Tonic GABAergic Inhibition Research Articles

Allosteric Modulation of αβδ GABAA Receptors

GABAA receptors mediate the majority of the fast inhibition in the mature brain and play an important role in the pathogenesis of many neurological and psychiatric disorders. The αβδ GABAA receptor localizes extra- or perisynaptically and mediates GABAergic tonic inhibition. Compared with synaptically localized αβγ receptors, αβδ receptors are more sensitive to GABA, display relatively slower desensitization and exhibit lower efficacy to GABA agonism. Interestingly, αβδ receptors can be positively modulated by a variety of structurally different compounds, even at saturating GABA concentrations. This review focuses on allosteric modulation of recombinant αβδ receptor currents and αβδ receptor-mediated tonic currents by anesthetics and ethanol. The possible mechanisms for the positive modulation of αβδ receptors by these compounds will also be discussed.

Transplantation of GABAergic Neurons from ESCs Attenuates Tactile Hypersensitivity Following Spinal Cord Injury

Abstract We investigated the therapeutic potential of mouse ESC-derived gamma-amino butyric acid (GABA)ergic neurons (∼74% of total neurons in vitro) to reduce neuropathic pain following spinal cord injury (SCI) in rats. Spinal cord hemisection at the T13 segment, which is used as a rat SCI pain model, induced tactile hypersensitivity of the hind paw, as evidenced by decreased paw withdrawal thresholds in response to von Frey filaments, and also induced hyperexcitability of wide dynamic range neurons in the lumbar spinal cord in response to natural cutaneous stimuli. At 2 weeks posthemisection, GABAergic neurons (500,000 cells) were transplanted into the subarachnoid space of the spinal lumbar enlargement via a modified lumbar puncture technique. The transplantation of GABAergic neurons led to long-term attenuation of hemisection-induced tactile hypersensitivity and neuronal hyperexcitability as compared with vehicle-treated controls. These attenuations were reversed by the application of bicuculline and CGP52432, GABA-A and GABA-B receptor antagonists, respectively, but not by application of the serotonergic receptor antagonist methylsergide, indicating a specific restoration of spinal GABAergic inhibition. Histological data from sections of the lumbar cord in grafts demonstrated that 43.5% of surviving engrafted cells were neurons and located densely in the lower-medial portion of the dorsal funiculi in the spinal white matter. Among the observed neurons, 26.2% were GABAergic. The results suggest that subarachnoid transplantation of ESC-derived GABAergic neurons appear to restore spinal GABAergic inhibitory tone and can be a promising strategy to treat SCI-induced pain.

Tonic GABAergic control of mouse dentate granule cells during postnatal development

The dentate gyrus is the main hippocampal input structure receiving strong excitatory cortical afferents via the perforant path. Therefore, inhibition at this 'hippocampal gate' is important, particularly during postnatal development, when the hippocampal network is prone to seizures. The present study describes the development of tonic GABAergic inhibition in mouse dentate gyrus. A prominent tonic GABAergic component was already present at early postnatal stages (postnatal day 3), in contrast to the slowly developing phasic postsynaptic GABAergic currents. Tonic currents were mediated by GABA(A) receptors containing α(5)- and δ-subunits, which are sensitive to low ambient GABA concentrations. The extracellular GABA level was determined by synaptic GABA release and GABA uptake via the GABA transporter 1. The contribution of these main regulatory components was surprisingly stable during postnatal granule cell maturation. Throughout postnatal development, tonic GABAergic signals were inhibitory. They increased the action potential threshold of granule cells and reduced network excitability, starting as early as postnatal day 3. Thus, tonic inhibition is already functional at early developmental stages and plays a key role in regulating the excitation/inhibition balance of both the adult and the maturing dentate gyrus.

D1 and D2 dopamine receptors in separate circuits cooperate to drive associative long-term potentiation in the prefrontal cortex

Dopamine release associated with motivational arousal is thought to drive goal-directed learning and consolidation of acquired memories. This dopamine hypothesis of learning and motivation directly suggests that dopamine is necessary for modifications of excitatory synapses in dopamine terminal fields, including the prefrontal cortex (PFC), to "stamp in" posttrial memory traces. It is unknown how such enabling occurs in native circuits tightly controlled by GABAergic inhibitory tone. Here we report that dopamine, via both D1-class receptors (D1Rs) and D2-class receptors (D2Rs), enables the induction of spike timing-dependent long-term potentiation (t-LTP) in layer V PFC pyramidal neurons over a "window" of more than 30 ms that is otherwise closed under intact inhibitory constraint. Dopamine acts at D2Rs in local GABAergic interneurons to suppress inhibitory transmission, gating the induction of t-LTP. Moreover, dopamine activates postsynaptic D1Rs in excitatory synapses to allow t-LTP induction at a substantially extended, normally ineffective, timing interval (+30 ms), thus increasing the associability of prepost coincident stimuli. Although the D2R-mediated disinhibition alone is sufficient to gate t-LTP at a normal timing (+10 ms), t-LTP at +30 ms requires concurrent activation of both D1Rs and D2Rs. Our results illustrate a previously unrecognized circuit-level mechanism by which dopamine receptors in separate microcircuits cooperate to drive Hebbian synaptic plasticity across a significant temporal window under intact inhibition. This mechanism should be important in functioning of interconnected PFC microcircuits, in which D1Rs and D2Rs are not colocalized but their coactivation is necessary.

Alcohol- and Alcohol Antagonist-Sensitive Human GABAA Receptors: Tracking δ Subunit Incorporation into Functional Receptors

GABA(A) receptors (GABA(A)Rs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic δ subunit-containing GABA(A)Rs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant α4β3δ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of α4β3δ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating δ subunits into functional receptors. To track δ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the δ subunit is replaced by an alanine residue found at the homologous position in γ subunits. We demonstrate that the δH68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive α4β3δ receptors. The extent of enhancement of α4β3δH68A receptors by 1 μM diazepam, 30 mM EtOH, and 1 μM β-carboline-3-carboxy ethyl ester (but not 1 μM Zn(2+) block) is correlated in individual recordings, suggesting that δ subunit incorporation into recombinant GABA(A)Rs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that δ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native δ subunit-containing GABA(A)Rs.

Glycinergic and GABAergic tonic inhibition fine tune inhibitory control in regionally distinct subpopulations of dorsal horn neurons

Inhibition mediated by glycine and GABA in the spinal cord dorsal horn is essential for controlling sensitivity to painful stimuli. Loss of inhibition results in hyperalgesia, a sensitized response to a painful stimulus, and allodynia, a pain-like response to an innocuous stimulus like touch. The latter is due, in part, to disinhibition of an excitatory polysynaptic pathway linking low threshold touch input to pain projection neurons. This critical impact of disinhibition raises the issue of what regulates the activity of inhibitory interneurons in the dorsal horn under non-pathological conditions. We have found that inhibitory neurons throughout lamina I-III, identified by the GAD67 promoter-driven EGFP, are tonically inhibited by glycine or GABA in a regionally distinct way that is mirrored by their inhibitory synaptic input. This tonic inhibition strongly modifies action potential firing properties. Surprisingly, we found that inhibitory neurons at the lamina II/III border are under tonic glycinergic control and receive synapses that are predominantly glycinergic. Futhermore, this tonic glycinergic inhibition remains strong as the mice mature postnatally. Interestingly, GlyT1, the glial glycine transporter, regulates the strength of tonic glycinergic inhibition of these glycine-dominant neurons. The more dorsal lamina I and IIo inhibitory neurons are mainly under control by tonic GABA action and receive synapses that are predominantly GABAergic. Our work supports the hypothesis that tonic glycine inhibition controls the inhibitory circuitry deep in lamina II that is likely to be responsible for separating low threshold input from high threshold output neurons of lamina I.

Impact of gestational and lactational phthalate exposure on hypothalamic content of amino acid neurotransmitters and FSH secretion in peripubertal male rats

This study investigated the effect of the pre- and perinatal exposure to di-(2-ethylhexyl) phthalate (DEHP) on the neuroendocrine parameters that regulate reproduction in peripubertal male rats. DEHP at dose of 3 and 30 mg/kg bw/day was administered orally to female rat since pregnancy onset until weaning. The male litters were sacrificed at 30 days of age to determine gonadotropin serum level and the hypothalamic contents of the amino acids aspartate and gamma-aminobutyric acid. No changes in gonadotropin, aspartate and gamma-aminobutyric acid levels were detected at the low dose. DEHP 30 mg/kg bw/day reduced testis weight and serum FSH, in correlation with a significant increase in the inhibitory GABAergic tone and a reduction in the stimulatory effect of aspartate on gonadotropin level. This study provides unknown data regarding changes in the hypothalamic contents of the amino acid neurotransmitters, which are involved in the neuroendocrine regulation of reproductive axis, in peripubertal male rat offspring from dams exposed to DEHP during gestational and lactational periods. This could be related with the gonadotropin modifications also here described.

Pharmacological studies reveal novel aspects of the versatility of GABAA receptors

When focusing on specific tasks of GABAergic transmission, it is not very common to realize an extraordinary multi-functionality of the GABAergic system. In the present article, I would like to emphasize some aspects of the amazing versatility of GABAA receptors (GABAARs) and, on this background, to highlight novel findings by Mortensen et al. (2010) in a recent issue of The Journal of Physiology relating to distinct kinetics and pharmacology of synaptic and extrasynaptic GABAARs.

Glucose prevents the fall in ventromedial hypothalamic GABA that is required for full activation of glucose counterregulatory responses during hypoglycemia

Local delivery of glucose into a critical glucose-sensing region within the brain, the ventromedial hypothalamus (VMH), can suppress glucose counterregulatory responses to systemic hypoglycemia. Here, we investigated whether this suppression was accomplished through changes in GABA output in the VMH. Sprague-Dawley rats had catheters and guide cannulas implanted. Eight to ten days later, microdialysis-microinjection probes were inserted into the VMH, and they were dialyzed with varying concentrations of glucose from 0 to 100 mM. Two groups of rats were microdialyzed with 100 mM glucose and microinjected with either the K(ATP) channel opener diazoxide or a GABA(A) receptor antagonist. These animals were then subjected to a hyperinsulinemic-hypoglycemic glucose clamp. As expected, perfusion of glucose into the VMH suppressed the counterregulatory responses. Extracellular VMH GABA levels positively correlated with the concentration of glucose in the perfusate. In turn, extracellular GABA concentrations in the VMH were inversely related to the degree of counterregulatory hormone release. Of note, microinjection of either diazoxide or the GABA(A) receptor antagonist reversed the suppressive effects of VMH glucose delivery on counterregulatory responses. Some GABAergic neurons in the VMH respond to changes in local glucose concentration. Glucose in the VMH dose-dependently stimulates GABA release, and this in turn dose-dependently suppresses the glucagon and epinephrine responses to hypoglycemia. These data suggest that during hypoglycemia a decrease in glucose concentration within the VMH may provide an important signal that rapidly inactivates VMH GABAergic neurons, reducing inhibitory GABAergic tone, which in turn enhances the counterregulatory responses to hypoglycemia.

Increased tonic GABA(A) receptor mediated signaling in dentate gyrus of SSADH deficient mice

Event Abstract Back to Event Increased tonic GABA(A) receptor mediated signaling in dentate gyrus of SSADH deficient mice Zita Dósa1*, Jose L. Nieto-Gonzalez1, Michael K. Gibson2 and Kimmo Jensen1 1 Aarhus University, Synaptic Physiology Laboratory, Department of Physiology and Biophysics, Denmark 2 Michigan Technological University, Department of Biological Sciences, United States Excessive γ-aminobutyric acid (GABA) during brain development may lead to structural and functional changes in the brain tissue. This is the case in succinic semialdehyde dehydrogenase (SSADH) deficiency, which is an inherited neurometabolic disorder of GABA catabolism. Since SSADH participates in the breakdown of GABA, this condition leads to elevated GABA levels, but also increased γ-hydroxybutyric acid (GHB) in the brain, which also activates GABA receptors. SSADH deficient patients display neuropsychiatric symptoms, ataxia, and epileptic seizures, and SSADH knock-out mice were recently engineered to model the human disorder. Aims: The purpose of this study was to determine how the SSADH gene dosage impacts single-neuron physiology, since this might offer insight into how SSADH regulates the extracellular GABA levels in physiological and pathophysiological situations. Methods: To investigate the activation of GABA(A) receptors in SSADH deficiency, we performed whole-cell patch-clamp recordings in SSADH wild-type, heterozygous, and knock-out mice. Results: Both in dentate gyrus granule cells and interneurons, we found an increase in extrasynaptic GABA(A) receptor-mediated tonic current. On the other hand, the frequency and waveform of spontaneous inhibitory postsynaptic currents (sIPSCs) arising from GABAergic activity showed no significant alteration in these cells. Decreases in the SSADH gene dosage led to non-linear increases in GABA(A) receptor mediated neurotransmission in these mice. Conclusion: We conclude that SSADH deficiency preferentially leads to increased tonic GABAergic inhibition in the dentate gyrus, which may play a role for the pathophysiological activity in cortical networks. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Cellular neuroscience Citation: Dósa Z, Nieto-Gonzalez JL, Gibson MK and Jensen K (2010). Increased tonic GABA(A) receptor mediated signaling in dentate gyrus of SSADH deficient mice. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00023 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 16 Apr 2010; Published Online: 16 Apr 2010. * Correspondence: Zita Dósa, Aarhus University, Synaptic Physiology Laboratory, Department of Physiology and Biophysics, Aarhus, Denmark, zd@fi.au.dk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Zita Dósa Jose L Nieto-Gonzalez Michael K Gibson Kimmo Jensen Google Zita Dósa Jose L Nieto-Gonzalez Michael K Gibson Kimmo Jensen Google Scholar Zita Dósa Jose L Nieto-Gonzalez Michael K Gibson Kimmo Jensen PubMed Zita Dósa Jose L Nieto-Gonzalez Michael K Gibson Kimmo Jensen Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Regulation of tonic GABA inhibitory function, presympathetic neuronal activity and sympathetic outflow from the paraventricular nucleus by astroglial GABA transporters

Neuronal activity in the hypothalamic paraventricular nucleus (PVN), as well as sympathetic outflow from the PVN, is basally restrained by a GABAergic inhibitory tone. We recently showed that two complementary GABA(A) receptor-mediated modalities underlie inhibition of PVN neuronal activity: a synaptic, quantal inhibitory modality (IPSCs, I(phasic)) and a sustained, non-inactivating modality (I(tonic)). Here, we investigated the role of neuronal and/or glial GABA transporters (GATs) in modulating these inhibitory modalities, and assessed their impact on the activity of RVLM-projecting PVN neurons (PVN-RVLM neurons), and on PVN influence of renal sympathetic nerve activity (RSNA). Patch-clamp recordings were obtained from retrogradely labelled PVN-RVLM neurons in a slice preparation. The non-selective GAT blocker nipecotic acid (100-300 microM) caused a large increase in GABA(A)I(tonic), and reduced IPSC frequency. These effects were replicated by beta-alanine (100 microM), but not by SKF 89976A (30 microM), relatively selective blockers of GAT3 and GAT1 isoforms, respectively. Similar effects were evoked by the gliotoxin L-alpha-aminodipic acid (2 mM). GAT blockade attenuated the firing activity of PVN-RVLM neurons. Moreover, PVN microinjections of nipecotic acid in the whole animal diminished ongoing RSNA. A robust GAT3 immunoreactivity was observed in the PVN, which partially colocalized with the glial marker GFAP. Altogether, our results indicate that by modulating ambient GABA levels and the efficacy of GABA(A)I(tonic), PVN GATs, of a likely glial location, contribute to setting a basal tone of PVN-RVLM firing activity, and PVN-driven RSNA.

New insights on the role of gephyrin in regulating both phasic and tonic GABAergic inhibition in rat hippocampal neurons in culture

Gephyrin is a tubulin-binding protein that acts as a scaffold for clustering glycine and GABA A receptors at postsynaptic sites. In this study, the role of gephyrin on GABA A receptor function was assessed at the post-translational level, using gephyrin-specific single chain antibody fragments (scFv-gephyrin). When expressed in cultured rat hippocampal neurons as a fusion protein containing a nuclear localization signal, scFv-gephyrin were able to remove endogenous gephyrin from GABA A receptor clusters. Immunocytochemical experiments revealed a significant reduction in the number of synaptic γ2-subunit containing GABA A receptors and a significant decrease in the density of the GABAergic presynaptic marker vesicular GABA transporter (VGAT). These effects were associated with a slow down of the onset kinetics, a reduction in the amplitude and in the frequency of miniature inhibitory postsynaptic currents (mIPSCs). The quantitative analysis of current responses to ultrafast application of GABA suggested that changes in onset kinetics resulted from modifications in the microscopic gating of GABA A receptors and in particular from a reduced entry into the desensitized state. In addition, hampering gephyrin function with scFv-gephyrin induced a significant reduction in GABA A receptor-mediated tonic conductance. This effect was probably dependent on the decrease in GABAergic innervation and in GABA release from presynaptic nerve terminals. These results indicate that gephyrin is essential not only for maintaining synaptic GABA A receptor clusters in the right position but also for regulating both phasic and tonic inhibition.

Involvement of gamma amino butyric acid (GABA) in the postnatal function of the GnRH pulse generator as determined on the basis of GnRH and GnRH-receptor gene expression in the hypothalamus and the pituitary

In many species the GnRH pulse generator functions early postnatally to become arrested during infancy. In rats highly variable LH levels in 15-day-old animals are suggestive that LH is being released by the pituitary in pulses whereas between day 20 after birth and puberty LH levels are low indicating that the GnRH pulse generator is arrested. In the present study we show on the basis of consecutively withdrawn blood samples in 15-day-old animals that LH pulses are indeed present at that age. The proper function of GnRH receptors in the pituitary is crucially dependent on pulsatile GnRH release from the hypothalamus. In addition, GnRH receptors have been demonstrated in the medial preoptic area and in the mediobasal hypothalamus of adult rats. In 15-day-old animals the functional GnRH pulse generator results in upregulated GnRH receptor gene expression as demonstrated by quantitative RT-PCR. It is not known what neural mechanisms are involved in turning the GnRH pulse generator off during infancy and a GABAergic brake has been discussed. Indeed, when 30-day-old animals were injected with the GABA-A receptor blocking drug bicuculline, this resulted in increased serum LH levels indicating that a tonic GABAergic inhibition is indeed operative at this age.

Role of GABA transporter 3 in GABAergic synaptic transmission at striatal output neurons

Striatal GABAergic signaling has been shown to be essential for basal ganglia output and proper motor performance. In the mouse neostriatum GABA transporter 1 (GAT-1) was previously found to assist in the clearance of GABA from the extracellular space and influence both phasic and tonic GABAergic inhibition of medium-sized striatal output neurons (SONs). It currently remains unknown whether GAT subtypes other than GAT-1 participate in the modulation of GABAergic transmission in this brain structure. In this study, we aimed at assessing the role of GAT-3 in the mouse neostriatum. To this end, we recorded GABAergic inhibitory postsynaptic currents (IPSCs) from SONs in brain slices at different developmental stages (postnatal days (P) 7-9, 12-14, and 28-34) using the whole-cell patch-clamp technique. When applied under control conditions, SNAP-5114 (40 microM), a specific GAT-3 blocker, did not affect miniature or evoked IPSCs (m/eIPSCs) and produced no significant effect on tonic GABAA receptor-mediated conductances in SONs. However, in the presence of NO-711 (10 microM), a specific GAT-1 blocker, SNAP-5114 reduced mIPSC frequencies without affecting mIPSC amplitudes or kinetics. In addition, SNAP-5114 reduced the mean amplitude of eIPSCs and increased the paired-pulse ratio. These effects were entirely abolished by CGP55845 (1 microM), a specific GABAB receptor blocker, indicating that they were mediated by presynaptic GABAB receptors. Similar results were obtained from all age groups. We conclude that GAT-3 is functionally expressed in the mouse neostriatum. Whereas an acute block of GAT-3 under resting conditions is fully compensated for by GAT-1, GAT-3 might provide an additional uptake capacity when neuronal activity and GABA release are increased.

Thermal Hyperalgesia via Supraspinal Mechanisms in Mice Lacking Glutamate Decarboxylase 65

Gamma-aminobutyric acid, which is synthesized by two isoforms of glutamate decarboxylase (GAD), inhibits the transfer of nociceptive signals from primary afferent fibers to the central nervous system. However, the roles of a 65-kDa isoform of GAD (GAD65)-mediated GABA in nociceptive processing are less clear. This study tested whether partial reductions in GABAergic inhibitory tone by GAD65 gene knockout [GAD65(-/-)] would contribute to the regulation of pain threshold in mice. Experiments were performed on male wild-type (WT) mice and GAD65(-/-) mice. Acute nociception and inflammatory pain tests were compared between WT mice and GAD65(-/-) mice. GABA(A) receptor-mediated inhibitory postsynaptic currents were also examined by use of the whole-cell patch-clamp method in somatosensory cortical neurons in brain slices. In the hot plate test, which reflects supraspinal sensory integration, a significant reduction in the latency was observed for GAD65(-/-) mice. Intraperitoneal administration of the GABA transporter 1 inhibitor, 1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (C(21)H(22)N(2)O(3).HCl; NO-711), dose-dependently prolonged the latency in both genotypes, suggesting that GABA concentration contributes to acute thermal nociception. However, there was no genotype difference in responses to the tail-immersion test or the von Frey test, indicating that spinal reflex and mechanical nociception are kept intact in GAD65(-/-) mice. There was no genotype difference in responses to chemical inflammatory nociception (formalin test and carrageenan test). Although properties of the phasic component of inhibitory postsynaptic currents were similar in both genotypes, tonic inhibition was significantly reduced in GAD65(-/-) mice. These results support the hypothesis that GAD65-mediated GABA synthesis plays relatively small but significant roles in nociceptive processing via supraspinal mechanisms.

Regulation of sympathetic nerve activity and arterial pressure by the rostral ventrolateral medulla in obese Zucker rats.

Adult obese Zucker rats (OZR) have elevated sympathetic nerve activity (SNA) and arterial pressure (AP) with exaggerated sympatho‐excitatory responses (somatic and diving reflex) compared to lean Zucker rats (LZR). The present study sought to determine whether the altered control of SNA and AP in OZR could be due to enhanced glutamatergic or reduced GABAergic control of the rostral ventrolateral medulla (RVLM). Adult rats (14‐17 wks) were anesthetized with urethane (1.5 g/kg, iv), ventilated, and paralyzed for recording of SNA and AP. Microinjections of glutamate (1 nmol/100 nl) were performed into the RVLM to locate sites for maximal increases in SNA and AP (1.4 ‐ 1.8 mm rostral to calamus, 1.7 ‐ 2.1 mm lateral to midline, 2.7 ‐ 3.1 mm ventral; tip angled 20° rostrally). Baseline mean AP of OZR was significantly higher than LZR (n=6, 124 ± 2 mmHg in OZR vs n=6, 108 ± 6 mmHg in LZR, P<0.05). The OZR had larger maximal increases in AP (n=6, 30 ± 3 mmHg in OZR vs n=6, 20 ± 2 mmHg in LZR, P<0.05) and SNA (n=6, 74 ± 8 % of baseline in OZR vs n=6, 58 ± 8 % in LZR) than LZR. After the blockade of GABAA receptors in the RVLM (gabazine; 100 pmol/100nl) the increases in AP (n=6, 58 ± 11 mmHg in OZR vs n=6, 42 ± 7 mmHg in LZR) and SNA (n=6, 83 ± 17 % of baseline in OZR vs n=6, 84 ± 21 % in LZR) were similar between groups. These results suggest that the RVLM is more easily activated by glutamate, but that tonic GABAergic inhibition of the RVLM is not altered in OZR compared to LZR.Grant Support: NIH R01HL086759

Pregnancy decreases GABAergic inhibition of the hypothalamic paraventricular nucleus

Depressor responses to peripheral or central infusion of Angiotensin II type 1 (AT 1) receptor antagonists (AT 1X) are greater in pregnant (P) compared to nonpregnant (NP) animals. AT 1 and ionotropic excitatory amino acid (EAA) receptors contribute to pressor responses to GABA A receptor blockade with bicuculline (Bic) in the paraventricular nucleus (PVN) of male rats. Therefore, we hypothesized that GABAergic inhibition is decreased and AT 1 receptors play a greater excitatory role in the PVN of P versus NP rats. Unilateral microinjection of Bic was performed before (Bic 1), after AT 1X (Bic 2), and after AT 1X + EAA blockade (kynurenate, Kyn) (Bic 3) in the PVN. Increases in mean arterial pressure (MAP: NP = 20 ± 2; P = 12 ± 2 mmHg), heart rate (HR: NP = 57 ± 6; P = 19 ± 6 beats/min) and renal sympathetic nerve activity (RSNA: NP = 70 ± 9; P = 33 ± 7%) due to Bic (Bic 1) were attenuated in P rats. Responses to AT 1X and Kyn alone were insignificant in both groups. In NP rats, AT 1X attenuated (+ 12 ± 4 mmHg), and AT 1X + Kyn further decreased the pressor response to Bic in the PVN (+ 6 ± 2 mmHg). In P rats AT 1X reduced the pressor response to Bic (+ 5 ± 1 mm Hg), and Kyn had no additional effect (+ 3 ± 1 mmHg). Effects of PVN Bic to alter the autospectra of RSNA were suppressed by prior AT 1X and Kyn in both groups. Thus, tonic GABAergic inhibition is decreased and the contribution of AT 1 receptors in the PVN may be greater in P rats.

Altered Responses to Propofol, but Not Ketamine, in Mice Deficient in the 65-Kilodalton Isoform of Glutamate Decarboxylase

GABA is synthesized by two isoforms of glutamate decarboxylase (GAD), GAD65, and GAD67. However, the relative contributions of GAD65-mediated GABA synthesis to the in vivo actions of anesthetics remain unknown. To address this issue, we used mice deficient in the 65-kDa isoform of GAD and tested the hypothesis that partial reduction of GABA content in GAD65-deficient mice [GAD65(-/-)] would contribute to hypnotic and immobilizing actions of the anesthetics. The open field test, loss of righting reflex (LORR), loss of tail-pinch withdrawal response (LTWR), and locomotor activity were compared between wild-type (WT) mice and GAD65(-/-) mice. Effects of general anesthetics on both phasic and tonic GABAergic currents were examined using the patch-clamp method in frontal cortex pyramidal neurons in brain slices. The duration of propofol (100 mg/kg i.p.)-induced LORR and the duration of propofol (150 mg/kg i.p.)-induced LTWR in GAD65(-/-) mice were significantly reduced compared with WT mice. In contrast, no difference was seen for ketamine. Preinjection of the GABA transporter 1 inhibitor, NO-711 (C(21)H(22)N(2)O(3).HCl) (0.75 mg/kg i.p.), reinstated diminished actions of propofol in GAD65(-/-) mice. Cortical pyramidal neurons in GAD65(-/-) mice had smaller tonic conductances, and propofol-induced enhancement of tonic inhibition was smaller than in WT mice, suggesting that genotype differences in GAD65-mediated GABAergic inhibitory tone may be, at least in part, a cellular basis underlying behavioral differences. In conclusion, GAD65(-/-) mice show a diminished response to propofol, but not ketamine, indicating that GAD65-mediated GABA synthesis plays an important role in hypnotic and immobilizing actions of propofol.

Modeling the Gabaergic Action of Etomidate on the Thalamocortical System

We have used a computational model of the thalamocortical system to investigate the effects of a GABAergic anesthetic (etomidate) on cerebral cortical and thalamic neuronal function. We examined the effects of phasic and tonic inhibition, as well as the relative importance of anesthetic action in the thalamus and cortex. The amount of phasic GABAergic inhibition was adjusted in the model to simulate etomidate concentrations of between 0.25 and 2 microM, with the concentration range producing unconsciousness assumed to be between 0.25 and 0.5 microM. In addition, we modeled tonic inhibition separately, and then phasic and tonic inhibition together. We also introduced phasic and tonic inhibition into the cerebral cortex and thalamus separately to determine the relative importance of each of these structures to anesthetic-induced depression of the thalamocortical system. Phasic inhibition decreased cortical neuronal firing by 11%-18% in the 0.25-0.5 microM range and by 38% at 2 microM. Tonic inhibition produced similar depression (11%-21%) in the 0.25-0.5 microM range but 65% depression at 2 microM; phasic and tonic inhibition combined produced the most inhibition (76% depression at 2 microM). When the thalamus and cortex were separately subjected to phasic and tonic inhibition, cortical firing rates decreased less compared to when both structures were targeted. In the 0.25-0.5 microM range, cortical firing rate was minimally affected when etomidate action was simulated in the thalamus only. This computational model of the thalamocortical system indicated that tonic GABAergic inhibition seems to be more important than phasic GABAergic inhibition (especially at larger etomidate concentrations), although both combined had the most effect on cerebral cortical firing rates. Furthermore, etomidate action in the thalamus, by itself, does not likely explain etomidate-induced unconsciousness.

Altered tonic GABAergic inhibition of amygdala neurons in PRIP1-KO mice

Altered tonic GABAergic inhibition of amygdala neurons in PRIP1-KO mice