Research ReportPresynaptic D1 heteroreceptors and mGlu autoreceptors act at individual cortical release sites to modify glutamate release

- Release from (1 µm) synaptic sites identified in cortical cultures was imaged by two methods.
- iGluSnFR is more sensitive than sypHx2 to detect electrically stimulated glutamate release.
- The number of release sites was increased by D1 receptor and mGluR2/3 activation reduced the number of active sites.

The aim of this work was to study release of glutamic acid (GLU) from one-axon terminal or bouton at-a-time using cortical neurons grown in vitro to study the effect of presynaptic auto- and heteroreceptor stimulation. Neurons were infected with release reporters SypHx2 or iGluSnFR at 7 or 3 days-in-vitro (DIV) respectively. At 13-15 DIV single synaptic boutons were identified from images obtained from a confocal scanning microscope before and after field electrical stimulation. We further stimulated release by raising intracellular levels of cAMP with forskolin (10 µM). Forskolin-mediated effects were dependent on protein kinase A (PKA) and did not result from an increase in endocytosis, but rather from an increase in the size of the vesicle readily releasable pool. Once iGluSnFR was confirmed as more sensitive than SypHx2, it was used to study the participation of presynaptic auto- and heteroreceptors on GLU release. Although most receptor agonizts (carbamylcholine, nicotine, dopamine D2, BDNF) did not affect electrically stimulated GLU release, a significant increase was observed in the presence of metabotropic D1/D5 heteroreceptor agonist (SKF38393 10 µM) that was reversed by PKA inhibitors. Interestingly, stimulation of group II metabotropic mGLU2/3 autoreceptors (LY379268 50 nM) induced a decrease in GLU release that was reversed by the specific mGLU2/3 receptor antagonist (LY341495 1 µM) and also by PKA inhibitors (KT5720 200 nM and PKI14-22 400 nM). These changes in release probability at individual release sites suggest another level of control of the distribution of transmitter substances in cortical tissue.