David J. Rademacher

Downregulation of Endocannabinoid Signaling in the Hippocampus Following Chronic Unpredictable Stress

Deficits in cognitive functioning and flexibility are seen following both chronic stress and modulation of endogenous cannabinoid (eCB) signaling. Here, we investigated whether alterations in eCB signaling might contribute to the cognitive impairments induced by chronic stress. Chronic stress impaired reversal learning and induced perseveratory behavior in the Morris water maze without significant effect on task acquisition. These cognitive impairments were reversed by exogenous cannabinoid administration, suggesting deficient eCB signaling underlies these phenomena. In line with this hypothesis, chronic stress downregulated CB1 receptor expression and significantly reduced the content of the endocannabinoid 2-arachidonylglycerol within the hippocampus. CB1 receptor density and 2-arachidonylglycerol content were unaffected in the limbic forebrain. These data suggest that stress-induced downregulation of hippocampal eCB signaling contributes to problems in behavioral flexibility and could play a role in the development of perseveratory and ruminatory behaviors in stress-related neuropsychiatric disorders.

Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signalling.

The role of endocannabinoid (eCB) signalling in restraint stress‐induced neuronal activation was studied. Male mice exposed to 30 min of restraint exhibit increased Fos protein within prefrontal cortex (PFC), lateral septum (LS), nucleus accumbens (Acb) and medial amygdala. SR141716 (2 mg/kg) itself had no effect on Fos but pretreatment with SR141716 significantly potentiated restraint‐induced Fos expression in cingulate, LS and Acb. SR141716 also significantly increased the time spent in active escape behaviours during the restraint. In restraint‐habituated mice (mice exposed to four previous restraint episodes), the fifth restraint exposure resulted in decreased expression of active escape behaviours compared to the first exposure and only induced Fos protein in the central and medial amygdala. Administration of SR141716 prior to the fifth restraint episode resulted in greater potentiation of restraint‐induced Fos induction than the first; significant increases occurred within all regions of PFC examined, LS and Acb. Brain regional eCB content was measured immediately after restraint. N‐arachidonylethanolamine content within the amygdala was significantly decreased after both restraint episodes. 2‐Arachidonylglycerol content was significantly increased in both the limbic forebrain and amygdala after the fifth restraint but not the first. Restraint had no effect on cerebellar eCB content. These data suggest that eCB activation of CB1 receptors opposes the behavioural and neuronal responses to aversive stimuli. Because repeated homotypic stress increased both limbic 2‐AG and resulted in a greater effect of SR141716 on limbic Fos expression, we hypothesize that increased CB1 receptor activity contributes to the expression of habituation to homotypic stress.

Effects of acute and repeated restraint stress on endocannabinoid content in the amygdala, ventral striatum, and medial prefrontal cortex in mice.

Endocannabinoid signaling has been implicated in habituation to repeated stress. The hypothesis that repeated exposures to stress alters endocannabinoid signaling in the limbic circuit was tested by restraining male mice for 30 min/day for 1, 7, or 10 days and measuring brain endocannabinoid content. Amygdalar N-arachidonylethanolamine was decreased after 1, 7, and 10 restraint episodes; 2-arachidonylglycerol was increased after the 10th restraint. A similar pattern occurred in the medial prefrontal cortex (mPFC): N-arachidonylethanolamine was decreased after the 7th and 10th restraints and 2-arachidonylglycerol was increased after the 10th restraint. In the ventral striatum, the pattern reversed: N-arachidonylethanolamine was increased after the 10th restraint and 2-arachidonylglycerol was decreased after the 7th restraint. Palmitoylethanolamide contents changed in parallel with N-arachidonylethanolamine in the amygdala and ventral striatum. A single restraint episode did not affect the activity of fatty acid amide hydrolase (FAAH) in any of the brain regions examined. After the 10th restraint, both V(max) and K(m) for N-arachidonylethanolamine were increased in the mPFC; while only the V(max) was increased in the amygdala. On the other hand, the V(max) of FAAH was decreased in ventral striatum after the 10th restraint. After the 10th restraint, the maximum velocity for 2-oleoylglycerol hydrolysis was increased in mPFC; no other changes in 2-oleoylglycerol hydrolysis occurred. Repeated exposure to restraint produced no changes in CB(1) receptor density in any of the areas examined. These studies are consistent with the hypothesis that stress exposure alters endocannabinoid signaling in the brain and that alterations in endocannabinoid signaling occur during habituation to stress.

Anandamide content is increased and CB1 cannabinoid receptor blockade is protective during transient, focal cerebral ischemia.

The role of endocannabinoid signaling in the response of the brain to injury is tantalizing but not clear. In this study, transient middle cerebral artery occlusion (MCAo) was used to produce ischemia/reperfusion injury. Brain content of N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol were determined during MCAo. Whole brain AEA content was significantly increased after 30, 60 and 120 min MCAo compared with sham-operated brain. The increase in AEA was localized to the ischemic hemisphere after 30 min MCAo, but at 60 and 120 min, was also increased in the contralateral hemisphere. 2-Arachidonoylglycerol content was unaffected by MCAo. In a second set of studies, injury was assessed 24 h after 2 h MCAo. Rats administered a single dose (3 mg/kg) of the cannabinoid receptor type 1 (CB1) receptor antagonist SR141716 prior to MCAo exhibited a 50% reduction in infarct volume and a 40% improvement in neurological function compared with vehicle control. A second CB1 receptor antagonist, LY320135 (6 mg/kg), also significantly improved neurological function. The CB1 receptor agonist, WIN 55212-2 (0.1-1 mg/kg) did not affect either infarct volume or neurological score.

The general anesthetic propofol increases brain N‐arachidonylethanolamine (anandamide) content and inhibits fatty acid amide hydrolase

Propofol (2,6‐diisopropylphenol) is widely used as a general anesthetic and for the maintenance of long‐term sedation. We have tested the hypothesis that propofol alters endocannabinoid brain content and that this effect contributes to its sedative properties. A sedating dose of propofol in mice produced a significant increase in the whole‐brain content of the endocannabinoid, N‐arachidonylethanolamine (anandamide), when administered intraperitoneally in either Intralipid or emulphor‐ethanol vehicles. In vitro, propofol is a competitive inhibitor (IC50 52 μM; 95% confidence interval 31, 87) of fatty acid amide hydrolase (FAAH), which catalyzes the degradation of anandamide. Within a series of propofol analogs, the critical structural determinants of FAAH inhibition and sedation were found to overlap. Other intravenous general anesthetics, including midazolam, ketamine, etomidate, and thiopental, do not affect FAAH activity at sedative‐relevant concentrations. Thiopental, however, is a noncompetitive inhibitor of FAAH at a concentration of 2 mM. Pretreatment of mice with the CB1 receptor antagonist SR141716 (1 mg kg−1, i.p.) significantly reduced the number of mice that lost their righting reflex in response to propofol. Pretreatment of mice with the CB1 receptor agonist, Win 55212‐2 (1 mg kg−1, i.p.), significantly potentiated the loss of righting reflex produced by propofol. These data indicate that CB1 receptor activity contributes to the sedative properties of propofol. These data suggest that propofol activation of the endocannabinoid system, possibly via inhibition of anandamide catabolism, contributes to the sedative properties of propofol and that FAAH could be a novel target for anesthetic development.

Interactions Between Endocannabinoids and Stress-Induced Decreased Sensitivity to Natural Reward

Since endocannabinoids modulate reward processing and the stress response, we tested the hypothesis that endocannabinoids regulate stress-induced decreased sensitivity to natural reward. Restraint was used to produce stress-induced reductions in sucrose consumption and preference in male mice. Central cannabinoid receptor (CB(1)) signaling was modulated pharmacologically prior to the application of stress. The preference for sucrose over water was significantly decreased in mice exposed to restraint. Treatment of mice with a cannabinoid receptor agonist (CP55940) or fatty acid amide hydrolase inhibitor (URB597) attenuated, while the CB(1) receptor antagonist/inverse agonist, rimonabant (SR141716), enhanced, stress-induced decreases in sucrose preference. These data are consistent with a tonically active, stress-inhibitory role for the CB(1) receptor. Mice treated with 10 daily episodes of restraint showed reduced sucrose preference that was unaffected by CP55940 and URB597. However, rimonabant produced a greater reduction in sucrose preference on day 10 compared to day 1. These data suggest that on day 10, endocannabinoid signaling is maximally activated and essential for reward sensitivity. The findings of the present study indicate that the CB(1)/endocannabinoid signaling system is an important allostatic mediator that both modulates the responses of mice to stress and is itself modulated by stress.