Andrea G. Hohmann

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.

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.

Pre- and postsynaptic distribution of cannabinoid and mu opioid receptors in rat spinal cord.

In vitro receptor binding and quantitative autoradiography were used to assess the pre- and postsynaptic distribution of cannabinoid receptors in the cervical dorsal horn of the rat spinal cord. An extensive unilateral dorsal rhizotomy was performed across seven or eight successive spinal segments from C3 to T1 or T2. The densities of cannabinoid and mu opioid receptors in the central (C6) spinal segment were assessed 2, 4, 8, and 16 days post rhizotomy and compared with those of untreated rats. Rhizotomy induced approximately a 50% ipsilateral loss in the [3H]CP55,940 binding to spinal cannabinoid receptors that was maximal at 8 days post-rhizotomy. By comparison, the binding of [3H][d-Ala2-MePhe4, Gly-ol5]enkephalin (DAMGO) to mu receptors was depleted approximately 60% in near-adjacent sections. By contrast, changes in [3H]CP55,940 binding contralateral to the deafferentation were largely absent at all post-lesion delays. These data suggest that under conditions in which a spinal segment is completely deafferented, approximately 50% of cannabinoid receptors in the cervical (C6) dorsal horn reside presynaptically on central terminals of primary afferents. The present data provide anatomical evidence for presynaptic as well as postsynaptic localization of cannabinoid receptors in the spinal dorsal horn.

Inhibition of noxious stimulus-evoked activity of spinal cord dorsal horn neurons by the cannabinoid WIN 55,212-2.

The effects of a potent synthetic cannabinoid WIN 55,212-2 on nociceptive responses of wide dynamic range (WDR) neurons in the lumbar spinal cord were investigated in anesthetized rats. WDR neurons were identified by their responses to innocuous brushing and to a range of pressure stimuli from innocuous to noxious. Noxious pressure was applied to regions of the ipsilateral hind paw corresponding to the receptive field of the neuron. WIN 55,212-2 (125 micrograms/kg and 250 micrograms/kg, i.v.) produced a profound inhibition of firing evoked by the noxious pressure stimulus. By contrast, the cannabinoid did not alter the evoked activity of non-nociceptive neurons in response to non-noxious levels of stimulation. Treatment with either vehicle or the inactive enantiomer WIN 55,212-3 (250 micrograms/kg) failed to alter noxious stimulus-evoked activity of WDR neurons. These data provide direct evidence for cannabinoid-mediated inhibition of pain neurotransmission in the spinal dorsal horn. The site of action for these effects remains to be determined.

The neurobiology of cannabinoid analgesia

The discovery of cannabinoid receptors and their putative endogenous ligands raises questions as to the nature of the effects produced by cannabinoids on neural circuits that mediate pain and whether endogenous cannabinoids produced by the brain or in the periphery serve naturally to modulate pain. A sizable body of previous work showed that cannabinoid agonists suppress pain behavior in a variety of models of acute and chronic pain. However, at appropriate doses, cannabinoids also profoundly suppress motor behavior (see Sañudo-Peña et al., this volume), which complicates the interpretation of behavioral analgesia since a motor response is the endpoint of virtually all such studies. Studies conducted in this laboratory used biochemical and neurophysiological measures to determine whether cannabinoids suppress nociceptive neurotransmission. The results showed that cannabinoids suppress nociceptive neurotransmission at the level of the spinal cord and the thalamus. These effects are reversible, receptor mediated, selective for painful as opposed to nonpainful somatic stimuli, and track the behavioral analgesia both in time course and potency.

Suppression of noxious stimulus-evoked expression of Fos protein-like immunoreactivity in rat spinal cord by a selective cannabinoid agonist.

In rats, cannabinoids inhibit behavioral responses to noxious stimulation with a potency and efficacy similar to that of morphine. However, because cannabinoids depress motor function, it has not been possible to state beyond any doubt that these effects were related to a dampening of noxious sensory input. Therefore, c-fos immunocytochemistry was used to explore the possibility that cannabinoids reduce behavioral responses to noxious stimuli by decreasing spinal processing of nociceptive inputs. Rats received systemic injections of the potent and selective cannabinoid agonist WIN 55,212-2, the receptor-inactive enantiomer WIN 55,212-3 or vehicle prior to observations in a model of tonic pain, the formalin test. As demonstrated previously, plantar injections of formalin led to lifting and licking of the injected paw, with two peaks of activity occurring at 5 and 30 min after injection. The cannabinoid agonist suppressed these pain responses and produced a reduction in mobility. Immunocytochemical processing of sections with an antibody to the Fos protein revealed that the cannabinoid markedly suppressed pain-evoked c-fos expression in the superficial and neck regions of the spinal dorsal horn, but not in the nucleus proprius. Decreased expression of c-fos also occurred in the ventral horn. The specificity of this effect and its probable mediation by cannabinoid receptors are suggested by three findings: (i) the suppression by the drug of both behavioral and immunocytochemical responses to pain was dose-dependent; (ii) neither the behavioral nor the immunocytochemical response to the noxious stimulus was significantly affected by the receptor-inactive enantiomer of the agonist; (iii) animals rendered tolerant to cannabinoids by repeated injections of the agonist showed reduced responses to the drug. These findings suggest that cannabinoids inhibit the spinal processing of nociceptive stimuli and support the notion that endogenous cannabinoids may act naturally to modify pain trnasmission within the central nervous system.

Cannabinoid modulation of wide dynamic range neurons in the lumbar dorsal horn of the rat by spinally administered WIN55,212-2.

The effects of spinally administered cannabinoids on nociceptive responses of wide dynamic range (WDR) neurons in the lumbar spinal cord were investigated in urethane-anesthetized rats. Noxious thermal stimulation was applied with a Peltier device to regions of the ipsilateral hindpaw corresponding to the receptive fields of isolated neurons. WIN55,212-2 (100 microg, i.t.), applied topically on the dorsal spinal surface, suppressed noxious heat-evoked activity in spinal WDR neurons. By contrast, responsiveness was unchanged following administration of either vehicle or WIN55,212-3, the receptor-inactive enantiomer. WIN55,212-2, administered intrathecally to separate rats, produced antinociceptive effects in the tail-flick test with a time course and efficacy that paralleled the suppression of noxious heat-evoked activity. These results suggest that cannabinoid modulation of spinal nociceptive processing involves direct actions in the spinal dorsal horn and is related to the antinociceptive effects of intrathecally administered cannabinoids.

Evidence for a role of endogenous cannabinoids in the modulation of acute and tonic pain sensitivity

The competitive CB1 receptor antagonist SR141716A was used to test the hypothesis that endogenous cannabinoids modulate tonic pain sensitivity. Pretreatment with the antagonist significantly enhanced the response to a chemical nociceptive stimulus in the formalin test. Postreatment with the antagonist 5 min following the induction of tonic pain produced hyperalgesia during the tonic phase only. These findings suggest that endogenous cannabinoids serve naturally to modulate the maintenance of pain following repeated noxious stimulation.

Autoradiographic distribution of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine (DTG) binding sites in guinea pig brain: a comparative study

Binding studies suggested the selectivity of (+)-pentazocine for sigma receptors, and subsequent synthesis and testing of [3H](+)-pentazocine confirmed its high potency and selectivity for sigma sites. Newer data have demonstrated the selectivity of (+)-pentazocine for a subtype of the sigma receptor called sigma-1. Based on these findings, the distribution of [3H](+)-pentazocine binding sites in the guinea pig brain was examined using in vitro autoradiography. [3H](+)-Pentazocine binding was high in the cingulate cortex, dorsal diagonal band, periaqueductal gray, cerebellum and cranial nerve nuclei. It was relatively low in the nucleus accumbens, neocortical areas and caudate nucleus. A significant correlation was found between the binding of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine, a selective sigma ligand across brain regions. However, certain nuclei exhibited markedly different ratios of binding of the two ligands. Since DTG is not selective for the sigma subtypes, while (+)-pentazocine is selective for the sigma-1 type, the data are suggestive of relative differences in the distributions of sigma-1 and sigma-2 sites.

Regional variation in the ratio of σ1 to σ2 binding in rat brain

Abstract In vitro binding experiments were performed to determine whether known subtypes of the putative σ receptor exhibit a differential distribution across brain regions and species. Rat brains were dissected into nine regions, pooled, and used to prepare membranes for ligand binding studies. Whole guinea pig brains were prepared in an identical manner for comparison to rat. σ1 Receptors were labeled with [3H](+)-pentazocine. σ2 Receptors were labeled with [3H]1,3-di-o-tolylguanidine (DTG) in the presence of 1 μM dextrallorphan to mask σ1 sites. Non-specific binding was determined in the presence of 10 μM haloperidol. Filtration and scintillation spectroscopy provided the binding values. The experiment revealed marked variation in the ratio of σ2 to σ1 binding across brain regions ranging from a low of 1.63 in the hindbrain to 3.51 in the cerebellum, that result mainly differences in the density of the receptors. Scatchard analysis on membranes derived from the hindbrain and cortex suggested that the effects were due primarily to regional differences in densities of receptor subtypes rather than different affinities. Guinea pig brain showed a marked preponderance of σ1 receptors with a ratio (σ2/σ1) of 0.67. These findings demonstrate that σ1 and σ2 receptors are differentially distributed in rat brain.