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.     

Synaptic transmission in the striatum: from plasticity to neurodegeneration

Striatal neurones receive myriad of synaptic inputs originating from different sources. Massive afferents from all areas of the cortex and the thalamus represent the most important source of excitatory amino acids, whereas the nigrostriatal pathway and intrinsic circuits provide the striatum with dopamine, acetylcholine, GABA, nitric oxide and adenosine. All these neurotransmitter systems interact each other and with voltage-dependent conductances to regulate the efficacy of the synaptic transmission within this nucleus. The integrative action exerted by striatal projection neurones on this converging information dictates the final output of the striatum to the other basal ganglia structures. Recent morphological, immunohistochemical and electrophysiological findings demonstrated that the striatum also contains different interneurones, whose role in physiological and pathological conditions represents an intriguing challenge in these years. The use of the in vitro brain slice preparation has allowed not only the detailed investigation of the direct pre- and postsynaptic electrophysiological actions of several neurotransmitters in striatal neurones, but also the understanding of their role in two different forms of corticostriatal synaptic plasticity, long-term depression and long-term potentiation. These long-lasting changes in the efficacy of excitatory transmission have been proposed to represent the cellular basis of some forms of motor learning and are altered in animal models of human basal ganglia disorders, such as Parkinson's disease. The striatum also expresses high sensitivity to hypoxic-aglycemic insults. During these pathological conditions, striatal synaptic transmission is altered depending on presynaptic inhibition of transmitter release and opposite membrane potential changes occur in projection neurones and in cholinergic interneurones. These ionic mechanisms might partially explain the selective neuronal vulnerability observed in the striatum during global ischemia and Huntington's disease.     

Transient receptor potential vanilloid 1 channels control acetylcholine/2-arachidonoylglicerol coupling in the striatum

The neurotransmitter acetylcholine (Ach) controls both excitatory and inhibitory synaptic transmission in the striatum. Here, we investigated the involvement of the endocannabinoid system in Ach-mediated inhibition of striatal GABA transmission, and the potential role of transient receptor potential vanilloid 1 (TRPV1) channels in the control of Ach-endocannabinoid coupling. We found that inhibition of Ach degradation and direct pharmacological stimulation of muscarinic M1 receptors reduced striatal inhibitory postsynaptic currents (IPSCs) through the stimulation of 2-arachidonoylglicerol (2AG) synthesis and the activation of cannabinoid CB1 receptors. The effects of M1 receptor activation on IPSCs were occlusive with those of metabotropic glutamate receptor 5 stimulation, and were prevented in the presence of capsaicin, agonist of TRPV1 channels. Elevation of anandamide (AEA) tone with URB597, a blocker of fatty acid amide hydrolase, mimicked the effects of capsaicin, indicating that endogenous AEA acts as an endovanilloid substance in the control of M1-dependent 2AG-mediated synaptic effects in the striatum. Accordingly, both capsaicin and URB597 effects were absent in mice lacking TRPV1 channels. Pharmacological interventions targeting AEA metabolism and TRPV1 channels might be considered alternative therapeutic routes in disorders of striatal cholinergic or endocannabinoid neurotransmission.     

Endocannabinoids mediate synaptic plasticity at glutamatergic synapses on spiny neurons within a basal ganglia nucleus necessary for song learning

Activation of type 1 cannabinoid receptors (CB(1)R) in many central nervous system structures induces both short- and long-term changes in synaptic transmission. Within mammalian striatum, endocannabinoids (eCB) are one of several mechanisms that induce synaptic plasticity at glutamatergic terminals onto medium spiny neurons. Striatal synaptic plasticity may contribute a critical component of adaptive motor coordination and procedural learning. Songbirds are advantageous for studying the neural mechanisms of motor learning because they possess a neural pathway necessary for song learning and adult song plasticity that includes a striato-pallidal nucleus, area X (homologous to a portion of mammalian basal ganglia). Recent findings suggest that eCBs contribute to vocal development. For example, dense CB(1)R expression in song control nuclei peaks around the closure of the sensori-motor integration phase of song development. Also, systemic administration of a CB(1)R agonist during vocal development impairs song learning. Here we test whether activation of CB(1)R alters excitatory synaptic input on spiny neurons in area X of adult male zebra finches. Application of the CB(1)R agonist WIN55212-2 decreased excitatory postsynaptic current (EPSC) amplitude; that decrease was blocked by the CB(1)R antagonist AM251. Guided by eCB experiments in mammalian striatum, we tested and verified that at least two mechanisms indirectly activate CB(1)Rs through eCBs in area X. First, activation of group I metabotropic glutamate receptors with the agonist 3,5-dihydroxyphenylglycine (DHPG) induced a CB(1)R-mediated reduction in EPSC amplitude. Second, we observed that a 10 s postsynaptic depolarization induced a calcium-mediated, eCB-dependent decrease in synaptic strength that resisted rescue with late CB(1)R blockade. Together, these results show that eCB modulation occurs at inputs to area X spiny neurons and could influence motor learning and production.     

Cannabinoids depress inhibitory synaptic inputs received by layer 2/3 pyramidal neurons of the neocortex

Using whole cell voltage-clamp recordings we investigated the effects of a synthetic cannabinoid (WIN55,212-2) on inhibitory inputs received by layer 2/3 pyramidal neurons in slices of the mouse auditory cortex. Activation of the type 1 cannabinoid receptor (CB1R) with WIN55,212-2 reliably reduced the amplitude of GABAergic inhibitory postsynaptic currents evoked by extracellular stimulation within layer 2/3. The suppression of this inhibition was blocked and reversed by the highly selective CB1R antagonist AM251, confirming a CB1R-mediated inhibition. Pairing evoked inhibitory postsynaptic currents (IPSCs) at short interstimulus intervals while applying WIN55,212-2 resulted in an increase in paired-pulse facilitation suggesting that the probability of GABA release was reduced. A presynaptic site of cannabinoid action was verified by an observed decrease in the frequency with no change in the amplitude or kinetics of action potential-independent postsynaptic currents (mIPSCs). When Cd(2+) was added or Ca(2+) was omitted from the recording solution, the remaining fraction of Ca(2+)-independent mIPSCs did not respond to WIN55,212-2. These data suggest that cannabinoids are capable of suppressing the inhibition of neocortical pyramidal neurons by depressing Ca(2+)-dependent GABA release from local interneurons.     

Subcellular localization of type 1 cannabinoid receptors in the rat basal ganglia

Endocannabinoids, acting via type 1 cannabinoid receptors (CB1), are known to be involved in short-term synaptic plasticity via retrograde signaling. Strong depolarization of the postsynaptic neurons is followed by the endocannabinoid-mediated activation of presynaptic CB1 receptors, which suppresses GABA and/or glutamate release. This phenomenon is termed depolarization-induced suppression of inhibition (DSI) or excitation (DSE), respectively. Although both phenomena have been reported to be present in the basal ganglia, the anatomical substrate for these actions has not been clearly identified. Here we investigate the high-resolution subcellular localization of CB1 receptors in the nucleus accumbens, striatum, globus pallidus and substantia nigra, as well as in the internal capsule, where the striato-nigral and pallido-nigral pathways are located. In all examined nuclei of the basal ganglia, we found that CB1 receptors were located on the membrane of axon terminals and preterminal axons. Electron microscopic examination revealed that the majority of these axon terminals were GABAergic, giving rise to mostly symmetrical synapses. Interestingly, preterminal axons showed far more intense staining for CB1, especially in the globus pallidus and substantia nigra, whereas their terminals were only faintly stained. Non-varicose, thin unmyelinated fibers in the internal capsule also showed strong CB1-labeling, and were embedded in bundles of myelinated CB1-negative axons. The majority of CB1 receptors labeled by immunogold particles were located in the axonal plasma membrane (92.3%), apparently capable of signaling cannabinoid actions. CB1 receptors in this location cannot directly modulate transmitter release, because the release sites are several hundred micrometers away. Interestingly, both the CB1 agonist, WIN55,212-2, as well as its antagonist, AM251, were able to block action potential generation, but via a CB1 independent mechanism, since the effects remained intact in CB1 knockout animals. Thus, our electrophysiological data suggest that these receptors are unable to influence action potential propagation, thus they may not be functional at these sites, but are likely being transported to the terminal fields. The present data are consistent with a role of endocannabinoids in the control of GABA, but not glutamate, release in the basal ganglia via presynaptic CB1 receptors, but also call the attention to possible non-CB1-mediated effects of widely used cannabinoid ligands on action potential generation.     

Localization of central cannabinoid CB1 receptor messenger RNA in neuronal subpopulations of rat dorsal root ganglia: a double-label in situ hybridization study

In situ hybridization histochemistry was used to show the distribution of messenger RNA for central cannabinoid CB 1 receptors in dorsal root ganglia of the rat. CB1 messenger RNA was highly expressed in neuronal subpopulations of rat dorsal root ganglia. The phenotypes of neurons that express messenger RNA for CB1 were subsequently examined by combining a 35S-labeled ribonucleotide probe for CB1 messenger RNA with digoxigenin-labeled riboprobes for preprotachykinin A (substance P precursor), alpha-calcitonin gene-related peptide and preprosomatostatin (somatostatin precursor) messenger RNAs. Qualitative examination revealed expression of CBI messenger RNA predominantly in medium-and large-sized cells distributed throughout the dorsal root ganglia. The majority of neurons expressing substance P messenger RNA were CB1 messenger RNA negative and smaller in size than the CB1 messenger RNA-positive cells. Only 13% of substance P messenger RNA-positive cells expressed CB1 messenger RNA. A similar degree of co-localization was observed with alpha-calcitonin gene-related peptide: 10% of cells expressing messenger RNA for this neuropeptide were CB1 messenger RNA positive. Co-localization of CB1 and somatostatin messenger RNAs was observed in less than 0.5% of somatostatin messenger RNA-positive cells. The data suggest that subpopulations of neurons in rat dorsal root ganglia are capable of synthesizing cannabinoid receptors and inserting them on terminals in the superficial dorsal horn. These findings provide anatomical evidence for cannabinoid modulation of primary afferent transmission. Although an anatomical basis for cannabinoid-mediated suppression of release of neurogenic peptides from nociceptive primary afferents is provided, our results demonstrate that the majority of CB messenger RNA-positive neurons in the dorsal root ganglia contain transmitters and/or neuromodulators other than the neuropeptides examined herein.     

Mechanisms of cannabinoid-receptor-mediated inhibition of synaptic transmission in cultured hippocampal pyramidal neurons.

Cannabinoids, such as marijuana, are known to impair learning and memory perhaps through their actions in the hippocampus where cannabinoid receptors are expressed at high density. Although cannabinoid receptor activation decreases glutamatergic synaptic transmission in cultured hippocampal neurons, the mechanisms of this action are not known. Cannabinoid receptor activation also inhibits calcium channels that support neurotransmitter release in these cells, making modulation of these channels a candidate for cannabinoid-receptor-mediated effects on synaptic transmission. Whole cell patch-clamp recordings of glutamatergic neurons cultured from the CA1 and CA3 regions of the hippocampus were used to identify the mechanisms of the effects of cannabinoids on synaptic transmission. Cannabinoid receptor activation reduced excitatory postsynaptic current (EPSC) size by approximately 50% but had no effect on the amplitude of spontaneous miniature EPSCs (mEPSCs). This reduction in EPSC size was accompanied by an increase in paired-pulse facilitation measured in low (1 mM) extracellular calcium and by a decrease in paired-pulse depression measured in normal (2.5 mM) extracellular calcium. Together, these results strongly support the hypothesis that cannabinoid receptor activation decreases EPSC size by reducing release of neurotransmitter presynaptically while having no effect on postsynaptic sensitivity to glutamate. Further experiments were done to identify the molecular mechanisms underlying this cannabinoid-receptor-mediated decrease in neurotransmitter release. Cannabinoid receptor activation had no effect on the size of the presynaptic pool of readily releasable neurotransmitter-filled vesicles, eliminating reduction in pool size as a mechanism for cannabinoid-receptor-mediated effects. After blockade of Q- and N-type calcium channels with omega-agatoxin TK and omega-conotoxin GVIA; however, activation of cannabinoid receptors reduced EPSC size by only 14%. These results indicate that cannabinoid receptor activation reduces the probability that neurotransmitter will be released in response to an action potential via an inhibition of presynaptic Q- and N-type calcium channels. This molecular mechanism most likely contributes to the impairment of learning and memory produced by cannabinoids and may participate in the analgesic, antiemetic, and anticonvulsive effects of these drugs as well.     

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.     

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.     

Postsynaptic endocannabinoid release is critical to long-term depression in the striatum

The striatum functions critically in movement control and habit formation. The development and function of cortical input to the striatum are thought to be regulated by activity-dependent plasticity of corticostriatal glutamatergic synapses. Here we show that the induction of a form of striatal synaptic plasticity, long-term depression (LTD), is dependent on activation of the CB1 cannabinoid receptor. LTD was facilitated by blocking cellular endocannabinoid uptake, and postsynaptic loading of anandamide (AEA) produced presynaptic depression. The endocannabinoid necessary for striatal LTD is thus likely to be released postsynaptically as a retrograde messenger. These findings demonstrate a new role for endocannabinoids in the induction of long-term synaptic plasticity in a circuit necessary for habit formation and motor control.     

Metabotropic glutamate receptors and striatal synaptic plasticity: implications for neurological diseases

Long- and short-term changes in the efficacy of synaptic transmission are known as synaptic plasticity. Phenomena such as long-term depression (LTD) and long-term potentiation (LTP) are two classical forms of synaptic plasticity that are expressed in several brain areas, including the striatum. Bi-directional changes in corticostriatal synaptic transmission, i.e. LTD and LTP, have been proposed to represent the cellular mechanisms underlying the physiological processes of motor learning and behavior. In parallel, other forms of synaptic plasticity induced by different experimental pathological conditions have been described in the striatum; these changes are presumed to represent the cellular processes underlying several neurological disorders, including Parkinson's disease and Huntington's chorea. A considerable number of receptor and post-receptor systems participate in the mechanisms of synaptic plasticity in the striatum, where glutamate plays a primary role through its ionotropic and metabotropic receptors (mGluRs). These latter constitute a group of recently characterized molecules, which have been shown to modulate synaptic transmission by acting on cellular excitability, ionic conductances and neurotransmitter release. These receptors have also been involved in several neuronal pathophysiological processes. The role of mGluRs in synaptic transmission and synaptic plasticity has been recently deeply studied and characterized in the striatum, in both physiological and pathological conditions. These findings open new and interesting perspectives in the study of basal ganglia function, and introduce new possible pharmacological approaches for the treatment of neurological disorders in which mGluRs have been experimentally involved.     

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.     

Somatostatin presynaptically inhibits both GABA and glutamate release onto rat basal forebrain cholinergic neurons

A whole cell patch-clamp study was carried out in slices obtained from young rat brain to elucidate the roles of somatostatin in the modulation of synaptic transmission onto cholinergic neurons in the basal forebrain (BF), a region that contains cholinergic and GABAergic corticopetal neurons and somatostatin (SS)-containing local circuit neurons. Cholinergic neurons within the BF were identified by in vivo prelabeling with Cy3 IgG. Because in many cases SS is contained in GABAergic neurons in the CNS, we investigated whether exogenously applied SS can influence GABAergic transmission onto cholinergic neurons. Bath application of somatostatin (1 muM) reduced the amplitude of the evoked GABAergic inhibitory presynaptic currents (IPSCs) in cholinergic neurons. SS also reduced the frequency of miniature IPSCs (mIPSCs) without affecting their amplitude distribution. SS-induced effect on the mIPSC frequency was significantly larger in the solution containing 7.2 mM Ca(2+) than in the standard (2.4 mM Ca(2+)) external solution. Similar effects were observed in the case of non-NMDA glutamatergic excitatory postsynaptic currents (EPSCs). SS inhibited the amplitude of evoked EPSCs and reduced the frequency of miniature EPSCs dependent on the external Ca(2+) concentration with no effect on their amplitude distribution. Pharmacological analyses using SS-receptor subtype-specific drugs suggest that SS-induced action of the IPSCs is mediated mostly by the sst(2) subtype, whereas sst subtypes mediating SS-induced inhibition of EPSCs are mainly sst(1) or sst(4). These findings suggest that SS presynaptically inhibits both GABA and glutamate release onto BF cholinergic neurons in a Ca(2+)-dependent way, and that SS-induced effect on IPSCs and EPSCs are mediated by different sst subtypes.     

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.     

Inflammation reduces the contribution of N-type calcium channels to primary afferent synaptic transmission onto NK1 receptor-positive lamina I neurons in the rat dorsal horn

N-type calcium channels contribute to the release of glutamate from primary afferent terminals synapsing onto nocisponsive neurons in the dorsal horn of the spinal cord, but little is known of functional adaptations to these channels in persistent pain states. Subtype-selective conotoxins and other drugs were used to determine the role of different calcium channel types in a rat model of inflammatory pain. Electrically evoked primary afferent synapses onto lumber dorsal horn neurons were examined three days after induction of inflammation with intraplantar complete Freund's adjuvant. The maximal inhibitory effect of the N-type calcium channel blockers, ω-conotoxins CVID and MVIIA, were attenuated in NK1 receptor-positive lamina I neurons after inflammation, but the potency of CVID was unchanged. This was associated with reduced inhibition of the frequency of asynchronous-evoked synaptic events by CVID studied in the presence of extracellular strontium, suggesting reduced N-type channel contribution to primary afferent synapses after inflammation. After application of CVID, the relative contributions of P/Q and L channels to primary afferent transmission and the residual current were unchanged by inflammation, suggesting the adaptation was specific to N-type channels. Blocking T-type channels did not affect synaptic amplitude under control or inflamed conditions. Reduction of N-type channel contribution to primary afferent transmission was selective for NK1 receptor-positive neurons identified by post hoc immunohistochemistry and did not occur at synapses in laminae II_o or II_i, or inhibitory synapses. These results suggest that inflammation selectively downregulates N-type channels in the terminals of primary afferents synapsing onto (presumed) nociceptive lamina I NK1 receptor-positive neurons.     

Altered CB1 receptor and endocannabinoid levels precede motor symptom onset in a transgenic mouse model of Huntington's disease

Huntington's disease (HD) is an inherited neurodegenerative disease characterised by cell dysfunction and death in the basal ganglia and cortex. Currently there are no effective pharmacological treatments available. Loss of cannabinoid CB1 receptor ligand binding in key brain regions is detected early in HD in human postmortem tissue [Glass M, Dragunow M, Faull RL (2000) The pattern of neurodegeneration in Huntington's disease: a comparative study of cannabinoid, dopamine, adenosine and GABA(A) receptor alterations in the human basal ganglia in Huntington's disease. Neuroscience 97:505–519]. In HD transgenic mice environmental enrichment upregulates the CB1 receptors and slows disease progression [Glass M, van Dellen A, Blakemore C, Hannan AJ, Faull RL (2004) Delayed onset of Huntington's disease in mice in an enriched environment correlates with delayed loss of cannabinoid CB1 receptors. Neuroscience 123:207–212]. These findings, combined with data from lesion studies have led to the suggestion that activation of cannabinoid receptors may be protective. However, studies suggest that CB1 mRNA may be decreased early in the disease progression in HD mice, making this a poor drug target. We have therefore performed a detailed analysis of CB1 receptor ligand binding, protein, gene expression and levels of endocannabinoids just prior to motor symptom onset (12 weeks of age) in R6/1 transgenic mice. We demonstrate that R6/1 mice exhibit a 27% decrease in CB1 mRNA in the striatum compared to wild type (WT). Total protein levels, determined by immunohistochemistry, are not significantly different to WT in the striatum or globus pallidus, but are significantly decreased by 19% in the substantia nigra. CB1 receptor ligand binding demonstrates significant but small decreases (<20%) in all basal ganglia regions evaluated. The levels of the endocannabinoid 2-arachidonoyl glycerol are significantly increased in the cortex (147%) while anandamide is significantly decreased in the hippocampus to 67% of WT. Decreases are also apparent in the ligand binding of neuronal D1 and D2 dopamine receptors co-located with CB1, while there is no change in GABA_A receptor ligand binding. These results suggest that in this R6/1 mouse colony at 12 weeks there are only very small changes in CB1 protein and receptors and thus this would be an appropriate time point to evaluate therapeutic interventions.     

Delayed onset of Huntington's disease in mice in an enriched environment correlates with delayed loss of cannabinoid CB1 receptors

Huntington's disease (HD) is a late onset progressive genetic disorder characterised by motor dysfunction, personality changes, dementia and premature death. The disease is caused by an unstable expanded trinucleotide (CAG) repeat encoding a polyglutamine stretch in the IT15 gene for huntingtin, a protein of unknown function. Transgenic mice expressing exon one of the human HD gene with an expanded polyglutamine region develop many features of human HD. Exposure of these mice to an "enriched" environment delays the onset of motor disorders and slows disease progression [Nature 404 (2000) 721]. We have compared the levels of receptor binding of a range of basal ganglia neurotransmitter receptors believed to be important in HD, in normal mice and R6/1 transgenic HD mice housed in either enriched or standard laboratory environments. HD mice housed in a normal environment show a loss of cannabinoid CB1 and dopamine D1 and D2 receptors in the striatum and the corresponding output nuclei of the basal ganglia. HD mice exposed to an enriched environment show equivalent loss of D1 and D2 receptors as their "non-enriched" counterparts; in contrast, the "enriched" mice show significantly less depletion of CB1 receptors. In the brains of humans diagnosed with HD cannabinoid CB1 receptors are selectively lost from the basal ganglia output nuclei prior to the development of other identifiable neuropathology [Neuroscience 97 (2000) 505]. Our results therefore show that an enhanced environment slows the rate of loss of one of the first identifiable neurochemical deficits of HD. This suggests that delaying the loss of CB1 receptors, either by environmental stimulation or pharmacologically, may be beneficial in delaying disease progression in HD patients.