The vanilloid receptor (VR1) protein functions both as a receptor for capsaicin and a transducer of noxious thermal stimuli. To determine the expression and targetting of this protein, we have generated antisera against both the amino and carboxy termini of VR1. Within the dorsal root and trigeminal ganglia of rats, VR1-immunoreactivity (VR1-ir) was restricted to small and medium sized neurons. VR1-ir was transported into both the central and peripheral processes of these primary afferent neurons, as evidenced by: (i) the presence of VR1-ir in nerve fibres and terminals in lamina I and lamina II of the superficial dorsal horn, and the association of VR1-ir with small diameter nerve fibres in the skin and cornea; (ii) the reduction of VR1-ir in the spinal cord after dorsal rhizotomy; and (iii) the accumulation of VR1-ir proximal to sciatic nerve ligation. At the ultrastructural level, VR1-ir was associated with plasma membranes of neuronal perikarya in dorsal root ganglia and nerve terminals in the dorsal horn. VR1-ir was also seen in nerve fibres and terminals in the spinal trigeminal nucleus and nucleus of the solitary tract. Within a large proportion of dorsal root ganglion neurons and the terminals of their axons, VR1-ir was colocalized with staining for the P2X3 purinoceptor, and with binding sites for the lectin IB4. Surprisingly, VR1-ir did not coexist substantially in nerve fibres and terminals that contain substance P and calcitonin gene-related peptide, suggesting complex mechanisms for the release of these neuropeptides in response to capsaicin application.

Opioid receptors regulate neuronal activity by both pre- and postsynaptic mechanisms. We recently reported that the cloned delta- opioid receptor (DOR1) is primarily targeted to axons, suggesting a presynaptic role. In the present study we have studied the distribution and targeting of another opioid receptor, the mu-opioid receptor (MOR1), by raising anti-peptide antisera to the C-terminal peptide of MOR1. The specificity of the antisera was determined by analysis of transfected cells, Western blots, and immunoisolation studies. Immunohistochemistry showed that MOR1 immunoreactivity was enriched in many brain areas including cerebral cortex, striatum, hippocampus, locus coeruleus, and the superficial laminae of the dorsal horn. Moreover, MOR1-expressing neurons seem to target this receptor preferentially to their somatodendritic domain as determined by double- labeling experiments with MAP2. However, discrete populations of neurons target MOR1 to their axons, including some primary afferent neurons that express DOR1. In many regions enkephalin-containing axons were complementary to MOR1, suggesting by their proximity that enkephalins may be physiologically relevant ligands for this receptor. Thus, these results provide a morphological basis for understanding pre- and postsynaptic functions mediated by MOR1.

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.

The initial pain from tissue damage may result from the release of cytoplasmic components that act upon nociceptors, the sensors for pain. ATP was proposed to fill this role because it elicits pain when applied intradermally and may be the active compound in cytoplasmic fractions that cause pain. Moreover, ATP opens ligand-gated ion channels (P2X receptors) in sensory neurons and only sensory neurons express messenger RNA for the P2X3 receptor. To test whether ATP contributes to nociception, we developed a tissue culture system that allows comparison of nociceptive (tooth-pulp afferent) and non-nociceptive (muscle-stretch receptor) rat sensory neurons. Low concentrations of ATP evoked action potentials and large inward currents in both types of neuron. Nociceptors had currents that were similar to those of heterologously expressed channels containing P2X3 subunits, and had P2X3 immunoreactivity in their sensory endings and cell bodies. Stretch receptors had currents that differed from those of P2X3 channels, and had no P2X3 immunoreactivity. These results support the theory that P2X3 receptors mediate a form of nociception, but also suggest non-nociceptive roles for ATP in sensory neurons.

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