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).     

Endogenous cannabinoid signaling is required for voluntary exercise‐induced enhancement of progenitor cell proliferation in the hippocampus

Voluntary exercise and endogenous cannabinoid activity have independently been shown to regulate hippocampal plasticity. The aim of the current study was to determine whether the endocannabinoid system is regulated by voluntary exercise and if these changes contribute to exercise‐induced enhancement of cell proliferation. In Experiment 1, 8 days of free access to a running wheel increased the agonist binding site density of the cannabinoid CB₁ receptor; CB₁ receptor‐mediated GTPγS binding; and the tissue content of the endocannabinoid anandamide in the hippocampus but not in the prefrontal cortex. In Experiment 2, the CB₁ receptor antagonist AM251 (1 mg kg⁻¹) was administered daily to animals given free access to a running wheel for 8 days, after which cell proliferation in the hippocampus was examined through immunohistochemical analysis of the cell cycle protein Ki‐67. Voluntary exercise increased proliferation of progenitor cells, as evidenced by the increase in the number of Ki‐67 positive cells in the granule cell layer of the dentate gyrus (DG) in the hippocampus. However, this effect was abrogated by concurrent treatment with AM251, indicating that the increase in endocannabinoid signaling in the hippocampus is required for the exercise‐induced increase in cell proliferation. These data demonstrate that the endocannabinoid system in the hippocampus is sensitive to environmental change and suggest that it is a mediator of experience‐induced plasticity. © 2009 Wiley‐Liss, Inc.     

Changes in the cannabinoids receptors in rats following treatment with antidepressants

The endocannabinoid (eCB) system plays a significant role in the pathophysiology of depression. The potential participation of this system in the mechanism of action of antidepressants has been highlighted in recent years. The aim of this study was to investigate the expression of cannabinoid (CB) receptors using Western blot and CB₁ receptor density using autoradiography after acute or chronic administration of antidepressant drugs [imipramine (IMI, 15 mg/kg), escitalopram (ESC, 10 mg/kg) and tianeptine (TIA, 10 mg/kg)]. Antidepressants given chronically elevated CB₁ receptor density in the cortical structures and hippocampal areas, while a decrease of CB₁ receptor density was observed in the striatum after IMI and ESC treatment. The CB₁ receptor expression decreases in the dorsal striatum after chronic administration of IMI and ESC or the receptor rise in the hippocampus after chronic ESC and TIA treatment were confirmed using Western blot analyses. An increase in the CB₂ receptor expression was observed in the cortical structures and hippocampus after chronic administration of ESC and TIA, while a decrease in this expression was noted in the striatum and cerebellum after chronic IMI treatment. Our results provide clear evidence that the antidepressant exposures provoke some modulations within the eCB system through CB receptors.     

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.     

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.     

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.     

Opposing local effects of endocannabinoids on the activity of noradrenergic neurons and release of noradrenaline: relevance for their role in depression and in the actions of CB1 receptor antagonists

There is strong evidence that endocannabinoids modulate signaling of serotonin and noradrenaline, which play key roles in the pathophysiology and treatment of anxiety and depression. Most pharmacological and genetic, human and rodent studies suggest that the presence of under-functioning endocannabinoid type-1 (CB₁) receptors is associated with increased anxiety and elevated extracellular serotonin concentration. In contrast, noradrenaline is presumably implicated in the mediation of depression-type symptoms of CB₁ receptor antagonists. Evidence shows that most CB₁ receptors located on axons and terminals of GABA-ergic, serotonergic or glutamatergic neurons stimulate the activity of noradrenergic neurons. In contrast, those located on noradrenergic axons and terminals inhibit noradrenaline release efficiently. In this latter process, excitatory ionotropic or G protein-coupled receptors, such as the NMDA, alpha1 and beta1 adrenergic receptors, activate local endocannabinoid synthesis at postsynaptic sites and stimulate retrograde endocannabinoid neurotransmission acting on CB₁ receptors of noradrenergic terminals. The underlying mechanisms include calcium signal generation, which activates enzymes that increase the synthesis of both anandamide and 2-arachidonoylglycerol, while G_q/11 protein activation also increases the formation of 2-arachidonoylglycerol from diacylglycerol during the signaling process. In addition, other non-CB₁ receptor endocannabinoid targets such as CB₂, transient receptor potential vanilloid subtype, peroxisome proliferator-activated receptor-alpha and possibly GPR55 can also mediate some of the endocannabinoid effects. In conclusion, both neuronal activation and neurotransmitter release depend on the in situ synthesized endocannabinoids and thus, local endocannabinoid concentrations in different brain areas may be crucial in the net effect, namely in the regulation of neurons located postsynaptically to the noradrenergic synapse.     

Altered responses of dopamine D3 receptor null mice to excitotoxic or anxiogenic stimuli: Possible involvement of the endocannabinoid and endovanilloid systems

Dopamine and the endocannabinoids, anandamide and 2-arachidonoylglycerol, interact at several levels in the brain, with the involvement of both cannabinoid CB₁ receptors and transient receptor potential vanilloid type-1 (TRPV1) channels (which are alternative anandamide receptors). Using pharmacological, immunohistochemical and analytical approaches, we investigated the response of dopamine D₃ receptor null (D3R^(−/−)) mice in models of epilepsy and anxiety, in relation to their brain endocannabinoid and endovanilloid tone. Compared to wild-type mice, D3R^(−/−) mice exhibited a delayed onset of clonic seizures, enhanced survival time, reduced mortality rate and more sensitivity to anticonvulsant effects of diazepam after intraperitoneal administration of picrotoxin (7 mg/kg), and a less anxious-like behaviour in the elevated plus maze test. D3R^(−/−) mice also exhibited different endocannabinoid and TRPV1, but not CB₁, levels in the hippocampus, nucleus accumbens, amygdala and striatum. Given the role played by CB₁ and TRPV1 in neuroprotection and anxiety, and based on data obtained here with pharmacological tools, we suggest that the alterations of endocannabinoid and endovanilloid tone found in D3R^(−/−) mice might account for part of their altered responses to excitotoxic and anxiogenic stimuli.     

Effects of electroconvulsive seizures and antidepressant drugs on brain-derived neurotrophic factor protein in rat brain.

BACKGROUND: The antidepressant-like effects of brain-derived neurotrophic factor (BDNF) infusions in brain, and the upregulation of BDNF mRNA and its receptor in rats exposed to electroconvulsive seizure (ECS) and antidepressants, suggested a role for increased BDNF protein. METHODS: We measured BDNF protein levels with a two-site enzyme-linked immunosorbent assay (ELISA) in six brain regions of adult male rats that received daily ECS or daily injections of antidepressant drugs. RESULTS: The BDNF ELISA method was validated by the 50% loss of BDNF protein in the brains of +/- BDNF knockout mice, the 60%-100% recovery of spiked recombinant BDNF, and by the amounts and regional variations of BDNF measured in the six brain regions. Ten consecutive daily exposures to ECS increased BDNF protein in the parietal cortex (219%), entorhinal cortex (153%), hippocampus (132%), frontal cortex (94%), neostriatum (67%), and septum (29%). BDNF increased gradually in the hippocampus and frontal cortex, with a peak response by the fourth day of ECS. Increases peaked at 15 hours after the last ECS and lasted at least 3 days thereafter. Two weeks of daily injections with the monoamine (MAO)-A and -B inhibitor tranylcypromine (8-10 mg/kg, IP) increased BDNF by 15% in the frontal cortex, and 3 weeks treatment increased it by 18% in the frontal cortex and by 29% in the neostriatum. Tranylcypromine, fluoxetine, and desmethylimipramine did not elevate BDNF in the hippocampus. CONCLUSIONS: Elevations in BDNF protein in brain are consistent with the greater treatment efficacy of ECS and MAO inhibitors in drug-resistant major depressive disorder and may be predictive for the antidepressant action of the more highly efficacious interventions.     

Effects of olanzapine, fluoxetine and olanzapine/fluoxetine on creatine kinase activity in rat brain

Recently, a fixed combination of the atypical antipsychotic olanzapine and the serotonin selective reuptake inhibitor (SSRI) fluoxetine has been approved in the US for the treatment of bipolar I depression. In this work, we evaluated the effect of acute and chronic administration of fluoxetine, olanzapine and the combination of fluoxetine/olanzapine on creatine kinase (CK) activity in the brain of rats. For acute treatment, adult male Wistar rats received one single injection of olanzapine (3 or 6 mg/kg) and/or fluoxetine (12.5 or 25 mg/kg). For chronic treatment, adult male Wistar rats received daily injections of olanzapine (3 or 6 mg/kg) and/or fluoxetine (12.5 or 25 mg/kg) for 28 days. In the present study we observed that acute administration of OLZ inhibited CK activity in cerebellum and prefrontal cortex. The acute administration of FLX inhibited creatine kinase in cerebellum, prefrontal cortex, hippocampus, striatum and cerebral cortex. In the chronic treatment, when the animals were killed 2 h after the last injection a decrease in creatine kinase activity after FLX administration, alone or in combination with OLZ, in cerebellum, prefrontal cortex, hippocampus, striatum and cerebral cortex of rats occurred. However, when the animals were killed 24 h after the last injection, we found no alterations in the enzyme. Although it is difficult to extrapolate our findings to the human condition, the inhibition of creatine kinase activity by these drugs may be associated to the occurrence of some side effects of OLZ and FLX.     

Chronic fluoxetine treatment upregulates 5-HT uptake sites and 5-HT2 receptors in rat brain: An autoradiographic study

This study was undertaken to investigate the effect of chronic treatment with fluoxetine, a selective serotonin uptake inhibitor used widely in the treatment of depression, on the distribution and density of 5-HT uptake sites, 5-HT₂ receptors, and vesicular amine uptake sites in rat brain. Fluoxetine (10 mg/kg i. p.) was administered daily for 21 days. The density of 5-HT uptake sites labelled by [³H]paroxetine, 5-HT₂ receptors labelled by [³H]ketanserin in presence of tetrabenazine and vesicular amine uptake sites labelled by [³H]ketanserin in the presence of mianserin were measured by quantitative autoradiography in 22 areas of rat brain, using coronal tissue sections. Chronic administration of fluoxetine produced significant increases in the density of 5-HT uptake sites in layers of frontoparietal cortex (by 32–43%), of striate cortex (by 55%), in CA1 field of hippocampus (by 111%) and in superior colliculus (by 20%). Fluoxetine treatment also resulted in upregulation of 5-HT₂ receptors in layers of frontparietal cortex (31–38%) and in CA2-3 fields of hippocampus (by 39%). The density of tetrabenazine-sensitive vesicular amine uptake sites in the caudate-putamen was also significantly increased (by 66%). The observed alterations in 5-HT uptake site and 5-HT₂ receptor densities are likely a part of adaptive neuronal changes that occur after chronic administration of fluoxetine and may be related to the antidepressant effect of the drug. © 1993 Wiley-Liss, Inc.     

Endocannabinoid modulation of inflammatory hyperalgesia in the IFN-α mouse model of depression

Depression is a well-recognised effect of long-term treatment with interferon-alpha (IFN-α), a widely used treatment for chronic viral hepatitis and malignancy. In addition to the emotional disturbances, high incidences of painful symptoms such as headache and joint pain have also been reported following IFN-α treatment. The endocannabinoid system plays an important role in emotional and nociceptive processing, however it is unknown whether repeated IFN-α administration induces alterations in this system. The present study investigated nociceptive responding in the IFN-α-induced mouse model of depression and associated changes in the endocannabinoid system. Furthermore, the effects of modulating peripheral endocannabinoid tone on inflammatory pain-related behaviour in the IFN-α model was examined. Repeated IFN-α administration (8000 IU/g/day) to male C57/Bl6 mice increased immobility in the forced swim test and reduced sucrose preference, without altering body weight gain or locomotor activity, confirming development of the depressive-like phenotype. There was no effect of repeated IFN-α administration on latency to respond in the hot plate test on day 4 or 7 of treatment, however, formalin-evoked nociceptive behaviour was significantly increased in IFN-α treated mice following 8 days of IFN-α administration. 2-Arachidonoyl glycerol (2-AG) levels in the periaqueductal grey (PAG) and rostroventromedial medulla (RVM), and anandamide (AEA) levels in the RVM, were significantly increased in IFN-α-, but not saline-, treated mice following formalin administration. There was no change in endocannabinoid levels in the prefrontal cortex, spinal cord or paw tissue between saline- or IFNα-treated mice in the presence or absence of formalin. Furthermore, repeated IFN-α and/or formalin administration did not alter mRNA expression of genes encoding the endocannabinoid catabolic enzymes (fatty acid amide hydrolase or monoacylglycerol lipase) or endocannabinoid receptor targets (CB_1, CB₂ or PPARs) in the brain, spinal cord or paw tissue. Intra plantar administration of PF3845 (1 μg/10 μl) or MJN110 (1 μg/10 μl), inhibitors of AEA and 2-AG catabolism respectively, attenuated formalin-evoked hyperalgesia in IFN-α, but not saline-, treated mice. In summary, increasing peripheral endocannabinoid tone attenuates inflammatory hyperalgesia induced following repeated IFN-α administration. These data provide support for the endocannabinoid system in mediating and modulating heightened pain responding associated with IFNα-induced depression.     

Expression of CB<Subscript>1</Subscript> cannabinoid receptor in the anterior cingulate cortex in schizophrenia,bipolar disorder,and major depression

Summary The human endogenous cannabinoid system is an appealing target in the investigation of psychiatric disorders. In schizophrenia, endocannabinoids and their receptors are involved in the pathology of the disease. Previous studies reported an increased radioligand binding to cannabinoid receptors 1 (CB₁) in schizophrenia, both in the dorsolateral prefrontal cortex and in the anterior cingulate cortex (ACC). We analyzed the expression of the CB₁ receptors in the ACC at the protein level using immunohistochemistry. In a quantitative postmortem study, 60 patients suffering from schizophrenia, bipolar disorder, major depression and controls were included. Numerical densities of neurons and glial cells immunopositive for CB₁ receptors were evaluated. No evidence of an increased or decreased density of CB₁ receptor immunopositive cells in schizophrenia or bipolar disorder was found. In major depression, CB₁ receptor immunopositive glial cells in the grey matter were decreased. Furthermore, our data show that different medications have an impact on the expression of CB₁ receptors in the ACC.     

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.     

Involvement of endocannabinoids in antidepressant and anti-compulsive effect of fluoxetine in mice

Endocannabinoid analogues exhibit antidepressant and anti-compulsive like effects similar to that of serotonin selective reuptake inhibitors (SSRIs) indicating a parallelism between the effects of serotonin and endocannabinoids. Therefore, the present study was designed to investigate the role of endocannabinoids in the antidepressant and anti-compulsive like effect of fluoxetine using mice model of forced swim test (FST) and marble-burying behavior (MBB). The results revealed that intracerebroventricular injections of endocannabinoid analogues, anandamide, a CB₁ agonist (AEA: 1-20 μg/mouse); AM404, an anandamide transport inhibitor (0.1-10 μg/mouse); and URB597, a fatty acid amide hydrolase inhibitor (0.05-10 μg/mouse) produced antidepressant-like effect dose-dependently, whereas influenced the MBB in a biphasic manner (produced a U-shaped dose-response curve). Fluoxetine (2.5-20 mg/kg, i.p.) dose dependently decreased the immobility time as well as burying behavior. Co-administration of sub-effective dose of fluoxetine (2.5 mg/kg, i.p.) potentiated the effect of sub-effective dose of AEA (0.5 μg/mouse, i.c.v.), AM404 (0.05 μg/mouse, i.c.v) or URB597 (0.01 μg/mouse, i.c.v) in both the paradigms. Interestingly, pretreatment with AM251, a CB₁ antagonist, blocked the effect of fluoxetine in FST and MBB at a dose (1 μg/mouse, i.c.v) that per se had no effect on either parameter. Similar effects were obtained with endocannabinoid analogues in AM251 pretreated mice. However, AM251 increased the burying behavior in MBB at a highest dose tested (5 μg/mouse). None of the treatments had any influence on locomotor activity. Thus, the study indicates an interaction between endocannabinoid and serotonergic system in regulation of depressive and compulsive-like behavior.     

Anticonvulsant effect of cannabidiol in the pentylenetetrazole model: Pharmacological mechanisms,electroencephalographic profile,and brain cytokine levels

Cannabidiol (CBD), the main nonpsychotomimetic compound from Cannabis sativa, inhibits experimental seizures in animal models and alleviates certain types of intractable epilepsies in patients. Its pharmacological profile, however, is still uncertain. Here we tested the hypothesis that CBD anticonvulsant mechanisms are prevented by cannabinoid (CB₁ and CB₂) and vanilloid (TRPV1) receptor blockers. We also investigated its effects on electroencephalographic (EEG) activity and hippocampal cytokines in the pentylenetetrazole (PTZ) model. Pretreatment with CBD (60 mg/kg) attenuated seizures induced by intraperitoneal, subcutaneous, and intravenous PTZ administration in mice. The effects were reversed by CB₁, CB₂, and TRPV1 selective antagonists (AM251, AM630, and SB366791, respectively). Additionally, CBD delayed seizure sensitization resulting from repeated PTZ administration (kindling). This cannabinoid also prevented PTZ-induced EEG activity and interleukin-6 increase in prefrontal cortex. In conclusion, the robust anticonvulsant effects of CBD may result from multiple pharmacological mechanisms, including facilitation of endocannabinoid signaling and TRPV1 mechanisms. These findings advance our understanding on CBD inhibition of seizures, EEG activity, and cytokine actions, with potential implications for the development of new treatments for certain epileptic syndromes.     

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.     

CB1 modulation of hormone secretion, neuronal activation and mRNA expression following extracellular volume expansion

The endocannabinoid system includes important signaling molecules that are involved in several homeostatic and neuroendocrine functions. In the present study, we evaluated the effects of the type 1 cannabinoid (CB₁) receptor antagonist, rimonabant (10 mg/kg, p.o.), on hormone secretion, neuronal activation and mRNA expression in the hypothalamus following isotonic (I-) or hypertonic (H-) extracellular volume expansion (EVE). The total nitrate content in the PVN and SON was also assessed under the same experimental conditions. Our results showed that OT and AVP plasma concentrations were increased in response to H-EVE, while decreased AVP levels were found following I-EVE. Accordingly, both I- and H-EVE stimulated oxytocinergic neuronal activation, as evidenced by the increased number of c-Fos/OT double labeled neurons in the hypothalamus. The vasopressinergic cells of the PVN and SON, however, were only activated in response to H-EVE. Furthermore, increased amounts of both AVP and OT mRNAs were found in the hypothalamus following EVE. Pretreatment with rimonabant significantly potentiated hormone secretion and also vasopressinergic and oxytocinergic neuronal activation induced by EVE, although decreased AVP and OT mRNA expression was found in the hypothalami of rimonabant pretreated groups. In addition, the nitrate content in the PVN and SON was not altered in response to EVE or rimonabant pretreatment. Taken together, these results suggest that the CB₁ receptor may modulate several events that contribute to the development of appropriate responses to increased fluid volume and osmolality.     

Lifespan changes in cannabinoid 1 receptor mRNA expression in the female C57BL/6J mouse brain

Many brain functions that underlie behavior, cognition, and emotions vary with age, as does susceptibility to neuropsychological disorders. The expression of specific genes that are involved in these functions, such as the genes encoding for oxytocin, its receptors, and apolipoprotein D, varies with age across different brain regions. The cannabinoid 1 receptor (CB₁R) is one of the most widely spread G-protein coupled receptors in the central nervous system and is increasingly recognized for its important contribution to various brain functions. Although changes in CB₁R expression with age have been reported in the male mouse brain, they have not been well investigated in the female brain. Here, we used fluorescence in situ hybridization to target CB₁R mRNA in the whole brains of female C57BL/6J mice aged 4, 6, 12, 52 (12 months) and 86 weeks (20 months), and quantified CB₁R-positive cells in 36 brain regions across the whole brain. The results showed that CB₁R-positive cells number changed with age. Specifically, CB₁R expression increased with age in some subregions of the cortex, decreased with age in the lateral septal area, and reached its lowest level at 52 weeks in the thalamus, hypothalamus, and hindbrain subregions. Cluster analysis revealed that some brain regions shared similar temporal characteristics in CB₁R-positive cell number across the lifespan. Our results provide evidence that investigation of the neural basis of age-related characteristics of female brain functions is not only warranted but required.     

Fluoxetine increases norepinephrine release in rat hypothalamus as measured by tissue levels of MHPG-SO4 and microdialysis in conscious rats

Summary The selective serotonin uptake inhibitor fluoxetine (10mg/kg i.p.) increased tissue levels of the norepinephrine metabolite 3-methoxy-4-hydroxyphenylethylene glycol sulfate (MHPG-SO₄) in rat hypothalamus, indicating an increased release of norepinephrine. Microdialysis studies in conscious rats showed that fluoxetine (10 mg/kg i.p.) increased extracellular concentrations of norepinephrine as well as serotonin in the hypothalamus. In contrast, desipramine (10mg/kg i.p.) increased extracellular concentration of norepinephrine but not serotonin in the hypothalamus. Consistent with its mechanism of being a selective serotonin uptake inhibitor, local perfusion of fluoxetine (10M) caused a 7-fold increase in hypothalamic extracellular serotonin and a small non-significant increase in extracellular norepinephrine. The subsequent systemic injection of fluoxetine (10mg/kg s.c.) after local perfusion caused a 3-fold increase in extracellular norepinephrine, indicating that fluoxetine's action leading to an increase in extracellular norepinephrine was not occurring in the terminal areas of the hypothalamus but elsewhere in the brain, possibly cell bodies in the locus coeruleus.