Sexual dimorphic distribution of cannabinoid 1 receptor mRNA in adult C57BL/6J mice

The cannabinoid 1 receptor (CB₁R) is the most abundant G protein-coupled receptor in the brain and plays crucial roles in emotion and behavior by modulating or mediating synaptic transmission and plasticity. Differences in CB₁R density between male and female rodents may be associated with distinct behavioral phenotypes. In the rat brain, CB₁R expression is significantly lower in the prefrontal cortex and amygdala of estrus females than in males. However, differences in CB₁R distribution due to sex over the whole mouse brain are still largely unknown. Here, we systemically investigated the expression of CB₁R mRNA in the brains of both male and female adult C57BL/6J mice using fluorescence in situ hybridization. There were significantly more CB₁R positive cells in males than in females in the orbital cortex, insular cortex, cingulate cortex, piriform cortex, secondary visual cortex, caudate putamen (striatum), and ventral hippocampal CA1. There were significantly more CB₁R mRNA cells in females than males in the fornix and dorsal hypothalamus. However, in some regions, strong hybridization signals without sex differences were detected, such as in the motor cortex, septum, medial habenular nucleus, and inferior colliculus. Moreover, female mice displayed different CB₁R mRNA expression patterns in the medial amygdala, basolateral amygdala, and parabrachial nucleus during different phases of the estrous cycle. These findings provide a basis for understanding sexual dimorphism in physiological and pathological brain functions related to CB₁R.     

Methamphetamine neurotoxicity increases brain expression and alters behavioral functions of CB1 cannabinoid receptors

Cannabis is the most common secondary illicit substance in methamphetamine (METH) users, yet the outcomes of the concurrent consumption of both substances remain elusive. Capitalizing on recent findings on the implication of CB₁ cannabinoid receptors in the behavioral effects of METH, we hypothesized that METH-induced neurotoxicity may alter the brain expression of CB₁, thereby affecting its role in behavioral functions. To test this possibility, we subjected rats to a well-characterized model of METH neurotoxicity (4 mg/kg, subcutaneous × 4 injections, 2 h apart), and analyzed their CB₁ receptor brain expression three weeks later. METH exposure resulted in significant enhancements of CB₁ receptor expression across several brain regions, including prefrontal cortex, caudate-putamen, basolateral amygdala, CA1 hippocampal region and perirhinal cortex. In parallel, a different group of METH-exposed rats was used to explore the responsiveness to the potent cannabinoid agonist WIN 55,212-2 (WIN) (0.5-1 mg/kg, intraperitoneal), within several paradigms for the assessment of emotional and cognitive functions, such as open field, object exploration and recognition, and startle reflex. WIN induced anxiolytic-like effects in METH-exposed rats and anxiogenic-like effects in saline-treated controls. Furthermore, METH-exposed animals exhibited a significantly lower impact of WIN on the attenuation of exploratory behaviors and short-term (90 min) recognition memory. Conversely, METH neurotoxicity did not significantly affect WIN-induced reductions in locomotor activity, exploration time and acoustic startle. These results suggest that METH neurotoxicity may alter the vulnerability to select behavioral effects of cannabis, by inducing distinct regional variations in the expression of CB₁ receptors.     

Identification of BiP as a CB1 Receptor-Interacting Protein That Fine-Tunes Cannabinoid Signaling in the Mouse Brain

Cannabinoids, the bioactive constituents of cannabis, exert a wide array of effects on the brain by engaging Type 1 cannabinoid receptor (CB₁R). Accruing evidence supports that cannabinoid action relies on context-dependent factors, such as the biological characteristics of the target cell, suggesting that cell population-intrinsic molecular cues modulate CB₁R-dependent signaling. Here, by using a yeast two-hybrid-based high-throughput screening, we identified BiP as a potential CB₁R-interacting protein. We next found that CB₁R and BiP interact specifically in vitro, and mapped the interaction site within the CB₁R C-terminal (intracellular) domain and the BiP C-terminal (substrate-binding) domain-α. BiP selectively shaped agonist-evoked CB₁R signaling by blocking an “alternative” G_q/11 protein-dependent signaling module while leaving the “classical” G_i/o protein-dependent inhibition of the cAMP pathway unaffected. In situ proximity ligation assays conducted on brain samples from various genetic mouse models of conditional loss or gain of CB₁R expression allowed to map CB₁R-BiP complexes selectively on terminals of GABAergic neurons. Behavioral studies using cannabinoid-treated male BiP^+/− mice supported that CB₁R-BiP complexes modulate cannabinoid-evoked anxiety, one of the most frequent undesired effects of cannabis. Together, by identifying BiP as a CB₁R-interacting protein that controls receptor function in a signaling pathway- and neuron population-selective manner, our findings may help to understand the striking context-dependent actions of cannabis in the brain.SIGNIFICANCE STATEMENT Cannabis use is increasing worldwide, so innovative studies aimed to understand its complex mechanism of neurobiological action are warranted. Here, we found that cannabinoid CB₁ receptor (CB₁R), the primary molecular target of the bioactive constituents of cannabis, interacts specifically with an intracellular protein called BiP. The interaction between CB₁R and BiP occurs selectively on terminals of GABAergic (inhibitory) neurons, and induces a remarkable shift in the CB₁R-associated signaling profile. Behavioral studies conducted in mice support that CB₁R-BiP complexes act as fine-tuners of anxiety, one of the most frequent undesired effects of cannabis use. Our findings open a new conceptual framework to understand the striking context-dependent pharmacological actions of cannabis in the brain.     

Downregulation of cannabinoid receptor 1 from neuropeptide Y interneurons in the basal ganglia of patients with Huntington's disease and mouse models

Cannabinoid receptor 1 (CB₁ receptor) controls several neuronal functions, including neurotransmitter release, synaptic plasticity, gene expression and neuronal viability. Downregulation of CB₁ expression in the basal ganglia of patients with Huntington's disease (HD) and animal models represents one of the earliest molecular events induced by mutant huntingtin (mHtt). This early disruption of neuronal CB₁ signaling is thought to contribute to HD symptoms and neurodegeneration. Here we determined whether CB₁ downregulation measured in patients with HD and mouse models was ubiquitous or restricted to specific striatal neuronal subpopulations. Using unbiased semi‐quantitative immunohistochemistry, we confirmed previous studies showing that CB₁ expression is downregulated in medium spiny neurons of the indirect pathway, and found that CB₁ is also downregulated in neuropeptide Y (NPY)/neuronal nitric oxide synthase (nNOS)‐expressing interneurons while remaining unchanged in parvalbumin‐ and calretinin‐expressing interneurons. CB₁ downregulation in striatal NPY/nNOS‐expressing interneurons occurs in R6/2 mice, Hdh^Q150/Q150 mice and the caudate nucleus of patients with HD. In R6/2 mice, CB₁ downregulation in NPY/nNOS‐expressing interneurons correlates with diffuse expression of mHtt in the soma. This downregulation also occludes the ability of cannabinoid agonists to activate the pro‐survival signaling molecule cAMP response element‐binding protein in NPY/nNOS‐expressing interneurons. Loss of CB₁ signaling in NPY/nNOS‐expressing interneurons could contribute to the impairment of basal ganglia functions linked to HD.     

Statistical parametric mapping reveals regional alterations in cannabinoid CB1 receptor distribution and G-protein activation in the 3D reconstructed epileptic rat brain

Purpose: The endocannabinoid system is known to modulate seizure activity in several in vivo and in vitro models, and CB₁‐receptor activation is anticonvulsant in the rat pilocarpine model of acquired epilepsy (AE). In these epileptic rats, a unique redistribution of the CB₁ receptor occurs within the hippocampus; however, an anatomically inclusive analysis of the effect of status epilepticus (SE)–induced AE on CB₁ receptors has not been thoroughly evaluated. Therefore, statistical parametric mapping (SPM), a whole‐brain unbiased approach, was used to study the long‐term effect of pilocarpine‐induced SE on CB₁‐receptor binding and G‐protein activation in rats with AE. Methods: Serial coronal sections from control and epileptic rats were cut at equal intervals throughout the neuraxis and processed for [³H]WIN55,212‐2 (WIN) autoradiography, WIN‐stimulated [³⁵S]GTPγS autoradiography, and CB₁‐receptor immunohistochemistry (IHC). The autoradiographic techniques were evaluated with both region of interest (ROI) and SPM analyses. Key Findings: In rats with AE, regionally specific increases in CB₁‐receptor binding and activity were detected in cortex, discrete thalamic nuclei, and other regions including caudate‐putamen and septum, and confirmed by IHC. However, CB₁ receptors were unaltered in several brain regions, including substantia nigra and cerebellum, and did not exhibit regional decreases in rats with AE. Significance: This study provides the first comprehensive evaluation of the regional distribution of changes in CB₁‐receptor expression, binding, and G‐protein activation in the rat pilocarpine model of AE. These regions may ultimately serve as targets for cannabinomimetic compounds or manipulation of the endocannabinoid system in epileptic brain.     

WAG/Rij rats show a reduced expression of CB1 receptors in thalamic nuclei and respond to the CB1 receptor agonist,R(+)WIN55,212‐2, with a reduced incidence of spike‐wave discharges

Purpose: Genetically epileptic WAG/Rij rats develop spontaneous absence‐like seizures after 3 months of age. We used WAG/Rij rats to examine whether absence seizures are associated with changes in the expression of type‐1 cannabinoid (CB₁) receptors. Methods: Receptor expression was examined by in situ hybridization and western blot analysis in various brain regions of “presymptomatic” 2‐month old and “symptomatic” 8‐month‐old WAG/Rij rats relative to age‐matched nonepileptic control rats. Furthermore, we examined whether pharmacologic activation of CB₁ receptor affects absence seizures. We recorded spontaneous spike‐wave discharges (SWDs) in 8‐month old WAG/Rij rats systemically injected with the potent CB₁ receptor agonist, R(+)WIN55,212‐2 (3–12 mg/kg, s.c.), given alone or combined with the CB₁ receptor antagonist/inverse agonist, AM251 (12 mg/kg, s.c.). Results: Data showed a reduction of CB₁ receptor mRNA and protein levels in the reticular thalamic nucleus, and a reduction in CB₁ receptor protein levels in ventral basal thalamic nuclei of 8‐month‐old WAG/Rij rats, as compared with age‐matched ACI control rats. In vivo, R(+)WIN55,212‐2 caused a dose‐dependent reduction in the frequency of SWDs in the first 3 h after the injection. This was followed by a late increase in the mean SWD duration, which suggests a biphasic modulation of SWDs by CB₁ receptor agonists. Both effects were reversed or attenuated when R(+)WIN55,212‐2 was combined with AM251. Discussion: These data indicate that the development of absence seizures is associated with plastic modifications of CB₁ receptors within the thalamic‐cortical‐thalamic network, and raise the interesting possibility that CB₁ receptors are targeted by novel antiabsence drugs.     

Rising stars: Modulation of brain functions by astroglial type‐1 cannabinoid receptors

The type‐1‐cannabinoid (CB₁) receptor is amongst the most widely expressed G protein‐coupled receptors in the brain. In few decades, CB₁ receptors have been shown to regulate a large array of functions from brain cell development and survival to complex cognitive processes. Understanding the cellular mechanisms underlying these functions of CB₁ is complex due to the heterogeneity of the brain cell types on which the receptor is expressed. Although the large majority of CB₁ receptors act on neurons, early studies pointed to a direct control of CB₁ receptors over astroglial functions including brain energy supply and neuroprotection. In line with the growing concept of the tripartite synapse highlighting astrocytes as direct players in synaptic plasticity, astroglial CB₁ receptor signaling recently emerged as the mediator of several forms of synaptic plasticity associated to important cognitive functions. Here, we shortly review the current knowledge on CB₁ receptor‐mediated astroglial functions. This functional spectrum is large and most of the mechanisms by which CB₁ receptors control astrocytes, as well as their consequences in vivo, are still unknown, requiring innovative approaches to improve this new cannabinoid research field. GLIA 2015;63:353–364     

In vivo SPECT and ex vivo autoradiographic brain imaging of the novel selective CB1 receptor antagonist radioligand [125I]SD7015 in CB1 knock-out and wildtype mouse

We aimed to evaluate the novel high-affinity and relatively lipophilic CB₁ receptor (CB₁R) antagonist radioligand [¹²⁵I]SD7015 for SPECT imaging of CB₁Rs in vivo using the multiplexed multipinhole dedicated small animal SPECT/CT system, NanoSPECT/CT^PLUS (Mediso, Budapest, Hungary), in knock-out CB₁ receptor knock-out (CB₁R-/-) and wildtype mice. In order to exclude possible differences in cerebral blood flow between the two types of animals, HMPAO SPECT scans were performed, whereas in order to confirm the brain uptake differences of the radioligand between knock-out mice and wildtype mice, in vivo scans were complemented with ex vivo autoradiographic measurements using the brains of the same animals. With SPECT/CT imaging, we measured the brain uptake of radioactivity, using %SUV (% standardised uptake values) in CB₁R-/- mice (n = 3) and C57BL6 wildtype mice (n = 7) under urethane anaesthesia after injecting [¹²⁵I]SD7015 intravenously or intraperitoneally. The Brookhaven Laboratory mouse MRI atlas was fused to the SPECT/CT images by using a combination of rigid and non-rigid algorithms in the Mediso Fusion™ (Mediso, Budapest, Hungary) and VivoQuant (inviCRO, Boston, MA, USA) softwares. Phosphor imager plate autoradiography (ARG) was performed on 4 μm-thin cryostat sections of the excised brains. %SUV was 8.6 ± 3.6 (average ± SD) in CB₁R-/- mice and 22.1 ± 12.4 in wildtype mice between 2 and 4 h after injection (p < 0.05). ARG of identically taken sections from wildtype mouse brain showed moderate radioactivity uptake when compared with the in vivo images, with a clear difference between grey matter and white matter, whereas ARG in CB₁R(-/-) mice showed practically no radioactivity uptake. [¹²⁵I]SD7015 enters the mouse brain in sufficient amount to enable SPECT imaging. Brain radioactivity distribution largely coincides with that of the known CB₁R expression pattern in rodent brain. We conclude that [¹²⁵I]SD7015 should be a useful SPECT radioligand for studying brain CB₁R in mouse and rat disease models.     

Antibodies to cannabinoid type 1 receptor co‐react with stomatin‐like protein 2 in mouse brain mitochondria

Anti‐cannabinoid type 1 receptor (CB₁) polyclonal antibodies are widely used to detect the presence of CB₁ in a variety of brain cells and their organelles, including neuronal mitochondria. Surprisingly, we found that anti‐CB₁ sera, in parallel with CB₁, also recognize the mitochondrial protein stomatin‐like protein 2. In addition, we show that the previously reported effect of synthetic cannabinoid WIN 55,212‐2 on mitochondrial complex III respiration is not detectable in purified mitochondrial preparations. Thus, our study indicates that a direct relationship between endocannabinoid signaling and mitochondrial functions in the cerebral cortex seems unlikely, and that caution should be taken interpreting findings obtained using anti‐CB₁ antibodies.     

Localization of the cannabinoid type‐1 receptor in subcellular astrocyte compartments of mutant mouse hippocampus

Astroglial type‐1 cannabinoid (CB₁) receptors are involved in synaptic transmission, plasticity and behavior by interfering with the so‐called tripartite synapse formed by pre‐ and post‐synaptic neuronal elements and surrounding astrocyte processes. However, little is known concerning the subcellular distribution of astroglial CB₁ receptors. In particular, brain CB₁ receptors are mostly localized at cells' plasmalemma, but recent evidence indicates their functional presence in mitochondrial membranes. Whether CB₁ receptors are present in astroglial mitochondria has remained unknown. To investigate this issue, we included conditional knock‐out mice lacking astroglial CB₁ receptor expression specifically in glial fibrillary acidic protein (GFAP)‐containing astrocytes (GFAP‐CB₁‐KO mice) and also generated genetic rescue mice to re‐express CB₁ receptors exclusively in astrocytes (GFAP‐CB₁‐RS). To better identify astroglial structures by immunoelectron microscopy, global CB₁ knock‐out (CB₁‐KO) mice and wild‐type (CB₁‐WT) littermates were intra‐hippocampally injected with an adeno‐associated virus expressing humanized renilla green fluorescent protein (hrGFP) under the control of human GFAP promoter to generate GFAPhrGFP‐CB₁‐KO and ‐WT mice, respectively. Furthermore, double immunogold (for CB₁) and immunoperoxidase (for GFAP or hrGFP) revealed that CB₁ receptors are present in astroglial mitochondria from different hippocampal regions of CB₁‐WT, GFAP‐CB₁‐RS and GFAPhrGFP‐CB₁‐WT mice. Only non‐specific gold particles were detected in mouse hippocampi lacking CB₁ receptors. Altogether, we demonstrated the existence of a precise molecular architecture of the CB₁ receptor in astrocytes that will have to be taken into account in evaluating the functional activity of cannabinergic signaling at the tripartite synapse.     

D2 autoreceptor switches CB2 receptor effects on [3H]-dopamine release in the striatum

CB₂ receptors (CB₂R) are expressed in midbrain neurons. To evidence the control of dopamine release in dorsal striatum by CB₂R, we performed experiments of [³H]-dopamine release in dorsal striatal slices. We found a paradoxical increase in K⁺-induced [³H]-dopamine release by CB₂R activation with GW 833972A and JWH 133 two selective agonist. To understand the mechanism involved, we tested for a role of the D₂ autoreceptor in this effect; because in pallidal structures, the inhibitory effect of CB₁ receptors (CB₁R) on GABA release is switched to a stimulatory effect by D₂ receptors (D₂R). We found that the blockade of D₂ autoreceptors with sulpiride prevented the stimulatory effect of CB₂R activation; in fact, under this condition, CB₂R decreased dopamine release, indicating the role of the D₂ autoreceptor in the paradoxical increase. We also found that the effect occurs in nigrostriatal terminals, since lesions with 6-OH dopamine in the middle forebrain bundle prevented CB₂R effects on release. In addition, D₂–CB₂R interaction promoted cAMP accumulation, and the increase in [³H]-dopamine release was prevented by PKA blockade. D₂–CB₂R coprecipitation and proximity ligation assay studies indicated a close interaction of receptors that could participate in the observed effects. Finally, intrastriatal injection of CB₂R agonist induced contralateral turning in amphetamine-treated rats, which was prevented by sulpiride, indicating the role of the interaction in motor behavior. Thus, these data indicate that the D₂ autoreceptor switches, from inhibitory to stimulatory, the CB₂R effects on dopamine release, involving the cAMP → PKA pathway in nigrostriatal terminals.     

Signaling molecules targeting cannabinoid receptors: Hemopressin and related peptides

Cannabinoid receptors (CBRs) are part of the endocannabinoid system, which is involved in various physiological processes such as nociception, inflammation, appetite, stress, and emotion regulation. Many studies have linked the endocannabinoid system to neuroinflammatory and neurodegenerative disorders such as Parkinson's disease, Huntington's chorea, Alzheimer's disease, and multiple sclerosis. Hemopressin [Hp; a fragment of the hemoglobin α1 chain (95–103 amino acids)] and related peptides [VD-Hpα and RVD-Hpα] are peptides that bind to CBRs. Hp acts as an inverse agonist to CB₁ receptor (CB₁R), VD-Hpα acts as an agonist to CB₁R, and RVD-Hpα acts as a negative allosteric modulator of CB₁R and a positive allosteric modulator of CB₂R. Because of the critical roles of CBRs in numerous physiological processes, it is appealing to use Hp and related peptides for therapeutic purposes. This review discusses their discovery, structure, metabolism, brain exposure, self-assembly characteristics, pharmacological characterization, and pharmacological activities.     

Neuronal and glial localization of the cannabinoid-1 receptor in the superficial spinal dorsal horn of the rodent spinal cord

A long line of experimental evidence indicates that endogenous cannabinoid mechanisms play important roles in nociceptive information processing in various areas of the nervous system including the spinal cord. Although it is extensively documented that the cannabinoid‐1 receptor (CB₁‐R) is strongly expressed in the superficial spinal dorsal horn, its cellular distribution is poorly defined, hampering our interpretation of the effect of cannabinoids on pain processing spinal neural circuits. Thus, we investigated the cellular distribution of CB₁‐Rs in laminae I and II of the rodent spinal dorsal horn with immunocytochemical methods. Axonal varicosities revealed a strong immunoreactivity for CB₁‐R, but no CB₁‐R expression was observed on dendrites and perikarya of neurons. Investigating the co‐localization of CB₁‐R with markers of peptidergic and non‐peptidergic primary afferents, and axon terminals of putative glutamatergic and GABAergic spinal neurons we found that nearly half of the peptidergic (immunoreactive for calcitonin gene‐related peptide) and more than 20% of the non‐peptidergic (binding isolectin B4) nociceptive primary afferents, more than one‐third and approximately 20% of the axon terminals of putative glutamatergic (immunoreactive for vesicular glutamate transporter 2) and GABAergic (immunoreactive for glutamic acid decarboxylase; GAD65 and/or GAD67) spinal interneurons, respectively, were positively stained for CB₁‐R. In addition to axon terminals, almost half of the astrocytic (immunoreactive for glial fibrillary acidic protein) and nearly 80% of microglial (immunoreactive for CD11b) profiles were also immunolabeled for CB₁‐R. The findings suggest that the activity‐dependent release of endogenous cannabinoids activates a complex signaling mechanism in pain processing spinal neural circuits into which both neurons and glial cells may contribute.     

The neuroprotective effect of cannabidiol in an in vitro model of newborn hypoxic–ischemic brain damage in mice is mediated by CB2 and adenosine receptors

To investigate the mechanisms involved in cannabidiol (CBD)-induced neuroprotection in hypoxic–ischemic (HI) immature brain, forebrain slices from newborn mice underwent oxygen and glucose deprivation in the presence of vehicle, or CBD alone or with selective antagonists of cannabinoid CB₁ and CB₂, and adenosine A₁ and A₂ receptors. CBD reduced acute (LDH efflux to the incubation medium) and apoptotic (caspase-9 concentration in tissue) HI brain damage by reducing glutamate and IL-6 concentration, and TNFα, COX-2, and iNOS expression. CBD effects were reversed by the CB₂ antagonist AM630 and by the A_2A antagonist SCH58261. The A_1A antagonist DPCPX only counteracted the CBD reduction of glutamate release, while the CB₁ antagonist SR141716 did not modify any effect of CBD. In conclusion, CBD induces robust neuroprotection in immature brain, by acting on some of the major mechanisms underlying HI cell death; these effects are mediated by CB₂ and adenosine, mainly A_2A, receptors.     

Neuropathological characteristics and alteration of the dopamine D2 receptor in hypoxic-ischemic basal ganglia necrosis

The neuropathological characteristics and alteration of the dopamine D₂ receptor (D₂R) were investigated in 27 cases of hypoxic-ischemic basal ganglia necrosis (BGN) by means of neuropathological and immunohistochemical methods. Perinatal hypoxic-ischemic BGN manifested neuronal karyorrhexis as well as eosinophilia, karyorrhexis being more predominant in preterm infants and eosinophilia more predominant in full-term infants. Immunoreactivity to D₂R was detected in the cytoplasm and dendrites of small and large neurons in the basal ganglia, and increased with neuronal maturation during the late gestational period in normal human basal ganglia. The number of D₂R-positive neurons was smaller in all cases of acute BGN than that in controls, the areas of decreased D₂R-positive neurons corresponding to the damaged regions observed on HE staining. Furthermore, neurons showed high expression of D₂R in a few cases of remote BGN, suggesting some plasticity as to the recovery of D₂R. Thus, the neuropathological characteristics of perinatal hypoxic-ischemic BGN may be related to neuronal maturation during different developmental stages in each region, and D₂R development may play a role in the basal ganglia vulnerability to hypoxic-ischemia.     

Anatomical characterization of the cannabinoid CB1 receptor in cell‐type–specific mutant mouse rescue models

Type 1 cannabinoid (CB₁) receptors are widely distributed in the brain. Their physiological roles depend on their distribution pattern, which differs remarkably among cell types. Hence, subcellular compartments with little but functionally relevant CB₁ receptors can be overlooked, fostering an incomplete mapping. To overcome this, knockin mice with cell‐type–specific rescue of CB₁ receptors have emerged as excellent tools for investigating CB₁ receptors’ cell‐type–specific localization and sufficient functional role with no bias. However, to know whether these rescue mice maintain endogenous CB₁ receptor expression level, detailed anatomical studies are necessary. The subcellular distribution of hippocampal CB₁ receptors of rescue mice that express the gene exclusively in dorsal telencephalic glutamatergic neurons (Glu‐CB₁‐RS) or GABAergic neurons (GABA‐CB₁‐RS) was studied by immunoelectron microscopy. Results were compared with conditional CB₁ receptor knockout lines. As expected, CB₁ immunoparticles appeared at presynaptic plasmalemma, making asymmetric and symmetric synapses. In the hippocampal CA1 stratum radiatum, the values of the CB₁ receptor‐immunopositive excitatory and inhibitory synapses were Glu‐CB₁‐RS, 21.89% (glutamatergic terminals); 2.38% (GABAergic terminals); GABA‐CB₁‐RS, 1.92% (glutamatergic terminals); 77.92% (GABAergic terminals). The proportion of CB₁ receptor‐immunopositive excitatory and inhibitory synapses in the inner one‐third of the dentate molecular layer was Glu‐CB₁‐RS, 53.19% (glutamatergic terminals); 2.30% (GABAergic terminals); GABA‐CB₁‐RS, 3.19% (glutamatergic terminals); 85.07% (GABAergic terminals). Taken together, Glu‐CB₁‐RS and GABA‐CB₁‐RS mice show the usual CB₁ receptor distribution and expression in hippocampal cell types with specific rescue of the receptor, thus being ideal for in‐depth anatomical and functional investigations of the endocannabinoid system. J. Comp. Neurol. 525:302–318, 2017. © 2016 Wiley Periodicals, Inc.     

Changes in the Brain Endocannabinoid System in Rat Models of Depression

A growing body of evidence implicates the endocannabinoid (eCB) system in the pathophysiology of depression. The aim of this study was to investigate the influence of changes in the eCB system, such as levels of neuromodulators, eCB synthesizing and degrading enzymes, and cannabinoid (CB) receptors, in different brain structures in animal models of depression using behavioral and biochemical analyses. Both models used, i.e., bulbectomized (OBX) and Wistar Kyoto (WKY) rats, were characterized at the behavioral level by increased immobility time. In the OBX rats, anandamide (AEA) levels were decreased in the prefrontal cortex, hippocampus, and striatum and increased in the nucleus accumbens, while 2-arachidonoylglycerol (2-AG) levels were increased in the prefrontal cortex and decreased in the nucleus accumbens with parallel changes in the expression of eCB metabolizing enzymes in several structures. It was also observed that CB₁ receptor expression decreased in the hippocampus, dorsal striatum, and nucleus accumbens, and CB₂ receptor expression decreased in the prefrontal cortex and hippocampus. In WKY rats, the levels of eCBs were reduced in the prefrontal cortex (2-AG) and dorsal striatum (AEA) and increased in the prefrontal cortex (AEA) with different changes in the expression of eCB metabolizing enzymes, while the CB₁ receptor density was increased in several brain regions. These findings suggest that dysregulation in the eCB system is implicated in the pathogenesis of depression, although neurochemical changes were linked to the particular brain structure and the factor inducing depression (surgical removal of the olfactory bulbs vs. genetic modulation).     

A role for the endocannabinoid system in exercise‐induced spatial memory enhancement in mice

It is well known that physical exercise has positive effects on cognitive functions and hippocampal plasticity. However, the underlying mechanisms have remained to be further investigated. Here we investigated the hypothesis that the memory‐enhancement promoted by physical exercise relies on facilitation of the endocannabinoid system. We observed that the spatial memory tested in the object location paradigm did not persist in sedentary mice, but could be improved by 1 week of treadmill running. In addition, exercise up‐regulated CB₁ receptor and BDNF expression in the hippocampus. To verify if these changes required CB₁ activation, we treated the mice with the selective antagonist, AM251, before each period of physical activity. In line with our hypothesis, this drug prevented the exercise‐induced memory enhancement and BDNF expression. Furthermore, AM251 reduced CB₁ expression. To test if facilitating the endocannabinoid system signaling would mimic the alterations observed after exercise, we treated sedentary animals during 1 week with the anandamide‐hydrolysis inhibitor, URB597. Mice treated with this drug recognized the object in a new location and have increased levels of CB₁ and BDNF expression in the hippocampus, showing that potentiating the endocanabinoid system equally benefits memory. In conclusion, the favorable effects of exercise upon spatial memory and BDNF expression depend on facilitation of CB₁ receptor signaling, which can be mimic by inhibition of anandamide hydrolysis in sedentary animals. Our results suggest that, at least in part, the promnesic effect of the exercise is dependent of CB1 receptor activation and is mediated by BDNF. © 2013 Wiley Periodicals, Inc.     

Early maternal deprivation induces gender‐dependent changes on the expression of hippocampal CB1 and CB2 cannabinoid receptors of neonatal rats

Early maternal deprivation (MD) in rats (24 h, postnatal day 9–10) is a model for neurodevelopmental stress. There are some data proving that MD affects the endocannabinoid system (ECS) in a gender‐dependent manner, and that these changes may account for the proposed schizophrenia‐like phenotype of MD rats. The impact of MD on cannabinoid receptor distribution in the hippocampus is unknown. The aim of this study is to evaluate the expression of CB₁ and CB₂ receptors in diverse relevant subregions (DG, CA1, and CA3) of the hippocampus in 13‐day‐old rats by immunohistochemistry and densitometry. MD induced a significant decrease in CB₁ immunoreactivity (more marked in males than in females), which was mainly associated with fibers in the strata pyramidale and radiatum of CA1 and in the strata oriens, pyramidale, and radiatum of CA3. In contrast, MD males and females showed a significant increase in CB₂ immunoreactivity in the three hippocampal areas analyzed that was detected in neuropil and puncta in the stratum oriens of CA1 and CA3, and in the polymorphic cell layer of the dentate gyrus. A marked sex dimorphism was observed in CA3, with females exhibiting higher CB₁ immunoreactivity than males, and in dentate gyrus, with females exhibiting lower CB₂ immunoreactivity than males. These results point to a clear association between developmental stress and dysregulation of the ECS. The present MD procedure may provide an interesting experimental model to further address the role of the ECS in neurodevelopmental mental illnesses such as schizophrenia. © 2008 Wiley‐Liss, Inc.     

Impaired hippocampal glucoregulation in the cannabinoid CB1 receptor knockout mice as revealed by an optimized in vitro experimental approach

Several techniques exist to study the rate of glucose uptake and metabolism in the brain but most of them are not sufficiently robust to permit extensive pharmacological analysis. Here we optimized an in vitro measurement of the simultaneous accumulation of the metabolizable and non-metabolizable ³H and ¹⁴C d-glucose analogues; permitting convenient large-scale studies on glucose uptake and metabolism in brain slices. Next, we performed an extensive pharmacological characterization on the putative glucoregulator role of the endocannabinoid system in the hippocampal slices of the rat, and the wild-type and the CB₁ cannabinoid receptor (CB₁R) knockout mice.We observed that ³H-3-O-methylglucose is a poor substrate to measure glucose uptake in the hippocampus. ³H-2-deoxyglucose is a better substrate but its uptake is still lower than that of ¹⁴C-U-d-glucose, from which the slices constantly metabolize and dissipate ¹⁴C atoms. Thus, uptake and the metabolism values are not to be used as standalones but as differences between a control and a treatment.The CB₁R knockout mice exhibited ∼10% less glucose uptake and glucose carbon atom dissipation in comparison with the wild-type mice. This may represent congenital defects as acute treatments of the rat and mouse slices with cannabinoid agonists, antagonists and inhibitors of endocannabinoid uptake/metabolism failed to induce robust changes in either the uptake or the metabolism of glucose.In summary, we report here an optimized technique ideal to complement other metabolic approaches of high spatiotemporal resolution. This technique allowed us concluding that CB₁Rs are at least indirectly involved in hippocampal glucoregulation.