Than-Vinh Dam

Quantitative autoradiographic distribution of multiple neurokinin binding sites in rat spinal cord

As a means of evaluating the role of neurokinins (NKs) in spinal function, the present study examines the quantitative autoradiographic distribution in the rat spinal cord of [125I]Bolton-Hunter-substance P, (2-[125I]iodohistidyl1)-neurokinin A and [125I]Bolton-Hunter-eledoisin as respective radioligands for NK-1, NK-2 and NK-3 receptors. These putative NK receptor sub-types are clearly differentially distributed at the various levels of the spinal cord. NK-1 sites represent the most abundant population of spinal NK receptors. They are most concentrated in the dorsal and ventromedial borders of the dorsal horn, the intermediolateral nucleus of the thoracic cord and the phrenic motor nucleus in the cervical ventral horn. NK-2 and NK-3 sites are also present in the spinal cord, although in much lower quantities than NK-1 sites. NK-2 sites are mostly found along the dorsal and ventromedial borders of the dorsal horn, in a narrow band connecting the two lateral horns of the thoracic cord, around the central canal of the lumbar and sacral segments and lamina IX of the cervical ventral horn. NK-3 sites are most dense in the dorsal border of the dorsal horn, with moderate amounts in the lateral horn of the thoracic cord and around the central canal of lumbar and sacral segments. The differential distribution of these 3 classes of NK sites in the spinal cord suggests that each NK receptor sub-type could mediate specific sensory, autonomic and/or motor functions at the spinal level.

Visualization of neurokinin-3 receptor sites in rat brain using the highly selective ligand [3H]senktide.

The autoradiographic distribution of the neurokinin (NK)-3 receptor sub-type was visualized in the rat brain using [3H]senktide, a highly selective ligand, [3H]Senktide apparently binds to a single class of high affinity (Kd = 2.8 +/- 1.0 nM), low capacity (Bmax = 31.2 +/- 3.0 fmol/mg protein) sites in rat brain cortex. The ligand selectivity pattern reveals that eledoisin and senktide are potent competitors of both [3H]senktide and [125I]Bolton-Hunter eledoisin binding sites demonstrating the NK-3 nature of these sites. Autoradiographic data show that [3H]senktide binding sites are concentrated in mid-cortical layers, supraoptic nucleus, zona incerta, basolateral nucleus of the amygdala and interpeduncular nucleus. Much lower densities of binding are seen in most other areas such as the caudate-putamen and cerebellum. This distribution is similar, but not identical, to that previously reported for NK-3 sites using less selective ligands. It is most likely because less selective probes also bind to other classes of NK receptors. The higher selectivity of [3H]senktide is thus an important advantage for the precise characterization of NK-3 receptor binding parameters.

Evidence for the existence of serotonin type-2 receptors on cholinergic terminals in rat cortex.

Levels of serotonin type-2 receptors and choline acetyltransferase (ChAT) were measured in various rat brain regions following unilateral lesions of the nucleus basalis magnocellularis (nBM). As expected, lesions of the nBM markedly decrease cortical ChAT activity. Moreover, [3H]ketanserin (serotonin type-2) binding is significantly decreased in lamina IV of the anterior and middle cortex on the lesioned vs control side. [3H]ketanserin binding in the striatum is not affected by lesions of the nBM. Autoradiograms of [3H]ketanserin binding in lesioned animals show similar results. This suggests that at least a certain proportion of serotonin type-2 receptor binding sites are located on cholinergic terminals in lamina IV of the rat cortex. Therefore, further investigations on cholinergic-serotonergic interactions in the cortex and their possible involvement in senile dementia of the Alzheimers type appear to be highly relevant.

Evidence for the existence of three classes of neurokinin receptors in brain. Differential ontogeny of neurokinin-1, neurokinin-2 and neurokinin-3 binding sites in rat cerebral cortex ☆

The autoradiographic distribution of the 3 neurokinin (NK) receptor sub-types, NK-1, NK-2 and NK-3, was compared in rat cerebral cortex during post-natal development using [125I]Bolton-Hunter-substance P, (2-[125I]iodohistidyl1)neurokinin A and [125I]Bolton-Hunter-eledoisin as respective radioligands. Throughout brain development, NK-1 receptor sites are present in low densities with some enrichment seen in lamina III while NK-3 binding sites are concentrated in layers IV and V. However, it appears that NK-2 receptors are mostly expressed in lamina VI and only during the first two postnatal weeks. These results demonstrate further the existence and differential ontogeny of 3 classes of NK receptors in rat brain cortex.

Multiple tachykinin receptors in guinea pig brain. High densities of substance K (neurokinin A) binding sites in the substantia nigra

Receptor binding characteristics of labelled substance P (SP) and substance K (SK) were investigated in guinea pig membrane preparations and by in vitro receptor autoradiography. The two ligands bind to single class of sites with Kd of 2.1 nM and Bmax of 37.0 fmol/mg protein for SP and 10.6 nM and 50.3 fmol/mg protein for SK. Ligand selectivity patterns markedly differ for the two ligands. On SP binding sites, SP greater than physalaemin greater than eledoisin greater than SK greater than kassinin greater than Neuromedin K. On the other hand, SK greater than kassinin greater than Neuromedin K greater than eledoisin greater than SP greater than physalaemin on SK binding sites. The autoradiographic distribution of SP and SK binding sites is also different. While the cerebral cortex, substantia nigra and cerebellum contain very low to low densities of SP sites, they are all rich in SK binding sites. These data clearly demonstrate the existence of at least two classes of tachykinin binding sites in guinea pig brain. Moreover, the high density of SK binding sites present in the substantia nigra suggests possible physiological roles for SK in this region.

Autoradiographic distribution of brain neurokinin-1/substance P receptors using a highly selective ligand [3H]-[Sar9,Met(O2)11]-substance P

The autoradiographic distribution of neurokinin (NK)-1 receptors was visualized in the rat brain using the highly selective ligand, [3H]-[Sar9,Met(O2)11]-substance P. This ligand apparently binds to a single class of high affinity (Kd = 1.4 +/- 0.5 nM), low capacity (Bmax = 160 +/- 3.0 fmol/mg protein) sites in rat brain membrane preparations. The ligand selectivity profile reveals that substance P (SP) and unlabeled [Sar9,Met(O2)11]-SP are potent competitors of [3H]-[Sar9,Met(O2)11]-SP binding while NK-2 and NK-3 analogues are virtually inactive demonstrating the selectivity of this radioligand for the NK-1 receptor class. Autoradiographic data show that [3H]-[Sar9,Met(O2)11]-SP binding sites are broadly but discretely distributed in rat brain, the highest densities of sites being located in the external plexiform layer of the olfactory bulb, striatum, olfactory tubercule, amygdala-hippocampal area, endopiriform and entorhinal cortices, superior colliculus, locus coeruleus and substantia gelatinosa of the spinal cord. This distribution is similar, but not identical, to that previously reported for NK-1 sites using less selective ligands such as [125I]Bolton-Hunter SP. For example, some difference in labelling patterns are observed in the hippocampal formation. This could be explained by the existence of NK-1 receptor subtypes, only one of them being recognized by [3H]-[Sar9,Met(O2)11]-SP or by the greater selectivity of this radioligand for NK-1 over NK-2 and NK-3 receptor classes.

Effects of dorsal rhizotomy on neurokinin receptor sub-types in the rat spinal cord: a quantitative autoradiographic study.

Although abundant evidence suggests a major role for substance P (SP) and other neurokinins (NK) in the transmission of nociceptive information, it is not known whether the various NK receptor classes are differentially located in the substantia gelatinosa of the spinal cord where primary afferent fibres mostly terminate. In order to investigate this issue, we studied the effects of unilateral dorsal rhizotomy on binding of 125I-Bolton-Hunter-SP, (2-[125I]iodohistidyl1)-neurokinin A, and 125I-Bolton-Hunter-eledoisin as respective radioligands for the NK-1, NK-2 and NK-3 receptor sub-types. Seven, 14, 21 and 28 days following unilateral lumbosacral dorsal horn deafferentiation, NK receptor binding parameters were evaluated using quantitative receptor autoradiography. Rhizotomy produced an increase in the densities of NK-1, NK-2 and NK-3 binding sites in the superficial laminae of the dorsal horn. Increases were maximal at 14 days, post-operatively, for both NK-1 and NK-2 sites; slight recovery being observed thereafter. For NK-3 sites, unilateral rhizotomy induced a progressive increase in binding without evidence of recovery over time, at least up to 28 days post-lesion. NK-1 receptor binding parameters around the central canal and in the ventral horn were not affected by the dorsal rhizotomy. These data suggest that all 3 NK receptor classes are located post-synaptically to afferent fiber terminals in laminae I, II and X of the dorsal horn of the spinal cord.

Pharmacological characterization and autoradiographic localization of substance P receptors in guinea pig brain

[3H]Substance P ([3H]SP) was used to characterize substance P (SP) receptor binding sites in guinea pig brain using membrane preparations and in vitro receptor autoradiography. Curvilinear Scatchard analysis shows that [3H]SP binds to a high affinity site (Kd = 0.5 nM) with a Bmax of 16.4 fmol/mg protein and a low affinity site (Kd = 29.6 nM) with a Bmax of 189.1 fmol/mg protein. Monovalent cations generally inhibit [3H]SP binding while divalent cations substantially increased it. The ligand selectivity pattern is generally similar to the one observed in rat brain membrane preparation with SP being more potent than SP fragments and other tachykinins. However, the potency of various nucleotides is different with GMP-PNP greater than GDP greater than GTP. The autoradiographic distribution of [3H]SP binding sites shows that high amounts of sites are present in the hippocampus, striatum, olfactory bulb, central nucleus of the amygdala, certain thalamic nuclei and superior colliculus. The cortex is moderately enriched in [3H]SP binding sites while the substantia nigra contains only very low amounts of sites. Thus, the autoradiographic distribution of SP binding sites is fairly similar in both rat and guinea pig brain.

Comparative Distribution of Receptor Types in the Mammalian Brain

The tachykinins (or neurokinins [NK]) are a group of structurally related peptides found in many regions of central nervous system, as well as in various peripheral tissues (Erspamer, 1981; Pernow, 1983; Maggio, 1988). Mammalian NKs are derived from the preprotachykinin I and II genes (PPT-I and PPT-II), two different but related genes (Nakanishi, 1987; Krause et al., 1989). Substance P (SP), neurokinin A (NKA), and the NKA-derived peptides, neuropeptide K, neuropeptide γ(NPγ), and NKA (3–10) are encoded by mRNAs resulting from PPT-I gene transcription, whereas neurokinin B (NKB) is derived from the maturation of the PPT-II gene (Nakanishi, 1987; Krause et al., 1989). These various NKs produce their physiological effects by activating specific membrane receptors.

Variability of chronic antidepressant treatments on beta-adrenergic receptor sites

It has been proposed that down-regulation of beta-adrenergic receptors may be related to the therapeutic action of antidepressants. To further characterize the role of beta-adrenergic receptor regulation in the actions of antidepressants, the effects of chronic treatment with desipramine, zimelidine and bupropion on beta-adrenergic receptors in rat brain membrane preparations were studied. Chronic treatment with desipramine, but not zimelidine and bupropion decreased significantly the density of beta-adrenergic receptors. These data suggest that clinically effective antidepressants may vary considerably in their ability to down-regulate beta-adrenergic receptors after chronic treatment.