Catecholaminergic neurons in synaptic connections with pre-Bötzinger complex neurons in the rostral ventrolateral medulla in normoxic and daily acute intermittent hypoxic rats.

The rostral ventrolateral medulla (RVLM) contains cardiovascular-related catecholaminergic neurons and respiratory-related pre-Bötzinger complex (pre-BötC) neurons, which are intermingled and functionally connected for coordinating cardiorespiratory activities. Daily acute intermittent hypoxia (dAIH) is known to elicit respiratory plasticity. However, it is unclear if the catecholaminergic neurons directly synapse onto pre-BötC neurons, and if the local circuitry exhibits plasticity when exposed to dAIH. The present study was aimed to determine the synaptic phenotypes between dopamine-ß-hydroxylase (DßH)-immunoreactive (ir) catecholaminergic neurons and neurokinin 1 receptor (NK1R)-ir pre-BötC neurons, and the effect of dAIH on the neuronal network. Immunofluorescence histochemistry was used to reveal immunoreactivities of DßH and NK1R in the RVLM of normoxic and dAIH rats. Synaptic phenotypes were examined with double-labeling immunoelectron microscopy. We found that DßH immunoreactivity was expressed in somata and processes, some of which were in close apposition to NK1R-ir pre-BötC neurons. DßH-ir gold particles were localized to somata, dendrites, and axonal terminals. DßH-ir terminals formed asymmetric synapses, and occasionally, symmetric synapses in the pre-BötC, featuring the local circuitry. Of the synapses, 28% in normoxic and 29.6% in dAIH groups were apposed to NK1R-ir dendrites. Significant increases in DßH expression and NK1R-ir processes were found in the dAIH group. Moreover, the area and number of processes in close appositions were significantly elevated, strongly suggesting that dAIH induced plasticity with increased connections and interactions between the cardiovascular- and respiratory-related neurons in the local circuitry. In conclusion, asymmetric synapses are predominant in the crosstalk between catecholaminergic and pre-BötC neurons in the RVLM, elaborating excitatory transmission driving the coupling of cardiorespiratory activities. The neural network manifests plasticity in response to dAIH challenge.

Synaptic relationship between somatostatin- and neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats.

The pre-Bötzinger complex (pre-BötC) in the ventrolateral medulla oblongata is critical for the generation of respiratory rhythm in mammals. Somatostatin (SST) and neurokinin 1 receptor (NK1R) immunoreactivity have been used as markers of the pre-BötC. SST immunoreactivity almost completely overlaps with small fusiform NK1R-immunoreactive (ir) neurons, the presumed rhythmogenic neurons, but not with large multipolar NK1R-ir neurons. Understanding the neurochemical characteristics, especially the synaptic relationship of SST/NK1R-ir neurons within the pre-BötC network is essential in providing cellular and structural bases for understanding their physiological significance. This work has not been documented so far. We found that SST immunoreactivity was highly expressed in terminals, somas, and primary dendrites in the pre-BötC. Besides the small fusiform neurons, a small population of medium-sized NK1R-ir neurons also colocalized with SST. Large NK1R-ir neurons were not SST-ir, but received somatostatinergic inputs. SST-ir terminals were glutamatergic or GABAergic, and synapsed with NK1R-ir neurons. Most of synapses between them were of the symmetric type, indicating their inhibitory nature. Asymmetric synapses were evident between SST-ir terminals and NK1R-ir dendrites, strongly suggesting an excitatory innervation from the presumed rhythmogenic neurons as these neurons are glutamatergic. We speculate that SST-mediated excitatory and inhibitory synaptic transmission onto NK1R-ir rhythmogenic and follower neurons synchronizes their activity to contribute to respiratory rhythmogenesis and control.

Neurokinin-1 receptors in cholinergic neurons of the rat ventral pallidum have a predominantly dendritic distribution that is affected by apomorphine when combined with startle-evoking auditory stimulation.

Cholinergic neurons of the basal forebrain are implicated in startle reflex inhibition by a prior weak stimulus often referred to as prepulse inhibition (PPI) and used as an index of sensorimotor gating deficits in schizophrenia. Gating deficits can be produced in rodent models by acute systemic administration of apomorphine, a non-selective dopamine D1 and D2 receptor agonist that also affects trafficking of neurokinin-1 (NK(1)) receptors induced by startle evoking auditory stimulation (AS) in midbrain neurons. We used electron microscopic immunolabeling of NK(1) receptors and the vesicular acetylcholine transporter (VAchT) to test the hypothesis that the subcellular distributions of these receptors in cholinergic neurons of the rat ventral pallidum are subject to a similar regulation. In vehicle controls, NK(1) immunogold was often seen near cytoplasmic endomembranes in somata and large dendrites, but was more equally distributed in cytoplasmic and plasmalemmal compartments of medium dendrites, and principally located on the plasma membrane of small dendrites. These labeling patterns appeared to be largely independent of whether the NK(1) receptor was co-expressed with VAchT, however only the medium and small VAchT-labeled dendrites showed significant treatment-specific differences in NK(1) immunogold distributions. The NK(1) receptor immunogold particle density on the plasma membrane of medium cholinergic dendrites was significantly enhanced by combined apomorphine and AS, while neither alone affected either the plasmalemmal density or the equality of the plasmalemmal and cytoplasmic distributions of NK(1) receptors in these dendrites. Small cholinergic dendrites showed a significant AS-induced increase in both the plasmalemmal and cytoplasmic density of NK(1) gold particles, and an apomorphine-induced disruption of the preferential plasmalemmal targeting of the NK(1) receptors. These results provide ultrastructural evidence that NK(1) receptors in cholinergic neurons of the ventral pallidum have subcellular locations and plasticity conducive to active involvement in dopamine-dependent sensorimotor processing.

Hippocampal CA3 pyramidal cells selectively innervate aspiny interneurons.

The specific connectivity among principal cells and interneurons determines the flow of activity in neuronal networks. To elucidate the connections between hippocampal principal cells and various classes of interneurons, CA3 pyramidal cells were intracellularly labelled with biocytin in anaesthetized rats and the three-dimensional distribution of their axon collaterals was reconstructed. The sections were double-stained for substance P receptor (SPR)- or metabotropic glutamate receptor 1alpha (mGluR-1alpha)-immunoreactivity to investigate interneuron targets of the CA3 pyramidal cells. SPR-containing interneurons represent a large portion of the GABAergic population, including spiny and aspiny classes. Axon terminals of CA3 pyramidal cells contacted SPR-positive interneuron dendrites in the hilus and in all hippocampal strata in both CA3 and CA1 regions (7.16% of all boutons). The majority of axons formed single contacts (87.5%), but multiple contacts (up to six) on single target neurons were also found. CA3 pyramidal cell axon collaterals innervated several types of morphologically different aspiny SPR-positive interneurons. In contrast, spiny SPR-interneurons or mGluR-1alpha-positive interneurons in the hilus, CA3 and CA1 regions were rarely contacted by the filled pyramidal cells. These findings indicate a strong target selection of CA3 pyramidal cells favouring the activation of aspiny classes of interneurons.

Cellular and subcellular localization of neurokinin-1 and neurokinin-3 receptors in primate globus pallidus.

The primate globus pallidus receives massive innervations from GABAergic striatal neurons that co-release the neuropeptide substance P (SP). To expand our knowledge regarding SP interaction at pallidal level, we used single and double antigen retrieval methods to study the cellular and subcellular localization of SP and its high-affinity receptors neurokinin-1 (NK-1R) and neurokinin-3 (NK-3R) in the globus pallidus of the squirrel monkey (Saimiri sciureus). At the light microscopic level, a large number of neurons and fibers located in both the external (GPe) and internal (GPi) segments of the globus pallidus expressed NK-1R or NK-3R immunoreactivity. At the electron microscopic level, both NK-1R and NK-3R were mainly associated with intracellular sites or located at extrasynaptic positions on the plasma membrane. Presynaptic axon terminals forming symmetric and asymmetric synapses occasionally contained NK-1R and NK-3R. Neurokinin receptors were also observed in a proportion of SP-immunoreactive axon terminals, but these terminals preferentially expressed NK-3R. The pattern of distribution of NK-1R and NK-3R in GPe and GPi indicates that SP effects at pallidal level are mediated through postsynaptic receptor as well as presynaptic autoreceptors and heteroreceptors. These morphological data suggest that, either alone or in conjunction with GABA, SP could have a wide range of effects at pallidal level. This neuroactive peptide may influence in a significant manner the integration and treatment of neural information that flows through the basal ganglia.

Substance P and Neurokinin 1 receptor - expression is affected in the ileum of mice with mutation in the W locus.

The tachykinin substance P (SP) acts on the gut muscle coat via its preferred receptor, neurokinin 1 (NK1r). In the mouse ileum, NK1r-immunoreactivity (NK1r-IR) was detected in neurons, in the interstitial cells of Cajal at the deep muscular plexus (ICC-DMP) and the myoid cells of the villi. SP-IR was detected in neurons and varicose nerve fibers, which were especially numerous at the DMP and closely associated with the ICC-DMP. In mice with a mutation in the W locus (ckit mutant animals), innervation is suggested to be normal although few studies have actually tested this hypothesis. Indeed, studies demonstrating ICC-DMP integrity are lacking and whether SP- and NK1r-IR are normal in these animals has not been investigated. Our aim was to perform an immunohistochemical study on the ileum of a strain of heterozygous mice with a mutation in the W locus, the W(e/+) mice, to test this hypothesis. SP-IR nerve fibers were significantly more numerous than in wild type mice; NK1r-IR was clustered on the plasma membrane and also intracytoplasmatic in the neurons, but absent in the ICC-DMP. The richness in SP-IR nerve fibers and the NK1r-IR distribution in the neurons, similar to that of activated cells, might be attempts to compensate for the SP preferred receptor absence at the ICC-DMP. In conclusion, SP content and NK1r expression are noticeably different in c-kit mutants with respect to wild type mice, and probably causing an anomalous tachykininergic control of intestinal motility. Physiological studies on Wmutant mice have to take into account that innervation in this animal model is affected by the c-kit mutation.

Synaptic connections of cone bipolar cells that express the neurokinin 1 receptor in the rabbit retina.

We have investigated and further characterized, in the rabbit retina, the synaptic connectivity of the ON-type cone bipolar cells that are immunoreactive for an antibody against the neurokinin-1 receptor (NK1R). NK1R-immunoreactive bipolar cell axons terminate in stratum 4 of the inner plexiform layer. The axons of NK1R-positive bipolar cells receive synaptic inputs from amacrine cells through conventional synapses and from putative AII amacrine cells via gap junctions. The major outputs from NK1R-positive bipolar cells make contacts with amacrine cell processes. The most frequent postsynaptic dyads comprise two amacrine cell processes. Double-labeling experiments with antibodies against NK1R and either calretinin or glycine have demonstrated that NK1R-immunoreactive bipolar cells form gap junctions with AII amacrine cells. Thus, NK1R-positive cone bipolar cells, together with calbindin-positive cone bipolar cells, may play an important role in transferring rod signals to the ON-type ganglion cells of the cone pathway in the rabbit retina.

A group of neurokinin-1 receptor-immunoreactive neurons expressing phospho-extracellular signal-regulated protein kinases in the pre-Bötzinger complex of rats.

Extracellular-signal-regulated kinases (ERKs) were described originally as critical regulators of cell growth and differentiation. ERKs have received increasing attention as important regulators of neuronal function, especially in synaptic plasticity and memory. We found a group of phospho-ERK1/2 (pERK1/2)-immunoreactive (ir) neurons in the ventrolateral medulla oblongata of adult rats. In the pre-Botzinger complex (pre-BotC) region, identified by neurokinin-1 receptor (NK1R) immunoreactivity, the distribution of pERK1/2-ir neurons seemed to overlap with that of NK1R-ir neurons. A detailed study combining immunofluorescence and immunogold-silver staining was designed to determine whether ERK-ir neurons were NK1R-ir and related to respiratory function in the pre-BotC. The pERK1/2 immunoreactivity was present mainly in somas and processes of small- to medium-sized neurons. Of pERK1/2-ir neurons in the pre-BotC, 19.7% were NK1R-ir. Some of the small, fusiform, NK1R-ir neurons, the putative rhythmogenic neurons, were also pERK1/2-ir. Under the electron microscope, immunogold particles indicative of pERK1/2 immunoreactivity were found in somas and dendrites in pre-BotC neurons. They were associated mainly with rough endoplasmic reticula and ribosomes. Immunogold particles could also be found in nuclei, but were absent from nucleoli. The existence of pERK1/2 in a subtype of NK1R-ir pre-BotC neurons implies that the pERK cascade may play a role in normal respiratory activity in adult rats. The pERK cascade may also participate in sympathoexcitatory activity, given that 42.2% of pERK1/2-ir neurons expressed tyrosine hydroxylase, a marker of C1 adrenergic neurons in the ventrolateral medulla oblongata.

Immunocytochemical localization of the neurokinin 1 receptor in rat dental pulp.

The dentin-pulp complex is a peripheral end-organ supplied by dense sensory nerve fibers. Substance P, a representative neuropeptide widely distributed in the dental pulp, has been reported to play roles in pain transmission and the amplification of inflammation. We analyzed here the expression of the neurokinin 1 (NK1) receptor, preferentially activated by substance P, using immunocytochemistry in rat dental pulp at both the light and electron microscopic levels. Conspicuous NK1 receptor immunoreactivity was found in the odontoblasts; immunolabelings were present at their plasma membrane and endosomal structures, especially in their cytoplasmic processes. Immunoreactions for NK1 receptor were also detectable in a part of the nerve terminals associated with the cytoplasmic processes of the odontoblasts. Furthermore, the endothelial cells of capillaries and post-capillary venules and the fibroblasts were labeled with the NK1 receptor in the subodontoblast layer. These findings suggest that pulpal cells and nerve fibers are targets for substance P that mediate multiple functions, including a vasoactive function and the regulation of vascular permeability as well as the modulation of pain transmission.

Specific innervation of aromatase neurons by substance P fibers in the dorsal horn of the spinal cord in quail.

The enzyme aromatase catalyzes the production of estrogens in the dorsal horn of the spinal cord where most of the nociceptive primary afferent fibers terminate. Numerous estrogen receptors are present in this area and the control of spinal aromatase activity is thought to play an important role in the estrogenic control of nociception. The coexistence of aromatase and nociceptive terminals suggests a role for aromatase cells in pain-related processes, but whether terminals releasing nociceptive neuropeptides (e.g., substance P) actually contact aromatase neurons is unknown and the factors that control spinal aromatase activity have not yet been identified. In the present study we analyzed by double-label immunocytochemistry the distribution in the Japanese quail spinal cord, of aromatase and of substance P or its receptor (neurokinin 1 receptor). All antigens were mainly localized in laminae I and II as observed in mammals. Most aromatase neurons were colocalized with neurokinin 1 receptors and were in close apposition with substance P-immunoreactive fibers. These results suggest that aromatase neurons are responsive to noxious stimulation and may participate in the control of nociception. Furthermore, spinal aromatase activity could be controlled by substance P through a regulation of the aromatase gene transcription as reported for the mouse diencephalon and/or through neurokinin 1 receptor-dependent phosphorylation of the aromatase protein.

GABAergic and glycinergic synapses onto neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats: light and electron microscopic studies.

The pre-Bötzinger complex (preBötC) in the ventrolateral medulla is thought to be the kernel for respiratory rhythm generation. Neurons in the preBötC contain intense neurokinin-1 receptor (NK1R) immunoreactivity. Some of these neurons in the adult preBötC are presumed to be the pre-inspiratory interneurons that are essential for generating respiratory rhythm in the neonate. Chloride-mediated synaptic inhibition is critical for rhythmogenesis in the adult. The present study used immunofluorescence histochemistry and immunogold-silver staining to determine the inhibitory synaptic relationship between glutamic acid decarboxylase (GAD)- or glycine transporter 2 (GlyT2)-immunoreactive (ir) boutons and NK1R-ir neurons in the preBötC of adult rats. Under the confocal microscope, we found that GAD- and GlyT2-ir boutons were in close apposition to NK1R-ir somas and dendrites in the preBötC. Under the electron microscope, GAD- and GlyT2-ir terminals were in close apposition to NK1R-ir somas and dendrites. Symmetric synapses were identified between GAD- or GlyT2-ir terminals and NK1R-ir neurons. A total of 51.6% GAD-ir and 38.2% GlyT2-ir terminals were found to contact or make synapses with NK1R-ir profiles, respectively. GAD- and GlyT2-ir terminals synapsed not only upon NK1R-ir neurons but also upon NK1R immuno-negative neurons. NK1R-ir neurons received both symmetric (presumed inhibitory) and asymmetric (presumed excitatory) synapses. Thus, the present findings provide the morphological basis for inhibitory inputs to NK1R-ir neurons in the preBötC, consistent with the suggestion that chloride-mediated synaptic inhibition may contribute importantly to rhythm generation by controlling the membrane potential trajectory and resetting rhythmic bursting of the kernel neurons in the adult.

Synaptic association of dopaminergic axon terminals and neurokinin-1 receptor-expressing intrinsic neurons in the striatum of the rat.

We examined if axon terminals of dopaminergic neurons might make synapses upon neurokinin-1 receptor (NK1R)-expressing intrinsic neurons in the rat striatum. In a double-immunocytochemical ultrastructural study, dopaminergic terminals were labeled by the immunoperoxidase method for tyrosine hydroxylase (TH), while NK1R-immunoreactivity (-IR) was revealed by the immunogold-silver labeling method. Some TH-immunoreactive (-ir) axon terminals formed synapses of the symmetric or intermediate type on NK1R-ir neuronal profiles; usually on dendritic profiles and rarely on somatic profiles. It was further confirmed by means of triple-immunofluorescence histochemistry that NK1R-ir neurons in close association with TH-ir axon terminals showed nitric oxide synthase (NOS)- or vesicular acetylcholine transporter-IR. Since NK1R-expressing striatal neurons are segregated into cholinergic and somatostatin/NOS-containing intrinsic neurons (Brain Res. 631 (1993) 297; Neurosci. Lett. 310 (2001) 109), the present results indicate that dopaminergic neurons make synapses upon these intrinsic neurons in the striatum.

MOR-1-immunoreactive neurons in the dorsal horn of the rat spinal cord: evidence for nonsynaptic innervation by substance P-containing primary afferents and for selective activation by noxious thermal stimuli.

A direct action of mu-opioid agonists on neurons in the spinal dorsal horn is thought to contribute to opiate-induced analgesia. In this study we have investigated neurons that express the mu-opioid receptor MOR-1 in rat spinal cord to provide further evidence about their role in nociceptive processing. MOR-1-immunoreactive cells were largely restricted to lamina II, where they comprised approximately 10% of the neuronal population. The cells received few contacts from nonpeptidergic unmyelinated afferents, but many from substance P-containing afferents. However, electron microscopy revealed that most of these contacts were not associated with synapses. None of the MOR-1 cells in lamina II expressed the neurokinin 1 receptor; however, the mu-selective opioid peptide endomorphin-2 was present in the majority (62-82%) of substance P axons that contacted them. Noxious thermal stimulation of the foot induced c-Fos expression in approximately 15% of MOR-1 cells in the medial third of the ipsilateral dorsal horn at mid-lumbar level. However, following pinching of the foot or intraplantar injection of formalin very few MOR-1 cells expressed c-Fos, and for intraplantar formalin injection this result was not altered significantly by pretreatment with systemic naloxone. Although these findings indicate that at least some of the neurons in lamina II with MOR-1 are activated by noxious thermal stimulation, the results do not support the hypothesis that the cells have a role in transmitting nociceptive information following acute mechanical or chemical noxious stimuli.

Alterations of neurokinin receptors and interstitial cells of Cajal during and after jejunal inflammation induced by Nippostrongylus brasiliensis in the rat.

Substance P (SP) and its receptors NK1 and NK2 are widely expressed in the intestinal wall by neurones, interstitial cells of Cajal (ICC) and smooth muscle cells. Changes in SP and/or its NK receptors have been documented during experimental inflammation in animals or inflammatory bowel diseases in humans, but the data concern the acute phase of the inflammatory process. We determined immunohistochemically whether NK receptors and SP were altered in the muscle coat during jejunal inflammation induced by the nematode Nippostrongylus brasiliensis and whether these alterations persisted when inflammation had spontaneously resolved 30 days postinfection. An ultrastructural analysis was also conducted on ICC, nerves and muscle. At day 14, when inflammation peaked, there was a reduction in NK1 receptors in myenteric neurones and in SP-immunoreactive nerve endings. There were also ultrastructural anomalies in synaptic vesicles and NK2 receptor loss in the circular muscle layer. The SP decrease persisted at day 30, whereas neurones and circular muscle cells re-expressed NK1 and NK2 receptors, respectively. The ICC at the deep muscular plexus, located near to the inflammatory site, underwent alterations leading to their complete loss at day 30. These morphological changes are probably associated with impairment in tachykinergic control of jejunal functions leading to the alterations of motility and sensitivity to distension already described in these animals.

Relationship between postsynaptic NK(1) receptor distribution and nerve terminals innervating myenteric neurons in the guinea-pig ileum.

The amounts of neurokinin 1 (NK(1)) receptor immunolabelling on the membranes of myenteric cell bodies at appositions with tachykinin-immunoreactive nerve terminals, other nerve terminals, and glial cells were compared at the ultrastructural level using pre-embedding, double-label immunocytochemistry. NK(1) receptor immunoreactivity was revealed using silver-intensified, 1 nm gold, and tachykinin-immunoreactive nerve terminals were revealed using diaminobenzidine. The density of NK(1) receptor immunolabelling (silver particles per length of cell membrane) on the membrane at appositions with tachykinin-immunoreactive nerve terminals was not significantly different from that at appositions with other (nonimmunoreactive) nerve terminals or with glial cells. Synaptic specializations ("active zones") were present at a small proportion of the appositions between NK(1) receptor-immunoreactive cell bodies and tachykinin-immunoreactive or other nerve terminals. The density of NK(1) receptor immunolabelling at synaptic specializations was lower than that at regions of appositions where no synaptic specializations were present. The presence of NK(1) receptor on the cell surface in areas not directly apposed to tachykinin-containing nerve terminals suggests that tachykinins that diffuse away from their site of release may still exert an action via NK(1) receptors. Although NK(1) receptors do not appear to be targetted to particular sites on the surfaces of myenteric nerve cell bodies and proximal dendrites, they are reduced in density at regions of the membrane-forming synaptic specializations.

mu-Opioid receptors often colocalize with the substance P receptor (NK1) in the trigeminal dorsal horn.

Substance P (SP) is a peptide that is present in unmyelinated primary afferents to the dorsal horn and is released in response to painful or noxious stimuli. Opiates active at the mu-opiate receptor (MOR) produce antinociception, in part, through modulation of responses to SP. MOR ligands may either inhibit the release of SP or reduce the excitatory responses of second-order neurons to SP. We examined potential functional sites for interactions between SP and MOR with dual electron microscopic immmunocytochemical localization of the SP receptor (NK1) and MOR in rat trigeminal dorsal horn. We also examined the relationship between SP-containing profiles and NK1-bearing profiles. We found that 56% of SP-immunoreactive terminals contact NK1 dendrites, whereas 34% of NK1-immunoreactive dendrites receive SP afferents. This result indicates that there is not a significant mismatch between sites of SP release and available NK1 receptors, although receptive neurons may contain receptors at sites distant from the peptide release site. With regard to opioid receptors, we found that many MOR-immunoreactive dendrites also contain NK1 (32%), whereas a smaller proportion of NK1-immunoreactive dendrites contain MOR (17%). Few NK1 dendrites (2%) were contacted by MOR-immunoreactive afferents. These results provide the first direct evidence that MORs are on the same neurons as NK1 receptors, suggesting that MOR ligands directly modulate SP-induced nociceptive responses primarily at postsynaptic sites, rather than through inhibition of SP release from primary afferents. This colocalization of NK1 and MORs has significant implications for the development of pain therapies targeted at these nociceptive neurons.

Cells in laminae III and IV of the rat spinal cord which possess the neurokinin-1 receptor receive monosynaptic input from myelinated primary afferents.

We have previously demonstrated that neurons which have cell bodies in laminae III or IV of the rat spinal cord, dendrites that enter the superficial laminae and which possess the neurokinin-1 receptor receive a major synaptic input from substance P-containing primary afferent axons. In this study we set out to determine whether these cells also receive monosynaptic input from myelinated primary afferents by using transganglionic transport of the B subunit of cholera toxin to identify the central terminals of myelinated afferents from the sciatic nerve. Dual-immunofluorescence and confocal microscopy revealed apparent contacts between labelled primary afferent terminals and all of the neurokinin-1 receptor-immunoreactive cells examined, although these contacts were much less numerous than those which the cells receive from substance P-containing primary afferents. By using a combined confocal and electron microscopic technique we were able to confirm that synapses were present at some of the contacts between primary afferents and neurokinin-1 receptor-immunoreactive neurons. These results suggest that cells of this type will have wide-dynamic range receptive fields, but with a relatively strong input from nociceptors.

Neurokinin-1 receptor-immunoreactive sympathetic preganglionic neurons: target specificity and ultrastructure.

Substance P is involved in cardiovascular control at the spinal cord level, where it acts through neurokinin-1 receptors. In this study we used immunocytochemistry and retrograde tracing to investigate the presence of the neurokinin-1 receptor and its ultrastructural localization in rat sympathetic preganglionic neurons that project to the superior cervical ganglion or the adrenal medulla. Immunofluorescence for the neurokinin-1 receptor outlined the somatic and dendritic surfaces of neurons in autonomic subnuclei of spinal cord segments T1-T12, whereas immunofluorescence for the tracer, cholera toxin B subunit, filled retrogradely labelled cells. There was a significant difference in the proportion of neurokinin-1 receptor-immunoreactive sympathetic preganglionic neurons supplying the superior cervical ganglion and the adrenal medulla. Thirty-eight percent of the neurons that projected to the superior cervical ganglion were immunoreactive for the neurokinin-1 receptor compared to 70% of neurons innervating the adrenal medulla. Of neurons projecting to the superior cervical ganglion, significantly different proportions showed neurokinin-1 receptor immunoreactivity in spinal cord segment T1 (15%) versus segments T2 T6 (45%). At the ultrastructural level, neurokinin-1 receptor staining occurred predominantly on the inner leaflets of the plasma membranes of retrogradely labelled sympathetic preganglionic neurons. Deposits of intracellular label were often observed in dendrites and in the rough endoplasmic reticulum and Golgi apparatus of cell bodies. Neurokinin-1 receptor immunoreactivity was present at many, but not all, synapses as well as at non-synaptic sites, and occurred at synapses with substance P-positive as well as substance P-negative nerve fibres. Only 37% of the substance P synapses occurred on neurokinin-1-immunoreactive neurons in the intermediolateral cell column. These results show that presence of the neurokinin-1 receptor in sympathetic preganglionic neurons is related to their target. The ultrastructural localization of the receptor suggests that sympathetic preganglionic neurons may be affected (i) by substance P released at neurokinin-1 receptor-immunoreactive synapses, (ii) by other tachykinins (e.g., neurokinin A), which co-localize in substance P fibres in the intermediolateral cell column, acting through other neurokinin receptors, and (iii) by substance P that diffuses to neurokinin-1 receptors from distant sites.

Topographic analysis of the S7 binding subsite of the tachykinin neurokinin-1 receptor.

Conformationally and configurationally restricted rotameric probes of phenylalanine have been incorporated in the sequence of substance P (SP)-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2-for analyzing the binding pockets of Phe7 (S7) and Phe8 (S8), in the neurokinin-1 receptor. These analogues of phenylalanine are (2S. 3R)- and (2S, 3S)-indanylglycines, E- and Z-alpha, beta-dehydrophenylalanines, and 2(S)-alpha, beta-cyclopropylphenylalanines [delta E Phe. delta Z Phe. inverted delta E2 (S) Phe, and inverted delta Z 2 (S) Phe]. Binding data obtained with either conformationally (Ing diastereoisomers) or configurationally (delta E Phe, delta Z Phe) probes have unveiled large differences in the binding potencies of these rotameric probes. With the support of nmr data and energy calculations done on these SP-substituted analogues, we attempt to answer questions inherent to such study. First, none of these six probes prevents the formation of bioactive conformation(s) of the backbone of SP. Second, both diastereoisomers (S, S) and (S, R) of indanylglycine preferentially adopt, in the sequence of SP, the gauche (-) and trans side-chain orientations, respectively, as previously postulated from energy calculations with model peptides. However, in solution, the difference in energy between these rotamers included in the sequence of SP, compared to model peptides, is small since the other rotamer can be detected in [(2S, 3R)Ing7]SP. Finally, from this study we can hypothesize that the large variations observed in the affinities of Phe7 substituted analogues of SP must come from steric hindrance in the S7 binding site, which drastically restricts the space filling around the C alpha-C beta bond of residue 7.