Chronic ethanol induces a pro-inflammatory switch in interleukin-1ß regulation of GABAergic signaling in the medial prefrontal cortex of male mice.

Neuroimmune pathways regulate brain function to influence complex behavior and play a role in several neuropsychiatric diseases, including alcohol use disorder (AUD). In particular, the interleukin-1 (IL-1) system has emerged as a key regulator of the brain's response to ethanol (alcohol). Here we investigated the mechanisms underlying ethanol-induced neuroadaptation of IL-1ß signaling at GABAergic synapses in the prelimbic region of the medial prefrontal cortex (mPFC), an area responsible for integrating contextual information to mediate conflicting motivational drives. We exposed C57BL/6J male mice to the chronic intermittent ethanol vapor-2 bottle choice paradigm (CIE-2BC) to induce ethanol dependence, and conducted ex vivo electrophysiology and molecular analyses. We found that the IL-1 system regulates basal mPFC function through its actions at inhibitory synapses on prelimbic layer 2/3 pyramidal neurons. IL-1ß can selectively recruit either neuroprotective (PI3K/Akt) or pro-inflammatory (MyD88/p38 MAPK) mechanisms to produce opposing synaptic effects. In ethanol naïve conditions, there was a strong PI3K/Akt bias leading to a disinhibition of pyramidal neurons. Ethanol dependence produced opposite IL-1 effects - enhanced local inhibition via a switch in IL-1ß signaling to the canonical pro-inflammatory MyD88 pathway. Ethanol dependence also increased cellular IL-1ß in the mPFC, while decreasing expression of downstream effectors (Akt, p38 MAPK). Thus, IL-1ß may represent a key neural substrate in ethanol-induced cortical dysfunction. As the IL-1 receptor antagonist (kineret) is already FDA-approved for other diseases, this work underscores the high therapeutic potential of IL-1 signaling/neuroimmune-based treatments for AUD.

Role of TLR4 in the Modulation of Central Amygdala GABA Transmission by CRF Following Restraint Stress.

AIMS: Stress induces neuroimmune responses via Toll-like receptor 4 (TLR4) activation. Here, we investigated the role of TLR4 in the effects of the stress peptide corticotropin-releasing factor (CRF) on GABAergic transmission in the central nucleus of the amygdala (CeA) following restraint stress. METHODS: Tlr4 knock out (KO) and wild-type rats were exposed to no stress (naïve), a single restraint stress (1 h) or repeated restraint stress (1 h per day for 3 consecutive days). After 1 h recovery from the final stress session, whole-cell patch-clamp electrophysiology was used to investigate the effects of CRF (200 nM) on CeA GABAA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs). RESULTS: TLR4 does not regulate baseline GABAergic transmission in the CeA of naive and stress-treated animals. However, CRF significantly increased the mean sIPSC frequencies (indicating enhanced GABA release) across all genotypes and stress treatments, except for the Tlr4 KO rats that experienced repeated restraint stress. CONCLUSIONS: Overall, our results suggest a limited role for TLR4 in CRF's modulation of CeA GABAergic synapses in naïve and single stress rats, though TLR4-deficient rats that experienced repeated psychological stress exhibit a blunted CRF cellular response. SHORT SUMMARY: TLR4 has a limited role in CRF's activation of the CeA under basal conditions, but interacts with the CRF system to regulate GABAergic synapse function in animals that experience repeated psychological stress.

Role of the IL-1 receptor antagonist in ethanol-induced regulation of GABAergic transmission in the central amygdala.

The IL-1 receptor antagonist (IL-1ra), encoded by the Il1rn gene, is an endogenous antagonist of the IL-1 receptor. Studies of Il1rn knockout (KO) and wild type (WT) mice identified differences in several ethanol-related behaviors, some of which may be mediated by GABAergic transmission in the central nucleus of the amygdala (CeA). In this study we examined phasic (both evoked and spontaneous) and tonic GABAergic transmission in the CeA of Il1rn KO and WT mice and the ethanol sensitivity of these GABAergic synapses. The mean amplitude of baseline evoked GABAA-inhibitory postsynaptic potentials (IPSPs), and the baseline frequency of spontaneous GABAA-inhibitory postsynaptic currents (sIPSCs), but not the frequency of miniature GABAA-IPSCs (mIPSCs), were significantly increased in KO compared to WT mice, indicating enhanced presynaptic action potential-dependent GABA release in the CeA of KO mice. In KO mice, we also found a cell-type specific switch in the ongoing tonic GABAA receptor conductance such that the tonic conductance in low threshold bursting (LTB) neurons is lost and a tonic conductance in late spiking (LS) neurons appears. Notably, the ethanol-induced facilitation of evoked and spontaneous GABA release was lost in most of the CeA neurons from KO compared to WT mice. Ethanol superfusion increased the sIPSC rise and decay times in both KO and WT mice, suggesting ethanol-induced postsynaptic effects. The pretreatment of CeA slices with exogenous IL-1ra (Kineret; 100ng/ml) returned sIPSC frequency in KO mice to the levels found in WT. Importantly, Kineret also restored ethanol-induced potentiation of the sIPSC frequency in the KO mice. These results show that IL-1ra regulates baseline GABAergic transmission in the CeA and is critical for the ethanol effects at these synapses.

Chronic ethanol alters adrenergic receptor gene expression and produces cognitive deficits in male mice.

Hyperkateifia and stress-induced alcohol cravings drive relapse in individuals with alcohol use disorder (AUD). The brain stress signal norepinephrine (also known as noradrenaline) tightly controls cognitive and affective behavior and was thought to be broadly dysregulated with AUD. The locus coeruleus (LC) is a major source of forebrain norepinephrine, and it was recently discovered that the LC sends distinct projections to addiction-associated regions suggesting that alcohol-induced noradrenergic changes may be more brain region-specific than originally thought. Here we investigated whether ethanol dependence alters adrenergic receptor gene expression in the medial prefrontal cortex (mPFC) and central amgydala (CeA), as these regions mediate the cognitive impairment and negative affective state of ethanol withdrawal. We exposed male C57BL/6J mice to the chronic intermittent ethanol vapor-2 bottle choice paradigm (CIE-2BC) to induce ethanol dependence, and assessed reference memory, anxiety-like behavior and adrenergic receptor transcript levels during 3-6 days of withdrawal. Dependence bidirectionally altered mouse brain α1 and ß receptor mRNA levels, potentially leading to reduced mPFC adrenergic signaling and enhanced noradrenergic influence over the CeA. These brain region-specific gene expression changes were accompanied by long-term retention deficits and a shift in search strategy in a modified Barnes maze task, as well as greater spontaneous digging behavior and hyponeophagia. Current clinical studies are evaluating adrenergic compounds as a treatment for AUD-associated hyperkatefia, and our findings can contribute to the refinement of these therapies by increasing understanding of the specific neural systems and symptoms that may be targeted.

PACAP regulation of central amygdala GABAergic synapses is altered by restraint stress.

The pituitary adenylate cyclase-activating polypeptide (PACAP) system plays a central role in the brain's emotional response to psychological stress by activating cellular processes and circuits associated with threat exposure. The neuropeptide PACAP and its main receptor PAC1 are expressed in the rodent central amygdala (CeA), a brain region critical in negative emotional processing, and CeA PACAPergic signaling drives anxiogenic and stress coping behaviors. Despite this behavioral evidence, PACAP's effects on neuronal activity within the medial subdivision of the CeA (CeM, the major output nucleus for the entire amygdala complex) during basal conditions and after psychological stress remain unknown. Therefore, in the present study, male Wistar rats were subjected to either restraint stress or control conditions, and PACAPergic regulation of CeM cellular function was assessed using immunohistochemistry and whole-cell patch-clamp electrophysiology. Our results demonstrate that PACAP-38 potentiates GABA release in the CeM of naïve rats, via its actions at presynaptic PAC1. Basal PAC1 activity also enhances GABA release in an action potential-dependent manner. Notably, PACAP-38's facilitation of CeM GABA release was attenuated after a single restraint stress session, but after repeated sessions returned to the level observed in naïve animals. A single restraint session also significantly decreased PAC1 levels in the CeM, with repeated restraint sessions producing a slight recovery. Collectively our data reveal that PACAP/PAC1 signaling enhances inhibitory control of the CeM and that psychological stress can modulate this influence to potentially disinhibit downstream effector regions that mediate anxiety and stress-related behaviors. This article is part of the special issue on 'Neuropeptides'.

The role of transcription factors cyclic-AMP responsive element modulator (CREM) and inducible cyclic-AMP early repressor (ICER) in epileptogenesis.

Alterations in the brain that contribute to the development of epilepsy, also called epileptogenesis, are not well understood, which makes it difficult to develop strategies for preventing epilepsy. Here we have studied the role of the CRE binding transcription factors, cyclic-AMP responsive element modulator (CREM) and inducible cyclic-AMP early repressor (ICER), in the development of epilepsy following pilocarpine induced status epilepticus (SE) in mice. Following SE, ICER mRNA and protein are increased in neurons. The increase in ICER, however, is not necessary for neuronal injury following SE as pilocarpine treatment induces equivalent neuronal injury in pyramidal neurons of wild type and CREM/ICER null mice. Following SE, the CREM/ICER null mice develop a more severe epileptic phenotype experiencing approximately threefold more frequent spontaneous seizures. Together these data suggest that the increase in ICER mRNA following SE may have a role in suppressing the severity of epilepsy.

P/Q-type voltage-gated calcium channels mediate the ethanol and CRF sensitivity of central amygdala GABAergic synapses.

The central amygdala (CeA) GABAergic system is hypothesized to drive the development of alcohol dependence, due to its pivotal roles in the reinforcing actions of alcohol and the expression of negative emotion, anxiety and stress. Recent work has also identified an important role for the CeA corticotropin-releasing factor (CRF) system in the interaction between anxiety/stress and alcohol dependence. We have previously shown that acute alcohol and CRF each increase action potential-independent GABA release in the CeA via their actions at presynaptic CRF type 1 receptors (CRF1s); however, the shared mechanism employed by these two compounds requires further investigation. Here we report that acute alcohol interacts with the CRF/CRF1 system, such that CRF and alcohol act via presynaptic CRF1s and P/Q-type voltage-gated calcium channels to promote vesicular GABA release and that both compounds occlude the effects of each other at these synapses. Chronic alcohol exposure does not alter P/Q-type voltage-gated calcium channel membrane abundance or this CRF1/P/Q-type voltage-gated calcium channel mechanism of acute alcohol-induced GABA release, indicating that alcohol engages this molecular mechanism at CeA GABAergic synapses throughout the transition to dependence. Thus, P/Q-type voltage-gated calcium channels, like CRF1s, are key regulators of the effects of alcohol on GABAergic signaling in the CeA.

Alcohol induces synaptotagmin 1 expression in neurons via activation of heat shock factor 1.

Many synapses within the central nervous system are sensitive to ethanol. Although alcohol is known to affect the probability of neurotransmitter release in specific brain regions, the effects of alcohol on the underlying synaptic vesicle fusion machinery have been little studied. To identify a potential pathway by which ethanol can regulate neurotransmitter release, we investigated the effects of acute alcohol exposure (1-24 h) on the expression of the gene encoding synaptotagmin 1 (Syt1), a synaptic protein that binds calcium to directly trigger vesicle fusion. Syt1 was identified in a microarray screen as a gene that may be sensitive to alcohol and heat shock. We found that Syt1 mRNA and protein expression are rapidly and robustly up-regulated by ethanol in mouse cortical neurons, and that the distribution of Syt1 protein along neuronal processes is also altered. Syt1 mRNA up-regulation is dependent on the activation of the transcription factor heat shock factor 1 (HSF1). The transfection of a constitutively active Hsf1 construct into neurons stimulates Syt1 transcription, while transfection of Hsf1 small interfering RNA (siRNA) or a constitutively inactive Hsf1 construct into neurons attenuates the induction of Syt1 by ethanol. This suggests that the activation of HSF1 can induce Syt1 expression and that this may be a mechanism by which alcohol regulates neurotransmitter release during brief exposures. Further analysis revealed that a subset of the genes encoding the core synaptic vesicle fusion (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor; SNARE) proteins share this property of induction by ethanol, suggesting that alcohol may trigger a specific coordinated adaptation in synaptic function. This molecular mechanism could explain some of the changes in synaptic function that occur following alcohol administration and may be an important step in the process of neuronal adaptation to alcohol.