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The distribution of neuropeptide FF receptors (NPFF(1) and NPFF(2)) was analyzed throughout the central nervous system of rodents (rat, mouse, Octodon degus, and guinea pig), rabbit, and marmoset monkey brains, representing three orders of mammals. Quantitative in vitro receptor autoradiography with [(125)I]EYF ([(125)I]EYWSLAAPQRF-NH(2)) and [(125)I]YVP ([(125)I]YVPNLPQRF-NH(2)) as specific radioligands for NPFF(2) and NPFF(1) receptors, respectively, was used. The NPFF(2) receptor is predominantly expressed in all species, except in the central nervous system of Octodon degus, in which it is undetectable. The density of the NPFF(1) subtype is low in rat and mice, moderate in octodon, rabbit, and monkey, and relatively high in the guinea pig. The present study reveals prominent species differences in the NPFF receptors expression in the brain. The distribution pattern of NPFF(2) receptors in the diencephalon and the superficial layers of the spinal cord is consistent with a hypothesized potential role for NPFF in the modulation of sensory input and opioid analgesia. In contrast, the constant presence of NPFF(1) receptors in the septum, the nucleus of the tractus solitarius, and the hypothalamus suggest its participation in neuroendocrine functions.

Although neuropeptide FF (NPFF) is generally considered an anti-opioid, its intrathecal administration produces analgesia. In the present study, the stable analog 1DMe ([D.Tyr(1), (NMe)Phe(3)]neuropeptide FF) was used in quantitative autoradiographic experiments in combination with surgical and chemical lesions to precisely localize NPFF receptors in the rat spinal cord. Ligation of lumbar dorsal spinal roots revealed the presence of NPFF receptors in dorsal root fibers and it induced a significant accumulation of [(125)I]1DMe-specific binding on the side peripheral to the ligature, demonstrating that a population of NPFF receptors is synthesized in dorsal root ganglia and migrates anterogradely towards primary afferent nerve endings. Complete mid-thoracic spinal cord transection failed to modify the [(125)I]1DMe labeling density in the dorsal horn, indicating that NPFF receptors are not located on the descending fiber terminals. In contrast, unilateral microinjections of kainic acid into the dorsal horn dramatically reduced [(125)I]1DMe-specific binding in the superficial layers, revealing localization of a population of NPFF receptors on the spinal intrinsic neurons. NPFF receptor binding was not modified during the development of spinal opioid tolerance. The pre- and postsynaptic localization of spinal NPFF receptors provide further support for heterogeneity in the pain modulation by NPFF and related agonists.

The anatomical localization of neuropeptide FF receptors was examined by in vitro autoradiography techniques in rat brain sections by using [125I][D.Tyr1, (NMe)Phe3]NPFF. The specific binding of [125I][D.Tyr1, (NMe)Phe3]NPFF reached 90% of the total binding at 0.05 nM in rat spinal cord sections. Up to 40% of the specific binding of[125I][D.Tyr1, (NMe)Phe3]NPFF to rat spinal cord sections was still detectable following fixation with glutaraldehyde. Afterwards, the distribution of NPFF receptors was studied by light microscopy and their densities by microdensitometry with an image analysis system. In the light microscope, [125I][D.Tyr1, (NMe)Phe3]NPFF labelling appeared more or less uniformly distributed over nerve-cell bodies and surrounding neuropil. High concentrations of binding sites were detected in the presubiculum, parafascicular thalamic nucleus, gracile nucleus, spinal trigeminal tract nucleus, and a number of brainstem nuclei, with virtually no labelling in the cerebellum. In several areas a rostrocaudal gradient of sites concentration was observed. Neuropeptide FF receptors are well-placed to control incoming sensory and autonomic information processing. In contrast, the more recently developed areas of the forebrain possessed low density of sites. The distribution of [125I][D.Tyr1, (NMe)Phe3]NPFF binding sites should suggest anatomical substrates for the actions of neuropeptide FF.

The distribution of β-opioid binding sites was studied by quantitative autoradiography in rat brain and spinal cord using the highly selective ligand [125I][D. Ala2]deltorphin-I. The binding properties of [125I][D. Ala2]deltorphin-I were investigated by microdensitometry of autoradiographic films with the aid of a computerassisted image-analysis system. [125I][D. Ala2]deltorphin-I appeared to interact with a single class of sites in all brain areas (KD = 0.9 nM). In 23 regions tested, whatever the δ site concentration, DTLET, a δ agonist, appears to be 2 orders of magnitude more effective than DAGO, a μ agonist, in inhibiting specific [125I][D. Ala2]deltorphin-I binding. The distribution of [125I][D. Ala2]deltorphin-I sites is globally consistent with that of other δ ligands and does not support the existence of a δ-receptor subtype recognized by [D. Ala2]deltorphin-I. [125I][D. Ala2]deltorphin-I binding sites were highly confined, exhibiting selective localization in the neocortex and a diffuse pattern in the striatum, accumbens nucleus, claustrum, layer of bulb, amygdaloid nucleus, pontine nuclei, and inferior colliculus. In several areas a rostro-caudal gradient of site concentration was indicated. [D. Ala2]deltorphin-I binding sites were also present in the substantia gelatinosa at all levels of the spinal cord and, unexpectedly, in deeper laminae and the ventral horn. These results demonstrate the ability of [125I][D. Ala2]deltorphin-I to characterize low concentrations of binding sites and to reveal new localizations of δ receptors. © 1993 Wiley-Liss, Inc.