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.

The Use of Miniature Specimens to Determine Local Properties and Fracture Behavior of LPBF-Processed Inconel 718 in as-Deposited and Post-Treated States.

This paper summarizes the assessment of directional anisotropy in local mechanical properties for Laser Powder Bed Fusion (LPBF) IN-718 bulk samples via the use of miniature samples excised from the bulk for both as-deposited and post-treated states. The quasi-static tensile properties at room temperature are investigated at several different locations along the build direction and at different orientations for both considered states. A comparison between the excised miniature tensile specimens and standard-sized sample results have also been conducted and exhibit very good agreement. Significant anisotropy is present in mechanical properties at different build heights for the as-deposited state, while the post-treated material exhibited more homogenous properties, both along the height and for different sampling orientations. However, significant reductions (e.g., >30%) in the strength (Yield, UTS) along with a significant increase in the reduction in area at fracture is found for post-processed materials. Metallography and fractography analyses were conducted in order to begin to determine the source(s) of this anisotropy for the as-deposited state.

Comparison of Pregabalin and Ketotifen in treatment of uremic pruritus in hemodialysis patients.

This study aims to compare the efficiency of Pregabalin and Ketotifen in treatment of uremic pruritus in hemodialysis (HD) patients. Thirty HD patients were randomly divided into two groups: A (Pregabalin 50 mg three times a day) and B (Ketotifen 1 mg twice a day). Efficacy of treatment and quality of life were weekly evaluated by visual analogue scale (VAS) and Itchy Quality of life, respectively. There was no significant difference between the two groups regarding demographic features, laboratory data, quality of life, and VAS before treatment. In the second week of treatment, the pruritus intensity was significantly lower in the Pregabalin group than the Ketotifen group (p = 0.026). The mean of life quality was significantly lower in Ketotifen than Pregabalin group in weeks 1, 2, and 4 (p = 0.001, p = 0.001, and p = 0.036, respectively). There was no significant difference between the two groups regarding the side effects of drugs. This study showed that a higher dose of Pregabalin could be a more effective treatment than Ketotifen without additive side effects in improving the quality of life in dialysis patients.

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.

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.

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.

A Critical Review on Metallic Glasses as Structural Materials for Cardiovascular Stent Applications.

Functional and mechanical properties of novel biomaterials must be carefully evaluated to guarantee long-term biocompatibility and structural integrity of implantable medical devices. Owing to the combination of metallic bonding and amorphous structure, metallic glasses (MGs) exhibit extraordinary properties superior to conventional crystalline metallic alloys, placing them at the frontier of biomaterials research. MGs have potential to improve corrosion resistance, biocompatibility, strength, and longevity of biomedical implants, and hence are promising materials for cardiovascular stent applications. Nevertheless, while functional properties and biocompatibility of MGs have been widely investigated and validated, a solid understanding of their mechanical performance during different stages in stent applications is still scarce. In this review, we provide a brief, yet comprehensive account on the general aspects of MGs regarding their formation, processing, structure, mechanical, and chemical properties. More specifically, we focus on the additive manufacturing (AM) of MGs, their outstanding high strength and resilience, and their fatigue properties. The interconnection between processing, structure and mechanical behaviour of MGs is highlighted. We further review the main categories of cardiovascular stents, the required mechanical properties of each category, and the conventional materials have been using to address these requirements. Then, we bridge between the mechanical requirements of stents, structural properties of MGs, and the corresponding stent design caveats. In particular, we discuss our recent findings on the feasibility of using MGs in self-expandable stents where our results show that a metallic glass based aortic stent can be crimped without mechanical failure. We further justify the safe deployment of this stent in human descending aorta. It is our intent with this review to inspire biodevice developers toward the realization of MG-based stents.

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.

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.

Essential oil composition of Eucalyptus microtheca and Eucalyptus viminalis.

OBJECTIVE: Eucalyptus (Fam. Myrtaceae) is a medicinal plant and various Eucalyptus species possess potent pharmacological actions against diabetes, hepatotoxicity, and inflammation. This study aims to investigate essential oil composition from leaves and flowers of E. microtheca and E. viminalis leaves growing in the Southeast of Iran. MATERIALS AND METHODS: The aerial parts of these plants were collected from Zahedan, Sistan and Baluchestan province, Iran in 2013. After drying the plant materials in the shade, the chemical composition of the essential oils was obtained by hydro-distillation method using a Clevenger-type apparatus and analyzed by GC/MS. RESULTS: In the essential oil of E. microtheca leaves, 101 compounds representing 100%, were identified. Among them, α-phellandrene (16.487%), aromadendrene (12.773%), α-pinene (6.752%), globulol (5.997%), ledene (5.665%), P-cymen (5.251%), and ß-pinene (5.006%) were the major constituents. In the oil of E. microtheca flowers, 88 compounds representing 100%, were identified in which α-pinene (16.246%), O-cymen (13.522%), ß-pinene (11.082%), aromadendrene (7.444%), α-phellandrene (7.006%), globulol (5.419%), and 9-octadecenamide (5.414%) were the major components. Sixty six compounds representing 100% were identified in the oil of E. viminalis leaves. The major compounds were 1, 8-cineole (57.757%), α-pinene (13.379%), limonene (5.443%), and globulol (3.054%). CONCLUSION: The results showed the essential oils from the aerial parts of Eucalyptus species are a cheap source for the commercial isolation of α-phellandrene, α-pinene, and 1, 8-cineole compounds to be used in medicinal and food products. Furthermore, these plants could be an alternative source of insecticide agents.

Molecular and functional diversity of GABA-A receptors in the enteric nervous system of the mouse colon.

The enteric nervous system (ENS) provides the intrinsic neural control of the gastrointestinal tract (GIT) and regulates virtually all GI functions. Altered neuronal activity within the ENS underlies various GI disorders with stress being a key contributing factor. Thus, elucidating the expression and function of the neurotransmitter systems, which determine neuronal excitability within the ENS, such as the GABA-GABAA receptor (GABAAR) system, could reveal novel therapeutic targets for such GI disorders. Molecular and functionally diverse GABAARs modulate rapid GABAergic-mediated regulation of neuronal excitability throughout the nervous system. However, the cellular and subcellular GABAAR subunit expression patterns within neurochemically defined cellular circuits of the mouse ENS, together with the functional contribution of GABAAR subtypes to GI contractility remains to be determined. Immunohistochemical analyses revealed that immunoreactivity for the GABAAR gamma (γ) 2 and alphas (α) 1, 2, 3 subunits was located on somatodendritic surfaces of neurochemically distinct myenteric plexus neurons, while being on axonal compartments of submucosal plexus neurons. In contrast, immunoreactivity for the α4-5 subunits was only detected in myenteric plexus neurons. Furthermore, α-γ2 subunit immunoreactivity was located on non-neuronal interstitial cells of Cajal. In organ bath studies, GABAAR subtype-specific ligands had contrasting effects on the force and frequency of spontaneous colonic longitudinal smooth muscle contractions. Finally, enhancement of γ2-GABAAR function with alprazolam reversed the stress-induced increase in the force of spontaneous colonic contractions. The study demonstrates the molecular and functional diversity of the GABAAR system within the mouse colon providing a framework for developing GABAAR-based therapeutics in GI disorders.

Localization of NG2 immunoreactive neuroglia cells in the rat locus coeruleus and their plasticity in response to stress.

The locus coeruleus (LC) nucleus modulates adaptive behavioral responses to stress and dysregulation of LC neuronal activity is implicated in stress-induced mental illnesses. The LC is composed primarily of noradrenergic neurons together with various glial populations. A neuroglia cell-type largely unexplored within the LC is the NG2 cell. NG2 cells serve primarily as oligodendrocyte precursor cells throughout the brain. However, some NG2 cells are in synaptic contact with neurons suggesting a role in information processing. The aim of this study was to neurochemically and anatomically characterize NG2 cells within the rat LC. Furthermore, since NG2 cells have been shown to proliferate in response to traumatic brain injury, we investigated whether such NG2 cells plasticity also occurs in response to emotive insults such as stress. Immunohistochemistry and confocal microscopy revealed that NG2 cells were enriched within the pontine region occupied by the LC. Close inspection revealed that a sub-population of NG2 cells were located within unique indentations of LC noradrenergic somata and were immunoreactive for the neuronal marker NeuN whilst NG2 cell processes formed close appositions with clusters immunoreactive for the inhibitory synaptic marker proteins gephyrin and the GABA-A receptor alpha3-subunit, on noradrenergic dendrites. In addition, LC NG2 cell processes were decorated with vesicular glutamate transporter 2 immunoreactive puncta. Finally, 10 days of repeated restraint stress significantly increased the density of NG2 cells within the LC. The study demonstrates that NG2 IR cells are integral components of the LC cellular network and they exhibit plasticity as a result of emotive challenges.

Dysfunctional astrocytic and synaptic regulation of hypothalamic glutamatergic transmission in a mouse model of early-life adversity: relevance to neurosteroids and programming of the stress response.

Adverse early-life experiences, such as poor maternal care, program an abnormal stress response that may involve an altered balance between excitatory and inhibitory signals. Here, we explored how early-life stress (ELS) affects excitatory and inhibitory transmission in corticotrophin-releasing factor (CRF)-expressing dorsal-medial (mpd) neurons of the neonatal mouse hypothalamus. We report that ELS associates with enhanced excitatory glutamatergic transmission that is manifested as an increased frequency of synaptic events and increased extrasynaptic conductance, with the latter associated with dysfunctional astrocytic regulation of glutamate levels. The neurosteroid 5α-pregnan-3α-ol-20-one (5α3α-THPROG) is an endogenous, positive modulator of GABAA receptors (GABAARs) that is abundant during brain development and rises rapidly during acute stress, thereby enhancing inhibition to curtail stress-induced activation of the hypothalamic-pituitary-adrenocortical axis. In control mpd neurons, 5α3α-THPROG potently suppressed neuronal discharge, but this action was greatly compromised by prior ELS exposure. This neurosteroid insensitivity did not primarily result from perturbations of GABAergic inhibition, but rather arose functionally from the increased excitatory drive onto mpd neurons. Previous reports indicated that mice (dams) lacking the GABAAR δ subunit (δ(0/0)) exhibit altered maternal behavior. Intriguingly, δ(0/0) offspring showed some hallmarks of abnormal maternal care that were further exacerbated by ELS. Moreover, in common with ELS, mpd neurons of δ(0/0) pups exhibited increased synaptic and extrasynaptic glutamatergic transmission and consequently a blunted neurosteroid suppression of neuronal firing. This study reveals that increased synaptic and tonic glutamatergic transmission may be a common maladaptation to ELS, leading to enhanced excitation of CRF-releasing neurons, and identifies neurosteroids as putative early regulators of the stress neurocircuitry.