title: Ultrastructural evidence for synaptic contacts between cortical noradrenergic afferents and endocannabinoid-synthesizing post-synaptic neurons.
abstract: Endocannabinoids (eCBs) are involved in a myriad of physiological processes that are mediated through the activation of cannabinoid receptors, which are ubiquitously distributed within the nervous system. One neurochemical target at which cannabinoids interact to have global effects on behavior is brain noradrenergic circuitry. We, and others, have previously shown that CB type 1 receptors (CB1r) are positioned to pre-synaptically modulate norepinephrine (NE) release in the rat frontal cortex (FC). Diacylglycerol lipase (DGL) is a key enzyme in the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). While DGL-α is expressed in the FC in the rat brain, it is not known whether noradrenergic afferents target neurons expressing synthesizing enzymes for the endocannabinoid, 2-AG. In the present study, we employed high-resolution neuroanatomical approaches to better define cellular sites for interactions between noradrenergic afferents and FC neurons expressing DGL-α. Immunofluorescence microscopy showed close appositions between processes containing the norepinephrine transporter (NET) or dopamine-β-hydroxylase (DβH) and cortical neurons expressing DGL-α-immunoreactivity. Ultrastructural analysis using immunogold-silver labeling for DGL-α and immunoperoxidase labeling for NET or DβH confirmed that NET-labeled axon terminals were directly apposed to FC somata and dendritic processes that exhibited DGL-α-immunoreactivity. Finally, tissue sections were processed for immunohistochemical detection of DGL-α, CB1r and DβH. Triple label immunofluorescence revealed that CB1r and DβH were co-localized in common cellular profiles and these were in close association with DGL-α. Taken together, these data provide anatomical evidence for direct synaptic associations between noradrenergic afferents and cortical neurons exhibiting endocannabinoid synthesizing machinery.
authors.affiliation: Department of Pharmacology and Physiology, College of Medicine, Drexel University, Philadelphia, PA 19102, United States. Electronic address: Beverly.Reyes@drexelmed.edu.
authors.forename: B A S
authors.initials: BA
authors.lastname: Reyes
authors.affiliation: Department of Pharmacology and Physiology, College of Medicine, Drexel University, Philadelphia, PA 19102, United States.
authors.forename: N A
authors.initials: NA
authors.lastname: Heldt
authors.affiliation: Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN 47405, United States.
authors.forename: K
authors.initials: K
authors.lastname: Mackie
authors.affiliation: Department of Pharmacology and Physiology, College of Medicine, Drexel University, Philadelphia, PA 19102, United States.
authors.forename: E J
authors.initials: EJ
authors.lastname: Van Bockstaele
mesh_terms: D000818:Animals; D002540:Cerebral Cortex / Q000166:cytology*; D003712:Dendrites / Q000000981:diagnostic imaging / Q000378:metabolism; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism; D063388:Endocannabinoids / Q000378:metabolism*; D008071:Lipoprotein Lipase / Q000378:metabolism; D008297:Male; D046529:Microscopy, Electron, Transmission; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure*; D009638:Norepinephrine / Q000378:metabolism*; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000378:metabolism; D017684:Oncorhynchus kisutch; D017729:Presynaptic Terminals / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D043884:Receptor, Cannabinoid, CB1 / Q000378:metabolism; D013569:Synapses / Q000378:metabolism / Q000648:ultrastructure*; D014463:Ultrasonography
chemical_list: D063388:Endocannabinoids; D050484:Norepinephrine Plasma Membrane Transport Proteins; D043884:Receptor, Cannabinoid, CB1; D004299:Dopamine beta-Hydroxylase; D008071:Lipoprotein Lipase; D009638:Norepinephrine
keywords: cannabinoid receptor type 1; diacylglycerol lipase; dopamine-β-hydroxylase; electron microscopy; norepinephrine transporter