ER-GCaMP6f: An Endoplasmic Reticulum-Targeted Genetic Probe to Measure Calcium Activity in Astrocytic Processes.

Ca2+ is a major second messenger involved in cellular and subcellular signaling in a wide range of cells, including astrocytes, which use calcium ions to communicate with other cells in the brain. Even though a variety of genetically encoded Ca2+ indicators have been developed to study astrocyte calcium signaling, understanding the dynamics of endoplasmic reticulum calcium signaling is greatly limited by the currently available tools. To address this, we developed an endoplasmic reticulum-targeted calcium indicator, ER-GCaMP6f, which is anchored to the cytosolic side of the organelle and measures signaling that occurs in close proximity to the endoplasmic reticulum of astrocytes. Using a combination of confocal and super-resolution microscopy techniques, we demonstrate the localization of the indicator in the endoplasmic reticulum in both cell soma and processes of astrocytes. Combining ER-GCaMP6f with total internal reflection fluorescence microscopy, we show that Ca2+ fluctuations in small astrocytic processes can be detected, which are otherwise not observable with existing indicators and standard wide-field and confocal techniques. We also compared the ER-GCaMP6f indicator against currently used plasma membrane-tethered and cytosolic GCaMP6f indicators. ER-GCaMP6f identifies dynamics in calcium signaling of endoplasmic reticulum resident receptors that are missed by plasma membrane-anchored indicators. We also generated an adeno-associated virus (AAV5) and demonstrate that ER-GCaMP6f can be expressed in vivo and by measured calcium activity in brain slices. ER-GCaMP6f provides a powerful tool to study calcium signaling in close proximity to the endoplasmic reticulum in astrocyte cell soma and processes both in culture and in brain slices.

Identification of Novel Protein Targets of Dimethyl Fumarate Modification in Neurons and Astrocytes Reveals Actions Independent of Nrf2 Stabilization.

The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown. Here, we investigated other intracellular thiol residues that may also be irreversibly modified by DMF in neurons and astrocytes. Using mass spectrometry, we identified 24 novel proteins that were modified by DMF in neurons and astrocytes, including cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2). Using an in vitro functional assay, we demonstrated that DMF-modified cofilin-1 loses its activity and generates less monomeric actin, potentially inhibiting its cytoskeletal remodeling activity, which could be beneficial in the modulation of myelination during RRMS. DMF modification of tubulin did not significantly impact axonal lysosomal trafficking. We found that the oxygen consumption rate of N1E-115 neurons and the levels of proteins related to mitochondrial energy production were only slightly affected by the highest doses of DMF, confirming that DMF treatment does not impair cellular respiratory function. In summary, our work provides new insights into the mechanisms supporting the neuroprotective and remyelination benefits associated with DMF treatment in addition to the antioxidant response by Nrf2.

[3H]Dopamine Uptake through the Dopamine and Norepinephrine Transporters is Decreased in the Prefrontal Cortex of Transgenic Mice Expressing HIV-1 Transactivator of Transcription Protein.

Dysregulation of dopamine neurotransmission has been linked to the development of human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND). To investigate the mechanisms underlying this phenomenon, this study used an inducible HIV-1 transactivator of transcription (Tat) transgenic (iTat-tg) mouse model, which demonstrates brain-specific Tat expression induced by administration of doxycycline. We found that induction of Tat expression in the iTat-tg mice for either 7 or 14 days resulted in a decrease (∼30%) in the V max of [3H]dopamine uptake via both the dopamine transporter (DAT) and norepinephrine transporter (NET) in the prefrontal cortex (PFC), which was comparable to the magnitude (∼35%) of the decrease in B max for [3H]WIN 35,428 and [3H]nisoxetine binding to DAT and NET, respectively. The decreased V max was not accompanied by a reduction of total or plasma membrane expression of DAT and NET. Consistent with the decreased V max for DAT and NET in the PFC, the current study also found an increase in the tissue content of DA and dihydroxyphenylacetic acid in the PFC of iTat-tg mice after 7 days' administration of doxycycline. Electrophysiological recordings in layer V pyramidal neurons of the prelimbic cortex from iTat-tg mice found a significant reduction in action potential firing, which was not sensitive to selective inhibitors for DAT and NET, respectively. These findings provide a molecular basis for using the iTat-tg mouse model in the studies of NeuroHIV. Determining the mechanistic basis underlying the interaction between Tat and DAT/NET may reveal novel therapeutic possibilities for preventing the increase in comorbid conditions as well as HAND. SIGNIFICANCE STATEMENT: Human immunodeficiency virus (HIV)-1 infection disrupts dopaminergic neurotransmission, leading to HIV-associated neurocognitive disorders (HANDs). Based on our in vitro and in vivo studies, dopamine uptake via both dopamine and norepinephrine transporters is decreased in the prefrontal cortex of HIV-1 Tat transgenic mice, which is consistent with the increased dopamine and dihydroxyphenylacetic acid contents in this brain region. Thus, these plasma membrane transporters are an important potential target for therapeutic intervention for patients with HAND.

Dopamine Triggers CTCF-Dependent Morphological and Genomic Remodeling of Astrocytes.

Dopamine is critical for processing of reward and etiology of drug addiction. Astrocytes throughout the brain express dopamine receptors, but consequences of astrocytic dopamine receptor signaling are not well established. We found that extracellular dopamine triggered rapid concentration-dependent stellation of astrocytic processes that was not a result of dopamine oxidation but instead relied on both cAMP-dependent and cAMP-independent dopamine receptor signaling. This was accompanied by reduced duration and increased frequency of astrocytic Ca2+ transients, but little effect on astrocytic voltage-gated potassium channel currents. To isolate possible mechanisms underlying these structural and functional changes, we used whole-genome RNA sequencing and found prominent dopamine-induced enrichment of genes containing the CCCTC-binding factor (CTCF) motif, suggesting involvement of chromatin restructuring in the nucleus. CTCF binding to promoter sites bidirectionally regulates gene transcription and depends on activation of poly-ADP-ribose polymerase 1 (PARP1). Accordingly, antagonism of PARP1 occluded dopamine-induced changes, whereas a PARP1 agonist facilitated dopamine-induced changes on its own. These results indicate that astrocyte response to elevated dopamine involves PARP1-mediated CTCF genomic restructuring and concerted expression of gene networks. Our findings propose epigenetic regulation of chromatin landscape as a critical factor in the rapid astrocyte response to dopamine.SIGNIFICANCE STATEMENT Although dopamine is widely recognized for its role in modulating neuronal responses both in healthy and disease states, little is known about dopamine effects at non-neuronal cells in the brain. To address this gap, we performed whole-genome sequencing of astrocytes exposed to elevated extracellular dopamine and combined it with evaluation of effects on astrocyte morphology and function. We demonstrate a temporally dynamic pattern of genomic plasticity that triggers pronounced changes in astrocyte morphology and function. We further show that this plasticity depends on activation of genes sensitive to DNA-binding protein CTCF. Our results propose that a broad pattern of astrocyte responses to dopamine specifically relies on CTCF-dependent gene networks.

Activation of glucagon-like peptide-1 receptors in the nucleus accumbens attenuates cocaine seeking in rats.

Recent evidence indicates that activation of glucagon-like peptide-1 (GLP-1) receptors reduces cocaine-mediated behaviors and cocaine-evoked dopamine release in the nucleus accumbens (NAc). However, no studies have examined the role of NAc GLP-1 receptors in the reinstatement of cocaine-seeking behavior, an animal model of relapse. Here, we show that systemic infusion of a behaviorally relevant dose of the GLP-1 receptor agonist exendin-4 penetrated the brain and localized with neurons and astrocytes in the NAc. Administration of exendin-4 directly into the NAc core and shell subregions significantly attenuated cocaine priming-induced reinstatement of drug-seeking behavior. These effects were not due to deficits in operant responding or suppression of locomotor activity as intra-accumbal exendin-4 administration had no effect on sucrose-seeking behavior. To determine the effects of GLP-1 receptor activation on neuronal excitability, exendin-4 was bath applied to ex vivo NAc slices from cocaine-experienced and saline-experienced rats following extinction of cocaine-taking behavior. Exendin-4 increased the frequency of action potential firing of NAc core and shell medium spiny neurons in cocaine-experienced rats while no effect was observed in saline controls. In contrast, exendin-4 did not affect the frequency or amplitude of spontaneous excitatory postsynaptic currents or alter the paired-pulse ratios of evoked excitatory postsynaptic currents. These effects were not associated with altered expression of GLP-1 receptors in the NAc following cocaine self-administration. Taken together, these findings indicate that increased activation of GLP-1 receptors in the NAc during cocaine abstinence increases intrinsic, but not synaptic, excitability of medium spiny neurons and is sufficient to reduce cocaine-seeking behavior.

Glucagon-like peptide-1 receptor activation in the nucleus accumbens core suppresses feeding by increasing glutamatergic AMPA/kainate signaling.

Glucagon-like peptide-1 receptor (GLP-1R) activation in the nucleus accumbens (NAc) core is pharmacologically and physiologically relevant for regulating palatable food intake. Here, we assess whether GLP-1R signaling in the NAc core of rats modulates GABAergic medium spiny neurons (MSNs) through presynaptic-glutamatergic and/or presynaptic-dopaminergic signaling to control feeding. First, ex vivo fast-scan cyclic voltammetry showed that the GLP-1R agonist exendin-4 (Ex-4) does not alter dopamine release in the NAc core. Instead, support for a glutamatergic mechanism was provided by ex vivo electrophysiological analyses showing that Ex-4 activates presynaptic GLP-1Rs in the NAc core to increase the activity of MSNs via a glutamatergic, AMPA/kainate receptor-mediated mechanism, indicated by increased mEPSC frequency and decreased paired pulse ratio in core MSNs. Only a small, direct excitatory effect on MSNs by Ex-4 was observed, suggesting that the contribution of postsynaptic GLP-1R to MSN activity is minimal. The behavioral relevance of the electrophysiological data was confirmed by the finding that intracore injection of the AMPA/kainate receptor antagonist CNQX attenuated the ability of NAc core GLP-1R activation by Ex-4 microinjection to suppress food intake and body weight gain; in contrast, intracore NMDA receptor blockade by AP-5 did not inhibit the energy balance effects of NAc core Ex-4. Together, these data provide evidence for a novel glutamatergic, but not dopaminergic, mechanism by which NAc core GLP-1Rs promote negative energy balance.

Extrasynaptic targeting of NMDA receptors following D1 dopamine receptor activation and cocaine self-administration.

We previously showed that after repeated exposure to cocaine, D1-like dopamine receptor (D1DR) stimulation reverses plastic changes of AMPA receptor-mediated signaling in the nucleus accumbens shell. However, there is little information on the impact of cocaine self-administration on D1-NMDA receptor interactions in this brain region. Here, using whole-cell patch-clamp recordings, we assessed whether cocaine self-administration alters the effects of D1DR stimulation on synaptic and extrasynaptic NMDA receptors (NMDARs). In slices from cocaine-naive rats, pretreatment with a D1DR agonist decreased synaptic NMDAR-mediated currents and increased the contribution of extrasynaptic NMDARs. In contrast, neither cocaine self-administration alone nor cocaine experience followed by D1DR stimulation had an effect on synaptic or extrasynaptic NMDAR signaling. Activation of extrasynaptic NMDARs relies on the availability of extracellular glutamate, which is regulated primarily by glutamate transporters. In cocaine-experienced animals, relative to cocaine-naive rats, administration of a glutamate reuptake blocker, DL-threo-ß-benzyloxyaspartic acid, revealed increased extrasynaptic NMDAR activity and stronger baseline activity of glutamate uptake transporters. In cocaine-naive rats, the D1DR-mediated increase in extrasynaptic NMDAR signaling was independent of the activity of glutamate reuptake transporters. Together, these results indicate that cocaine experience blunts the influence of D1DRs on synaptic and extrasynaptic NMDAR signaling. Additionally, prior cocaine self-administration limits activation of the extrasynaptic NMDAR pool by increasing glutamate reuptake. These findings outline a pattern of adaptive interactions between D1DRs and NMDARs in the nucleus accumbens shell and demonstrate upregulation of extrasynaptic NMDAR signaling as a novel consequence of cocaine self-administration.

Dynamic effects of ventral hippocampal NRG3/ERBB4 signaling on nicotine withdrawal-induced responses.

Tobacco smoking remains a leading cause of preventable death in the United States, with approximately a 5% success rate for smokers attempting to quit. High relapse rates have been linked to several genetic factors, indicating that the mechanistic relationship between genes and drugs of abuse is a valuable avenue for the development of novel smoking cessation therapies. For example, various single nucleotide polymorphisms (SNPs) in the gene for neuregulin 3 (NRG3) and its cognate receptor, the receptor tyrosine-protein kinase erbB-4 (ERBB4), have been linked to nicotine addiction. Our lab has previously shown that ERBB4 plays a role in anxiety-like behavior during nicotine withdrawal (WD); however, the neuronal mechanisms and circuit-specific effects of NRG3-ERBB4 signaling during nicotine and WD are unknown. The present study utilizes genetic, biochemical, and functional approaches to examine the anxiety-related behavioral and functional role of NRG3-ERBB4 signaling, specifically in the ventral hippocampus (VH) of male and female mice. We report that 24hWD from nicotine is associated with altered synaptic expression of VH NRG3 and ERBB4, and genetic disruption of VH ErbB4 leads to an elimination of anxiety-like behaviors induced during 24hWD. Moreover, we observed attenuation of GABAergic transmission as well as alterations in Ca2+-dependent network activity in the ventral CA1 area of VH ErbB4 knock-down mice during 24hWD. Our findings further highlight contributions of the NRG3-ERBB4 signaling pathway to anxiety-related behaviors seen during nicotine WD.

Voltage-gated potassium channels control extended access cocaine seeking: a role for nucleus accumbens astrocytes.

Dopaminergic signaling in the nucleus accumbens shell (NAc) regulates neuronal activity relevant to reward-related learning, including cocaine-associated behaviors. Although astrocytes respond to dopamine and cocaine with structural changes, the impact of dopamine and cocaine on astrocyte functional plasticity has not been widely studied. Specifically, behavioral implications of voltage-gated channel activity in the canonically non-excitable astrocytes are not known. We characterized potassium channel function in NAc astrocytes following exposure to exogenous dopamine or cocaine self-administration training under short (2 h/day) and extended (6 h/day) access schedules. Electrophysiological, Ca2+ imaging, mRNA, and mass spectrometry tools were used for molecular characterization. Behavioral effects were examined after NAc-targeted microinjections of channel antagonists and astroglial toxins. Exogenous dopamine increased activity of currents mediated by voltage-gated (Kv7) channels in NAc astrocytes. This was associated with a ~5-fold increase in expression of Kcnq2 transcript level in homogenized NAc micropunches. Matrix-assisted laser desorption/ionization mass spectrometry revealed increased NAc dopamine levels in extended access, relative to short access, rats. Kv7 inhibition selectively increased frequency and amplitude of astrocyte intracellular Ca2+ transients in NAc of extended access rats. Inhibition of Kv7 channels in the NAc attenuated cocaine-seeking in extended access rats only, an effect that was occluded by microinjection of the astrocyte metabolic poison, fluorocitrate. These results suggest that voltage-gated K+ channel signaling in NAc astrocytes is behaviorally relevant, support Kv7-mediated regulation of astrocyte Ca2+ signals, and propose novel mechanisms of neuroglial interactions relevant to drug use.

Deterministic and stochastic neuronal contributions to distinct synchronous CA3 network bursts.

Computational studies have suggested that stochastic, deterministic, and mixed processes all could be possible determinants of spontaneous, synchronous network bursts. In the present study, using multicellular calcium imaging coupled with fast confocal microscopy, we describe neuronal behavior underlying spontaneous network bursts in developing rat and mouse hippocampal area CA3 networks. Two primary burst types were studied: giant depolarizing potentials (GDPs) and spontaneous interictal bursts recorded in bicuculline, a GABA(A) receptor antagonist. Analysis of the simultaneous behavior of multiple CA3 neurons during synchronous GDPs revealed a repeatable activation order from burst to burst. This was validated using several statistical methods, including high Kendall's coefficient of concordance values for firing order during GDPs, high Pearson's correlations of cellular activation times between burst pairs, and latent class analysis, which revealed a population of 5-6% of CA3 neurons reliably firing very early during GDPs. In contrast, neuronal firing order during interictal bursts appeared homogeneous, with no particular cells repeatedly leading or lagging during these synchronous events. We conclude that GDPs activate via a deterministic mechanism, with distinct, repeatable roles for subsets of neurons during burst generation, while interictal bursts appear to be stochastic events with cells assuming interchangeable roles in the generation of these events.

The food intake-suppressive effects of glucagon-like peptide-1 receptor signaling in the ventral tegmental area are mediated by AMPA/kainate receptors.

Glucagon-like peptide-1 receptor (GLP-1R) activation in the ventral tegmental area (VTA) is physiologically relevant for the control of palatable food intake. Here, we tested whether the food intake-suppressive effects of VTA GLP-1R activation are mediated by glutamatergic signaling within the VTA. Intra-VTA injections of the GLP-1R agonist exendin-4 (Ex-4) reduced palatable high-fat food intake in rats primarily by reducing meal size; these effects were mediated in part via glutamatergic AMPA/kainate but not NMDA receptor signaling. Additional behavioral data indicated that GLP-1R expressed specifically within the VTA can partially mediate the intake- and body weight-suppressive effects of systemically administered Ex-4, offering the intriguing possibility that this receptor population may be clinically relevant for food intake control. Intra-VTA Ex-4 rapidly increased tyrosine hydroxylase levels within the VTA, suggesting that GLP-1R activation modulates VTA dopaminergic signaling. Further evidence for this hypothesis was provided by electrophysiological data showing that Ex-4 increased the frequency of AMPA-mediated currents and reduced the paired/pulse ratio in VTA dopamine neurons. Together, these data provide novel mechanisms by which GLP-1R agonists in the mesolimbic reward system control for palatable food intake.

Dynamic Effects of Ventral Hippocampal NRG3/ERBB4 Signaling on Nicotine Withdrawal-Induced Responses.

Tobacco smoking remains a leading cause of preventable death in the United States, with a less than 5% success rate for smokers attempting to quit. High relapse rates have been linked to several genetic factors, indicating that the mechanistic relationship between genes and drugs of abuse is a valuable avenue for the development of novel smoking cessation therapies. For example, various single nucleotide polymorphisms (SNPs) in the gene for neuregulin 3 (NRG3) and its cognate receptor, the receptor tyrosine-protein kinase erbB-4 (ERBB4), have been linked to nicotine addiction. Our lab has previously shown that ERBB4 plays a role in anxiety-like behavior during nicotine withdrawal (WD); however, the neuronal mechanisms and circuit-specific effects of NRG3-ERBB4 signaling during nicotine and WD are unknown. The present study utilizes genetic, biochemical, and functional approaches to examine the anxiety-related behavioral and functional role of NRG3-ERBB4 signaling, specifically in the ventral hippocampus (VH). We report that 24hWD from nicotine is associated with altered synaptic expression of VH NRG3 and ERBB4, and genetic disruption of VH ErbB4 leads to an elimination of anxiety-like behaviors induced during 24hWD. Moreover, we observed attenuation of GABAergic transmission as well as alterations in Ca2+-dependent network activity in the ventral CA1 area of VH ErbB4 knock-down mice during 24hWD. Our findings further highlight contributions of the NRG3-ERBB4 signaling pathway to anxiety-related behaviors seen during nicotine WD.

Study of Calcium Signaling in Astrocytes with a Novel Endoplasmic Reticulum-Targeted GCaMP Sensor.

The endoplasmic reticulum (ER), the major organelle for the storage of Ca2+ , maintains a concentration of Ca2+ much higher than in the cytosol or other subcellular organelles, such as the mitochondria. A variety of tools have been developed for measuring Ca2+ activity in neuronal and glial cells, but most of these sensors target either the plasma membrane (PM) or the cytosol. Though these sensors are important for measuring Ca2+ transients, they lack the capability to measure activity in the periphery of the ER or to measure low-amplitude events resulting from Ca2+ exchange between the ER and other organelles, such as the mitochondria. We recently developed an ER-targeted GCaMP6f anchored to the cytosolic side of the ER that can measure minute calcium exchange occurring in this region. In this article, we discuss detailed methods to characterize the ER-GCaMP6f sensor, utilize it for calcium imaging in cultured astrocytes, and assess its expression and calcium imaging in astrocytes in rodent brains. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expression and characterization of ER-GCaMP6f Support Protocol 1: ER-GCaMP6f-expressing stable cell line generation Basic Protocol 2: In vitro calcium imaging with ER-GCaMP6f Support Protocol 2: Imaging of drug-induced calcium activity Alternate Protocol 1: Transduction of astrocytes with ER-GCaMP6f AAV Alternate Protocol 2: Calcium imaging of astrocytes with Fluo-4 AM Basic Protocol 3: In vivo ER-GCaMP6f expression and slice calcium imaging Support Protocol 3: Pharmacological studies with 2-APB in brain slices.

Cerebellar nicotinic cholinergic receptors are intrinsic to the cerebellum: implications for diverse functional roles.

Although recent studies have delineated the specific nicotinic subtypes present in the mammalian cerebellum, very little is known about their location or function within the cerebellum. This is of increased interest since nicotinic receptors (nAChRs) in the cerebellum have recently been implicated in the pathology of autism spectrum disorders. To begin to better understand the roles of these heteromeric nAChRs in the cerebellar circuitry and their therapeutic potential as targets for drug development, we used various chemical and stereotaxic lesion models in conjunction with slice electrophysiology to examine how specific heteromeric nAChR subtypes may influence the surrounding cerebellar circuitry. Using subunit-specific immunoprecipitation of radiolabeled nAChRs in the cerebella following N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride, p-chloroamphetamine, and pendunculotomy lesions, we show that most, if not all, cerebellar nicotinic receptors are present in cells within the cerebellum itself and not in extracerebellar afferents. Furthermore, we demonstrate that the ß4-containing, but not the ß2-containing, nAChRs intrinsic to the cerebellum can regulate inhibitory synaptic efficacy at two major classes of cerebellar neurons. These tandem findings suggest that nAChRs may present a potential drug target for disorders involving the cerebellum.

Differential tonic GABA conductances in striatal medium spiny neurons.

Medium spiny neurons (MSNs) provide the principal output for the dorsal striatum. Those that express dopamine D2 receptors (D2+) project to the globus pallidus external and are thought to inhibit movement, whereas those that express dopamine D1 receptors (D1+) project to the substantia nigra pars reticulata and are thought to facilitate movement. Whole-cell and outside-out patch recordings in slices from bacterial artificial chromosome transgenic mice examined the role of GABA(A) receptor-mediated currents in dopamine receptor D1+ striatonigral and D2+ striatopallidal MSNs. Although inhibitory synaptic currents were similar between the two neuronal populations, D2+ MSNs showed greater GABA(A) receptor-mediated tonic currents. TTX application abolished the tonic current to a similar extent as GABA(A) antagonists, suggesting a synaptic origin of the ambient GABA. Low GABA concentrations produced larger whole-cell responses and longer GABA channel openings in D2+ than in D1+ MSNs. Recordings from MSNs in alpha1-/- mice and pharmacological analysis of tonic currents suggested greater expression of alpha5-containing GABA(A) receptors in D2+ than in D1+ MSNs. As a number of disorders such as Parkinson's disease, Huntington's chorea, and tardive dyskinesia arise from an imbalance between these two pathways, the GABA(A) receptors responsible for tonic currents in D2+ MSNs may be a potential target for therapeutic intervention.

Altered gating of Kv1.4 in the nucleus accumbens suppresses motivation for reward.

Deficient motivation contributes to numerous psychiatric disorders, including withdrawal from drug use, depression, schizophrenia, and others. Nucleus accumbens (NAc) has been implicated in motivated behavior, but it remains unclear whether motivational drive is linked to discrete neurobiological mechanisms within the NAc. To examine this, we profiled cohorts of Sprague-Dawley rats in a test of motivation to consume sucrose. We found that substantial variability in willingness to exert effort for reward was not associated with operant responding under low-effort conditions or stress levels. Instead, effort-based motivation was mirrored by a divergent NAc shell transcriptome with differential regulation at potassium and dopamine signaling genes. Functionally, motivation was inversely related to excitability of NAc principal neurons. Furthermore, neuronal and behavioral outputs associated with low motivation were linked to faster inactivation of a voltage-gated potassium channel, Kv1.4. These results raise the prospect of targeting Kv1.4 gating in psychiatric conditions associated with motivational dysfunction.

Microglia morphology and proinflammatory signaling in the nucleus accumbens during nicotine withdrawal.

Smoking is the largest preventable cause of death and disease in the United States. However, <5% of quit attempts are successful, underscoring the urgent need for novel therapeutics. Microglia are one untapped therapeutic target. While previous studies have shown that microglia mediate both inflammatory responses in the brain and brain plasticity, little is known regarding their role in nicotine dependence and withdrawal phenotypes. Here, we examined microglial changes in the striatum-a mesolimbic region implicated in the rewarding effects of drugs and the affective disruptions occurring during withdrawal. We show that both nicotine and withdrawal induce microglial morphological changes; however, proinflammatory effects and anxiogenic behaviors were observed only during nicotine withdrawal. Pharmacological microglial depletion during withdrawal prevented these effects. These results define differential effects of nicotine and withdrawal on inflammatory signaling in the brain, laying the groundwork for development of future smoking cessation therapeutics.

Neuregulin 3 Signaling Mediates Nicotine-Dependent Synaptic Plasticity in the Orbitofrontal Cortex and Cognition.

Neuregulin 3 (NRG3) and ErbB4 have been linked to nicotine addiction; however, the neuronal mechanisms and behavioral consequences of NRG3-ErbB4 sensitivity to nicotine remain elusive. Recent literature suggests that relapse to smoking is due to a lack of impulsive control, which is thought to be due to altered functioning within the orbitofrontal cortex (OFC). Therefore, we examined circuitry changes within this structure following nicotine application. We report that nicotine controls synaptic plasticity in the OFC through NRG3/ErbB4-dependent regulation of GABAergic inhibition. We observed that both nicotine and NRG3 facilitated the conversion of long-term potentiation into long-term depression at cortical layer 3/5 synapses. Induction of long-term depression by nicotine relied on nicotinic receptor activation and key regulators of NRG3 signaling: (1) release of intracellular calcium, (2) activation of the BACE1 beta-secretase, and (3) ErbB4 receptor activation. Nicotine-induced synaptic plasticity was also associated with accumulation of intracellular GABA and was completely blocked by GABAA/GABAB antagonists. To test whether these mechanisms underlie OFC-dependent behavior, we evaluated the effects of nicotine in the go/no-go task. Nicotine-impaired stimulus discrimination in this task was rescued by pharmacologic disruption of the NRG3 receptor, ErbB4. Altogether, our data indicate that nicotine-induced synaptic plasticity in the OFC and cognitive changes depend on NRG3-ErbB4 signaling. We propose that nicotine activation of this pathway may contribute to nicotine addiction, particularly in individuals with genetic variation in NRG3.

Deletion of the GABA(A) receptor alpha1 subunit increases tonic GABA(A) receptor current: a role for GABA uptake transporters.

The loss of more than half the number of GABA(A) receptors yet lack of pronounced phenotype in mice lacking the gene for the GABA(A) alpha1 subunit is somewhat paradoxical. We explored the role of tonic GABA(A) receptor-mediated current as a target of compensatory regulation in the alpha1 knock-out (-/-) mice. A 62% increase of tonic current was observed in the cerebellar granule cells (CGCs) of alpha1-/- compared with wild-type (+/+) mice along with a 67% increase of baseline current variance. Examination of whole-cell currents evoked by low concentrations of GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol suggested no upregulation of alpha6 and delta subunit-containing GABA(A) receptors in the alpha1-/-, confirming previous biochemical studies. Single-channel current openings were on average 32% shorter in the alpha1-/- neurons. Single-channel conductance and frequency of opening were not different between genotypes. Tonic current induced by application of the GABA transporter GAT-1 blocker NO711 (1-[2([(diphenylmethylene)imino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride) was significantly larger in the alpha1-/-, suggesting an increase of ambient GABA concentration. Experiments done with a known concentration of extracellular GABA complemented by a series of biochemical experiments revealed a reduction of GAT activity in alpha1-/- without an identifiable reduction of GAT-1 or GAT-3 protein. We report increased tonic GABA(A) receptor-mediated current in the alpha1-/- CGCs as a novel compensatory mechanism. Our data establish a role for GABA transporters as regulators of neuronal excitability in this and relevant models and examine other tonic conductance-regulating mechanisms responsible for the adaptive response of the cerebellar network to a deletion of a major synaptic GABA(A) receptor subunit.