Cannabinoid receptors are localized to noradrenergic axon terminals in the rat frontal cortex

Abstract

Cannabinoid agonists exert complex actions on modulatory neurotransmitters involved in attention and cognition. Previous studies have demonstrated that acute systemic administration of the synthetic cannabinoid agonist, WIN 55,212-2, increases norepinephrine efflux in the rat frontal cortex. In an effort to elucidate whether cannabinoid (CB1) receptors are positioned to presynaptically modulate norepinephrine release in the frontal cortex, immunocytochemical detection of the CB1 receptor and the catecholamine-synthesizing enzyme dopamine-beta-hydroxylase (DbetaH) was performed using confocal immunofluorescence microscopy and immunoelectron microscopy in rat brain. Fluorescence microscopy analysis of dually labeled tissue sections from the frontal cortex indicated that individual axonal processes exhibited both CB1 receptor and DbetaH immunoreactivities. Ultrastructural analysis confirmed that one-third of axon terminals containing CB1 immunolabeling also exhibited DbetaH labeling. Cortical neurons were also found to be targeted by separately labeled CB1- and DbetaH-containing axon terminals. In conclusion, the present neuroanatomical data suggest that cortical norepinephrine release may be modulated, in part, by CB1 receptors that are presynaptically distributed on noradrenergic axon terminals.

Figure 1

A. Low magnification brightfield photomicrograph showing immunoperoxidase labeling for the CB1 receptor in the rat frontal cortex. The region denoted within the black box is shown at higher magnification in panel B. The asterisks denotes a blood vessel that can be identified at higher magnification in panel B. The frontal cortical area indicated by the black box, defined as the infralimbic cortex, was the region sampled for ultrastructural analysis. Arrows indicate dorsal (D) and medial (M) orientation of the tissue section. Bar = 100 μm. B. High magnification image of the same section shown in panel A showing CB1 immunoperoxidase labeling in the frontal cortex. CB1 receptor labeling is localized abundantly to varicose processes in layers II, III and VI of the rat frontal cortex. Arrows indicate labeled fibers and processes. In panels A and B, cc indicated the corpus callosum. C. High magnification darkfield illuminated photomicrograph showing the noradrenergic synthesizing enzyme dopamine-β-hydroxylase (DßH) in varicose processes in a separate section of tissue through the same region illustrated in panel A. Localization of DßH was conducted using immunoperoxidase detection. White arrows depict DßH-labeled beaded processes in the frontal cortex. Bar for B and C = 100 μm.

Figure 2

A–F: Confocal microscopy photomicrographs showing sections processed for the immunocytochemical detection of the catecholamine synthesizing enzyme, DßH (panel A, D) and CB1 receptors (panel B, E) in coronal sections from the infralimbic area of the frontal cortex. Abundant labeling in varicose processes was present in the frontal cortex for both antigens. Processes containing DßH alone can be seen at the long white arrows while fibers containing CB1 alone are shown at arrowheads. Panels C and F are merged images. Yellow labeling denotes co-localization of both CB1 and DßH in the same process. Magnification A–C: 20x, D–F: 40x oil G. Histogram analysis demonstrating the co-localization of DßH (red) and CB1 receptor (green). Intensity of labeling for DßH is seen on the x-axis (CH2-T1) and intensity of labeling for the CB1 receptor is on the y-axis (CH3-T2). Colors in the histogram represent pixel frequency and intensity.

Figure 3

Electron micrographs showing localization of CB1 and DßH in axon terminals (t) in the frontal cortex. A. Dense peroxidase labeling for CB1 can be identified in a cellular process (CB1-p) denoted at black arrows. The CB1-p is apposed to an unlabeled terminal (ut). Another ut nearby contacts a spine (sp). The DßH-t is apposed by a profile resembling a glial process that is immunoreactive for CB1. B. Co-localization of gold-silver labeling for DßH (arrowheads) and peroxidase labeling (indicated by long black arrows) for CB1 in the same axon terminal. The DßH + CB1 terminal is apposed to an unlabeled dendrite (ud). Several unlabeled terminals can be seen in the neuropil. C. Reversal of immunolabels indicates similar patterns of immunolabeling. Gold-silver labeling for the CB1 receptor (arrowheads) and peroxidase labeling for DßH (long black arrows) can be seen in the same terminal. Gold-silver labeling for the CB1 receptor shows localization (CB1-t) to the plasma membrane of the axon terminal. D. A CB1 receptor immunoperoxidase-labeled terminal and a DßH immunogold-labeled terminal converge (DßH-t) onto a common dendrite. m: mitochondria. ut: unlabeled terminal. sp: spine.

Figure 4

Schematic illustration depicting cellular associations of CB1 labeled terminals (CB1R) with dopamine-β-hydroxylase labeled terminals, dendrites, and unlabeled axon terminals. Percentages reflect associations of at least one-fourth of the plasmalemma of CB1 labeled profiles that are directly apposed to different cellular elements in the microenvironment.