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.     

Novel cannabinoid-sensitive receptor mediates inhibition of glutamatergic synaptic transmission in the hippocampus

Psychoactive effects of cannabinoids are thought to be mediated, at least in part, by suppression of both glutamate and GABA release via CB1 cannabinoid receptor. Two types of cannabinoid receptor (CB1 and CB2) have been cloned so far. The CB1 receptors are abundantly expressed in the nervous system, whereas CB2 receptors are limited to lymphoid organs (Matsuda et al., 1990; Munro et al., 1993). Immunocytochemical and electrophysiological studies revealed that in the hippocampus CB1 receptors are expressed on axon terminals of GABAergic inhibitory interneurons (Tsou et al., 1999; Katona et al., 1999) and activation of these receptors decreases GABA release (Hájos et al., 2000). Other physiological studies pointed out the involvement of CB1 receptors in the modulation of hippocampal glutamatergic synaptic transmission and long-term potentiation (Stella et al., 1997; Misner and Sullivan, 1999), but anatomical studies could not confirm the existence of CB1 receptors on glutamatergic terminals. Here we examined cannabinoid actions on both glutamatergic and GABAergic synaptic transmission in the hippocampus of wild type (CB1+/+) and CB1 receptor knockout mice (CB1-/-). The synthetic cannabinoid agonist WIN55,212-2 reduced the amplitudes of excitatory postsynaptic currents in both wild type and CB1-/- mice, while inhibitory postsynaptic currents were decreased only in wild type mice, but not in CB1-/- animals. Our findings are consistent with a CB1 cannabinoid receptor-dependent modulation of GABAergic postsynaptic currents, but a novel cannabinoid-sensitive receptor must be responsible for the inhibition of glutamatergic neurotransmission.     

Retrograde signaling changes the venue of postsynaptic inhibition in rat substantia nigra

Both endocannabinoids through cannabinoid receptor type I (CB1) receptors and dopamine through dopamine receptor type D1 receptors modulate postsynaptic inhibition in substantia nigra by changing GABA release from striatonigral terminals. By recording from visually identified pars compacta and pars reticulata neurons we searched for a possible co-release and interaction of endocannabinoids and dopamine. Depolarization of a neuron in pars reticulata or in pars compacta transiently suppressed evoked synaptic currents which were blocked by GABA(A) receptor antagonists (inhibitory postsynaptic currents [IPSCs]). This depolarization-induced suppression of inhibition (DSI) was abrogated by the cannabinoid CB1 receptor antagonist AM251 (1 microM). A correlation existed between the degree of DSI and the degree of reduction of evoked IPSCs by the CB1 receptor agonist WIN55,212-2 (1 microM). The cholinergic receptor agonist carbachol (0.5-5 microM) enhanced DSI, but suppression of spontaneous IPSCs was barely detectable pointing to the existence of GABA release sites without CB1 receptors. In dopamine, but not in GABAergic neurons DSI was enhanced by the dopamine D1 receptor antagonist SCH23390 (3-10 microM). Both the antagonist for CB1 receptors and the antagonist for dopamine D1 receptors enhanced or reduced, respectively, the amplitudes of evoked IPSCs. This tonic influence persisted if the receptor for the other ligand was blocked. We conclude that endocannabinoids and dopamine can be co-released. Retrograde signaling through endocannabinoids and dopamine changes inhibition independently from each other. Activation of dopamine D1 receptors emphasizes extrinsic inhibition and activation of CB1 receptors promotes intrinsic inhibition.     

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.     

Direct actions of cannabinoids on synaptic transmission in the nucleus accumbens: a comparison with opioids

The nucleus accumbens (NAc) represents a critical site for the rewarding and addictive properties of several classes of abused drugs. The medium spiny GABAergic projection neurons (MSNs) in the NAc receive innervation from intrinsic GABAergic interneurons and glutamatergic innervation from extrinsic sources. Both GABA and glutamate release onto MSNs are inhibited by drugs of abuse, suggesting that this action may contribute to their rewarding properties. To investigate the actions of cannabinoids in the NAc, we performed whole cell recordings from MSNs located in the shell region in rat brain slices. The cannabinoid agonist WIN 55,212-2 (1 microM) had no effect on the resting membrane potential, input resistance, or whole cell conductance, suggesting no direct postsynaptic effects. Evoked glutamatergic excitatory postsynaptic currents (EPSCs) were inhibited to a much greater extent by [Tyr-D-Ala(2), N-CH(3)-Phe(4), Gly-ol-enkephalin] (DAMGO, approximately 35%) than by WIN 55,212-2 (<20%), and an analysis of miniature EPSCs suggested that the effects of DAMGO were presynaptic, whereas those of WIN 55,212-2 were postsynaptic. However, electrically evoked GABAergic inhibitory postsynaptic currents (evIPSCs), were reduced by WIN 55,212-2 in every neuron tested (EC(50) = 123 nM; 60% maximal inhibition), and the inhibition of IPSCs by WIN 55,212-2 was completely antagonized by the CB1 receptor antagonist SR141716A (1 microM). In contrast evIPSCs were inhibited in approximately 50% of MSNs by the mu/delta opioid agonist D-Ala(2)-methionine(2)-enkephalinamide and were completely unaffected by a selective mu-opioid receptor agonist (DAMGO). WIN 55,212-2 also increased paired-pulse facilitation of the evIPSCs and did not alter the amplitudes of tetrodotoxin-resistant miniature IPSCs, suggesting a presynaptic action. Taken together, these data suggest that cannabinoids and opioids differentially modulate inhibitory and excitatory synaptic transmission in the NAc and that the abuse liability of marijuana may be related to the direct actions of cannabinoids in this structure.     

GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus

Cannabinoids have been shown to disrupt memory processes in mammals including humans. Although the CB1 neuronal cannabinoid receptor was identified several years ago, neuronal network mechanisms mediating cannabinoid effects are still controversial in animals, and even more obscure in humans. In the present study, the localization of CB1 receptors was investigated at the cellular and subcellular levels in the human hippocampus, using control post mortem and epileptic lobectomy tissue. The latter tissue was also used for [³H]GABA release experiments, testing the predictions of the anatomical data. Detectable expression of CB1 was confined to interneurons, most of which were found to be cholecystokinin-containing basket cells. CB1-positive cell bodies showed immunostaining in their perinuclear cytoplasm, but not in their somadendritic plasmamembrane. CB1-immunoreactive axon terminals densely covered the entire hippocampus, forming symmetrical synapses characteristic of GABAergic boutons. Human temporal lobectomy samples were used in the release experiments, as they were similar to the controls regarding cellular and subcellular distribution of CB1 receptors. We found that the CB1 receptor agonist, WIN 55,212-2, strongly reduced [³H]GABA release, and this effect was fully prevented by the specific CB1 receptor antagonist SR 141716A.

This unique expression pattern and the presynaptic modulation of GABA release suggests a conserved role for CB1 receptors in controlling inhibitory networks of the hippocampus that are responsible for the generation and maintenance of fast and slow oscillatory patterns. Therefore, a likely mechanism by which cannabinoids may impair memory and associational processes is an alteration of the fine-tuning of synchronized, rhythmic population events.     

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 CB1 receptor signaling dichotomously modulates inhibitory and excitatory synaptic transmission in rat inner retina

In the inner retina, ganglion cells (RGCs) integrate and process excitatory signal from bipolar cells (BCs) and inhibitory signal from amacrine cells (ACs). Using multiple labeling immunohistochemistry, we first revealed the expression of the cannabinoid CB1 receptor (CB1R) at the terminals of ACs and BCs in rat retina. By patch-clamp techniques, we then showed how the activation of this receptor dichotomously regulated miniature inhibitory postsynaptic currents (mIPSCs), mediated by GABA_A receptors and glycine receptors, and miniature excitatory postsynaptic currents (mEPSCs), mediated by AMPA receptors, of RGCs in rat retinal slices. WIN55212-2 (WIN), a CB1R agonist, reduced the mIPSC frequency due to an inhibition of L-type Ca²⁺ channels no matter whether AMPA receptors were blocked. In contrast, WIN reduced the mEPSC frequency by suppressing T-type Ca²⁺ channels only when inhibitory inputs to RGCs were present, which could be in part due to less T-type Ca²⁺ channels of cone BCs, presynaptic to RGCs, being in an inactivation state under such condition. This unique feature of CB1R-mediated retrograde regulation provides a novel mechanism for modulating excitatory synaptic transmission in the inner retina. Moreover, depolarization of RGCs suppressed mIPSCs of these cells, an effect that was eliminated by the CB1R antagonist SR141716, suggesting that endocannabinoid is indeed released from RGCs.     

Lack of depolarization-induced suppression of inhibition (DSI) in layer 2/3 interneurons that receive cannabinoid-sensitive inhibitory inputs

In layer 2/3 of neocortex, brief trains of action potentials in pyramidal neurons (PNs) induce the mobilization of endogenous cannabinoids (eCBs), resulting in a depression of GABA release from the terminals of inhibitory interneurons (INs). This depolarization-induced suppression of inhibition (DSI) is mediated by activation of the type 1 cannabinoid receptor (CB1) on presynaptic terminals of a subset of INs. However, it is not clear whether CB1 receptors are also expressed at synapses between INs, and whether INs can release eCBs in response to depolarization. In the present studies, brain slices containing somatosensory cortex were prepared from 14- to 21-day-old CD-1 mice. Whole cell recordings were obtained from layer 2/3 PNs and from INs classified as regular spiking nonpyramidal, irregular spiking, or fast spiking. For all three classes of INs, the cannabinoid agonist WIN55,212-2 suppressed inhibitory synaptic activity, similar to the effect seen in PNs. In addition, trains of action potentials in PNs resulted in significant DSI. In INs, however, DSI was not seen in any cell type, even with prolonged high-frequency spike trains that produced calcium increases comparable to that seen with DSI induction in PNs. In addition, blocking eCB reuptake with AM404, which enhanced DSI in PNs, failed to unmask any DSI in INs. Thus the lack of DSI in INs does not appear to be due to an insufficient increase in intracellular calcium or enhanced reuptake. These results suggest that layer 2/3 INs receive CB1-expressing inhibitory inputs, but that eCBs are not released by these INs.     

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.     

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.     

Protracted postnatal development of inhibitory synaptic transmission in rat hippocampal area CA1 neurons

In the CNS, inhibitory synaptic function undergoes profound transformation during early postnatal development. This is due to variations in the subunit composition of subsynaptic GABA(A) receptors (GABA(A)Rs) at differing developmental stages as well as other factors. These include changes in the driving force for chloride-mediated conductances as well as the quantity and/or cleft lifetime of released neurotransmitter. The present study was undertaken to investigate the nature and time course of developmental maturation of GABAergic synaptic function in hippocampal CA1 pyramidal neurons. In neonatal [postnatal day (P) 1-7] and immature (P8-14) CA1 neurons, miniature inhibitory postsynaptic currents (mIPSCs) were significantly larger, were less frequent, and had slower kinetics compared with mIPSCs recorded in more mature neurons. Adult mIPSC kinetics were achieved by the third postnatal week in CA1 neurons. However, despite this apparent maturation of mIPSC kinetics, significant differences in modulation of mIPSCs by allosteric agonists in adolescent (P15-21) neurons were still evident. Diazepam (1-300 nM) and zolpidem (200 nM) increased the amplitude of mIPSCs in adolescent but not adult neurons. Both drugs increased mIPSC decay times equally at both ages. These differential agonist effects on mIPSC amplitude suggest that in adolescent CA1 neurons, inhibitory synapses operate differently than adult synapses and function as if subsynaptic receptors are not fully occupied by quantal release of GABA. Rapid agonist application experiments on perisomatic patches pulled from adolescent neurons provided additional support for this hypothesis. In GABA(A)R currents recorded in these patches, benzodiazepine amplitude augmentation effects were evident only when nonsaturating GABA concentrations were applied. Furthermore nonstationary noise analysis of mIPSCs in P15-21 neurons revealed that zolpidem-induced mIPSC augmentation was not due to an increase in single-channel conductance of subsynaptic GABA(A)Rs but rather to an increase in the number of open channels responding to a single GABA quantum, further supporting the hypothesis that synaptic receptors may not be saturated during synaptic function in adolescent neurons. These data demonstrate that inhibitory synaptic transmission undergoes a markedly protracted postnatal maturation in rat CA1 pyramidal neurons. In the first two postnatal weeks, mIPSCs are large in amplitude, are slow, and occur infrequently. By the third postnatal week, mIPSCs have matured kinetically but retain distinct responses to modulatory drugs, possibly reflecting continued immaturity in synaptic structure and function persisting through adolescence.     

Cannabinoid receptor activation differentially modulates ion channels in photoreceptors of the tiger salamander

Cannabinoid CB1 receptors have been detected in retinas of numerous species, with prominent labeling in photoreceptor terminals of the chick and monkey. CB1 labeling is well-conserved across species, suggesting that CB1 receptors might also be present in photoreceptors of the tiger salamander. Synaptic transmission in vertebrate photoreceptors is mediated by L-type calcium currents-currents that are modulated by CB1 receptors in bipolar cells of the tiger salamander. Presence of CB1 receptors in photoreceptor terminals would therefore be consistent with presynaptic modulation of synaptic transmission, a role seen for cannabinoids in other parts of the brain. Here we report immunohistochemical and electrophysiological evidence for the presence of functional CB1 receptors in rod and cone photoreceptors of the tiger salamander. The cannabinoid receptor agonist WIN 55212-2 enhances calcium currents of rod photoreceptors by 39% but decreases calcium currents of large single cones by 50%. In addition, WIN 55212-2 suppresses potassium currents of rods and large single cones by 44 and 48%, respectively. Thus functional CB1 receptors, present in the terminals of rod and cone photoreceptors, differentially modulate calcium and potassium currents in rods and large single cones. CB1 receptors are therefore well positioned to modulate neurotransmitter release at the first synapse of the visual system.     

Cannabinoid CB(1) receptor expression and affinity in the rat hippocampus following bilateral vestibular deafferentation

Numerous studies have shown that bilateral vestibular deafferentation (BVD) results in spatial memory deficits and hippocampal dysfunction in rats and humans. Since cannabinoid CB(1) receptors are well known to regulate synaptic plasticity in the hippocampus, we investigated whether BVD resulted in changes in CB(1) receptor expression and affinity in the rat hippocampus at 1, 3 and 7 days post-surgery, using a combination of Western blotting and radioligand binding. Using Western blotting, we found that CB(1) receptor expression was significantly lower in BVD animals compared to sham controls only in the CA3 area across the 3 time points (P=0.03). CB(1) receptor expression decreased significantly over time for both the BVD and sham animals (P=0.000). The radioligand binding assays showed no significant change in the IC(50) of the CB(1) receptor for the cannabinoid CB(1)/CB(2) receptor agonist, WIN55,212-2. These results suggest that the CB(1) receptor down-regulates in the CA3 region of the hippocampus following BVD, but with no changes in the affinity of the CB(1) receptor for WIN55,212-2.     

Cannabinoids suppress synaptic input to neurones of the rat dorsal motor nucleus of the vagus nerve

Cannabinoids bind central type 1 receptors (CB1R) and modify autonomic functions, including feeding and anti-emetic behaviours, when administered peripherally or into the dorsal vagal complex. Western blots and immunohistochemistry indicated the expression of CB1R in the rat dorsal vagal complex, and tissue polymerase chain reaction confirmed that CB1R message was made within the region. To identify a cellular substrate for the central autonomic effects of cannabinoids, whole-cell patch-clamp recordings were made in brainstem slices to determine the effects of CB1R activation on synaptic transmission to neurones of the dorsal motor nucleus of the vagus (DMV). A subset of these neurones was identified as gastric related after being labelled retrogradely from the stomach. The CB1R agonists WIN55,212-2 and anandamide decreased the frequency of spontaneous excitatory or inhibitory postsynaptic currents in a concentration-related fashion, an effect that persisted in the presence of tetrodotoxin. Paired pulse ratios of electrically evoked postsynaptic currents were also increased by WIN55,212-2. The effects of  WIN55,212-2 were sensitive to the selective CB1R antagonist AM251. Cannabinoid agonist effects on synaptic input originating from neurones in the nucleus tractus solitarius (NTS) were determined by evoking activity in the NTS with local glutamate application. Excitatory and inhibitory synaptic inputs arising from the NTS were attenuated by WIN55,212-2. Our results indicate that cannabinoids inhibit transfer of synaptic information to the DMV, including that arising from the NTS, in part by acting at receptors located on presynaptic terminals contacting DMV neurones. Inhibition of synaptic input to DMV neurones is likely to contribute to the suppression of visceral motor responses by cannabinoids.     

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.     

Antinociceptive effect of intrathecal cannabinoid receptor agonist WIN 55,212-2 in a rat bone tumor pain model

Bone tumor pain is a poorly controlled pain comprising background and severe pain on moving or weight-bearing postures that decreases the quality of life for cancer patients; thus, more effective analgesics are clearly needed. This study evaluated the efficacy of a cannabinoid (CB) receptor agonist (WIN 55,212-2) on bone tumor pain in the spinal cords of rats, and clarified the roles of the CB1 and CB2 receptors in WIN 55,212-2-induced antinociception at the spinal level. Bone tumor pain was induced by injecting MRMT-1 tumor cells (1×10(5)) into the right tibias of female Sprague-Dawley rats under sevoflurane anesthesia. Bone tumor development was monitored radiologically. Under sevoflurane anesthesia, a polyethylene catheter was inserted into the intrathecal space for drug administration. To assess pain, the withdrawal threshold was measured by applying a von Frey filament to the tumor cell inoculation site. The effect of intrathecal WIN 55,212-2 was investigated. Next, the WIN 55,212-2-mediated antinociception was reversed using CB1 (AM 251) and CB2 (AM 630) receptor antagonists. The intratibial injection of MRMT-1 tumor cells produced radiologically confirmed bone tumors. The paw withdrawal threshold decreased significantly (mechanical allodynia) with tumor development; however, intrathecal WIN 55,212-2 dose-dependently increased the withdrawal threshold. The antinociceptive effect of WIN 55,212-2 was reversed by both CB1 and CB2 receptor antagonists. Intrathecal WIN 55,212-2 reduced bone tumor-related pain behavior mediated via spinal CB1 and CB2 receptors. Therefore, spinal CB receptor agonists may be novel analgesics in the treatment of bone tumor pain.     

5-HT1B receptor-mediated presynaptic inhibition of GABA release in the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) receives a dense serotonergic innervation that modulates photic input to the SCN via serotonin 1B (5-HT1B) presynaptic receptors on retinal glutamatergic terminals. However, the majority of 5-HT1B binding sites in the SCN are located on nonretinal terminals and most axonal terminals in the SCN are GABAergic. We therefore tested the hypothesis that 5-HT1B receptors might also be located on SCN GABAergic terminals by examining the effects of the highly selective 5-HT1B receptor agonist CP-93,129 on SCN miniature inhibitory postsynaptic currents (mIPSCs). Whole cell patch-clamp recordings of mIPSCs were obtained from rat and mouse SCN neurons in hypothalamic slices. Using CsCl-containing microelectrodes with QX314, we isolated mPSCs that were sensitive to the GABAA receptor antagonist, bicuculline. Bath application of CP-93,129 (1 microM) decreased the frequency of mIPSCs by an average of 22% (n = 7) in rat SCN neurons and by an average of 30% (n = 8) in mouse SCN neurons with no clear effect on mIPSC amplitude. In mice lacking functional 5-HT1B receptors, CP-93,129 (1 microM) had no clear effect on the frequency or the amplitude of mIPSCs recorded in any of the cells tested (n = 4). The decrease in the frequency of mIPSCs of SCN neurons produced by the selective 5-HT1B receptor agonist CP-93,129 is consistent with the interpretation that 5-HT1B receptors are located on GABA terminals in the SCN and that 5-HT inhibits GABA release via a 5-HT1B presynaptic receptor-mediated mechanism.     

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.     

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.