Antagonistic properties of caged GABA compounds used for activation of GABAA receptors in neocortical pyramidal neurons

Caged photolysable compounds have served to be pivotal to neuroscientific investigations; allowing the cognizing of molecular kinetics and properties of neuronal micro-machinery such as neurotransmitter receptors. Precision in terms of temporal and spatial resolution of neurotransmitter release endowed by photolysis has multitudinal applicabilities in the realm of GABAA receptors (GABAARs), their neuronal niche and effects on neuronal and network activity. Caged compounds, in their caged form, may display certain unideal traits such as undesired interactions with the system and antagonistic activity on the target receptor. This study aims to reevaluate the GABAAR antagonistic actions of caged Rubi-GABA, which was found to antagonize these receptors at significantly lower concentrations than those reported in the literature. Furthermore, this study electrophysiologically characterizes the possible antagonistic properties of a novel quinoline-derived UV-photolysable caged GABA compound, 8 DMAQ GABA, whose activity, in its caged form appears to have a much more favorable antagonism profile compared to the widely used RuBi-GABA. To assess the antagonistic effects of these compounds on GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs) patch-clamp recordings were carried out in the whole-cell voltage clamp configuration on cortical layer 2/3 cortical pyramidal neurons in acute neocortical slices prepared from 16-18 day-old rat rats. The results of this study indicate a revised antagonism profile for caged Rubi-GABA, with marked GABAAR toxicity in the low micromolar range. The study also scrutinizes the photo-kinetic properties of both caged GABA compounds and reveals that the rate of GABA release from 8-DMAQ is slower than from RuBi-GABA

Distinct Modulation of Spontaneous and GABA-Evoked Gating by Flurazepam Shapes Cross-Talk Between Agonist-Free and Liganded GABAA Receptor Activity

GABAA receptors (GABAARs) play a crucial inhibitory role in the CNS. Benzodiazepines (BDZs) are positive modulators of specific subtypes of GABAARs, but the underlying mechanism remains obscure. Early studies demonstrated the major impact of BDZs on binding and more recent investigations indicated gating, but it is unclear which transitions are affected. Moreover, the upregulation of GABAAR spontaneous activity by BDZs indicates their impact on receptor gating but the underlying mechanisms remain unknown. Herein, we investigated the effect of a BDZ (flurazepam) on the spontaneous and GABA-induced activity for wild-type (WT, α1β2γ2) and mutated (at the orthosteric binding site α1F64) GABAARs. Surprisingly, in spite of the localization at the binding site, these mutations increased the spontaneous activity. Flurazepam (FLU) upregulated this activity for mutants and WT receptors to a similar extent by affecting opening/closing transitions. Spontaneous activity affected GABA-evoked currents and is manifested as an overshoot after agonist removal that depended on the modulation by BDZs. We explain the mechanism of this phenomenon as a cross-desensitization of ligand-activated and spontaneously active receptors. Moreover, due to spontaneous activity, FLU-pretreatment and co-application (agonist + FLU) protocols yielded distinct results. We provide also the first evidence that GABAAR may enter the desensitized state in the absence of GABA in a FLU-dependent manner. Based on our data and model simulations, we propose that FLU affects agonist-induced gating by modifying primarily preactivation and desensitization. We conclude that the mechanisms of modulation of spontaneous and ligand-activated GABAAR activity concerns gating but distinct transitions are affected in spontaneous and agonist-evoked activity

Acute neuroinflammation leads to disruption of neuronal chloride regulation and consequent hyperexcitability in the dentate gyrus

Publisher Copyright: © 2023 The AuthorsNeuroinflammation is a salient part of diverse neurological and psychiatric pathologies that associate with neuronal hyperexcitability, but the underlying molecular and cellular mechanisms remain to be identified. Here, we show that peripheral injection of lipopolysaccharide (LPS) renders the dentate gyrus (DG) hyperexcitable to perforant pathway stimulation in vivo and increases the internal spiking propensity of dentate granule cells (DGCs) in vitro 24 h post-injection (hpi). In parallel, LPS leads to a prominent downregulation of chloride extrusion via KCC2 and to the emergence of NKCC1-mediated chloride uptake in DGCs under experimental conditions optimized to detect specific changes in transporter efficacy. These data show that acute neuroinflammation leads to disruption of neuronal chloride regulation, which unequivocally results in a loss of GABAergic inhibition in the DGCs, collapsing the gating function of the DG. The present work provides a mechanistic explanation for neuroinflammation-driven hyperexcitability and consequent cognitive disturbance.Peer reviewe

Expression patterns of NKCC1 in neurons and non-neuronal cells during cortico-hippocampal development

The Na-K-2Cl cotransporter NKCC1 is widely expressed in cells within and outside the brain. However, our understanding of its roles in brain functions throughout development, as well as in neuropsychiatric and neurological disorders, has been severely hindered by the lack of reliable data on its developmental and (sub)cellular expression patterns. We provide here the first properly controlled analysis of NKCC1 protein expression in various cell types of the mouse brain using custom-made antibodies and an NKCC1 knock-out validated immunohistochemical procedure, with parallel data based on advanced mRNA approaches. NKCC1 protein and mRNA are expressed at remarkably high levels in oligodendrocytes. In immature neurons, NKCC1 protein was located in the somata, whereas in adult neurons, only NKCC1 mRNA could be clearly detected. NKCC1 immunoreactivity is also seen in microglia, astrocytes, developing pericytes, and in progenitor cells of the dentate gyrus. Finally, a differential expression of NKCC1 splice variants was observed, with NKCC1a predominating in non-neuronal cells and NKCC1b in neurons. Taken together, our data provide a cellular basis for understanding NKCC1 functions in the brain and enable the identification of major limitations and promises in the development of neuron-targeting NKCC1-blockers.Peer reviewe