Sex differences in the neurokinin B system in the human infundibular nucleus.

CONTEXT: The recent report that loss-of-function mutations in either the gene encoding neurokinin B (NKB) or its receptor (NK3R) produce gonadotropin deficiencies in humans strongly points to NKB as a key regulator of GnRH release. OBJECTIVES: We used NKB immunohistochemistry on postmortem human brain tissue to determine: 1) whether the human NKB system in the infundibular nucleus (INF) is sexually dimorphic; 2) at what stage in development the infundibular NKB system would diverge between men and women; 3) whether this putative structural difference is reversed in male-to-female (MtF) transsexual people; and 4) whether menopause is accompanied by changes in infundibular NKB immunoreactivity. METHODS: NKB immunohistochemical staining was performed on postmortem hypothalamus material of both sexes from the infant/pubertal period into the elderly period and from MtF transsexuals. RESULTS: Quantitative analysis demonstrated that the human NKB system exhibits a robust female-dominant sexual dimorphism in the INF. During the first years after birth, both sexes displayed a moderate and equivalent level of NKB immunoreactivity in the INF. The adult features emerged progressively around puberty until adulthood, where the female-dominant sex difference appeared and continued into old age. In MtF transsexuals, a female-typical NKB immunoreactivity was observed. Finally, in postmenopausal women, there was a significant increase in NKB immunoreactivity compared to premenopausal women. CONCLUSION: Our results indicate that certain sex differences do not emerge until adulthood when activated by sex steroid hormones and the likely involvement of the human infundibular NKB system in the negative and positive feedback of estrogen on GnRH secretion.

Analysis of the expression of neurokinin B, kisspeptin, and their cognate receptors NK3R and KISS1R in the human female genital tract.

OBJECTIVE: To investigate the presence of neurokinin B (NKB)/NK(3) receptor (NK(3)R) and kisspeptin/KISS1 receptor (KISS1R) messenger RNA (mRNA) and proteins throughout the human female genital tract. DESIGN: In vitro study. SETTING: Academic research laboratories and academic hospitals. PATIENT(S): Fifteen reproductive-age women and 16 postmenopausal women provided fresh samples of uterus, ovary, or oviduct, and 12 women provided archival samples of endometrium or oviduct. INTERVENTION(S): Fresh and archival samples of uterus, ovary, and oviduct obtained from reproductive-age and postmenopausal women. MAIN OUTCOME MEASURE(S): Results of reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry to investigate the pattern of expression of NKB/NK(3)R and kisspeptin/KISS1R in target tissues. RESULT(S): Expression of the genes encoding NKB (TAC3) and NK(3)R (TACR3), and kisspeptin (KISS1) and its receptor (KISS1R) was found in the uterus, ovary, and oviduct. Both NKB and NK(3)R immunoreactivity was detected in the endometrium, the oviduct, and the ovary, with marked expression in endometrial and oviductal epithelial cells, where intense coexpression of kisspeptin and KISS1R was also detected. Positive staining for NKB and NK(3)R was found in the myometrium where, in contrast, kisspeptin and KISS1R were absent. CONCLUSION(S): NKB/NK(3)R and kisspeptin/KISS1R are present in female peripheral reproductive tissues with colocalization of both systems in some non-neuronal cell populations of the human female genital tract. Our findings are compatible with a potential modulatory role of NKB and kisspeptin at peripheral reproductive tissues.

Upregulation of tachykinin NK-1 and NK-3 receptor binding sites in the spinal cord of spontaneously hypertensive rat: impact on the autonomic control of blood pressure.

1 Effects of intrathecally (i.t.) injected tachykinin NK-1 and -3 receptor agonists and antagonists were measured on mean arterial blood pressure (MAP) and heart rate (HR) in awake unrestrained spontaneously hypertensive rats (SHR,15-week-old) and age-matched Wistar Kyoto rats (WKY). Quantitative in vitro autoradiography was also performed on the lower thoracic spinal cord of both strains and Wistar rats using specific radioligands for NK-1 receptor ([(125)I]HPP[Arg(3),Sar(9),Met(O(2))(11)]SP (3-11)) and NK-3 receptor ([(125)I]HPP-Asp-Asp-Phe-N-MePhe-Gly-Leu-Met-NH(2)). 2 The NK-1 agonist [Sar(9),Met(O(2))(11)]SP (650 and 6500 pmol) decreased MAP and increased HR in WKY. The fall in MAP was blunted in SHR and substituted by increases in MAP (65-6500 pmol) and more sustained tachycardia. The NK-3 agonist senktide (6.5-65 pmol) evoked marked increases in MAP and HR (SHR>>>WKY), yet this response was rapidly desensitized. Cardiovascular effects of [Sar(9),Met(O(2))(11)]SP (650 pmol) and senktide (6.5 pmol) were selectively blocked by the prior i.t. injection of LY303870 (NK-1 antagonist, 65 nmol) and SB235375 (NK-3 antagonist, 6.5 nmol), respectively. Antagonists had no direct effect on MAP and HR in both strains. 3 Densities of NK-1 and -3 receptor binding sites were significantly increased in all laminae of the spinal cord in SHR when compared to control WKY and Wistar rats. The dissociation constant was however not affected in SHR for both NK-1 (K(d)=2.5 nM) and NK-3 (K(d)=5 nM) receptors. 4 Data highlight an upregulation of NK-1 and -3 receptor binding sites in the thoracic spinal cord of SHR that may contribute to the hypersensitivity of the pressor response to agonists and to the greater sympathetic activity seen in this model of arterial hypertension.

Distribution and pharmacological characterization of primate NK-1 and NK-3 tachykinin receptors in the central nervous system of the rhesus monkey.

Much attention has focused on tachykinin receptors as therapeutic targets for neuropsychiatric disorders, although their expressional distributions in the primate central nervous system (CNS) remain unclear. We cloned the genes encoding the NK-1 and NK-3 tachykinin receptors (referred to as rmNK-1 and rmNK-3) from the rhesus monkey (Macaca mulatta) brain and examined their pharmacological profiles and regional distributions in the CNS. The deduced rmNK-1 amino-acid sequence differed by only two amino acids from the human NK-1 (hNK-1). The deduced rmNK-3 amino-acid sequence was two amino acids shorter than human NK-3 (hNK-3), with a seven-amino-acid difference in sequence. Ligand binding studies revealed that the affinity of rmNK-1 to substance P (SP) was comparable to that of hNK-1 in cell lines that expressed individual receptors stably. Nonpeptide antagonists had similar effects on the binding of rmNK-1 and hNK-1. Affinity of rmNK-3 for NKB was stronger than for SP and the IC50 value was comparable with that of hNK-3. Ca2+ imaging showed that activations of both rmNK-1 and rmNK-3 by specific ligands, SP and senktide, induced increased intracellular Ca2+ in cell lines that stably expressed individual primate tachykinin receptors. The amounts of rmNK-1 and rmNK-3 mRNAs were quantitatively determined in the monkey CNS. The expression of rmNK-1 was observed in all of the cortical and subcortical regions, including the hippocampus and the amygdala. The putamen contained the most NK-1 mRNA in the brain, with less rmNK-3 mRNA found in the cortex compared to rmNK-1 mRNA. In the monkey hippocampus and amygdala, rmNK-1 mRNA was present at markedly higher concentrations than rmNK-3 mRNA. The present results provide an insight into the distinct physiological nature and significance of the NK-1 and NK-3 tachykinin systems in the primate CNS. These findings are indispensable for establishing model systems in the search for a subtype-specific tachykinin receptor agonist and antagonist for the treatment of neuropsychiatric disorders.

Autoradiographic distribution of tachykinin NK2 binding sites in the rat brain: comparison with NK1 and NK3 binding sites.

The autoradiographic distribution of tachykinin NK(2) binding sites was determined in the adult rat brain using [(125)I]neurokinin A in the presence of either senktide (NK(3) agonist) and [Pro(9)]substance P (NK(1) agonist) or senktide and SR 140333 (NK(1) antagonist). Indeed, this radioligand labels two subtypes of NK(1) binding sites (which present a high affinity not only for SP but also for neurokinin A, neuropeptide K and neuropeptide gamma) as well as NK(3) binding sites. The distribution of NK(2) binding sites was also compared with those of NK(1) and NK(3) binding sites, these sites being labeled with [(125)I]Bolton and Hunter substance P and [(125)I]Bolton and Hunter eledoisin, respectively. In agreement with our results obtained with membranes from various brain structures, NK(2)-sensitive [(125)I]neurokinin A labeling was mainly observed in few structures including the dorsal and ventral hippocampus, the septum, the thalamus and the prefrontal cortex. The density of NK(2) binding sites was weak when compared with those of NK(1) and NK(3) binding sites. Marked differences were observed in the distributions of NK(1), NK(2) and NK(3) binding sites. These results are discussed taking into consideration differences or similarities between the distributions of NK(2)-sensitive [(125)I]neurokinin A binding sites and of their endogenous ligands (neurokinin A, neuropeptide K and neuropeptide gamma) but also local NK(2) agonist responses blocked by NK(2) antagonists. Insights on the roles of endogenous tachykinins in several brain functions are also discussed on the basis of the respective distributions of different neurokinin binding sites.

Implication of nigral tachykinin NK3 receptors in the maintenance of hypertension in spontaneously hypertensive rats: a pharmacologic and autoradiographic study.

1. The role of nigral tachykinin NK(1), NK(2) and NK(3) receptors in central cardiovascular regulation was studied by measuring the effects of selective agonists and antagonists on mean arterial pressure (MAP) and heart rate (HR) after bilateral microinjection into the substantia nigra of spontaneously hypertensive rats (SHR). Quantitative in vitro autoradiography was also performed in the midbrain of SHR and Wistar-Kyoto (WKY) with the NK(3) receptor ligand [(125)I]-HPP-Senktide. 2. Tachycardia was elicited by the NK(1) ([Sar(9),Met(O(2))(11)]SP) and NK(2) ([betaAla(8)]NKA(4-10)) agonists at 25 and 100 pmol while the NK(3) agonist (senktide, 50 and 100 pmol) had no significant effect. The three agonists had no effect on behaviour, and increases in MAP were elicited by the NK(1) agonist only. 3. Whereas antagonists at NK(1) (RP 67580, 500 pmol) and NK(2) (SR 48968, 500 pmol) receptors had no significant effect on MAP and HR, the NK(3) antagonist (R-820, 500 pmol) reduced MAP for over 3 h in SHR. That anti-hypertensive effect did not occur after intracerebroventricular or intravenous injection of R-820. Also, R-820 had no cardiovascular effect in WKY. 4. The affinity (K(D): 0.7 nM) and densities of specific NK(3) receptor binding sites measured in the substantia nigra, ventral tegmental area, hippocampus and amygdala were not significantly different in SHR and WKY. 5. It is concluded that endogenous tachykinins exert a tonic activity on NK(3) receptors in the substantia nigra of SHR to maintain high blood pressure. Hence, nigral tachykinin NK(3) receptors may represent a promising therapeutic target in the treatment of arterial hypertension.

Immunocytochemical description of five bipolar cell types of the mouse retina.

With the ever-growing number of transgenic mice being used in vision research, a precise knowledge of the cellular organization of the mouse retina is required. As with the cat, rabbit, rat, and primate retinae, as many as 10 cone bipolar types and one rod bipolar type can be expected to exist in the mouse retina; however, they still have to be defined. In the current study, several immunocytochemical markers were applied to sections of mouse retina, and the labeling of bipolar cells was studied using confocal microscopy and electron microscopy. By using antibodies against the neurokinin-3 receptor NK3R; the plasma membrane calcium ATPase1 (PMCA1); and the calcium (Ca)-binding proteins CaB1, CaB5, caldendrin, and recoverin, three different OFF-cone bipolar cells could be identified. One type of ON-cone bipolar cell was identified through its immunoreactivity for CaB5 and PMCA1. Rod bipolar cells, comparable in morphology to those of other mammalian retinae, expressed protein kinase Calpha and CaB5. It was also shown that putative OFF-cone bipolar cells receive light signals through flat contacts at the cone pedicle base, whereas ON-cone bipolar signaling involves invaginating contacts. The distribution of the kainate receptor subunit GluR5 was studied by confocal and electron microscopy. GluR5 was expressed at flat bipolar cell contacts; however, it appears to be involved with only certain types of OFF-cone bipolar cells. This suggests that different bipolar cell types receive their light signals through different sets of glutamate receptors.

Comparative distribution of NK1, NK2, and NK3 receptors in the rat brainstem auditory nuclei.

While the distribution of substance P in the auditory system is well illustrated, the localization of its receptors has not yet been documented. The goal of our study was to characterize the distribution of the tachykinin receptors NK1-R, NK2-R and NK3-R in the brainstem auditory nuclei of the adult rat using immunohistochemical techniques. The immunoreactivity of the neurokinin receptors was found to be widely distributed in most neurons of the cochlear nucleus (CN), the lateral superior olive (LSO), the medial nucleus of the trapezoid body (MNTB) and in the inferior colliculus (IC). Immunoreactivity was generally confined to post-synaptic targets (neuronal cell body and proximal or primary dendrites) in all auditory nuclei. However, unlike brainstem nuclei, the IC showed, in addition to neuronal cell body staining, a positive axonal immunolabeling (axons and pre-synaptic terminals) with the anti-NK1-R antibody. This axonal staining, revealing a pre-synaptic expression of NK1-R, is in good agreement with the known presence of substance P in the IC neurons. The absence of axonal staining in the superior olivary complex nuclei which projects afferent to the IC indicated that the NK1-R labeled axons are rather intrinsic IC fibers or descending thalamic projections to the IC. Overall, the wide distribution of the three types of tachykinin receptors observed in the present study argues for an important role of tachykinin neuropeptides in the central auditory system.

Neurokinin NK1- and NK3-immunoreactive neurons in serotonergic cell groups in the rat brain.

Interactions are known to occur in the brain between serotonin (5-HT) and substance P (SP). To investigate whether SP can directly influence serotonergic neurons, double immunohistochemical labelings were performed on rat brain sections with NK1 or NK3 affinity-purified antibodies and a 5-HT monoclonal antibody. It was found that the vast majority of serotonergic cell bodies do not colocalize NK1 or NK3 labeling. Only in the central linear nucleus and ventral part of the dorsal raphe nucleus were a few serotonergic neurons double-labeled for NK1 receptors (15 and 0.8% of serotonergic neurons, respectively). It is suggested that serotonergic neurons are not major direct targets for SP in the rat brain.

Localization of neurokinin B receptor in mouse gastrointestinal tract.

AIM: To observe the location of neurokinin receptor (NK3r) in the mouse gastrointestinal tract. METHODS: The abdomens of 8 male Kunming mice were opened under anaesthesia with sodium pentobarbital. The exposed gut organs were kept moisture and temperature at the same time. Then the esophagus,jejulum, ileum, and colon, etc were respectively cut and the segments from the stomach to the distal colon were opened along the mesenteric border. A circular 4mm-6mm enteric part(pieces of 1 cm(2) were to be prepared) and mucosa and submucosa were removed, then the longitudinal muscle layer was pulled off from the circular muscle layer under microphotograph. They were rinsed in 50 nmol x L(-1) potassium phosphate-buffered saline(PBS). Immunohistochemistry and immunoreactive fluorescence were used in the staining procedures. RESULTS: There was NK3r-Like(-Li) positive material on the smooth muscle cells of the esophagus, stomach, and intestines and other regions. The nerve cell bodies with immunoreactivity for NK3r were mainly distributed in the submucousal nerve plexus or myenteric nerve plexus of the gastrointestinal tract except for the esophagus, stomach and rectum. The reaction product was located on the surface of the nerve cell plasma. It was occasionally observed in the cell plasma endosomes, but was very weakly stained. Among the NK3-like positive neurons in the plexus,the morphological type in many neurons appeared like Dogiel II type cells. Some neuron cell bodies were big, having many profiles, some were long ones or having grading structure. Cell body diameter was about 10 microm-46 microm and 8 microm-42 microm in myenteric plexus and submucous plexus. CONCLUSION: This study not only described the distribution of neurokinin B receptor in the mouse gut, but also provided a morphological basis for deducing the functional identity of the NK3r-LI immunoreactivity neurons, suggesting the possibility that these neurons were closely related to gastrointestinal tract contraction and relaxing activity.

Cholinergic neurons expressing neuromedin K receptor (NK3) in the basal forebrain of the rat: a double immunofluorescence study.

By using a double immunofluorescence method we have examined the distribution of cholinergic neurons expressing neuromedin K receptor (NK3) in the rat brain and spinal cord. The distribution of neuromedin K receptor-like immunoreactive neurons completely overlapped with that of choline acetyltransferase-positive neurons in certain regions of the basal forebrain, e.g. the medial septal nucleus, nucleus of the diagonal band of Broca, magnocellular preoptic nucleus and substantia innominata. Partially overlapping distributions of neuromedin K receptor-like immunoreactive and choline acetyltransferase-positive neurons were found in the basal nucleus of Meynert, globus pallidus, ventral pallidum of the forebrain, tegmental nuclei of the pons and dorsal motor nucleus of the vagus. Neurons showing both neuromedin K receptor-like and choline acetyltransferase immunoreactivities, however, were found predominantly in the medial septal nucleus, nucleus of the diagonal band of Broca and magnocellular preoptic nucleus of the basal forebrain: 66-80% of these choline acetyltransferase-positive neurons displayed neuromedin K receptor-like immunoreactivity. Neurons showing both neuromedin K receptor-like and choline acetyltransferase immunoreactivities were hardly detected in other aforementioned regions of the forebrain, brainstem and spinal cord. The present study has provided morphological evidence for direct physiological modulation or regulation of cholinergic neurons by tachykinins through the neuromedin K receptor in the basal forebrain of rats.

Immunohistochemical demonstration of the NK(1) tachykinin receptor on muscle and epithelia in guinea pig intestine.

BACKGROUND AND AIMS: Previous immunohistochemical studies failed to reveal neurokinin (NK)(1) tachykinin receptors on intestinal muscle, despite convincing pharmacologic data indicating their presence. This study aimed to apply optimal immunohistochemical methods to reveal the receptors. METHODS: NK(1)-receptor immunoreactivity was examined by confocal microscopy in tissue incubated with or without 10(-7) mol/L substance P (SP), 10(-7) mol/L SP plus 10(-6) mol/L NK(1) receptor antagonist (CP99994), or with fluorescent cyanine 3.18 (Cy3) SP. RESULTS: Without incubation, NK(1)-receptor immunoreactivity was strong on muscle of the rectum and distal colon and weak in proximal colon and small intestine. NK(1) receptor was located on the surface of muscle cells in all gut regions. Exposure to SP increased the intensity of immunoreactivity, and the receptor moved into the cytoplasm. Mobilization of the receptor by SP was blocked by the NK(1)-receptor antagonist CP99994. Cy3-SP was internalized by muscle cells and colocalized with the receptor. NK(1)-receptor immunoreactivity occurred on crypt epithelial cells in the small intestine and the base of glands in the proximal colon. CONCLUSIONS: The NK(1) receptor occurs on the external muscle throughout the small and large intestines. SP binds and triggers NK(1)-receptor aggregation and internalization in the muscle.

NK(3) receptors in the feline nucleus tractus solitarius are not involved with the muscle pressor response.

Isometric muscle contractions cause an increase in mean arterial pressure and heart rate. Previously, we showed that substance P (SP) is released from sites in the feline medial nucleus tractus solitarius (mNTS) in response to isometric muscle contractions, and that it most likely interacted with NK(1) tachykinin receptors at these sites. This study was undertaken to determine whether other tachykinin receptors in this area of the brainstem are involved with the muscle pressor response. Receptor autoradiography, using [(125)I]Bolton-Hunter SP and [(125)I] [MePhe(7)] neurokinin B to label NK(1) and NK(3) receptors, respectively, indicated that NK(3) tachykinin receptors are as abundant as NK(1) and NK(3) receptors, respectively, indicated that NK(3) tachykinin receptors are as abundant as NK(1) receptors in this region of the feline brainstem Injections of the specific NK(3) receptor antagonist, SR 142801 (0.1 to 10 microM) into the mNTS did not modify the pressor response or the heart rate response to isometric muscle contractions. Injection of SR142801 into the NTS prior to the injection of the NK(1) antagonist, GR82334 did not affect the action of GR82334 to attenuate the muscle pressor reflex. We conclude that NK(3) receptors in the NTS are not involved with the regulation of cardiovascular function during activation of the muscle pressor response.

Retinal dopaminergic neurons (A17) expressing neuromedin K receptor (NK(3)): a double immunocytochemical study in the rat.

By using a double immunofluorescence method we examined the distribution of dopaminergic neurons (A17) expressing neuromedin K receptor (NKR, NK(3)) in the rat retina. The distribution of NKR-like immunoreactive (-LI) neurons partially overlapped that of tyrosine hydroxylase (TH)-LI neurons in the inner retina of section and flat-mount preparation. Neurons showing both TH- and NKR-like immunoreactivities were found in the retina (A17): 100% of these TH-LI neurons displayed NKR-like immunoreactivity, and they constituted about 3.5% of total NKR-LI neurons. The majority of double-labeled neurons with TH- and NKR-like immunoreactivities were distributed in the proximal inner nuclear layer and the upper part of inner plexiform layer of the retina, and characterized with appearance of amacrine cells. The present study has provided morphological evidence for direct physiological modulation of dopaminergic neurons by tachykinins through NKR in the rat retina (A17).

NK1, NK2 and NK3 tachykinin receptor localization and tachykinin distribution in the ileum of the mouse.

Tachykinin receptors NK1r, NK2r and NK3r bind tachykinins with different affinities and share pharmacological and molecular differences among animal species. NK1r, NK2r, NK3r and tachykinin (SP/NKA) distribution was studied by immunohistochemistry in the ileum of mouse since no data are available for this species. The results were then compared to those obtained in the rat and guinea pig either by us or by others to ascertain interspecies similarities and/or differences. NK1r- and NK3r-immunoreactivity (IR) were detected in neurons and NK1r-IR in the interstitial cells of Cajal at the deep muscular plexus. At variance with rat and guinea pig, NK1r-IR was also found in the myoid cells of the villi, while NK2r-IR was never detected in nerve varicosities. This latter datum suggests that the NK2r does not play a presynaptic role in the mouse. Unexpectedly, a high NK2r-IR and the presence of NK3r-IR were observed at the inner portion of the circular muscle layer in the mouse as well as in the rat and guinea pig, demonstrating a subregional distribution of these receptors. Tachykinin distribution did not show noticeable species-related differences. The present findings show species-related differences in the tachykinin receptor distribution that might be related to a different tachykinin control of intestinal motility.

Presynaptic nicotinic acetylcholine receptors in the myenteric plexus of guinea pig intestine.

Presynaptic nicotinic acetylcholine receptors (nAChRs) were studied in myenteric plexus preparations from guinea pig ileum using intracellular electrophysiological methods. Microapplication of nicotine (1 mM) caused a biphasic depolarization in all AH neurons (n = 30) and in 36 of 49 S neurons. Cytisine (1 mM) caused fast depolarizations in S neurons and no response in AH neurons. Mecamylamine (10 microM) blocked all responses caused by nicotine and cytisine. TTX (0.3 microM) blocked slow excitatory synaptic potentials in S and AH neurons but had no effect on fast depolarizations caused by nicotine. Nicotine-induced slow depolarizations were reduced by TTX in two of twelve AH neurons (79% inhibition) and four of nine S neurons (90+/-12% inhibition). Slow nicotine-induced depolarizations in the remaining neurons were TTX resistant. TTX-resistant slow depolarizations were inhibited after neurokinin receptor 3 desensitization caused by senktide (0.1 microM); senktide desensitization inhibited the slow nicotine-induced depolarization by 81+/-5% and 63+/-15% in AH and S neurons, respectively. A low-calcium and high-magnesium solution blocked nicotine-induced slow depolarizations in AH neurons. In conclusion, presynaptic nAChRs mediate the release of substance P and/or neurokinin A to cause slow depolarizations of myenteric neurons.

Mesencephalic dopaminergic neurons expressing neuromedin K receptor (NK3): a double immunocytochemical study in the rat.

By using a double immunocytochemical method we examined the distribution of dopaminergic neurons expressing neuromedin K receptor (NKR; NK3) in the rat brain. The distribution of NKR-like immunoreactive (-LI) neurons completely overlapped that of tyrosine hydroxylase (TH)-LI neurons in the retrorubral field (A8), substantia nigra (A9), ventral tegmental area and nucleus raphe linealis (A10). Completely or partially overlapping distributions of NKR- and TH-LI neurons were found in certain regions of the hypothalamus (A11-A15) and olfactory bulb (A16). Neurons showing both NKR- and TH-like immunoreactivities, however, were only found in A8-A10: All of the NKR-LI neurons displayed TH-like immunoreactivity, and about 71-86% of the TH-LI neurons expressed NKR-like immunoreactivity. The present results provided morphological evidence for physiological modulation of dopaminergic neurons by tachykinins through NKR in A8-A10.

Immunoelectron microscopic localization of NK-3 receptor in the rat spinal cord.

The immunocytochemical localization of tachykinin NK-3 receptor (NK-3R) was studied in rat spinal cord using a mouse monoclonal antibody directed against the C-terminus peptide (434-465) of the human NK-3R. Light microscopic labeling prevailed in lamina II and lamina X. Under the electron microscope, the immunolabeling in lamina II involved mainly dendritic spines inside glomeruli, preterminal axons and axon terminals. This suggests that neurokinin B can modulate primary afferent, sensory information, both post- and pre-synaptically. These actions are likely to involve extrasynaptic diffusion of the peptide, since NK-3R immunoreactivity was not found in association with postjunctional differentiations.

Localisation of neurokinin 3 (NK3) receptor immunoreactivity in the rat gastrointestinal tract.

The localisation of the neurokinin 3 receptor (NK3r) in the rat gastrointestinal tract has been studied by using a polyclonal antiserum against the C-terminal portion (amino acids 388-452) of the rat NK3r. In the oesophagus, immunoreactivity for the NK3r was found on smooth muscle cells of the muscularis mucosae. NK3r immunoreactivity was not present on muscle cells of other regions. Nerve cell bodies immunoreactive for NK3r were seen in the myenteric and submucous plexuses of the small and large intestine, but not in the stomach or oesophagus. Immunoreactivity was largely confined to nerve cell surfaces. The reaction product was on the cell soma and initial parts of axons. Reactivity was not seen on nerve terminals. Immunoreactive nerve cells had Dogiel Type II morphology. Patterns of co-localisation of NK3r and immunoreactivity for other markers were examined in the ileum, to provide a basis from which to deduce the functional identity of NK3r-immunoreactive nerve cells. Most of the NK3r-immunoreactive nerve cells were also immunoreactive for the calcium-binding proteins, calretinin and calbindin, and all were immunoreactive for the NK1 receptor (NK1r). Nerve cells that were immunoreactive for nitric oxide synthase were not immunoreactive for either NK3r or NK1r. The projections of the calbindin and calretinin neurons were determined by nerve lesion studies. Their morphology, projections to the mucosa and other ganglia and immunoreactivity for the calcium-binding proteins suggest that the NK3r-immunoreactive neurons are intrinsic sensory neurons.

Relationship of NK3 receptor-immunoreactivity to subpopulations of neurons in rat spinal cord.

The distribution of immunoreactivity to the neurokinin3 receptor (NK3R) was examined in segments C7, T11-12, L1-2, and L4-6 of the rat spinal cord. NK3R immunoreactivity was visualized by using two antisera generated against sequences of amino acids contained in the C-terminal region of the NK3R. NK3R-immunoreactive cells were numerous in the substantia gelatinosa of all spinal segments examined as well as the dorsal commissural nucleus of spinal segments L1-2. Isolated, immunoreactive cells were scattered throughout other regions of the spinal cord. The relationship of NK3R-immunoreactivity with neurons was demonstrated by colocalization with microtubule associated protein 2-immunoreactivity in individual cells. Within neurons, NK3R-immunoreactivity was associated predominately with the plasma membrane of cell bodies and dendrites. Within the substantia gelatinosa, 86% of nitric oxide synthase (NOS)-immunoreactive neurons were also NK3R-immunoreactive. Although NOS-immunoreactive neurons were found throughout all other regions of the spinal cord in the segments examined, these were not NK3R-immunoreactive. When preganglionic sympathetic neurons in spinal segments T11-12 and L1-2 were visualized by intraperitoneal injection of Fluorogold, less than 1% of the Fluorogold-labeled neurons were also immunoreactive for NK3R. The large number of NK3R-immunoreactive neurons in the substantia gelatinosa suggests that some effects of tachykinins on somatosensation may be mediated by NK3R.