A novel mechanism strengthens GABAergic inhibition in the hypothalamic PVN in proportion to the level of synaptic L‐glutamate spillover

Abstract

Perhaps the most robust property of pre‐sympathetic neurons (PSNs) of the hypothalamic paraventricular nucleus (PVN) in unstressed animals is their state of near quiescence. Although discharge quiescence has been strongly linked to the dominance of GABAergic synaptic inhibition, mechanisms that govern the strength of local GABAergic inhibition remain to be fully elucidated. Here, whole cell patch‐clamp recordings were made from identified parvocellular PVN neurons in brain slices and GABAergic inhibitory postsynaptic currents (IPSCs) were evoked by electrical stimuli delivered to the surrounding peri‐nuclear zone. To investigate the impact of GABA synthesis and vesicular packaging on GABAergic IPSC amplitude, we compared eIPSC amplitude before and after bath application of the GABA synthetic substrate L‐glutamate (L‐Glu; 100 μM, 10 min). Within 3–5 minutes of L‐Glu washout, eIPSC amplitude suddenly and significantly increased (+23 ± 6%; P<0.002). This response was unaffected by selective inhibition of glial L‐Glu uptake with dihydrokainate (DHK; 250 μM), but was nearly abolished (−77 ± 8.5%) by DL‐threo‐β‐benzyloxyaspartic acid (TBOA; 100 μM), a pan‐specific inhibitor of L‐glutamate transporters, suggesting that strengthening of GABAergic inhibition by L‐Glu depends on uptake by the neuronal L‐Glu transporter EAAT3. Similar results were obtained when local synaptic activity was increased by bath perfusion with high K+ (10 mM) aCSF. In current‐clamp recordings of spontaneous firing, high bath K+ caused a delayed reduction of discharge (1.58 ± 0.59Hz to 1.18 ± 0.51Hz; P<0.03) that was not observed when depolarizing current was injected to mimic the spiking response to high K+. These findings suggest that GABAergic IPSC strengthening is sufficient to reduce PVN neuronal discharge. High frequency stimulation‐induced GABAergic synaptic depression/vesicular depletion revealed that exposure to bath L‐Glu did not increase quantal content (i.e., the number of GABA vesicles released during each IPSC), but enhance recovery of IPSC amplitude by +36 pA within 10 s. These data indicate that bath L‐Glu selectively increases the number of GABA molecules contained within each GABA vesicle. In anesthetized rats (n=6), acute blockade of GABA‐A receptors in the PVN with picrotoxin (PTX) increased renal sympathetic nerve activity (+23%). Prior PVN injection of L‐Glu greatly amplified the PTX response (+72%), which is consistent with increased functional inhibitory tonus. Collectively, our findings indicate that GABA synaptic vesicles are “over‐filled” when GABA is synthesized from L‐Glu under conditions of heightened L‐Glu availability. This process depends on EAAT3‐mediated L‐Glu uptake, likely into GABA terminals. As over‐filled vesicles transition to the readily releasable pool, the amplitude of IPSCs increases, allowing synaptic inhibitory tonus to increase in proportion to the level of synaptic L‐Glu spillover. This local mechanism is postulated to help prevent neuronal hyper‐activation under conditions of heightened glutamatergic excitation.Support or Funding InformationNIH HL088052 (GMT)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.