Mode of action of cannabinoids on nociceptive nerve endings

In recent years, cannabinoids have emerged as attractive alternatives or supplements to therapy for chronic pain states. However, in humans the activation of cannabinoid receptors in neurons of the central nervous system is associated with psychotropic side effects, temporary memory impairment and dependence, which arise via the effects of cannabinoids on forebrain circuits. For clinical exploitation of the analgesic properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesic effects. The cannabinoid receptor family currently includes two cloned metabotropic receptors: CB1, CB2 and possibly GPR55 which are distributed widely across many key loci in pain-modulating pathways, including the peripheral terminals of primary afferents. Modulation of transducer ion channels expressed at nociceptive terminals occurs upon activation of metabotropic cannabinoid receptors, but direct cannabinoid action on ion channels involved in sensory transduction or regulation of neuron excitability likely contributes to the peripheral cannabinoid effects.     

Cannabinoid receptors undergo axonal flow in sensory nerves.

Cannabinoids modulate nociceptive processing through central and peripheral mechanisms. The present study was conducted to evaluate axonal flow of cannabinoid receptors from the dorsal root ganglion to the periphery and to identify the putative involvement of CB1 and/or CB2 receptor subtypes. The sciatic nerve was tightly ligated to dam the flow of cannabinoid receptors to the periphery. The densities of cannabinoid receptors proximal and distal to one or two tightly constrictive ligatures was evaluated using in vitro receptor binding and high-resolution emulsion autoradiography. In both models, [3H]CP55,940 binding accumulated proximal as opposed to distal to the ligature. These data indicate that axonal transport of cannabinoid receptors to the periphery was occluded by tight constriction of the sciatic nerve. In situ hybridization histochemistry revealed that dorsal root ganglia cells synthesize CB1 but not CB2 receptor messenger RNA. By contrast, CB2 messenger RNA was highly expressed in sections of rat spleen that were processed together with the dorsal root ganglia, as previously described. These data demonstrate that neuronal cannabinoid CB1 receptors are synthesized in cells of the dorsal root ganglia and inserted on terminals in the periphery.     

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.     

Endocannabinoid-dependent LTD in a nociceptive synapse requires activation of a presynaptic TRPV-like receptor

Recent studies have found that some forms of endocannabinoid-dependent synaptic plasticity in the hippocampus are mediated through activation of transient potential receptor vanilloid (TRPV) receptors instead of cannabinoid receptors CB1 or CB2. The potential role for synaptic localization of TRPV receptors during endocannabinoid modulation of nociceptive synapses was examined in the leech CNS where it is possible to record from the same pair of neurons from one preparation to the next. Long-term depression (LTD) in the monosynaptic connection between the nociceptive (N) sensory neuron and the longitudinal (L) motor neuron was found to be endocannabinoid-dependent given that this depression was blocked by RHC-80267, an inhibitor of DAG lipase that is required for 2-arachidonoyl glycerol (2AG) synthesis. Intracellular injection of a second DAG lipase inhibitor, tetrahyrdolipstatin (THL) was also able to block this endocannabinoid-dependent LTD (ecLTD) when injected postsynaptically but not presynaptically. N-to-L ecLTD was also inhibited by the TRPV1 antagonists capsazepine and SB 366791. Bath application of 2AG or the TRPV1 agonists capsaicin and resiniferatoxin mimicked LTD and both capsaicin- and 2AG-induced depression were blocked by capsazepine. In addition, pretreatment with 2AG or capsaicin occluded subsequent expression of LTD induced by repetitive activity. Presynaptic, but not postsynaptic, intracellular injection of capsazepine blocked both activity- and 2AG-induced ecLTD, suggesting that a presynaptic TRPV-like receptor in the leech mediated this form of synaptic plasticity. These findings potentially extend the role ecLTD to nociceptive synapses and suggest that invertebrate synapses, which are thought to lack CB1/CB2 receptor orthologues, utilize a TRPV-like protein as an endocannabinoid receptor.     

Characterisation of cannabinoid 1 receptor expression in the perikarya, and peripheral and spinal processes of primary sensory neurons

The cannabinoid 1 (CB1) receptor is expressed by a sub-population of primary sensory neurons. However, data on the neurochemical identity of the CB1 receptor-expressing cells, and CB1 receptor expression by the peripheral and central terminals of these neurons are inconsistent and limited. We characterised CB1 receptor expression in dorsal root ganglia (DRG) and spinal cord at the lumbar 4–5 level, as well as in the urinary bladder and glabrous skin of the hindpaw. About 1/3 of DRG neurons exhibited immunopositivity for the CB1 receptor, the majority of which showed positivity for the nociceptive markers calcitonin gene-related peptide (CGRP) or/and Griffonia (bandeiraea) simplicifolia IB4 isolectin-binding. Virtually all CB1 receptor-immunostained fibres showed immunopositivity for CGRP in the skin, while very few did in the urinary bladder. No CB1 receptor-immunopositive nerve fibres were IB4 positive in either peripheral tissue. Spinal laminae I and II-outer showed the highest density of CB1 receptor-immunopositive punctae, the majority of which showed positivity for CGRP or/and IB4 binding. These data indicate that a major sub-population of nociceptive primary sensory neurons expresses CB1 receptors that are transported to both peripheral and central terminals of these cells. Therefore, the present data suggest that manipulation of endogenous CB1 receptor agonist levels in these areas may significantly reduce nociceptive input into the spinal cord.     

Cannabinoid agonist, CP 55,940, prevents capsaicin-induced sensitization of spinal cord dorsal horn neurons

Low doses of cannabinoids applied intrathecally attenuate capsaicin-evoked heat and mechanical hyperalgesia via CB1 receptors. Although cannabinoids produce antinociception, in part, by attenuating responses of nociceptive neurons in the spinal cord, few studies have examined the effect of cannabinoids on sensitization of spinal neurons. We therefore investigated whether a cannabinoid receptor agonist, CP 55,940, attenuated excitation and sensitization of spinal nociceptive neurons produced by intraplantar injection of 0.1% capsaicin (10 microl). In rats, wide-dynamic-range (WDR) and high-threshold (HT) neurons were classified according to responses evoked by mechanical stimuli of varying intensity. CP 55,940 (10 microg in 50 microl) or vehicle was applied directly to the spinal cord and responses to mechanical (von Frey monofilament) and heat stimuli were recorded 10 min after drug treatment. CP 55,940 alone did not alter responses to mechanical stimuli; however the enhanced responses to mechanical stimuli after injection of capsaicin into the receptive field were dose dependently attenuated in both HT and WDR neurons. Vehicle-treated neurons increased their response to 300.6 +/- 52.1% of baseline after capsaicin, whereas CP 55,940-treated neurons responded at 153.0 +/- 27.1% of baseline. The effects of CP 55,940 on sensitization to heat were less pronounced; however, CP 55,940 attenuated the capsaicin-evoked decrease in heat threshold in HT neurons. The attenuation by CP 55,940 of sensitization to mechanical stimuli was blocked by pretreatment of the spinal cord with the CB1 receptor antagonist, SR141716A. These studies demonstrate that cannabinoid application to the spinal cord prevents central sensitization.     

Cannabinoid suppression of noxious heat-evoked activity in wide dynamic range neurons in the lumbar dorsal horn of the rat

The effects of cannabinoid agonists on noxious heat-evoked firing of 62 spinal wide dynamic range (WDR) neurons were examined in urethan-anesthetized rats (1 cell/animal). Noxious thermal stimulation was applied with a Peltier device to the receptive fields in the ipsilateral hindpaw of isolated WDR neurons. To assess the site of action, cannabinoids were administered systemically in intact and spinally transected rats and intraventricularly. Both the aminoalkylindole cannabinoid WIN55,212-2 (125 microg/kg iv) and the bicyclic cannabinoid CP55,940 (125 microg/kg iv) suppressed noxious heat-evoked activity. Responses evoked by mild pressure in nonnociceptive neurons were not altered by CP55,940 (125 microg/kg iv), consistent with previous observations with another cannabinoid agonist, WIN55,212-2. The cannabinoid induced-suppression of noxious heat-evoked activity was blocked by pretreatment with SR141716A (1 mg/kg iv), a competitive antagonist for central cannabinoid CB1 receptors. By contrast, intravenous administration of either vehicle or the receptor-inactive enantiomer WIN55,212-3 (125 microg/kg) failed to alter noxious heat-evoked activity. The suppression of noxious heat-evoked activity induced by WIN55,212-2 in the lumbar dorsal horn of intact animals was markedly attenuated in spinal rats. Moreover, intraventricular administration of WIN55,212-2 suppressed noxious heat-evoked activity in spinal WDR neurons. By contrast, both vehicle and enantiomer were inactive. These findings suggest that cannabinoids selectively modulate the activity of nociceptive neurons in the spinal dorsal horn by actions at CB1 receptors. This modulation represents a suppression of pain neurotransmission because the inhibitory effects are selective for pain-sensitive neurons and are observed with different modalities of noxious stimulation. The data also provide converging lines of evidence for a role for descending antinociceptive mechanisms in cannabinoid modulation of spinal nociceptive processing.     

The effects of cannabinoids on the brain.

Cannabinoids have a long history of consumption for recreational and medical reasons. The primary active constituent of the hemp plant Cannabis sativa is delta9-tetrahydrocannabinol (delta9-THC). In humans, psychoactive cannabinoids produce euphoria, enhancement of sensory perception, tachycardia, antinociception, difficulties in concentration and impairment of memory. The cognitive deficiencies seem to persist after withdrawal. The toxicity of marijuana has been underestimated for a long time, since recent findings revealed delta9-THC-induced cell death with shrinkage of neurons and DNA fragmentation in the hippocampus. The acute effects of cannabinoids as well as the development of tolerance are mediated by G protein-coupled cannabinoid receptors. The CB1 receptor and its splice variant CB1A, are found predominantly in the brain with highest densities in the hippocampus, cerebellum and striatum. The CB2 receptor is found predominantly in the spleen and in haemopoietic cells and has only 44% overall nucleotide sequence identity with the CB1 receptor. The existence of this receptor provided the molecular basis for the immunosuppressive actions of marijuana. The CB1 receptor mediates inhibition of adenylate cyclase, inhibition of N- and P/Q-type calcium channels, stimulation of potassium channels, and activation of mitogen-activated protein kinase. The CB2 receptor mediates inhibition of adenylate cyclase and activation of mitogen-activated protein kinase. The discovery of endogenous cannabinoid receptor ligands, anandamide (N-arachidonylethanolamine) and 2-arachidonylglycerol made the notion of a central cannabinoid neuromodulatory system plausible. Anandamide is released from neurons upon depolarization through a mechanism that requires calcium-dependent cleavage from a phospholipid precursor in neuronal membranes. The release of anandamide is followed by rapid uptake into the plasma and hydrolysis by fatty-acid amidohydrolase. The psychoactive cannabinoids increase the activity of dopaminergic neurons in the ventral tegmental area-mesolimbic pathway. Since these dopaminergic circuits are known to play a pivotal role in mediating the reinforcing (rewarding) effects of the most drugs of abuse, the enhanced dopaminergic drive elicited by the cannabinoids is thought to underlie the reinforcing and abuse properties of marijuana. Thus, cannabinoids share a final common neuronal action with other major drugs of abuse such as morphine, ethanol and nicotine in producing facilitation of the mesolimbic dopamine system.     

Cannabinoid receptors and pain

Mammalian tissues contain at least two types of cannabinoid receptor, CB(1) and CB(2), both coupled to G proteins. CB(1) receptors are expressed mainly by neurones of the central and peripheral nervous system whereas CB(2) receptors occur centrally and peripherally in certain non-neuronal tissues, particularly in immune cells. The existence of endogenous ligands for cannabinoid receptors has also been demonstrated. The discovery of this 'endocannabinoid system' has prompted the development of a range of novel cannabinoid receptor agonists and antagonists, including several that show marked selectivity for CB(1) or CB(2) receptors. It has also been paralleled by a renewed interest in cannabinoid-induced antinociception. This review summarizes current knowledge about the ability of cannabinoids to produce antinociception in animal models of acute pain as well as about the ability of these drugs to suppress signs of tonic pain induced in animals by nerve damage or by the injection of an inflammatory agent. Particular attention is paid to the types of pain against which cannabinoids may be effective, the distribution pattern of cannabinoid receptors in central and peripheral pain pathways and the part that these receptors play in cannabinoid-induced antinociception. The possibility that antinociception can be mediated by cannabinoid receptors other than CB(1) and CB(2) receptors, for example CB(2)-like receptors, is also discussed as is the evidence firstly that one endogenous cannabinoid, anandamide, produces antinociception through mechanisms that differ from those of other types of cannabinoid, for example by acting on vanilloid receptors, and secondly that the endocannabinoid system has physiological and/or pathophysiological roles in the modulation of pain.     

Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors

Although endocannabinoids constitute one of the first lines of defense against pain, the anatomical locus and the precise receptor mechanisms underlying cannabinergic modulation of pain are uncertain. Clinical exploitation of the system is severely hindered by the cognitive deficits, memory impairment, motor disturbances and psychotropic effects resulting from the central actions of cannabinoids. We deleted the type 1 cannabinoid receptor (CB1) specifically in nociceptive neurons localized in the peripheral nervous system of mice, preserving its expression in the CNS, and analyzed these genetically modified mice in preclinical models of inflammatory and neuropathic pain. The nociceptor-specific loss of CB1 substantially reduced the analgesia produced by local and systemic, but not intrathecal, delivery of cannabinoids. We conclude that the contribution of CB1-type receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia is paramount, which should enable the development of peripherally acting CB1 analgesic agonists without any central side effects.     

A light and electron microscopic study of the CB1 cannabinoid receptor in the primate spinal cord

The distribution of cannabinoid receptors was studied in the monkey spinal cord by immunocytochemistry and electron microscopy, using an antibody to the CB1 brain cannabinoid receptor. Large numbers of labelled neurons were observed in all portions of the grey matter of the spinal cord. These included small diameter 9–16µm neurons in the dorsal horn, larger (40–60µm) neurons in the intermediate grey, and very large (60–100µm), motor neurons in the ventral horn. Reaction product was observed in dendrites postsynaptic to unlabelled axon terminals. Since cannabinoid receptor activation decreases neuronal excitability by several mechanisms, including inhibition of voltage dependent calcium channels, the dense staining of CB1 in dorsal horn neurons suggests that CB1 could reduce calcium influx through such channels in these neurons. This, in turn, could decrease calcium-dependent changes in synaptic transmission and decrease sensitisation to nociceptive stimuli in these neurons. Similarly, the dense staining of CB1 in ventral horn cells suggests that cannabinoid receptors could limit calcium influx through voltage dependent calcium channels in these neurons, and could be significant in terms of neuroprotection to these neurons.     

Cannabinoids modulate the P-type high-voltage-activated calcium currents in purkinje neurons

Endocannabinoids released by postsynaptic cells inhibit neurotransmitter release in many central synapses by activating presynaptic cannabinoid CB1 receptors. In particular, in the cerebellum, endocannabinoids inhibit synaptic transmission at granule cell to Purkinje cell synapses by modulating presynaptic calcium influx via N-, P/Q-, and R-type calcium channels. Using whole cell patch-clamp techniques, we show that in addition to this presynaptic action, both synthetic and endogenous cannabinoids inhibit P-type calcium currents in isolated rat Purkinje neurons independent of CB1 receptor activation. The IC50 for the anandamide (AEA)-induced inhibition of P-current peak amplitude was 1.04 +/- 0.04 microM. In addition, we demonstrate that all the tested cannabinoids in a physiologically relevant range of concentrations strongly accelerate inactivation of P currents. The effects of AEA cannot be attributed to the metabolism of AEA because a nonhydrolyzing analogue of AEA, methanandamide inhibited P-type currents with a similar efficacy. All effects of cannabinoids on P-type Ca2+ currents were insensitive to antagonists of CB1 cannabinoid or vanilloid TRPV1 receptors. In cerebellar slices, WIN 55,212-2 significantly affected spontaneous firing of Purkinje neurons in the presence of CB1 receptor antagonist, in a manner similar to that of a specific P-type channel antagonist, indicating a possible functional implication of the direct effects of cannabinoids on P current. Taken together these findings demonstrate a functionally important direct action of cannabinoids on P-type calcium currents.     

The agonists for nociceptors are ubiquitous, but the modulators are specific: P2X receptors in the sensory neurons are modulated by cannabinoids

P2X2 and P2X3 receptors expressed in mammalian sensory neurons participate in nociception. Cannabinoid receptors modulate nociceptive processing in various models of pain. They are also expressed in nociceptive sensory neurons. We have examined the effect of cannabinoids on the slow P2X2 and P2X2/3 receptors in the cells isolated from nodosal and dorsal root ganglia of rat. The study was carried out by means of the whole-cell patch clamp and rapid superfusion methods. We have found that both endogenous and synthetic cannabinoids (anandamide, WIN55,212-2, and (R)-(+)-methanandamide) inhibit the slow response to ATP mediated by P2X2 and P2X2/3 receptors in a majority of tested neurons. This inhibition was significant but only partial: anandamide (0.5–1 μM) inhibited the response to 51±21% of control. In the remaining minority of tested neurons, the response was transiently facilitated. The effect of cannabinoids appears to be mediated via cannabinoid CB₁ receptors: it was reversibly inhibited by selective CB₁ antagonist, SR141716A (10 μM). Introduction of cyclic AMP (0.5 mM) into the cell potently facilitated the inhibitory effect of cannabinoids: the ATP-activated current was inhibited to 13±10% of control. These data indicate that cannabinoids may inhibit nociceptive responses produced by P2X receptors.     

Cannabinoids attenuate depolarization-dependent Ca2+ influx in intermediate-size primary afferent neurons of adult rats

CB1 receptors have been localized to primary afferent neurons, but little is known about the direct effect of cannabinoids on these neurons. The depolarization-evoked increase in the concentration of free intracellular calcium ([Ca(2+)](i)), measured by microfluorimetry, was used as a bioassay for the effect of cannabinoids on isolated, adult rat primary afferent neurons 20-28 h after dissociation of dorsal root ganglia. Cannabinoid agonists CP 55,940 (100 nM) and WIN 55,212-2 (1 microM) had no effect on the mean K(+)-evoked increase in [Ca(2+)](i) in neurons with a somal area<800 microm(2), but the ligands attenuated the evoked increase in [Ca(2+)](i) by 35% in neurons defined as intermediate in size (800-1500 microm(2)). The effects of CP 55,940 and WIN 55,212-2 were mediated by the CB1 receptor on the basis of relative effective concentrations, blockade by the CB1 receptor antagonist SR141716A and lack of effect of WIN 55,212-3. Intermediate-size neurons rarely responded to capsaicin (100 nM). Although cannabinoid agonists generally did not inhibit depolarization-evoked increases in [Ca(2+)](i) in small neurons, immunocytochemical studies indicated that CB1 receptor-immunoreactivity occurred in this population. CB1 receptor-immunoreactive neurons ranged in size from 227 to 2995 microm(2) (mean somal area of 1044 microm(2)). In double labeling studies, CB1 receptor-immunoreactivity co-localized with labeling for calcitonin gene-related peptide and RT97, a marker for myelination, in some primary afferent neurons.The decrease in evoked Ca(2+) influx indicates that cannabinoids decrease conductance through voltage-dependent calcium channels in a subpopulation of primary afferent neurons. Modulation of calcium channels is one mechanism by which cannabinoids may decrease transmitter release from primary afferent neurons. An effect on voltage-dependent calcium channels, however, represents only one possible effect of cannabinoids on primary afferent neurons. Identifying the mechanisms by which cannabinoids modulate nociceptive neurons will increase our understanding of how cannabinoids produce anti-nociception in normal animals and animals with tissue injury.     

Selective cannabinoid CB1 receptor activation inhibits spinal nociceptive transmission in vivo.

Cannabinoid1 (CB1) receptors are located at CNS sites, including the spinal cord, involved in somatosensory processing. Analgesia is one of the tetrad of behaviors associated with cannabinoid agonists. Here, effects of a potent cannabinoid CB1 receptor agonist arachidonyl-2-chloroethylamide (ACEA) on evoked responses of dorsal horn neurons in anesthetized rats were investigated. Extracellular recordings of convergent dorsal horn neurons were made in halothane anesthetized Sprague-Dawley rats (n = 16). Effects of spinal application of ACEA on electrically evoked responses of dorsal horn neurons were studied. Mean maximal effects of 0.5, 5, 50, and 500 ng/50 microl ACEA on the C-fiber-mediated postdischarge response were 79 +/- 6, 62 +/- 10, and 54 +/- 7% (P < 0.01), 45 +/- 6% (P < 0.01), of control, respectively. ACEA (500 ng/50 microl) also reduced the C-fiber-evoked nonpotentiated responses of neurons (59 +/- 9% of control, P < 0.05) and Adelta-fiber-evoked responses of neurons (68 +/- 10% of control, P < 0.01). Minor effects of ACEA on Abeta-fiber-evoked responses were observed. Spinal pre-administration of the selective CB1 receptor antagonist SR141716A (0.01 microg/50 microl) significantly reduced effects of ACEA (500 ng/50 microl) on postdischarge responses of dorsal horn neurons. This study demonstrates that spinal CB1 receptors modulate the transmission of C- and Adelta-fiber-evoked responses in anesthetized rats; this may reflect pre- and/or postsynaptic effects of cannabinoids on nociceptive transmission. CB1 receptors inhibit synaptic release of glutamate in rat dorsolateral striatum, a similar mechanism of action may underlie the effects of ACEA on noxious evoked responses of spinal neurons reported here.     

Cannabis and cannabinoids: pharmacology and rationale for clinical use

It is now known that there are at least two types of cannabinoid receptors. These are CB1 receptors, present mainly on central and peripheral neurones, and CB2 receptors, present mainly on immune cells. Endogenous cannabinoid receptor agonists ('endocannabinoids') have also been identified. The discovery of this 'endogenous cannabinoid system' has led to the development of selective CB1 and CB2 receptor ligands and fueled renewed interest in the clinical potential of cannabinoids. Two cannabinoid CB1 receptor agonists are already used clinically, as antiemetics or as appetite stimulants. These are D 9 - tetrahydrocannabinol (THC) and nabilone. Other possible uses for CB1 receptor agonists include the suppression of muscle spasm/spasticity associated with multiple sclerosis or spinal cord injury, the relief of chronic pain and the management of glaucoma and bronchial asthma. CB1 receptor antagonists may also have clinical applications, e. g. as appetite suppressants and in the management of schizophrenia or disorders of cognition and memory. So too may CB2 receptor ligands and drugs that activate cannabinoid receptors indirectly by augmenting endocannabinoid levels at cannabinoid receptors. When taken orally, THC seems to undergo variable absorption and to have a narrow 'therapeutic window' (dose range in which it is effective without producing significant unwanted effects). This makes it difficult to predict an oral dose that will be both effective and tolerable to a patient and indicates a need for better cannabinoid formulations and modes of administration. For the therapeutic potential of cannabis or CB1 receptor agonists to be fully exploited, it will be important to establish objectively and conclusively (a) whether these agents have efficacy against selected symptoms that is of clinical significance and, if so, whether the benefits outweigh the risks, (b) whether cannabis has therapeutic advantages over individual cannabinoids, (c) whether there is a need for additional drug treatments to manage any of the disorders against which cannabinoids are effective, and (d) whether it will be possible to develop drugs that have reduced psychotropic activity and yet retain the ability to act through CB1 receptors to produce their sought-after effects.     

Cannabinoid-induced presynaptic inhibition of glutamatergic EPSCs in substantia gelatinosa neurons of the rat spinal cord.

The effect of cannabinoids on excitatory transmission in the substantia gelatinosa was investigated using intracellular recording from visually identified neurons in a transverse slice preparation of the juvenile rat spinal cord. In the presence of strychnine and bicuculline, perfusion of the cannabinoid receptor agonist WIN55,212-2 reduced the frequency and the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). Furthermore, the frequency of miniature EPSCs (mEPSCs) was also decreased by WIN55,212-2, whereas their amplitude was not affected. Similar effects were reproduced using the endogenous cannabinoid ligand anandamide. The effects of both agonists were blocked by the selective CB(1) receptor antagonist SR141716A. Electrical stimulation of high-threshold fibers in the dorsal root evoked a monosynaptic EPSC in lamina II neurons. In the presence of WIN55,212-2, the amplitude of the evoked EPSC (eEPSCs) was reduced, and the paired-pulse ratio was increased. The reduction of the eEPSC following CB(1) receptor activation was unlikely to have a postsynaptic origin because the response to AMPA, in the presence of 1 microM TTX, was unchanged. To investigate the specificity of this synaptic inhibition, we selectively activated the nociceptive C fibers with capsaicin, which induced a strong increase in the frequency of EPSCs. In the presence of WIN55,212-2, the response to capsaicin was diminished. In conclusion, these results strongly suggest a presynaptic location for CB(1) receptors whose activation results in inhibition of glutamate release in the spinal dorsal horn. The strong inhibitory effect of cannabinoids on C fibers may thereby contribute to the modulation of the spinal excitatory transmission, thus producing analgesia at the spinal level.     

Cannabinoid 1 receptors are expressed in nociceptive primary sensory neurons

Expression of cannabinoid 1 (CB1) and vanilloid 1 (VR1) receptor proteins was studied in adult, cultured rat dorsal root ganglion neurons. Immunostaining of CB1 receptors alone produced labelling in 57±2% of the cultured dorsal root ganglion neurons (n=3 cultures). The area of the labelled cells was between 200 and 800 μm² with an average of 527±68 μm². VR1 immunolabelling revealed immunopositivity in 42±6% of the total population of dorsal root ganglion neurons. Cells showing VR1-like immunopositivity had an area between 200 and 600 μm². The mean area of the VR1-like immunopositive neurons was 376±61 μm². Double immunostaining with antisera raised against the CB1 and VR1 receptor proteins, showed a high degree of co-expression between CB1 and VR1 receptors. An average of 82±3% of the CB1-like immunopositive cells also showed VR1-like immunoreactivity (n=3 cultures) while 98±2% of the VR1-like immunolabelled neurons showed CB1 receptor-like immunostaining (n=3 cultures). Our data suggests that nociceptive primary sensory neurons co-express CB1 and VR1 receptors to a very high degree. We propose that this may provide an anatomical basis for a powerful combination of VR1 mediated excitation and CB1-mediated inhibition of nociceptive responses at central and peripheral terminals of nociceptive primary afferents.     

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.