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. 2015 Sep 10:303:323-37.
doi: 10.1016/j.neuroscience.2015.07.009. Epub 2015 Jul 8.

Ultrastructural evidence for synaptic contacts between cortical noradrenergic afferents and endocannabinoid-synthesizing post-synaptic neurons

B A S Reyes  1 , N A Heldt  2 , K Mackie  3 , E J Van Bockstaele  2
Affiliations

Ultrastructural evidence for synaptic contacts between cortical noradrenergic afferents and endocannabinoid-synthesizing post-synaptic neurons

B A S Reyes et al. Neuroscience. .

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.

Keywords: cannabinoid receptor type 1; diacylglycerol lipase; dopamine-β-hydroxylase; electron microscopy; norepinephrine transporter.

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Figures

Figure 1
Figure 1
Confocal fluorescence photomicrographs showing immunolabeling of diacylglycerol lipase-α (DGL-α) in the frontal cortex with respect to noradrenergic afferents as visualized by the presence of dopamine-β-hydroxylase (DβH) or the norepinephrine transporter (NET) (Panels A-F). A. DβH was detected using a rhodamine isothiocyanate-conjugated secondary antibody (TRITC donkey anti-mouse; red). B. DGL-α was detected using a fluorescein isothiocyanate-conjugated secondary antibody (FITC donkey anti-rabbit; green). C. Merged image of panels A and B. The inset shows a higher magnification view of the area outlined by the boxed region showing close associations between DβH (arrowhead) and DGL-α (arrow). D. NET (red) was detected using a TRITC donkey anti-mouse secondary antibody. E. DGL-α (green) was detected using a FITC donkey anti-rabbit secondary antibody. F. Merged image of panels D and E. The inset shows a higher magnification view of the area outlined by the boxed region showing close associations of NET (arrowhead) with DGL-α (arrow). Scale bars = 100 μm.
Figure 2
Figure 2
Electron micrographs showing immunoperoxidase labeling for dopamine-β-hydroxylase (DβH) in an axon terminal and immunogold-silver labeling for diacylglycerol lipase-α (DGL-α) in a dendrite in the frontal cortex. A-B. Adjacent sections depicting a DβH-t that forms a symmetric synapse (arrowheads) with a DGL-d. Arrows point to immunogold-silver labeling. m: mitochondria; ut: unlabeled terminal. Scale bars = 0.50 μm.
Figure 3
Figure 3
Electron micrographs showing immunoperoxidase labeling for the norepinephrine transporter (NET) in axon terminals and immunogold-silver labeling for diacylglycerol lipase-α (DGL-α) in dendrites in the the frontal cortex. A. Two NET-labeled axon terminals (NET-t) are shown in the same field as a DGL-α-labeled dendrite (DGL-d). B. Immunoperoxidase labeling can be seen in a NET-immunoreactive axon terminal (NET-t) contacting (double arrows) a DGL-d. C. Dense immunoperoxidase labeling can be seen in a NET-t that forms a symmetric synapse (double arrowheads) with a DGL-d. Also shown are two unlabeled axon terminals (ut) that form a contact with the same DGL-d. D. A dense NET-t contacts (arrowhead) a DGL-d. Black arrows point to immunogold-silver labeling for DGL-α throughout. m: mitochondria; ut: unlabeled terminal. Scale bars = 0.50 μm
Figure 4
Figure 4
Electron micrographs showing immunoperoxidase labeling for the norepinephrine transporter (NET) in axon terminals and immunogold-silver labeling for diacylglycerol lipase-α (DGL-α) in the frontal cortex (FC). A-B. NET-labeled axon terminals (NET-t) are directly contacting perikarya containing immunogold silver labeling for DGL-α (DGL-s). Arrows point to immunogold-silver labeling. Scale bar, 0.50 μm.
Figure 5
Figure 5
Confocal fluorescence micrographs showing cannabinoid receptor type 1 (CB1r, A), 1,2 diacylglycerol lipase-α (DGL-α, B) and dopamine-β-hydroxylase (DβH, C) in the rat frontal cortex (FC). CB1r was detected using a rhodamine isothiocyanate-conjugated secondary antibody (TRITC donkey anti-guinea pig; red) and DGL-α was detected using a fluorescein isothiocyanate-conjugated secondary antibody (FITC-donkey anti-rabbit; green). DβH-labeling was detected using Cy5 donkey anti-mouse secondary antibody (blue). CB1r and DβH appeared punctate throughout. The merged image (D) shows co-localization of CB1r and DβH in the same processes (white thin arrows) and in close proximity to DGL-α (arrowhead) in the FC. Thick white arrows indicate single-labeling (CB1r, DGL or DβH). The inset shows a higher magnification view of the area outlined by the boxed region showing close associations between DβH/CB1r and DGL-α. Scale bars, 100 μm.
Figure 6
Figure 6
Schematic diagram depicting proposed mechanisms underlying modulation of NE afferents by the eCB system. Noradrenergic axon terminals that express CB1r may co-localize inhibitory transmitters, such as gamma-amino butyric acid (Hajos et al., 2000; Ranganathan and D'Souza, 2006), or may co-localize excitatory transmitters such as glutamate (Katona et al., 2006; Kawamura et al., 2006). Excitatory ionotropic or G protein-coupled receptor activation (via excitatory amino acid, alpha1 or beta-adrenergic receptors), stimulate calcium production and engage intracellular pathways that contribute to local eCB synthesis and release. Following activation, 2-AG may diffuse into the synapse and bind to CB1r located on noradrenergic terminals that co-express inhibitory or excitatory transmitters. Functional consequences of eCB modulation of cortical afferents include inhibition of inhibitory neurotransmitter release as cannabinoids have been shown to reduce inhibitory neurotransmitter efflux (Trettel and Levine, 2002; Zamberletti et al., 2014) and increased excitatory neurotransmitter release (Galanopoulos et al., 2011). Once NE is released, the possibility exists that it could bind to pre-synaptically distributed alpha2 adrenergic receptors that are coupled to inhibitory Gi proteins, and through their activation, further tonically inhibit NE. In addition to alterations in NE, retrograde suppression of inhibitory neurotransmitter release and increases in excitatory neurotransmitter release in FC may affect synaptic integration and cortical neuronal activity. Furthermore, it is also feasible that NE activates the beta adrenergic receptor thereby increasing neuronal activity by activation of voltage-gated calcium channels (Yu et al., 2015) thereby increasing calcium influx resulting in neuronal depolarization and consequently increased 2-AG production.
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