Jonathon C. Arnold

Adolescent rats find repeated Delta(9)-THC less aversive than adult rats but display greater residual cognitive deficits and changes in hippocampal protein expression following exposure.

The current study examined whether adolescent rats are more vulnerable than adult rats to the lasting adverse effects of cannabinoid exposure on brain and behavior. Male Wistar rats were repeatedly exposed to Δ-9-tetrahydrocannabinol (Δ9-THC, 5 mg/kg i.p.) in a place-conditioning paradigm during either the adolescent (post-natal day 28+) or adult (post-natal day 60+) developmental stages. Adult rats avoided a Δ9-THC-paired environment after either four or eight pairings and this avoidance persisted for at least 16 days following the final Δ9-THC injection. In contrast, adolescent rats showed no significant place aversion. Adult Δ9-THC-treated rats produced more vocalizations than adolescent rats when handled during the intoxicated state, also suggesting greater drug-induced aversion. After a 10–15 day washout, both adult and adolescent Δ9-THC pretreated rats showed decreased social interaction, while only Δ9-THC pretreated adolescent rats showed significantly impaired object recognition memory. Seventeen days following their last Δ9-THC injection, rats were euthanased and hippocampal tissue processed using two-dimensional gel electrophoresis proteomics. There was no evidence of residual Δ9-THC being present in blood at this time. Proteomic analysis uncovered 27 proteins, many involved in regulating oxidative stress/mitochondrial functioning and cytoarchitecture, which were differentially expressed in adolescent Δ9-THC pretreated rats relative to adolescent controls. In adults, only 10 hippocampal proteins were differentially expressed in Δ9-THC compared to vehicle-pretreated controls. Overall these findings suggest that adolescent rats find repeated Δ9-THC exposure less aversive than adults, but that cannabinoid exposure causes greater lasting memory deficits and hippocampal alterations in adolescent than adult rats.

A behavioural comparison of acute and chronic Δ9-tetrahydrocannabinol and cannabidiol in C57BL/6JArc mice

Cannabis contains over 70 unique compounds and its abuse is linked to an increased risk of developing schizophrenia. The behavioural profiles of the psychotropic cannabis constituent Delta9-tetrahydrocannabinol (Delta9-THC) and the non-psychotomimetic constituent cannabidiol (CBD) were investigated with a battery of behavioural tests relevant to anxiety and positive, negative and cognitive symptoms of schizophrenia. Male adult C57BL/6JArc mice were given 21 daily intraperitoneal injections of vehicle, Delta9-THC (0.3, 1, 3 or 10 mg/kg) or CBD (1, 5, 10 or 50 mg/kg). Delta9-THC produced the classic cannabinoid CB1 receptor-mediated tetrad of hypolocomotion, analgesia, catalepsy and hypothermia while CBD had modest hyperthermic effects. While sedative at this dose, Delta9-THC (10 mg/kg) produced locomotor-independent anxiogenic effects in the open-field and light-dark tests. Chronic CBD produced moderate anxiolytic-like effects in the open-field test at 50 mg/kg and in the light-dark test at a low dose (1 mg/kg). Acute and chronic Delta9-THC (10 mg/kg) decreased the startle response while CBD had no effect. Prepulse inhibition was increased by acute treatment with Delta9-THC (0.3, 3 and 10 mg/kg) or CBD (1, 5 and 50 mg/kg) and by chronic CBD (1 mg/kg). Chronic CBD (50 mg/kg) attenuated dexamphetamine (5 mg/kg)-induced hyperlocomotion, suggesting an antipsychotic-like action for this cannabinoid. Chronic Delta9-THC decreased locomotor activity before and after dexamphetamine administration suggesting functional antagonism of the locomotor stimulant effect. These data provide the first evidence of anxiolytic- and antipsychotic-like effects of chronic but not acute CBD in C57BL/6JArc mice, extending findings from acute studies in other inbred mouse strains and rats.

Systemically administered oxytocin decreases methamphetamine activation of the subthalamic nucleus and accumbens core and stimulates oxytocinergic neurons in the hypothalamus

Recent preclinical evidence indicates that the neuropeptide oxytocin may have potential in the treatment of drug dependence and drug withdrawal. Oxytocin reduces methamphetamine self‐administration, conditioned place preference and hyperactivity in rodents. However, it is unclear how oxytocin acts in the brain to produce such effects. The present study examined how patterns of neural activation produced by methamphetamine were modified by co‐administered oxytocin. Male Sprague‐Dawley rats were pretreated with either 2 mg/kg oxytocin (IP) or saline and then injected with either 2 mg/kg methamphetamine (IP) or saline. After injection, locomotor activity was measured for 80 minutes prior to perfusion. As in previous studies, co‐administered oxytocin significantly reduced methamphetamine‐induced behaviors. Strikingly, oxytocin significantly reduced methamphetamine‐induced Fos expression in two regions of the basal ganglia: the subthalamic nucleus and the nucleus accumbens core. The subthalamic nucleus is of particular interest given emerging evidence for this structure in compulsive, addiction‐relevant behaviors. When administered alone, oxytocin increased Fos expression in several regions, most notably in the oxytocin‐synthesizing neurons of the supraoptic nucleus and paraventricular nucleus of the hypothalamus. This provides new evidence for central actions of peripheral oxytocin and suggests a self‐stimulation effect of exogenous oxytocin on its own hypothalamic circuitry. Overall, these results give further insight into the way in which oxytocin might moderate compulsive behaviors and demonstrate the capacity of peripherally administered oxytocin to induce widespread central effects.

Analysis of cannabis seizures in NSW, Australia: cannabis potency and cannabinoid profile.

Recent analysis of the cannabinoid content of cannabis plants suggests a shift towards use of high potency plant material with high levels of Δ9-tetrahydrocannabinol (THC) and low levels of other phytocannabinoids, particularly cannabidiol (CBD). Use of this type of cannabis is thought by some to predispose to greater adverse outcomes on mental health and fewer therapeutic benefits. Australia has one of the highest per capita rates of cannabis use in the world yet there has been no previous systematic analysis of the cannabis being used. In the present study we examined the cannabinoid content of 206 cannabis samples that had been confiscated by police from recreational users holding 15 g of cannabis or less, under the New South Wales “Cannabis Cautioning” scheme. A further 26 “Known Provenance” samples were analysed that had been seized by police from larger indoor or outdoor cultivation sites rather than from street level users. An HPLC method was used to determine the content of 9 cannabinoids: THC, CBD, cannabigerol (CBG), and their plant-based carboxylic acid precursors THC-A, CBD-A and CBG-A, as well as cannabichromene (CBC), cannabinol (CBN) and tetrahydrocannabivarin (THC-V). The “Cannabis Cautioning” samples showed high mean THC content (THC+THC-A = 14.88%) and low mean CBD content (CBD+CBD-A = 0.14%). A modest level of CBG was detected (CBG+CBG-A = 1.18%) and very low levels of CBC, CBN and THC-V (<0.1%). “Known Provenance” samples showed no significant differences in THC content between those seized from indoor versus outdoor cultivation sites. The present analysis echoes trends reported in other countries towards the use of high potency cannabis with very low CBD content. The implications for public health outcomes and harm reduction strategies are discussed.

Adolescent Oxytocin Exposure Causes Persistent Reductions in Anxiety and Alcohol Consumption and Enhances Sociability in Rats

Previous studies have suggested that administration of oxytocin (OT) can have modulatory effects on social and anxiety-like behavior in mammals that may endure beyond the time of acute OT administration. The current study examined whether repeated administration of OT to male Wistar rats (n = 48) during a key developmental epoch (early adolescence) altered their physiology and behavior in later-life. Group housed rats were given intraperitoneal injections of either 1 mg/kg OT or vehicle during early adolescence (post natal-days [PND] 33–42). OT treatment caused a transient inhibition of body weight gain that recovered quickly after the cessation of treatment. At PND 50, the rats pre-treated with OT displayed less anxiety-like behavior on the emergence test, while at PND 55 they showed greater levels of social interaction. A subgroup of OT pre-treated rats examined at PND 63 showed a strong trend towards increased plasma OT levels, and also displayed significantly increased OT receptor mRNA in the hypothalamus. Rats pre-treated with OT and their controls showed similar induction of beer intake in daily 70 min test sessions (PND 63 onwards) in which the alcohol concentration of beer was gradually increased across days from 0.44% to 4.44%. However, when given ad libitum access to beer in their home cages from PND 72 onwards (early adulthood), consumption of beer but not water was significantly less in the OT pre-treated rats. A “booster” shot of OT (1 mg/kg) given after 25 days of ad libitum access to beer had a strong acute inhibitory effect on beer intake without affecting water intake. Overall these results suggest that exogenous OT administered during adolescence can have subtle yet enduring effects on anxiety, sociability and the motivation to consume alcohol. Such effects may reflect the inherent neuroplasticity of brain OT systems and a feed-forward effect whereby exogenous OT upregulates endogenous OT systems.

Heterozygous neuregulin 1 mice display greater baseline and Δ9-tetrahydrocannabinol-induced c-Fos expression

Cannabis use may increase the risk of developing schizophrenia by precipitating the disorder in genetically vulnerable individuals. Neuregulin 1 (NRG1) is a schizophrenia susceptibility gene and mutant mice heterozygous for the transmembrane domain of this gene (Nrg1 HET mice) exhibit a schizophrenia-related phenotype. We have recently shown that Nrg1 HET mice are more sensitive to the behavioral effects of the main psychoactive constituent of cannabis, Delta(9)-tetrahydrocannabinol (THC). In the present study, we examined the effects of THC (10 mg/kg i.p.) on neuronal activity in Nrg1 HET mice and wild type-like (WT) mice using c-Fos immunohistochemistry. In the lateral septum, THC selectively increased c-Fos expression in Nrg1 HET mice with no corresponding effect being observed in WT mice. In addition, THC promoted a greater increase in c-Fos expression in Nrg1 HET mice than WT mice in the central nucleus of the amygdala, the bed nucleus of the stria terminalis and the paraventricular nucleus of the hypothalamus. Consistent with Nrg1 HET mice exhibiting a schizophrenia-related phenotype, these mice expressed greater drug-free levels of c-Fos in two regions thought to be involved in schizophrenia, the shell of the nucleus accumbens and the lateral septum. Interestingly, the effects of genotype on c-Fos expression, drug-free or following THC exposure, were only observed when animals experienced behavioral testing prior to perfusion. This suggests an interaction with stress was necessary for the promotion of these effects. These data provide neurobiological correlates for the enhanced behavioral sensitivity of Nrg1 HET mice to THC and reinforce the existence of cannabinoid-neuregulin 1 interactions in the CNS. This research may enhance our understanding of how genetic factors increase individual vulnerability to schizophrenia and cannabis-induced psychosis.

The role of endocannabinoid transmission in cocaine addiction

Research is beginning to outline a role for the endocannabinoid system in cocaine addiction. Human and animal studies indicate that exogenous cannabinoids modulate the acute rewarding effects of cocaine. These studies, however, cannot directly investigate the necessity of endocannabinoid transmission in cocaine addiction. Studies that do offer a direct assessment show that neither pharmacological antagonism nor deletion of the CB1 receptor alters the acute rewarding effects of cocaine. In contrast, CB1 receptors appear to be involved in the association of cocaine reward with environmental cues and reinstatement of cocaine self-administration. Together, these results point to CB1 receptor antagonists as potential anti-craving compounds in the treatment of cocaine addiction. Given the limitations of human population studies, animal research may be useful in discerning causal inferences between cannabis and cocaine use. While animal research suggests cannabis use may precipitate cocaine relapse, cross-sensitization between cannabinoids and cocaine has not been demonstrated and CB1 receptors do not mediate behavioral sensitization to cocaine. The effect of acute or chronic cocaine on endocannabinoid transmission in reward-related areas of the brain is relatively under-researched. Acute cocaine administration increases anandamide levels in the striatum, an effect that is mediated by dopamine D2-like receptors. Conversely, chronic cocaine exposure has no effect on anandamide, but decreases 2-arachidonylglycerol levels in the limbic forebrain. This review highlights research indicating that the endocannabinoid system may subserve certain aspects of cocaine addiction and suggests avenues for future investigation.

The distribution of cannabinoid-induced Fos expression in rat brain: differences between the Lewis and Wistar strain.

Previous studies have suggested that cannabis-like drugs produce mainly aversive and anxiogenic effects in Wistar strain rats, but rewarding effects in Lewis strain rats. In the present study we compared Fos expression, body temperature effects and behavioral effects elicited by the cannabinoid CB(1) receptor agonist CP 55,940 in Lewis and Wistar rats. Both a moderate (50 microg/kg) and a high (250 microg/kg) dose level were used. The 250 microg/kg dose caused locomotor suppression, hypothermia and catalepsy in both strains, but with a significantly greater effect in Wistar rats. The 50 microg/kg dose provoked moderate hypothermia and locomotor suppression but in Wistar rats only. CP 55,940 caused significant Fos immunoreactivity in 24 out of 33 brain regions examined. The most dense expression was seen in the paraventricular nucleus of the hypothalamus, the islands of Calleja, the lateral septum (ventral), the central nucleus of the amygdala, the bed nucleus of the stria terminalis (lateral division) and the ventrolateral periaqueductal gray. Despite having a similar distribution of CP 55,940-induced Fos expression, Lewis rats showed less overall Fos expression than Wistars in nearly every brain region counted. This held equally true for anxiety-related brain structures (e.g. central nucleus of the amygdala, periaqueductal gray and the paraventricular nucleus of the hypothalamus) and reward-related sites (nucleus accumbens and pedunculopontine tegmental nucleus). In a further experiment, Wistar rats and Lewis rats did not differ in the amount of Fos immunoreactivity produced by cocaine (15 mg/kg). These results indicate that Lewis rats are less sensitive to the behavioral, physiological and neural effects of cannabinoids. The exact mechanism underlying this subsensitivity requires further investigation.

Resilience and reduced c-Fos expression in P2X7 receptor knockout mice exposed to repeated forced swim test.

There is considerable evidence suggesting genetic factors play an important role in the pathophysiology of depression, possibly by increasing susceptibility to repeated environmental stressors. Recent linkage studies have associated a polymorphism of the gene coding for the P2X7 receptor (P2X7R) with both major depressive disorder and bipolar disorder. Here we assessed whether P2X7 deletion affected the behavioural and neural response to repeated stress. P2X7R knockout (P2X7-/-) mice were subjected to the forced swim test for three consecutive days and neuronal activation in response to the third exposure was assessed using c-Fos immunohistochemistry. In addition, anxiety was evaluated in another group of P2X7-/- mice using the elevated plus maze (EPM) and light dark emergence (LDE) tests. Equivalent levels of immobility were observed in P2X7-/- mice and wild-type (WT) mice on the first exposure to forced swim, but much greater immobility was seen in WT mice on second and third exposures. This suggests that P2X7-/- mice exhibit an impaired adaptive coping response to repeated stress. Reinforcing this view, c-Fos expression in the dentate gyrus of the hippocampus and in the basolateral amygdala was seen in WT mice but not P2X7-/- mice following repeated forced swim. In addition, decreased locomotor activity was detected in P2X7-/- mice without any specific effects on anxiety in the LDE test. However, P2X7-/- mice showed greater anxiety-like behaviour in the EPM. These data suggest that the P2X7R may be involved in the adaptive mechanisms elicited by exposure to repeated environmental stressors that leads to the development of depression-like behaviours. This suggests that P2X7R antagonists may be useful therapeutics for the treatment of major depression, possibly by increasing resilience in the face of repeated stress.

Effects of pre-exposure and co-administration of the cannabinoid receptor agonist CP 55,940 on behavioral sensitization to cocaine

Rats given cocaine (15 mg/kg, i.p.) every second day over a 2-week period displayed a progressively greater locomotor response to the drug over days indicating behavioral sensitization. When the cannabinoid receptor agonist CP 55,940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hyd roxypropyl)cyclohexanol) (10, 25 or 50 microg/kg) was administered under a similar regime, no such sensitization was observed. Rather, the two highest doses of CP 55,940 (25 and 50 microg/kg) caused locomotor suppression that lasted throughout administration. When rats pre-exposed 10 times to CP 55,940 were challenged with cocaine (15 mg/kg), no exaggerated locomotor response to cocaine was evident relative to non pre-exposed rats. When these rats were subsequently re-tested with CP 55,940, the cannabinoid continued to produce a dose-dependent suppression of locomotor activity. Finally, when CP 55,940 (50 microg/kg) was co-administered with cocaine, it significantly reduced the locomotor hyperactivity produced by the drug but did not block the development of behavioral sensitization. These results show that CP 55,940 does not sensitize locomotor activity with repeated administration in the same way as cocaine, and that pre-exposure or concurrent exposure to CP 55,940 does not enhance sensitivity to the subsequent behavioral effects of cocaine.