Inhibitory and excitatory synaptic neuroadaptations in the diazepam tolerant brain.

Benzodiazepine (BZ) drugs treat seizures, anxiety, insomnia, and alcohol withdrawal by potentiating γ2 subunit containing GABA type A receptors (GABAARs). BZ clinical use is hampered by tolerance and withdrawal symptoms including heightened seizure susceptibility, panic, and sleep disturbances. Here, we investigated inhibitory GABAergic and excitatory glutamatergic plasticity in mice tolerant to benzodiazepine sedation. Repeated diazepam (DZP) treatment diminished sedative effects and decreased DZP potentiation of GABAAR synaptic currents without impacting overall synaptic inhibition. While DZP did not alter γ2-GABAAR subunit composition, there was a redistribution of extrasynaptic GABAARs to synapses, resulting in higher levels of synaptic BZ-insensitive α4-containing GABAARs and a concomitant reduction in tonic inhibition. Conversely, excitatory glutamatergic synaptic transmission was increased, and NMDAR subunits were upregulated at synaptic and total protein levels. Quantitative proteomics further revealed cortex neuroadaptations of key pro-excitatory mediators and synaptic plasticity pathways highlighted by Ca2+/calmodulin-dependent protein kinase II (CAMKII), MAPK, and PKC signaling. Thus, reduced inhibitory GABAergic tone and elevated glutamatergic neurotransmission contribute to disrupted excitation/inhibition balance and reduced BZ therapeutic power with benzodiazepine tolerance.

Long-term α5 GABA A receptor negative allosteric modulator treatment reduces NMDAR-mediated neuronal excitation and maintains basal neuronal inhibition.

α5 subunit-containing GABA type-A receptors (α5 GABAARs) are enriched in the hippocampus and play critical roles in neurodevelopment, synaptic plasticity, and cognition. α5 GABAAR preferring negative allosteric modulators (α5 NAMs) show promise mitigating cognitive impairment in preclinical studies of conditions characterized by excess GABAergic inhibition, including Down syndrome and memory deficits post-anesthesia. However, previous studies have primarily focused on acute application or single-dose α5 NAM treatment. Here, we measured the effects of chronic (7-day) in vitro treatment with L-655,708 (L6), a highly selective α5 NAM, on glutamatergic and GABAergic synapses in rat hippocampal neurons. We previously showed that 2-day in vitro treatment with L6 enhanced synaptic levels of the glutamate NMDA receptor (NMDAR) GluN2A subunit without modifying surface α5 GABAAR expression, inhibitory synapse function, or L6 sensitivity. We hypothesized that chronic L6 treatment would further increase synaptic GluN2A subunit levels while maintaining GABAergic inhibition and L6 efficacy, thus increasing neuronal excitation and glutamate-evoked intracellular calcium responses. Immunofluorescence experiments revealed that 7-day L6 treatment slightly increased the synaptic levels of gephyrin and surface α5 GABAARs. Functional studies showed that chronic α5 NAM treatment did not alter inhibition or α5 NAM sensitivity. Surprisingly, chronic L6 exposure decreased surface levels of GluN2A and GluN2B subunits, concurrent with reduced NMDAR-mediated neuronal excitation as seen by faster synaptic decay rates and reduced glutamate-evoked calcium responses. Together, these results show that chronic in vitro treatment with an α5 NAM leads to subtle homeostatic changes in inhibitory and excitatory synapses that suggest an overall dampening of excitability.

The Yin and Yang of GABAergic and Glutamatergic Synaptic Plasticity: Opposites in Balance by Crosstalking Mechanisms.

Synaptic plasticity is a critical process that regulates neuronal activity by allowing neurons to adjust their synaptic strength in response to changes in activity. Despite the high proximity of excitatory glutamatergic and inhibitory GABAergic postsynaptic zones and their functional integration within dendritic regions, concurrent plasticity has historically been underassessed. Growing evidence for pathological disruptions in the excitation and inhibition (E/I) balance in neurological and neurodevelopmental disorders indicates the need for an improved, more "holistic" understanding of synaptic interplay. There continues to be a long-standing focus on the persistent strengthening of excitation (excitatory long-term potentiation; eLTP) and its role in learning and memory, although the importance of inhibitory long-term potentiation (iLTP) and depression (iLTD) has become increasingly apparent. Emerging evidence further points to a dynamic dialogue between excitatory and inhibitory synapses, but much remains to be understood regarding the mechanisms and extent of this exchange. In this mini-review, we explore the role calcium signaling and synaptic crosstalk play in regulating postsynaptic plasticity and neuronal excitability. We examine current knowledge on GABAergic and glutamatergic synapse responses to perturbances in activity, with a focus on postsynaptic plasticity induced by short-term pharmacological treatments which act to either enhance or reduce neuronal excitability via ionotropic receptor regulation in neuronal culture. To delve deeper into potential mechanisms of synaptic crosstalk, we discuss the influence of synaptic activity on key regulatory proteins, including kinases, phosphatases, and synaptic structural/scaffolding proteins. Finally, we briefly suggest avenues for future research to better understand the crosstalk between glutamatergic and GABAergic synapses.

Sustained treatment with an α5 GABA A receptor negative allosteric modulator delays excitatory circuit development while maintaining GABAergic neurotransmission.

α5 subunit GABA type A receptor (GABAAR) preferring negative allosteric modulators (NAMs) are cognitive enhancers with antidepressant-like effects. α5-NAM success in treating mouse models of neurodevelopmental disorders with excessive inhibition have led to Phase 2 clinical trials for Down syndrome. Despite in vivo efficacy, no study has examined the effects of continued α5-NAM treatment on inhibitory and excitatory synapse plasticity to identify mechanisms of action. Here we used L-655,708, an imidazobenzodiazepine that acts as a highly selective but weak α5-NAM, to investigate the impact of sustained treatment on hippocampal neuron synapse and dendrite development. We show that 2-day pharmacological reduction of α5-GABAAR signaling from DIV12-14, when GABAARs contribute to depolarization, delays dendritic spine maturation and the NMDA receptor (NMDAR) GluN2B/GluN2A developmental shift. In contrast, α5-NAM treatment from DIV19-21, when hyperpolarizing GABAAR signaling predominates, enhances surface synaptic GluN2A while decreasing GluN2B. Despite changes in NMDAR subtype surface levels and localization, total levels of key excitatory synapse proteins were largely unchanged, and mEPSCs were unaltered. Importantly, 2-day α5-NAM treatment does not alter the total surface levels or distribution of α5-GABAARs, reduce the gephyrin inhibitory synaptic scaffold, or impair phasic or tonic inhibition. Furthermore, α5-NAM inhibition of the GABAAR tonic current in mature neurons is maintained after 2-day α5-NAM treatment, suggesting reduced tolerance liability, in contrast to other clinically relevant GABAAR-targeting drugs such as benzodiazepines. Together, these results show that α5-GABAARs contribute to dendritic spine maturation and excitatory synapse development via a NMDAR dependent mechanism without perturbing overall neuronal excitability.

Anterograde trafficking signals in GABAA subunits are required for functional expression.

Pentameric GABAA receptors are composed from 19 possible subunits. The GABAA ß subunit is unique because the ß1 and ß3 subunits can assemble and traffic to the cell surface as homomers, whereas most of the other subunits, including ß2, are heteromers. The intracellular domain (ICD) of the GABAA subunits has been implicated in targeting and clustering GABAA receptors at the plasma membrane. Here, we sought to test whether and how the ICD is involved in functional expression of the ß3 subunit. Since θ is the most homologous to ß but does not form homomers, we created two reciprocal chimeric subunits, swapping the ICD between the ß3 and θ subunits, and expressed them in HEK293 cells. Surface expression was detected with immunofluorescence and functional expression was quantified using whole-cell patch-clamp recording with fast perfusion. Results indicate that, unlike ß3, neither the ß3/θIC nor the θ/ß3IC chimera can traffic to the plasma membrane when expressed alone; however, when expressed in combination with either wild-type α3 or ß3, the ß3/θIC chimera was functionally expressed. This suggests that the ICD of α3 and ß3 each contain essential anterograde trafficking signals that are required to overcome ER retention of assembled GABAA homo- or heteropentamers.