VGluT3-expressing CCK-positive basket cells construct invaginating synapses enriched with endocannabinoid signaling proteins in particular cortical and cortex-like amygdaloid regions of mouse brains

Abstract

Invaginating synapses in the basal amygdala are a unique type of GABAergic synapses equipped with molecular-anatomical organization specialized for 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid signaling. Cholecystokinin (CCK)-positive basket cell terminals protrude into pyramidal cell somata and form invaginating synapses, where apposing presynaptic and postsynaptic elements are highly loaded with cannabinoid receptor CB 1 or 2-AG synthetic enzyme diacylglycerol lipase-α (DGLα), respectively. The present study scrutinized their neurochemical and neuroanatomical phenotypes in adult mouse telencephalon. In the basal amygdala, vesicular glu-tamate transporter-3 (VGluT3) was transcribed in one-fourth of CB 1 -expressing GABAergic interneurons. The majority of VGluT3-positive CB 1 -expressing basket cell terminals apposed DGLα clusters, whereas the majority of VGluT3-negative ones did not. Importantly, VGluT3-positive basket cell terminals selectively constructed invaginating synapses. GABA A receptors accumulated on the postsynaptic membrane of invaginating synapses, whereas metabotropic glutamate receptor-5 (mGluR 5) was widely distributed on the somatodendritic surface of pyramidal cells. Moreover, CCK 2 receptor (CCK 2 R) was highly transcribed in pyramidal cells. In cortical regions, pyramidal cells equipped with such VGluT3/CB 1 /DGLα-accumulated invaginating synapses were found at variable frequencies depending on the subregions. Therefore, in addition to extreme proximity of CB 1 - and DGLα-loaded presynaptic and postsynaptic elements, tripartite transmitter phenotype of GABA/glutamate/CCK is the common neurochemical feature of invaginating synapses, suggesting that glutamate, CCK, or both can promote 2-AG synthesis through activating Gα q/11 protein-coupled mGluR 5 and CCK 2 R. These molecular configurations led us to hypothesize that invaginating synapses might be evolved to provide some specific mechanisms of induction, regulation, and cooperativity for 2-AG-mediated retrograde signaling in particular cortical and cortex-like amygdaloid regions.