Current awareness

Function and pharmacology of multiple GABA_A receptor subunits

Alpha-casozepine (α CZP), a tryptic hydrolysate of milk casein is a decapeptide shown to promote sleep and produce anxiolytic or anticonvulsant activity. Intriguingly, studies indicate structural similarities to benzodiazepine (BZD)-like molecules (e.g., diazepam), resulting in positive modulation of γ-aminobutyric acid A type (GABA_A) receptors. However, some unexplained anomalous behaviour of α-CZP includes 1) 1000 times less affinity for BZD site on GABA_A receptor in vitro conditions, whereas in vivo it showed 10-fold increased affinity when compared to diazepam; 2) anxiolytic effects were observed only in stressed conditions and 3) unlike diazepam, it failed to exhibit dependence or habituation. Interestingly, neurosteroids like allopregnanolone or its analogues that are synthesized de novo have both genomic and non-genomic actions. The rapid nongenomic neuronal inhibition of these compounds is mediated by GABA_A receptors through autocrine and paracrine actions. Studies have shown that changes in the levels of neurosteroids during acute (rise) and chronic stress (decreased), consequently, altering the senetivity of GABA_A receptor subunits. Neurosteroids even at low concentration (nanomolar range) potentiate the response of GABA indirectly, while at higher concentrations they directly activate the receptor-channel complex. Interestingly, coadministration of neurosteroids and BZPs has shown not only to prevent the development of tolerance of BZP and augmented recovery from BZP withdrawal anxiety and hyperactivity in mice. The combination also produced synergetic anxiolytic effects. Taken together, the evidence suggests possible implications of neurosteroids in the actions of CZP via BZD receptors. The present hypothesis brings out the possible role of neurosteroids and the various factors that might participate in CZP-induce anxiolytic effects.

Changes in functional GABA_AR signaling in hippocampus have previously been evaluated using pre-clinical animal models of either diffuse brain injury or extreme focal brain injury that precludes measurement of cells located ipsilateral to injury. As a result, there is little information about the status of functional GABA_AR signaling in dentate granule cells (DGCs) located ipsilateral to focal brain injury, where significant cellular changes have been documented. We used whole-cell patch-clamp recordings from hippocampal slices to measure changes in GABA_ARs in dentate granule cells (DGCs) at 1–2, 3–5, and 8–13 weeks after controlled cortical impact (CCI) brain injury. Synaptic and tonic GABA_AR currents (I_TonicGABA) were measured in DGCs at baseline conditions and during application of the GABA_AR agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride (THIP) to assess in the function of δ subunit-containing GABA_ARs. DGCs ipsilateral to CCI exhibited no changes in the amplitude of resting I_TonicGABA relative to DGCs after sham-injury or contralateral to CCI. In contrast, there was a significant reduction in the THIP-evoked I_TonicGABA in DGCs ipsilateral to CCI at both time-points. Tonic GABAergic inhibition of DGCs ipsilateral to injury also exhibited reduced responsiveness to the neurosteroid THDOC. I_TonicGABA in DGCs ipsilateral to CCI did not exhibit a change in sensitivity to L655,708, an inverse agonist with selectivity for α₅ subunit-containing GABA_ARs, suggesting a lack of functional change in GABA_ARs containing this subunit. At the 8–13 week time-point, gene expression of GABA_AR subunits expected to contribute to I_TonicGABA (i.e., α₄, α₅ and δ) was not significantly altered by CCI injury in isolated dentate gyrus. Collectively, these results demonstrate enduring functional changes in I_TonicGABA in DGCs ipsilateral to focal brain injury that occur independent of altered gene expression.

Auditory cortex (AI) shows age-related decreases in pre-synaptic markers for gamma-aminobutyric acid (GABA) and degraded AI neuronal response properties. Previous studies find age-related increases in spontaneous and driven activity, decreased spectral and directional sensitivity, and impaired novelty detection. The present study examined expression of GABA_A receptor (GABA_AR) subunit message, protein, and quantitative GABA_AR binding in young, middle-aged, and aged rat AI, with comparisons with adjoining parietal cortex. Significant loss of GABA_AR α₁ subunit message across AI layers was observed in middle-aged and aged rats and α₁ subunit protein levels declined in layers II and III. Age-related increases in GABA_AR α₃ subunit message and protein levels were observed in certain AI layers. GABA_AR subunits, including β₁, β₂, γ₁, γ_2s, and γ_2L, primarily, but not exclusively, showed age-related declines at the message and protein levels. The ability of GABA to modulate [³H]t-butylbicycloorthobenzoate binding in the chloride channel showed age-related decreases in peak binding and changes in desensitization kinetics. Collectively, age-related changes in GABA_AR subunit composition would alter the magnitude and temporal properties of inhibitory synaptic transmission and could underpin observed age-related functional changes seen in the elderly.

There are several modulatory sites at GABA_A receptors, which mediate the actions of many drugs, among them benzodiazepine. Three kinds of allosteric modulators act through the benzodiazepine binding site: positive (agonist), neutral (antagonist), and negative (inverse agonist). The goal of the present study was to examine the influence of the inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) acting on α GABA_A receptor and compare its dose-response effects on memory and depression-like behavior. We independently studied the effects of DMCM (0.05–1.0 mg/kg) on retention versus acquisition of active avoidance and depression-like behavior in the forced swim test. Throughout the study, drugs were given intraperitoneally, 30 min before testing. ANOVA has showed that treatment with DMCM significantly affected retrieval of avoidance response (p < 0.05), exerted promnesic effects in inverted U-shape manner. Dunnett's test indicated that the DMCM avoidance-facilitatory dose was 0.1 mg/kg. At the dose facilitating retrieval of avoidance memory, DMCM significantly (p < 0.05, comparison of regression coefficients by Student's t-test) and progressively increased acquisition rate during 5 days training, compared to the saline group. In forced swim test, ANOVA indicated statistically significant effects of DMCM (p < 0.05). Dunnett's analysis showed that DMCM significantly decreased immobility time at the dose of 0.1 mg/kg, exerted acute antidepressant-like effects. Our results experimentally support the findings that under certain circumstances, nonselective benzodiazepine site inverse agonists, produce memory-enhancing and antidepressant-like effects. The molecular and neuronal substrates linking the actions of specific GABA-benzodiazepine receptor complex subunits remains to be further elucidated.

Centrally acting Angiotensin II AT₁ receptor blockers (ARBs) protect from stress-induced disorders and decrease anxiety in a model of inflammatory stress, the systemic injection of bacterial endotoxin lipopolysaccharide (LPS). In order to better understand the anxiolytic effect of ARBs, we treated rats with LPS (50 μg/kg) with or without 3 days of pretreatment with the ARB candesartan (1 mg/kg/day), and studied cortical benzodiazepine (BZ) and corticotrophin-releasing factor (CRF) receptors. We compared the cortical BZ and CRF receptors expression pattern induced by LPS with that produced in restraint stress. Inflammation stress produced a generalized increase in cortical BZ₁ receptors and reduced mRNA expression of the GABA_A receptor γ₂ subunit in cingulate cortex; changes were prevented by candesartan pretreatment. Moreover, restraint stress produced similar increases in cortical BZ₁ receptor binding, and candesartan prevented these changes. Treatment with candesartan alone increased cortical BZ₁ binding, and decreased γ₂ subunit mRNA expression in the cingulate cortex. Conversely, we did not find changes in CRF₁ receptor expression in any of the cortical areas studied, either after inflammation or restraint stress. Cortical CRF₂ receptor binding was undetectable, but CRF₂ mRNA expression was decreased by inflammation stress, a change prevented by candesartan. We conclude that stress promotes rapid and widespread changes in cortical BZ₁ receptor expression; and that the stress-induced BZ₁ receptor expression is under the control of AT₁ receptor activity. The results suggest that the anti-anxiety effect of ARBs may be associated with their capacity to regulate stress-induced alterations in cortical BZ₁ receptors.

Clinical studies have suggested that children experiencing a febrile seizure (FS) before the age of 1 year have persistent deficits, but it is unknown whether these seizures lead to permanent cortical reorganization and alterations in function. A FS on the background of increased genetic seizure susceptibility may also lead to negative long-term consequences. Alterations in neocortical motor map expression provide a measure of neocortical reorganization and have been reported in both adults with frontal lobe epilepsy and following seizure induction in experimental models. The objectives of the present study were to determine whether 1) an infantile FS leads to changes to motor map expression in adulthood; 2) long-term cortical reorganization is a function of the age at FS or genetic seizure susceptibility; and 3) different levels of GABA_A or glutamate receptor subunits or cation-chloride-co-transporters (CCCs) at the time of FS correlate with alterations to motor map expression.

FSs were induced in postnatal day 10 (P10) or P14 Long–Evans (LE) rats or in P14 seizure-prone FAST rats by the administration of the bacterial endotoxin lipopolysaccharide (LPS) and a subconvulsant dose of kainic acid. Ten weeks later intracortical microstimulation was performed to generate motor maps of forelimb movement representations. Sensorimotor neocortex samples were also dissected from naïve P10 FAST and P10 and P14 LE pups for western blotting with antibodies against various GABA_A, NMDA, and AMPA receptor subunits and for CCCs.

Adult FAST rats had larger motor maps with lower stimulation thresholds after a FS at P14, while adult LE rats had significantly lower map stimulation thresholds but similar sized maps after a FS at P10 compared to controls. There were no differences in neocortical motor map size or stimulation thresholds in adult LE rats after a FS at P14. Both P10 LE and P14 FAST rats had significantly lower levels of the GABA_A receptor α1 subunit, higher levels of the α2 subunit, and a higher NKCC1/KCC2 ratio in the sensorimotor cortex compared with the P14 LE rat. In addition, the P14 FAST rats had lower levels of the GluR2 and NR2A receptor subunits in the sensorimotor cortex compared with the P14 LE rats.

A single infantile FS can have long-term effects on neocortical reorganization in younger individuals and those with underlying seizure susceptibility. These changes may be related to an increased level of excitability in the neocortex of younger or genetically seizure-prone rats, as suggested by immaturity of their GABAergic and CCC systems. Given the high incidence of FSs in children, it will be important to gain a better understanding of how age and genetic seizure predisposition may contribute to the long-term sequelae of these events.