Tapping into the endocannabinoid system to ameliorate acute inflammatory flares and associated pain in mouse knee joints

Introduction

During the progression of rheumatoid arthritis (RA), there are frequent but intermittent flares in which the joint becomes acutely inflamed and painful. Although a number of drug therapies are currently used to treat RA, their effectiveness is variable and side effects are common. Endocannabinoids have the potential to ameliorate joint pain and inflammation, but these beneficial effects are limited by their rapid degradation. One enzyme responsible for endocannabinoid breakdown is fatty acid amide hydrolase (FAAH). The present study examined whether URB597, a potent and selective FAAH inhibitor, could alter inflammation and pain in a mouse model of acute synovitis.

Methods

Acute joint inflammation was induced in male C57BL/6 mice by intra-articular injection of 2% kaolin/2% carrageenan. After 24 hr, articular leukocyte kinetics and blood flow were used as measures of inflammation, while hindlimb weight bearing and von Frey hair algesiometry were used as measures of joint pain. The effects of local URB597 administration were then determined in the presence or absence of either the cannabinoid (CB)1 receptor antagonist AM251, or the CB2 receptor antagonist AM630.

Results

URB597 decreased leukocyte rolling and adhesion, as well as inflammation-induced hyperaemia. However, these effects were only apparent at low doses and the effects of URB597 were absent at higher doses. In addition to the anti-inflammatory effects of URB597, fatty acid amide hydrolase (FAAH) inhibition improved both hindlimb weight bearing and von Frey hair withdrawal thresholds. The anti-inflammatory effects of URB597 on leukocyte rolling and vascular perfusion were blocked by both CB1 and CB2 antagonism, while the effect on leukocyte adherence was independent of cannabinoid receptor activation. The analgesic effects of URB597 were CB1 mediated.

Conclusions

These results suggest that the endocannabinoid system of the joint can be harnessed to decrease acute inflammatory reactions and the concomitant pain associated with these episodes.     

Medial prefrontal cortex endocannabinoid system modulates baroreflex activity through CB(1) receptors

Neural reflex mechanisms, such as the baroreflex, are involved in the regulation of cardiovascular system activity. Previous results from our group (Resstel LB, Correa FM. Medial prefrontal cortex NMDA receptors and nitric oxide modulate the parasympathetic component of the baroreflex. Eur J Neurosci 23: 481-488, 2006) have shown that glutamatergic synapses in the ventral portion of the medial prefrontal cortex (vMPFC) modulate baroreflex activity. Moreover, glutamatergic neurotransmission in the vMPFC can be modulated by the endocannabinoids system (eCBs), particularly the endocannabinoid anandamide, through presynaptic CB(1) receptor activation. Therefore, in the present study, we investigated eCBs receptors that are present in the vMPFC, and more specifically whether CB(1) receptors modulate baroreflex activity. We found that bilateral microinjection of the CB(1) receptor antagonist AM251 (100 or 300 pmol/200 nl) into the vMPFC increased baroreflex activity in unanesthetized rats. Moreover, bilateral microinjection of either the anandamide transporter inhibitor AM404 (100 pmol/200 nl) or the inhibitor of the enzyme fatty acid amide hydrolase that degrades anandamide, URB597 (100 pmol/200 nl), into the MPFC decreased baroreflex activity. Finally, pretreatment of the vMPFC with an ineffective dose of AM251 (10 pmol/200 nl) was able to block baroreflex effects of both AM404 and URB597. Taken together, our results support the view that the eCBs in the vMPFC is involved in the modulation of baroreflex activity through the activation of CB(1) receptors, which modulate local glutamate release.     

L-Type Calcium Channel Mediates Anticonvulsant Effect of Cannabinoids in Acute and Chronic Murine Models of Seizure

The anticonvulsant activities of cannabinoid compounds have been shown in various models of seizure and epilepsy. At least, part of antiseizure effects of cannabinoid compounds is mediated through calcium (Ca²⁺) channels. The L-type Ca²⁺ channels have been shown to be important in various epilepsy models. However, there is no data regarding the role of L-type Ca²⁺ channels in protective action of cannabinoids on acute and chronic models of seizure. In this study, the effects of cannabinoid compounds and L-type Ca²⁺ channels blockers, either alone or in combination were investigated using acute model of pentylenetetrazole (PTZ)-induced seizure in mice and chronic model electrical kindling of amygdala in rats. Pretreatment of mice with both cannabinoid CB1 receptor agonist arachidonyl-2′-chloroethylamide (ACEA) and endocannabinoid degradating enzyme inhibitor cyclohexylcarbamic acid 3′-carbamoyl-biphenyl-3-yl ester (URB597) produced a protective effect against PTZ-induced seizure. Administration of various doses of the two L-type Ca²⁺ channel blockers verapamil and diltiazem did not alter PTZ-induced seizure threshold. However, co-administration of verapamil and either ACEA or URB597 attenuated the protective effect of cannabinoid compounds against PTZ-induced seizure. Also, pretreatment of mice with diltiazem blocked the anticonvulsant activity of both ACEA and URB597. Moreover, (R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate (WIN55,212-2), the non-selective cannabinoid CB1 and CB2 receptor agonist showed anticonvulsant effect in amygdala-kindled rats. However, co-administration of WIN55,212-2 and verapamil attenuated the protective properties of WIN55,212-2. Our results showed that the anticonvulsant activity of cannabinoid compounds is mediated, at least in part, by L-type Ca²⁺ channels in these two models of convulsion and epilepsy.     

Modulation of anticonvulsant effects of cannabinoid compounds by GABA-A receptor agonist in acute pentylenetetrazole model of seizure in rat

Cannabinoid system plays an important role in controlling neuronal excitability and brain function. On the other hand, modulation of gamma-aminobutyric acid (GABA) transmission is one of the initial strategies for the treatment of seizure. The aim of the present study was to evaluate possible interaction between cannabinoidergic and GABAergic systems in pentylenetetrazole (PTZ)-induced acute seizure in rat. Drugs were administered by intracerebroventricular (i.c.v.) administration 20 min before a single intraperitoneal (i.p.) injection of PTZ and the latency to the first generalized tonic-clonic seizure was measured. Both the cannabinoid receptor agonist WIN55212-2 (10, 30, 50 and 100 μg/rat) and the GABA-A receptor agonist isoguvacine (IGN; 10, 30 and 50 μg/rat) significantly increased the latency of seizure occurrence. Moreover, the fatty acid amide hydrolase inhibitor URB597 showed no anticonvulsive effect while the monoacyl glycerol lipase (MAGL) inhibitor URB602 (10, 50 and 100 μg/rat) protected rats against PTZ-induced seizure. Moreover, co-administration of IGN and cannabinoid compounds attenuated the anticonvulsant action of both WIN55212-2 and IGN in this model of seizure. Our data suggests that exogenous cannabinoid WIN55212-2 and MAGL inhibitor URB602 imply their antiseizure action in part through common brain receptorial system. Moreover, the antagonistic interaction of cannabinoids and IGN in protection against PTZ-induced seizure could suggest the involvement of GABAergic system in their anticonvulsant action.     

Chronic ethanol treatment potentiates ethanol-induced increases in interstitial nucleus accumbens endocannabinoid levels in rats

We employed in vivo microdialysis to characterize the effect of an ethanol challenge injection on endocannabinoid levels in the nucleus accumbens of ethanol-naïve and chronic ethanol-treated rats. Ethanol (0.75 and 2 g/kg, i.p.) dose-dependently increased dialysate 2-arachidonoylglycerol (to a maximum 157 ± 20% of baseline) and decreased anandamide (to a minimum 52 ± 9% of baseline) in ethanol-naïve rats. The endocannabinoid clearance inhibitor N -(4-hydrophenyl) arachidonoylamide (AM404; 3 mg/kg) potentiated ethanol effects on 2-arachidonoylglycerol levels but did not alter ethanol-induced decreases in anandamide. AM404 alone did not alter dialysate levels of either endocannabinoid. Then, we characterized the effect of ethanol challenge on nucleus accumbens endocannabinoid levels in rats previously maintained on an ethanol-containing liquid diet. Ethanol challenge produced a greater and more prolonged increase in 2-arachidonoylglycerol (to a maximum 394 ± 135% of baseline) in ethanol-experienced than in ethanol-naïve rats. The profile in ethanol-experienced rats was similar to that produced by AM404 pre-treatment in ethanol-naïve rats. AM404 in ethanol-experienced rats led to a further enhancement in the 2-arachidonoylglycerol response to ethanol challenge (to a maximum 704 ± 174% of baseline). Our findings demonstrate that ethanol-induced increases in nucleus accumbens 2-arachidonoylglycerol are potentiated in animals with a history of ethanol consumption.     

Full Inhibition of Spinal FAAH Leads to TRPV1-Mediated Analgesic Effects in Neuropathic Rats and Possible Lipoxygenase-Mediated Remodeling of Anandamide Metabolism

Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats.     

Differences in spontaneously avoiding or approaching mice reflect differences in CB1-mediated signaling of dorsal striatal transmission

Approach or avoidance behaviors are accompanied by perceptual vigilance for, affective reactivity to and behavioral predisposition towards rewarding or punitive stimuli, respectively. We detected three subpopulations of C57BL/6J mice that responded with avoiding, balancing or approaching behaviors not induced by any experimental manipulation but spontaneously displayed in an approach/avoidance conflict task. Although the detailed neuronal mechanisms underlying the balancing between approach and avoidance are not fully clarified, there is growing evidence that endocannabinoid system (ECS) plays a critical role in the control of these balancing actions. The sensitivity of dorsal striatal synapses to the activation of cannabinoid CB1 receptors was investigated in the subpopulations of spontaneously avoiding, balancing or approaching mice. Avoiding animals displayed decreased control of CB1 receptors on GABAergic striatal transmission and in parallel increase of behavioral inhibition. Conversely, approaching animals exhibited increased control of CB1 receptors and in parallel increase of explorative behavior. Balancing animals reacted with balanced responses between approach and avoidance patterns. Treating avoiding animals with URB597 (fatty acid amide hydrolase inhibitor) or approaching animals with AM251 (CB1 receptor inverse agonist) reverted their respective behavioral and electrophysiological patterns. Therefore, enhanced or reduced CB1-mediated control on dorsal striatal transmission represents the synaptic hallmark of the approach or avoidance behavior, respectively. Thus, the opposite spontaneous responses to conflicting stimuli are modulated by a different involvement of endocannabinoid signaling of dorsal striatal neurons in the range of temperamental traits related to individual differences.     

Pharmacological characterization of receptor types mediating the dilator action of anandamide on blood vessels of the rat knee joint

This study investigates the actions of N-(2-hydroxyethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (anandamide) on blood flow of the rat knee joint. Topical bolus administration of anandamide (10-1000 nmol) onto the exposed knee joint capsules produced dose-dependent increases in the knee joint blood flow. Various antagonists were tested on the vasodilator response to 100 nmol anandamide. Capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide), an antagonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor, given at 10 and 100 nmol, suppressed the response by a maximum of 71%. A cannabinoid CB(1) receptor antagonist AM281 (10 nmol) and a CB(2) receptor antagonist AM630 (10 nmol) shortened its duration from 15 min to 5 min. O-1918 (1 nmol), an antagonist of the putative endothelial anandamide/abnormal-cannabidiol receptor, on its own or combined with capsazepine and the two cannabinoid receptor antagonists produced 38% and 24% inhibition on the peak vasodilator response to anandamide, respectively. URB597 (1 nmol), an inhibitor of fatty acid amide hydrolase (FAAH) suppressed the response by 40%, and an anandamide transporter inhibitor [N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide] (AM404; 1 nmol) or a cyclo-oxygenase (COX) inhibitor flurbiprofen (20 nmol) abolished the response. These findings suggest the vasodilator action of anandamide in the rat knee joint involved hydrolysis of the compound by FAAH, production of COX-derived eicosanoid(s), activation of TRPV1 receptors, and a small component involved activation of endothelial anandamide/abnormal-cannabidiol receptors; a minor delayed dilator response was mediated by activation of conventional cannabinoid receptors.     

Compounds acting at the endocannabinoid and/or endovanilloid systems reduce hyperkinesia in a rat model of Huntington's disease

We have recently reported that the administration of AM404, an inhibitor of the endocannabinoid re-uptake process, which also has affinity for the vanilloid VR1 receptors, is able to reduce hyperkinesia, and causes recovery from neurochemical deficits, in a rat model of Huntington's disease (HD) generated by bilateral intrastriatal injections of 3-nitropropionic acid (3NP). In the present study, we wanted to explore the mechanism(s) by which AM404 produces its antihyperkinetic effect in 3NP-lesioned rats by employing several experimental approaches. First, we tried to block the effects of AM404 with selective antagonists for the CB1 or VR1 receptors, i.e. SR141716A and capsazepine, respectively. We found that the reduction caused by AM404 of the increased ambulation exhibited by 3NP-lesioned rats in the open-field test was reversed when the animals had been pre-treated with capsazepine but not with SR141716A, thus suggesting a major role of VR1 receptors in the antihyperkinetic effects of AM404. However, despite the lack of behavioral effects of the CB1 receptor antagonist, the pretreatment with this compound abolished the recovery of neurochemical [gamma-aminobutyric acid (GABA) and dopamine] deficits in the caudate- putamen caused by AM404, as also did capsazepine. In a second group of studies, we wanted to explore the potential antihyperkinetic effects of various compounds which, compared to AM404, exhibit more selectivity for either the endovanilloid or the endocannabinoid systems. First, we tested VDM11 or AM374, two selective inhibitors or the endocannabinoid re-uptake or hydrolysis, respectively. Both compounds were mostly unable to reduce hyperkinesia in 3NP-lesioned rats, although VDM11 produced a certain motor depression, and AM374 exhibited a trend to stimulate ambulation, in control rats. We also tested the effects of selective direct agonists for VR1 (capsaicin) or CB1 (CP55,940) receptors. Capsaicin exhibited a strong antihyperkinetic activity and, moreover, was able to attenuate the reductions in dopamine and GABA transmission provoked by the 3NP lesion, whereas CP55,940 had also antihyperkinetic activity but was unable to cause recovery of either dopamine or GABA deficits in the basal ganglia. In summary, our data indicate a major role for VR1 receptors, as compared to CB1 receptors, in the antihyperkinetic effects and the recovery of neurochemical deficits caused in 3NP-lesioned rats by compounds that activate both CB1 and VR1 receptors, either directly or via manipulation of the levels of endogenous agonists.     

Prenatal stress and peripubertal stimulation of the endocannabinoid system differentially regulate emotional responses and brain metabolism in mice

The central endocannabinoid system (ECS) and the hypothalamic-pituitary-adrenal-axis mediate individual responses to emotionally salient stimuli. Their altered developmental adjustment may relate to the emergence of emotional disturbances. Although environmental influences regulate the individual phenotype throughout the entire lifespan, their effects may result particularly persistent during plastic developmental stages (e.g. prenatal life and adolescence). Here, we investigated whether prenatal stress - in the form of gestational exposure to corticosterone supplemented in the maternal drinking water (100 mg/l) during the last week of pregnancy - combined with a pharmacological stimulation of the ECS during adolescence (daily fatty acid amide hydrolase URB597 i.p. administration - 0.4 mg/kg - between postnatal days 29-38), influenced adult mouse emotional behaviour and brain metabolism measured through in vivo quantitative magnetic resonance spectroscopy. Compared to control mice, URB597-treated subjects showed, in the short-term, reduced locomotion and, in the long term, reduced motivation to execute operant responses to obtain palatable rewards paralleled by reduced levels of inositol and taurine in the prefrontal cortex. Adult mice exposed to prenatal corticosterone showed increased behavioural anxiety and reduced locomotion in the elevated zero maze, and altered brain metabolism (increased glutamate and reduced taurine in the hippocampus; reduced inositol and N-Acetyl-Aspartate in the hypothalamus). Present data further corroborate the view that prenatal stress and pharmacological ECS stimulation during adolescence persistently regulate emotional responses in adulthood. Yet, whilst we hypothesized these factors to be interactive in nature, we observed that the consequences of prenatal corticosterone administration were independent from those of ECS drug-induced stimulation during adolescence.     

Release of Endocannabinoids into the Cerebrospinal Fluid during the Induction of the Trigemino-Hypoglossal Reflex in Rats

The endocannabinoid system (ECS) plays an important role in pain processing and modulation. Since the specific effects of endocannabinoids within the orofacial area are largely unknown, we aimed to determine whether an increase in the endocannabinoid concentration in the cerebrospinal fluid (CSF) caused by the peripheral administration of the FAAH inhibitor URB597 and tooth pulp stimulation would affect the transmission of impulses between the sensory and motor centers localized in the vicinity of the third and fourth cerebral ventricles. The study objectives were evaluated on rats using a method that allowed the recording of the amplitude of evoked tongue jerks (ETJ) in response to noxious tooth pulp stimulation and URB597 treatment. The amplitude of ETJ was a measure of the effect of endocannabinoids on the neural structures. The concentrations of the endocannabinoids tested (AEA and 2-AG) were determined in the CSF, along with the expression of the cannabinoid receptors (CB1 and CB2) in the tissues of the mesencephalon, thalamus, and hypothalamus. We demonstrated that anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), was significantly increased in the CSF after treatment with a FAAH inhibitor, while tooth pulp stimulation had no effect on the AEA and 2-AG concentrations in the CSF. We also found positive correlations between the CSF AEA concentration and cannabinoid receptor type 1 (CB1R) expression in the brain, and between 2-AG and cannabinoid receptor type 2 (CB2R), and negative correlations between the CSF concentration of AEA and brain CB2R expression, and between 2-AG and CB1R. Our study shows that endogenous AEA, which diffuses through the cerebroventricular ependyma into CSF and exerts a modulatory effect mediated by CB1Rs, alters the properties of neurons in the trigeminal sensory nuclei, interneurons, and motoneurons of the hypoglossal nerve. In addition, our findings may be consistent with the emerging concept that AEA and 2-AG have different regulatory mechanisms because they are involved differently in orofacial pain. We also suggest that FAAH inhibition may offer a therapeutic approach to the treatment of orofacial pain.     

Receptor-independent depression of DA and 5-HT uptake by cannabinoids in rat neocortex--involvement of Na(+)/K(+)-ATPase

There is evidence that cannabinoids modulate the reuptake of some neurotransmitters in the central nervous system. In this study, we investigated the effects of the synthetic cannabinoid receptor agonist WIN55212-2, the endocannabinoid anandamide and the chemically related arachidonic acid on serotonin (5-HT) and dopamine (DA) uptake into rat neocortical synaptosomes. At micromolar concentrations, anandamide and arachidonic acid produced steep inhibition curves with Hill coefficients above unity. WIN55212-2 inhibited both DA and 5-HT uptake with Hill coefficients near unity, also within the micromolar range. The effect of WIN55212-2 was not mediated by cannabinoid receptors, since the CB1 receptor antagonist AM251 failed to diminish uptake inhibition by WIN55212-2 and since the Ki estimates of WIN55212-2 were outside the range of the dissociation constants of WIN55212-2 at both CB1 and CB2 receptors. A 100-fold higher concentration of DA, respectively 5-HT, did not induce a shift to the right of the WIN55212-2 concentration-inhibition curves, suggesting a carrier-independent mechanism. The Na(+)/K(+)-ATPase inhibitor ouabain concentration dependently inhibited 5-HT uptake. Possible drug effects on commercial Na(+)/K(+)-ATPase and synaptosomal ATP consumption were investigated using an ATP bioluminescence assay. Ouabain inhibited both commercial and synaptosomal Na(+)/K(+)-ATPase. WIN55212-2 had no effect on commercial Na(+)/K(+)-ATPase, but inhibited synaptosomal ATP consumption. Anandamide produced a sharp decrease in the activity of commercial Na(+)/K(+)-ATPase and on synaptosomal ATP consumption. Presence of ouabain significantly reduced the inhibitory effect of anandamide on synaptosomal ATP consumption, whereas the effect of WIN55212-2 remained unchanged. Our results show that cannabinoids and arachidonic acid inhibit DA and 5-HT uptake into rat neocortical synaptosomes. This effect is neither cannabinoid receptor-mediated nor due to competitive inhibition of membrane transporters, but is partly effected by a decreased Na(+)/K(+)-ATPase activity.     

Hydrolysis of 2-arachidonoylglycerol in Tetrahymena thermophila. Identification and partial characterization of a Monoacylglycerol Lipase-like enzyme

Tetrahymena thermophila is a model organism for molecular and cellular biology. Previous studies from our group showed that Tetrahymena contains major components of the endocannabinoid system, such as various endocannabinoids and FAAH. In mammalian cells the endocannabinoid 2-arachidonoylglycerol is inactivated mainly by MAGL. In this study we showed that 2-arachidonoylglycerol and 2-oleoylglycerol are hydrolyzed by the combined actions of MAGL and FAAH. MAGL-like activity was examined in the presence of FAAH specific inhibitors, URB597 or AM374 and showed optimum pH of 8-9, apparent K(M) of 14.1μM and V(max) of 5.8nmol/min×mg. The enzyme was present in membrane bound and cytosolic isoforms; molecular mass was determined at ～45 and ～40kDa. MAGL and FAAH could also inactivate endogenous signaling lipids, which might play an important role in Tetrahymena as suggested in mammals. Tetrahymena could be used as a model system for testing drugs targeting enzymes of the endocannabinoid system.     

Involvement of the suprachiasmatic nucleus in diurnal ACTH and corticosterone responsiveness to stress

We explored the contribution of the suprachiasmatic nucleus (SCN) in ACTH and corticosterone (CORT) diurnal responsiveness of the rat to restraint stress applied either in the morning (AM) or in the evening (PM). Ablation of the SCN caused the diurnal rhythmicity of the CORT response to disappear but had no effects on AM vs. PM differences in the ACTH response. Stress-response curves in SCN-lesioned rats that had prestress levels of CORT either in the AM range or in the PM range, when compared with those obtained for AM and PM controls, showed that the SCN differentially regulates the stress response depending on the underlying secretory activity of the adrenal cortex. When basal CORT secretion is at its lowest, the SCN inhibits CORT responsiveness to stress by controlling pituitary corticotrophs; but when it is at its highest, it has a permissive action that will bypass the hypophysis and reach the adrenals to adjust the response of the gland to ACTH.     

Gender-dependent cellular and biochemical effects of maternal deprivation on the hippocampus of neonatal rats: a possible role for the endocannabinoid system

Adult animals submitted to a single prolonged episode of maternal deprivation (MD) [24 h, postnatal days (PND) 9-10] show behavioral alterations that resemble specific symptoms of schizophrenia. These behavioral impairments may be related to neuronal loss in the hippocampus triggered by elevated glucocorticoids. Furthermore, our previous data suggested functional relationships between MD stress and the endocannabinoid system. In this study, we addressed the effects of MD on hippocampal glial cells and the possible relationship with changes in plasma corticosterone (CORT) levels. In addition, we investigated the putative involvement of the endocannabinoid system by evaluating (a) the effects of MD on hippocampal levels of endocannabinoids (b) The modulation of MD effects by two inhibitors of endocannabinoids inactivation, the fatty acid amide hydrolase inhibitor N-arachidonoyl-serotonin (AA-5-HT), and the endocannabinoid reuptake inhibitor, OMDM-2. Drug treatments were administered once daily from PND 7 to PND 12 at a dose of 5 mg/kg, and the animals were sacrificed at PND 13. MD induced increased CORT levels in both genders. MD males also showed an increased number of astrocytes in CA1 and CA3 areas and a significant increase in hippocampal 2-arachidonoylglycerol. The cannabinoid compounds reversed the endocrine and cellular effects of maternal deprivation. We provide direct evidence for gender-dependent cellular and biochemical effects of MD on developmental hippocampus, including changes in the endocannabinoid system.     

Early Endogenous Activation of CB1 and CB2 Receptors after Spinal Cord Injury Is a Protective Response Involved in Spontaneous Recovery

Spinal cord injury (SCI) induces a cascade of processes that may further expand the damage (secondary injury) or, alternatively, may be part of a safeguard response. Here we show that after a moderate-severe contusive SCI in rats there is a significant and very early increase in the spinal cord content of the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamide (anandamide, AEA). Since 2-AG and AEA act through CB1 and CB2 cannabinoid receptors, we administered at 20 minutes after lesion a single injection of their respective antagonists AM281 and AM630 alone or in combination to block the effects of this early endocannabinoid accumulation. We observed that AM281, AM630 or AM281 plus AM630 administration impairs the spontaneous motor recovery of rats according to the Basso-Beattie-Bresnahan (BBB) locomotor scale. However, blockade of CB1, CB2 or both receptors produced different effects at the histopathological level. Thus, AM630 administration results at 90 days after lesion in increased MHC-II expression by spinal cord microglia/monocytes and reduced number of serotoninergic fibres in lumbar spinal cord (below the lesion). AM281 exerted the same effects but also increased oedema volume estimated by MRI. Co-administration of AM281 and AM630 produced the effects observed with the administration of either AM281 or AM630 and also reduced white matter and myelin preservation and enhanced microgliosis in the epicentre. Overall, our results suggest that the endocannabinoids acting through CB1 and CB2 receptors are part of an early neuroprotective response triggered after SCI that is involved in the spontaneous recovery after an incomplete lesion.     

Timed Daily Administration of Prolactin and Corticosteroid Hormone Reduces Murine Tumor Growth and Enhances Immune Reactivity

In the present study, we investigated the time-dependent interactive effects of daily injections of prolactin (PRL) and corticosterone (CORT) on the activation of lymphocyte function and inhibition of tumor growth in vivo in mice. BALB/c mice were injected subcutaneously with EMT-6 fibrosarcoma cells (a murine connective tissue tumor cell derived from mammary gland), and then different groups of animals were treated with PRL (1μg/g body weight [BW] ip) at Oh, 4h, 8h, 12h, 16h, or 20h after CRT (1 μg/g BW ip) daily for 10 days. Different control groups were vehicle treated or treated with either hormone alone. Mice were kept in constant light 1 week before and during injections and in a 14:10 light-dark cycle thereafter. Tumor progression was monitored for up to 21 days after the cessation of treatment, and thereafter spleen lymphocytes were harvested and tested for mitogen-triggered proliferation. Prolactin administration at 8h or 16-20h after cortico-steroid treatment reduced tumor volume by 77% and 49%, respectively, relative to vehicle-treated controls. Other time relations of hormone treatment were ineffectual. Further studies indicated that the immunosuppressant cyclosporin A (CSA) substantially stimulated tumor growth; this effect was completely abrogated by a simultaneous 8h related hormone treatment. However, the 8h hormone treatment was ineffective in inhibiting tumor growth in T-cell-deficient nude mice. Spleen lymphocytes from tumor-bearing (TB) mice showed an elevated basal proliferative capacity stimulated by concanav-alin A (ConA; a stimulus for T-cell proliferation) and lipopolysaccharide (LPS; a stimulus for B-cell proliferation) compared to non-TB mice. Spleen lymphocytes from TB mice treated with CORT and PRL at 8h intervals exhibited an increased spontaneous (as well as LPS- and ConA- triggered) proliferation (by 104%, 48%, and 70%, respectively) compared with vehicle control TB mice. Fluorescence-activated cell sorting (FACS) analysis of splenocytes from hormone-treated animals indicated a 34-100% increase in the CD4⁺ (e.g., T helper cell) population. Treatment of animals with either hormone alone did not inhibit tumor growth or stimulate immune function relative to vehicle controls. The daily rhythms of plasma PRL, CORT, and thyroxine were all substantially altered by the presence of tumor in these mice. These results indicate that appropriately timed daily treatment of PRL and CORT can attenuate tumor growth, in part, via activation of antitumor immune mechanisms. Collectively, these data suggest that circadian neuroen-docrine activities must be temporally organized appropriately to inhibit tumor growth.     

Timed Daily Administration of Prolactin and Corticosteroid Hormone Reduces Murine Tumor Growth and Enhances Immune Reactivity

In the present study, we investigated the time-dependent interactive effects of daily injections of prolactin (PRL) and corticosterone (CORT) on the activation of lymphocyte function and inhibition of tumor growth in vivo in mice. BALB/c mice were injected subcutaneously with EMT-6 fibrosarcoma cells (a murine connective tissue tumor cell derived from mammary gland), and then different groups of animals were treated with PRL (1μg/g body weight [BW] ip) at Oh, 4h, 8h, 12h, 16h, or 20h after CRT (1 μg/g BW ip) daily for 10 days. Different control groups were vehicle treated or treated with either hormone alone. Mice were kept in constant light 1 week before and during injections and in a 14:10 light-dark cycle thereafter. Tumor progression was monitored for up to 21 days after the cessation of treatment, and thereafter spleen lymphocytes were harvested and tested for mitogen-triggered proliferation. Prolactin administration at 8h or 16-20h after cortico-steroid treatment reduced tumor volume by 77% and 49%, respectively, relative to vehicle-treated controls. Other time relations of hormone treatment were ineffectual. Further studies indicated that the immunosuppressant cyclosporin A (CSA) substantially stimulated tumor growth; this effect was completely abrogated by a simultaneous 8h related hormone treatment. However, the 8h hormone treatment was ineffective in inhibiting tumor growth in T-cell-deficient nude mice. Spleen lymphocytes from tumor-bearing (TB) mice showed an elevated basal proliferative capacity stimulated by concanav-alin A (ConA; a stimulus for T-cell proliferation) and lipopolysaccharide (LPS; a stimulus for B-cell proliferation) compared to non-TB mice. Spleen lymphocytes from TB mice treated with CORT and PRL at 8h intervals exhibited an increased spontaneous (as well as LPS- and ConA- triggered) proliferation (by 104%, 48%, and 70%, respectively) compared with vehicle control TB mice. Fluorescence-activated cell sorting (FACS) analysis of splenocytes from hormone-treated animals indicated a 34-100% increase in the CD4⁺ (e.g., T helper cell) population. Treatment of animals with either hormone alone did not inhibit tumor growth or stimulate immune function relative to vehicle controls. The daily rhythms of plasma PRL, CORT, and thyroxine were all substantially altered by the presence of tumor in these mice. These results indicate that appropriately timed daily treatment of PRL and CORT can attenuate tumor growth, in part, via activation of antitumor immune mechanisms. Collectively, these data suggest that circadian neuroen-docrine activities must be temporally organized appropriately to inhibit tumor growth.     

Short-term tolerance to morphine: effects of diazepam, phenobarbital, and amphetamine

Short-term tolerance to morphine, which can be demonstrated in as little as 3 hours after a single administration of the opiate, was examined in animals chronically pretreated with diazepam, phenobarbital, or amphetamine. Tail-flick latency in mice and changes in plasma corticosterone in rats were the parameters tested in these experiments. Rats primed with either saline or morphine, 10 mg/kg, were injected 3 hours subsequently with morphine, 5 mg/kg. Those primed with saline showed the characteristic plasma corticosterone elevation following morphine, when serial blood samples were examined, whereas those previously treated with morphine did not. Mice were primed with saline or either of two doses of morphine, 30 or 100 mg/kg, 3.5 hours prior to estimation of tail-flick latency and ED50 determinations. Mice primed with either dose of morphine had significantly higher ED50's than those primed with saline. Chronic treatment with diazepam or amphetamine in either species did not significantly alter short-term tolerance development by either parameter. However, with phenobarbital pretreatment, the plasma corticosterone response was attenuated and short-term tolerance to morphine's analgesic effects did not occur. Further studies in morphine-pelleted mice showed that analgesic tolerance occurred similarly in all groups. This suggests that barbiturates may delay the process.     

Endocannabinoid Signaling Regulates Sleep Stability

The hypnogenic properties of cannabis have been recognized for centuries, but endogenous cannabinoid (endocannabinoid) regulation of vigilance states is poorly characterized. We report findings from a series of experiments in mice measuring sleep with polysomnography after various systemic pharmacological manipulations of the endocannabinoid system. Rapid, unbiased scoring of vigilance states was achieved using an automated algorithm that we devised and validated. Increasing endocannabinoid tone with a selective inhibitor of monoacyglycerol lipase (JZL184) or fatty acid amide hydrolase (AM3506) produced a transient increase in non-rapid eye movement (NREM) sleep due to an augmentation of the length of NREM bouts (NREM stability). Similarly, direct activation of type 1 cannabinoid (CB1) receptors with CP47,497 increased NREM stability, but both CP47,497 and JZL184 had a secondary effect that reduced NREM sleep time and stability. This secondary response to these drugs was similar to the early effect of CB1 blockade with the antagonist/inverse agonist AM281, which fragmented NREM sleep. The magnitude of the effects produced by JZL184 and AM281 were dependent on the time of day this drug was administered. While activation of CB1 resulted in only a slight reduction in gamma power, CB1 blockade had dramatic effects on broadband power in the EEG, particularly at low frequencies. However, CB1 blockade did not significantly reduce the rebound in NREM sleep following total sleep deprivation. These results support the hypothesis that endocannabinoid signaling through CB1 is necessary for NREM stability but it is not necessary for sleep homeostasis.