α5GABAA Receptors Mediate Tonic Inhibition in the Spinal Cord Dorsal Horn and Contribute to the Resolution Of Hyperalgesia.

Neuronal inhibition mediated by GABAA receptors constrains nociceptive processing in the spinal cord, and loss of GABAergic inhibition can produce allodynia and hyperalgesia. Extrasynaptic α5 subunit-containing GABAA receptors (α5GABAA Rs) generate a tonic conductance that inhibits neuronal activity and constrains learning and memory; however, it is unclear whether α5GABAA Rs similarly generate a tonic conductance in the spinal cord dorsal horn to constrain nociception. We assessed the distribution of α5GABAA Rs in the spinal cord dorsal horn by immunohistochemical analysis, and the activity and function of α5GABAA Rs in neurons of the superficial dorsal horn using electrophysiological and behavioral approaches in male, null-mutant mice lacking the GABAA R α5 subunit (Gabra5-/-) and wild-type mice (WT). The expression of α5GABAA Rs in the superficial dorsal horn followed a laminar pattern of distribution, with a higher expression in lamina II than lamina I. Similarly, the tonic GABAA current in lamina II neurons had a larger contribution from α5GABAA Rs than in lamina I, with no significant contribution of these receptors to synaptic GABAA current. In behavioural tests, WT and Gabra5-/- mice exhibited similar acute thermal and mechanical nociception, and similar mechanical sensitization immediately following intraplantar capsaicin or Complete Freund's Adjuvant (CFA). However, Gabra5-/- mice showed prolonged recovery from sensitization in these models, and increased responses in the late phase of the formalin test. Overall, our data suggest that tonically-active α5GABAA Rs in the spinal cord dorsal horn accelerate the resolution of hyperalgesia and may therefore serve as a novel therapeutic target to promote recovery from pathological pain. © 2016 Wiley Periodicals, Inc.

Inhibition of α5 γ-Aminobutyric acid type A receptors restores recognition memory after general anesthesia.

BACKGROUND: General anesthetics cause cognitive deficits that persist much longer than would be expected on the basis of their pharmacokinetics. The cellular mechanisms underlying these postanesthetic cognitive deficits remain unknown. γ-Aminobutyric acid type A (GABA(A)) receptors are principal targets for most anesthetics. In particular, the α5GABA(A) receptor subtype has been implicated in acute memory blockade during anesthesia and memory deficits in the early postoperative period. We first sought to determine whether working memory and short-term recognition memory are impaired after isoflurane anesthesia. The second aim of the study was to determine whether memory deficits after isoflurane can be reversed by inhibiting α5GABA(A) receptors. We also sought to determine whether the expression of α5GABA(A) receptors is necessary for the development of memory dysfunction after isoflurane. Lastly, the effect of sevoflurane on memory was studied. METHODS: Wild-type and α5GABA(A) receptor null-mutant (Gabra5-/-) mice were treated with isoflurane (1.3%; 1 minimum alveolar concentration [MAC]) or sevoflurane (2.3%; 1 MAC) or vehicle gas for 1 hour. Memory performance was assessed with a novel object recognition task. Mice were trained on the recognition task either 24 hours or 72 hours after isoflurane anesthesia. Working memory and short-term memory were tested 1 minute and 1 hour after training, respectively. To determine whether inhibition of α5GABA(A) receptors reverses memory deficits, we treated a subset of mice with L-655,708 (0.35 mg/kg or 0.7 mg/kg) 23.5 hours after isoflurane and 30 minutes before behavioral training. RESULTS: Short-term memory was impaired in wild-type mice 24 hours after isoflurane as evidenced by a decrease in the discrimination ratio (control 0.66 ± 0.03 vs isoflurane 0.51 ± 0.03, P = 0.0005). In contrast, working memory was not impaired by isoflurane (control 0.68 ± 0.05 vs isoflurane 0.67 ± 0.04, P = 0.979). The deficit in short-term memory was fully reversed by L-655,708 (effect of isoflurane × L-655,708, F(2,102) = 3.59, P = 0.032; isoflurane 0.51 ± 0.03 vs isoflurane + L-655,708 at 0.35 mg/kg 0.67 ± 0.03, P < 0.05). By 72 hours, the deficits in short-term memory resolved spontaneously (control 0.65 ± 0.05 vs isoflurane 0.60 ± 0.04, P = 0.441). Gabra5-/- mice showed no short-term memory deficits 24 hours after isoflurane (effect of isoflurane F(1,47) = 0.375, P = 0.544). Sevoflurane also caused memory deficits 24 hours after anesthesia, as evidence by a reduction in the discrimination ratio (control 0.63 ± 0.02 vs sevoflurane 0.53 ± 0.03, P = 0.039). CONCLUSIONS: Inhalational anesthetics cause deficits in anterograde recognition memory. This proof-of-concept study shows that α5GABA(A) receptors are necessary for the development of postanesthetic deficits in recognition memory and that these receptors can be targeted to restore memory even after the anesthetic has been eliminated.

Alpha5GABAA receptor activity sets the threshold for long-term potentiation and constrains hippocampus-dependent memory.

Synaptic plasticity, which is the neuronal substrate for many forms of hippocampus-dependent learning, is attenuated by GABA type A receptor (GABA(A)R)-mediated inhibition. The prevailing notion is that a synaptic or phasic form of GABAergic inhibition regulates synaptic plasticity; however, little is known about the role of GABA(A)R subtypes that generate a tonic or persistent inhibitory conductance. We studied the regulation of synaptic plasticity by alpha5 subunit-containing GABA(A)Rs (alpha5GABA(A)Rs), which generate a tonic inhibitory conductance in CA1 pyramidal neurons using electrophysiological recordings of field and whole-cell potentials in hippocampal slices from both wild-type and null mutant mice for the alpha5 subunit of the GABA(A)R (Gabra5(-/-) mice). In addition, the strength of fear-associated memory was studied. The results showed that alpha5GABA(A)R activity raises the threshold for induction of long-term potentiation in a highly specific band of stimulation frequencies (10-20 Hz) through mechanisms that are predominantly independent of inhibitory synaptic transmission. The deletion or pharmacological inhibition of alpha5GABA(A)Rs caused no change in baseline membrane potential or input resistance but increased depolarization during 10 Hz stimulation. The encoding of hippocampus-dependent memory was regulated by alpha5GABA(A)Rs but only under specific conditions that generate moderate but not robust forms of fear-associated learning. Thus, under specific conditions, alpha5GABA(A)R activity predominates over synaptic inhibition in modifying the strength of both synaptic plasticity in vitro and certain forms of memory in vivo.

Short-term memory impairment after isoflurane in mice is prevented by the α5 γ-aminobutyric acid type A receptor inverse agonist L-655,708.

BACKGROUND: Memory blockade is an essential component of the anesthetic state. However, postanesthesia memory deficits represent an undesirable and poorly understood adverse effect. Inhibitory α5 subunit-containing γ-aminobutyric acid subtype A receptors (α5GABAA) are known to play a critical role in memory processes and are highly sensitive to positive modulation by anesthetics. We postulated that inhibiting the activity of α5GABAA receptors during isoflurane anesthesia would prevent memory deficits in the early postanesthesia period. METHODS: Mice were pretreated with L-655,708, an α5GABAA receptor-selective inverse agonist, or vehicle. They were then exposed to isoflurane for 1 h (1.3%, or 1 minimum alveolar concentration, or air-oxygen control). Then, either 1 or 24 h later, mice were conditioned in fear-associated contextual and cued learning paradigms. In addition, the effect of L-655,708 on the immobilizing dose of isoflurane was studied. Motor coordination, sedation, anxiety, and the concentration of isoflurane in the brain at 5 min, 1 h, and 24 h after isoflurane were also examined. RESULTS: Motor and sensory function recovered within minutes after termination of isoflurane administration. In contrast, a robust deficit in contextual fear memory persisted for at least 24 h. The α5GABAA receptor inverse agonist, L-655,708, completely prevented memory deficits without changing the immobilizing dose of isoflurane. Trace concentrations of isoflurane were measured in the brain 24 h after treatment. CONCLUSIONS: Memory deficits occurred long after the sedative, analgesic, and anxiolytic effects of isoflurane subsided. L-655,708 prevented memory deficit, suggesting that an isoflurane interaction at α5GABAA receptors contributes to memory impairment during the early postanesthesia period.

Gabapentin increases expression of δ subunit-containing GABAA receptors.

BACKGROUND: Gabapentin is a structural analog of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Its anticonvulsant, analgesic and anxiolytic properties suggest that it increases GABAergic inhibition; however, the molecular basis for these effects is unknown as gabapentin does not directly modify GABA type A (GABAA) receptor function, nor does it modify synaptic inhibition. Here, we postulated that gabapentin increases expression of δ subunit-containing GABAA (δGABAA) receptors that generate a tonic inhibitory conductance in multiple brain regions including the cerebellum and hippocampus. METHODS: Cell-surface biotinylation, Western blotting, electrophysiologic recordings, behavioral assays, high-performance liquid chromatography and gas chromatography-mass spectrometry studies were performed using mouse models. FINDINGS: Gabapentin enhanced expression of δGABAA receptors and increased a tonic inhibitory conductance in neurons. This increased expression likely contributes to GABAergic effects as gabapentin caused ataxia and anxiolysis in wild-type mice but not δ subunit null-mutant mice. In contrast, the antinociceptive properties of gabapentin were observed in both genotypes. Levels of GABAA receptor agonists and neurosteroids in the brain were not altered by gabapentin. INTERPRETATION: These results provide compelling evidence to account for the GABAergic properties of gabapentin. Since reduced expression of δGABAA receptor occurs in several disorders, gabapentin may have much broader therapeutic applications than is currently recognized. FUND: Supported by a Foundation Grant (FDN-154312) from the Canadian Institutes of Health Research (to B.A.O.); a NSERC Discovery Grant (RGPIN-2016-05538), a Canada Research Chair in Sensory Plasticity and Reconsolidation, and funding from the University of Toronto Centre for the Study of Pain (to R.P.B.).

Social transmission and buffering of synaptic changes after stress.

Stress can trigger enduring changes in neural circuits and synapses. The behavioral and hormonal consequences of stress can also be transmitted to others, but whether this transmitted stress has similar effects on synapses is not known. We found that authentic stress and transmitted stress in mice primed paraventricular nucleus of the hypothalamus (PVN) corticotropin-releasing hormone (CRH) neurons, enabling the induction of metaplasticity at glutamate synapses. In female mice that were subjected to authentic stress, this metaplasticity was diminished following interactions with a naive partner. Transmission from the stressed subject to the naive partner required the activation of PVN CRH neurons in both subject and partner to drive and detect the release of a putative alarm pheromone from the stressed mouse. Finally, metaplasticity could be transmitted sequentially from the stressed subject to multiple partners. Our findings demonstrate that transmitted stress has the same lasting effects on glutamate synapses as authentic stress and reveal an unexpected role for PVN CRH neurons in transmitting distress signals among individuals.

α5GABAA receptor deficiency causes autism-like behaviors.

The prevalence of autism spectrum disorders (ASDs), which affect over 1% of the population, has increased twofold in recent years. Reduced expression of GABAA receptors has been observed in postmortem brain tissue and neuroimaging of individuals with ASDs. We found that deletion of the gene for the α5 subunit of the GABAA receptor caused robust autism-like behaviors in mice, including reduced social contacts and vocalizations. Screening of human exome sequencing data from 396 ASD subjects revealed potential missense mutations in GABRA5 and in RDX, the gene for the α5GABAA receptor-anchoring protein radixin, further supporting a α5GABAA receptor deficiency in ASDs.

Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABA(A) receptor δ subunit in cerebellar granule neurons and delays motor development in rats.

Exposure to ethanol (EtOH) during fetal development can lead to long-lasting alterations, including deficits in fine motor skills and motor learning. Studies suggest that these are, in part, a consequence of cerebellar damage. Cerebellar granule neurons (CGNs) are the gateway of information into the cerebellar cortex. Functionally, CGNs are heavily regulated by phasic and tonic GABAergic inhibition from Golgi cell interneurons; however, the effect of EtOH exposure on the development of GABAergic transmission in immature CGNs has not been investigated. To model EtOH exposure during the 3rd trimester-equivalent of human pregnancy, neonatal pups were exposed intermittently to high levels of vaporized EtOH from postnatal day (P) 2 to P12. This exposure gradually increased pup serum EtOH concentrations (SECs) to ∼60 mM (∼0.28 g/dl) during the 4 h of exposure. EtOH levels gradually decreased to baseline 8 h after the end of exposure. Surprisingly, basal tonic and phasic GABAergic currents in CGNs were not significantly affected by postnatal alcohol exposure (PAE). However, PAE increased δ subunit expression at P28 as detected by immunohistochemical and western blot analyses. Also, electrophysiological studies with an agonist that is highly selective for δ-containing GABA(A) receptors, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP), showed an increase in THIP-induced tonic current. Behavioral studies of PAE rats did not reveal any deficits in motor coordination, except for a delay in the acquisition of the mid-air righting reflex that was apparent at P15 to P18. These findings demonstrate that repeated intermittent exposure to high levels of EtOH during the equivalent of the last trimester of human pregnancy has significant but relatively subtle effects on motor coordination and GABAergic transmission in CGNs in rats.

Sustained increase in α5GABAA receptor function impairs memory after anesthesia.

Many patients who undergo general anesthesia and surgery experience cognitive dysfunction, particularly memory deficits that can persist for days to months. The mechanisms underlying this postoperative cognitive dysfunction in the adult brain remain poorly understood. Depression of brain function during anesthesia is attributed primarily to increased activity of γ-aminobutyric acid type A receptors (GABA(A)Rs), and it is assumed that once the anesthetic drug is eliminated, the activity of GABA(A)Rs rapidly returns to baseline and these receptors no longer impair memory. Here, using a murine model, we found that a single in vivo treatment with the injectable anesthetic etomidate increased a tonic inhibitory current generated by α5 subunit-containing GABA(A)Rs (α5GABA(A)Rs) and cell-surface expression of α5GABA(A)Rs for at least 1 week. The sustained increase in α5GABA(A)R activity impaired memory performance and synaptic plasticity in the hippocampus. Inhibition of α5GABA(A)Rs completely reversed the memory deficits after anesthesia. Similarly, the inhaled anesthetic isoflurane triggered a persistent increase in tonic current and cell-surface expression of α5GABA(A)Rs. Thus, α5GABA(A)R function does not return to baseline after the anesthetic is eliminated, suggesting a mechanism to account for persistent memory deficits after general anesthesia.

Hyperpolarization-activated current (In) is reduced in hippocampal neurons from Gabra5-/- mice.

Changes in the expression of γ-aminobutyric acid type A (GABAA) receptors can either drive or mediate homeostatic alterations in neuronal excitability. A homeostatic relationship between α5 subunit-containing GABAA (α5GABAA) receptors that generate a tonic inhibitory conductance, and HCN channels that generate a hyperpolarization-activated cation current (Ih) was recently described for cortical neurons, where a reduction in Ih was accompanied by a reciprocal increase in the expression of α5GABAA receptors resulting in the preservation of dendritosomatic synaptic function. Here, we report that in mice that lack the α5 subunit gene (Gabra5-/-), cultured embryonic hippocampal pyramidal neurons and ex vivo CA1 hippocampal neurons unexpectedly exhibited a decrease in Ih current density (by 40% and 28%, respectively), compared with neurons from wild-type (WT) mice. The resting membrane potential and membrane hyperpolarization induced by blockade of Ih with ZD-7288 were similar in cultured WT and Gabra5-/- neurons. In contrast, membrane hyperpolarization measured after a train of action potentials was lower in Gabra5-/- neurons than in WT neurons. Also, membrane impedance measured in response to low frequency stimulation was greater in cultured Gabra5-/- neurons. Finally, the expression of HCN1 protein that generates Ih was reduced by 41% in the hippocampus of Gabra5-/- mice. These data indicate that loss of a tonic GABAergic inhibitory conductance was followed by a compensatory reduction in Ih. The results further suggest that the maintenance of resting membrane potential is preferentially maintained in mature and immature hippocampal neurons through the homeostatic co-regulation of structurally and biophysically distinct cation and anion channels.

Memory deficits induced by inflammation are regulated by α5-subunit-containing GABAA receptors.

Systemic inflammation causes learning and memory deficits through mechanisms that remain poorly understood. Here, we studied the pathogenesis of memory loss associated with inflammation and found that we could reverse memory deficits by pharmacologically inhibiting α5-subunit-containing γ-aminobutyric acid type A (α5GABA(A)) receptors and deleting the gene associated with the α5 subunit. Acute inflammation reduces long-term potentiation, a synaptic correlate of memory, in hippocampal slices from wild-type mice, and this reduction was reversed by inhibition of α5GABA(A) receptor function. A tonic inhibitory current generated by α5GABA(A) receptors in hippocampal neurons was increased by the key proinflammatory cytokine interleukin-1ß through a p38 mitogen-activated protein kinase signaling pathway. Interleukin-1ß also increased the surface expression of α5GABA(A) receptors in the hippocampus. Collectively, these results show that α5GABA(A) receptor activity increases during inflammation and that this increase is critical for inflammation-induced memory deficits.

The sedative but not the memory-blocking properties of ethanol are modulated by α5-subunit-containing γ-aminobutyric acid type A receptors.

The precise mechanisms underlying the memory-blocking properties of ethanol are unknown, in part because ethanol targets a wide array of neurotransmitter receptors and transporters. The aim of this study was to determine whether the memory loss caused by ethanol is mediated, in part, by α5 subunit-containing γ-aminobutyric acid subtype A receptors. These receptors have been implicated in learning and memory processes and are targets for a variety of neurodepressive drugs. Also, since these receptors generate a tonic inhibitory current in hippocampal pyramidal neurons, we examined whether concentrations of ethanol that block memory in vivo increased the tonic current using whole-cell patch-clamp recordings in hippocampal neurons. Null mutant mice lacking the α5 subunit (Gabra5-/-) and wild-type mice were equally impaired in contextual fear conditioning by moderate (1mg/kg) and high (1.5mg/kg) doses of ethanol. The higher dose of ethanol also reduced auditory delay fear conditioning to the same extent in the two genotypes. Interestingly, wild-type mice were more sensitive than Gabra5-/- mice to the sedative effects of low (0.5mg/kg) and moderate (1mg/kg) doses of ethanol in the open-field task. Concentrations of ethanol that impaired memory performance in vivo did not increase the amplitude of the tonic current. Together, the results suggest that the α5-subunit containing γ-aminobutyric acid subtype A receptors are not direct targets for positive modulation by ethanol nor do they contribute to ethanol-induced memory loss. In contrast, these receptors may contribute to the sedative properties of ethanol.