therapeutically manipulated to oppose drug-induced synaptic changes and behavior.

Investigating role of ASIC2 in synaptic and behavioral responses to drugs of abuse.

Drugs of abuse produce rearrangements at glutamatergic synapses thought to contribute to drug-reinforced behaviors. Acid-Sensing Ion Channels (ASICs) have been suggested to oppose these effects, largely due to observations in mice lacking the ASIC1A subunit. However, the ASIC2A and ASIC2B subunits are known to interact with ASIC1A, and their potential roles in drugs of abuse have not yet been investigated. Therefore, we tested the effects of disrupting ASIC2 subunits in mice exposed to drugs of abuse. We found conditioned place preference (CPP) to both cocaine and morphine were increased in Asic2 -/- mice, which is similar to what was observed in Asic1a -/- mice. Because nucleus accumbens core (NAcc) is an important site of ASIC1A action, we examined expression of ASIC2 subunits there. By western blot ASIC2A was readily detected in wild-type mice, while ASIC2B was not, suggesting ASIC2A is the predominant subunit in nucleus accumbens core. An adeno-associated virus vector (AAV) was used to drive recombinant ASIC2A expression in nucleus accumbens core of Asic2 -/- mice, resulting in near normal protein levels. Moreover, recombinant ASIC2A integrated with endogenous ASIC1A subunits to form functional channels in medium spiny neurons (MSNs). However, unlike ASIC1A, region-restricted restoration of ASIC2A in nucleus accumbens core was not sufficient to affect cocaine or morphine conditioned place preference, suggesting effects of ASIC2 differ from those of ASIC1A. Supporting this contrast, we found that AMPA receptor subunit composition and the ratio of AMPA receptor-mediated current to NMDA receptor-mediated current (AMPAR/NMDAR) were normal in Asic2 -/- mice and responded to cocaine withdrawal similarly to wild-type animals. However, disruption of ASIC2 significantly altered dendritic spine morphology, and these effects differed from those reported previously in mice lacking ASIC1A. We conclude that ASIC2 plays an important role in drug-reinforced behavior, and that its mechanisms of action may differ from ASIC1A.

Carbonic anhydrase 4 disruption decreases synaptic and behavioral adaptations induced by cocaine withdrawal.

Cocaine use followed by withdrawal induces synaptic changes in nucleus accumbens (NAc), which are thought to underlie subsequent drug-seeking behaviors and relapse. Previous studies suggest that cocaine-induced synaptic changes depend on acid-sensing ion channels (ASICs). Here, we investigated potential involvement of carbonic anhydrase 4 (CA4), an extracellular pH-buffering enzyme. We examined effects of CA4 in mice on ASIC-mediated synaptic transmission in medium spiny neurons (MSNs) in NAc, as well as on cocaine-induced synaptic changes and behavior. We found that CA4 is expressed in the NAc and present in synaptosomes. Disrupting CA4 either globally, or locally, increased ASIC-mediated synaptic currents in NAc MSNs and protected against cocaine withdrawal-induced changes in synapses and cocaine-seeking behavior. These findings raise the possibility that CA4 might be a previously unidentified therapeutic target for addiction and relapse.

Inflammation, NF-κB, and Chronic Diseases: How are They Linked?

Most chronic diseases, caused by lifestyle factors, appear to be linked to inflammation. Inflammation is activated mechanistically, and nuclear factor-κB (NF-κB) is a significant mediator. NF-κB, one of the most studied transcription factors, was first identified in the nucleus of B lymphocytes almost three decades ago. This protein has a key function in regulating the human immune system, and its dysregulation has been linked to many chronic diseases including asthma, cancer, diabetes, rheumatoid arthritis, inflammation, and neurological disorders. Physiologically, many cytokines have been discovered that activate NF-κB. Pathologically, environmental carcinogens such as cigarette smoke, radiation, bacteria, and viruses can also activate this transcription factor. NF-κB activation controls expression of more than 500 genes, and most are deleterious to the human body when dysregulated. More than 70,000 articles have been published regarding NF-κB. This review emphasizes the upside and downside of NF-κB in normal and disease conditions and the ways in which we can control this critical transcription factor in patients.

Upside and Downside of Tumor Necrosis Factor Blockers for Treatment of Immune/Inflammatory Diseases.

Tumor necrosis factor (TNF)-α, the most potent proinflammatory cytokine discovered to date, was first isolated in 1984 from human macrophage cells. Initially, it was thought to be a protein that was cytotoxic to tumor cells. But later, it was regarded as an agent that promotes inflammation and other chronic diseases found in humans. Currently, we know that the TNF superfamily (TNFS) has 19 members that perform a wide variety of functions via > 40 TNF receptors. Of TNFS members, TNF-α has been studied extensively and was found to be implicated in numerous autoimmune diseases, such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriasis, systemic lupus erythematosus, juvenile idiopathic arthritis, and diabetes. Thus, agents that can inhibit TNF-α have great potential for prevention and treatment of chronic diseases. To date, the U.S. Food and Drug Administration has approved many TNF-α blockers, such as etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab. These agents can block TNF-α actions and be used to treat different diseases. However, the uses of TNF-α blockers are not without serious adverse effects. Therefore, natural TNF-α blockers are best for developing safe, efficacious, and affordable agents for prevention and treatment of chronic diseases. The current review details the TNFS, functions of TNF-α in normal and disease conditions, roles of TNF-α blockers, and advantages and disadvantages.

Attenuation of cocaine seeking in rats via enhancement of infralimbic cortical activity using stable step-function opsins.

RATIONALE: The infralimbic cortex (IL) and its downstream projection target the nucleus accumbens shell (NAshell) mediate the active suppression of cocaine-seeking behavior. Although an optogenetic approach would be beneficial for stimulating the IL and its efferents to study their role during reinstatement of cocaine seeking, the use of channelrhodopsin introduces significant difficulties, as optimal stimulation parameters are not known. OBJECTIVES: The present experiments utilized a stable step-function opsin (SSFO) to potentiate endogenous activity in the IL and in IL terminals in the NAshell during cocaine-seeking tests to determine how these manipulations affect cocaine-seeking behaviors. METHODS: Rats first underwent 6-h access cocaine self-administration followed by 21-27 days in the homecage. Rats then underwent cue-induced and cocaine-primed drug-seeking tests during which the optogenetic manipulation was given. The same rats then underwent extinction training, followed by cue-induced and cocaine-primed reinstatements. RESULTS: Potentiation of endogenous IL activity did not significantly alter cue-induced or cocaine-primed drug seeking following the homecage period. However, following extinction training, enhancement of endogenous IL activity attenuated cue-induced reinstatement by 35% and cocaine-primed reinstatement by 53%. Stimulation of IL terminals in the NAshell did not consistently alter cocaine-seeking behavior. CONCLUSION: These results suggest the utility of an SSFO-based approach for enhancing activity in a structure without driving specific patterns of neuronal firing. However, the utility of an SSFO-based approach for axon terminal stimulation remains unclear. Moreover, these results suggest that the ability of the IL to reduce cocaine seeking depends, at least in part, on rats first having undergone extinction training.

Extinction of Cocaine Seeking Requires a Window of Infralimbic Pyramidal Neuron Activity after Unreinforced Lever Presses.

The infralimbic cortex (IL) mediates extinction learning and the active suppression of cocaine-seeking behavior. However, the precise temporal relationship among IL activity, lever pressing, and extinction learning is unclear. To address this issue, we used activity-guided optogenetics in male Sprague Dawley rats to silence IL pyramidal neurons optically for 20 s immediately after unreinforced lever presses during early extinction training after cocaine self-administration. Optical inhibition of the IL increased active lever pressing during shortened extinction sessions, but did not alter the retention of the extinction learning as assessed in ensuing extinction sessions with no optical inhibition. During subsequent cued reinstatement sessions, rats that had previously received optical inhibition during the extinction sessions showed increased cocaine-seeking behavior. These findings appeared to be specific to inhibition during the post-lever press period because IL inhibition given in a noncontingent, pseudorandom manner during extinction sessions did not produce the same effects. Illumination alone (i.e., with no opsin expression) and food-seeking control experiments also failed to produce the same effects. In another experiment, IL inhibition after lever presses during cued reinstatement sessions increased cocaine seeking during those sessions. Finally, inhibition of the prelimbic cortex immediately after unreinforced lever presses during shortened extinction sessions decreased lever pressing during these sessions, but had no effect on subsequent reinstatement. These results indicate that IL activity immediately after unreinforced lever presses is necessary for normal extinction of cocaine seeking, suggesting that critical encoding of the new contingencies between a lever press and a cocaine reward occurs during that period.SIGNIFICANCE STATEMENT The infralimbic cortex (IL) contributes to the extinction of cocaine-seeking behavior, but the precise relationship among IL activity, lever pressing during extinction, and extinction learning has not been elucidated using traditional methods. Using a closed-loop optogenetic approach, we found that selective inhibition of the IL immediately after unreinforced lever pressing impaired within-session extinction learning and promoted the subsequent cued reinstatement of cocaine seeking. These studies suggest that IL activity immediately after the instrumental response during extinction learning of cocaine seeking encodes information required for such learning and that altering such activity produces long-lasting changes in subsequent measures of cocaine craving/relapse.

The NMDA receptor GluN2C subunit controls cortical excitatory-inhibitory balance, neuronal oscillations and cognitive function.

Despite strong evidence for NMDA receptor (NMDAR) hypofunction as an underlying factor for cognitive disorders, the precise roles of various NMDAR subtypes remains unknown. The GluN2C-containing NMDARs exhibit unique biophysical properties and expression pattern, and lower expression of GluN2C subunit has been reported in postmortem brains from schizophrenia patients. We found that loss of GluN2C subunit leads to a shift in cortical excitatory-inhibitory balance towards greater inhibition. Specifically, pyramidal neurons in the medial prefrontal cortex (mPFC) of GluN2C knockout mice have reduced mEPSC frequency and dendritic spine density and a contrasting higher frequency of mIPSCs. In addition a greater number of perisomatic GAD67 puncta was observed suggesting a potential increase in parvalbumin interneuron inputs. At a network level the GluN2C knockout mice were found to have a more robust increase in power of oscillations in response to NMDAR blocker MK-801. Furthermore, GluN2C heterozygous and knockout mice exhibited abnormalities in cognition and sensorimotor gating. Our results demonstrate that loss of GluN2C subunit leads to cortical excitatory-inhibitory imbalance and abnormal neuronal oscillations associated with neurodevelopmental disorders.

Glutamate Delta-1 Receptor Regulates Metabotropic Glutamate Receptor 5 Signaling in the Hippocampus.

The delta family of ionotropic glutamate receptors consists of glutamate delta-1 (GluD1) and glutamate delta-2 receptors. We have previously shown that GluD1 knockout mice exhibit features of developmental delay, including impaired spine pruning and switch in the N-methyl-D-aspartate receptor subunit, which are relevant to autism and other neurodevelopmental disorders. Here, we identified a novel role of GluD1 in regulating metabotropic glutamate receptor 5 (mGlu5) signaling in the hippocampus. Immunohistochemical analysis demonstrated colocalization of mGlu5 with GluD1 punctas in the hippocampus. Additionally, GluD1 protein coimmunoprecipitated with mGlu5 in the hippocampal membrane fraction, as well as when overexpressed in human embryonic kidney 293 cells, demonstrating that GluD1 and mGlu5 may cooperate in a signaling complex. The interaction of mGlu5 with scaffold protein effector Homer, which regulates mechanistic target of rapamycin (mTOR) signaling, was abnormal both under basal conditions and in response to mGlu1/5 agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) in GluD1 knockout mice. The basal levels of phosphorylated mTOR and protein kinase B, the signaling proteins downstream of mGlu5 activation, were higher in GluD1 knockout mice, and no further increase was induced by DHPG. We also observed higher basal protein translation and an absence of DHPG-induced increase in GluD1 knockout mice. In accordance with a role of mGlu5-mediated mTOR signaling in synaptic plasticity, DHPG-induced internalization of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits was impaired in the GluD1 knockout mice. These results demonstrate that GluD1 interacts with mGlu5, and loss of GluD1 impairs normal mGlu5 signaling potentially by dysregulating coupling to its effector. These studies identify a novel role of the enigmatic GluD1 subunit in hippocampal function.

Essential role of GluD1 in dendritic spine development and GluN2B to GluN2A NMDAR subunit switch in the cortex and hippocampus reveals ability of GluN2B inhibition in correcting hyperconnectivity.

The glutamate delta-1 (GluD1) receptor is highly expressed in the forebrain. We have previously shown that loss of GluD1 leads to social and cognitive deficits in mice, however, its role in synaptic development and neurotransmission remains poorly understood. Here we report that GluD1 is enriched in the medial prefrontal cortex (mPFC) and GluD1 knockout mice exhibit a higher dendritic spine number, greater excitatory neurotransmission as well as higher number of synapses in mPFC. In addition abnormalities in the LIMK1-cofilin signaling, which regulates spine dynamics, and a lower ratio of GluN2A/GluN2B expression was observed in the mPFC in GluD1 knockout mice. Analysis of the GluD1 knockout CA1 hippocampus similarly indicated the presence of higher spine number and synapses and altered LIMK1-cofilin signaling. We found that systemic administration of an N-methyl-d-aspartate (NMDA) receptor partial agonist d-cycloserine (DCS) at a high-dose, but not at a low-dose, and a GluN2B-selective inhibitor Ro-25-6981 partially normalized the abnormalities in LIMK1-cofilin signaling and reduced excess spine number in mPFC and hippocampus. The molecular effects of high-dose DCS and GluN2B inhibitor correlated with their ability to reduce the higher stereotyped behavior and depression-like behavior in GluD1 knockout mice. Together these findings demonstrate a critical requirement for GluD1 in normal spine development in the cortex and hippocampus. Moreover, these results identify inhibition of GluN2B-containing receptors as a mechanism for reducing excess dendritic spines and stereotyped behavior which may have therapeutic value in certain neurodevelopmental disorders such as autism.