GABA Release Research Articles

Diminished inhibitory efficacy of GABA on PVN neurons in AngII‐salt hypertension reflects a KCC2‐independent increase of intracellular Cl‐ concentration

The hypothalamic PVN receives glutamatergic excitation from sodium and AngII sensing regions of the forebrain along with dense GABAergic inhibition from the surrounding peri-nuclear zone (PNZ). Previously, we found that synaptic glutamate release in PVN enhances GABA inhibition, but whether forebrain inputs can do so in the setting of hypertension (HTN) is unknown. Studies indicate that a high salt diet alone does not raise mean arterial pressure (MAP), while AngII treatment raises MAP in proportion to the level of salt intake. This suggests that whereas excitatory inputs to PVN driven by high salt intake alone cannot overcome PVN GABA inhibition, AngII driven inputs can. The notion that GABA inhibition of PVN is strengthened forebrain inputs in AngII-salt HTN is contrary to a prior report that GABA inhibition in AngII-salt HTN is reduced. To reconcile these seemingly disparate findings, we compared mechanisms regulating PVN GABA inhibition in mice consuming a normal salt (0.4% NaCl) diet (NSD), a high salt (4% NaCl) diet (HSD) or a HSD with AngII (600 ng/kg/min, s.c.) infusion (AngII-salt HTN). Using gramicidin perforated patch recordings, we determined that the GABA equilibrium potential (EGABA) of PVN neurons was not different between NSD (-67.2 ± 6.3 mV, n=3 cells) and HSD (-53.7 ± 15.6 mV, n=4 cells) mice, but was significantly depolarized in mice with AngII-salt HTN (-32.9 ± 8.8 mV, n=6 cells)(P=0.0033). This indicates that in AngII-salt HTN the inhibitory effect of GABA on PVN neurons decreases as [Cl-]i increases. Normally, [Cl-]i is held low by activities of KCC2 and NKCC1, which promote neuronal Cl- efflux and influx, respectively. In brain slices, KCC2 inhibition (VU0463271, 10 µM) rapidly depolarized PVN EGABA, consistent with blunting of GABA inhibition. Accordingly, PVN inhibition of KCC2 (VU0463271, 100 pmol, 25 nL) in vivo increased renal SNA, suggesting GABA was switched from an inhibitory to an excitatory transmitter. Consistent with this, PVN GABA-AR activation (muscimol, 50 pmol, 25 nL) during KCC2 inhibition increased renal SNA. Thus, KCC2 appears critical for maintaining low [Cl-]i and a hyperpolarized EGABA in PVN. In light of this, it would seem likely that elevated PVN [Cl-]i in AngII-salt HTN reflects a relative loss of KCC2 Cl- efflux. To investigate this, western blot analysis of PVN punches was performed and revealed that expression of KCC2 and NKCC1 was increased and decreased, respectively, both in mice consuming a HSD alone and mice with AngII-salt HTN. These changes were unexpected as they are predicted to drive [Cl-]i low, not high. Therefore, expression changes appear aimed at buffering elevated [Cl-]i caused by an alternative mechanism, possibly excess Cl- influx through GABA-AR, reflecting increased, not decreased, GABA release in AngII-salt HTN. Similar expression changes in HSD mice suggests that high salt intake generates a permissive state of Cl- ionic plasticity that blunts PVN GABA inhibition and facilitates AngII-salt HTN.

Apical intercalated cell cluster: A distinct sensory regulator in the amygdala.

GABAergic neurons regulate different aspects of information processing in the amygdala. Among these are clusters of intercalated cells (ITCs), which have been implicated in fear-related behaviors. Although a few of the ITC clusters have been studied, the functional role of apical ITCs (apITCs) is unknown. Here, we combine monosynaptic rabies tracing with optogenetics and demonstrate that apITCs receive synaptic input from medial geniculate nucleus (MGm), posterior intralaminar nucleus (PIN), and medial dorsal nucleus of the thalamus and from a diverse range of cortical areas including temporal association, entorhinal, insular, piriform, and somatosensory cortex. Upon fear learning, PIN/MGm inputs are strengthened, indicative of their involvement in fear behaviors. 3-D reconstruction of apITCs reveals local arborization and innervation of the dorsal striatum and lateral amygdala. We further show that apITCs provide sensory feedforward inhibition to LA principal cells, a putative mechanism for controlling plasticity during fear learning.

Regulation of hippocamposeptal input within the medial septum/diagonal band of Broca

The medial septum/diagonal band of Broca (MS/DBB) receives direct GABAergic input from the hippocampus via hippocamposeptal (HS) projection neurons as part of a reciprocal loop that mediates cognition and is altered in Alzheimer's disease. Cholinergic and GABAergic interactions occur throughout the MS/DBB, but it is not known how HS GABA release is impacted by these circuits. Most HS neurons contain somatostatin (SST), so to evoke HS GABA release we expressed Cre-dependent mCherry/channelrhodopisin-2 (ChR2) in the hippocampi of SST-IRES-Cre mice and then used optogenetics to stimulate HS fibers while performing whole-cell patch clamp recordings from MS/DBB neurons in acute slices. We found that the acetylcholine receptor (AChR) agonist carbachol and the GABAB receptor (GABABR) agonist baclofen significantly decreased HS GABA release in the MS/DBB. Carbachol's effects were blocked by eliminating local GABAergic activity or inhibiting GABABRs, indicating that it was indirectly decreasing HS GABA release by increasing GABAergic tone. There was no effect of acute exposure to amyloid-β on HS GABA release. Repetitive stimulation of HS fibers increased spontaneous GABA release in the MS/DBB, revealing that HS projections can modulate local GABAergic tone. These results show that HS GABA release has far-reaching impacts on overall levels of inhibition in the MS/DBB and is under regulatory control by cholinergic and GABAergic activity. This bidirectional modulation of GABA release from local and HS projections in the MS/DBB will likely have profound impact not only on activity within the MS/DBB, but also on output to the hippocampus and hippocampal-dependent learning and memory.

The Pathological Role of Astrocytic MAOB in Parkinsonism Revealed by Genetic Ablation and Over-expression of MAOB.

The cause of Parkinson’s disease has been traditionally believed to be the dopaminergic neuronal death in the substantia nigra pars compacta (SNpc). This traditional view has been recently challenged by the proposal that reactive astrocytes serve as key players in the pathology of Parkinson’s disease through excessive GABA release. This aberrant astrocytic GABA is synthesized by the enzymatic action of monoamine oxidase B (MAOB), whose pharmacological inhibition and gene-silencing are reported to significantly alleviate parkinsonian motor symptoms in animal models of Parkinson’s disease. However, whether genetic ablation and over-expression of MAOB can bidirectionally regulate parkinsonian motor symptoms has not been tested. Here we demonstrate that genetic ablation of MAOB blocks the MPTP-induced augmentation of astrocytic GABA-mediated tonic inhibition of neighboring dopaminergic neurons as well as parkinsonian motor symptoms, indicating the necessity of MAOB for parkinsonian motor symptoms. Furthermore, we demonstrate that GFAP-MAOB transgenic mice, in which MAOB is over-expressed under the GFAP promoter for astrocyte-specific over-expression, display exacerbated MPTP-induced tonic inhibition and parkinsonian motor symptoms compared to wild-type mice, indicating the importance of astrocytic MAOB for parkinsonian motor symptoms. Our study provides genetic pieces of evidence for the causal link between the pathological role of astrocytic MAOB-dependent tonic GABA synthesis and parkinsonian motor symptoms.

Epilepsy channelopathies go neddy: stabilizing NaV1.1 channels by neddylation.

Loss-of-function mutations of SCN1A encoding the pore-forming α subunit of the NaV1.1 neuronal sodium channel cause a severe developmental epileptic encephalopathy, Dravet syndrome (DS). In this issue of the JCI, Chen, Luo, Gao, et al. describe a phenocopy for DS in mice deficient for posttranslational conjugation with neural precursor cell expressed, developmentally downregulated 8 (NEDD8) (neddylation), selectively engineered in inhibitory interneurons. Pursuing the possibility that this phenotype is also caused by loss of NaV1.1, Chen, Luo, Gao, and colleagues show that interneuron excitability and GABA release are impaired, NaV1.1 degradation rate is increased with a commensurate decrease of NaV1.1 protein, and NaV1.1 is a substrate for neddylation. These findings establish neddylation as a mechanism for stabilizing NaV1.1 subunits and suggest another pathomechanism for epileptic sodium channelopathy.

Neddylation stabilizes Nav1.1 to maintain interneuron excitability and prevent seizures in murine epilepsy models

The excitability of interneurons requires Nav1.1, the α subunit of voltage-gated sodium channel. Nav1.1 deficiency and mutations reduce interneuron excitability, a major pathological mechanism for epilepsy syndromes. However, the regulatory mechanisms of Nav1.1 expression remain unclear. Here we provide evidence that neddylation is critical to Nav1.1 stability. Mutant mice lacking Nae1, an obligatory component of the E1 ligase for neddylation, in parvalbumin-positive interneurons (PVINs) exhibited spontaneous epileptic seizures and premature death. Electrophysiological studies indicate that Nae1 deletion reduced in PVIN excitability and GABA release, and consequently increased the network excitability of pyramidal neurons (PyNs). Further analysis revealed a reduction in sodium current density, not a change in channel property, in mutant PVINs and decreased Nav1.1 protein level. These results suggest that insufficient neddylation in PVINs reduces Nav1.1 stability and thus the excitability of PVINs; ensuing increased PyN activity causes seizures in mice. In agreement, Nav1.1 was found reduced by proteomic analysis that revealed abnormality in synapses and metabolic pathways. Our findings, for the first time, described a role of neddylation in maintaining Nav1.1 stability for PVIN excitability and reveal a new mechanism in pathogenesis of epilepsy.

Molecular Basis of GABA Hypofunction in Adolescent Schizophrenia-Like Animals.

Schizophrenia is a neurodevelopmental disorder that NMDA receptor (NMDAR) hypofunction appears centrally involved. Schizophrenia typically emerges in adolescence or early adulthood. Electrophysiological and several neurochemical changes have linked the GABA deficits to abnormal behaviors induced by NMDAR hypofunction. However, few studies have systematically investigated the molecular basis of GABA deficits, especially during adolescence. To address this issue, we transiently administrated MK-801 to mice on PND 10, which exhibited schizophrenia-relevant deficits in adolescence. Slice recording showed reduced GABA transmission and PVI+ hypofunction, indicating GABAergic hypofunction. Cortical proteomic evaluation combined with analysis of single cell data from the Allen Brain showed that various metabolic processes were enriched in top ranks and differentially altered in excitatory neurons, GABAergic interneurons, and glial cells. Notably, the GABA-related amino acid metabolic process was disturbed in both astrocytes and interneurons, in which we found a downregulated set of GABA-related proteins (GAD65, SYNPR, DBI, GAT3, SN1, and CPT1A). They synergistically regulate GABA synthesis, release, reuptake, and replenishment. Their downregulation indicates impaired GABA cycle and homeostasis regulated by interneuron-astrocyte communication in adolescence. Our findings on molecular basis of GABA deficits could provide potential drug targets of GABAergic rescue for early prevention and intervention.

Adenosine and Kynurenic Acid Interactions: Possible Relevance for Schizophrenia Treatment?

Adenosine and Kynurenic Acid Interactions: Possible Relevance for Schizophrenia Treatment?

Positive allosteric modulation of the mu-opioid receptor produces analgesia with reduced side effects

Positive allosteric modulators (PAMs) of the mu-opioid receptor (MOR) have been hypothesized as potentially safer analgesics than traditional opioid drugs. This is based on the idea that PAMs will promote the action of endogenous opioid peptides while preserving their temporal and spatial release patterns and so have an improved therapeutic index. However, this hypothesis has never been tested. Here, we show that a mu-PAM, BMS-986122, enhances the ability of the endogenous opioid Methionine-enkephalin (Met-Enk) to stimulate G protein activity in mouse brain homogenates without activity on its own and to enhance G protein activation to a greater extent than β-arrestin recruitment in Chinese hamster ovary (CHO) cells expressing human mu-opioid receptors. Moreover, BMS-986122 increases the potency of Met-Enk to inhibit GABA release in the periaqueductal gray, an important site for antinociception. We describe in vivo experiments demonstrating that the mu-PAM produces antinociception in mouse models of acute noxious heat pain as well as inflammatory pain. These effects are blocked by MOR antagonists and are consistent with the hypothesis that in vivo mu-PAMs enhance the activity of endogenous opioid peptides. Because BMS-986122 does not bind to the orthosteric site and has no inherent agonist action at endogenously expressed levels of MOR, it produces a reduced level of morphine-like side effects of constipation, reward as measured by conditioned place preference, and respiratory depression. These data provide a rationale for the further exploration of the action and safety of mu-PAMs as an innovative approach to pain management.

Elevated expression of complement C4 in the mouse prefrontal cortex causes schizophrenia-associated phenotypes.

Accumulating evidence supports immune involvement in the pathogenesis of schizophrenia, a severe psychiatric disorder. In particular, high expression variants of C4, a gene of the innate immune complement system, were shown to confer susceptibility to schizophrenia. However, how elevated C4 expression may impact brain circuits remains largely unknown. We used in utero electroporation to overexpress C4 in the mouse prefrontal cortex. We found reduced glutamatergic input to pyramidal cells of juvenile and adult, but not of newborn C4-overexpressing (C4-OE) mice, together with decreased spine density, which mirrors spine loss observed in the schizophrenic cortex. Using time-lapse two-photon imaging in vivo, we observed that these deficits were associated with decreased dendritic spine gain and elimination in juvenile C4-OE mice, which may reflect poor formation and/or stabilization of immature spines. In juvenile and adult C4-OE mice, we found evidence for NMDA receptor hypofunction, another schizophrenia-associated phenotype, and synaptic accumulation of calcium-permeable AMPA receptors. Alterations in cortical GABAergic networks have been repeatedly associated with schizophrenia. We found that functional GABAergic transmission was reduced in C4-OE mice, in line with diminished GABA release probability from parvalbumin interneurons, lower GAD67 expression, and decreased intrinsic excitability in parvalbumin interneurons. These cellular abnormalities were associated with working memory impairment. Our results substantiate the causal relationship between an immunogenetic risk factor and several distinct cortical endophenotypes of schizophrenia and shed light on the underlying cellular mechanisms.

Projection-dependent heterogeneity of cerebellar granule cell calcium responses

Cerebellar granule cells (GCs) relay mossy fiber (MF) inputs to Purkinje cell dendrites via their axons, the parallel fibers (PFs), which are individually located at a given sublayer of the molecular layer (ML). Although a certain degree of heterogeneity among GCs has been recently reported, variability of GC responses to MF inputs has never been associated with their most notable structural variability, location of their projecting PFs in the ML. Here, we utilize an adeno-associated virus (AAV)-mediated labeling technique that enables us to categorize GCs according to the location of their PFs, and compare the Ca2+ responses to MF stimulations between three groups of GCs, consisting of either GCs having PFs at the deep (D-GCs), middle (M-GCs), or superficial (S-GCs) sublayer. Our structural analysis revealed that there was no correlation between position of GC soma in the GC layer and location of its PF in the ML, confirming that our AAV-mediated labeling was important to test the projection-dependent variability of the Ca2+ responses in GCs. We then found that the Ca2+ responses of D-GCs differed from those of M-GCs. Pharmacological experiments implied that the different Ca2+ responses were mainly attributable to varied distributions of GABAA receptors (GABAARs) at the synaptic and extrasynaptic regions of GC dendrites. In addition to GABAAR distributions, amounts of extrasynaptic NMDA receptors appear to be also varied, because Ca2+ responses were different between D-GCs and M-GCs when glutamate spillover was enhanced. Whereas the Ca2+ responses of S-GCs were mostly equivalent to those of D-GCs and M-GCs, the blockade of GABA uptake resulted in larger Ca2+ responses in S-GCs compared with D-GCs and M-GCs, implying existence of mechanisms leading to more excitability in S-GCs with increased GABA release. Thus, this study reveals MF stimulation-mediated non-uniform Ca2+ responses in the cerebellar GCs associated with the location of their PFs in the ML, and raises a possibility that combination of inherent functional variability of GCs and their specific axonal projection contributes to the information processing through the GCs.

Potential mechanism of GABA secretion in response to the activation of GluK1-containing kainate receptors

Hippocampal GABAergic neurons are subdivided into more than 20 subtypes that are distinguished by features and functions. We have previously described the subpopulation of GABAergic neurons, which can be identified in hippocampal cell culture by the calcium response to the application of domoic acid (DoA), an agonist of kainate receptors (KARs). Here, we investigate the features of DoA-sensitive neurons and their GABA release mechanism in response to KARs activation. We demonstrate that DoA-sensitive GABAergic neurons express GluK1-containing KARs because ATPA, a selective agonist of GluK1-containing receptors, induces the calcium response exclusively in these GABAergic neurons. Our experiments also show that NASPM, previously considered a selective antagonist of calcium-permeable AMPARs, blocks calcium-permeable KARs. We established using NASPM that GluK1-containing receptors of the studied population of GABAergic neurons are calcium-permeable, and their activation is required for GABA release, at least in particular synapses. Notably, GABA release occurs even in the presence of tetrodotoxin, indicating that propagation of the depolarizing stimulus is not required for GABA release in this case. Thus, our data demonstrate that the activation of GluK1-containing calcium-permeable KARs mediates the GABA release by the studied subpopulation of GABAergic neurons.

Amino acid systems in the interpeduncular nucleus are altered in a sex-dependent manner during nicotine withdrawal.

Prior work in male rodents established that the medial habenula-interpeduncular nucleus (MHb-IPN) pathway modulates nicotine withdrawal. Specifically, withdrawal severity has been closely associated with inhibitory tone in the IPN via interneurons that release γ-aminobutyric acid (GABA). Inhibitory tone in the IPN is regulated by projections from the MHb that co-release glutamate and acetylcholine. Within the IPN, inhibitory tone is also regulated via corticotropin-releasing factor type 1 (CRF1) receptors that control GABA release from local interneurons. This study extends previous work by comparing sex differences in GABA, glutamate, as well serotonin levels in the IPN during precipitated nicotine withdrawal. Sex differences in withdrawal-induced neurochemical effects were also compared following systemic administration of a CRF1 receptor antagonist. The results revealed that there were no group differences in serotonin levels in the IPN. A major finding was that females displayed a larger withdrawal-induced increases in GABA levels in the IPN than males. Also, withdrawal increased IPN glutamate levels in a similar manner in females and males. Blockade of CRF1 receptors produced a larger suppression of the withdrawal-induced increases in GABA levels in the IPN of females versus males, an effect that was likely related to the robust increase in glutamate following administration of the CRF1 receptor antagonist in females. These data suggest that amino acid systems in the IPN modulate sex differences in the behavioral effects of nicotine withdrawal. Furthermore, our data imply that medications that target stress-induced activation of the IPN may reduce withdrawal severity, particularly in females.

Insulin differentially modulates GABA signalling in hippocampal neurons and, in an age-dependent manner, normalizes GABA-activated currents in the tg-APPSwe mouse model of Alzheimer's disease.

We examined if tonic γ-aminobutyric acid (GABA)-activated currents in primary hippocampal neurons were modulated by insulin in wild-type and tg-APPSwe mice, an Alzheimer's disease (AD) model. GABA-activated currents were recorded in dentate gyrus (DG) granule cells and CA3 pyramidal neurons in hippocampal brain slices, from 8 to 10weeks old (young) wild-type mice and in dorsal DG granule cells in adult, 5-6 and 10-12 (aged) months old wild-type and tg-APPSwe mice, in the absence or presence of insulin, by whole-cell patch-clamp electrophysiology. In young mice, insulin (1nmol/L) enhanced the total spontaneous inhibitory postsynaptic current (sIPSCT ) density in both dorsal and ventral DG granule cells. The extrasynaptic current density was only increased by insulin in dorsal CA3 pyramidal neurons. In absence of action potentials, insulin enhanced DG granule cells and dorsal CA3 pyramidal neurons miniature IPSC (mIPSC) frequency, consistent with insulin regulation of presynaptic GABA release. sIPSCT densities in DG granule cells were similar in wild-type and tg-APPSwe mice at 5-6months but significantly decreased in aged tg-APPSwe mice where insulin normalized currents to wild-type levels. The extrasynaptic current density was increased in tg-APPSwe mice relative to wild-type littermates but, only in aged tg-APPSwe mice did insulin decrease and normalize the current. Insulin effects on GABA signalling in hippocampal neurons are selective while multifaceted and context-based. Not only is the response to insulin related to cell-type, hippocampal axis-location, age of animals and disease but also to the subtype of neuronal inhibition involved, synaptic or extrasynaptic GABAA receptors-activated currents.

Targeting angiotensin type-2 receptors located on pressor neurons in the nucleus of the solitary tract to relieve hypertension in mice.

These studies evaluate whether angiotensin type-2 receptors (AT2Rs) that are expressed on γ-aminobutyric acid (GABA) neurons in the nucleus of the solitary tract (NTS) represent a novel endogenous blood pressure-lowering mechanism. Experiments combined advanced genetic and neuroanatomical techniques, pharmacology, electrophysiology, and optogenetics in mice to define the structure and cardiovascular-related function of NTS neurons that contain AT2R. Using mice with Cre-recombinase directed to the AT2R gene, we discovered that optogenetic stimulation of AT2R-expressing neurons in the NTS increases GABA release and blood pressure. To evaluate the role of the receptor, per se, in cardiovascular regulation, we chronically delivered C21, a selective AT2R agonist, into the brains of normotensive mice and found that central AT2R activation reduces GABA-related gene expression and blunts the pressor responses induced by optogenetic excitation of NTS AT2R neurons. Next, using in situ hybridization, we found that the levels of Agtr2 mRNAs in GABAergic NTS neurons rise during experimentally induced hypertension, and we hypothesized that this increased expression may be exploited to ameliorate the disease. Consistent with this, final experiments revealed that central administration of C21 attenuates hypertension, an effect that is abolished in mice lacking AT2R in GABAergic NTS neurons. These studies unveil novel hindbrain circuits that maintain arterial blood pressure, and reveal a specific population of AT2R that can be engaged to alleviate hypertension. The implication is that these discrete receptors may serve as an access point for activating an endogenous depressor circuit.

Tetanus and tetanus neurotoxin: From peripheral uptake to central nervous tissue targets.

Tetanus is a deadly but preventable disease caused by a protein neurotoxin produced by Clostridium tetani. Spores of C. tetani may contaminate a necrotic wound and germinate into a vegetative bacterium that releases a toxin, termed tetanus neurotoxin (TeNT). TeNT enters the general circulation, binds to peripheral motor neurons and sensory neurons, and is transported retroaxonally to the spinal cord. It then enters inhibitory interneurons and blocks the release of glycine or GABA causing a spastic paralysis. This review attempts to correlate the metalloprotease activity of TeNT and its trafficking and localization into the vertebrate body to the nature and sequence of appearance of the symptoms of tetanus.

Long-Range GABAergic Inhibition Modulates Spatiotemporal Dynamics of the Output Neurons in the Olfactory Bulb.

Local interneurons of the olfactory bulb (OB) are densely innervated by long-range GABAergic neurons from the basal forebrain (BF), suggesting that this top-down inhibition regulates early processing in the olfactory system. However, how GABAergic inputs modulate the OB output neurons, the mitral/tufted cells, is unknown. Here, in male and female mice acute brain slices, we show that optogenetic activation of BF GABAergic inputs produced distinct local circuit effects that can influence the activity of mitral/tufted cells in the spatiotemporal domains. Activation of the GABAergic axons produced a fast disinhibition of mitral/tufted cells consistent with a rapid and synchronous release of GABA onto local interneurons in the glomerular and inframitral circuits of the OB, which also reduced the spike precision of mitral/tufted cells in response to simulated stimuli. In addition, BF GABAergic inhibition modulated local oscillations in a layer-specific manner. The intensity of locally evoked θ oscillations was decreased on activation of top-down inhibition in the glomerular circuit, while evoked γ oscillations were reduced by inhibition of granule cells. Furthermore, BF GABAergic input reduced dendrodendritic inhibition in mitral/tufted cells. Together, these results suggest that long-range GABAergic neurons from the BF are well suited to influence temporal and spatial aspects of processing by OB circuits.SIGNIFICANCE STATEMENT Disruption of GABAergic inhibition from the basal forebrain (BF) to the olfactory bulb (OB) impairs the discrimination of similar odors, yet how this centrifugal inhibition influences neuronal circuits in the OB remains unclear. Here, we show that the BF GABAergic neurons exclusively target local inhibitory neurons in the OB, having a functional disinhibitory effect on the output neurons, the mitral cells. Phasic inhibition by BF GABAergic neurons reduces spike precision of mitral cells and lowers the intensity of oscillatory activity in the OB, while directly modulating the extent of dendrodendritic inhibition. These circuit-level effects of this centrifugal inhibition can influence the temporal and spatial dynamics of odor coding in the OB.

Enhancing GABAergic signaling ameliorates aberrant gamma oscillations of olfactory bulb in AD mouse models

BackgroundBefore the deposition of amyloid-beta plaques and the onset of learning memory deficits, patients with Alzheimer’s disease (AD) experience olfactory dysfunction, typified by a reduced ability to detect, discriminate, and identify odors. Rodent models of AD, such as the Tg2576 and APP/PS1 mice, also display impaired olfaction, accompanied by aberrant in vivo or in vitro gamma rhythms in the olfactory pathway. However, the mechanistic relationships between the electrophysiological, biochemical and behavioral phenomena remain unclear.MethodsTo address the above issues in AD models, we conducted in vivo measurement of local field potential (LFP) with a combination of in vitro electro-olfactogram (EOG), whole-cell patch and field recordings to evaluate oscillatory and synaptic function and pharmacological regulation in the olfactory pathway, particularly in the olfactory bulb (OB). Levels of protein involved in excitation and inhibition of the OB were investigated by western blotting and fluorescence staining, while behavioral studies assessed olfaction and memory function.ResultsLFP measurements demonstrated an increase in gamma oscillations in the OB accompanied by altered olfactory behavior in both APP/PS1 and 3xTg mice at 3–5 months old, i.e. an age before the onset of plaque formation. Fewer olfactory sensory neurons (OSNs) and a reduced EOG contributed to a decrease in the excitatory responses of M/T cells, suggesting a decreased ability of M/T cells to trigger interneuron GABA release indicated by altered paired-pulse ratio (PPR), a presynaptic parameter. Postsynaptically, there was a compensatory increase in levels of GABAAR α1 and β3 subunits and subsequent higher amplitude of inhibitory responses. Strikingly, the GABA uptake inhibitor tiagabine (TGB) ameliorated abnormal gamma oscillations and levels of GABAAR subunits, suggesting a potential therapeutic strategy for early AD symptoms. These findings reveal increased gamma oscillations in the OB as a core indicator prior to onset of AD and uncover mechanisms underlying aberrant gamma activity in the OB.ConclusionsThis study suggests that the concomitant dysfunction of both olfactory behavior and gamma oscillations have important implications for early AD diagnosis: in particular, awareness of aberrant GABAergic signaling mechanisms might both aid diagnosis and suggest therapeutic strategies for olfactory damage in AD.

Moderate adolescent chronic intermittent ethanol exposure sex-dependently disrupts synaptic transmission and kappa opioid receptor function in the basolateral amygdala of adult rats

Adolescent alcohol exposure is associated with many consequences in adulthood, including altered affective and reward-related behaviors. However, the long-term neurological disruptions underlying these behaviors are not fully understood. Shifts in the excitatory/inhibitory balance in the basolateral amygdala (BLA) relate to the expression of these behaviors and changes to BLA physiology are seen during withdrawal immediately following adolescent ethanol exposure, but no studies have examined whether these changes persist long-term. The kappa opioid receptor (KOR) neuromodulatory system mediates negative affective behaviors, and alterations of this system are implicated in behavioral changes following adult and adolescent chronic ethanol exposure. In the BLA, the KOR system undergoes functional changes across development, but whether BLA KOR function is disrupted by adolescent ethanol exposure is unknown. In this study, male and female Sprague-Dawley rats were exposed to a vapor model of moderate adolescent chronic intermittent ethanol (aCIE) and assessed for long-term effects on GABAergic and glutamatergic neurotransmission within the adult BLA and KOR modulation of these systems. aCIE exposure increased presynaptic glutamate transmission in females but had no effect in males or on GABA transmission in either sex. Additionally, aCIE exposure disrupted male KOR modulation of GABA release, with no effects in females or on glutamate transmission. These data suggest that aCIE produces sex-dependent and long-term changes to BLA physiology and KOR function. This is the first study to examine these persistent adaptations following adolescent alcohol exposure and opens a broad avenue for future investigation into other adolescent ethanol-induced disruptions of these systems.

66108 Central Cholinergic Synapse Formation in Optimized Primary Septal “Hippocampal Co” cultures

ABSTRACT IMPACT: Optimization of primary septal-hippocampal co-cultures facilitates studying central cholinergic synapse formation and dysfunction OBJECTIVES/GOALS: Septal cholinergic innervation to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer’s disease. To understand the molecular events underlying this loss, we optimized a primary septal-hippocampal co-culture system that facilitates the study of central cholinergic synapses. METHODS/STUDY POPULATION: We developed an optimized in vitro septal-hippocampal co-culture system modified from previously published protocols. Briefly, hippocampal and septal tissue were harvested from embryonic day 19 (E19) Sprague-Dawley rats, digested with 0.1% trypsin, and an equal number of cells from each region plated onto coverslips coated with poly-D-lysine and laminin at a final density of 300 cells/mm2. We use immunostaining with validated primary antibodies and a fluorescent binding assay, together with confocal microscopy, to determine the structure of cholinergic synapses that are 1) native, 2) mammalian, 3) CNS derived, 4) comprised of physiological synaptic partners, and 5) developmentally mature. RESULTS/ANTICIPATED RESULTS: After DIV21, co-cultures maintained a healthy morphology. A subpopulation of neurons strongly expressed the cholinergic markers vesicular ACh transporter (vAChT), choline acetyltransferase (ChAT), and the high-affinity choline transporter (ChT1), whereas most neurons lacked vAChT expression and were presumably glutamatergic or GABAergic. The percentage of cholinergic neurons attained in the co-culture is ˜5-7%. The size of these cholinergic neurons is strikingly similar to that reported for BFCNs in the intact brain (mean 30μm, range 18-43μm). All sampled cholinergic neurons (28/28 neurons) also expressed molecular machinery necessary for GABA release. Staining for a cholinergic postsynaptic marker shows that 63% of the contacts made with are synaptic. DISCUSSION/SIGNIFICANCE OF FINDINGS: Primary septal-hippocampal co-cultured neurons have not been exploited extensively in the field, perhaps due to the difficulty in maintaining such cultures for extended periods. Here, we optimized an in vitro septal-hippocampal co-culture system, a powerful tool to comprehensively analyze central cholinergic synapse formation and dysfunction.