Abstracts

Rationale: Hippocampal GABAergic synapses are typically associated with inhibitory function. However, our previous study demonstrated that individual interneurons could evoke GABA_A responses with variable reversal potentials (E_GABA) onto the same pyramidal neuron. This was observed by puffing glutamate onto GABAergic interneurons, and simultaneously using whole-cell patch-clamp to record the E_GABA from neighboring cell. Notably, 12.5% interneurons exhibited E_GABA values more depolarized than the resting membrane potential, indicating a potentially excitatory GABA_A effect. Despite this observation, the extent and functional significance of E_GABA heterogeneity remain poorly characterized. Additionally, glutamate puffing lacks temporal precision and is labor intensive.

Methods: To overcome these limitations, we combined optogenetics with whole-cell patch-clamp recordings to assess how individual interneurons influence nearby pyramidal neurons in organotypic hippocampal slice cultures (OHSC), an ex vivo model in which slicing-induced trauma leads to spontaneous epileptiform activity. To selectively target interneurons, we delivered a Cre-dependent AAV encoding st-CoChR, an excitatory light-gated cation channel that can be activated at 920nm, to OHSCs from P6-P8 DLX-Cre pups on the day of preparation. Recordings were performed at or after day in vitro 7.

Results: We first validated st-CoChR activation by optically stimulating and simultaneously recording from st-CoChR-expressing interneurons. Pulses of 5 ms, 10 ms, and 20 ms at 920 nm were tested; a 5 ms pulse reliably evoked single action potentials (n=3 cells). In separate experiments, we optically stimulated st-CoChR-expressing interneurons while recording from neighboring st-CoChR-negative pyramidal neurons. These recordings revealed robust IPSPs with variable amplitudes, suggesting heterogeneity in inhibitory synaptic strength and/or E_GABA.

Conclusions: Future experiments will focus on stimulating individual st-CoChR-expressing interneurons in CA1 and DG while recording IPSPs and E_GABA from neighboring cells using gramicidin perforated-patch techniques, which preserve intracellular chloride concentrations for more accurate E_GABA measurements. To distinguish developmental effects from those driven by seizures, we will compare inhibitory connectivity in CA1 and DG at DIV2 and DIV10, with a subset of slices treated with kynurenic acid to suppress epileptiform activity. These studies aim to characterize the heterogeneity of inhibitory synapses and how seizures alters inhibitory signaling. Understanding this variability may inform strategies to restore inhibitory function in the epileptic brain.

Funding: NS116852, NS112538