Excitatory Amino Acid Transporters and Presynaptic Afferent Metabotropic Glutamate Receptors (mGluRs) Limit Synaptic Currents via Control of Presynaptic Calcium and Extracellular Glutamate Kinetics
Authors: Martinez Diana , Hermann Gerlinda E , Hasser Eileen M , Rogers Richard C , Kline David D 
Published: June 2021

The nucleus tractus solitarii (nTS) is the first central integration site for visceral reflexes including the chemoafferent reflex. Sensory afferent signals are transmitted to the nTS via release of glutamate. Extracellular glutamate is removed from the synaptic cleft via Excitatory Amino Acid Transporters (EAATs), thus limiting glutamate receptor activation. We have shown that EAAT block with the general antagonist DL‐threo‐β‐Benzyloxyaspartic acid (DL‐TBOA) reduced the amplitude of afferent (TS)‐evoked EPSC (TS‐EPSC). Interestingly, both activation of Group II/III mGluRs and chronic intermittent hypoxia (CIH), a model of obstructive sleep apnea, also decrease TS‐EPSC amplitude. The present study sought to identify the contribution of presynaptic Group II/III mGluRs to the decrease in TS‐EPSC amplitude following EAAT block and CIH. To accomplish this goal, we used a combination of electrophysiology, novel live cell imaging with a fluorescent intensity based glutamate reporter (iGluSnFR), and presynaptic afferent Ca2+ imaging. Male Sprague‐Dawley rats (3–6 weeks) underwent 10 days normoxia (Norm, 21% O2) or CIH (alternating 21% O2 and 6% O2, 8h/day) followed by brain slice generation. Prior to slice generation a subset of rats received either nTS microinjection of AAV‐hSyn‐iGluSnFR followed by 3 weeks of expression, or nodose ganglia injection of the Ca2+ tracer Calcium Green Dextran‐1 and allowed 5 days for anterograde nTS transport. TS‐EPSC amplitude in monosynaptic nTS neurons, extracellular glutamate concentration [[Glu]e, [change in fluorescence intensity, (DF/F)%]] or presynaptic Ca2+ [(DF/F)%] was monitored during 20 Hz stimulation. Responses were examined in aCSF, EAAT block (TFB‐TBOA, 500 nM) alone, and during block of EAAT and Group II/III receptors (eGlu and MSOP 200 μM). In Norm‐exposed rats, EAAT block reduced TS‐EPSC amplitude, and antagonism of mGluR‐II/III limited the EAAT‐induced depression. To confirm a presynaptic mechanism, presynaptic Ca2+ was measured; Ca2+ entry decreased after EAAT block but was restored to control levels after EAAT‐mGluR‐II/III block. To examine the consequence of reduced afferent Ca2+, which is responsible for attenuated TS‐EPSCs, we examined [Glu]e. EAAT block increased [Glu]e and additional mGluR‐II/III block further enhanced [Glu]e and dramatically slowed its decay kinetics. In contrast to Norm, in CIH‐exposed rats, TS‐EPSC amplitude was decreased and EAAT block increased TS‐EPSC amplitude. Following CIH, in aCSF afferent‐evoked Glu release was reduced compared to Norm. EAAT block produced only a modest increase in [Glu]e, further mGluR‐II/III block dramatically enhanced [Glu]e when compared to EAAT block alone, and slowed its removal. These data suggest that EAATs regulate [Glu]e, glutamate kinetics and the amplitude of TS‐EPSCs through mGluR Group II/III. CIH reduces EAAT influence on synaptic activity and mGluRs contribute to the reduced TS‐EPSC.Support or Funding InformationSupported by RO1 HL128454This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.