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.

Cortical adrenoceptor expression, function and adaptation under conditions of cannabinoid receptor deletion

“…Immunoperoxidase detection of CB1 receptor antibody was conducted in tissue sections obtained from mice with CB1 receptor deletion in parallel with the tissue sections obtained from the wild-type mice. CB1 receptor immunoperoxidase labeling was detected in multiple brain regions in WT mice including the cerebral cortex and hippocampus (not shown) consistent with previous studies (Katona et al, 2006; Scavone et al, 2010; Fitzgerald et al, 2012; Cathel et al, 2014; Reyes et al, 2015). Furthermore, the specificity of CB1 receptor was also demonstrated using immunoblotting where a protein of the expected molecular size was detected (Suarez et al, 2008).…”

“…Tissue sections obtained from the rat forebrain incubated in this solution did not exhibit CB1 receptor immunoreactivity compared with tissue sections in which standard immunohistochemistry was performed. In addition, preabsorption experiment was also performed using Western blot analysis in which CB1 receptor immunoreactivity was also abolished (Scavone et al, 2010; Reyes et al, 2015). Tissue sections obtained from the rat forebrain incubated in this solution did not exhibit CB1 receptor immunoreactivity compared with tissue sections in which standard immunohistochemistry was performed.…”

“…Tissue sections obtained from the rat forebrain incubated in this solution did not exhibit CB1 receptor immunoreactivity compared with tissue sections in which standard immunohistochemistry was performed. In addition, preabsorption experiment was also performed using Western blot analysis in which CB1 receptor immunoreactivity was also abolished (Scavone et al, 2010; Reyes et al, 2015). The monoclonal antibody against HA-α2A-adrenoceptor was generated in mouse against the 12-amino-acid peptide CYPYDVPDYASL The specificity of the TH antibody has been examined by preabsorption of the antibody with a high concentration of TH (Van Bockstaele and Pickel, 1993).…”

“…Another caveat associated with the procedure is the specificity of immunogold-silver labeling. This was controlled by quantifying profiles that contained a minimum of two immunogold-silver particles (Carvalho et al, 2010; Cathel et al, 2014; Oropeza et al, 2007; Reyes et al, 2006; Reyes et al, 2015; Scavone et al, 2010; Van Bockstaele and Pickel, 1993). …”

Cannabis: A Treasure Trove or Pandora's Box?

“…CB 1 Rs are also present in noradrenergic varicose terminals of the frontal cortex [748,749]. The source of the endocannabinoid 2-AG to activate monoaminergic fibres in the frontal cortex is likely the cortical neurons [750], thus retrograde endocannabinoid control is also relevant for monoamines. CB 1 R activation inhibits the electrically and chemically evoked release of serotonin and noradrenaline in the cerebral cortex including the frontal cortex and the hippocampus [396,722,751,752].…”