A high seizure burden increases several prostaglandin species in the hippocampus of a Scn1a+/- mouse model of Dravet syndrome.

Dravet syndrome is an intractable epilepsy with a high seizure burden that is resistant to current anti-seizure medications. There is evidence that neuroinflammation plays a role in epilepsy and seizures, however few studies have specifically examined neuroinflammation in Dravet syndrome under conditions of a higher seizure burden. Here we used an established genetic mouse model of Dravet syndrome (Scn1a+/- mice), to examine whether a higher seizure burden impacts the number and morphology of microglia in the hippocampus. Moreover, we examined whether a high seizure burden influences classical inflammatory mediators in this brain region. Scn1a+/- mice with a high seizure burden induced by thermal priming displayed a localised reduction in microglial cell density in the granule cell layer and subgranular zone of the dentate gyrus, regions important to postnatal neurogenesis. However, microglial cell number and morphology remained unchanged in other hippocampal subfields. The high seizure burden in Scn1a+/- mice did not affect hippocampal mRNA expression of classical inflammatory mediators such as interleukin 1ß and tumour necrosis factor α, but increased cyclooxygenase 2 (COX-2) expression. We then quantified hippocampal levels of prostanoids that arise from COX-2 mediated metabolism of fatty acids and found that Scn1a+/- mice with a high seizure burden displayed increased hippocampal concentrations of numerous prostaglandins, notably PGF2α, PGE2, PGD2, and 6-K-PGF1A, compared to Scn1a+/- mice with a low seizure burden. In conclusion, a high seizure burden increased hippocampal concentrations of various prostaglandin mediators in a mouse model of Dravet syndrome. Future studies could interrogate the prostaglandin pathways to further better understand their role in the pathophysiology of Dravet syndrome.

Medical cannabis use in Australia seven years after legalisation: findings from the online Cannabis as Medicine Survey 2022-2023 (CAMS-22).

BACKGROUND: Cannabis was legalised for medical purposes in 2016. Uptake was initially slow, but since 2019 there has been a large increase in the number of Australians who have been prescribed cannabis for medical reasons. Yet a significant number of consumers continue to treat their medical conditions via illicitly-sourced cannabis. Little is known about how these two groups of medical cannabis consumers differ. METHODS: The anonymous Cannabis-As-Medicine Survey 2022-2023 (CAMS-22) was available for completion online from December 2022 to April 2023 to adult Australians who had used cannabis to treat a medical condition in the previous year. Recruitment occurred through social media, consumer forums, and medical practices. Questions included demographic characteristics, patterns of cannabis use, conditions treated, and self-rated effectiveness. RESULTS: Of the 3323 respondents included in these analyses, 2352 (73%) mainly used prescribed medical cannabis, 871 (27%) mainly used illicit. Prescribed users were significantly more likely than illicit users to have had their health condition diagnosed (OR = 1.7, 95% CI 1.3, 2.2), to consume their cannabis via oral (OR = 1.9; CI 1.5, 2.4) or vaporised (OR = 5.2; CI 4.0, 6.8) routes, and to be sure of the composition of their medical cannabis (OR = 25.0; CI 16.7, 50.0). Prescribed users were significantly less likely to have used cannabis non-medically before medical use (OR = 0.6, CI 0.5, 0.7), consume cannabis via smoked routes (OR = 0.2, CI 0.1, 0.2), and to report any side effects (OR = 0.1; CI 0.1, 0.2). The most common conditions among both prescribed and illicit users were pain (37%), mental health (36%), and sleep (15%) conditions. Prescribed users were significantly more likely to use cannabis to mainly treat a pain (OR = 1.3; CI 1.1, 1.5) or sleep condition (OR = 1.4; CI 1.1, 1.7) and less likely to treat a mental health condition (OR = 0.8; CI 0.7, 0.9). There were no between-group differences in effectiveness with 97% saying medical cannabis had improved their symptoms. CONCLUSIONS: From a harm-reduction perspective there is much to recommend prescribed medical cannabis; it has fewer side-effects than illicit, is used more safely (oral or vaporised versus smoked routes), gives consumers greater certainty regarding the composition and quality of their medicine, and does not risk exposure to the criminal justice system. Of concern, however, is the apparent willingness of prescribers to prescribe for indications for which there is limited evidence of efficacy, such as mental health and sleep conditions.

Medical cannabis use in Australia: consumer experiences from the online cannabis as medicine survey 2020 (CAMS-20).

BACKGROUND: Australia has had a framework for legal medicinal cannabis since 2016, yet prior online surveys in 2016 and 2018 indicated that most consumers continued to use illicit medical cannabis products. Regulatory data indicate an increase in the prescription of medicinal cannabis since 2019, and this survey examines consumer experiences of prescribed and illicit medical cannabis (MC) use in Australia. METHODS: A cross-sectional anonymous online survey was administered September 2020 to January 2021. Recruitment via social media, professional and consumer forums, and medical practices. Participant eligibility: ≥ 18 years; used a cannabis product for self-identified medical reason(s) in the past year, and resident in Australia. Outcome measures included consumer characteristics, conditions treated, source and patterns of MC use, and perspectives on accessing MC. RESULTS: Of the 1600 participants (mean age 46.4 ± 14.3 years, 53% male), 62.4% (n = 999) reported using only illicit and 37.6% (n = 601) used prescribed MC in the past year. MC was used on a median of 28 (IQR: 12, 28) of the past 28 days and cost $AUD 74 ± 72 weekly (median = $40, IQR: $7, $100). Prescribed participants were more likely to treat pain conditions than those using illicit MC (52% v 40%, OR = 1.7, 1.3-2.1) and less likely to treat sleep conditions (6% v 11%, OR = 0.5, 0.3-0.8), with mental health conditions also a common indication in both groups (26%, 31%). Prescribed MC was consumed predominately by oral routes (72%), whereas illicit MC was most commonly smoked (41%). Prescribed MC was 'mainly THC' (26%), 'equal THC/CBD' (40%), 'mainly CBD' (31%) and 'uncertain' (3%), while 34% of those using illicit MC were 'uncertain' of the cannabinoid profile. Cost and difficulties finding medical practitioners to prescribe remain significant barriers to accessing prescribed MC, and few (10.8%) described the existing model for accessing prescribed MC as 'straightforward or easy'. CONCLUSIONS: There has been a notable shift from illicit to prescribed MC by many consumers compared to prior surveys. Consumers using prescribed MC reported a range of advantages compared to illicit MC, including safer routes of administration, and greater certainty regarding access and composition of products.

Citalopram and Cannabidiol: In Vitro and In Vivo Evidence of Pharmacokinetic Interactions Relevant to the Treatment of Anxiety Disorders in Young People.

BACKGROUND: Cannabidiol (CBD), a major nonintoxicating constituent of cannabis, exhibits anxiolytic properties in preclinical and human studies and is of interest as a novel intervention for treating anxiety disorders. Existing first-line pharmacotherapies for these disorders include selective serotonin reuptake inhibitor and other antidepressants. Cannabidiol has well-described inhibitory action on cytochrome P450 (CYP450) drug-metabolizing enzymes and significant drug-drug interactions (DDIs) between CBD and various anticonvulsant medications (eg, clobazam) have been described in the treatment of epilepsy. Here, we examined the likelihood of DDIs when CBD is added to medications prescribed in the treatment of anxiety. METHODS: The effect of CBD on CYP450-mediated metabolism of the commonly used antidepressants fluoxetine, sertraline, citalopram, and mirtazapine were examined in vitro. Cannabidiol-citalopram interactions were also examined in vivo in patients (n = 6) with anxiety disorders on stable treatment with citalopram or escitalopram who received ascending daily doses of adjunctive CBD (200-800 mg) over 12 weeks in a recent clinical trial. RESULTS: Cannabidiol minimally affected the metabolism of sertraline, fluoxetine, and mirtazapine in vitro. However, CBD significantly inhibited CYP3A4 and CYP2C19-mediated metabolism of citalopram and its stereoisomer escitalopram at physiologically relevant concentrations, suggesting a possible in vivo DDI. In patients on citalopram or escitalopram, the addition of CBD significantly increased citalopram plasma concentrations, although it was uncertain whether this also increased selective serotonin reuptake inhibitor-mediated adverse events. CONCLUSIONS: Further pharmacokinetic examination of the interaction between CBD and citalopram/escitalopram is clearly warranted, and clinicians should be vigilant around the possibility of treatment-emergent adverse effects when CBD is introduced to patients taking these antidepressants.

The effect of daily aerobic cycling exercise on sleep quality during inpatient cannabis withdrawal: A randomised controlled trial.

Sleep disturbance is a common symptom encountered by cannabis-dependent individuals abstaining from cannabis use. In the present study, we investigated the effect of daily aerobic cycling exercise versus control stretching on sleep quality during inpatient cannabis withdrawal in treatment-seeking dependent cannabis users. The protocol incorporated three consecutive phases: a 4-Day (4-Night) (at-home) 'Baseline' phase, a 6-Day (5-Night) 'Treatment' phase (within a 7-Day inpatient hospital stay) and a 3-Day (4-Night) (at-home) 'Post-Treatment' phase. Participants performed 35 min of monitored activity per day during the Treatment phase. The intervention group (n = 19) cycled at ~60% aerobic capacity (VO2max ), while the control group (n = 12) performed a stretching routine. Objective sleep quality was measured nightly throughout the study using wrist actigraphy ratings of subjective sleep quality were also recorded during the Treatment phase. There were no group differences in sleep measures during the Baseline phase (all p > .05). Objective sleep onset latency increased from the Baseline to the Treatment phase in the control (stretching) group (p = .042). In contrast, the Cycling group exhibited improvements in sleep duration (p = .008) and sleep efficiency (p = .023) during the Treatment phase compared to the Baseline phase. Cycling also increased sleep duration (p = .005), decreased average wake bout (p = .040) and tended to increase sleep efficiency (p = .051) compared to stretching during the Treatment phase. Subjective sleep quality ratings did not differ between groups (p > .10). These preliminary findings suggest that moderate-intensity aerobic exercise may attenuate the sleep disturbances associated with cannabis withdrawal.

Cannabichromene and Δ9-Tetrahydrocannabinolic Acid Identified as Lactate Dehydrogenase-A Inhibitors by in Silico and in Vitro Screening.

Cannabis sativa contains >120 phytocannabinoids, but our understanding of these compounds is limited. Determining the molecular modes of action of the phytocannabinoids may assist in their therapeutic development. Ligand-based virtual screening was used to suggest novel protein targets for phytocannabinoids. The similarity ensemble approach, a virtual screening tool, was applied to target identification for the phytocannabinoids as a class and predicted a possible interaction with the lactate dehydrogenase (LDH) family of enzymes. In order to evaluate this in silico prediction, a panel of 18 phytocannabinoids was screened against two LDH isozymes (LDHA and LDHB) in vitro. Cannabichromene (CBC) and Δ9-tetrahydrocannabinolic acid (Δ9-THCA) inhibited LDHA via a noncompetitive mode of inhibition with respect to pyruvate, with Ki values of 8.5 and 6.5 µM, respectively. In silico modeling was then used to predict the binding site for CBC and Δ9-THCA. Both were proposed to bind within the nicotinamide pocket, overlapping the binding site of the cofactor NADH, which is consistent with the noncompetitive modes of inhibition. Stemming from our in silico screen, CBC and Δ9-THCA were identified as inhibitors of LDHA, a novel molecular target that may contribute to their therapeutic effects.

Functional genomics of epilepsy-associated mutations in the GABAA receptor subunits reveal that one mutation impairs function and two are catastrophic.

A number of epilepsy-causing mutations have recently been identified in the genes of the α1, ß3, and γ2 subunits comprising the γ-aminobutyric acid type A (GABAA) receptor. These mutations are typically dominant, and in certain cases, such as the α1 and ß3 subunits, they may lead to a mix of receptors at the cell surface that contain no mutant subunits, a single mutated subunit, or two mutated subunits. To determine the effects of mutations in a single subunit or in two subunits on receptor activation, we created a concatenated protein assembly that links all five subunits of the α1ß3γ2 receptor and expresses them in the correct orientation. We created nine separate receptor variants with a single-mutant subunit and four receptors containing two subunits of the γ2R323Q, ß3D120N, ß3T157M, ß3Y302C, and ß3S254F epilepsy-causing mutations. We found that the singly mutated γ2R323Q subunit impairs GABA activation of the receptor by reducing GABA potency. A single ß3D120N, ß3T157M, or ß3Y302C mutation also substantially impaired receptor activation, and two copies of these mutants within a receptor were catastrophic. Of note, an effect of the ß3S254F mutation on GABA potency depended on the location of this mutant subunit within the receptor, possibly because of the membrane environment surrounding the transmembrane region of the receptor. Our results highlight that precise functional genomic analyses of GABAA receptor mutations using concatenated constructs can identify receptors with an intermediate phenotype that contribute to epileptic phenotypes and that are potential drug targets for precision medicine approaches.

Cannabidiol disrupts conditioned fear expression and cannabidiolic acid reduces trauma-induced anxiety-related behaviour in mice.

The major phytocannabinoid cannabidiol (CBD) has anxiolytic properties and lacks tetrahydrocannabinol-like psychoactivity. Cannabidiolic acid (CBDA) is the acidic precursor to CBD, and this compound appears more potent than CBD in animal models of emesis, pain and epilepsy. In this short report, we aimed to examine whether CBDA is more potent than CBD in disrupting expression of conditioned fear and generalised anxiety-related behaviour induced by Pavlovian fear conditioning. Mice underwent fear conditioning and 24 h later were administered CBD and CBDA before testing for fear expression and generalized anxiety-like behaviour. We found that CBD and CBDA had dissociable effects; while CBD but not CBDA disrupted cued fear memory expression, CBDA but not CBD normalized trauma-induced generalized anxiety-related behaviour. Neither phytocannabinoid affected contextual fear expression. Our findings form the basis for future experiments examining whether phytocannabinoids, alone and in combination, are effective in these mouse models of fear and anxiety.

Medical cannabis use in the Australian community following introduction of legal access: the 2018-2019 Online Cross-Sectional Cannabis as Medicine Survey (CAMS-18).

BACKGROUND: In 2016, the Australian federal government passed legislation enabling a range of cannabis-based products to be prescribed to patients by registered healthcare professionals. An online survey conducted immediately prior to these legislative changes found that the vast majority of respondents at the time were illicitly sourcing cannabis plant matter, smoking was the preferred route of administration and mental health, chronic pain, and sleep conditions were the most frequently cited reasons for medical cannabis use. This manuscript reports the results of a follow-up survey conducted in 2018-2019, the Cannabis As Medicine Survey (CAMS-18). The goal of this second questionnaire was to examine patterns of use and consumer perspectives regarding medical cannabis use in Australia, 2 years after the introduction of legal access pathways. METHODS: Anonymous online cross-sectional survey with convenience sample, recruited mainly through online media between September 2018 and March 2019. Participants were adults (18 years or over) residing in Australia who reported using a cannabis product for self-identified therapeutic reasons during the preceding 12 months. The survey measured consumer characteristics, indications and patterns of medical cannabis use, routes and frequency of administration, perceived benefits and harms, experiences and preferred models of access to medical cannabis. RESULTS: Data were available for 1388 respondents. The main categories of condition being treated with medical cannabis were pain (36.4%), mental health (32.8%), sleep (9.2%), neurological (5.2%) and cancer (3.8%). Respondents reported using medical cannabis on 15.8 (11.2) days in the past 28, by inhaled (71.4%) or oral (26.5%) routes and spending AUD$82.27 ($101.27) per week. There were high levels of self-reported effectiveness, but also high rates of side effects. There was uncertainty regarding the composition of illicit cannabinoid products and concerns regarding their possible contamination. Few respondents (2.7%) had accessed legally prescribed medical cannabis, with the main perceived barriers being cost, disinterest from the medical profession and stigma regarding cannabis use. CONCLUSIONS: Chronic pain, mental health and sleep remain the main clinical conditions for which consumers report using medical cannabis. Despite 2 years of legal availability, most consumers in Australia reported accessing illicit cannabis products, with uncertainty regarding the quality or composition of cannabis products.

Prescribing medicinal cannabis.

The Australian Federal Government legalised access to medicinal cannabis in 2016 More than 100 different cannabis products are now available to prescribe. Most are oral preparations (oils) or capsules containing delta-9-tetrahydrocannabinol or cannabidiol. Dried-flower products are also available As most products are unregistered drugs, prescribing requires approval under the Therapeutic Goods Administration Special Access Scheme-B or Authorised Prescriber Scheme Special Access Scheme Category B applications can be made online, with approval usually being given within 24­48 hours. However, supply chain problems may delay dispensing by the pharmacy By the end of 2019, over 28,000 prescribing approvals had been issued to patients, involving more than 1400 doctors, mostly GPs. More than 70,000 approvals are projected by the end of 2020 Most prescriptions are for chronic non-cancer pain, anxiety, cancer-related symptoms, epilepsy and other neurological disorders. However, the evidence supporting some indications is limited Many doctors are cautious about prescribing cannabis. While serious adverse events are rare, there are legitimate concerns around driving, cognitive impairment and drug dependence with products containing delta-9-tetrahydrocannabinol. Cannabidiol-only products pose fewer risks

Microglial cell hyper-ramification and neuronal dendritic spine loss in the hippocampus and medial prefrontal cortex in a mouse model of PTSD.

Few animal models exist that successfully reproduce several core associative and non-associative behaviours relevant to post-traumatic stress disorder (PTSD), such as long-lasting fear reactions, hyperarousal, and subtle attentional and cognitive dysfunction. As such, these models may lack the face validity required to adequately model pathophysiological features of PTSD such as CNS grey matter loss and neuroinflammation. Here we aimed to investigate in a mouse model of PTSD whether contextual fear conditioning associated with a relatively high intensity footshock exposure induces loss of neuronal dendritic spines in various corticolimbic brain regions, as their regression may help explain grey matter reductions in PTSD patients. Further, we aimed to observe whether these changes were accompanied by alterations in microglial cell number and morphology, and increased expression of complement factors implicated in the mediation of microglial cell-mediated engulfment of dendritic spines. Adult male C57Bl6J mice were exposed to a single electric footshock and subsequently underwent phenotyping of various PTSD-relevant behaviours in the short (day 2-4) and longer-term (day 29-31). 32 days post-exposure the brains of these animals were subjected to Golgi staining of dendritic spines, microglial cell Iba-1 immunohistochemistry and immunofluorescent staining of the complement factors C1q and C4. Shock exposure promoted a lasting contextual fear response, decreased locomotor activity, exaggerated acoustic startle responses indicative of hyperarousal, and a short-term facilitation of sensorimotor gating function. The shock triggered loss of dendritic spines on pyramidal neurons was accompanied by increased microglial cell number and complexity in the medial prefrontal cortex and dorsal hippocampus, but not in the amygdala. Shock also increased expression of C1q in the pyramidal layer of the CA1 region of the hippocampus but not in other brain regions. The present study further elaborates on the face and construct validity of a mouse model of PTSD and provides a good foundation to explore potential molecular interactions between microglia and dendritic spines.

Coadministered cannabidiol and clobazam: Preclinical evidence for both pharmacodynamic and pharmacokinetic interactions.

OBJECTIVE: Cannabidiol (CBD) has been approved by the US Food and Drug Administration (FDA) to treat intractable childhood epilepsies, such as Dravet syndrome and Lennox-Gastaut syndrome. However, the intrinsic anticonvulsant activity of CBD has been questioned due to a pharmacokinetic interaction between CBD and a first-line medication, clobazam. This recognized interaction has led to speculation that the anticonvulsant efficacy of CBD may simply reflect CBD augmenting clobazam exposure. The present study aimed to address the nature of the interaction between CBD and clobazam. METHODS: We examined whether CBD inhibits human CYP3A4 and CYP2C19 mediated metabolism of clobazam and N-desmethylclobazam (N-CLB), respectively, and performed studies assessing the effects of CBD on brain and plasma pharmacokinetics of clobazam in mice. We then used the Scn1a+/- mouse model of Dravet syndrome to examine how CBD and clobazam interact. We compared anticonvulsant effects of CBD-clobazam combination therapy to monotherapy against thermally-induced seizures, spontaneous seizures and mortality in Scn1a+/- mice. In addition, we used Xenopus oocytes expressing γ-aminobutyric acid (GABA)A receptors to investigate the activity of GABAA receptors when treated with CBD and clobazam together. RESULTS: CBD potently inhibited CYP3A4 mediated metabolism of clobazam and CYP2C19 mediated metabolism of N-CLB. Combination CBD-clobazam treatment resulted in greater anticonvulsant efficacy in Scn1a+/- mice, but only when an anticonvulsant dose of CBD was used. It is important to note that a sub-anticonvulsant dose of CBD did not promote greater anticonvulsant effects despite increasing plasma clobazam concentrations. In addition, we delineated a novel pharmacodynamic mechanism where CBD and clobazam together enhanced inhibitory GABAA receptor activation. SIGNIFICANCE: Our study highlights the involvement of both pharmacodynamic and pharmacokinetic interactions between CBD and clobazam that may contribute to its efficacy in Dravet syndrome.

Pharmacokinetics of Phytocannabinoid Acids and Anticonvulsant Effect of Cannabidiolic Acid in a Mouse Model of Dravet Syndrome.

Cannabis sativa produces a complex mixture of many bioactive molecules including terpenophenolic compounds known as phytocannabinoids. Phytocannabinoids come in neutral forms (e.g., Δ9-tetrahydrocannabinol, THC; cannabidiol, CBD; etc.) or as acid precursors, which are dominant in the plant (e.g., Δ9-tetrahydrocannabinolic acid, THCA; cannabidiolic acid, CBDA; etc.). There is increasing interest in unlocking the therapeutic applications of the phytocannabinoid acids; however, the present understanding of the basic pharmacology of phytocannabinoid acids is limited. Herein the brain and plasma pharmacokinetic profiles of CBDA, THCA, cannabichromenic acid (CBCA), cannabidivarinic acid (CBDVA), cannabigerolic acid (CBGA), and cannabigerovarinic acid (CBGVA) were examined following intraperitoneal administration in mice. Next it was examined whether CBDA was anticonvulsant in a mouse model of Dravet syndrome (Scn1aRX/+ mice). All the phytocannabinoid acids investigated were rapidly absorbed with plasma tmax values of between 15 and 45 min and had relatively short half-lives (<4 h). The brain-plasma ratios for the acids were very low at ≤0.04. However, when CBDA was administered in an alternate Tween 80-based vehicle, it exhibited a brain-plasma ratio of 1.9. The anticonvulsant potential of CBDA was examined using this vehicle, and it was found that CBDA significantly increased the temperature threshold at which the Scn1aRX/+ mice had a generalized tonic-clonic seizure.

Medicinal cannabis in Australia, 2016: the Cannabis as Medicine Survey (CAMS-16).

OBJECTIVE: To explore patterns of cannabis use for medical purposes in Australia immediately prior to the 2016 legislation for frameworks for medical cannabis use. Design, setting: Anonymous online survey with convenience sample, April-October 2016. Participants were recruited through online media and at professional and consumer forums. PARTICIPANTS: Adults (at least 18 years of age) who reported using a cannabis product for self-identified medical or therapeutic reasons during the preceding 12 months. MAIN OUTCOME MEASURES: Consumer characteristics; indications and patterns of medical cannabis use; perceived benefits and harms; views on appropriate availability of medical cannabis. RESULTS: Most of the 1748 participants were men (68.1%) and employed (56.6%), with a mean age of 37.9 years (SD, 13.4 years) and mean reported period of medical cannabis use of 9.8 years (SD, 12.5 years). The most frequent reasons for medical cannabis use were anxiety (50.7%), back pain (50.0%), depression (49.3%), and sleep problems (43.5%). Respondents had used medical cannabis on a mean of 19.9 of the previous 28 days (SD, 10.0 days), spending a mean $68.60 (SD, $85.00) per week, and 83.4% had inhaled the substance. Participants reported high levels of clinical effectiveness and frequent side effects, including drowsiness, ocular irritation, lethargy and memory impairment; 17% met DSM-5 criteria for moderate or severe cannabis use disorder. Many reported harms or concerns related to the illicit status of cannabis. Participants believed that medical cannabis should be integrated into mainstream health care, and that products should be required to meet consistency and safety standards. CONCLUSION: Illicitly sourced cannabis is used to treat a broad range of medical conditions in Australia. Future models of prescribed medical cannabis take consumer patterns of use and demand into consideration.

Genetic deletion of P-glycoprotein alters stress responsivity and increases depression-like behavior, social withdrawal and microglial activation in the hippocampus of female mice.

P-glycoprotein (P-gp) is an ABC transporter expressed at the blood brain barrier and regulates the brain uptake of various xenobiotics and endogenous mediators including glucocorticoid hormones which are critically important to the stress response. Moreover, P-gp is expressed on microglia, the brain's immune cells, which are activated by stressors and have an emerging role in psychiatric disorders. We therefore hypothesised that germline P-gp deletion in mice might alter the behavioral and microglial response to stressors. Female P-gp knockout mice displayed an unusual, frantic anxiety response to intraperitoneal injection stress in the light-dark test. They also tended to display reduced conditioned fear responses compared to wild-type (WT) mice in a paradigm where a single electric foot-shock stressor was paired to a context. Foot-shock stress reduced social interaction and decreased microglia cell density in the amygdala which was not varied by P-gp genotype. Independently of stressor exposure, female P-gp deficient mice displayed increased depression-like behavior, idiosyncratic darting behavior, age-related social withdrawal and hyperactivity, facilitated sensorimotor gating and altered startle reactivity. In addition, P-gp deletion increased microglia cell density in the CA3 region of the hippocampus, and the microglial cells exhibited a reactive, hypo-ramified morphology. Further, female P-gp KO mice displayed increased glucocorticoid receptor (GR) expression in the hippocampus. In conclusion, this research shows that germline P-gp deletion affected various behaviors of relevance to psychiatric conditions, and that altered microglial cell activity and enhanced GR expression in the hippocampus may play a role in mediating these behaviors.

Cannabinoids and healthy ageing: the potential for extending healthspan and lifespan in preclinical models with an emphasis on Caenorhabditis elegans.

There is a significant global upsurge in the number and proportion of older persons in the population. With this comes an increasing prevalence of age-related conditions which pose a major challenge to healthcare systems. The development of anti-ageing treatments may help meet this challenge by targeting the ageing process which is a common denominator to many health problems. Cannabis-like compounds (cannabinoids) are reported to improve quality of life and general well-being in human trials, and there is increasing preclinical research highlighting that they have anti-ageing activity. Moreover, preclinical evidence suggests that endogenous cannabinoids regulate ageing processes. Here, we review the anti-ageing effects of the cannabinoids in various model systems, including the most extensively studied nematode model, Caenorhabditis elegans. These studies highlight that the cannabinoids lengthen healthspan and lifespan, with emerging evidence that they may also hinder the development of cellular senescence. The non-psychoactive cannabinoid cannabidiol (CBD) shows particular promise, with mechanistic studies demonstrating it may work through autophagy induction and activation of antioxidative systems. Furthermore, CBD improves healthspan parameters such as diminishing age-related behavioural dysfunction in models of both healthy and accelerated ageing. Translation into mammalian systems provides an important next step. Moreover, looking beyond CBD, future studies could probe the multitude of other cannabis constituents for their anti-ageing activity.

Transmembrane domain Nrg1 mutant mice show altered susceptibility to the neurobehavioural actions of repeated THC exposure in adolescence.

Heavy cannabis abuse increases the risk of developing schizophrenia. Adolescents appear particularly vulnerable to the development of psychosis-like symptoms after cannabis use. To test whether the schizophrenia candidate gene neuregulin 1 (NRG1) modulates the effects of cannabinoids in adolescence, we tested male adolescent heterozygous transmembrane domain Nrg1 mutant (Nrg1 TM HET) mice and wild type-like littermates (WT) for their neurobehavioural response to repeated Δ(9)-tetrahydrocannabinol (THC, 10 mg/kg i.p. for 21 d starting on post-natal day 31). During treatment and 48 h after treatment withdrawal, we assessed several behavioural parameters relevant to schizophrenia. After behavioural testing we measured autoradiographic CB(1), 5-HT(2A) and NMDA receptor binding. The hyperlocomotor phenotype typical of Nrg1 mutants emerged after drug withdrawal and was more pronounced in vehicle than THC-treated Nrg1 TM HET mice. All mice were equally sensitive to THC-induced suppression of locomotion. However, mutant mice appeared protected against inhibiting effects of repeated THC on investigative social behaviours. Neither THC nor Nrg1 genotype altered prepulse inhibition. Repeated adolescent THC promoted differential effects on CB(1) and 5-HT(2A) receptor binding in the substantia nigra and insular cortex respectively, decreasing binding in WT while increasing it in Nrg1 TM HET mice. THC also selectively affected 5-HT(2A) receptor binding in several other regions in WT mice, whereas NMDA receptor binding was only affected in mutant mice. Overall, Nrg1 mutation does not appear to increase the induction of psychotomimetic symptoms by repeated adolescent THC exposure but may attenuate some of its actions on social behaviour and schizophrenia-relevant neurotransmitter receptor profiles.

The safety and efficacy of low oral doses of cannabidiol: An evaluation of the evidence.

Global interest in the non-intoxicating cannabis constituent, cannabidiol (CBD), is increasing with claims of therapeutic effects across a diversity of health conditions. At present, there is sufficient clinical trial evidence to support the use of high oral doses of CBD (e.g., 10-50 mg/kg) in treating intractable childhood epilepsies. However, a question remains as to whether "low-dose" CBD products confer any therapeutic benefits. This is an important question to answer, as low-dose CBD products are widely available in many countries, often as nutraceutical formulations. The present review therefore evaluated the efficacy and safety of low oral doses of CBD. The review includes interventional studies that measured the clinical efficacy in any health condition and/or safety and tolerability of oral CBD dosed at less than or equal to 400 mg per day in adult populations (i.e., ≥18 years of age). Studies were excluded if the product administered had a Δ9 -tetrahydrocannabinol content greater than 2.0%. Therapeutic benefits of CBD became more clearly evident at doses greater than or equal to 300 mg. Increased dosing from 60 to 400 mg/day did not appear to be associated with an increased frequency of adverse effects. At doses of 300-400 mg, there is evidence of efficacy with respect to reduced anxiety, as well as anti-addiction effects in drug-dependent individuals. More marginal and less consistent therapeutic effects on insomnia, neurological disorders, and chronic pain were also apparent. Larger more robust clinical trials are needed to confirm the therapeutic potential of lower (i.e., <300 mg/day) oral doses of CBD.

Ictal vocalizations in the Scn1a+/- mouse model of Dravet syndrome.

OBJECTIVE: Ictal vocalizations have shown diagnostic utility in epilepsy patients. Audio recordings of seizures have also been used for seizure detection. The present study aimed to determine whether generalized tonic-clonic seizures in the Scn1a+/- mouse model of Dravet syndrome are associated with either audible mouse squeaks or ultrasonic vocalizations. METHODS: Acoustic recordings were captured from group-housed Scn1a+/- mice undergoing video-monitoring to quantify spontaneous seizure frequency. We generated audio clips (n = 129) during a generalized tonic-clonic seizure (GTCS) that included 30 seconds immediately prior to the GTCS (preictal) and 30 seconds following the conclusion of the seizure (postictal). Nonseizure clips (n = 129) were also exported from the acoustic recordings. A blinded reviewer manually reviewed the audio clips, and vocalizations were identified as either an audible (<20 kHz) mouse squeak or ultrasonic (>20 kHz). RESULTS: Spontaneous GTCS in Scn1a+/- mice were associated with a significantly higher number of total vocalizations. The number of audible mouse squeaks was significantly greater with GTCS activity. Nearly all (98%) the seizure clips contained ultrasonic vocalizations, whereas ultrasonic vocalizations were present in only 57% of nonseizure clips. The ultrasonic vocalizations emitted in the seizure clips were at a significantly higher frequency and were nearly twice as long in duration as those emitted in the nonseizure clips. Audible mouse squeaks were primarily emitted during the preictal phase. The greatest number of ultrasonic vocalizations was detected during the ictal phase. SIGNIFICANCE: Our study shows that ictal vocalizations are exhibited by Scn1a+/- mice. Quantitative audio analysis could be developed as a seizure detection tool for the Scn1a+/- mouse model of Dravet syndrome.