Medicinally, Cannabis acts as an analgesic, anti-emetic and appetite stimulant. Recreationally, it is an anxiolytic producing a sense of euphoria. However, Cannabis has also been identified as a risk factor for inducing acute psychoses in healthy individual and schizophrenia in individuals susceptible to mental illness. Schizophrenia is a chronic mental disorder with cognitive, emotional and behavioral disturbances, affecting ~1% of the global population. Te complex psychiatric condition is manifested through an array of negative symptoms, such as anhedonia and alogia, as well as through positive symptoms like disordered thoughts and catatonia. Psychosis, comprising of episodic delusions and hallucinations, is an additional symptom of schizophrenia and may also be brought on by illness, extreme stress or drug use. Drugs that trigger psychotic symptoms in humans, such as the hallucinogenic phencyclidine and the sedative ketamine, have been found to initiate repetitive stereotyped circling when administered to zebrafsh. Interestingly, rotational swimming has not been observed following administration of other psychotropics like lysergic acid , 3,4,5-Trimethoxyphenethylamine or 3,4-Methylenedioxymethamphetamine. Consequently, this distinct behavioral stereotypy has been attributed to a mechanism shared between PCP and ketamine, namely antagonism of the glutamate N-methyl-D-aspartate receptor. Glutamate is the predominant excitatory neurotransmitter in the central nervous system and acts as a precursor to the main inhibitory neurotransmitter γ‐aminobutyric acid. Together they work to maintain an excitation/inhibition balance. Inhibition of the ionotropic NMDAR impedes further excitatory signaling and may give rise to the repetitive stereotyped circling. Tis NMDAR hypofunction is also a prominent clinical hallmark of psychosis and schizophrenia, and thus the animal behavior stereotypy has potential as a measure of psychosis-like behavior. Hereafter,ebb flow the psychosis-like behavior refers to the circling behavior as a psychopharmacological response relevant to human psychosis.
Similar to PCP and ketamine, the main psychoactive component of Cannabis, Δ9-tetrahydrocannabinol , causes circling in rats. THC binds Gi/o-protein coupled cannabinoid receptors in the brain and periphery although CB2R expression has also been reported in the midbrain dopamine neurons. Since administration of the CB1R antagonist SR-141716 eliminates the THC-induced circling in rats, the behavior is hypothesized to be mediated through CB1R. Among a broad range of downstream effects, CB1R activation inhibits NMDAR signaling, suggesting a comparable mechanism behind the repetitive stereotyped circling as NMDAR antagonists. However, another pivotal target of THC relating to its rewarding effects is the brain’s dopaminergic system. Although the exact signal transduction path remains unknown, THC increases dopamine signaling along the mesolimbic pathway from the midbrain ventral tegmental area to the nucleus accumbens of the forebrain. In addition to the above-mentioned glutamate hypothesis of schizophrenia, there is also long-standing empirical support for hyperactive DA signaling as a basis for psychosis etiology. Given the high prevalence of Cannabis use and its influence on both glutamatergic and dopaminergic neurotransmission, animal models of its psychotomimetic effects are valuable tools for elucidating the endocannabinoid system’s role in psychosis. Zebrafsh have a highly conserved eCBS and display neurobehavioral similarities with rodents following NMDAR antagonism. As zebrafsh lack DA neuronal expression in the midbrain, DA neurons in the basal diencephalon are a proposed functional counterpart to the mammalian mesolimbic DA system. Te considerable homology between the zebrafsh CNS and the human CNS, combined with their rapid development, accessibility to molecular genetic dissection and in vivo imaging, make them an attractive choice in the biomedical field as they permit high-throughput screenings of genetic and pharmacological manipulations of embryos, larvae and adults.
Making use of these beneficial traits, this present study firstly aims to produce and quantify THC’s effect on zebrafsh stereotyped behavior using a newly developed computational method to quantify the Repetition Index . Secondly, the effect of neurotransmitter imbalance on the behavioral stereotypy was investigated through co-administrations of THC with NMDAR agonist NMDA and GABAA receptor antagonist pentylenetetrazol respectively. Thirdly, to validate if the behavioral stereotypy is mediated via CB1R or CB2R, THC was tested with the selective CB1R inverse agonist AM251 and with the selective CB2R inverse agonist AM630. Finally, to determine if the circular swimming is indicative of a psychotic state, THC was co-administered with the antipsychotic sulpiride. Overall, a zebrafsh model of THC-induced behavioral stereotypies is presented, which is a valuable tool for future in-depth studies of the mechanistic relationship between Cannabis use and risk of mental illness at cellular and molecular levels.Using a new analytical method that we have developed, this study demonstrated that 1 μM THC administration in adult zebrafsh triggered a shift from typical navigational locomotor patterns to a repetitive circling behavior, which was ameliorated by the antipsychotic sulpiride . This behavioral phenotype appears analogous to THC’s efect in rats and the efect of NMDAR antagonists in zebrafsh models of psychosis. Notably, it did not occur in the ethanol control group or in the experimental conditions without THC. Harnessing this behavioral stereotypy through a quantitative measure of RI rather than through manual scoring, eliminates issues of experimenter bias and broadens the possibilities of standardized screens of antipsychotic drugs and for clarifying the enigmatic relationship between endocannabinoids and psychosis/schizophrenia. Cannabis has had a medicinal role for millennia and has lower dependence potential compared to other common drugs of abuse like nicotine or alcohol. Teories connecting Cannabis-use and psychotic episodes began to surface in the 1980s and since then, research has put forward bidirectional associations between Cannabis consumption and psychosis, where high frequency use, early onset of use and use of Cannabis containing high THC concentrations act as mediating factors.
Te susceptibility to psychosis-like symptoms varies across Cannabis consumers as it involves a complex interplay between environmental factors and genetic predispositions. Polymorphisms of genes involved in DA metabolism, e.g. COMT and DAT1, are of reoccurring interest as they may increase the vulnerability to neuronal over-excitation by DA in the prefrontal cortex and give rise to executive dysfunctions and psychoses. As cannabinoids increase dopaminergic signaling, by interrupting glutamate and GABA neurotransmission, Cannabis-use may entail long-term risks in those with dysfunctional DA metabolism. Cannabis is an atypical drug with contradicting responses, especially in zebrafsh where there are reports of anxiogenic effects in adults and biphasic responses in larvae depending on the dosage. Here we present a concentration-dependent THC-induced behavioral stereotypy which is partially attenuated by NMDA, in a nonlinear fashion . This hints of an indirect glutamate modulation of the behavioral phenotype in question, corroborating previous zebrafsh studies with the NMDAR antagonists PCP, ketamine and MK-801. Tepharmacological amplifcation of NMDAR excitation and thereby an increased glutamate release, may have counteracted THC’s NMDAR antagonism. Likewise, inhibiting GABAAR using PTZ showed trends of lowering the RI . A combined depression of glutamate by THC and GABA by PTZ could have maintained the excitation/ inhibition balance of the CNS and prevented repetitive circular locomotion. However, the potent nature of PTZ caused convulsions at 2 mM . Therefore, RI reductions could be due to a general PTZ efect on locomotion and not a direct counteraction of THC. Expanding the dose response analysis of THC, NMDA and PTZ and performing absorption, distribution, metabolism and excretion analysis in zebrafsh will shed further light on the observed concentration-dependent effects. Regardless of the possible THC-mediated shift in CNS excitation/inhibition balance, THC’s effect on the current behavioral phenotype appeared to be CB1R-independent and CB2R-dependent in zebrafsh. Te CB1R specific inverse agonist AM251 was ineffective at lowering the RI when co-administered with THC, to a value not significantly different from the control condition . This was surprising as it contradicts CB1R’s central role in cannabinoid modulation of rodent locomotion, cognition, behavior and reports of CB1R antagonists reversing THC’s effects. CB1R is also known to directly regulate NMDAR via the HINT1 protein,cannabis drying and is colocalized with cholecystokinin basket cells, a type of GABA interneuron in the PFC. Trough these interactions, CB1R agonists may diminish NMDAR activity and inhibit GABA release from CCK-basket cells, leading to a disinhibition of excitatory pyramidal cells. Consequently, downstream DA excitation is potentiated and causes an imbalance in cortical functioning, which is a clinical feature of schizophrenia. Despite the multitude of CB1R pathways for THC to exert its efects on glutamate, GABA and downstream DA signaling, reports of THC as a multi-target ligand may better explain the non-CB1R mediated THC behavioral stereotypy. Te CB2R inverse agonist AM630 given with 1 μM THC reduced the frequency of circling and significantly lowered the mean RI of 1 μM THC alone to a RI not significantly different from the controls . In addition, AM630 prevented the THC-related reduction in velocity during immersion . CB2R modulation of zebrafsh locomotion is complex, as larvae lacking CB2R have been shown to swim less in light periods and more in dark. Te CB2R knockouts also avoided open spaces, thereby displaying an anxiety-like behavior compared to WT larvae. Zebrafsh carry two CB2R duplicates , as opposed to one CB1R, that could exhibit different functional activities compared to CB2R of other species. Although CB2R are mainly expressed in immune cells of the peripheral nervous system, their expression has also been reported in the central nervous system, e.g., midbrain dopamine neurons. Associations between the single nucleotide polymorphisms rs12744386 and rs2501432, which impair the function of the CNR2 gene encoding CB2R, and an enhanced risk of schizophrenia have been reported.
Additionally, reduced reflex responses in the pre-pulse inhibition test, where a sub-threshold stimulus precedes a startle stimulus, have been established in both schizophrenic patients and in mice lacking CB2R.This warrants future experiments with adult zebrafsh lacking CB2R and structurally dissimilar CB2R antagonists to further examine the CB2R’s potential action in the phenotype of interest and psychosis. Promising support for the circular swimming mimicking schizophrenia-like symptoms was obtained in the sulpiride tests . Sulpiride is an atypical antipsychotic that inhibits central DA D2 receptors and acts to dampen the disorder’s DA hyperactivity. Both 10 μM and 100 μM sulpiride with 1 μM THC lowered the mean RI of 1 μM THC alone to a RI not significantly different from the controls . Importantly, sulpiride alone and with THC did not significantly influence the overall velocity of the fish . Atypical antipsychotics have been successful in reversing additional aspects of schizophrenia-like behavior, such as cognitive impairment and social withdrawal, induced by NMDAR antagonist MK-801 in zebrafsh and rats . One of the downstream effects of their serotonergic and dopaminergic antagonism is NMDAR activation via d-serine release in the PFC. Te polypharmacology of atypical antipsychotics may therefore explain their efficacy, by simultaneously targeting the DA hypothesis and the glutamate hypothesis of schizophrenia. Similarly, THC’s discussed mechanisms of action are also intertwined with both hypotheses, making it difficult to pinpoint a direct cause-effect relationship . Future co-treatments of THC with other atypical antipsychotics, such as clozapine, will further strengthen these notions. With any animal model of complex disorders and diseases there is always the question of face validity and construct validity, i.e., how well the model resembles and measures the illness. One approach to address the complexity issues in gene-behavior interactions is to focus on endophenotypes, which concentrate on a specific heritable characteristic and its circuitry such as the PPI deficit in schizophrenia. Future experiments to further strengthen the THC-induced behavioral stereotypy as an endophenotype of psychosis include tests in zebrafsh lacking CB2R or carrying mutations linked to psychosis or addiction. Another limitation of using a newly established analytical method is that it lacks validation across different data sets. Further optimization of our newly developed algorithm and machine learning would allow better detection and extraction of repetitive patterns and bridge the gap between distinct behavior detected by the human eye and patterns detected by the computer. Tailored RI measures for abnormal repetitive behaviors can greatly improve assays such as the current one and lay a foundation for an automated analysis with standardized behavioral endpoints. This in turn can assist in further validating the behavioral stereotypy as an endophenotype for THC-induced psychosis. From there, the search for its genetic underpinnings and pharmacological interventions can be pursued. In conclusion, zebrafsh engage in intriguing concentration-dependent swimming patterns when immersed in THC, which share characteristics with other animal models of drug induced psychosis- and schizophrenia-like behaviors.