Anti-inflammatory property of the cannabinoid agonist WIN-55212-2 in a rodent model of chronic brain inflammation

Cannabinoid receptors (CBr) stimulation induces numerous central and peripheral effects. A growing interest in the beneficial properties of manipulating the endocannabinoid system has led to the possible involvement of CBr in the control of brain inflammation. In the present study we examined the effect of the CBr agonist, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4benzoxazin-6-yl]-1-naphthalenyl-methanone mesylate (WIN-55212-2), on microglial activation and spatial memory performance, using a well-characterized animal model of chronic brain inflammation produced by the infusion of lipopolysaccharide (LPS, 250 ng/h for 3 weeks) into the fourth ventricle of young rats. WIN-55212-2 (0.5 or 1.0 mg/kg/day, i.p.) was administered for 3 weeks. During the third week of treatment, spatial memory ability was examined using the Morris water-maze task. We found that 0.5 and 1 mg/kg WIN-55212-2 reduced the number of LPS-activated microglia, while 1 mg/kg WIN-55212-2 potentiated the LPS-induced impairment of performance in the water maze task. Cannabinoid receptors 1 were not expressed by microglia and astrocytes, suggesting an indirect effect of WIN-55212-2 on microglia activation and memory impairment. Our results emphasize the potential use of CBr agonists in the regulation of inflammatory processes within the brain; this knowledge may lead to the use of CBr agonists in the treatment of neurodegenerative diseases associated with chronic neuroinflammation, such as Alzheimer disease.     

Modulation of electrically evoked acetylcholine release through cannabinoid CB1 receptors: evidence for an endocannabinoid tone in the human neocortex

Cannabinoids are known to inhibit neurotransmitter release in the CNS through CB1 receptors. The present study compares the effects of synthetic cannabinoids on acetylcholine (ACh) release in human and mice neocortex. We further investigated a possible endocannabinoid tone on CB1 receptors in human neocortex caused by endogenous agonists like anandamide or 2-arachidonylglycerol. Brain slices, incubated with [3H]-choline, were superfused and stimulated electrically under autoinhibition-free conditions to evoke tritium overflow assumed to represent ACh release. The first series of experiments was performed with 26 pulses, 60 mA, at 0.1 Hz. In mice neocortical slices, the cannabinoid receptor agonist WIN55212-2 decreased ACh release (pIC50=6.68, I(max)=67%). In the human neocortex the concentration-response curve of WIN55212-2 was bell-shaped and flat (I(max observed) approximately 30%). The estimated maximum possible inhibition, however, was much larger: I(max derived)=79%. Lec, the negative logarithm (lg) of the biophase concentration of endocannabinoids in 'WIN55212-2 units,' was -6.52, the pKd of WIN55212-2 was 7.47. The CB1 receptor antagonist/inverse agonist SR141716 enhanced ACh release in the human neocortex (by 38%) and prevented the inhibitory effect of WIN55212-2. The concentration-response curve of WIN55212-2 was changed in its shape including a shift to the right due to the presence of SR141716. A pA2 of this antagonist between 11.60 and 11.18 was obtained. SR141716 alone had no effect in mice neocortical slices. A partial agonist without inverse agonistic activity, O-1184, enhanced ACh release in the human neocortex. The endocannabinoid uptake-inhibitor AM404 decreased ACh release in human, but not in mice, neocortical slices. Change of the stimulation parameters (eight trains of pseudo-one-pulse bursts (4 pulses, 76 mA, 100 Hz), spaced by 45 s intervals) led to a stronger inhibitory effect of WIN55212-2, and abolished the disinhibitory effect of SR141716 and O-1184. The results show that activation of CB1 cannabinoid receptors leads to inhibition of ACh release in the human and mouse neocortex. The endocannabinoid tone is high in the human, but not in the mouse neocortex and is dependent on neuronal activity. SR141716 acts as a competitive CB1 receptor antagonist.     

Functional CB1 receptors are broadly expressed in neocortical GABAergic and glutamatergic neurons

The cannabinoid receptor CB1 is found in abundance in brain neurons, whereas CB2 is essentially expressed outside the brain. In the neocortex, CB1 is observed predominantly on large cholecystokinin (CCK)-expressing interneurons. However, physiological evidence suggests that functional CB1 are present on other neocortical neuronal types. We investigated the expression of CB1 and CB2 in identified neurons of rat neocortical slices using single-cell RT-PCR. We found that 63% of somatostatin (SST)-expressing and 69% of vasoactive intestinal polypeptide (VIP)-expressing interneurons co-expressed CB1. As much as 49% of pyramidal neurons expressed CB1. In contrast, CB2 was observed in a small proportion of neocortical neurons. We performed whole cell recordings of pyramidal neurons to corroborate our molecular findings. Inhibitory postsynaptic currents (IPSCs) induced by a mixed muscarinic/nicotinic cholinergic agonist showed depolarization-induced suppression of inhibition and were decreased by the CB1 agonist WIN-55212-2 (WIN-2), suggesting that interneurons excited by cholinergic agonists (mainly SST and VIP neurons) possess CB1. IPSCs elicited by a nicotinic receptor agonist were also reduced in the presence of WIN-2, suggesting that neurons excited by nicotinic agonists (mainly VIP neurons) indeed possess CB1. WIN-2 largely decreased excitatory postsynaptic currents evoked by intracortical electrical stimulation, pointing at the presence of CB1 on glutamatergic pyramidal neurons. All WIN-2 effects were strongly reduced by the CB1 antagonist AM 251. We conclude that CB1 is expressed in various neocortical neuronal populations, including glutamatergic neurons. Our combined molecular and physiological data suggest that CB1 widely mediates endocannabinoid effects on glutamatergic and GABAergic transmission to modulate cortical networks.     

Cannabinoids inhibit excitatory neurotransmission in the substantia nigra pars reticulata

The substantia nigra pars reticulata belongs to the brain regions with the highest density of CB(1) cannabinoid receptors. Since the level of CB(1) receptor messenger RNA is very low in the pars reticulata, most of the receptors are probably localized on terminals of afferent axons. The hypothesis was tested that terminals of glutamatergic afferents of substantia nigra pars reticulata neurons possess CB(1) cannnabinoid receptors, the activation of which presynaptically modulates neurotransmission.Rat midbrain slices were superfused and the electrophysiological properties of substantia nigra pars reticulata neurons were studied with the patch-clamp technique. Focal electrical stimulation in the presence of bicuculline evoked excitatory postsynaptic currents mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate glutamate receptors. The excitatory postsynaptic currents were reduced by the metabotropic glutamate receptor agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD; 10(-4)M). The mixed CB(1)/CB(2) cannabinoid receptor agonists R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2, 3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone (WIN55212-2; 10(-8)-10(-5)M) and (-)-cis-3-[2-hydroxy-4-(1, 1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940; 10(-6)M) also produced inhibition. The maximal inhibition by WIN55212-2 was 54+/-6%. The CB(1) cannabinoid antagonist N-piperidino-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716A; 10(-6)M) prevented the effect of WIN55212-2, but had no effect when superfused alone. WIN55212-2 (10(-6)M) increased the amplitude ratio of two excitatory postsynaptic currents evoked with an interstimulus interval of 100ms. Currents evoked by short ejection of glutamate on to the surface of the slices were not changed by WIN55212-2.The results show that activation of CB(1) cannabinoid receptors inhibits glutamatergic synaptic transmission between afferent axons and neurons in the substantia nigra pars reticulata. The lack of effect of the cannabinoids on glutamate-evoked currents and the increase of the paired-pulse ratio indicate that the mechanism of action is presynaptic inhibition of transmitter release.     

Cannabinoids inhibit striatonigral GABAergic neurotransmission in the mouse

The substantia nigra pars reticulata (SNR) belongs to the brain regions with the highest density of CB(1) cannabinoid receptors. Anatomical studies indicate that the great majority of CB(1) receptors in the SNR are localized on terminals of GABAergic axons arriving from the caudate-putamen (striatonigral axons). The aim of the present experiments was to clarify the role of CB(1) receptors on terminals of striatonigral axons.Oblique sagittal slices, including the caudate-putamen and the substantia nigra, were prepared from brains of young mice. Electrical stimulation in the caudate-putamen elicited GABAergic inhibitory postsynaptic currents (IPSCs) in the SNR, which were studied by patch-clamp techniques. The long latency of IPSCs (14+/-1 ms) suggests that striatonigral axons were indeed activated within the caudate-putamen. The synthetic CB(1)/CB(2) cannabinoid receptor agonist WIN55212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate; 10(-5) M) decreased the amplitude of IPSCs by 93+/-1%. CP55940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; 10(-5) M), another CB(1)/CB(2) receptor agonist, also reduced IPSC amplitude, by 76+/-4%. The CB(1) cannabinoid receptor antagonist SR141716A (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide; 10(-6) M) prevented the inhibition produced by WIN55212-2 (10(-5) M). Depolarization of SNR neurons led to suppression of IPSCs; this suppression was prevented by SR141716A (10(-6) M). Three observations indicate that the agonists inhibited neurotransmission presynaptically. (1) CP55940 (10(-5) M) enhanced the ratio of amplitudes of two IPSCs which were elicited by two electrical stimuli 100 ms apart (paired pulses). (2) WIN55212-2 (10(-5) M) did not change the amplitude of miniature IPSCs recorded in the presence of tetrodotoxin. (3) WIN55212-2 (10(-5) M) also had no effect on currents elicited in SNR neurons by ejection of the GABA(A) receptor agonist muscimol from a pipet.In summary, we have established a method which allows selective examination of GABAergic neurotransmission between striatonigral axons and SNR neurons. Using this method, the function of CB(1) cannabinoid receptors on terminals of striatonigral axons was unequivocally clarified. Activation of these receptors causes strong presynaptic inhibition of GABAergic neurotransmission between striatonigral axons and SNR neurons. This effect may be one explanation of the catalepsy observed in animals after cannabinoid administration. Endocannabinoids released from SNR neurons can modulate striatonigral neurotransmission by inhibiting GABA release from terminals of striatonigral axons.     

Cannabinoid and kappa opioid receptors reduce potassium K current via activation of G(s) proteins in cultured hippocampal neurons

The current study showed that potassium K current (I(K)), which is evoked at depolarizing potentials between -30 and +40 mV in cultured hippocampal neurons, was significantly reduced by exposure to the CB1 cannabinoid receptor agonist WIN 55,212-2 (WIN-2). WIN-2 (20-40 nM) produced an average 45% decrease in I(K) amplitude across all voltage steps, which was prevented by SR141716A, the CB1 receptor antagonist. The cannabinoid receptor has previously been shown to be G(i/o) protein-linked to several cellular processes; however, the decrease in I(K) was unaffected by modulators of G(i/o) proteins and agents that alter levels of protein kinase A. In contrast, CB1 receptor-mediated or direct activation of G(s) proteins with cholera toxin (CTX) produced the same decrease in I(K) amplitude as WIN-2, and the latter was blocked in CTX-treated cells. G(s) protein inhibition via GDPbetaS also eliminated the effects of WIN-2 on I(K). Consistent with this outcome, activation of protein kinase C (PKC) by arachidonic acid produced similar effects to WIN-2 and CTX. Kappa opioid receptor agonists, which also reduce I(K) amplitude via G(s) proteins, were compared with WIN-2 actions on I(K.) The kappa receptor agonist U50,488 reduced I(K) amplitude in the same manner as WIN-2, while the kappa receptor antagonist, nor-binaltorphimine, actually increased I(K) amplitude and significantly reduced the effect of co-administered WIN-2. The results indicate that CB1 and kappa receptor activation is additive with respect to I(K) amplitude, suggesting that CB1 and kappa receptors share a common G(s) protein signaling pathway involving PKC.     

Endocannabinoids contribute to metabotropic glutamate receptor-mediated inhibition of GABA release onto hippocampal CA3 pyramidal neurons in an isolated neuron/bouton preparation

Retrograde synaptic signaling by endogenous cannabinoids (endocannabinoids) is a recently discovered form of neuromodulation in various brain regions. In hippocampus, it is well known that endocannabinoids suppress presynaptic inhibitory neurotransmitter release in CA1 region. However, endocannabinoid signaling in CA3 region remains to be examined. Here we investigated whether presynaptic inhibition can be caused by activation of postsynaptic group I metabotropic glutamate receptors (mGluRs) and following presynaptic cannabinoid receptor type 1 (CB1 receptor) using mechanically dissociated rat hippocampal CA3 pyramidal neurons with adherent functional synaptic boutons. Application of group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) reversibly suppressed spontaneous inhibitory postsynaptic currents (IPSCs). In the presence of tetrodotoxin (TTX), frequency of miniature IPSCs was significantly reduced by DHPG, while there were no significant changes in minimum quantal size and sensitivity of postsynaptic GABA_A receptors to the GABA_A receptor agonist muscimol, indicating that this suppression was caused by a decrease in GABA release from presynaptic nerve terminals. Application of CB1 synthetic agonist WIN55212-2 (mesylate(R)-(+)-[2,3-dihydro-5-methyl-3-[4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone) or endocannabinoid 2-arachidonoylglycerol also suppressed the spontaneous IPSC. The inhibitory effect of DHPG on spontaneous IPSCs was abolished by SR-141716 (5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide), a CB1 receptor antagonist. Furthermore, postsynaptic application of GDP-βS blocked the DHPG-induced inhibition of spontaneous IPSCs, indicating the involvement of endcannabinoid-mediated retrograde synaptic signaling. These results provide solid evidence for retrograde signaling from postsynaptic group I mGluRs to presynaptic CB1 receptors, which induces presynaptic inhibition of GABA release in rat hippocampal CA3 region.     

Cannabinoid receptor modulation of synapses received by cerebellar Purkinje cells

The high density of cannabinoid receptors in the cerebellum and the degradation of motor coordination produced by cannabinoid intoxication suggest that synaptic transmission in the cerebellum may be strongly regulated by cannabinoid receptors. Therefore the effects of exogenous cannabinoids on synapses received by Purkinje cells were investigated in rat cerebellar slices. Parallel fiber-evoked (PF) excitatory postsynaptic currents (EPSCs) were strongly inhibited by bath application of the cannabinoid receptor agonist WIN 55212-2 (5 microM, 12% of baseline EPSC amplitude). This effect was completely blocked by the cannabinoid CB1 receptor antagonist SR 141716. It is unlikely that this was the result of alterations in axonal excitability because fiber volley velocity and kinetics were unchanged and a cannabinoid-induced decrease in fiber volley amplitude was very minor (93% of baseline). WIN 55212-2 had no effect on the amplitude or frequency of spontaneously occurring miniature EPSCs (mEPSCs), suggesting that the effect of CB1 receptor activation on PF EPSCs was presynaptically expressed, but giving no evidence for modulation of release processes after Ca(2+) influx. EPSCs evoked by climbing fiber (CF) stimulation were less powerfully attenuated by WIN 55212-2 (5 microM, 74% of baseline). Large, action potential-dependent, spontaneously occurring inhibitory postsynaptic currents (sIPSCs) were either severely reduced in amplitude (<25% of baseline) or eliminated. Miniature IPSCs (mIPSCs) were reduced in frequency (52% of baseline) but not in amplitude, demonstrating suppression of presynaptic vesicle release processes after Ca(2+) influx and suggesting an absence of postsynaptic modulation. The decrease in mIPSC frequency was not large enough to account for the decrease in sIPSC amplitude, suggesting that presynaptic voltage-gated channel modulation was also involved. Thus, while CB1 receptor activation reduced neurotransmitter release at all major classes of Purkinje cell synapses, this was not accomplished by a single molecular mechanism. At excitatory synapses, cannabinoid suppression of neurotransmitter release was mediated by modulation of voltage-gated channels in the presynaptic axon terminal. At inhibitory synapses, in addition to modulation of presynaptic voltage-gated channels, suppression of the downstream vesicle release machinery also played a large role.     

The synthetic cannabinoid WIN 55212-2 differentially modulates thigmotaxis but not spatial learning in adolescent and adult animals

Unlike Δ(9)-THC, the synthetic compound WIN 55212-2 (WIN) is a full agonist of endogenous cannabinoid receptors. Previous work has shown Δ(9)-THC to affect adolescent and adult animals differently on numerous behavioral measures of spatial memory, anxiety, and locomotor activity. However, far less is known about the developmental and neurobehavioral effects of WIN. To address this, we assessed the effect of WIN (1mg/kg) on spatial learning in adolescent and adult rats using the Morris water maze. While all animals demonstrated decreased swim distance across days, WIN affected adolescents and adults differently. It improved performance in adolescents and resulted in a nearly significant performance decrement in adults. However, these effects were significantly related to thigmotaxis, which declined across days in the water maze testing protocol. WIN reduced thigmotaxis on days 1 and 2 (but not days 3-5) only in adolescents. The effect of age, treatment, and the age×treatment interaction was eliminated after controlling for thigmotaxis. These results indicate that WIN affects thigmotaxis rather than spatial reference memory. More importantly, these findings indicate a dissociation between the developmental effects of THC and the synthetic CB1 receptor agonist, WIN 55212-2. We suggest that the role of thigmotaxis be carefully evaluated in future neurodevelopmental studies of spatial learning, especially those investigating the endocannabinoid system.     

Caffeine attenuates lipopolysaccharide-induced neuroinflammation

Caffeine is an antagonist at A1 and A2A adenosine receptors and epidemiological evidence suggests that caffeine consumption reduces the risk of Alzheimer's and Parkinson's diseases. Neuroinflammation plays a role in the etiology of these diseases and caffeine may provide protection through the modulation of inflammation. Adenosine has a known role in the propagation of inflammation and caffeine may reduce microglia activation directly by blocking adenosine receptors on microglia. Chronic neuroinflammation is associated with an increase in extracellular levels of glutamate and drugs that limit the effects of glutamate at neuronal receptors have been shown to indirectly reduce the neuroinflammatory response of microglia cells. A1 and A2A receptors have been shown to regulate the pre-synaptic release of glutamate, therefore, caffeine may also reduce neuroinflammation via its ability to regulate glutamate release. Caffeine was administered at various doses to young rats with experimentally induced neuroinflammation by chronic infusion of lipopolysaccharide (LPS) over two or four weeks into the 4th ventricle and to aged rats with naturally elevated levels of microglia activation. Caffeine attenuated the number of activated microglia within the hippocampus of animals with LPS-induced and age-related inflammation.     

A beneficial aspect of a CB1 cannabinoid receptor antagonist: SR141716A is a potent inhibitor of macrophage infection by the intracellular pathogen Brucella suis

The psychoactive component of marijuana, delta9-tetrahydrocannabinol (THC) suppresses different functions of immunocytes, including the antimicrobicidal activity of macrophages. The triggering of cannabinoid receptors of CB1 and CB2 subtypes present on leukocytes may account for these effects. We investigated the influence of specific CB1 or CB2 receptor antagonists (SR141716A and SR144528, respectively) and nonselective CB1/CB2 cannabinoid receptor agonists (CP55,940 or WIN 55212-2) on macrophage infection by Brucella suis, an intracellular gram-negative bacteria. None of the compounds tested affected bacterial phagocytosis. By contrast, the intracellular multiplication of Brucella was dose-dependently inhibited in cells treated with 10-500 nM SR141716A and 1 microM SR141716A-induced cells exerted a potent microbicidal effect against the bacteria. SR144528, CP55,940, or WIN 55212-2 did not affect (or slightly potentiated) the growth of phagocytized bacteria. However, CP55,940 or WIN 55212-2 reversed the SR141716A-mediated effect, which strongly suggested an involvement of macrophage CB1 receptors in the phenomenon. SR141716A was able to pre-activate macrophages and to trigger an activation signal that inhibited Brucella development. The participation of endogenous cannabinoid ligand(s) in Brucella infection was discussed. Finally, our data show that SR141716A up-regulates the antimicrobial properties of macrophages in vitro and might be a pharmaceutical compound useful for counteracting the development of intramacrophagic gram-negative bacteria.     

Cannabinoid receptor and N-acyl phosphatidylethanolamine phospholipase D--evidence for altered expression in multiple sclerosis

Cannabinoids have been shown to have a beneficial effect in both animal models of multiple sclerosis (MS) and human disease, although the mechanisms of action are unclear. We examined expression of the major cannabinoid receptors [(CBRs) cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2)] and a key enzyme involved in synthesis of the endocannabinoid anandamide [N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] in autopsy brain samples from patients with MS. CB1 was expressed in neurons, injured axons, oligodendrocytes, macrophages/microglia, some astrocytes, endothelial cells, smooth muscle cells and pericytes. CB2 and NAPE-PLD were localized to cerebral endothelial cells, pericytes, smooth muscle cells, astrocytes and macrophages/microglia. NAPE-PLD immunoreactivity was also seen in neurons. Endothelial CB2 expression was greatest in chronic inactive plaques, and in areas was seen in segments of endothelium where the endothelial expression of adhesion molecules (VCAM-1 and ICAM-1) was focally undetectable, and was often expressed in areas of blood-brain barrier damage. Vascular density was increased in chronic active plaques and normal-appearing white matter compared with controls. These data support findings from animal models which suggest a role for the endocannabinoid system in the MS, particularly in the regulation of endothelial leukocyte adhesion and the cellular response to injury.     

Redistribution of CB1 cannabinoid receptors during evolution of cholinergic basal forebrain territories and their cortical projection areas: a comparison between the gray mouse lemur (Microcebus murinus, primates) and rat

Endocannabinoid signaling, mediated by presynaptic CB1 cannabinoid receptors on neurons, is fundamental for the maintenance of synaptic plasticity by modulating neurotransmitter release from axon terminals. In the rodent basal forebrain, CB1 cannabinoid receptor-like immunoreactivity is only harbored by a subpopulation of cholinergic projection neurons. However, endocannabinoid control of cholinergic output from the substantia innominata, coincident target innervation of cholinergic and CB1 cannabinoid receptor-containing afferents, and cholinergic regulation of endocannabinoid synthesis in the hippocampus suggest a significant cholinergic-endocannabinergic interplay. Given the functional importance of the cholinergic modulation of endocannabinoid signaling, here we studied CB1 cannabinoid receptor distribution in cholinergic basal forebrain territories and their cortical projection areas in a prosimian primate, the gray mouse lemur. Perisomatic CB1 cannabinoid receptor immunoreactivity was unequivocally present in non-cholinergic neurons of the olfactory tubercule, and in cholecystokinin-containing interneurons in layers 2/3 of the neocortex. Significantly, CB1 cannabinoid receptor-like immunoreactivity was localized to cholinergic perikarya in the magnocellular basal nucleus. However, cortical cholinergic terminals lacked detectable CB1 cannabinoid receptor levels. A dichotomy of CB1 cannabinoid receptor distribution in frontal (suprasylvian) and parietotemporal (subsylvian) cortices was apparent. In the frontal cortex, CB1 cannabinoid receptor-containing axons concentrated in layers 2/3 and layer 6, while layer 4 and layer 5 were essentially devoid of CB1 cannabinoid receptor immunoreactivity. In contrast, CB1 cannabinoid receptors decorated axons in all layers of the parietotemporal cortex with peak densities in layer 2 and layer 4. In the hippocampus, CB1 cannabinoid receptor-containing terminals concentrated around pyramidal cell somata and proximal dendrites in the CA1-CA3 areas, and granule cell dendrites in the molecular layer of the dentate gyrus. CB1 cannabinoid receptors frequently localized to inhibitory GABAergic terminals while leaving glutamatergic boutons unlabeled. Aging did not affect either the density or layer-specific distribution of CB1 cannabinoid receptor-immunoreactive processes. We concluded that organizing principles of CB1 cannabinoid receptor-containing neurons and their terminal fields within the basal forebrain are evolutionarily conserved between rodents and prosimian primates. In contrast, the areal expansion and cytoarchitectonic differentiation of neocortical subfields in primates is associated with differential cortical patterning of CB1 cannabinoid receptor-containing subcortical and intracortical afferents.     

The cannabinoid receptor agonist, WIN 55,212-2, inhibits cool-specific lamina I medullary dorsal horn neurons

Cannabinoid receptor agonists have been demonstrated to inhibit medullary and spinal cord dorsal horn nociceptive neurons. The effect of cannabinoids on thermoreceptive specific neurons in the spinal or medullary dorsal horn remains unknown. In the present study, single-unit recordings from the rat medullary dorsal horn were performed to examine the effect of a cannabinoid receptor agonists on cold-specific lamina I spinothalamic tract neurons. The cannabinoid CB1/CB2 receptor agonist, WIN 55,212-2 (WIN-2), was locally applied to the medullary dorsal horn and the neuronal activity evoked by cooling the receptive field was recorded. WIN-2 (1 microg/microl and 2 microg/microl) significantly attenuated cold-evoked activity. Co-administration of the CB1 receptor antagonist SR 141716 with WIN-2 did not affect cold-evoked activity. These results demonstrate a potential mechanism by which cannabinoids produce hypothermia, and also suggest that cannabinoids may affect non-noxious thermal discrimination.     

Autoradiography of receptor-activated G-proteins in post mortem human brain

The agonist-stimulated guanosine 5'-(gamma-[(35)S]thio)triphosphate binding assay was used to anatomically localize receptor-activated G-proteins by autoradiography in post mortem human brain. The optimal conditions for guanosine 5'-(gamma-[(35)S]thio)triphosphate binding to human brain sections were established in post mortem samples of the prefrontal cortex, hippocampus, basal ganglia, brainstem and cerebellar cortex. An excess of GDP (2mM) was required to decrease basal activity and obtain effective stimulation by specific agonists. guanosine 5'-(gamma-[(35)S]Thio)triphosphate binding was increased after stimulation with specific agonists of different G-protein-coupled receptors. They include cannabinoid (WIN55212-2), mu-opioid ([D-Ala(2),N-Me-Phe(4), Gly(5)-ol]enkephalin), serotonin-1A [(+/-)-8-hydroxy-2-(di-n-propylamino)tetralin] and serotonin-1B/1D (sumatriptan), cholinergic muscarinic receptors (carbachol) and alpha(2)-adrenoceptors (UK14304). Such stimulation reached 1458%, 440%, 188%, 219%, 61% and 339%, respectively, over the basal levels. In tissue sections, the use of the above-mentioned agonists (10(-4)M) showed patterns of anatomical distribution similar to those already described by receptor autoradiography, with high densities over the hippocampus (serotonin-1A receptors), cortex (alpha(2)-adrenoceptors) and striatum (mu-opioid receptors). The highest binding levels were reached with the cannabinoid receptor agonist in most of the analysed brain regions. Carbachol produced only moderate stimulation of those same regions. The blockage of agonist-stimulated guanosine 5'-(gamma-[(35)S]thio)triphosphate binding by selective antagonists verified that the effect was receptor mediated.This technique provides a method to identify modifications of the receptor-mediated activation of G-proteins in post mortem human brain with anatomical resolution. It also provides valuable information on the level of drug efficacy in the human species.     

Cannabinoids depress excitatory neurotransmission between the subthalamic nucleus and the globus pallidus

The globus pallidus receives its major glutamatergic input from the subthalamic nucleus and subthalamic nucleus neurons synthesize CB1 cannabinoid receptors. The hypothesis of the present work was that CB1 receptors are localized in terminals of subthalamo-pallidal glutamatergic axons and that their activation leads to presynaptic modulation of neurotransmission between these axons and globus pallidus neurons. Patch-clamp studies were carried out on oblique-sagittal mouse brain slices. The subthalamic nucleus was stimulated electrically and the resulting excitatory postsynaptic currents (EPSCs) were recorded in globus pallidus neurons. The mixed CB1/CB2 receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate (WIN55212-2; 3 x 10(-7) M) had no effect on EPSCs. WIN55212-2 (10(-5) M) decreased the amplitude of EPSCs by 44+/-8%. The inhibition by WIN55212-2 (10(-5) M) was prevented by the CB1 antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide (10(-6) M). WIN55212-2 (10(-5) M) did not change the amplitude of spontaneous EPSCs (sEPSCs) recorded in globus pallidus neurons but lowered their frequency. Moreover, WIN55212-2 (10(-5) M) had no effect on currents elicited by direct activation of postsynaptic receptors on globus pallidus neurons by glutamate (10(-3) M) ejected from a pipette. In a final series of experiments, the firing of subthalamic nucleus neurons was recorded; WIN55212-2 (10(-5) M) did not change the firing of these neurons. The results show that activation of CB1 receptors inhibits glutamatergic neurotransmission between the subthalamic nucleus and the globus pallidus. Lack of effect of cannabinoids on the amplitude of sEPSCs and on currents evoked by direct stimulation of postsynaptic glutamate receptors indicates that the mechanism is presynaptic inhibition of glutamate release from axon terminals. Cannabinoids seem to act preferentially presynaptically: in contrast to their action on axon terminals, they have no effect on somadendritic receptors regulating firing rate. Cannabinoids elicit catalepsy in vivo. The observed inhibition of glutamatergic neurotransmission in the globus pallidus would favor catalepsy.     

Ethanol desensitizes cannabinoid CB1 receptors modulating monoamine synthesis in the rat brain in vivo

The endocannabinoid system and the cannabinoid CB(1) receptors are involved in the development of ethanol tolerance and dependence. This study aimed to investigate the in vivo sensitivity of a CB(1) receptor agonist (WIN 55,212-2) modulating the synthesis of 3,4-dihydroxy-phenylalanine/dopamine/noradrenaline (DOPA/DA/NA) and that of 5-hydroxy-tryptophan/serotonin (5-HTP/5-HT) in rat brain after ethanol treatment and withdrawal. In control rats, WIN 55,212-2 (4 mg/kg, i.p., for 1h), through a mechanism sensible to the CB(1) antagonist SR 141716A, increased the synthesis of DOPA/NA in a slice of brainstem containing the locus ceruleus (250%) and in the hippocampus (64%), and it reduced DOPA/DA synthesis in the striatum (47%). WIN 55,212-2 also decreased the synthesis of 5-HTP/5-HT in the locus ceruleus (43%), hippocampus (35%) and striatum (35%). In the locus ceruleus of ethanol-treated rats, the stimulatory effect of WIN 55,212-2 on DOPA/NA synthesis was abolished (acute treatment) or markedly attenuated (53-55%, chronic treatment and withdrawal), whereas in the hippocampus this effect was reduced only in chronic ethanol-withdrawn rats (33%). In the striatum of ethanol-treated rats (acute, chronic and withdrawal), the inhibitory effect of WIN 55,212-2 on DOPA/DA synthesis was completely blunted or markedly reduced. Similarly, the inhibitory effect of WIN 55,212-2 on 5-HTP/5-HT synthesis was reduced or abolished in the three brain regions after chronic ethanol and during withdrawal. These results indicate that treatment with ethanol in rats induces a functional desensitization of CB(1) receptors modulating the synthesis of brain monoamines.     

Dopamine activation of endogenous cannabinoid signaling in dorsal striatum

We measured endogenous cannabinoid release in dorsal striatum of freely moving rats by microdialysis and gas chromatography/mass spectrometry. Neural activity stimulated the release of anandamide, but not of other endogenous cannabinoids such as 2-arachidonylglycerol. Moreover, anandamide release was increased eightfold over baseline after local administration of the D2-like (D2, D3, D4) dopamine receptor agonist quinpirole, a response that was prevented by the D2-like receptor antagonist raclopride. Administration of the D1-like (D1, D5) receptor agonist SKF38393 had no such effect. These results suggest that functional interactions between endocannabinoid and dopaminergic systems may contribute to striatal signaling. In agreement with this hypothesis, pretreatment with the cannabinoid antagonist SR141716A enhanced the stimulation of motor behavior elicited by systemic administration of quinpirole. The endocannabinoid system therefore may act as an inhibitory feedback mechanism countering dopamine-induced facilitation of motor activity.     

Characterization of cannabinoid-binding sites in zebrafish brain

We present here the pharmacological characterization of cannabinoid-binding sites in zebrafish brain homogenates using radiolabeled binding techniques. The nonselective agonist [3H]-CP55940 binds with high affinity (KD = 0.50+/-0.06 nM and a Bmax = 1047+/-36.01 fmol/mg protein), displaying one binding site. The slightly CB2 selective agonist [3H]-WIN55212-2 also binds with high affinity to zebrafish brain membranes displaying two different binding sites with affinities KD1 = 0.35+/-0.09 nM and KD2 = 105.81+/-66.36 nM. Competition binding assays using [3H]-WIN55212-2 and several unlabeled ligands were performed. WIN55212-2 significantly displaced the tritiated ligand binding showing the two binding sites observed with its tritiated homologous, while the slightly selective CB1 cannabinoid ligand HU-210, the nonselective cannabinoid ligand CP55940 and the endogenous cannabinoid ligand anandamide presented one binding site. Also, the functionality of these cannabinoid sites was analyzed using the known [35S]GTPgammaS assay. All the agonist used presented an agonist profile and the rank order for potency was HU-210 > WIN55212-2 > CP55940 >anandamide. Our results provide evidence that, although some of the typical cannabinoid ligands for mammalian receptors do not fully recognize the cannabinoid-binding sites in zebrafish brain, the activity of the endogenous zebrafish cannabinoid system might not significantly differ from that displayed by the cannabinoid system described in other species. Hence the study of zebrafish cannabinoid activity may contribute to an understanding of the endogenous cannabinoid system in higher vertebrates.     

Social isolation and chronic handling alter endocannabinoid signaling and behavioral reactivity to context in adult rats

Social deprivation in early life disrupts emotionality and attentional processes in humans. Rearing rats in isolation reproduces some of these abnormalities, which are attenuated by daily handling. However, the neurochemical mechanisms underlying these responses remain poorly understood. We hypothesized that post-weaning social isolation alters the endocannabinoid system, a neuromodulatory system that controls emotional responding. We characterized behavioral consequences of social isolation and evaluated whether handling would reverse social isolation-induced alterations in behavioral reactivity to context and the endocannabinoid system. At weaning, pups were single or group housed and concomitantly handled or not handled daily until adulthood. Rats were tested in emotionality- and attentional-sensitive behavioral assays (open field, elevated plus maze, startle and prepulse inhibition). Cannabinoid receptor densities and endocannabinoid levels were quantified in a separate group of rats. Social isolation negatively altered behavioral responding. Socially-isolated rats that were handled showed less deficits in the open field, elevated plus maze, and prepulse inhibition tests. Social isolation produced site-specific alterations (supraoptic nucleus, ventrolateral thalamus, rostral striatum) in cannabinoid receptor densities compared to group rearing. Handling altered the endocannabinoid system in neural circuitry controlling emotional expression. Handling altered endocannabinoid content (prefrontal and piriform cortices, nucleus accumbens) and cannabinoid receptor densities (lateral globus pallidus, cingulate and piriform cortices, hippocampus) in a region-specific manner. Some effects of social isolation on the endocannabinoid system were moderated by handling. Isolates were unresponsive to handling-induced increases in cannabinoid receptor densities (caudal striatum, anterior thalamus), but were sensitive to handling-induced changes in endocannabinoid content (piriform, prefrontal cortices), compared to group-reared rats. Our findings suggest alterations in the endocannabinoid system may contribute to the abnormal isolate phenotype. Handling modifies the endocannabinoid system and behavioral reactivity to context, but surmounts only some effects of social isolation. These data implicate a pivotal role for the endocannabinoid system in stress adaptation and emotionality-related disturbances.