Felipe V. Gomes

Adolescence as a period of vulnerability and intervention in schizophrenia: Insights from the MAM model.

Adolescence is a time of extensive neuroanatomical, functional and chemical reorganization of the brain, which parallels substantial maturational changes in behavior and cognition. Environmental factors that impinge on the timing of these developmental factors, including stress and drug exposure, increase the risk for psychiatric disorders. Indeed, antecedents to affective and psychotic disorders, which have clinical and pathophysiological overlap, are commonly associated with risk factors during adolescence that predispose to these disorders. In the context of schizophrenia, psychosis typically begins in late adolescence/early adulthood, which has been replicated by animal models. Rats exposed during gestational day (GD) 17 to the mitotoxin methylazoxymethanol acetate (MAM) exhibit behavioral, pharmacological, and anatomical characteristics consistent with an animal model of schizophrenia. Here we provide an overview of adolescent changes within the dopamine system and the PFC and review recent findings regarding the effects of stress and cannabis exposure during the peripubertal period as risk factors for the emergence of schizophrenia-like deficits. Finally, we discuss peripubertal interventions appearing to circumvent the emergence of adult schizophrenia-like deficits.

Effects of Pubertal Cannabinoid Administration on Attentional Set-Shifting and Dopaminergic Hyper-Responsivity in a Developmental Disruption Model of Schizophrenia

Background: Adolescent exposure to cannabinoids in vulnerable individuals is proposed to be a risk factor for psychiatric conditions later in life, particularly schizophrenia. Evidence from studies in animals has indicated that a combination of repeated pubertal cannabinoid administration with either neonatal prefrontocortical lesion, isolation rearing, or chronic NMDA receptor antagonism administration induces enhanced schizophrenia-like behavioral disruptions. The effects of adolescent exposure to CB1 receptor agonists, however, have not been tested in a developmental disruption model of schizophrenia. Methods: This was tested in the methylazoxymethanol (MAM) model, in which repeated treatment with the synthetic cannabinoid agonist WIN 55,212-2 (WIN; 1.2mg/kg) was extended over 25 days throughout puberty (postnatal days 40–65) in control and MAM rats. The rats received 20 injections, which were delivered irregularly to mimic the human condition. Adult rats were tested for attentional set-shifting task and locomotor response to amphetamine, which was compared with in vivo recording from ventral tegmental area (VTA) dopamine (DA) neurons. Results: MAM-treated rats showed impairment in the attentional set-shifting task, augmented locomotor response to amphetamine administration, and an increased number of spontaneously active DA neurons in the VTA. Interestingly, pubertal WIN treatment in normal animals induced similar changes at adulthood as those observed in MAM-treated rats, supporting the notion that adolescence exposure to cannabinoids may represent a risk factor for developing schizophrenia-like signs at adulthood. However, contrary to expectations, pubertal WIN administration did not exacerbate the behavioral and electrophysiological changes in MAM-treated rats beyond that observed in WIN-treated saline rats (Sal). Indeed, WIN treatment actually attenuated the locomotor response to amphetamine in MAM rats without impacting DA neuron activity states. Conclusions: Taken together, the present results indicate that the impact of cannabinoids during puberty/adolescence on schizophrenia models is more complex than may be predicted.

Plastic and Neuroprotective Mechanisms Involved in the Therapeutic Effects of Cannabidiol in Psychiatric Disorders

Beneficial effects of cannabidiol (CBD) have been described for a wide range of psychiatric disorders, including anxiety, psychosis, and depression. The mechanisms responsible for these effects, however, are still poorly understood. Similar to clinical antidepressant or atypical antipsychotic drugs, recent findings clearly indicate that CBD, either acutely or repeatedly administered, induces plastic changes. For example, CBD attenuates the decrease in hippocampal neurogenesis and dendrite spines density induced by chronic stress and prevents microglia activation and the decrease in the number of parvalbumin-positive GABA neurons in a pharmacological model of schizophrenia. More recently, it was found that CBD modulates cell fate regulatory pathways such as autophagy and others critical pathways for neuronal survival in neurodegenerative experimental models, suggesting the potential benefit of CBD treatment for psychiatric/cognitive symptoms associated with neurodegeneration. These changes and their possible association with CBD beneficial effects in psychiatric disorders are reviewed here.

Microglial Cells as a Link between Cannabinoids and the Immune Hypothesis of Psychiatric Disorders

Psychiatric disorders are one of the leading causes of disability worldwide. Although several therapeutic options are available, the exact mechanisms responsible for the genesis of these disorders remain to be fully elucidated. In the last decade, a body of evidence has supported the involvement of the immune system in the pathophysiology of these conditions. Microglial cells play a significant role in maintaining brain homeostasis and surveillance. Dysregulation of microglial functions has been associated with several psychiatric conditions. Cannabinoids regulate the brain–immune axis and inhibit microglial cell activation. Here, we summarized evidence supporting the hypothesis that microglial cells could be a target for cannabinoid influence on psychiatric disorders, such as anxiety, depression, schizophrenia, and stress-related disorders.

Prefrontal Cortex Dysfunction Increases Susceptibility to Schizophrenia-Like Changes Induced by Adolescent Stress Exposure.

Stress during adolescence is a risk factor for schizophrenia, and medial prefrontal cortex (mPFC) dysfunction is proposed to interfere with stress control, increasing the susceptibility to stress. We evaluated the impact of different stressful events during adolescence (restraint stress [RS], footshock [FS], or the combination of FS and RS) on behaviors correlated with schizophrenia in rats as adults. At adulthood, animals were tested for anxiety responses (elevated plus-maze), cognitive function (novel-object recognition test) and dopamine (DA) system responsivity (locomotor response to amphetamine and DA neuron activity in the ventral tegmental area (VTA) using in vivo electrophysiology). All adolescent stressors impaired weight gain and induced anxiety-like responses in adults. FS and FS + RS also disrupted cognitive function. Interestingly, only the combination of FS and RS induced a DA hyper-responsivity as indicated by augmented locomotor response to amphetamine and increased number of spontaneously active DA neurons which was confined to the lateral VTA. Additionally, prelimbic (pl) mPFC lesions triggered a DA hyper-responsivity in animals exposed to FS alone during adolescence. Our results are consistent with previous studies showing long-lasting changes induced by stressful events during adolescence. The impact on DA system activity, however, seems to depend on intense multiple stressors. Our data also suggest that plPFC dysfunction increases the vulnerability to stress in terms of changes in the DA system. Hence, stress during adolescence could be a precipitating factor for the transition to schizophrenia, and stress control at this vulnerable period may circumvent these changes to prevent the emergence of psychosis.

Adolescent Stress as a Driving Factor for Schizophrenia Development—A Basic Science Perspective

Schizophrenia has been associated with heightened stress responsivity in adolescence that precedes onset of psychosis. We now report that multiple stressors during adolescence in normal rats leads to deficits in adults analogous to that seen in schizophrenia patients. Moreover, impairment of stress control by lesion of the prelimbic prefontal cortex in adolescence caused previously subthreshold levels of stress to induce these deficit states when tested as adults. Thus, predisposition to stress hyper-responsivity, or exposure to substantial stressors, during adolescence can trigger a cascade of events that result in a schizophrenia-like profile in adults. This data can provide crucial information with respect to identifying markers for schizophrenia vulnerability early in life and, by mitigating the impact of stressors, prevent the transition to psychosis.

The Circuitry of Dopamine System Regulation and its Disruption in Schizophrenia: Insights Into Treatment and Prevention

Despite evidence for a role of the dopamine system in the pathophysiology of schizophrenia, there has not been substantial evidence that this disorder originates from a pathological change within the dopamine system itself. Current data from human imaging studies and preclinical investigations instead point to a disruption in afferent regulation of the dopamine system, with a focus on the hippocampus. We found that the hippocampus in the methylazoxymethanol acetate (MAM) rodent developmental disruption model of schizophrenia is hyperactive and dysrhythmic, possibly due to loss of parvalbumin interneurons, leading to a hyperresponsive dopamine system. Whereas current therapeutic approaches target dopamine receptor blockade, treatment at the site of pathology may be a more effective therapeutic avenue. This model also provided insights into potential means for prevention of schizophrenia. Specifically, given that stress is a risk factor in schizophrenia, and that stress can damage hippocampal parvalbumin interneurons, we tested whether alleviating stress early in life can effectively circumvent transition to schizophrenia-like states. Administering diazepam prepubertally at an antianxiety dose in MAM rats was effective at preventing the emergence of the hyperdopaminergic state in the adult. Moreover, multiple stressors applied to normal rats at the same time point resulted in pathology similar to the MAM rat. These data suggest that a genetic predisposition leading to stress hyper-responsivity, or exposure to substantial stressors, could be a primary factor leading to the emergence of schizophrenia later in life, and furthermore treating stress at a critical period may be effective in circumventing this transition.

Altered brain cannabinoid 1 receptor mRNA expression across postnatal development in the MAM model of schizophrenia

Altered cannabinoid 1 receptor (CB1R) expression has been reported in the brain of subjects with schizophrenia, a developmental mental illness that usually emerges in late adolescence/early adulthood. However, the developmental period at which changes in the CB1R expression appear in schizophrenia is unknown. To gain insight into this factor, we assessed the postnatal developmental trajectory of CB1R expression in the methylazoxymethanol (MAM) model of schizophrenia. Using in situ hybridization with film and grain analyses, CB1R messenger RNA (mRNA) levels were quantified in multiple brain regions, including the medial prefrontal cortex (mPFC), secondary motor cortex, dorsomedial and dorsolateral striatum, dorsal subregions and ventral subiculum of the hippocampus, of MAM-treated rats and normal controls at three developmental periods [juvenile - postnatal day (PD) 30; adolescence - PD45; and adulthood - PD85]. In all brain regions studied, CB1R mRNA levels were highest in juveniles and then decreased progressively toward adolescent and adult levels in control and MAM-treated rats. However, in MAM-treated rats, CB1R mRNA levels were lower in the mPFC at PD85 and higher in the dorsolateral striatum at PD45 and PD85 relative to controls. Cellular analyses confirmed the changes in CB1R mRNA expression in MAM-treated rats. These findings are in accordance with previous studies showing a decrease in the CB1R mRNA expression from juvenile period to adolescence to adulthood in cortical, striatal, and hippocampal regions. Additionally, similar to most of the schizophrenia-like signs observed in the MAM model, embryonic exposure to MAM leads to schizophrenia-related changes in CB1R mRNA expression that only emerge later in development.

13.2 CANNABIDIOL AS AN ANTIPSYCHOTIC DRUG

Abstract Background The phytocannabinoid cannabidiol (CBD) attenuates the psychotomimetic effects produced by high doses of delta-9-tetrahydrocannabinol (THC), the main component of the Cannabis sativa plant. Corroborating this effect, several preclinical and clinical studies indicate that CBD has antipsychotic properties. The mechanisms responsible for these properties, however, remain unknown (Campos et al., Philos Trans R Soc Lond B Biol Sci 367:3364–782013). We have recently found that repeated CBD administration prevents the behavioral impairments, measured in the pre-pulse inhibition, social interaction and novel object recognition tests, induced in mice by repeated treatment (28 days) with the NMDA receptor antagonist MK-801. CBD also prevented the neural (measured by delta-FosB) and microglia activation, and the decrease in the number of parvalbumin-positive neurons, observed in the medial prefrontal cortex (Gomes et al., Int J Neuropsychopharmacol 18(5)2014, Schizophr Res 164:155–63, 2015). Currently, we are investigating if CBD could also reverse these changes once they have been established and the possible mechanisms of this effect. Methods Male C57BL/6J mice received intraperitoneal injections of MK-801 (0.25, 0.5 or 1 mg/kg, twice a day) for 7 or 14 days. To determine if these treatments regime would induce acute and long-term deficits, the social interaction (SI) test was performed 1 or 8 days after the end of the MK-801 treatment. Twenty-four hours after the SI, animals were submitted to the novel object recognition (NOR) test. Having established that 14 days of MK-801 induce both acute (24 h after) and long-term (8 days after) behavioral deficits in the SI and NOR tests, we investigated if repeated treatment with CBD (15, 30 or 60 mg/kg daily, i.p.) would reverse these changes. CBD treatment began 24h after the end of the MK-801 treatment and lasted for 7 days. Repeated clozapine (1 mg/kg) was used as a positive control. Forty-eight hours after the last injection, animals were submitted to SI and, 24-h later, to the NOR test. In a second experiment, independent groups of mice received, before each CBD injection, AM251 (a CB1 receptor inverse agonist, 0.1–0.3 mg/kg), AM630 (a CB2 receptor inverse agonist, 0.1–0.3 mg/kg), or the 5HT1A receptor antagonist WAY100635 (0.1–0.3 mg/kg). The data were analyzed by ANOVA followed by the Newman-Keuls test. Results MK-801 (0.5 mg/kg) administration for 14 days, but not for 7 days, impaired SI and NOR. Repeated CBD or clozapine treatment reversed these impairments. CB1 and CB2 antagonists (AM251 and AM630, respectively) failed to change CBD effect. However, its effect was blocked by pretreatment with the 5HT1A receptor antagonist WAY100635. Discussion Our findings show that a 2-week treatment with the NMDA receptor antagonist MK801 impairs social interaction and novel object recognition, which have been associated with negative and cognitive symptoms of schizophrenia, respectively. These behavioral deficits last for at least one week and are reversed by the atypical antipsychotic clozapine or CBD, reinforcing the proposal that this latter drug has antipsychotic-like properties. CBD effects seem to depend on facilitation of 5HT1A-mediated neurotransmission.

O1.8. STRESS-INDUCED AMYGDALA HYPERACTIVITY LEADS TO INTERNEURON LOSS AND SCHIZOPHRENIA-LIKE PATHOLOGY IN A DEVELOPMENTAL DISRUPTION MODEL OF SCHIZOPHRENIA

Abstract Background Studies of the MAM rat model of schizophrenia show that rats exhibit higher anxiety levels, greater response to stress, and amygdala hyperactivity prepubertally before the emergence of the hyperdopaminergic state later in life. Furthermore, administration of diazepam prepubertally prevents this transition. These data suggest that MAM may predispose to psychosis via increased response to environmental stressors. If this is accurate, then one would predict that sufficiently strong stressors administered to normal rats during the critical period prepubertally would lead to psychosis-like state in the adult. Methods Rats are exposed to either 3 sessions of 1-hour restraint, 25 footshocks daily for 10 days, or both stressors delivered either at PD 31–40 (prepuberty) or PD 65–74 (adult) in intact rats and rats with prelimbic PFC (plPFC) lesions at PD25. Rats were tested for amphetamine locomotion, novel object recognition (NOR), and VTA DA neuron firing. Valproic acid (VPA) was administered to adults 5 days before and during the combined stressors to reopen the critical period. DREADD activation of the amygdala was also evaluated. Results While individual stressors prepubertally augmented anxiety and disrupted NOR in the adult, only the combined stressors resulted in amphetamine hyperlocomotion and increased DA neuron population activity similar to the MAM rats. plPFC lesions enabled the footshock alone to lead to anxiety, NOR deficits, and the hyperdopaminergic state in the adult. Given that stress is known to impact parvalbumin (PV) interneurons in the hippocampus when administered during the critical period in prepubertal rats, we tested the impact of opening the critical period in the adult rats with VPA. Normal rats given combined stressors at PD 65–74 showed attenuated DA neuron activity similar to that observed in depression models. However, administration of VPA caused the combined stressors to lead to the schizophrenia phenotype. This was accompanied by hippocampal hyperactivity driven by an overactive amygdala, since DREADD activation of the amygdala produced similar effects. Discussion These data suggest that factors that increase the response to environmental stressors during the prepubertal critical period lead to activation of the stress-activated amygdala-hippocampal pathway and PV interneuron loss, which leads to the hyperdopaminergic state in the adult thought to underlie psychosis. Furthermore, re-opening the critical period in the adult makes the adult sensitive to stress-induced psychosis. This suggests that controlling the impact of stress early in life in susceptible individuals may be an effective means to circumvent the transition to psychosis later in life.