Activation Of GABAB Receptors Research Articles

Cyclic analogues of α-conotoxin Vc1.1 inhibit colonic nociceptors and provide analgesia in a mouse model of chronic abdominal pain.

Patients with irritable bowel syndrome suffer from chronic visceral pain (CVP) and limited analgesic therapeutic options are currently available. We have shown that α-conotoxin Vc1.1 induced activation of GABAB receptors on the peripheral endings of colonic afferents and reduced nociceptive signalling from the viscera. However, the analgesic efficacy of more stable, cyclized versions of Vc1.1 on CVP remains to be determined. Using ex vivo colonic afferent preparations from mice, we determined the inhibitory actions of cyclized Vc1.1 (cVc1.1) and two cVc1.1 analogues on mouse colonic nociceptors in healthy and chronic visceral hypersensitivity (CVH) states. Using whole-cell patch clamp recordings, we also assessed the inhibitory actions of these peptides on the neuronal excitability of colonic innervating dorsal root ganglion neurons. In vivo, the analgesic efficacy of these analogues was assessed by determining the visceromotor response to colorectal distension in healthy and CVH mice. cVc1.1 and the cVc1.1 analogues, [C2H,C8F]cVc1.1 and [N9W]cVc1.1, all caused concentration-dependent inhibition of colonic nociceptors from healthy mice. Inhibition by these peptides was greater than those evoked by linear Vc1.1 and was substantially greater in colonic nociceptors from CVH mice. cVc1.1 also reduced excitability of colonic dorsal root ganglion neurons, with greater effect in CVH neurons. CVH mice treated with cVc1.1 intra-colonically displayed reduced pain responses to noxious colorectal distension compared with vehicle-treated CVH mice. Cyclic versions of Vc1.1 evoked significant anti-nociceptive actions in CVH states, suggesting that they could be novel candidates for treatment of CVP. This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Pre- and Postsynaptic Activation of GABAB Receptors Modulates Principal Cell Excitation in the Piriform Cortex

The piriform cortex (PC), like other cortical regions, normally operates in a state of dynamic equilibrium between excitation and inhibition. Here we examined the roles played by pre- and postsynaptic GABAB receptors in maintaining this equilibrium in the PC. Using whole-cell recordings in brain slices from the anterior PC of mice, we found that synaptic activation of postsynaptic GABAB receptors hyperpolarized the two major classes of layer 2 principal neurons and reduced the intrinsic electrical excitability of these neurons. Presynaptic GABAB receptors are expressed on the terminals of associational (intracortical) glutamatergic axons in the PC. Heterosynaptic activation of these receptors reduced excitatory associational inputs onto principal cells. Presynaptic GABAB receptors are also expressed on the axons of GABAergic interneurons in the PC, and blockade of these autoreceptors enhanced inhibitory inputs onto principal cells. Hence, presynaptic GABAB autoreceptors produce disinhibition of principal cells. To study the functional consequences of GABAB activation in vivo, we used 2-photon calcium imaging to simultaneously monitor the activity of ~200 layer 2 neurons. Superfusion of the GABAB agonist baclofen reduced spontaneous random firing but also promoted synchronous epileptiform activity. These findings suggest that, while GABAB activation can dampen excitability by engaging pre- and postsynaptic GABAB heteroreceptors on glutamatergic neurons, it can also promote excitability by disinhibiting principal cells by activating presynaptic GABAB autoreceptors on interneurons. Thus, depending on the dynamic balance of hetero- and autoinhibition, GABAB receptors can function as variable modulators of circuit excitability in the PC.

Presynaptic GABAA Receptors Modulate Thalamocortical Inputs in Layer 4 of Rat V1.

Fast inhibitory GABAergic transmission plays a fundamental role in neural circuits. Current theories of cortical function assume that fast GABAergic inhibition acts via GABAA receptors on postsynaptic neurons, while presynaptic effects of GABA depend on GABAB receptor activation. Manipulations of GABAA receptor activity in vivo produced different effects on cortical function, which were generally ascribed to the mode of action of a drug, more than its site of action. Here we show that in rodent primary visual cortex, α4-containing GABAA receptors can be located on subsets of glutamatergic and GABAergic presynaptic terminals and decrease synaptic transmission. Our data provide a novel mechanistic insight into the effects of changes in cortical inhibition; the ability to modulate inputs onto cortical circuits locally, via presynaptic regulation of release by GABAA receptors.

Effects of GABAB receptor activation on spatial cognitive function and hippocampal neurones in rat models of type 2 diabetes mellitus.

The present study was conducted with the aim being to investigate the effect γ-aminobutyric acid type B (GABAB) receptor activation have on spatial cognitive function and hippocampal neurones found in the rat models of type 2 diabetes mellitus (T2DM). T2DM rat models were then established, randomized, and subsequently assigned into normal control (NC), T2DM, T2DM + chemical grade propylene (CGP), T2DM + baclofen, and T2DM + CGP + baclofen groups. T2DM rats’ weight and blood sugar concentrations were monitored. The DMS-2 Morris water maze testing system was performed in order to figure out the spatial cognitive function of these rats. Reverse-transcription quantitative PCR (RT-qPCR) and Western blotting were also performed in order to detect GABAB mRNA and protein expressions. We used the Nissl staining method in order to detect the number of hippocampal neurones, TUNEL (terminal deoxyribonucleotidy transferase-mediated dUTP nick labeling) staining to detect cell apoptosis, and Western blotting method in order to measure the expressions of the apoptosis-related proteins (Bax, cytochrome c (Cyt-c), Caspase-3, and Bcl-2). In comparison with the T2DM group, the weight decreased, blood sugar concentration increased, and spatial cognitive function as well as hippocampal neurones were both impaired in the T2DM + CGP group, contrary to the rats in the T2DM + baclofen group who showed an opposite trend. The situation in the T2DM + CGP + baclofen group was better than that found in the T2DM + CGP group while proving to be more serious than that of the NC and T2DM + baclofen groups. Conclusively, activating the GABAB receptor improved spatial cognitive function and hippocampal neurones in the T2DM rats.

A novel \u03b1-conopeptide Eu1.6 inhibits N-type (CaV2.2) calcium channels and exhibits potent analgesic activity

We here describe a novel α-conopeptide, Eu1.6 from Conus eburneus, which exhibits strong anti-nociceptive activity by an unexpected mechanism of action. Unlike other α-conopeptides that largely target nicotinic acetylcholine receptors (nAChRs), Eu1.6 displayed only weak inhibitory activity at the α3β4 and α7 nAChR subtypes and TTX-resistant sodium channels, and no activity at TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons, or opiate receptors, VR1, KCNQ1, L- and T-type calcium channels expressed in HEK293 cells. However, Eu1.6 inhibited high voltage-activated N-type calcium channel currents in isolated mouse DRG neurons which was independent of GABAB receptor activation. In HEK293 cells expressing CaV2.2 channels alone, Eu1.6 reversibly inhibited depolarization-activated Ba2+ currents in a voltage- and state-dependent manner. Inhibition of CaV2.2 by Eu1.6 was concentration-dependent (IC50 ~1 nM). Significantly, systemic administration of Eu1.6 at doses of 2.5–5.0 μg/kg exhibited potent analgesic activities in rat partial sciatic nerve injury and chronic constriction injury pain models. Furthermore, Eu1.6 had no significant side-effect on spontaneous locomotor activity, cardiac and respiratory function, and drug dependence in mice. These findings suggest α-conopeptide Eu1.6 is a potent analgesic for the treatment of neuropathic and chronic pain and opens a novel option for future analgesic drug design.

Alleviation by GABAB Receptors of Neurotoxicity Mediated by Mitochondrial Permeability Transition Pore in Cultured Murine Cortical Neurons Exposed to N-Methyl-D-aspartate.

Mitochondrial permeability transition pore (PTP) is supposed to at least in part participate in molecular mechanisms underlying the neurotoxicity seen after overactivation of N-methyl-D-aspartate (NMDA) receptor (NMDAR) in neurons. In this study, we have evaluated whether activation of GABAB receptor (GABABR), which is linked to membrane G protein-coupled inwardly-rectifying K+ ion channels (GIRKs), leads to protection of the NMDA-induced neurotoxicity in a manner relevant to mitochondrial membrane depolarization in cultured embryonic mouse cortical neurons. The cationic fluorescent dye 3,3'-dipropylthiacarbocyanine was used for determination of mitochondrial membrane potential. The PTP opener salicylic acid induced a fluorescence increase with a vitality decrease in a manner sensitive to the PTP inhibitor ciclosporin, while ciclosporin alone was effective in significantly preventing both fluorescence increase and viability decrease by NMDA as seen with an NMDAR antagonist. The NMDA-induced fluorescence increase and viability decrease were similarly prevented by pretreatment with the GABABR agonist baclofen, but not by the GABAAR agonist muscimol, in a fashion sensitive to a GABABR antagonist. Moreover, the GIRK inhibitor tertiapin canceled the inhibition by baclofen of the NMDA-induced fluorescence increase. These results suggest that GABABR rather than GABAAR is protective against the NMDA-induced neurotoxicity mediated by mitochondrial PTP through a mechanism relevant to opening of membrane GIRKs in neurons.

Regional difference in spontaneous firing inhibition by GABAA and GABAB receptors in nigral dopamine neurons.

GABAergic control over dopamine (DA) neurons in the substantia nigra is crucial for determining firing rates and patterns. Although GABA activates both GABAA and GABAB receptors distributed throughout the somatodendritic tree, it is currently unclear how regional GABA receptors in the soma and dendritic compartments regulate spontaneous firing. Therefore, the objective of this study was to determine actions of regional GABA receptors on spontaneous firing in acutely dissociated DA neurons from the rat using patch-clamp and local GABA-uncaging techniques. Agonists and antagonists experiments showed that activation of either GABAA receptors or GABAB receptors in DA neurons is enough to completely abolish spontaneous firing. Local GABA-uncaging along the somatodendritic tree revealed that activation of regional GABA receptors limited within the soma, proximal, or distal dendritic region, can completely suppress spontaneous firing. However, activation of either GABAA or GABAB receptor equally suppressed spontaneous firing in the soma, whereas GABAB receptor inhibited spontaneous firing more strongly than GABAA receptor in the proximal and distal dendrites. These regional differences of GABA signals between the soma and dendritic compartments could contribute to our understanding of many diverse and complex actions of GABA in midbrain DA neurons.

Differential sensitivity of presynaptic and postsynaptic GABAB receptors in rat ventrolateral periaqueductal gray.

The ventrolateral periaqueductal gray (PAG) in the midbrain plays a key role in the descending pain modulatory system. γ-Aminobutyric acid type B (GABAB) receptors belong to a metabotropic receptor subfamily and mediate both presynaptic and postsynaptic effects in PAG. It has been well documented that activation of GABAB receptors yields analgesia in some PAG subdivisions. In the present study, employing whole-cell patch-clamp recordings on acute rat PAG slices, we simultaneously monitored the responses of presynaptic and postsynaptic GABAB receptors. We found that the GABAB agonist, baclofen, exhibits less efficacy and potency at GABAB postsynaptic versus presynaptic receptors. This sensitivity bias may contribute to synapse homeostasis and implicate a novel pharmacotherapy treatment.

Event-Related Potentials in the Visual Center of the Carp on Application of Baclofen to the Retina

The effects of activation of retinal GABAB receptors with baclofen on event-related potentials (ERP) arising in the primary visual center (the tectum) on illumination of the retina with white light and on electrical stimulation of the optic nerve were studied in dark-adapted carp. These studies showed that baclofen-induced decreases in the amplitude of the electroretinogram (ERG) b wave were accompanied by increases in ERP amplitude both to light stimuli and to electrical stimulation of the optic nerve. It is suggested that this increase is due to an increase in the response of tertiary retinal neurons to light and/or an increase in the excitability of tectum neurons due to a reduction in tonic influences from the visual afferent stream.

R-Baclofen Reverses Cognitive Deficits and Improves Social Interactions in Two Lines of 16p11.2 Deletion Mice

Human chromosome 16p11.2 microdeletion is among the most common gene copy number variations (CNVs) known to confer risk for intellectual disability (ID) and autism spectrum disorder (ASD) and affects an estimated 3 in 10 000 people. Caused by a single copy deletion of ~27 genes, 16p11.2 microdeletion syndrome is characterized by ID, impaired language, communication and socialization skills, and ASD. Studies in animal models where a single copy of the syntenic 16p11.2 region has been deleted have revealed morphological, behavioral, and electrophysiological abnormalities. Previous studies suggested the possibility of some overlap in the mechanisms of pathophysiology in 16p11.2 microdeletion syndrome and fragile X syndrome. Improvements in fragile X phenotypes have been observed following chronic treatment with R-baclofen, a selective agonist of GABAB receptors. We were therefore motivated to investigate the effects of chronic oral R-baclofen administration in two independently generated mouse models of 16p11.2 microdeletion syndrome. In studies performed across two independent laboratories, we found that chronic activation of GABAB receptors improved performance on a series of cognitive and social tasks known to be impaired in two different 16p11.2 deletion mouse models. Our findings suggest that R-baclofen may have clinical utility for some of the core symptoms of human 16p11.2 microdeletion syndrome.

Lithium reduces the span of G protein-activated K+ (GIRK) channel inhibition in hippocampal neurons.

Lithium (Li+ ) is one of the most widely used treatments for bipolar disorder (BD). However, the molecular and neuronal basis of BD, as well as the mechanisms of Li+ actions are poorly understood. Cellular and biochemical studies identified G proteins as being among the cellular targets for Li+ action, while genetic studies indicated an association with the KCNJ3 gene, which encodes the G protein-activated inwardly rectifying K+ (GIRK) channels. GIRK channels regulate neuronal excitability by mediating the inhibitory effects of multiple neurotransmitters and contribute to the resting potassium conductance. Here, we explored the effects of therapeutic dose of Li+ on neuronal excitability and the role of GIRK channels in Li+ actions. Effects of Li+ on excitability were studied in hippocampal brain slices using whole-cell electrophysiological recordings. A therapeutic dose of Li+ (1mM) dually regulated the function of GIRK channels in hippocampal slices. Li+ hyperpolarized the resting membrane potential of hippocampal CA1 pyramidal neurons and prolonged the latency to reach the action potential threshold and peak. These effects were abolished in the presence of tertiapin, a specific GIRK channel blocker, and at doses above the therapeutic window (2mM). In contrast, Li+ reduced GIRK channel opening induced by GABAB receptor (GABAB R) activation, causing reduced hyperpolarization of the membrane potential, attenuated reduction of input resistance, and a smaller decrease of neuronal firing. A therapeutic dose of Li+ reduces the span of GIRK channel-mediated inhibition due to enhancement of basal GIRK currents and inhibition of GABAB R evoked responses, providing an important link between Li+ action, neuronal excitability, and cellular and genetic targets of BD.

GABAB receptors suppress burst-firing in reticular thalamic neurons

ABSTRACTBurst-firing in thalamic neurons is known to play a key role in mediating thalamocortical (TC) oscillations that are associated with non-REM sleep and some types of epileptic seizure. Within the TC system the primary output of GABAergic neurons in the reticular thalamic nucleus (RTN) is thought to induce the de-inactivation of T-type calcium channels in thalamic relay (TR) neurons, promoting burst-firing drive to the cortex and the propagation of TC network activity. However, RTN neurons also project back onto other neurons within the RTN. The role of this putative negative feedback upon the RTN itself is less well understood, although is hypothesized to induce de-synchronization of RTN neuron firing leading to the suppression of TC oscillations. Here we tested two hypotheses concerning possible mechanisms underlying TC oscillation modulation. Firstly, we assessed the burst-firing behavior of RTN neurons in response to GABAB receptor activation using acute brain slices. The selective GABAB receptor agonist baclofen was found to induce suppression of burst-firing concurrent with effects on membrane input resistance. Secondly, RTN neurons express CaV3.2 and CaV3.3 T-type calcium channel isoforms known to contribute toward TC burst-firing and we examined the modulation of these channels by GABAB receptor activation. Utilizing exogenously expressed T-type channels we assessed whether GABAB receptor activation could directly alter T-type calcium channel properties. Overall, GABAB receptor activation had only modest effects on CaV3.2 and CaV3.3 isoforms. The only effect that could be predicted to suppress burst-firing was a hyperpolarized shift in the voltage-dependence of inactivation, potentially causing lower channel availability at membrane potentials critical for burst-firing. Conversely, other effects observed such as a hyperpolarized shift in the voltage-dependence of activation of both CaV3.2 and CaV3.3 as well as increased time constant of activation of the CaV3.3 isoform would be expected to enhance burst-firing. Together, we hypothesize that GABAB receptor activation mediates multiple downstream effectors that combined act to suppress burst-firing within the RTN. It appears unlikely that direct GABAB receptor-mediated modulation of T-type calcium channels is the major mechanistic contributor to this suppression.

Spreading Depression Promotes Astrocytic Calcium Oscillations and Enhances Gliotransmission to Hippocampal Neurons.

Spreading depression (SD) is a pathophysiological phenomenon characterized by propagating waves of profound neuronal and glial depolarization in central nervous system gray matter. Although SD is primarily mediated by neurons with a subsequent astrocytic response, it remains unclear how astrocytic activity is modulated after SD and how altered astrocyte signaling contribute to neuronal excitability. Here, we report that after the concurrent Ca2+ wave, SD enhanced astrocytic activity by promoting a secondary period of Ca2+ oscillations. SD-induced Ca2+ oscillations did not require the activation of metabotropic glutamate receptors or purinergic receptors; instead, they were mediated by the activation of GABAB receptors and 1,4,5-trisphosphate (IP3) receptors. Furthermore, SD increased the number of NMDA receptor-mediated slow inward currents (SICs) in CA1 pyramidal neurons. The frequency of SD-induced SICs was reduced by blockade of GABAB receptors or by limiting Ca2+ efflux from the ER. Selective inhibition of astrocytic Ca2+ signals by dialysis of BAPTA into astrocytes or by knocking out the astrocytic type of IP3 receptors suppressed SICs after SD. These results demonstrated a causative link between the SD-induced Ca2+ oscillations and the enhanced glutamatergic astrocyte-neuron signaling. Therefore, we conclude that SD enhances the astrocyte Ca2+ signals and further promotes gliotransmission and neuronal excitability.

A Cooperative Mechanism Involving Ca2+-Permeable AMPA Receptors and Retrograde Activation of GABAB Receptors in Interpeduncular Nucleus Plasticity

The medial habenula-interpeduncular nucleus (MHb-IPN) pathway, which connects the limbic forebrain to the midbrain, has recently been implicated in aversive behaviors. The MHb-IPN circuit is characterized by a unique topographical organization, an excitatory role of GABA, and a prominent co-release of neurotransmitters and neuropeptides. However, little is known about synaptic plasticity in this pathway. An application of a high-frequency stimulation resulted in a long-lasting potentiation of glutamate release in IPN neurons. Our experiments reveal that a Ca2+-permeable AMPA receptor (CPAR)-dependent release of GABA from IPN neurons and a retrograde activation of GABAB receptors on MHb terminals result in a long-lasting enhancement of glutamate release. Strikingly, adolescent IPN neurons lacked CPARs and exhibited an inability to undergo plasticity. In addition, fear conditioning suppressed an activity-dependent potentiation of MHb-IPN synapses, whereas fear extinction reversed this plasticity deficit, suggesting a role of the MHb-IPN synaptic plasticity in the regulation of aversive behaviors.

Abstract 2894: Epinephrine-induced gamma-secretase/Notch signaling in pancreatic cancer stem cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths with 5-year survivals below 10%. Cancer stem cells represent a small population of cells in PDAC and are thought to drive the development, progression, metastasis and drug resistance of this cancer. The Notch pathway regulates the self-renewal of pancreatic cancer stem cells as well as angiogenesis and drug resistance. Gamma-secretase cleaves the Notch-1 receptor, resulting in release of the Notch intracellular domain (NICD) that then translocates to the nucleus and activates the transcription of target genes. Aberrant Notch signaling activated by γ-secretase activity is associated with progression, angiogenesis and drug resistance of PDAC and is being explored as a target for PDAC therapy. Smoking and psychological stress are risk factors for PDAC. However, the potential modulation of γ-secretase/Notch by these risk factors has not been investigated to date. Nicotine and psychological stress both cause hyperactivity of the sympathetic nervous system and adrenal glands, resulting in increased blood and organ levels of the stress neurotransmitters norepinephrine (NOR) and epinephrine (EPI), which are the physiological agonists of beta-adrenergic receptors (β-ARs). In turn, β-ARs are coupled to the stimulatory G-protein Gαs that stimulates the formation of intracellular cAMP, which activates multiple signaling pathways in a cell type-specific manner. Using spheroid formation assays and Western blotting, our data show, -for the first time,- that the stress neurotransmitter Epi significantly increases the self-renewal of pancreatic cancer stem cells by increasing the expression of the activated forms (c-terminal) of the γ-secretase component presenilin1 and of Notch1 (NICD). These responses were inhibited by the β-AR antagonist (beta-blocker) propranolol and by the inhibitory neurotransmitter γ-aminobutyric acid (GABA) that inhibits cAMP formation via activation of Gαi-coupled GABAB receptors. These findings suggest that the γ-sexretasesecretase/Notch pathway in pancreatic cancer stem cells is under regulatory control by neurotransmitters, with Epi as stimulator and GABA as inhibitor. These data could open up new avenues for the prevention and therapy of PDAC. Additional experiments are currently underway to further investigate mechanistic aspects of this novel concept. Supported by RO1CA042829 with NIH. Note: This abstract was not presented at the meeting. Citation Format: Xuemin Xu, Chen Hu, Hildegard M. Schuller. Epinephrine-induced gamma-secretase/Notch signaling in pancreatic cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2894. doi:10.1158/1538-7445.AM2017-2894

Baclofen Reversed the Nicotine-Induced Anxiety-Like Behavior: A Beneficial Therapeutic Strategy for Smoking Cessation Aid

Background: Nicotine dependence is the most widespread form of substance abuse. Anxiety is one of the prominent symptoms in withdrawal syndromes of nicotine. Nucleus Accumbens (NAc) is a principle area which participates in several behavioral responses (e.g., anxiety), and several studies reported that receptors are involved in the modulation of anxiety. This study was aimed to examine the interaction of baclofen and nicotine on anxiety behavior in NAc shell. Materials and Methods: Rats weighing between 220 and 250g were used. The guide cannulas were implanted bilaterally into the shell of the NAc. Nicotine (0.025, 0.05 and 0.5 μg/rat) and baclofen (0.25, 0.5 and 2 μg/rat) and the co-administration of nicotine (0.025, 0.05 and 0.5μg/rat) with baclofen (0.25 μg/rat) were microinjected through the infusion cannula. The elevated plus-maze (EPM) was used to evaluate the anxiety-like parameters, and all data were recorded and analyzed. Results: Intra-NAc administration of nicotine (0.5μg/rat) decreased anxiety (P<0.001). Microinjections of baclofen (0.5 and 2 μg/rat) decreased anxiety (P< 0.001). However, there were no significant changes in anxiety-like parameters at the lowest dose of baclofen (0.25μg/rat). Co-administration of an ineffective dose of baclofen (0.25 μg/rat) with 3 doses of nicotine (0.025, 0.05 and 0.5 μg/rat) reversed the effects of nicotine in an effective dose (0.5μg/rat). Conclusion: Our result indicated that GABAergic agonist baclofen in ineffective doses could reverse the effects of nicotine on anxiety symptoms. Furthermore, our result suggested that enhancement of GABA transmission through activation of GABA-B receptors on NAc shell could reverse nicotine anxiety-related effects. It may be a strategy of treatment for smoking cessation aid.[GMJ.2017;6(2):118-27]

KCTD12 modulation of GABA(B) receptor function.

The molecular composition and functional diversity of native GABAB receptors (GABABR) are still poorly understood, thus hindering development of selective GABABR ligands. Potassium channel tetramerization domain‐containing protein (KCTD) 12 is a GABABR auxiliary subunit and mouse KCTD12 can alter GABABR function. In this study, we sought to characterize the effects of human KCTD12 on GABABR kinetics and pharmacology, using an automated electrophysiological assay. Seizure susceptibility and ethanol consumption were also investigated in a KCTD12 knockout mouse model. Human KCTD12 co‐expression altered the kinetics of GABABR‐mediated GIRK channels, speeding rates of both activation and desensitization. Analysis of concentration‐response curves showed that KCTD12 coexpression did not alter effects of the agonists GABA or baclofen on GABABR. KCTD12 coexpression enhanced the potentiating effects of the positive allosteric modulator CGP7930, and its effects on GABABR activation and desensitization. The function of KCTD12 in vivo was examined, using the KCTD12 knockout mouse model. The knockout mice were more resistant to a pentylenetetrazole proconvulsant challenge suggesting reduced seizure susceptibility. In the two bottle preference test, KCTD12 knockout mice demonstrated a reduced consumption at high ethanol concentrations. In summary, human KCTD12 accelerated the kinetics of GABABR in vitro, in a manner possibly sensitive to allosteric pharmacological modulation. This study also provides novel in vivo evidence that the interaction between KCTD12 and GABABR is of physiological significance, and may be a mechanism to more selectively modulate GABABR.

Differential surface density and modulatory effects of presynaptic GABAB receptors in hippocampal cholecystokinin and parvalbumin basket cells

The perisomatic domain of cortical neurons is under the control of two major GABAergic inhibitory interneuron types: regular-spiking cholecystokinin (CCK) basket cells (BCs) and fast-spiking parvalbumin (PV) BCs. CCK and PV BCs are different not only in their intrinsic physiological, anatomical and molecular characteristics, but also in their presynaptic modulation of their synaptic output. Most GABAergic terminals are known to contain GABAB receptors (GABABR), but their role in presynaptic inhibition and surface expression have not been comparatively characterized in the two BC types. To address this, we performed whole-cell recordings from CCK and PV BCs and postsynaptic pyramidal cells (PCs), as well as freeze-fracture replica-based quantitative immunogold electron microscopy of their synapses in the rat hippocampal CA1 area. Our results demonstrate that while both CCK and PV BCs contain functional presynaptic GABABRs, their modulatory effects and relative abundance are markedly different at these two synapses: GABA release is dramatically inhibited by the agonist baclofen at CCK BC synapses, whereas a moderate reduction in inhibitory transmission is observed at PV BC synapses. Furthermore, GABABR activation has divergent effects on synaptic dynamics: paired-pulse depression (PPD) is enhanced at CCK BC synapses, but abolished at PV BC synapses. Consistent with the quantitative differences in presynaptic inhibition, virtually all CCK BC terminals were found to contain GABABRs at high densities, but only 40% of PV BC axon terminals contain GABABRs at detectable levels. These findings add to an increasing list of differences between these two interneuron types, with implications for their network functions.

GABA-B Controls Persistent Na+ Current and Coupled Na+-Activated K+ Current.

The GABA-B receptor is densely expressed throughout the brain and has been implicated in many CNS functions and disorders, including addiction, epilepsy, spasticity, schizophrenia, anxiety, cognitive deficits, and depression, as well as various aspects of nervous system development. How one GABA-B receptor is involved in so many aspects of CNS function remains unanswered. Activation of GABA-B receptors is normally thought to produce inhibitory responses in the nervous system, but puzzling contradictory responses exist. Here we report that in rat mitral cells of the olfactory bulb, GABA-B receptor activation inhibits both the persistent sodium current (INaP) and the sodium-activated potassium current (IKNa), which is coupled to it. We find that the primary effect of GABA-B activation is to inhibit INaP, which has the secondary effect of inhibiting IKNa because of its dependence on persistent sodium entry for activation. This can have either a net excitatory or inhibitory effect depending on the balance of INaP/IKNa currents in neurons. In the olfactory bulb, the cell bodies of mitral cells are densely packed with sodium-activated potassium channels. These channels produce a large IKNa which, if constitutively active, would shunt any synaptic potentials traversing the soma before reaching the spike initiation zone. However, GABA-B receptor activation might have the net effect of reducing the IKNa blocking effect, thus enhancing the effectiveness of synaptic potentials.

Neuronal Chloride Regulation via KCC2 Is Modulated through a GABAB Receptor Protein Complex.

GABAB receptors are G-protein-coupled receptors that mediate inhibitory synaptic actions through a series of downstream target proteins. It is increasingly appreciated that the GABAB receptor forms part of larger signaling complexes, which enable the receptor to mediate multiple different effects within neurons. Here we report that GABAB receptors can physically associate with the potassium-chloride cotransporter protein, KCC2, which sets the driving force for the chloride-permeable ionotropic GABAA receptor in mature neurons. Using biochemical, molecular, and functional studies in rodent hippocampus, we show that activation of GABAB receptors results in a decrease in KCC2 function, which is associated with a reduction in the protein at the cell surface. These findings reveal a novel “crosstalk” between the GABA receptor systems, which can be recruited under conditions of high GABA release and which could be important for the regulation of inhibitory synaptic transmission.SIGNIFICANCE STATEMENT Synaptic inhibition in the brain is mediated by ionotropic GABAA receptors (GABAARs) and metabotropic GABAB receptors (GABABRs). To fully appreciate the function and regulation of these neurotransmitter receptors, we must understand their interactions with other proteins. We describe a novel association between the GABABR and the potassium-chloride cotransporter protein, KCC2. This association is significant because KCC2 sets the intracellular chloride concentration found in mature neurons and thereby establishes the driving force for the chloride-permeable GABAAR. We demonstrate that GABABR activation can regulate KCC2 at the cell surface in a manner that alters intracellular chloride and the reversal potential for the GABAAR. Our data therefore support an additional mechanism by which GABABRs are able to modulate fast synaptic inhibition.