# The Role of Calcium-Permeable Kainate and AMPA Receptors in the Leading Reaction of GABAergic Neurons to Excitation

## TL;DR

This study identifies a proactive GABAergic inhibition mechanism in the hippocampus that prevents hyperexcitability by using calcium-permeable kainate and AMPA receptors.

## Contribution

The paper discovers a subgroup of GABAergic neurons with calcium-permeable receptors that mediate early GABA release to prevent excitatory runaway activity.

## Key findings

- GABAergic neurons show earlier calcium responses to excitatory stimuli compared to glutamatergic neurons.
- CP-KARs on GABAergic neurons are responsible for the delayed excitation of glutamatergic neurons.
- Early-responding neurons in hippocampal slices are likely GABAergic and protect the network from hyperexcitation.

## Abstract

Excitable neurons are intrinsically capable of firing action potentials (AP), yet a state of hyperexcitability is prevented in the central nervous system by powerful GABAergic inhibition. For this inhibition to be effective, it must occur before excitatory signals can initiate runaway activity, implying the existence of a proactive control system. To test for such proactive inhibition, we used Ca2+ imaging and patch-clamp recording to measure how hippocampal neurons respond to depolarization and glutamatergic agonists. In mature hippocampal cultures (14 days in vitro (DIV)) and acute brain slices from two-month-old rats, neurons exhibited non-simultaneous responses to various excitatory stimuli, including KCl, NH4Cl, forskolin, domoic acid, and glutamate. We observed that the Ca2+ rise occurred significantly earlier in GABAergic neurons than in glutamatergic neurons. This delay in glutamatergic neurons was abolished by GABA(A) receptor inhibitors, suggesting a mechanism of preliminary γ-aminobutyric acid (GABA) release. We further found that these early-responding GABAergic neurons express calcium-permeable kainate and AMPA receptors (CP-KARs and CP-AMPARs). Application of domoic acid induced an immediate Ca2+ increase in neurons expressing these receptors, but a delayed response in others. Crucially, when domoic acid was applied in the presence of the AMPA receptor inhibitors NBQX or GYKI-52466, the response delay in glutamatergic neurons was significantly prolonged. This confirms that CP-KARs on GABAergic neurons are responsible for the delayed excitation of glutamatergic neurons. In hippocampal slices from two-month-old rats, depolarization with 50 mM KCl revealed two distinct neuronal populations based on their calcium dynamics: a majority group (presumably glutamatergic) exhibited fluctuating Ca2+ signals, while a minority (presumably GABAergic) showed a steady, advancing increase in [Ca2+]i. This distinction was reinforced by the application of domoic acid. The “advancing-response” neurons reacted to domoic acid with a similar prompt increase, whereas the “fluctuating-response” neurons displayed an even more delayed and fluctuating reaction (80 s delay). Therefore, we identify a subgroup of hippocampal neurons—in both slices and cultures—that respond to depolarization and domoic acid with an early [Ca2+]i signal. Consistent with our data from cultures, we conclude these early-responding neurons are GABAergic. Their early GABA release directly explains the delayed Ca2+ response observed in glutamatergic neurons. We propose that this proactive mechanism, mediated by CP-KARs on GABAergic neurons, is a primary means of protecting the network from hyperexcitation. Furthermore, the activity of these CP-KAR-expressing neurons is itself regulated by GABAergic neurons containing CP-AMPARs.