Cannabinoid CB1 Receptor Expression and Localization in the Dorsal Horn of Male and Female Rat and Human Spinal Cord.

Background: Preclinical and clinical evidence suggests that cannabis has potential analgesic properties. However, cannabinoid receptor expression and localization within spinal cord pain processing circuits remain to be characterized across sex and species. Aims: We aimed to investigate the differential expression of the cannabinoid type 1 (CB1) receptor across dorsal horn laminae and cell populations in male and female adult rats and humans. Methods: To investigate and quantify CB1 receptor expression in the spinal dorsal horn across species, we refined immunohistochemical procedures for successful rat and human fixed tissue staining and confocal imaging. Immunohistochemical results were complemented with analysis of CB1 gene (CNR1) expression within rodent and human dorsal horn using single-cell/nuclei RNA sequencing data sets. Results: We found that CB1 was preferentially localized to the neuropil within the superficial dorsal horn of both rats and humans, with CB1 somatic staining across dorsal horn laminae. CB1 receptor immunoreactivity was significantly higher in the superficial dorsal horn compared to the deeper dorsal horn laminae for both rats and humans, which was conserved across sex. Interestingly, we found that CB1 immunoreactivity was not primarily localized to peptidergic afferents in rats and humans and that CNR1 (CB1) but not CNR2 (CB2) was robustly expressed in dorsal horn neuron subpopulations of both rodents and humans. Conclusions: The conserved preferential expression of CB1 receptors in the superficial dorsal horn in male and female rodents and humans has significant implications for understanding the roles of this cannabinoid receptor in spinal mechanisms of nociception and analgesia.

Contexte: Les données probantes précliniques et cliniques indiquent que le cannabis possède des propriétés analgésiques potentielles. Cependant, l'expression et la localisation des récepteurs cannabinoïdes au sein des circuits de traitement de la douleur de la moelle épinière restent à caractériser selon le sexe et les espèces.Objectifs: Nous avons cherché à étudier l'expression différenciée du récepteur cannabinoïde de type 1 (CB1) dans les différentes couches de la corne dorsale et les populations cellulaires chez des rats et des êtres humains adultes de sexe masculin et féminin.Méthodes: Pour étudier et quantifier l'expression des récepteurs CB1 dans la corne dorsale de la moelle épinière chez différentes espèces, nous avons perfectionné les procédures d'immunohistochimie pour obtenir des résultats de coloration réussis sur des échantillons de tissus provenant de rats et d'êtres humains, ainsi que des images confocales. Les résultats immunohistochimiques ont été complétés par l'analyse de l'expression du gène CB1 (CNR1) dans la corne dorsale des rongeurs et des humains en utilisant des ensembles de données de séquençage d'ARN au niveau des cellules uniques et des noyaux.Résultats: Nous avons constaté que le CB1 était principalement localisé dans le neuropile au sein de la corne dorsale superficielle chez les rats et les humains, avec une coloration somatique du CB1 dans les différentes couches de la corne dorsale. Chez les deux espèces, l'immunoréactivité du récepteur CB1 était significativement plus élevée dans la couche superficielle de la corne dorsale par rapport aux couches plus profondes, indépendamment du sexe. De manière intéressante, nous avons constaté que l'immunoréactivité du CB1 n'était pas principalement localisée dans les afférences peptidergiques chez les rats et les humains. De plus, nous avons observé une forte expression du gène CNR1 (CB1), mais pas du CNR2 (CB2), au sein de sous-populations de neurones de la corne dorsale chez les rongeurs et les êtres humains.Conclusions: La localisation privilégiée et constante des récepteurs CB1 dans la couche superficielle de la corne dorsale chez les rongeurs et les humains, quel que soit leur sexe, revêt une importance majeure pour la compréhension des fonctions de ce récepteur des cannabinoïdes dans les mécanismes médullaires de la nociception et de l'analgésie.

Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain.

The prevalence and severity of many chronic pain syndromes differ across sex, and recent studies have identified differences in immune signalling within spinal nociceptive circuits as a potential mediator. Although it has been proposed that sex-specific pain mechanisms converge once they reach neurons within the superficial dorsal horn, direct investigations using rodent and human preclinical pain models have been lacking. Here, we discovered that in the Freund's adjuvant in vivo model of inflammatory pain, where both male and female rats display tactile allodynia, a pathological coupling between KCC2-dependent disinhibition and N-methyl-D-aspartate receptor (NMDAR) potentiation within superficial dorsal horn neurons was observed in male but not female rats. Unlike males, the neuroimmune mediator brain-derived neurotrophic factor (BDNF) failed to downregulate inhibitory signalling elements (KCC2 and STEP61) and upregulate excitatory elements (pFyn, GluN2B and pGluN2B) in female rats, resulting in no effect of ex vivo brain-derived neurotrophic factor on synaptic NMDAR responses in female lamina I neurons. Importantly, this sex difference in spinal pain processing was conserved from rodents to humans. As in rodents, ex vivo spinal treatment with BDNF downregulated markers of disinhibition and upregulated markers of facilitated excitation in superficial dorsal horn neurons from male but not female human organ donors. Ovariectomy in female rats recapitulated the male pathological pain neuronal phenotype, with BDNF driving a coupling between disinhibition and NMDAR potentiation in adult lamina I neurons following the prepubescent elimination of sex hormones in females. This discovery of sexual dimorphism in a central neuronal mechanism of chronic pain across species provides a foundational step towards a better understanding and treatment for pain in both sexes.

A systematic review of the effects of cannabis on cognition in people with multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic demyelinating disease which leads to sensory, motor, autonomic, and cognitive symptoms. Cannabis is a common way for persons with MS (pwMS) to seek symptomatic therapy. Given the capacity for both cannabis and MS to cause cognitive impairment, it is important to determine whether there is any negative impact when the two co-occur. The objective of this systematic review was to evaluate the effects of cannabis and medicinal cannabinoid products on cognition in pwMS in order to provide guidance to clinicians and enable them to make evidence-based recommendations regarding cannabis and cannabinoid products. METHODS: A systematic review was carried out searching common keyword combinations for cannabis and MS across five databases, producing 840 unique articles, 18 of which were included in a qualitative synthesis. RESULTS: Aggregate data from existing studies to date highlight potential impairments from chronic whole-plant cannabis use in commonly affected cognitive domains in multiple sclerosis, including attention and working memory, and to a lesser extent, visual memory, verbal memory, and executive function. Results also suggest that in the short-term, medicinal cannabinoid preparations do not significantly impair cognition and may even ameliorate cognitive symptoms in the context of obtrusive MS disease. The findings are limited by disparities in detail of cannabis use data reported across whole-plant cannabis publications. CONCLUSION: Existing literature on co-occurrence of cannabis use and MS lacks high quality evidence to recommend for or against cannabis and cannabinoid therapies for pwMS based on cognitive effects. Existing data suggest that cognition may be differentially impacted in pwMS depending on the type of product, the duration of use, and the indication. Future studies on whole-plant cannabis require comprehensive cannabis use data reporting including frequency, dosing, duration, and type of cannabis product. Future studies on medicinal cannabinoid products should be long-term to assess the effects of chronicity.

Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats.

N-methyl-D-aspartate receptors (NMDARs) are excitatory ionotropic glutamate receptors expressed throughout the CNS, including in the spinal dorsal horn. The GluN2 subtypes of NMDAR subunit, which include GluN2A, GluN2B, and GluN2D in the dorsal horn, confer NMDARs with structural and functional variability, enabling heterogeneity in synaptic transmission and plasticity. Despite essential roles for NMDARs in physiological and pathological pain processing, the distribution and function of these specific GluN2 isoforms across dorsal horn laminae remain poorly understood. Surprisingly, there is a complete lack of knowledge of GluN2 expression in female rodents. We, therefore, investigated the relative expression of specific GluN2 variants in the dorsal horn of lumbar (L4/L5) spinal cord from both male and female rats. In order to detect synaptic GluN2 isoforms, we used pepsin antigen-retrieval to unmask these highly cross-linked protein complexes. We found that GluN2B and GluN2D are preferentially localized to the pain-processing superficial regions of the dorsal horn in males, while only GluN2B is predominantly localized to the superficial dorsal horn of female rats. The GluN2A subunit is diffusely localized to neuropil throughout the dorsal horn of both males and females, while GluN2B and GluN2D immunolabelling are found both in the neuropil and on the soma of dorsal horn neurons. Finally, we identified an unexpected enhanced expression of GluN2B in the medial division of the superficial dorsal horn, but in males only. These sex-specific localization patterns of GluN2-NMDAR subunits across dorsal horn laminae have significant implications for the understanding of divergent spinal mechanisms of pain processing.

The impact of dextran sodium sulphate and probiotic pre-treatment in a murine model of Parkinson's disease.

BACKGROUND: Recent work has established that Parkinson's disease (PD) patients have an altered gut microbiome, along with signs of intestinal inflammation. This could help explain the high degree of gastric disturbances in PD patients, as well as potentially be linked to the migration of peripheral inflammatory factors into the brain. To our knowledge, this is the first study to examine microbiome alteration prior to the induction of a PD murine model. METHODS: We presently assessed whether pre-treatment with the probiotic, VSL #3, or the inflammatory inducer, dextran sodium sulphate (DSS), would influence the PD-like pathology provoked by a dual hit toxin model using lipopolysaccharide (LPS) and paraquat exposure. RESULTS: While VSL #3 has been reported to have anti-inflammatory effects, DSS is often used as a model of colitis because of the gut inflammation and the breach of the intestinal barrier that it induces. We found that VSL#3 did not have any significant effects (beyond a blunting of LPS paraquat-induced weight loss). However, the DSS treatment caused marked changes in the gut microbiome and was also associated with augmented behavioral and inflammatory outcomes. In fact, DSS markedly increased taxa belonging to the Bacteroidaceae and Porphyromonadaceae families but reduced those from Rikencellaceae and S24-7, as well as provoking colonic pro-inflammatory cytokine expression, consistent with an inflamed gut. The DSS also increased the impact of LPS plus paraquat upon microglial morphology, along with circulating lipocalin-2 (neutrophil marker) and IL-6. Yet, neither DSS nor VSL#3 influenced the loss of substantia nigra dopamine neurons or the astrocytic and cytoskeleton remodeling protein changes that were provoked by the LPS followed by paraquat treatment. CONCLUSIONS: These data suggest that disruption of the intestinal integrity and the associated microbiome can interact with systemic inflammatory events to promote widespread brain-gut changes that could be relevant for PD and at the very least, suggestive of novel neuro-immune communication.

Quantum dot conjugated saporin activates microglia and induces selective substantia nigra degeneration.

Parkinson's disease (PD) is characterized by profound microglial driven inflammatory processes and the loss of dopamine neurons of the substantia nigra (SNc). Both microglia and dopamine neurons that are affected in the SNc are particularly vulnerable to environmental toxicants and finding more selective ways of targeting these cell types is of importance. Quantum dots (QDs) might be a useful vehicle for selectively delivering toxicants to microglia and owing to their fluorescent capability, they can be microscopically tracked within the cell. Accordingly, we assessed the impact of QDs alone and QDs conjugated to the ribosomal toxin, saporin, upon SNc microglia and dopamine neurons. We found that intra-SNc infused QDs selectively entered microglia and induced morphological changes consistent with an activated state. QDs conjugated to saporin also caused a significant loss of dopamine neurons and motor coordination (on a rotarod test) deficits, along with an increase in the inflammatory microglial actin regulatory factors, WAVE2. These data suggest that QDs might be a viable route for toxicant delivery and also has an added advantage of being fluorescently visible. Ultimately, we found SNc neurons to be exceptionally vulnerable to QD-saporin and suggest that this could be a novel targeted approach to model PD-like inflammatory pathology.

Interactive effects of ghrelin and ketamine on forced swim performance: Implications for novel antidepressant strategies.

The efficacy of ketamine to alleviate depressive symptoms has promoted a wealth of research exploring alternate therapeutic targets for depression. Given the caveats of ketamine treatment taken together with the increasingly greater emphasis on combinatorial therapeutic approaches to depression, we sought to asses whether the hypothalamic "hunger hormone", ghrelin, would augment the effects of ketamine. Indeed, ghrelin has recently been found to possess antidepressant potential and may be especially effective against the metabolic and feeding deficits observed with depression. Two studies were performed: 1. mice were given an intraperitoneal injection of ghrelin (80µg/kg) or saline, followed by a saline or a low or high dose of ketamine (5 or 10mg/kg) and 2. mice received 10mg/kg of ketamine together with saline or the ghrelin receptor antagonist JMV2959 (3 or 6mg/kg) and Forced Swim Test (FST) performance was assessed. In both studies, ketamine alone reduced FST immobility. Similarly, ghrelin alone reduced swim immobility suggesting an antidepressant-like response. However, ghrelin did not augment the impact of ketamine when co-administered and in fact, it appeared to antagonize its actions at the lower dose. As well, JMV2959 did not significantly influence FST performance. These data confirm the antidepressant-like effects of ketamine and further suggest that ghrelin might have similar properties. Yet, our results caution against combinatorial treatment with these agents, probably owing to unexpected allosteric or other antagonist actions.