Co-localization of the cannabinoid type 1 receptor with corticotropin-releasing factor-containing afferents in the noradrenergic nucleus locus coeruleus: implications for the cognitive limb of the stress response

Abstract

The noradrenergic system has been shown to play a key role in the regulation of stress responses, arousal, mood, and emotional states. Corticotropin-releasing factor (CRF) is a primary mediator of stress-induced activation of noradrenergic neurons in the nucleus locus coeruleus (LC). The endocannabinoid (eCB) system also plays a key role in modulating stress responses, acting as an "anti-stress" neuro-mediator. In the present study, we investigated the cellular sites for interactions between the cannabinoid receptor type 1 (CB1r) and CRF in the LC. Immunofluorescence and high-resolution immunoelectron microscopy showed co-localization of CB1r and CRF in both the core and peri-LC areas. Semi-quantitative analysis revealed that 44% (208/468) of CRF-containing axon terminals in the core and 35% (104/294) in the peri-LC expressed CB1r, while 18% (85/468) of CRF-containing axon terminals in the core and 6.5% (19/294) in the peri-LC were presynaptic to CB1r-containing dendrites. In the LC core, CB1r + CRF axon terminals were more frequently of the symmetric (inhibitory) type; while in the peri-LC, a majority were of the asymmetric (excitatory) type. Triple label immunofluorescence results supported the ultrastructural analysis indicating that CB1r + CRF axon terminals contained either gamma amino butyric acid or glutamate. Finally, anterograde transport from the central nucleus of the amygdala revealed that CRF-amygdalar afferents projecting to the LC contain CB1r. Taken together, these results indicate that the eCB system is poised to directly modulate stress-integrative heterogeneous CRF afferents in the LC, some of which arise from limbic sources.

Sex differences in the effect of cannabinoid type 1 receptor deletion on locus coeruleus‐norepinephrine neurons and corticotropin releasing factor‐mediated responses

“…While electrophysiology experiments were carried out in CB1r-KO mice, Western blot and ELISA experiments were carried out in CB1r/CB2r-KO mice due to tissue availability. Many immunohistochemical studies have identified CB1r as an abundant protein in the LC, both directly on the noradrenergic neurons as well as on presynaptic afferents synapsing onto LC-NE dendrites and cell bodies (Scavone et al, 2010; Wyrofsky et al, 2017). While CB2r is traditionally thought to play a role in the periphery and immune responses (Castillo et al, 2012), growing accumulating evidence suggests that it does in fact play a role in neuronal signaling in some select brain regions (Van Sickle et al, 2005; Gong et al, 2006).…”

“…One of the most widely used recreational drugs in the world, cannabis, often causes feelings of relaxation and euphoria (Velez et al, 1989; Green et al, 2003; Di Marzo et al, 2004); however, chronic usage can be anxiogenic and produce dysphoria (Reilly et al, 1998). Via activation of cannabinoid type 1 receptors (CB1r), cannabinoids are known to affect attention and anxiety (Witkin et al, 2005; Pattij et al, 2008; Hill and Gorzalka, 2009), in part via actions on noradrenergic circuitry (Carvalho et al, 2010; Carvalho and Van Bockstaele, 2012; Wyrofsky et al, 2017). There are, however, some discrepancies within the cannabinoid field.…”

“…Immunofluorescence and immunoelectron microscopy studies have provided direct anatomical evidence for CB1r co-localization to noradrenergic terminals in the mPFC and localization to TH-containing neurons in the LC (Carvalho et al, 2010; Wyrofsky et al, 2017). Several studies have also shown that CB1r activation increases NE release.…”

“…Following a stressor, CRF is released from limbic and autonomic afferent sources such as the paraventricular nucleus of the hypothalamus, central nucleus of the amygdala, and bed nucleus of the stria terminalis (Van Bockstaele et al, 1996; Van Bockstaele et al, 1999; Van Bockstaele et al, 2001; Valentino and Van Bockstaele, 2008), and can lead to increases in LC-NE firing and dysregulation of NE release in target regions, including the mPFC (Curtis et al, 1996). We have recently shown that CB1r are positioned both pre- and post-synaptically with respect to CRF-containing afferents within the LC, providing a neural substrate for eCB modulation of CRF in this noradrenergic nucleus (Wyrofsky et al, 2017).…”

Endocannabinoids, stress signaling, and the locus coeruleus-norepinephrine system

“…Indeed, recent immunofluorescence studies from our laboratory have demonstrated that CB1r are co-localized with CRF in the rat LC (Fig. 3A) (Wyrofsky et al, 2017). Additionally, using Phaseolus Vulgaris Leucoagglutinin (PHAL) as an anterograde tracer injected into the amygdala, CB1r are directly positioned to regulate CRF release from amygdalar limbic afferents (Fig.…”