Neurosteroid Modulation of Synaptic and Extrasynaptic GABAA Receptors of the Mouse Nucleus Accumbens.

The recent approval of formulations of the endogenous neurosteroid allopregnanolone (brexanolone) and the synthetic neuroactive steroid SAGE-217 (zuranolone) to treat postpartum depression (PPD) has encouraged further research to elucidate why these potent enhancers of GABAAR function are clinically effective in this condition. Dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens are associated with reward/motivation and brain imaging studies report that individuals with PPD show reduced activity of this pathway in response to reward and infant engagement. However, the influence of neurosteroids on GABA-ergic transmission in the nucleus accumbens has received limited attention. Here, we investigate, in the medium spiny neurons (MSNs) of the mouse nucleus accumbens core, the effect of allopregnanolone, SAGE-217 and other endogenous and synthetic steroids of interest on fast phasic and tonic inhibition mediated by synaptic (α1/2ßγ2) and extrasynaptic (α4ßδ) GABAARs, respectively. We present evidence suggesting the resident tonic current results from the spontaneous opening of δ-GABAARs, where the steroid-enhanced tonic current is GABA-dependent. Furthermore, we demonstrate local neurosteroid synthesis in the accumbal slice preparation and reveal that GABA-ergic neurotransmission of MSNs is influenced by an endogenous neurosteroid tone. Given the dramatic fluctuations in allopregnanolone levels during pregnancy and postpartum, this neurosteroid-mediated local fine-tuning of GABAergic transmission in the MSNs will probably be perturbed.

Subunit-specific expression and function of AMPA receptors in the mouse locus coeruleus.

The locus coeruleus (LC) provides the principal supply of noradrenaline (NA) to the brain, thereby modulating an array of brain functions. The release of NA, and therefore its impact on the brain, is governed by LC neuronal excitability. Glutamatergic axons, from various brain regions, topographically innervate different LC sub-domains and directly alter LC excitability. However, it is currently unclear whether glutamate receptor sub-classes, such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, are divergently expressed throughout the LC. Immunohistochemistry and confocal microscopy were used to identify and localise individual GluA subunits in the mouse LC. Whole-cell patch clamp electrophysiology and subunit-preferring ligands were used to assess their impact on LC spontaneous firing rate (FR). GluA1 immunoreactive clusters were associated with puncta immunoreactive for VGLUT2 on somata, and VGLUT1 on distal dendrites. GluA4 was associated with these synaptic markers only in the distal dendrites. No specific signal was detected for the GluA2-3 subunits. The GluA1/2 receptor agonist (S)-CPW 399 increased LC FR, whilst the GluA1/3 receptor antagonist philanthotoxin-74 decreased it. 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA), a positive allosteric modulator of GluA3/4 receptors, had no significant effect on spontaneous FR. The data suggest distinct AMPA receptor subunits are targeted to different LC afferent inputs and have contrasting effects on spontaneous neuronal excitability. This precise expression profile could be a mechanism for LC neurons to integrate diverse information contained in various glutamate afferents.

Domain and cell type-specific immunolocalisation of voltage-gated potassium channels in the mouse striatum.

Diverse classes of voltage-gated potassium channels (Kv) are integral to the variety of electrical activity patterns that distinguish different classes of neurons in the brain. A feature of their heterogenous expression patterns is the highly precise manner in which specific cell types target their location within functionally specialised sub-cellular domains. Although Kv expression profiles in cortical brain regions are widely reported, their immunolocalisation in sub-cortical areas such as the striatum, and in associated diseases such as Parkinson's disease (PD), remain less well described. Therefore, the broad aims of this study were to provide a high resolution immunolocalisation analysis of various Kv subtypes within the mouse striatum and assess their potential plasticity in a model of PD. Immunohistochemistry and confocal microscopy revealed that immunoreactivity for Kv1.1, 1.2 and 1.4 overlapped to varying degrees with excitatory and inhibitory axonal marker proteins suggesting these Kv subtypes are targeted to axons innervating striatal medium spiny neurons (MSNs). Immunoreactivity for Kv1.3 strongly overlapped with signal for mitochondrial marker proteins in MSN somata and dendrites. Kv1.5 immunoreactivity was expressed in parvalbumin-immunopositive neurons whereas Kv1.6 was located in cells immunopositive for microglia. Signal for Kv2.1 was concentrated on the somatic and proximal dendritic plasma membrane of MSNs, whilst immunoreactivity for Kv4.2 was targeted to their distal dendritic regions. Finally, striatal Kv2.1 expression, at both the mRNA and protein levels, was decreased in alpha-synuclein overexpressing mice, yet increased in alpha-synuclein knockout mice, compared to wild-type counterparts. The data indicate a variety of Kv expression patterns that are distinctive to the striatum and susceptible to pathology that mirrors PD. Furthermore, these findings advance our understanding of the molecular diversity of various striatal cell types, and potentially have implications for the homeostatic changes of MSN excitability during associated medical conditions such as PD.

Adenosine is released during thalamic oscillations to provide negative feedback control.

Physiological oscillations in the cortico-thalamo-cortical loop occur during processes such as sleep, but these can become dysfunctional in pathological conditions such as absence epilepsy. The purine neuromodulator adenosine can act as an endogenous anticonvulsant: it is released into the extracellular space during convulsive seizures to activate A1 receptors suppressing on-going activity and delaying the occurrence of the next seizure. However, the role of adenosine in thalamic physiological and epileptiform oscillations is less clear. Here we have combined immunohistochemistry, electrophysiology, and fixed potential amperometry (FPA) biosensor measurements to characterise the release and actions of adenosine in thalamic oscillations measured in rodent slices. In the thalamus, A1 receptors are highly expressed particularly in the ventral basal (VB) thalamus and reticular thalamic nucleus (nRT) supporting a role for adenosine signalling in controlling oscillations. In agreement with previous studies, both adenosine and adenosine A1 receptor agonists inhibited thalamic oscillations in control (spindle-like) and in epileptic conditions. Here we have shown for the first time that both control and epileptiform oscillations are enhanced (i.e., increased number of oscillatory cycles) by blocking A1 receptors consistent with adenosine release occurring during oscillations. Although increases in extracellular adenosine could not be directly detected during control oscillations, clear increases in adenosine concentration could be detected with a biosensor during epileptiform oscillation activity. Thus, adenosine is released during thalamic oscillations and acts via A1 receptors to feedback and reduce thalamic oscillatory activity.

Syndapin-2 mediated transcytosis of amyloid-ß across the blood-brain barrier.

A deficient transport of amyloid-ß across the blood-brain barrier, and its diminished clearance from the brain, contribute to neurodegenerative and vascular pathologies, such as Alzheimer's disease and cerebral amyloid angiopathy, respectively. At the blood-brain barrier, amyloid-ß efflux transport is associated with the low-density lipoprotein receptor-related protein 1. However, the precise mechanisms governing amyloid-ß transport across the blood-brain barrier, in health and disease, remain to be fully understood. Recent evidence indicates that the low-density lipoprotein receptor-related protein 1 transcytosis occurs through a tubulation-mediated mechanism stabilized by syndapin-2. Here, we show that syndapin-2 is associated with amyloid-ß clearance via low-density lipoprotein receptor-related protein 1 across the blood-brain barrier. We further demonstrate that risk factors for Alzheimer's disease, amyloid-ß expression and ageing, are associated with a decline in the native expression of syndapin-2 within the brain endothelium. Our data reveals that syndapin-2-mediated pathway, and its balance with the endosomal sorting, are important for amyloid-ß clearance proposing a measure to evaluate Alzheimer's disease and ageing, as well as a target for counteracting amyloid-ß build-up. Moreover, we provide evidence for the impact of the avidity of amyloid-ß assemblies in their trafficking across the brain endothelium and in low-density lipoprotein receptor-related protein 1 expression levels, which may affect the overall clearance of amyloid-ß across the blood-brain barrier.

Specific Dystrophins Selectively Associate with Inhibitory and Excitatory Synapses of the Mouse Cerebellum and their Loss Alters Expression of P2X7 Purinoceptors and Pro-Inflammatory Mediators.

Duchenne muscular dystrophy (DMD) patients, having mutations of the DMD gene, present with a range of neuropsychiatric disorders, in addition to the quintessential muscle pathology. The neurobiological basis remains poorly understood because the contributions of different DMD gene products (dystrophins) to the different neural networks underlying such symptoms are yet to be fully characterised. While full-length dystrophin clusters in inhibitory synapses, with inhibitory neurotransmitter receptors, the precise subcellular expression of truncated DMD gene products with excitatory synapses remains unresolved. Furthermore, inflammation, involving P2X purinoceptor 7 (P2RX7) accompanies DMD muscle pathology, yet any association with brain dystrophins is yet to be established. The aim of this study was to investigate the comparative expression of different dystrophins, alongside ionotropic glutamate receptors and P2RX7s, within the cerebellar circuitry known to express different dystrophin isoforms. Immunoreactivity for truncated DMD gene products was targeted to Purkinje cell (PC) distal dendrites adjacent to, or overlapping with, signal for GluA1, GluA4, GluN2A, and GluD2 receptor subunits. P2X7R immunoreactivity was located in Bergmann glia profiles adjacent to PC-dystrophin immunoreactivity. Ablation of all DMD gene products coincided with decreased mRNA expression for Gria2, Gria3, and Grin2a and increased GluD2 immunoreactivity. Finally, dystrophin-null mice showed decreased brain mRNA expression of P2rx7 and several inflammatory mediators. The data suggest that PCs target different dystrophin isoforms to molecularly and functionally distinct populations of synapses. In contrast to muscle, dystrophinopathy in brain leads to the dampening of the local immune system.

The Free-movement pattern Y-maze: A cross-species measure of working memory and executive function.

Numerous neurodegenerative and psychiatric disorders are associated with deficits in executive functions such as working memory and cognitive flexibility. Progress in developing effective treatments for disorders may benefit from targeting these cognitive impairments, the success of which is predicated on the development of animal models with validated behavioural assays. Zebrafish offer a promising model for studying complex brain disorders, but tasks assessing executive function are lacking. The Free-movement pattern (FMP) Y-maze combines aspects of the common Y-maze assay, which exploits the inherent motivation of an organism to explore an unknown environment, with analysis based on a series of sequential two-choice discriminations. We validate the task as a measure of working memory and executive function by comparing task performance parameters in adult zebrafish treated with a range of glutamatergic, cholinergic and dopaminergic drugs known to impair working memory and cognitive flexibility. We demonstrate the cross-species validity of the task by assessing performance parameters in adapted versions of the task for mice and Drosophila, and finally a virtual version in humans, and identify remarkable commonalities between vertebrate species' navigation of the maze. Together, our results demonstrate that the FMP Y-maze is a sensitive assay for assessing working memory and cognitive flexibility across species from invertebrates to humans, providing a simple and widely applicable behavioural assay with exceptional translational relevance.

Identification of intraneuronal amyloid beta oligomers in locus coeruleus neurons of Alzheimer's patients and their potential impact on inhibitory neurotransmitter receptors and neuronal excitability.

AIMS: Amyloid ß-oligomers (AßO) are potent modulators of Alzheimer's pathology, yet their impact on one of the earliest brain regions to exhibit signs of the condition, the locus coeruleus (LC), remains to be determined. Of particular importance is whether AßO impact the spontaneous excitability of LC neurons. This parameter determines brain-wide noradrenaline (NA) release, and thus NA-mediated brain functions, including cognition, emotion and immune function, which are all compromised in Alzheimer's patients. Therefore, the aim of the study was to determine the expression profile of AßO in the LC of Alzheimer's patients and to probe their potential impact on the molecular and functional correlates of LC excitability, using a mouse model of increased Aß production (APP-PSEN1). METHODS AND RESULTS: Immunohistochemistry and confocal microscopy, using AßO-specific antibodies, confirmed LC AßO expression both intraneuronally and extracellularly in both Alzheimer's and APP-PSEN1 samples. Patch clamp electrophysiology recordings revealed that APP-PSEN1 LC neuronal hyperexcitability accompanied this AßO expression profile, arising from a diminished inhibitory effect of GABA due to impaired expression and function of the GABA-A receptor (GABAA R) α3 subunit. This altered LC α3-GABAA R expression profile overlapped with AßO expression in samples from both APP-PSEN1 mice and Alzheimer's patients. Finally, strychnine-sensitive glycine receptors (GlyRs) remained resilient to Aß-induced changes and their activation reversed LC hyperexcitability. CONCLUSIONS: The data suggest a close association between AßO and α3-GABAA Rs in the LC of Alzheimer's patients, and their potential to dysregulate LC activity, thereby contributing to the spectrum of pathology of the LC-NA system in this condition.

Developmental and age-dependent plasticity of GABAA receptors in the mouse colon: Implications in colonic motility and inflammation.

Lifelong functional plasticity of the gastrointestinal (GI) tract is essential for health, yet the underlying molecular mechanisms are poorly understood. The enteric nervous system (ENS) regulates all aspects of the gut function, via a range of neurotransmitter pathways, one of which is the GABA-GABAA receptor (GABAAR) system. We have previously shown that GABAA receptor subunits are differentially expressed within the ENS and are involved in regulating various GI functions. We have also shown that these receptors are involved in mediating stress-induced colonic inflammation. However, the expression and function of intestinal GABAARs, at different ages, is largely unexplored and was the focus of this study. Here we show that the impact of GABAAR activation on colonic contractility changes from early postnatal period through to late adulthood, in an age-dependant manner. We also show that the highest levels of expression for all GABAAR subunits is evident at postnatal day (P) 10 apart from the α3 subunit which increased with age. This increase in the α3 subunit expression in late adulthood (18 months old) is accompanied by an increase in the expression of inflammatory markers within the mouse colon. Finally, we demonstrate that the deletion of the α3 subunit prevents the increase in the expression of colonic inflammatory markers associated with healthy ageing. Collectively, the data provide the first demonstration of the molecular and functional plasticity of the GI GABAAR system over the course of a lifetime, and its possible role in mediating the age-induced colonic inflammation associated with healthy ageing.

A Synaptically Connected Hypothalamic Magnocellular Vasopressin-Locus Coeruleus Neuronal Circuit and Its Plasticity in Response to Emotional and Physiological Stress.

The locus coeruleus (LC)-norepinephrine (NE) system modulates a range of salient brain functions, including memory and response to stress. The LC-NE system is regulated by neurochemically diverse inputs, including a range of neuropeptides such as arginine-vasopressin (AVP). Whilst the origins of many of these LC inputs, their synaptic connectivity with LC neurons, and their contribution to LC-mediated brain functions, have been well characterized, this is not the case for the AVP-LC system. Therefore, our aims were to define the types of synapses formed by AVP+ fibers with LC neurons using immunohistochemistry together with confocal and transmission electron microscopy (TEM), the origins of such inputs, using retrograde tracers, and the plasticity of the LC AVP system in response to stress and spatial learning, using the maternal separation (MS) and Morris water maze (MWM) paradigms, respectively, in rat. Confocal microscopy revealed that AVP+ fibers contacting tyrosine hydroxylase (TH)+ LC neurons were also immunopositive for vesicular glutamate transporter 2, a marker of presynaptic glutamatergic axons. TEM confirmed that AVP+ axons formed Gray type I (asymmetric) synapses with TH+ dendrites thus confirming excitatory synaptic connections between these systems. Retrograde tracing revealed that these LC AVP+ fibers originate from hypothalamic vasopressinergic magnocellular neurosecretory neurons (AVPMNNs). MS induced a significant increase in the density of LC AVP+ fibers. Finally, AVPMNN circuit upregulation by water-deprivation improved MWM performance while increased Fos expression was found in LC and efferent regions such as hippocampus and prefrontal cortex, suggesting that AVPMMN projections to LC could integrate homeostatic responses modifying neuroplasticity.

Early-life stress influences acute and sensitized responses of adult mice to cocaine by interacting with GABAA α2 receptor expression.

Early-life stress (ELS) is known to exert long-term effects on brain function, with resulting deleterious consequences for several aspects of mental health, including the development of addiction to drugs of abuse. One potential mechanism in humans is suggested by findings that ELS interacts with polymorphisms of the GABRA2 gene, encoding α2 subunits of GABAA receptors, to increase the risk for both post-traumatic stress disorder and vulnerability to cocaine addiction. We used a mouse model, in which the amount of material for nest building was reduced during early postnatal life, to study interactions between ELS and expression of α2-containing GABAA receptors in influencing cocaine-related behaviour. Breeding of parents heterozygous for a deletion of α2 resulted in litters containing homozygous knockout (α2), heterozygous knockout (α2) and wild-type (α2) offspring. Following the ELS procedure, the mice were allowed to develop to adulthood before being tested for the acute effect of cocaine on locomotor stimulation, behavioural sensitization to repeated cocaine and to cocaine-conditioned activity. Exposure to ELS resulted in increased acute locomotor stimulant effects of cocaine across all genotypes, with the most marked effects in α2 mice (which also showed increased activity following vehicle). Repeated cocaine administration to nonstressed mice resulted in sensitization in α2 and α2 mice, but, in keeping with previous findings, not in α2 mice. Previous exposure to ELS reduced sensitization in α2 mice, albeit not significantly, and abolished sensitization in α2 mice. Conditioned activity was elevated following ELS in all animals, independently of genotype. Thus, while the enhanced acute effects of cocaine following ELS being most marked in α2 mice suggests a function of α2-containing GABAA receptors in protecting against stress, the interaction between ELS and genotype in influencing sensitization may be more in keeping with ELS reducing expression of α2-containing GABAA receptors. The ability of ELS to increase cocaine-conditioned locomotor activity appears to be independent of α2-containing GABAA receptors.

Early-life adversity selectively impairs α2-GABAA receptor expression in the mouse nucleus accumbens and influences the behavioral effects of cocaine.

Haplotypes of the Gabra2 gene encoding the α2-subunit of the GABAA receptor (GABAAR) are associated with drug abuse, suggesting that α2-GABAARs may play an important role in the circuitry underlying drug misuse. The genetic association of Gabra2 haplotypes with cocaine addiction appears to be evident primarily in individuals who had experienced childhood trauma. Given this association of childhood trauma, cocaine abuse and the Gabra2 haplotypes, we have explored in a mouse model of early life adversity (ELA) whether such events influence the behavioral effects of cocaine and if, as suggested by the human studies, α2-GABAARs in the nucleus accumbens (NAc) are involved in these perturbed behaviors. In adult mice prior ELA caused a selective decrease of accumbal α2-subunit mRNA, resulting in a selective decrease in the number and size of the α2-subunit (but not the α1-subunit) immunoreactive clusters in NAc core medium spiny neurons (MSNs). Functionally, in adult MSNs ELA decreased the amplitude and frequency of GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs), a profile similar to that of α2 "knock-out" (α2-/-) mice. Behaviourally, adult male ELA and α2-/- mice exhibited an enhanced locomotor response to acute cocaine and blunted sensitisation upon repeated cocaine administration, when compared to their appropriate controls. Collectively, these findings reveal a neurobiological mechanism which may relate to the clinical observation that early trauma increases the risk for substance abuse disorder (SAD) in individuals harbouring haplotypic variations in the Gabra2 gene.

Spatiotemporal Distribution of GABAA Receptor Subunits Within Layer II of Mouse Medial Entorhinal Cortex: Implications for Grid Cell Excitability.

GABAergic parvalbumin-expressing (PV+) interneurons provide powerful inhibitory modulation of grid cells in layer II of the medial entorhinal cortex (MEC LII). However, the molecular machinery through which PV+ cells regulate grid cell activity is poorly defined. PV+ interneurons impart inhibitory modulation primarily via GABA-A receptors (GABAARs). GABAARs are pentameric ion channels assembled from a repertoire of 19 subunits. Multiple subunit combinations result in a variety of receptor subtypes mediating functionally diverse postsynaptic inhibitory currents. Whilst the broad expression patterns of GABAAR subunits within the EC have been reported, those expressed by individual MEC LII cell types, in particular grid cells candidates, stellate and pyramidal cells, are less well described. Stellate and pyramidal cells are distinguished by their selective expression of reelin (RE+) and calbindin (CB+) respectively. Thus, the overall aim of this study was to provide a high resolution analysis of the major (α and γ) GABAAR subunits expressed in proximity to somato-dendritic PV+ boutons, on RE+ and CB+ cells, using immunohistochemistry, confocal microscopy and quantitative RT-PCR (qPCR). Clusters immunoreactive for the α1 and γ2 subunits decorated the somatic membranes of both RE+ and CB+ cells and were predominantly located in apposition to clusters immunoreactive for PV and vesicular GABA transporter (VGAT), suggesting expression in GABAergic synapses innervated by PV interneurons. Although intense α2 subunit-immunopositive clusters were evident in hippocampal fields located in close proximity to the EC, no specific signal was detected in MEC LII RE+ and CB+ profiles. Immunoreactivity for the α3 subunit was detected in all RE+ somata. In contrast, only a sub-population of CB+ cells was α3 immunopositive. These included CB-α3 cells which were both PV+ and PV-. Furthermore, α3 subunit mRNA and immunofluorescence decreased significantly between P 15 and P 25, a period implicated in the functional maturation of grid cells. Finally, α5 subunit immunoreactivity was detectable only on CB+ cells, not on RE+ cells. The present data demonstrates that physiologically distinct GABAAR subtypes are selectively expressed by CB+ and RE+ cells. This suggests that PV+ interneurons could utilize distinct postsynaptic signaling mechanisms to regulate the excitability of these different, candidate grid cell sub-populations.

GABAA Receptor Subtypes Regulate Stress-Induced Colon Inflammation in Mice.

BACKGROUND & AIMS: Psychological stress, in early life or adulthood, is a significant risk factor for inflammatory disorders, including inflammatory bowel diseases. However, little is known about the mechanisms by which emotional factors affect the immune system. γ-Aminobutyric acid type A receptors (GABAARs) regulate stress and inflammation, but it is not clear whether specific subtypes of GABAARs mediate stress-induced gastrointestinal inflammation. We investigated the roles of different GABAAR subtypes in mouse colon inflammation induced by 2 different forms of psychological stress. METHODS: C57BL/6J mice were exposed to early-life stress, and adult mice were exposed to acute-restraint stress; control mice were not exposed to either form of stress. We collected colon tissues and measured contractility using isometric tension recordings; colon inflammation, based on levels of cluster of differentiation 163 and tumor necrosis factor messenger RNA (mRNA) and protein and myeloperoxidase activity; and permeability, based on levels of tight junction protein 1 and occludin mRNA and protein. Mice were given fluorescently labeled dextran orally and systemic absorption was measured. We also performed studies of mice with disruption of the GABAAR subunit α3 gene (Gabra3-/- mice). RESULTS: Mice exposed to early-life stress had significantly altered GABAAR-mediated colonic contractility and impaired barrier function, and their colon tissue had increased levels of Gabra3 mRNA compared with control mice. Restraint stress led to colon inflammation in C57/BL6J mice but not Gabra3-/- mice. Colonic inflammation was induced in vitro by an α3-GABAAR agonist, showing a proinflammatory role for this receptor subtype. In contrast, α1/4/5-GABAAR ligands decreased the expression of colonic inflammatory markers. CONCLUSIONS: We found stress to increase expression of Gabra3 and induce inflammation in mouse colon, together with impaired barrier function. The in vitro pharmacologic activation of α3-GABAARs recapitulated colonic inflammation, whereas α1/4/5-GABAAR ligands were anti-inflammatory. These proteins might serve as therapeutic targets for treatment of colon inflammation or inflammatory bowel diseases.

TREK-1 Channel Expression in Smooth Muscle as a Target for Regulating Murine Intestinal Contractility: Therapeutic Implications for Motility Disorders.

Gastrointestinal (GI) motility disorders such as irritable bowel syndrome (IBS) can occur when coordinated smooth muscle contractility is disrupted. Potassium (K+) channels regulate GI smooth muscle tone and are key to GI tract relaxation, but their molecular and functional phenotypes are poorly described. Here we define the expression and functional roles of mechano-gated K2P channels in mouse ileum and colon. Expression and distribution of the K2P channel family were investigated using quantitative RT-PCR (qPCR), immunohistochemistry and confocal microscopy. The contribution of mechano-gated K2P channels to mouse intestinal muscle tension was studied pharmacologically using organ bath. Multiple K2P gene transcripts were detected in mouse ileum and colon whole tissue preparations. Immunohistochemistry confirmed TREK-1 expression was smooth muscle specific in both ileum and colon, whereas TREK-2 and TRAAK channels were detected in enteric neurons but not smooth muscle. In organ bath, mechano-gated K2P channel activators (Riluzole, BL-1249, flufenamic acid, and cinnamyl 1-3,4-dihydroxy-alpha-cyanocinnamate) induced relaxation of KCl and CCh pre-contracted ileum and colon tissues and reduced the amplitude of spontaneous contractions. These data reveal the specific expression of mechano-gated K2P channels in mouse ileum and colon tissues and highlight TREK-1, a smooth muscle specific K2P channel in GI tract, as a potential therapeutic target for combating motility pathologies arising from hyper-contractility.

Molecular Characterization of GABA-A Receptor Subunit Diversity within Major Peripheral Organs and Their Plasticity in Response to Early Life Psychosocial Stress.

Gamma aminobutyric acid (GABA) subtype A receptors (GABAARs) are integral membrane ion channels composed of five individual proteins or subunits. Up to 19 different GABAAR subunits (α1-6, ß1-3, γ1-3, δ, ε, θ, π, and ρ1-3) have been identified, resulting in anatomically, physiologically, and pharmacologically distinct multiple receptor subtypes, and therefore GABA-mediated inhibition, across the central nervous system (CNS). Additionally, GABAAR-modulating drugs are important tools in clinical medicine, although their use is limited by adverse effects. While significant advances have been made in terms of characterizing the GABAAR system within the brain, relatively less is known about the molecular phenotypes within the peripheral nervous system of major organ systems. This represents a potentially missed therapeutic opportunity in terms of utilizing or repurposing clinically available GABAAR drugs, as well as promising research compounds discarded due to their poor CNS penetrance, for the treatment of peripheral disorders. In addition, a broader understanding of the peripheral GABAAR subtype repertoires will contribute to the design of therapies which minimize peripheral side-effects when treating CNS disorders. We have recently provided a high resolution molecular and function characterization of the GABAARs within the enteric nervous system of the mouse colon. In this study, the aim was to determine the constituent GABAAR subunit expression profiles of the mouse bladder, heart, liver, kidney, lung, and stomach, using reverse transcription polymerase chain reaction and western blotting with brain as control. The data indicate that while some subunits are expressed widely across various organs (α3-5), others are restricted to individual organs (γ2, only stomach). Furthermore, we demonstrate complex organ-specific developmental expression plasticity of the transporters which determine the chloride gradient within cells, and therefore whether GABAAR activation has a depolarizing or hyperpolarizing effect. Finally, we demonstrate that prior exposure to early life psychosocial stress induces significant changes in peripheral GABAAR subunit expression and chloride transporters, in an organ- and subunit-specific manner. Collectively, the data demonstrate the molecular diversity of the peripheral GABAAR system and how this changes dynamically in response to life experience. This provides a molecular platform for functional analyses of the GABA-GABAAR system in health, and in diseases affecting various peripheral organs.

A GABAergic cell type in the lateral habenula links hypothalamic homeostatic and midbrain motivation circuits with sex steroid signaling.

The lateral habenula (LHb) has a key role in integrating a variety of neural circuits associated with reward and aversive behaviors. There is limited information about how the different cell types and neuronal circuits within the LHb coordinate physiological and motivational states. Here, we report a cell type in the medial division of the LHb (LHbM) in male rats that is distinguished by: (1) a molecular signature for GABAergic neurotransmission (Slc32a1/VGAT) and estrogen receptor (Esr1/ERα) expression, at both mRNA and protein levels, as well as the mRNA for vesicular glutamate transporter Slc17a6/VGLUT2, which we term the GABAergic estrogen-receptive neuron (GERN); (2) its axonal projection patterns, identified by in vivo juxtacellular labeling, to both local LHb and to midbrain modulatory systems; and (3) its somatic expression of receptors for vasopressin, serotonin and dopamine, and mRNA for orexin receptor 2. This cell type is anatomically located to receive afferents from midbrain reward (dopamine and serotonin) and hypothalamic water and energy homeostasis (vasopressin and orexin) circuits. These afferents shared the expression of estrogen synthase (aromatase) and VGLUT2, both in their somata and axon terminals. We demonstrate dynamic changes in LHbM VGAT+ cell density, dependent upon gonadal functional status, that closely correlate with motivational behavior in response to predator and forced swim stressors. The findings suggest that the homeostasis and reward-related glutamatergic convergent projecting pathways to LHbMC employ a localized neurosteroid signaling mechanism via axonal expression of aromatase, to act as a switch for GERN excitation/inhibition output prevalence, influencing depressive or motivated behavior.

Dynamic Modulation of Mouse Locus Coeruleus Neurons by Vasopressin 1a and 1b Receptors.

The locus coeruleus (LC) is a brainstem nucleus distinguished by its supply of noradrenaline throughout the central nervous system. Apart from modulating a range of brain functions, such as arousal, cognition and the stress response, LC neuronal excitability also corresponds to the activity of various peripheral systems, such as pelvic viscera and the cardiovascular system. Neurochemically diverse inputs set the tone for LC neuronal activity, which in turn modulates these adaptive physiological and behavioral responses essential for survival. One such LC afferent system which is poorly understood contains the neurohormone arginine-vasopressin (AVP). Here we provide the first demonstration of the molecular and functional characteristics of the LC-AVP system, by characterizing its receptor-specific modulation of identified LC neurons and plasticity in response to stress. High resolution confocal microscopy revealed that immunoreactivity for the AVP receptor 1b (V1b) was located on plasma membranes of noradrenergic and non-noradrenergic LC neurons. In contrast, immunoreactivity for the V1a receptor was exclusively located on LC noradrenergic neurons. No specific signal, either at the mRNA or protein level, was detected for the V2 receptor in the LC. Clusters immunoreactive for V1a-b were located in proximity to profiles immunoreactive for GABAergic and glutamatergic synaptic marker proteins. AVP immunopositive varicosities were also located adjacent to labeling for such synaptic markers. Whole-cell patch clamp electrophysiology revealed that the pharmacological activation of V1b receptors significantly increased the spontaneous activity of 45% (9/20) of recorded noradrenergic neurons, with the remaining 55% (11/20) of cells exhibiting a significant decrease in their basal firing patterns. Blockade of V1a and V1b receptors on their own significantly altered LC neuronal excitability in a similar heterogeneous manner, demonstrating that endogenous AVP sets the basal LC neuronal firing rates. Finally, exposing animals to acute stress increased V1b, but not V1a receptor expression, whilst decreasing AVP immunoreactivity. This study reveals the AVP-V1a-b system as a considerable component of the LC molecular architecture and regulator of LC activity. Since AVP primarily functions as a regulator of homeostasis, the data suggest a novel pathway by modulating the functioning of a brain region that is integral to mediating adaptive responses.

During postnatal development endogenous neurosteroids influence GABA-ergic neurotransmission of mouse cortical neurons.

As neuronal development progresses, GABAergic synaptic transmission undergoes a defined program of reconfiguration. For example, GABAA receptor (GABAAR)-mediated synaptic currents, (miniature inhibitory postsynaptic currents; mIPSCs), which initially exhibit a relatively slow decay phase, become progressively reduced in duration, thereby supporting the temporal resolution required for mature network activity. Here we report that during postnatal development of cortical layer 2/3 pyramidal neurons, GABAAR-mediated phasic inhibition is influenced by a resident neurosteroid tone, which wanes in the second postnatal week, resulting in the brief phasic events characteristic of mature neuronal signalling. Treatment of cortical slices with the immediate precursor of 5α-pregnan-3α-ol-20-one (5α3α), the GABAAR-inactive 5α-dihydroprogesterone, (5α-DHP), greatly prolonged the mIPSCs of P20 pyramidal neurons, demonstrating these more mature neurons retain the capacity to synthesize GABAAR-active neurosteroids, but now lack the endogenous steroid substrate. Previously, such developmental plasticity of phasic inhibition was ascribed to the expression of synaptic GABAARs incorporating the α1 subunit. However, the duration of mIPSCs recorded from L2/3 cortical neurons derived from α1 subunit deleted mice, were similarly under the developmental influence of a neurosteroid tone. In addition to principal cells, synaptic GABAARs of L2/3 interneurons were modulated by native neurosteroids in a development-dependent manner. In summary, local neurosteroids influence synaptic transmission during a crucial period of cortical neurodevelopment, findings which may be of importance for establishing normal network connectivity.

Localisation and stress-induced plasticity of GABAA receptor subunits within the cellular networks of the mouse dorsal raphe nucleus.

The dorsal raphe nucleus (DRN) provides the major source of serotonin to the central nervous system (CNS) and modulates diverse neural functions including mood. Furthermore, DRN cellular networks are engaged in the stress-response at the CNS level allowing for adaptive behavioural responses, whilst stress-induced dysregulation of DRN and serotonin release is implicated in psychiatric disorders. Therefore, identifying the molecules regulating DRN activity is fundamental to understand DRN function in health and disease. GABAA receptors (GABAARs) allow for brain region, cell type and subcellular domain-specific GABA-mediated inhibitory currents and are thus key regulators of neuronal activity. Yet, the GABAAR subtypes expressed within the neurochemically diverse cell types of the mouse DRN are poorly described. In this study, immunohistochemistry and confocal microscopy revealed that all serotonergic neurons expressed immunoreactivity for the GABAAR alpha2 and 3 subunits, although the respective signals were co-localised to varying degrees with inhibitory synaptic marker proteins. Only a topographically located sub-population of serotonergic neurons exhibited GABAAR alpha1 subunit immunoreactivity. However, all GABAergic as well as non-GABAergic, non-serotonergic neurons within the DRN expressed GABAAR alpha1 subunit immunoreactivity. Intriguingly, immunoreactivity for the GABAAR gamma2 subunit was enriched on GABAergic rather than serotonergic neurons. Finally, repeated restraint stress increased the expression of the GABAAR alpha3 subunit at the mRNA and protein level. The study demonstrates the identity and location of distinct GABAAR subunits within the cellular networks of the mouse DRN and that stress impacts on the expression levels of particular subunits at the gene and protein level.