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. 2012 Aug;236(2):327-35.
doi: 10.1016/j.expneurol.2012.05.016. Epub 2012 Jun 4.

Stress-induced sensitization of cortical adrenergic receptors following a history of cannabinoid exposure

B A S Reyes  1 , P Szot, C Sikkema, A M Cathel, L G Kirby, E J Van Bockstaele
Affiliations

Stress-induced sensitization of cortical adrenergic receptors following a history of cannabinoid exposure

B A S Reyes et al. Exp Neurol. 2012 Aug.

Abstract

The cannabinoid receptor agonist, WIN 55,212-2, increases extracellular norepinephrine levels in the rat frontal cortex under basal conditions, likely via desensitization of inhibitory α2-adrenergic receptors located on norepinephrine terminals. Here, the effect of WIN 55,212-2 on stress-induced norepinephrine release was assessed in the medial prefrontal cortex (mPFC), in adult male Sprague-Dawley rats using in vivo microdialysis. Systemic administration of WIN 55,212-2 30 min prior to stressor exposure prevented stress-induced cortical norepinephrine release induced by a single exposure to swim when compared to vehicle. To further probe cortical cannabinoid-adrenergic interactions, postsynaptic α2-adrenergic receptor (AR)-mediated responses were assessed in mPFC pyramidal neurons using electrophysiological analysis in an in vitro cortical slice preparation. We confirm prior studies showing that clonidine increases cortical pyramidal cell excitability and that this was unaffected by exposure to acute stress. WIN 55,212-2, via bath application, blocked postsynaptic α2-AR mediated responses in cortical neurons irrespective of exposure to stress. Interestingly, stress exposure prevented the desensitization of α2-AR mediated responses produced by a history of cannabinoid exposure. Together, these data indicate the stress-dependent nature of cannabinoid interactions via both pre- and postsynaptic ARs. In summary, microdialysis data indicate that cannabinoids restrain stress-induced cortical NE efflux. Electrophysiology data indicate that cannabinoids also restrain cortical cell excitability under basal conditions; however, stress interferes with these CB1-α2 AR interactions, potentially contributing to over-activation of pyramidal neurons in mPFC. Overall, cannabinoids are protective of the NE system and cortical excitability but stress can derail this protective effect, potentially contributing to stress-related psychopathology. These data add to the growing evidence of complex, stress-dependent modulation of monoaminergic systems by cannabinoids and support the potential use of cannabinoids in the treatment of stress-induced noradrenergic dysfunction.

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Figures

Figure 1
Figure 1
(A) A representative brightfield photomicrograph of a coronal section counterstained with pontamine sky blue dye showing histological verification of a microdialysis probe placement in the frontal cortex. Arrows depict the position of the implanted probe. Dorsal (D) and medial (M) orientation of the tissue section are indicated by arrows. Scale bar = 100 μm. Inset shows a schematic diagram adapted from the rat brain atlas of Paxinos (Paxinos and Watson, 1986) depicting the anterior posterior level of the representative microdialysis probe placement shown in panel A. Big arrow indicates the microdialysis probe placement. Arrows depict dorsal (D) and medial (M) orientation of the tissue section. Cl, claustrum; fmi, forceps minor corpus callosum.
Figure 2
Figure 2
The effect of pre-treatment with the CB1 receptor agonist WIN 55,212-2 on extracellular norepinephrine release in the rat PFC following a 15-minute exposure to swim was measured using in vivo microdialysis with HPLC-ED. Pre-treatment with 3.0 mg/kg of WIN 55,212-2 produced a significant increase in norepinephrine release in the PFC (1.20 ± 0.32 pg/25ml) at 20 minutes post drug administration. The increase in norepinephrine release observed in cannabinoid-treated animals was significant compared to extracellular norepinephrine release observed SR 141716A (a selective CB1 receptor antagonist) +WIN 55,212-2 and vehicle at 20 minutes post drug administration (P < 0.05). Exposure to acute swim stress (period of test illustrated by black bar) caused a marked increase in norepinephrine release in the PFC in vehicle (0.935 ± 0.17 pg/25ml). In cannabinoid-treated rats, the increase in norepinephrine release elicited by exposing the rats to swimming was attenuated (0.45 ± 0.13 pg/25ml) compared to vehicle (P < 0.05). *P < 0.05 vs SR 141716A+WIN and vehicle; # P < 0.05 vs WIN+swim.
Figure 3
Figure 3
Effect of WIN 55,212-2 on rat behavior during swim stress. Mean counts of immobility, swimming, and climbing behaviors are shown. Behaviors were sampled every 5 s during the first 5 min of the 15-min test period (n=5 rats per group). Rats were treated with vehicle (gray bars) or 3 mg/kg of WIN 55,212-2 (black bars) 60 minutes prior to the swim test. The swim test significantly increased the frequency of immobility counts during the swim sessions when compared to controls (*P < 0.001). This was accompanied by a slight decrease in climbing behavior (*P < 0.05) with no significant difference in swimming behavior (P = 0.24).
Figure 4
Figure 4
Layer V/VI pyramidal neuron in PFC: morphology and electrophysiology. Panel A shows a layer V/VI pyramidal neuron in PFC filled with biocytin and visualized using Alexa 488-conjugated streptavidin. This cell has a large (~20 μm) pyramidal-shaped cell body with a large apical dendrite (arrowhead) extending toward the brain surface which is characteristic of cortical pyramidal neurons. Clonidine (10 μM) increases excitability in a PFC pyramidal neuron from a 2 to 5 spike response to an 80 pA current step. Scale bar = 50 μM.
Figure 5
Figure 5
Swim stress does not affect α2-adrenergic receptor-mediated increase in PFC pyramidal neuron excitability. Clonidine (10 μM) significantly elevated mean pyramidal cell excitability (panel A; N = 6) in brain slices from naïve (non-stressed) subjects. In slices from animals exposed to a 15 min swim stress, this α2-adrenergic effect was unchanged (panel B; N = 12). The asterisk indicates a significant change from baseline by paired Student’s t-test (P < 0.05) and the pound sign indicates a significant difference from both baseline and vehicle by post-hoc Student-Newman-Keuls tests (P < 0.05). Data are represented as mean ± SEM.
Figure 6
Figure 6
Acute CB1 receptor stimulation blocks α2-adrenergic receptor-mediated increase in PFC pyramidal neuron excitability in brain slices from unstressed and stressed subjects. Acute in vitro pretreatment with the CB1 agonist, WIN 55,212-2 (1.0 μM) blocks clonidine (10 μM)-induced elevation of PFC pyramidal neuron excitability in brain slices from naïve (unstressed) subjects (N = 7; panel A) and in slices from subjects exposed to a 15 min swim stress(N = 11; panel B). Data are represented as mean ± SEM.
Figure 7
Figure 7
Chronic CB1 receptor stimulation blocks α2-adrenergic receptor-mediated increase in PFC pyramidal neuron excitability in brain slices from unstressed but not stressed subjects. Clonidine (10 μM) significantly increased PFC pyramidal neuron excitability in slices from either unstressed (N = 17; panel A) or stressed animals pretreated with chronic vehicle (N = 13; panel B). Chronic WIN 55,212-2 treatment (3 mg/kg, i.p., once daily for 7 days) blocked clonidine’s effect on PFC neuron excitability in unstressed subjects (N = 10; panel C). By contrast, swim exposure prevented this CB1-α2-adrenergic receptor interaction. In slices from subjects exposed to a 15 min swim stress, chronic WIN 55,212-2 treatment did not alter the ability of clonidine to increase PFC neuron excitability (N = 13; panel D). Asterisks indicate a significant change from baseline by paired t-test (* P < 0.05; ** P < 0.01). Data are represented as mean ± SEM.
Figure 8
Figure 8
Schematic diagram showing putative mechanisms underlying cannabinoid modulation of norepinephrine. Microdialysis and electrophysiology data indicate cannabinoid-induced de-sensitization of pre- and post-synaptic α2-ARs, respectively. Presynaptic α2-AR de-sensitization will lead to cannabinoid-induced increases in cortical NE efflux. Electrophysiological studies demonstrate blockade of α2-AR-mediated pyramidal cell excitability by WIN implicating de-sensitization of postsynaptic α2-ARs in the effect. Stress is impacting CB1-α2-AR interactions at both pre- and post-synaptic levels. In summary, the microdialysis study suggests that cannabinoids restrain stress-induced NE efflux. The electrophysiological studies suggest that cannabinoids restrain PFC cell excitability via de-sensitization of postsynaptic α2-ARs but stress can interfere with this interaction, potentially contributing to over-activation of pyramidal neurons in PFC. Alternatively, cannabinoids may desensitize α2-AR on GABA interneurons, resulting in increased GABA tone and potential restraint of both cortical excitability and NE efflux. If stress interferes with this desensitization, the effect would be decreased GABA tone, excess NE release and excitability of cortical neurons. Future studies are required to address local network interactions. Overall, cannabinoids are protective of the NE system and cortical excitability but stress can derail this protective effect, leading to psychopathology. Abbreviations: Acb-nucleus accumbens; α2-AR-α2-adrenergic receptor; aCSF-artificial cerebrospinal fluid; CB1-cannabinoid receptor 1; GABA-gamma amino butyric acid; HPLC-ED-high performance liquid chromatography with electrochemical detection; LC-locus coeruleus; mPFC-medial prefrontal cortex; NE-norepinephrine; PTSD-post-traumatic stress disorder; pyr-pyramidal neurons; TH-tyrosine hydroxylase;.
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