Glutamate spillover dynamically strengthens GABAergic synaptic inhibition of the hypothalamic paraventricular nucleus

Abstract

The hypothalamic paraventricular nucleus (PVN) is strongly inhibited by γ-aminobutyric acid (GABA) from the surrounding peri-nuclear zone (PNZ). Because glutamate mediates fast excitatory transmission and is substrate for GABA synthesis, we tested its capacity to dynamically strengthen GABA inhibition. In PVN slices from male mice, bath glutamate applied during ionotropic glutamate receptor blockade, increased PNZ-evoked inhibitory postsynaptic currents (eIPSCs) without affecting GABA-A receptor agonist currents or single channel conductance, implicating a presynaptic mechanism(s). Consistent with this interpretation, bath glutamate failed to strengthen IPSCs during pharmacological saturation of GABA-A receptors. Presynaptic analyses revealed that glutamate did not affect paired-pulse ratio, peak eIPSC variability, GABA vesicle recycling speed or readily releasable pool (RRP) size. Notably, glutamate-GABA strengthening (GGS) was unaffected by metabotropic glutamate receptor blockade and graded external Ca2+when normalized to baseline amplitude. GGS was prevented by pan- but not glial-specific inhibition of glutamate uptake and by inhibition of glutamic acid decarboxylase (GAD), indicating reliance on glutamate uptake by neuronal excitatory amino acid transporter 3 (EAAT3) and enzymatic conversion of glutamate to GABA. EAAT3 immunoreactivity was strongly localization to presumptive PVN GABA terminals. High bath K+also induced GGS, which was prevented by glutamate vesicle depletion, indicating that synaptic glutamate release strengthens PVN GABA inhibition. GGS suppressed PVN cell firing, indicating its functional significance. In sum, PVN GGS buffers neuronal excitation by apparent “over-filling” of vesicles with GABA synthesized from synaptically released glutamate. We posit that GGS protects against sustained PVN excitation and excitotoxicity whilst potentially aiding stress adaptation and habituation.Significance StatementHere we found that PVN GABA inhibition, which dominates glutamatergic excitation in the unstressed state, is rapidly strengthened by increased synaptic glutamate release. This negative-feedback homeostatic response reflects an apparent quantal mechanism with pharmacological inhibition studies implicating neuronal glutamate uptake by EAAT3 andde novoenzymatic synthesis of GABA, culminating in apparent GABA vesicle “over-filling”. This glutamate-GABA strengthening response restrains PVN neuronal discharge responses with potential to mitigate behavioral, endocrine, and autonomic responses to glutamatergic excitation during stress.