Glutamate strengthening of GABAergic inhibition in the hypothalamic PVN: Dependence on EAAT3‐mediated Glu uptake into local GABA terminals

Abstract

In unstressed animals, pre‐sympathetic neurons (PSNs) of the hypothalamic paraventricular nucleus (PVN) are held in a state of near discharge quiescence by the dominance of GABAergic inhibition. In brain slice studies we found that the amplitude of PSN GABAergic inhibitory postsynaptic currents (IPSCs) is increased by elevated ECF L‐glutamate (Glu). This Glu‐GABA strengthening (GGS) phenomenon, originally described in the hippocampus, can be elicited in PVN PSNs both by exposure to bath Glu (100 μM) or high K+ (+10 mM) for 10 min. PVN GGS is (1) mediated by a presynaptic mechanism that increases unitary quantal size (more GABA molecules/vesicle), (2) is unaffected by inhibition of glial GLT1‐mediated Glu uptake, and (3) is prevented by global inhibition of Glu uptake. Collectively, these findings indicate that PVN GGS depends on Glu uptake by the neuronal Glu transporter EAAT3. Importantly, bath Glu elicits a delayed GABA‐AR‐mediated suppression of PSN firing, indicating that the Glu‐induced increase of IPSC amplitude (i.e., GGS) is sufficient to regulate PSN discharge quiescence. Moreover, in vivo studies indicate that PVN Glu enhances the increase of renal SNA caused by local blockade of GABA‐AR, consistent with elevated Glu increasing functional GABAergic inhibitory tone in the PVN. Here we performed PVN immunohistochemistry and found EAAT3 localized diffusely within somatodendritic compartments and densely in puncta resembling synaptic terminals. EAAT3+ puncta co‐localized with GAD65 punctate staining, consistent with EAAT3 expression on local GABAergic synaptic terminals. To test dependence of GGS on EAAT3 uptake of Glu specifically by PVN GABA terminals, we used a CRISPR/Cas9 gene editing strategy in which spCas9 was expressed in a Cre‐dependent manner amongst vGAT+ (GABAergic) neurons. To knockdown (KD) EAAT3 specifically amongst GABAergic inputs to PVN, AAV1‐CAG‐EAAT3:sgRNA‐GFP was nanoinjected (400 nl) into the surrounding peri‐nuclear zone (PNZ). Western blot analysis of tissue punches (Cntrl: n=10, CRISPR EAAT3: n=12) revealed a nearly 40% loss of EAAT3 in PVN. Given that EAAT3 amongst non‐GABAergic (i.e., glutamatergic) PVN neurons were not targeted by our CRISPR/Cas9 strategy, loss of EAAT3 specifically amongst PVN‐projecting GABAergic PNZ neurons would likely have been much greater than ~40%. Crucially, strengthening of PNZ evoked GABAergic IPSCs by bath Glu was effectively abolished (before: −238 ± 10 pA, after: −226 ± 12 pA; n=3) by CRISPR EAAT3 KD amongst PNZ GABAergic neurons. Results indicate that EAAT3‐mediated Glu uptake into PVN GABA terminals actively defends and restores PSN quiescence following acute glutamatergic excitation.Support or Funding InformationNIH HL088052 (GMT)