Daniel Thomas Malone

Adolescent cannabis use and psychosis: epidemiology and neurodevelopmental models

Cannabis is one of the most widely used illicit drugs among adolescents, and most users first experiment with it in adolescence. Adolescence is a critical phase for brain development, characterized by neuronal maturation and rearrangement processes, such as myelination, synaptic pruning and dendritic plasticity. The endocannabinoid system plays an important role in fundamental brain developmental processes such as neuronal cell proliferation, migration and differentiation. Therefore changes in endocannabinoid activity during this specific developmental phase, induced by the psychoactive component of marijuana, Δ9‐tetrahydrocannabinol, might lead to subtle but lasting neurobiological changes that can affect brain functions and behaviour. In this review, we outline recent research into the endocannabinoid system focusing on the relationships between adolescent exposure to cannabinoids and increased risk for certain neuropsychiatric diseases such as schizophrenia, as highlighted by both human and animal studies. Particular emphasis will be given to the possible mechanisms by which adolescent cannabis consumption could render a person more susceptible to developing psychoses such as schizophrenia.

Biased Agonism and Biased Allosteric Modulation at the CB1 Cannabinoid Receptor.

CB1 cannabinoid receptors (CB1Rs) are attractive therapeutic targets for numerous central nervous system disorders. However, clinical application of cannabinoid ligands has been hampered owing to their adverse on-target effects. Ligand-biased signaling from, and allosteric modulation of, CB1Rs offer pharmacological approaches that may enable the development of improved CB1R drugs, through modulation of only therapeutically desirable CB1R signaling pathways. There is growing evidence that CB1Rs are subject to ligand-biased signaling and allosterism. Therefore, in the present study, we quantified ligand-biased signaling and allosteric modulation at CB1Rs. Cannabinoid agonists displayed distinct biased signaling profiles at CB1Rs. For instance, whereas 2-arachidonylglycerol and WIN55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone] showed little preference for inhibition of cAMP and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2), N-arachidonoylethanolamine (anandamide), methanandamide, CP55940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol], and HU-210 [11-hydroxy-Δ8-THC-dimethylheptyl] were biased toward cAMP inhibition. The small-molecule allosteric modulator Org27569 [5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide] displayed biased allosteric effects by blocking cAMP inhibition mediated by all cannabinoid ligands tested, at the same time having little or no effect on ERK1/2 phosphorylation mediated by a subset of these ligands. Org27569 also displayed negative binding cooperativity with [3H]SR141716A [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide]; however, it had minimal effects on binding of cannabinoid agonists. Furthermore, we highlight the need to validate the reported allosteric effects of the endogenous ligands lipoxin A4 and pregnenolone at CB1Rs. Pregnenolone but not lipoxin A4 displaced [3H]SR141716A, but there was no functional interaction between either of these ligands and cannabinoid agonists. This study demonstrates an approach to validating and quantifying ligand-biased signaling and allosteric modulation at CB1Rs, revealing ligand-biased “fingerprints” that may ultimately allow the development of improved CB1R-targeted therapies.

Cannabidiol reverses the reduction in social interaction produced by low dose Δ9-tetrahydrocannabinol in rats

While Delta(9)-tetrahydrocannabinol (THC) is the main psychoactive constituent of the cannabis plant, a non-psychoactive constituent is cannabidiol (CBD). CBD has been implicated as a potential treatment of a number of disorders including schizophrenia and epilepsy and has been included with THC in a 1:1 combination for the treatment of conditions such as neuropathic pain. This study investigated the effect of THC and CBD, alone or in combination, on some objective behaviours of rats in the open field. Pairs of rats were injected with CBD or vehicle followed by THC or vehicle and behaviour in the open field was assessed for 10 min. In vehicle pretreated rats THC (1 mg/kg) significantly reduced social interaction between rat pairs. Treatment with CBD had no significant effect alone, but pretreatment with CBD (20 mg/kg) reversed the THC-induced decreases in social interaction. A higher dose of THC (10 mg/kg) produced no significant effect on social interaction. However, the combination of high dose CBD and high dose THC significantly reduced social interaction between rat pairs, as well as producing a significant decrease in locomotor activity. This data suggests that CBD can reverse social withdrawal induced by low dose THC, but the combination of high dose THC and CBD impairs social interaction, possibly by decreasing locomotor activity.

Effect of cannabidiol in a MK-801-rodent model of aspects of Schizophrenia

Cannabidiol is a non-psychoactive phytocannabinoid which, based on several previous preclinical and clinical reports, is purported to have antipsychotic potential. The purpose of this investigation was to further investigate if these effects would be seen using an MK-801-induced rat model of aspects of schizophrenia. MK-801 is an NMDA receptor-antagonist known to produce hyperactivity, deficits in prepulse inhibition and social withdrawal, behaviours which correlate well with some of the positive, cognitive and negative symptoms of schizophrenia. Following a 4-day acclimatisation to the holding room, rats were acclimatised to startle chambers on day 5 and their prepulse inhibition (PPI) determined on day 6 following treatment with cannabidiol or vehicle and MK-801 or vehicle. On day 9, rats were acclimatised to the social interaction testing arena and on day 10, were tested for social interaction and locomotor activity following the same treatments. Cannabidiol treatment alone disrupted PPI and produced hyperactivity but had no effect on social behaviour. Cannabidiol had no effect on MK-801-induced disruption of PPI or hyperactivity but showed potential towards inhibiting MK-801-induced social withdrawal. As a comparator, we also tested the effect of the atypical antipsychotic clozapine which only partially reversed MK-801-induced disruption of PPI but was able to reverse MK-801-induced hyperactivity and social withdrawal. In conclusion, cannabidiol showed both propsychotic activity and partial antipsychotic activity in an MK-801-induced model of aspects of schizophrenia. Further behavioural studies would be required using a range of species, strains, animal models and testing paradigms to conclusively establish the antipsychotic potential of cannabidiol.

Consequences of early life MK-801 administration: Long-term behavioural effects and relevance to schizophrenia research

Animal models contribute significantly to advancing the understanding of schizophrenia neurobiology, in addition to being an important tool for the screening of antipsychotic potential of new compounds. However, the entire spectrum or all the symptoms manifested in schizophrenia cannot be straightforwardly reproduced in animals due to the complexity of the disorder, difference in mental capacities and behaviours, and the ability to quantify or measure the changes. Blockade of the NMDA receptor by the use of MK-801, a non-competitive NMDA receptor antagonist, during the early postnatal period has been proposed to be an experimental model which induces behavioural changes that mimic several aspects of the disorder. The long term behavioural profile arising from this early life manipulation is reviewed herein, with a specific focus on behaviours relevant to a schizophrenia-like condition. Some of the reported neurochemical changes are also compiled. Although this method may be suitable to model some aspects of schizophrenia in rodents, there are unmet areas which need to be addressed, notably the characterisation of its predictive value.

Current pharmacological models of social withdrawal in rats: relevance to schizophrenia.

Social dysfunction in schizophrenia is one of the core negative symptoms, which to date is not adequately addressed by treatment with both typical and atypical antipsychotics. A number of different pharmacological models of social withdrawal are used to mimic social dysfunction in rats, such as amphetamine, N-methyl-D-aspartic acid antagonists, cannabinergic and serotonergic receptor ligands. The purpose of this review is to discuss and compare these models of social withdrawal with a focus on their face, construct and predictive validities. Various techniques and strategies used to observe and analyze rodent social behaviour and other factors that are of relevance to this paradigm have also been examined. After comparing the reports, we are of the opinion that to improve replicability of any given model and its antipsychotic screening potential and the reliability of comparisons made, efforts need to be directed towards cross-laboratory standardization of variables that may confound experimental outcomes and cause discrepancies in results reported. In keeping with an earlier suggestion this may be facilitated through the creation of an online consortium for behavioural neuroscientists to share and compare methodologies, laboratory layouts and perhaps even raw data.

Cannabidiol and clozapine reverse MK-801-induced deficits in social interaction and hyperactivity in Sprague–Dawley rats:

Recently, a novel paradigm has been designed to assess social investigative behaviour in pairs of Sprague–Dawley rats, which involves physical separation whilst ensuring they are able to maintain contact through other social cues. We have modified this set-up in order to assess not just social behaviour but also locomotor activity of the rats. Results showed that the MK-801- (0.3 mg/kg) treated rats displayed reduced social investigative behaviour, hyperactivity as well as reduced attention span. Pretreatment with the phytocannabinoid cannabidiol (3 mg/kg) not only normalised social investigative behaviour but increased it beyond control levels. Pretreatment with clozapine (1, 3 mg/kg) also normalised social investigative behaviour. Both cannabidiol and clozapine inhibited MK-801-induced hyperactivity. However, there were no effects of pretreatment on impairments to attention span. Our findings reinforce several aspects of the validity of the MK-801-induced model of social withdrawal and hyperactivity and also support the use of this novel set-up for further investigations to assess the antipsychotic potential of novel compounds.

A two-hit model: behavioural investigation of the effect of combined neonatal MK-801 administration and isolation rearing in the rat.

This study combined two neurodevelopmental manipulations, neonatal MK-801 treatment and isolation rearing, to produce a ‘two-hit’ model and determine whether two hits induce a more robust behavioural phenotype of an animal model of aspects of schizophrenia compared with individual manipulations alone. The effect of clozapine was also assessed. Male Sprague-Dawley rats received 0.2 mg/kg MK-801 or saline intraperitoneally (i.p.) once daily on postnatal days (PNDs) 7–10 and were assigned to group or isolation rearing at weaning (PND 21). From PND 77, they received a vehicle or 5 mg/kg clozapine (i.p.) treatment regimen and were subjected to three prepulse inhibition (PPI) tests, a locomotor activity assessment and a novel object recognition task. MK-801-treated rats reared in isolation displayed robust PPI disruptions which were consistently manifested in all three tests. PPI deficits were also detected in saline-treated rats reared in isolation but not in all tests. Only the two-hit rats demonstrated hyperlocomotion and impaired object recognition memory. Clozapine restored PPI anomalies in the two-hit rats. The two-hit model showed greater psychotic-like effects than either neonatal MK-801 or isolation rearing alone. The preliminary predictive validity shown with clozapine suggests this model may be useful for predicting the efficacy of putative antipsychotics.

Male contraception via simultaneous knockout of α1A-adrenoceptors and P2X1-purinoceptors in mice

Significance The search for a viable male contraceptive target has been a medical challenge for many years. Most strategies have focused on hormonal or germ-line strategies to produce dysfunctional sperm that are incapable of fertilization. The problem with such approaches is that they have intolerable side effects such as affecting male sexual activity or causing long-term irreversible effects on fertility. In addition, some strategies may transmit detrimental changes to future offspring. This manuscript describes a male contraceptive target within the autonomic nervous system, which would not affect the long-term viability of sperm nor the sexual or general health of males. In addition, due to the nature of the target, the contraceptive has the potential to be orally administered. Therapeutic targets for male contraception are associated with numerous problems due to their focus on disrupting spermatogenesis or hormonal mechanisms to produce dysfunctional sperm. Here we describe the dual genetic deletion of α1A-adrenergic G protein-coupled receptors (adrenoceptors) and P2X1-purinoceptor ligand gated ion channels in male mice, thereby blocking sympathetically mediated sperm transport through the vas deferens during the emission phase of ejaculation. This modification produced 100% infertility without effects on sexual behavior or function. Sperm taken from the cauda epididymides of double knockout mice were microscopically normal and motile. Furthermore, double knockout sperm were capable of producing normal offspring following intracytoplasmic sperm injection into wild-type ova and implantation of the fertilized eggs into foster mothers. Blood pressure and baroreflex function was reduced in double knockout mice, but no more than single knockout of α1A-adrenoceptors alone. These results suggest that this autonomic method of male contraception appears free of major physiological and behavioral side effects. In addition, they provide conclusive proof of concept that pharmacological antagonism of the P2X1-purinoceptor and α1A-adrenoceptor provides a safe and effective therapeutic target for a nonhormonal, readily reversible male contraceptive.

Effect of testing conditions on the propsychotic action of MK-801 on prepulse inhibition, social behaviour and locomotor activity.

The present paper reports on two investigations designed with the aim of refining existing animal models representing several aspects of psychosis, used to evaluate antipsychotic potential of novel compounds. The aim of the first investigation was to determine the effect of habituating rats to the injection procedure on three behavioural testing paradigms, social interaction, locomotor activity and prepulse inhibition (PPI) of the acoustic startle response. Results showed that while there was no effect on social behaviour or locomotor activity, the habituating to injection procedure decreased startle magnitude. For the second study, the aim was to determine whether the order in which the tests were conducted would affect sensitivity to the effects of dizocilpine (MK-801), a non-competitive NMDA receptor antagonist known to induce social withdrawal, increase locomotor activity and disrupt PPI. Either social interaction or locomotor activity tests were carried out 3 days prior to PPI tests (protocol 1), or PPI tests were carried out 3 days prior to social interaction and locomotor activity tests (protocol 2). Results showed that protocol 2 rats were more sensitive to the social withdrawal-inducing, hyperlocomotive- and PPI-disruptive effects of MK-801. Based on these results, the testing conditions appear to have a significant influence on the outcome of experiments aimed at observing the propsychotic action of MK-801.