Inhibitory GABA Research Articles

Bestrophin1-mediated tonic GABA release from reactive astrocytes prevents the development of seizure-prone network in kainate-injected hippocampi.

Tonic extrasynaptic GABAA receptor (GABAA R) activation is under the tight control of tonic GABA release from astrocytes to maintain the brain's excitation/inhibition (E/I) balance; any slight E/I balance disturbance can cause serious pathological conditions including epileptic seizures. However, the pathophysiological role of tonic GABA release from astrocytes has not been tested in epileptic seizures. Here, we report that pharmacological or genetic intervention of the GABA-permeable Bestrophin-1 (Best1) channel prevented the generation of tonic GABA inhibition, disinhibiting CA1 pyramidal neuronal firing and augmenting seizure susceptibility in kainic acid (KA)-induced epileptic mice. Astrocyte-specific Best1 over-expression in KA-injected Best1 knockout mice fully restored the generation of tonic GABA inhibition and effectively suppressed seizure susceptibility. We demonstrate for the first time that tonic GABA from reactive astrocytes strongly contributes to the compensatory shift of E/I balance in epileptic hippocampi, serving as a good therapeutic target against altered E/I balance in epileptic seizures.

Cortical GABA in migraine with aura -an ultrashort echo magnetic resonance spectroscopy study

ObjectiveThe aim of this cross-sectional study was to investigate the cortical metabolite concentrations in patients suffering from migraine with aura (MWA). We hypothesized that occipital γ-aminobutyric acid (GABA) levels are lower in MWA patients.BackgroundRecent studies have indicated that a disturbance in the inhibitory GABA is involved in triggering the migraine aura. We aimed to explore this using a novel magnetic resonance spectroscopy sequence.MethodsUsing spin echo full intensity acquired localized spectroscopy on a Siemens 3 Tesla magnetic resonance scanner, we obtained occipital and parietal metabolite concentrations in 14 patients suffering from migraine with aura and a group of 16 matched healthy subjects.All scans were performed at Aarhus University Hospital, at the Center for Functionally Integrative Neuroscience (CFIN).ResultsNo difference was found in GABA/(Total creatine) levels in either the occipital cortex (p = 0.744) or in the somatosensory cortex (p = 0.305).ConclusionThese findings indicate that cortical GABA levels are normal in patients suffering from relatively few migraine attacks. Previous studies have reported that cortical GABA in patients with more frequent migraines is reduced; further investigation of the inhibitory system in migraine patients is warranted to determine the underlying mechanisms.

Identification of KCC2 Mutations in Human Epilepsy Suggests Strategies for Therapeutic Transporter Modulation.

Epilepsy is a common neurological disorder characterized by recurrent and unprovoked seizures thought to arise from impaired balance between neuronal excitation and inhibition. Our understanding of the neurophysiological mechanisms that render the brain epileptogenic remains incomplete, reflected by the lack of satisfactory treatments that can effectively prevent epileptic seizures without significant drug-related adverse effects. Type 2 K+-Cl− cotransporter (KCC2), encoded by SLC12A5, is important for chloride homeostasis and neuronal excitability. KCC2 dysfunction attenuates Cl− extrusion and impairs GABAergic inhibition, and can lead to neuronal hyperexcitability. Converging lines of evidence from human genetics have secured the link between KCC2 dysfunction and the development of epilepsy. Here, we review KCC2 mutations in human epilepsy and discuss potential therapeutic strategies based on the functional impact of these mutations. We suggest that a strategy of augmenting KCC2 activity by antagonizing its critical inhibitory phosphorylation sites may be a particularly efficacious method of facilitating Cl− extrusion and restoring GABA inhibition to treat medication-refractory epilepsy and other seizure disorders.

Cortical excitation-inhibition ratio mediates the effect of pre-attentive auditory processing deficits on interpersonal difficulties

Several lines of evidence identify aberrant excitatory-inhibitory neural processes across autism and schizophrenia spectrum disorders, particularly within the psychosocial domain. Such neural processes include increased excitatory glutamate and reduced inhibitory GABA concentrations, which may affect auditory pre-attentive processing as indexed by the mismatch negativity (MMN); thus, an excitation-inhibition imbalance might lead to aberrant MMN, which might in turn drive the relationship between the MMN and psychosocial difficulties. This research has the potential to enhance the neurochemical understanding of the relationship between electrophysiology (MMN) and behavioural/clinical measures (psychosocial difficulties).Thirty-eight adults (18 male, 18–40 years) completed the Schizotypal Personality Questionnaire (SPQ) and Autism-Spectrum Quotient (AQ). Glutamate and GABA concentrations in bilateral superior temporal cortex (STC) were quantified using proton magnetic resonance spectroscopy (1H-MRS) while auditory MMN to a duration deviant was measured with magnetoencephalography. Spearman correlations probed the relationships between STC glutamate/GABA ratios, MMN amplitude and latency, and AQ and SPQ dimensions. Mediation effects of glutamate/GABA ratios on the relationship between MMN and AQ-SPQ dimensions were probed using causal mediation analysis.Only SPQ-interpersonal and AQ-communication were significantly correlated with right hemisphere glutamate/GABA ratios and MMN latency (ps < 0.05), which were themselves correlated (p = .035). Two mediation models were investigated, with right MMN latency as predictor and SPQ-interpersonal and AQ-communication as outcome variables. Right STC glutamate/GABA ratios significantly mediated the relationship between MMN latency and SPQ-interpersonal scores, but only partially mediated the relationship between MMN latency and AQ-communication scores.These findings support the growing body of literature pointing toward an excitation-inhibition imbalance that is central to psychosocial functioning across multi-dimensional spectrum disorders, such as autism and schizophrenia, and provides neurochemical indicators of the processes that underlie psychosocial dysfunction.

Response Latency Tuning by Retinal Circuits Modulates Signal Efficiency

In the visual system, retinal ganglion cells (RGCs) of various subtypes encode preprocessed photoreceptor signals into a spike output which is then transmitted towards the brain through parallel feature pathways. Spike timing determines how each feature signal contributes to the output of downstream neurons in visual brain centers, thereby influencing efficiency in visual perception. In this study, we demonstrate a marked population-wide variability in RGC response latency that is independent of trial-to-trial variability and recording approach. RGC response latencies to simple visual stimuli vary considerably in a heterogenous cell population but remain reliable when RGCs of a single subtype are compared. This subtype specificity, however, vanishes when the retinal circuitry is bypassed via direct RGC electrical stimulation. This suggests that latency is primarily determined by the signaling speed through retinal pathways that provide subtype specific inputs to RGCs. In addition, response latency is significantly altered when GABA inhibition or gap junction signaling is disturbed, which further supports the key role of retinal microcircuits in latency tuning. Finally, modulation of stimulus parameters affects individual RGC response delays considerably. Based on these findings, we hypothesize that retinal microcircuits fine-tune RGC response latency, which in turn determines the context-dependent weighing of each signal and its contribution to visual perception.

Social hierarchy regulates ocular dominance plasticity in adult male mice.

We here show that social rank, as assessed by competition for a running wheel, influences ocular dominance plasticity in adult male mice. Dominant animals showed a clear ocular dominance shift after 4days of MD, whereas their submissive cagemates did not. NMDA receptor activation, reduced GABA inhibition, and serotonin transmission were necessary for this plasticity, but not sufficient to explain the difference between dominant and submissive animals. In contrast, prefrontal dopamine concentration was higher in dominant than submissive mice, and systemic manipulation of dopamine transmission bidirectionally changed ocular dominance plasticity. Thus, we could show that a social hierarchical relationship influences ocular dominance plasticity in the visual cortex via higher-order cortices, most likely the medial prefrontal cortex. Further studies will be needed to elucidate the precise mechanisms by which this regulation takes place.

Pharmacological and genetic inhibition of astrocytic GABA-transaminase enhances tonic GABA inhibition in the hippocampus

Pharmacological and genetic inhibition of astrocytic GABA-transaminase enhances tonic GABA inhibition in the hippocampus

Psychosocial deficits across autism and schizotypal spectra are interactively modulated by excitatory and inhibitory neurotransmission.

Continued human and animal research has strengthened evidence for aberrant excitatory-inhibitory neural processes underlying autism and schizophrenia spectrum disorder psychopathology, particularly psychosocial functioning, in clinical and nonclinical populations. We investigated the extent to which autistic traits and schizotypal dimensions were modulated by the interactive relationship between excitatory glutamate and inhibitory GABA neurotransmitter concentrations in the social processing area of the superior temporal cortex using proton magnetic resonance spectroscopy. In total, 38 non-clinical participants (20 females; age range = 18-35 years, mean (standard deviation) = 23.22 (5.52)) completed the autism spectrum quotient and schizotypal personality questionnaire, and underwent proton magnetic resonance spectroscopy to quantify glutamate and GABA concentrations in the right and left superior temporal cortex. Regression analyses revealed that glutamate and GABA interactively modulated autistic social skills and schizotypal interpersonal features (pcorr < 0.05), such that those with high right superior temporal cortex glutamate but low GABA concentrations exhibited poorer social and interpersonal skills. These findings evidence an excitation-inhibition imbalance that is specific to psychosocial features across the autism and schizophrenia spectra.

Amyotrophic lateral sclerosis: Origins traced to impaired balance between neural excitation and inhibition in the neonatal period.

Amyotrophic lateral sclerosis (ALS) is an adult onset disease but with an increasingly recognized preclinical prodrome. A wide spectrum of investigative approaches has identified loss of inhibitory function at the heart of ALS. In developing an explanation for the onset of ALS, it remains a consideration that ALS has its origins in neonatal derangement of the γ-aminobutyric acid (GABA)-ergic system, with delayed conversion from excitatory to mature inhibitory GABA and impaired excitation/inhibition balance. If this is so, the resulting chronic excitotoxicity could marginalize cortical network functioning very early in life, laying the path for neurodegeneration. The possibility that adult-onset neurodegenerative conditions might have their roots in early developmental derangements is worthy of consideration, particularly in relation to current models of disease pathogenesis. Unraveling the very early molecular events will be crucial in developing a better understanding of ALS and other adult neurodegenerative disorders. Muscle Nerve, 2019.

Dynamics of Changes in GABA and Catecholamines Contents and MAO-A Activity in Experimental Post-Traumatic Stress Disorder in Rats

Post-traumatic stress disorder (PTSD) develops as a delayed consequence of psychological trauma and is accompanied by characteristic changes in the levels of biogenic amines. The aim of the present work was to carry out a complex study of the dynamics of changes in measures of behavioral activity and noradrenaline and dopamine contents taking cognizance of the activity of the enzyme metabolizing them, i.e., monoamine oxidase A (MAO-A), and the level of the inhibitory transmitter GABA in PTSD. PTSD was modeled in Wistar rats using cat odor. Behavioral changes were studied in the elevated plus maze test. Contents of GABA and catecholamines were determined, along with MAO-A activity levels. The development of poststress anxiety was found to be linked with changes in neurotransmitter levels in the brain. The onset of anxiogenic disorders was preceded by an anxiolytic phase, associated on day 3 with an increase in the GABA level and on day 7 with a decrease in the noradrenaline level in the brain. Only on day 10 was an increase in the noradrenaline content seen, this being synchronized with increases in anxiety. Thus, a significant relationship between the development of behavioral signs of PTSD and an increase in noradrenaline content in the brain was identified.

The spiking and secretory activity of oxytocin neurones in response to osmotic stimulation: a computational model.

A quantitative model of oxytocin neurones that combines a spiking model, a model of stimulus-secretion coupling and a model of plasma clearance of oxytocin was tested. To test the model, a variety of sources of published data were used that relate either the electrical activity of oxytocin cells or the secretion of oxytocin to experimentally induced changes in plasma osmotic pressure. To use these data to test the model, the experimental challenges involved were computationally simulated. The model predictions closely matched the reported outcomes of the different experiments. Magnocellular vasopressin and oxytocin neurones in the rat hypothalamus project to the posterior pituitary, where they secrete their products into the bloodstream. In rodents, both vasopressin and oxytocin magnocellular neurones are osmoresponsive, and their increased spiking activity is mainly a consequence of an increased synaptic input from osmoresponsive neurons in regions adjacent to the anterior wall of the third ventricle. Osmotically stimulated vasopressin secretion promotes antidiuresis while oxytocin secretion promotes natriuresis. In this work we tested a previously published computational model of the spiking and secretion activity of oxytocin cells against published evidence of changes in spiking activity and plasma oxytocin concentration in response to different osmotic challenges. We show that integrating this oxytocin model with a simple model of the osmoresponsive inputs to oxytocin cells achieves a strikingly close match to diverse sources of data. Comparing model predictions with published data using bicuculline to block inhibitory GABA inputs supports the conclusion that inhibitory inputs and excitatory inputs are co-activated by osmotic stimuli. Finally, we studied how the gain of osmotically stimulated oxytocin release changes in the presence of a hypovolaemic stimulus, showing that this is best explained by an inhibition of an osmotically regulated inhibitory drive to the magnocellular neurones.

SAT-411 Prepubertal Tonic Gamma-Amino Butyric Acid (GABA) Inhibition Is Upstream of Neurokinin B (NKB), Kisspeptin, and Gonadotropin Releasing Hormone (GnRH) Neuronal Network in Male Rhesus Monkeys

The concept that GnRH release during the prepubertal period is under tonic central inhibition is well established in primates. Although it has been reported that the makorin RING finger protein 3 (MKRN3) gene plays a role in this process (1), neuronal substrates responsible for prepubertal tonic inhibition remain unclear. A series of studies in this lab have shown that GABA neurons are consistently inhibitory to GnRH and kisspeptin neurons in female prepubertal rhesus monkeys (2, 3, 4). Because we recently found that release of GnRH, kisspeptin and NKB in prepubertal male monkeys is significantly lower than those in pubertal monkeys and the pubertal increase in release of these neuropeptides is gonadal steroid independent, in the present study, we ask the question of whether GABA neurons play a role in central inhibition of GnRH release before puberty onset in male monkeys. As described previously, a microdialysis probe was inserted into the median eminence of the hypothalamus in 4 gonadally intact prepubertal male monkeys (15.1 ± 1.9 months of age) and the effects of GABAA receptor antagonist, bicuculline, on release of GnRH, kisspeptin and NKB were examined. The results indicate that bicuculline at 1 µM, but not vehicle, dramatically stimulated release of GnRH, kisspeptin, and NKB in prepubertal male monkeys. Subsequently, we examined whether the bicuculline-induced release of GnRH and kisspeptin was affected by the presence of NKB antagonist, SB222200, as previously we found that NKB signaling is upstream of kisspeptin and GnRH neurons in male monkeys (5). We found that inhibition of NKB signaling attenuated the bicuculline-induced release of GnRH and kisspeptin, suggesting that NKB neurons, in part, relay inhibitory GABA signals to GnRH and kisspeptin neurons. These results are interpreted to mean that in male monkeys, inhibitory GABA signaling plays a role in central inhibition during the prepubertal period and that inhibitory GABA signaling is upstream of the NKB, kisspeptin, and GnRH neuronal pathway. How “Central GABA Inhibition” is regulated by genes involved in this process, such as MKRN3, remains to be investigated.

Chronic sleep fragmentation enhances habenula cholinergic neural activity.

Sleep is essential to emotional health. Sleep disturbance, particularly REM sleep disturbance, profoundly impacts emotion regulation, but the underlying neural mechanisms remain elusive. Here we show that chronic REM sleep disturbance, achieved in mice by chronic sleep fragmentation (SF), enhanced neural activity in the medial habenula (mHb), a brain region increasingly implicated in negative affect. Specifically, after a 5-day SF procedure that selectively fragmented REM sleep, cholinergic output neurons (ChNs) in the mHb exhibited increased spontaneous firing rate and enhanced firing regularity in brain slices. The SF-induced firing changes remained intact upon inhibition of glutamate, GABA, acetylcholine, and histamine receptors, suggesting cell-autonomous mechanisms independent of synaptic transmissions. Moreover, the SF-induced hyperactivity was not because of enhanced intrinsic membrane excitability, but was accompanied by depolarized resting membrane potential in mHb ChNs. Furthermore, inhibition of TASK-3 (KCNK9) channels, a subtype of two-pore domain K+ channels, mimicked the SF effects by increasing the firing rate and regularity, as well as depolarizing the resting membrane potential in mHb ChNs in control-sleep mice. These effects of TASK-3 inhibition were absent in SF mice, suggesting reduced TASK-3 activity following SF. By contrast, inhibition of small-conductance Ca2+-activated K+ (SK) channels did not produce similar effects. Thus, SF compromised TASK-3 function in mHb ChNs, which likely led to depolarized resting membrane potential and increased spontaneous firing. These results not only demonstrate that selective REM sleep disturbance leads to hyperactivity of mHb ChNs, but also identify a key molecular substrate through which REM sleep disturbance may alter affect regulation.

BDNF shifts excitatory‐inhibitory balance in the paraventricular nucleus of the hypothalamus (PVN) to elevate blood pressure

The PVN is an important cardiovascular and autonomic center involved in sympathetic nerve activity and blood pressure (BP) regulation. Stimulation of PVN presympathetic neurons elevate sympathetic activity and BP via excitatory glutamatergic projections to the rostral ventrolateral medulla and the intermediolateral cell column of the spinal cord. Presympathetic neuronal activity is regulated by the balance of excitatory glutamatergic and inhibitory GABA‐ and noradrenergic signaling. BDNF is a neurotrophic factor capable of modulating glutamatergic, GABAergic and catecholaminergic (CA‐ergic) signaling in the CNS by changing expression and membrane trafficking of neurotransmitter receptors and transporters and by increasing the soma size of neurons. Within the PVN, BDNF expression is upregulated in response to physical and psychological stressors; therefore, BDNF may play a significant role in mediating stress‐induced changes in autonomic regulation of BP. Thus, we set out to examine the long‐term effects of BDNF overexpression within the PVN on NMDA‐, GABAA‐ and adrenergic receptor‐mediated BP mechanisms and its effect on PVN neuronal soma size. To test this, Sprague Dawley (SD) rats received bilateral PVN injections of AAV2 viral vectors expressing either GFP or BDNF. Three weeks later, BP and heart rate (HR) responses to PVN injections of AP5 (10 mM), an NMDA antagonist, gabazine (2 mM), a GABAA receptor antagonist, and isoprenaline (250 μM), a non‐selective β‐adrenergic agonist, were recorded under alpha chloralosed‐urethane anesthesia. To test if BDNF alters the soma size of PVN neurons, fixed PVN brain sections were analyzed in ImageJ to quantify the soma size of BDNF or GFP expressing neurons. Our results showed that in the BDNF group, peak decreases in BP and HR in response to AP5 were −21±4 mmHg and − 38±13 BPM, compared with −2±1 mmHg (p&lt;0.0001) and −3±1 BPM (p&lt;0.0001) in the GFP group. In response to gabazine, peak increases in BP and HR in the BDNF group were 52±3 mmHg and 85±19 BPM, compared with 91±6 mmHg (p&lt;0.01) and 20±3 BPM (p=0.06, n.s.) in the GFP group. In response to isoprenaline, peak decreases in BP and HR in the BDNF group were −8±1 mmHg and −19±12 BPM, compared with −35±2 mmHg (p&lt;0.001) and −34±9 BPM (p=0.39, n.s.) in the GFP group. Analysis of PVN neuronal dimensions showed that the BDNF treated group had a significantly larger average soma size of 220±11 μm2 compared to the GFP group of 122±6 μm2 (p&lt;0.0001). In summary, our findings indicate BDNF may alter the excitatory‐inhibitory balance in the PVN by enhancing glutamatergic and diminishing GABAergic and CA‐ergic signaling mechanisms within the PVN. In addition, BDNF‐induced increases in the volume and membrane surface area of PVN neurons could also contribute to long‐term changes in BP regulatory mechanisms.Support or Funding InformationSupported by R01 HL133211‐01A1 and UVM start‐up funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Age-Dependent Electrocorticogram Dynamics and Epileptogenic Responsiveness in Rats Subjected to Prenatal Hypoxia

Using electrocorticogram (ECoG) analysis, we compared age-related dynamics of general neuronal activity and convulsive epileptiform responsiveness induced by intracortical microinjections of 4-aminopyridine (4-AP) in control Wistar rats and those subjected to prenatal hypoxia (Hx; E14; 7% O₂, 3 h). The studies were carried out in three age periods roughly corresponding to childhood (P20–27), adolescence (P30–45), and adulthood (P90–120). It was found that in the process of postnatal development of the control rats, the peak of the ECoG power spectrum density (PSD) of the theta rhythm during wakefulness shifted from the low to the higher frequency, while in the Hx rats this shift had the opposite direction. Moreover, the Hx rats had different frequency characteristics of the ECoG PSD and longer episodes of spike-and-wave discharges caused by 4-AP injections compared to the controls. The total ECoG PSD of slow-wave sleep (1–5 Hz) was also dramatically decreased in the process of development of the Hx rats. Such alterations in PSD could be explained by the changes in balance of the excitation and inhibition processes in the cortical networks. Analyzing protein levels of neurotransmitter transporters in the brain structures of the Hx rats, we found that the content of the glutamate transporter EAAT1 was higher in the parietal cortex in all age groups of Hx rats while in the hippocampus it decreased during postnatal development compared to controls. Furthermore, the content of the vesicular acetylcholine transporter in the parietal cortex, and of the inhibitory GABA transporter 1 in the hippocampus, was also affected by prenatal Hx. These data suggest that prenatal Hx results in a shift in the excitatory and inhibitory balance in the rat cortex towards excitation, making the rat’s brain more vulnerable to the effects of proconvulsant drugs and predisposing animals to epileptogenesis during postnatal life.

GABAAR isoform and subunit structural motifs determine synaptic and extrasynaptic receptor localisation

GABAA receptors (GABAARs) are the principal inhibitory neurotransmitter receptors in the central nervous system. They control neuronal excitability by synaptic and tonic forms of inhibition mostly mediated by different receptor subtypes located in specific cell membrane subdomains. A consensus suggests that α1-3βγ comprise synaptic GABAARs, whilst extrasynaptic α4βδ, α5βγ and αβ isoforms largely underlie tonic inhibition. Although some structural features that enable the spatial segregation of receptors are known, the mobility of key synaptic and extrasynaptic GABAARs are less understood, and yet this is a key determinant of the efficacy of GABA inhibition. To address this aspect, we have incorporated functionally silent α-bungarotoxin binding sites (BBS) into prominent hippocampal GABAAR subunits which mediate synaptic and tonic inhibition. Using single particle tracking with quantum dots we demonstrate that GABAARs that are traditionally considered to mediate synaptic or tonic inhibition are all able to access inhibitory synapses. These isoforms have variable diffusion rates and are differentially retained upon entering the synaptic membrane subdomain. Interestingly, α2 and α4 subunits reside longer at synapses compared to α5 and δ subunits. Furthermore, a high proportion of extrasynaptic δ-containing receptors exhibited slower diffusion compared to δ subunits at synapses. A chimera formed from δ-subunits, with the intracellular domain of γ2L, reversed this behaviour. In addition, we observed that receptor activation affected the diffusion of extrasynaptic, but not of synaptic GABAARs. Overall, we conclude that the differential mobility profiles of key synaptic and extrasynaptic GABAARs are determined by receptor subunit composition and intracellular structural motifs.This article is part of the special issue entitled ‘Mobility and trafficking of neuronal membrane proteins’.

Dopaminergic-GABAergic interplay and alcohol binge drinking

The dopamine D3 receptor (D3R), in the nucleus accumbens (NAc), plays an important role in alcohol reward mechanisms. The major neuronal type within the NAc is the GABAergic medium spiny neuron (MSN), whose activity is regulated by dopaminergic inputs. We previously reported that genetic deletion or pharmacological blockade of D3R increases GABAA α6 subunit in the ventral striatum. Here we tested the hypothesis that D3R-dependent changes in GABAA α6 subunit in the NAc affect voluntary alcohol intake, by influencing the inhibitory transmission of MSNs.We performed in vivo and ex vivo experiments in D3R knockout (D3R −/−) mice and wild type littermates (D3R +/+). Ro 15-4513, a high affinity α6-GABAA ligand was used to study α6 activity.At baseline, NAc α6 expression was negligible in D3R+/+, whereas it was robust in D3R−/−; other relevant GABAA subunits were not changed. In situ hybridization and qPCR confirmed α6 subunit mRNA expression especially in the NAc. In the drinking-in-the-dark paradigm, systemic administration of Ro 15-4513 inhibited alcohol intake in D3R+/+, but increased it in D3R−/−; this was confirmed by intra-NAc administration of Ro 15-4513 and furosemide, a selective α6-GABAA antagonist. Whole-cell patch-clamp showed peak amplitudes of miniature inhibitory postsynaptic currents in NAc medium spiny neurons higher in D3R−/− compared to D3R+/+; Ro 15-4513 reduced the peak amplitude in the NAc of D3R−/−, but not in D3R+/+.We conclude that D3R-dependent enhanced expression of α6 GABAA subunit inhibits voluntary alcohol intake by increasing GABA inhibition in the NAc.

Optical Regulation of Class C GPCRs by Photoswitchable Orthogonal Remotely Tethered Ligands.

G protein-coupled receptors (GPCRs) respond to a wide range of extracellular cues to initiate complex downstream signaling cascades that control myriad aspects of cell function. Despite a long-standing appreciation of their importance to both basic physiology and disease treatment, it remains a major challenge to understand the dynamic activation patterns of GPCRs and the mechanisms by which they modulate biological processes at the molecular, cellular, and tissue levels. Unfortunately, classical methods of pharmacology and genetic knockout are often unable to provide the requisite precision needed to probe such questions. This is an especially pressing challenge for the class C GPCR family which includes receptors for the major excitatory and inhibitory neurotransmitters, glutamate and GABA, which signal in a rapid, spatially-delimited manner and contain many different subtypes whose roles are difficult to disentangle. The desire to manipulate class C GPCRs with spatiotemporal precision, genetic targeting, and subtype specificity has led to the development of a variety of photopharmacological tools. Of particular promise are the photoswitchable orthogonal remotely tethered ligands ("PORTLs") which attach to self-labeling tags that are genetically encoded into full length, wild-type metabotropic glutamate receptors (mGluRs) and allow the receptor to be liganded and un-liganded in response to different wavelengths of illumination. While powerful for studying class C GPCRs, a number of detailed considerations must be made when working with these tools. The protocol included here should provide a basis for the development, characterization, optimization, and application of PORTLs for a wide range of GPCRs.

Ketone body modulation of ligand-gated ion channels

Ketogenesis is a metabolic process wherein ketone bodies are produced from the breakdown of fatty acids. In humans, fatty acid catabolism results in the production of acetyl-CoA which can then be used to synthesize three ketone bodies: acetoacetate, acetone, and β-hydroxybutyrate. Ketogenesis occurs at a higher rate in situations of low blood glucose, such as during fasting, heavy alcohol consumption, and in situations of low insulin, as well as in individuals who follow a ‘ketogenic diet’ consisting of low carbohydrate and high fat intake. This diet has various therapeutic indications, including reduction of seizure likelihood in epileptic patients and alcohol withdrawal syndrome. However, the mechanisms underlying these therapeutic benefits are still unclear, with studies suggesting various mechanisms such as a shift in energy production in the brain, effects on neurotransmitter production, or effects on various protein targets. Two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes was used to investigate the actions of ketone bodies on three ionotropic receptors: GABAA, glycine, and NMDA receptors. While physiologically-relevant concentrations of acetone have little effect on inhibitory GABA or glycine receptors, β–hydroxybutyrate inhibits the effects of agonists of these receptors at concentrations achieved in vivo. Additionally, both acetone and β-hydroxybutyrate act as inhibitors of glutamate at the excitatory NMDA receptor. Due to the role of hyperexcitability in the pathogenesis of epilepsy and alcohol withdrawal, the inhibitory actions of acetone and β-hydroxybutyrate at NMDA receptors may underlie the therapeutic benefit of a ketogenic diet for these disorders.

Analgesic Effects of Triterpenoid Saponins From Stauntonia chinensis via Selective Increase in Inhibitory Synaptic Response in Mouse Cortical Neurons

Triterpenoid saponins from Stauntonia chinensis (TSS) are potential therapeutic agents because of its analgesic properties. However, the underlying mechanisms of the anti-nociceptive activity of TSS are largely unclear, especially in CNS. The present study confirmed the analgesic effect of TSS using four models of acute pain based on thermal or chemical stimuli. TSS treatment specifically impaired the threshold of thermal- and chemical-stimulated acute pain. Naloxone did not block the anti-nociceptive effects of TSS, which showed no participation of the opioid system. We investigated the electrical signal in cultured cortical neurons to explore whether TSS treatment directly affected synaptic transmission. TSS treatment selectively increased spontaneous inhibitory synaptic release and GABA induced charge transfer in mouse cortical neurons. The effects of TSS were maintained for at least 8 h in cultured neurons and in injected mice. Taken together, our results suggest that the analgesic role of TSS in cortex occurs via a particular increase in the inhibitory synaptic response at resting state, which supports TSS as a potential candidate for inflammatory pain relief.