Alicia Brusco

Cannabinoid CB2 receptors: immunohistochemical localization in rat brain.

Brain expression of CB2 cannabinoid receptors has been much less well established and characterized in comparison to the expression of brain CB1 receptors. Since CB2 receptors are intensely expressed in peripheral and immune tissues, expression in brain microglia has been anticipated. Nevertheless, we now describe expression of CB2-receptor-like immunoreactivity in brain in neuronal patterns that support broader CNS roles for this receptor. Two anti-CB2 affinity purified polyclonal antibodies were raised in rabbits immunized with peptide conjugates that corresponded to amino acids 1-33 and 20-33. Western blot analyses revealed specific bands that were identified using these sera and were absent when the sera were preadsorbed with 8.3 mug/ml of the immunizing peptides. These studies, and initial RT-PCR analyses of brain CB1 and CB2 mRNAs, also support the expression of brain CB2 receptor transcripts at levels much lower than those of CB1 receptors. CB2 cannabinoid receptor mRNA was clearly expressed in the cerebellum of wild type but not in CB2 knockout mice. CB2 immunostaining was detected in the interpolar part of spinal 5th nucleus of wild type but not in CB2 knockout mice, using a mouse C-terminal CB2 receptor antibody. Immunohistochemical analyses revealed abundant immunostaining for CB2 receptors in apparent neuronal and glial processes in a number of rat brain areas. Cerebellar Purkinje cells and hippocampal pyramidal cells revealed substantial immunoreactivity that was absent when sections were stained with preadsorbed sera. CB2 immunoreactivity was also observed in olfactory tubercle, islands of Calleja, cerebral cortex, striatum, thalamic nuclei, hippocampus, amygdala, substantia nigra, periaqueductal gray, paratrochlear nucleus, paralemniscal nucleus, red nucleus, pontine nuclei, inferior colliculus and the parvocellular portion of the medial vestibular nucleus. In-vitro, CB2 immunoreactivity was also present in hippocampal cell cultures. The multifocal expression of CB2 immunoreactivity in glial and neuronal patterns in a number of brain regions suggests reevaluation of the possible roles that CB2 receptors may play in the brain.

Discovery of the Presence and Functional Expression of Cannabinoid CB2 Receptors in Brain

Abstract:  Two well‐characterized cannabinoid receptors (CBrs), CB1 and CB2, mediate the effects of cannabinoids and marijuana use, with functional evidence for other CBrs. CB1 receptors are expressed primarily in brain and peripheral tissues. For over a decade several laboratories were unable to detect CB2 receptors in brain and were known to be intensely expressed in peripheral and immune tissues and have traditionally been referred to as peripheral CB2 CBrs. We have reported the discovery and functional presence of CB2 cannabinoid receptors in mammalian brain that may be involved in depression and drug abuse and this was supported by reports of identification of neuronal CB2 receptors that are involved in emesis. We used RT‐PCR, immunoblotting, hippocampal cultures, immunohistochemistry, transmission electron microscopy, and stereotaxic techniques with behavioral assays to determine the functional expression of CB2 CBrs in rat brain and mice brain exposed to chronic mild stress (CMS) or those treated with abused drugs. RT‐PCR analyses supported the expression of brain CB2 receptor transcripts at levels much lower than those of CB1 receptors. In situ hybridization revealed CB2 mRNA in cerebellar neurons of wild‐type but not of CB2 knockout mice. Abundant CB2 receptor immunoreactivity (iCB2) in neuronal and glial processes was detected in brain and CB2 expression was detected in neuron‐specific enolase (NSE) positive hippocampal cell cultures. The effect of direct CB2 antisense oligonucleotide injection into the brain and treatment with JWH015 in motor function and plus‐maze tests also demonstrated the functional presence of CB2 cannabinoid receptors in the central nervous system (CNS). Thus, contrary to the prevailing view that CB2 CBrs are restricted to peripheral tissues and predominantly in immune cells, we demonstrated that CB2 CBrs and their gene transcripts are widely distributed in the brain. This multifocal expression of CB2 immunoreactivity in brain suggests that CB2 receptors may play broader roles in the brain than previously anticipated and may be exploited as new targets in the treatment of depression and substance abuse.

Brain Neuronal CB2 Cannabinoid Receptors in Drug Abuse and Depression: From Mice to Human Subjects

Background Addiction and major depression are mental health problems associated with stressful events in life with high relapse and reoccurrence even after treatment. Many laboratories were not able to detect the presence of cannabinoid CB2 receptors (CB2-Rs) in healthy brains, but there has been demonstration of CB2-R expression in rat microglial cells and other brain associated cells during inflammation. Therefore, neuronal expression of CB2-Rs had been ambiguous and controversial and its role in depression and substance abuse is unknown. Methodology/Principal Findings In this study we tested the hypothesis that genetic variants of CB2 gene might be associated with depression in a human population and that alteration in CB2 gene expression may be involved in the effects of abused substances including opiates, cocaine and ethanol in rodents. Here we demonstrate that a high incidence of (Q63R) but not (H316Y) polymorphism in the CB2 gene was found in Japanese depressed subjects. CB2-Rs and their gene transcripts are expressed in the brains of naïve mice and are modulated following exposure to stressors and administration of abused drugs. Mice that developed alcohol preference had reduced CB2 gene expression and chronic treatment with JWH015 a putative CB2-R agonist, enhanced alcohol consumption in stressed but not in control mice. The direct intracerebroventricular microinjection of CB2 anti-sense oligonucleotide into the mouse brain reduced mouse aversions in the plus-maze test, indicating the functional presence of CB2-Rs in the brain that modifies behavior. We report for the using electron microscopy the sub cellular localization of CB2-Rs that are mainly on post-synaptic elements in rodent brain. Conclusions/Significance Our data demonstrate the functional expression of CB2-Rs in brain that may provide novel targets for the effects of cannabinoids in depression and substance abuse disorders beyond neuro-immunocannabinoid activity.

Functional Expression of Brain Neuronal CB2 Cannabinoid Receptors Are Involved in the Effects of Drugs of Abuse and in Depression

Major depression and addiction are mental health problems associated with stressful events in life with high relapse and recurrence even after treatment. Many laboratories were not able to detect the presence of CB2 cannabinoid receptors (CB2‐Rs) in healthy brains, but CB2‐R expression has been demonstrated in rat microglial cells and other brain‐associated cells during inflammation. Thus, neuronal expression of CB2‐Rs has been ambiguous and controversial, and its role in depression and substance abuse is unknown. In this study we tested the hypothesis that genetic variants of the CB2 gene might be associated with depression in a human population and that alteration in CB2 gene expression may be involved in the effects of abused substances, including opiates, cocaine, and ethanol, in rodents. Here we demonstrate that a high incidence of Q63R but not H316Y polymorphism in the CB2 gene was found in Japanese depressed subjects. CB2‐Rs and their gene transcripts are expressed in the brains of naïve mice and are modulated after exposure to stressors and administration of abused drugs. Mice that developed an alcohol preference had reduced CB2 gene expression, and chronic treatment with JWH015 a putative CB2‐R agonist, enhanced alcohol consumption in stressed but not in control mice. The direct intracerebroventricular microinjection of CB2 antisense oligonucleotide into the mouse brain reduced mouse aversions in the plus‐maze test, indicating the functional presence of CB2‐Rs in the brain that modifies behavior. Using electron microscopy we report the subcellular localization of CB2‐Rs that are mainly on postsynaptic elements in rodent brain. Our data demonstrate the functional expression of CB2‐Rs in the brain that may provide novel targets for the effects of cannabinoids in depression and substance abuse disorders beyond neuroimmunocannabinoid activity.

Postsynaptic localization of CB2 cannabinoid receptors in the rat hippocampus.

The expression of CB2 cannabinoid receptors (CB2‐Rs) in the brain and their neuronal function has now attracted research interest, since we and others have demonstrated the presence of CB2‐Rs in neuronal and glial cells in the brain. In this study, we show the subcellular distribution of CB2‐Rs in neuronal, glial, and endothelial cells in the rat hippocampus using immunohistochemical electron microscopy. Brain sections from the hippocampus were immunolabeled for CB2‐R, visualized, and analyzed by electron microscopy. We found that in neurons, CB2‐R immunoreactivity is present in the cell body as well as in large and medium‐sized dendrites. In the soma, the CB2‐R labeling is associated with the rough endoplasmic reticulum and Golgi apparatus demonstrating that CB2‐Rs are synthesized by hippocampal neurons. CB2‐R labeling in dendrites was observed in the cytoplasm and associated with the plasma membrane near the area of synaptic contact with axon terminals indicating a postsynaptic distribution of these receptors. In CB2‐R immunoreactive glial and endothelial cells, the labeling was also found to be associated with the plasma membrane. These results provide the first ultrastructural evidence that CB2‐Rs are mainly postsynaptic in the rat hippocampus. Synapse 62:944–949, 2008.

Astrocyte-neuron vulnerability to prenatal stress in the adult rat brain.

Chronic activation of the stress response during pregnancy has been shown to be injurious to the development of the offspring. We have previously demonstrated that restraint prenatal stress inflicted during the last week of pregnancy in rats increased dopamine and glutamate receptors in forebrain areas of the adult offsprings. In this study, the same prenatal insult was employed to assess morphological changes in astrocytes and in the dendritic arborization in frontal cortex, striatum, and hippocampus of the adult rat brain. On postnatal day 90, brains were processed for immunocytochemistry using primary antibodies to glial fibrillary acidic protein (GFAP; the main cytoskeletal astroglial protein), S100B protein (an astroglial‐derived neurotrophic factor), MAP‐2 (a microtubule‐associated protein present almost exclusively in dendrites), and synaptophysin (Syn; one major integral protein of the synaptic vesicles membrane). The results show a significant increase in the cell area of GFAP‐immunoreactive (‐IR) astrocytes, with high levels of S100B protein and a significant decrease in the relative area of MAP‐2‐IR neuronal processes in prenatally stressed adult rats. The expression of synaptophysin decreased in all areas studied. These results demonstrate that prenatal stress induces a long‐lasting astroglial reaction and a reduced dendritic arborization, with synaptic loss in the brain of adult offspring. In addition to the neurochemical alterations previously reported, these morphological changes might be underlying the behavioral and learning impairment previously observed in prenatally stressed rats.

Ultrastructural Localization of Neuronal Brain CB2 Cannabinoid Receptors

The functional expression of neuronal CB2 cannabinoid receptors (CB2‐Rs) in the brain has been controversial. We and others have now demonstrated that CB2‐Rs are expressed in neurons and glial cells in the brain. However, the subcellular localization of these receptors has not been characterized. In this study we used immunohistochemical electron microscopy to determine the subcellular distribution of CB2‐Rs in two brain regions. Brain sections from the CA1 hippocampal area and substantia nigra were immunostained for CB2‐Rs and analyzed by electron microscopy. In each region immunoperoxidase labeling for CB2‐Rs was detected in neurons as well as in glial and endothelial cells. In neuronal cells, CB2‐R immunoreactivity was observed in somata and large and medium‐sized dendrites. In the soma, the CB2‐R labeling was mainly associated with the rough endoplasmic reticulum and Golgi apparatus, suggesting its endogenous synthesis. In the dendrites, the CB2‐R labeling was observed in the cytoplasm and was associated with the plasma membrane near the area of synaptic contact with axon terminals, indicating a postsynaptic distribution of these receptors. In CB2‐Rs in immunoreactive glial and endothelial cells, the labeling was also found to be associated with the plasma membrane. In the substantia nigra, some unmyelinated axons were immunoreactive for CB2‐Rs, but we rarely found CB2‐R‐labeled axon terminals. These results extend our previous detection of postsynaptic cortical CB2‐Rs and provide additional ultrastructural evidence that CB2‐Rs are mainly postsynaptic in the CA1 area of the hippocampus and substantia nigra. The functional implication of pre‐ and/or postsynaptic localization of CB2‐Rs remains to be determined.

The 5HT1A receptor agonist, 8-OH-DPAT, protects neurons and reduces astroglial reaction after ischemic damage caused by cortical devascularization.

Serotonin 1A (5HT1A) receptor agonists have shown neuroprotective properties in different models of central nervous system injury. Activation of neuronal 5HT1A receptors appears to be involved in the neuroprotective effects. It remains to be elucidated if astroglial cells are responsive to the 5HT1A neuroprotective effects. The participation of astroglial S100B trophic factor has been proposed since 5HT1A activation leads to S100B release and nanomolar concentration level of this molecule showed pro-survival activity in neuronal cultures. Using the cortical devascularization model (CD; unilateral pial disruption), a procedure that results in localized ischemia without producing direct physical damage to brain tissue, we tested the effects of a full 5HT1A agonist, 8-OH-DPAT, or the antagonist WAY-100635 on cortical neuronal survival, astroglial cell response and S100B expression. Wistar rats were subjected to CD lesion which consisted of a craniotomy followed by physical damage to the underlying pial blood vessels. Two and twenty-four hours after the CD lesion, animals received intraperitoneally 8-OH-DPAT (1 mg/kg), WAY-100635 (1 mg/kg) or vehicle (sterile saline). At 3, 7 or 14 days post-lesion, animals were sacrificed and their brains processed for immunohistochemistry to detect GFAP, vimentin, MAP-2, S100B and nuclear Hoechst staining. S100B level in the brain cortex and serum was quantified by an ELISA assay. Serum S100B was considered an index of S100B release. 8-OH-DPAT treatment reduced neuronal death, dendrite loss, astroglial hypertrophy and hyperplasia. In contrast, WAY-100635 treatment increased these parameters of damage. S100B intracellular immunoreactivity in astrocytes and total S100B level showed long-lasting changes after the CD lesion and subsequent treatments depending on the 5HT1A activity. The level of serum S100B was increased in 8-OH-DPAT-treated animals. Increased damage observed in WAY-100635-treated animals supports the hypothesis that the protective 8-OH-DPAT action may be mediated by specific 5HT1A receptors. The reduction in astroglial hypertrophy and hyperplasia as well as long-term changes in S100B immunoreactivity and increased S100B release that we observed allows us to hypothesize that astroglial cells may play an important role in 5HT1A-mediated neuroprotection.

Neuronal and Glial Expression of the Multidrug Resistance Gene Product in an Experimental Epilepsy Model

Abstract1. Failure of anticonvulsive drugs to prevent seizures is a common complication of epilepsy treatment known as drug-refractory epilepsy but their causes are not well understood. It is hypothesized that the multidrug resistance P-glycoprotein (Pgp-170), the product of the MDR-1 gene that is normally expressed in several excretory tissues including the blood brain barrier, may be participating in the refractory epilepsy.2. Using two monoclonal antibodies against Pgp-170, we investigated the expression and cellular distribution of this protein in the rat brain during experimentally induced epilepsy. Repeated seizures were induced in male Wistar rats by daily administration of 3-mercaptopropionic acid (MP) 45 mg/kg i.p. for either 4 days (MP-4) or 7 days (MP-7). Control rats received an equivalent volume of vehicle. One day after the last injection, rats were sacrificed and brains were processed for immunohistochemistry for Pgp-170. As it was previously described, Pgp-170 immunostaining was observed in some brain capillary endothelial cells of animals from control group.3. Increased Pgp-170 immunoreactivity was detected in MP-treated animals. Besides the Pgp-170 expressed in blood vessels, neuronal, and glial immunostaining was detected in hippocampus, striatum, and cerebral cortex of MP-treated rats. Pgp-170 immunolabeled neurons and glial cells were observed in a nonhomogeneous distribution. MP-4 animals presented a very prominent Pgp-170 immunostaining in the capillary endothelium, surrounding astrocytes and some neighboring neurons while MP-7 group showed increased neuronal labeling.4. Our results demonstrate a selective increase in Pgp-170 immunoreactivity in the brain capillary endothelial cells, astrocytes, and neurons during repetitive MP-induced seizures.5. The role for this Pgp-170 overexpression in endothelium and astrocytes as a clearance mechanism in the refractory epilepsy, and the consequences of neuronal Pgp-170 expression remain to be disclosed.

Neuronal cytoskeleton and synaptic densities are altered after a chronic treatment with the cannabinoid receptor agonist WIN 55,212-2.

Cannabinoid CB1 receptors are the most abundant G-protein-coupled receptors in the brain. Its presynaptic location suggests a role for cannabinoids in modulating the release of neurotransmitters from axon terminals by retrograde signaling. The neuroprotective effects of cannabinoid agonists in animal models of ischemia, seizures, hypoxia, Multiple Sclerosis, Huntington and Parkinson disease have been demonstrated in several reports. The proposed mechanism for the neuroprotection ranges from antioxidant effects, reduction of microglial activation and anti-inflammatory reaction to receptor-mediated reduction of glutamate release. In the present work, we analyzed the morphological changes induced by a chronic treatment with the synthetic cannabinoid receptor agonist, WIN 55,212-2, in four brain regions where the CB1 cannabinoid receptor is present in high density: the CA1 hippocampal area, corpus striatum, cerebellum and frontal cortex. After a twice-daily treatment for 14 days with the cannabinoid receptor agonist (3 mg/kg sc, each dose) to male Wistar rats (150-170 g), the expression of neurofilaments (Nf-160 and Nf-200), microtubule-associated protein-2 (MAP-2), synaptophysin (Syn) and glial fibrillary acidic protein (GFAP) was studied by immunohistochemistry and digital image analysis. Ultrastructural study of the synapses was done using electron microscopy. After the treatment, a significant increase in the expression of neuronal cytoskeletal proteins (Nf-160, Nf-200, MAP-2) was observed, but we did not find changes in the expression of GFAP, the main astroglial cytoskeletal protein. In cerebellum, there was an increase in Syn expression and in the number of synaptic vesicles, while, in the hippocampus, an increase in the Syn expression and in the thickness of the postsynaptic densities was observed. The results obtained from these studies provide evidences on the absence of astroglial reaction and a sprouting phenomena induced by the WIN treatment that might be a key contributor to the long-term neuroprotective effects observed after cannabinoid treatments in different models of central nervous system (CNS) injury reported in the literature.