NK1 Receptor Immunoreactivity Research Articles

Treatment with the NK1 antagonist emend reduces blood brain barrier dysfunction and edema formation in an experimental model of brain tumors.

The neuropeptide substance P (SP) has been implicated in the disruption of the blood-brain barrier (BBB) and development of cerebral edema in acute brain injury. Cerebral edema accumulates rapidly around brain tumors and has been linked to several tumor-associated deficits. Currently, the standard treatment for peritumoral edema is the corticosteroid dexamethasone, prolonged use of which is associated with a number of deleterious side effects. As SP is reported to increase in many cancer types, this study examined whether SP plays a role in the genesis of brain peritumoral edema. A-375 human melanoma cells were injected into the right striatum of male Balb/c nude mice to induce brain tumor growth, with culture medium injected in animals serving as controls. At 2, 3 or 4 weeks following tumor cell inoculation, non-treated animals were perfusion fixed for immunohistochemical detection of Albumin, SP and NK1 receptor. A further subgroup of animals was treated with a daily injection of the NK1 antagonist Emend (3 mg/kg), dexamethasone (8 mg/kg) or saline vehicle at 3 weeks post-inoculation. Animals were sacrificed a week later to determine BBB permeability using Evan's Blue and brain water content. Non-treated animals demonstrated a significant increase in albumin, SP and NK1 receptor immunoreactivity in the peritumoral area as well as increased perivascular staining in the surrounding brain tissue. Brain water content and BBB permeability was significantly increased in tumor-inoculated animals when compared to controls (p<0.05). Treatment with Emend and dexamethasone reduced BBB permeability and brain water content when compared to vehicle-treated tumor-inoculated mice. The increase in peritumoral staining for both SP and the NK1 receptor, coupled with the reduction in brain water content and BBB permeability seen following treatment with the NK1 antagonist Emend, suggests that SP plays a role in the genesis of peritumoral edema, and thus warrants further investigation as a potential anti-edematous treatment.

Changes in substance P and NK1 receptor immunohistochemistry following human spinal cord injury

An immunohistological assessment of substance P (SP), its NK1 receptor and claudin-5 in human spinal cord injury (SCI) tissue. To determine whether SP and NK1 receptor immunoreactivity are altered following human traumatic SCI. Australia. SP has been implicated in the development of neurogenic inflammation and subsequent edema development following both traumatic brain injury and ischemic stroke. In these conditions, inhibition of its NK1 receptor has been shown to be neuroprotective as reflected in a reduction of edema and improved functional outcome. However, the role of SP following human SCI has not yet been assessed. Archived human SCI tissue was grouped according to survival times: control (no injury; n=5); immediate (death within an hour of the incident; n=6); 2-5 h (n=3); 3 days (n=5); 1 week (n=3); and 3-4 weeks (n=6). Sections were assessed for SP, its NK1 receptor and claudin-5 using immunohistochemical techniques. Following SCI, dorsal horn SP immunoreactivity demonstrated a profound decrease compared with control tissue, indicating the loss of SP with SCI. A marked increase in perivascular NK1 staining was demonstrated after SCI compared with control levels. No obvious change in claudin-5 immunoreactivity was present immediately following injury, however, by 1 week post-SCI, decreased levels were noted. This study demonstrates that severe acute traumatic human SCI results in decreased SP and an immediate increase in NK1 receptor immunoreactivity, suggesting that there is a neurogenic inflammatory component following human SCI.

Substance P as a Mediator of Neurogenic Inflammation after Balloon Compression Induced Spinal Cord Injury

Although clinical spinal cord injury (SCI) occurs within a closed environment, most experimental models of SCI create an open injury. Such an open environment precludes the measurement of intrathecal pressure (ITP), whose increase after SCI has been linked to the development of greater tissue damage and functional deficits. Raised ITP may be potentiated by edema, which we have recently shown to be associated with substance P (SP) induced neurogenic inflammation in both traumatic brain injury and stroke. The present study investigates whether SP plays a similar role as a mediator of neurogenic inflammation after SCI. A closed balloon compression injury was induced at T10 in New Zealand white rabbits. Animals were thereafter assessed for blood spinal cord barrier (BSCB) permeability, edema, ITP, histological outcome, and functional outcome from 5 h to 2 weeks post-SCI. The balloon compression model produced significant increases in BSCB permeability, edema, and ITP along with significant functional deficits that persisted for 2 weeks. Histological assessment demonstrated decreased SP immunoreactivity in the injured spinal cord while NK1 receptor immunoreactivity initially increased before returning to sham levels. In addition, aquaporin 4 immunoreactivity increased early post-SCI, implicating this water channel in the development of edema after SCI. The changes described in the present study support a role for SP as a mediator of neurogenic inflammation after SCI.

NK receptors, Substance P, Ano1 expression and ultrastructural features of the muscle coat in Cav-1−/− mouse ileum

Caveolin (Cav)-1 is an integral membrane protein of caveolae playing a crucial role in various signal transduction pathways. Caveolae represent the sites for calcium entry and storage especially in smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Cav-1−/− mice lack caveolae and show abnormalities in pacing and contractile activity of the small intestine. Presently, we investigated, by transmission electron microscopy (TEM) and immunohistochemistry, whether the absence of Cav-1 in Cav-1−/− mouse small intestine affects ICC, SMC and neuronal morphology, the expression of NK1 and NK2 receptors, and of Ano1 (also called Dog1 or TMEM16A), an essential molecule for slow wave activity in gastrointestinal muscles. ICC were also labelled with c-Kit and tachykinergic neurons with Substance P (SP). In Cav-1−/− mice: (i) ICC were Ano1-negative but maintained c-Kit expression, (ii) NK1 and NK2 receptor immunoreactivity was more intense and, in the SMC, mainly intracytoplasmatic, (iii) SP-immunoreactivity was significantly reduced. Under TEM: (i) ICC, SMC and telocytes lacked typical caveolae but had few and large flask-shaped vesicles we called large-sized caveolae; (ii) SMC and ICC contained an extraordinary high number of mitochondria, (iii) neurons were unchanged. To maintain intestinal motility, loss of caveolae and reduced calcium availability in Cav-1–knockout mice seem to be balanced by a highly increased number of mitochondria in ICC and SMC. Loss of Ano-1 expression, decrease of SP content and consequently overexpression of NK receptors suggest that all these molecules are Cav-1–associated proteins.

NK-receptors, Substance P, Ano1 expression and the ultrastructural features of the muscle coat are modified in the Cav-1-/- mouse ileum

Caveolin (Cav)-1 is an integral membrane protein of caveolae playing a crucial role in various signal transduction pathways. Caveolae represent the sites for calcium entry and storage especially in smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Cav-1-/- mice lack caveolae and show abnormalities in pacing and contractile activity of the small intestine. In particular, the absence of caveolae in ICC compromised their ability to maintain frequencies of contraction. Presently, we investigated, by transmission electron microscopy (TEM) and immunohistochemistry, whether the absence of Cav-1 in Cav-1-/- mouse small intestine affects ICC, SMC and neuronal morphology, the expression of NK1 and NK2 receptors, and of Ano1 (also called Dog1 or TMEM16A) an essential molecule for slow wave activity in gastrointestinal muscles. ICC were also labeled with c-Kit and tachykinergic neurons with Substance P (SP). Immunohistochemical results showed that in Cav-1-/- mice: i) ICC were Ano1-negative but maintained c-Kit expression, ii) NK1 and NK2 receptor immunoreactivity was increased and, in the SMC, mainly intracytoplasmatic, iii) SP-immunoreactivity was significantly reduced. Under TEM: i) ICC and SMC lacked typical caveolae but had few and large flask-shaped vesicles we called large-sized caveolae; ii) SMC and ICC contained an extraordinary high number of mitochondria; iii) neurons were unchanged. In conclusion, the present study shows important changes in SMC, ICC and neurons of the Cav-1-/- mice. Loss of Ano1 expression in the ICC and rearrangement of NK receptors in the SMC are interpretable as consequence of Cav-1/caveolae loss and possibly responsible for the impaired contractile activity. However, the impressive richness in mitochondria and the decrease in SP content might represent the ways to compensate the reduced calcium availability and the increased expression of NKr, allowing the maintenance of a certain cell function.

Neurokinin B Acts via the Neurokinin-3 Receptor in the Retrochiasmatic Area to Stimulate Luteinizing Hormone Secretion in Sheep

Recent data have demonstrated that mutations in the receptor for neurokinin B (NKB), the NK-3 receptor (NK3R), produce hypogonadotropic hypogonadism in humans. These data, together with reports that NKB expression increases after ovariectomy and in postmenopausal women, have led to the hypothesis that this tachykinin is an important stimulator of GnRH secretion. However, the NK3R agonist, senktide, inhibited LH secretion in rats and mice. In this study, we report that senktide stimulates LH secretion in ewes. A dramatic increase in LH concentrations to levels close to those observed during the preovulatory LH surge was observed after injection of 1 nmol senktide into the third ventricle during the follicular, but not in the luteal, phase. Similar increases in LH secretion occurred after insertion of microimplants containing this agonist into the retrochiasmatic area (RCh) in anestrous or follicular phase ewes. A low-dose microinjection (3 pmol) of senktide into the RCh produced a smaller but significant increase in LH concentrations in anestrous ewes. Moreover, NK3R immunoreactivity was clearly evident in the RCh, although it was not found in A15 dopaminergic cell bodies in this region. These data provide evidence that NKB stimulates LH (and presumably GnRH) secretion in ewes and point to the RCh as one important site of action. Based on these data, and the effects of NK3R mutations in humans, we hypothesize that NKB plays an important stimulatory role in the control of GnRH and LH secretion in nonrodent species.

Central injections of tachykinin NK3 receptor agonists inhibit salt appetite and cause translocation of the NK3 receptor to the nuclei of neurons in the paraventricular nucleus of the hypothalamus.

Central, intraventricular injections of tachykinin NK3 receptor (NK3R) agonists inhibit the ingestion of sodium containing solutions under need-free and need-induced conditions. NK3R are internalized within the cell following agonist binding and as such, the translocation of the receptor provides a means by which to identify NK3R expressing neurons that are activated by the intraventricular injection. Male, Charles River Laboratory rats were fitted with lateral ventricle cannulas and maintained on chow and water. Rats were administered intraventricular injections of isotonic saline or 200 ng senktide, a selective NK3R agonist, and then given access to 0.15 M NaCl. A separate group of rats were also treated with saline or senktide, and sacrificed 5, 20 or 120 min later, and the brains processed for NK3R immunoreactivity (IR). Intraventricular injections of senktide significantly suppressed the ingestion of NaCl during the first 20 min of the test. Confocal examination of the paraventricular nucleus of the hypothalamus (PVN) showed that in saline treated rats, NK3R IR was largely membrane bound but following senktide, NK3R IR was detected in punctate clusters within the cytoplasm within 10 min of the intraventricular injection. Interestingly, at 20 min, NK3R IR was detected within the cell nuclei. The presence of NK3R in the cell nuclei was confirmed by immunoelectron microscopy and Western blot. Thus, intraventricular injections of senktide activate NK3R expressed by PVN neurons and causes the internalization and translocation of the NK3R to the cell nuclei, where the protein may directly affect gene transcription.

Ablation of NK 1 receptor bearing neurons in the nucleus of the solitary tract blunts cardiovascular reflexes in awake rats

The nucleus of the solitary tract (NTS) receives primary afferents involved in cardiovascular regulation. We investigated the role of NK 1-receptor bearing neurons in the NTS on cardiovascular reflexes in awake rats fitted with chronic venous and arterial cannulae. These neurons were lesioned selectively with saporin conjugated with substance P (SP-SAP, 2 μM, bilateral injections of 20 nL in the subpostremal NTS, or 200 nL in both the subpostremal and the commissural NTS). Before, and 7 and 14 days after injection of SP-SAP, we measured changes in blood pressure and heart rate induced by i.v. injection of phenylephrine and nitroprusside (baroreceptor reflex), cyanide (arterial chemoreceptor reflex), and phenylbiguanide (Bezold-Jarisch reflex). The smaller injections with SP-SAP completely abolished NK1 receptor staining in the subpostremal NTS. The larger injections abolished NK1 receptor immunoreactivity in an area that extended from the commissural NTS to the rostral end of the subpostremal NTS. The lesions seemed to affect only a limited number of neurons, since neutral red stained sections did not show any obvious reduction in cell number. The smaller lesions reduced the gain of baroreflex bradycardia and the hypotension induced by phenylbiguanide. The larger lesions completely abolished the response to phenylbiguanide, blocked the baroreflex bradycardia induced by phenylephrine, severely blunted the baroreflex tachycardia, and blocked the bradycardia and reduced the hypertension induced by cyanide. Thus, these responses depend critically on NK 1-receptor bearing neurons in the NTS.

Morphologic evidence that neurokinin B modulates gonadotropin‐releasing hormone secretion via neurokinin 3 receptors in the rat median eminence

Recent studies suggest that arcuate neurokinin B (NKB) neurons play a role in the regulation of gonadotropin secretion, but there is little information on the relationship between these neurons and the hypothalamic reproductive axis. In the present study, dual-label fluorescent immunohistochemistry was used to visualize the relationship between gonadotropin-releasing hormone (GnRH) neurons and either proNKB or NK3 receptor (NK3R) immunoreactivity. Immunocytochemistry was also combined with i.p. injections of the fluorescent retrograde tracer aminostilbamidine to determine whether arcuate neuroendocrine neurons expressed either proNKB or NK3R. A dense interweaving and close apposition of GnRH and proNKB-immunoreactive (ir) fibers was observed within the rat median eminence, where GnRH axons expressed NK3R immunoreactivity. These data provide morphological evidence that NKB neurons could influence GnRH secretion via interaction with NK3R in the rat median eminence. Colocalization of GnRH and NK3R was also identified in fiber tracts converging within the organum vasculosum of the lamina terminalis. In contrast, only a small number (16%) of GnRH-ir somata exhibited NK3R staining. ProNKB and NK3R-ir somata were identified within the arcuate nucleus, but none of these neurons were labeled by aminostilbamidine. Thus, we found no evidence that arcuate NKB neurons project to the primary capillary plexus of the portal system. Arcuate neuroendocrine neurons, however, were surrounded and closely apposed by proNKB-ir puncta and fibers. These data suggest that NKB neurons could indirectly influence anterior pituitary function by inputs to arcuate neuroendocrine neurons, but through a receptor other than NK3R. Our results provide an anatomic framework for putative interactions between NKB neurons and the hypothalamic reproductive axis.

Elevated substance P (NK-1) receptor immunoreactivity in the cerebellum of seizure prone gerbil

In the present study, we performed a comparative analysis of the distribution of substance P (SP) receptor (NK-1) immunoreactivity in order to determine the characteristics of the SP system in the cerebelli of rat and gerbils. In the rat cerebellar cortex, only a few Purkinje cells exhibited weak NK-1 receptor immunoreactivity. Similar to the case of rat, NK-1 receptor immunoreactivity in the cerebellar cortex of seizure resistant (SR) gerbils was rarely detected. In contrast, in the cerebellar cortex of seizure sensitive (SS) gerbils, dendrites and cell bodies of Purkinje cell showed strong NK-1 receptor immunoreactivity. Similar to the cerebellar cortex, little NK-1 receptor immunoreactivity in deep cerebellar nuclei was observed in the rat. In SR gerbils, however, deep cerebellar nuclei showed weak NK-1 receptor immunoreactivity. NK-1 receptor immunoreactivity in the deep cerebellar nuclei of SS gerbils was markedly increased, as compared with SR gerbils. Based on the present data, we suggest that the SP system of cerebellar circuit in gerbil are different from rat, and over-expression of NK-1 receptor immunoreactivity in Purkinje cells of SS gerbils may be relevant to Purkinje cell loss induced by seizure activity.

Expression of the neurokinin 1 receptor in the mouse retina

Previous studies have revealed that the expression pattern of the neurokinin 1 receptor (the preferred receptor for substance P, SP) varies in different mammalian retinas. We investigated NK1 receptor expression in the mouse retina to provide background information for future studies in transgenic mice on SP functional roles in the retina. Mouse retinal sections were treated for single and double-label immunofluorescence. NK1 receptor immunoreactivity was in bipolar cells and in numerous amacrine cells. Double-label studies showed that NK1 receptor-expressing bipolar cells constituted a population of ON-type cone bipolar cells, since they were distinct from rod bipolar cells and contained glycine. They were nonrandomly distributed with highest density in central retina. These cells were similar and may correspond to the population of NK1 receptor-expressing bipolar cells of the rabbit retina. Different subsets of NK1 receptor-expressing amacrine cells were identified on the basis of the expression of selected neurotransmitter substances: i) about 23% of NK1 receptor-expressing amacrine cells also contained glycine; ii) the remaining 77% were likely to be GABAergic, although some inconsistency was observed in the GABA immunostaining obtained with two different GABA antibodies; iii) all dopaminergic amacrine cells also expressed NK1 receptors; iv) about one third of SP-containing amacrine cells also expressed NK1 receptors. These findings confirm and expand previous observations in rat and rabbit retinas. In particular, common to all three species is the expression of NK1 receptors in dopaminergic amacrine cells, indicating that SP neurotransmission may be a universal feature of the circuitry of the dopaminergic amacrine cell. Peculiar to the mouse retina is the presence of putative NK1 autoreceptors expressed by SP-containing amacrine cells.

Distinct regional distributions of NK1 and NK3 neurokinin receptor immunoreactivity in rat brainstem gustatory centers

Tachykinins and their receptors are present in gustatory centers, but little is known about tachykinin function in gustation. In this study, immunohistochemical localization of substance P and two centrally prevalent neurokinin receptors, NK1 and NK3, was carried out in the rostral nucleus of the solitary tract and the caudal parabrachial nucleus to evaluate regional receptor/ligand correspondences. All three proteins showed regional variations in labeling density that correlated with distinct sites in gustatory centers. In the rostral nucleus of the solitary tract, the relative densities of substance P and NK1 receptors varied in parallel across subnuclei, with both being moderate to dense in the dorsocentral, chemoresponsive zone. NK3 receptors had a distinct distribution in the caudal half of this zone, suggesting a unique role in processing taste input from the posterior tongue. In the caudal parabrachial nucleus, substance P and NK1 receptor immunoreactivities were dense in the pontine taste area, while NK3 receptor labeling was sparse. The external medial subnucleus had substantial NK3 receptor and substance P labeling, but little NK1 receptor immunoreactivity. These findings suggest that distinct tachykinin ligand/neurokinin receptor combinations may be important in local processing of information within brainstem gustatory centers.

Spinal NK1 receptor is upregulated after chronic bladder irritation

It has been suggested that there is a significant upregulation of the NK1 receptor (NK1R) on neurons in the dorsal spinal cord after long-term somatic inflammation. This upregulation appears to play a significant role in central sensitization in chronic pain states. However, it is not clear whether such a change is also observed after chronic visceral (bladder) inflammation. Changes in NK1R immunoreactivity after chronic bladder irritation were investigated in order to evaluate the existence of hypersensitive states in the spinal cord after chronic bladder irritation. Experiments were performed on a total of 12 adult female Sprague–Dawley rats. In six animals, cyclophosphamide (CPA) was administered intraperitoneally for 2 weeks. Another six animals were given intraperitoneal saline injections and served as the control group. After these treatments, immunohistochemical staining for NK1Rs and substance P in rat lumbosacral spinal cord was performed. In CPA-treated animals, NK1R-positive areas and staining intensity within the dorsal spinal cord were significantly increased in the L5 to S2 spinal cord areas, especially in the L6 and S1 segments. In the L6 spinal segment, CPA-treatment enhanced NK1R immunostaining in the medial and the lateral dorsal horn, as well as in the lateral laminae including the sacral parasympathetic nucleus to a lesser extent. In CPA-treated animals, substance P staining intensity increased in the same regions in which NK1R immunoreactivity was increased. This finding probably implies the upregulation of spinal NK1R and the occurrence of central sensitization within the spinal cord after chronic visceral inflammation.

Substance P stimulates inhibitory synaptic transmission in the guinea pig basolateral amygdala in vitro

To determine the physiological role of tachykinin NK1 receptors in the basolateral nucleus of the amygdala (BLN) we have studied the electrophysiological effects of substance P (SP) in the absence and presence of selective tachykinin receptor antagonists in guinea pig brain slices. Recordings were made from two populations of neurones; spiny pyramidal and stellate neurones, both thought to be projection neurones. Activation of NK1 receptors with SP increased the frequency of spontaneous inhibitory postsynaptic potentials in the majority of cells. This effect was blocked by bicuculline or tetrodotoxin, but not ionotropic glutamate receptor antagonists. The enhanced synaptic activity induced by SP was antagonised by the NK1 receptor antagonist L-760,735 but not by the less active enantiomer L-781,773 or the NK3 receptor antagonist L-769,927. Thus in the basolateral nucleus of the guinea pig amygdala, NK1 receptor activation preferentially stimulates inhibitory synaptic activity. Consistent with this observation, immunohistochemistry revealed NK1 receptor immunoreactivity to be largely restricted to a subset of GABA interneurones. These studies support a physiological role for SP in the regulation of pathways involved in the control of emotional behaviour.

Tachykinin NK1 and NK3 receptors in the prefrontal cortex of the human brain.

1. The tachykinins are neuropeptides found in both the central and peripheral nervous systems that play a role in inflammation and pain mechanisms and some autonomic reflexes and behaviours. 2. Although the distribution of the tachykinin receptors has been described in the brains of various animal species, little is known about the distribution of the NK1 and NK3 receptors in the human brain. 3. The present paper examines the distribution of the NK1 and NK3 receptors in the prefrontal cortex of formalin-fixed postmortem human brain tissue by immunohistochemical techniques. 4. The majority of NK1 receptor immunoreactivity appeared as a thin band of punctate staining at the pial surface, with dark brown dots of NK1 receptor immunoreactivity predominantly scattered across the mid to upper cortical layers. 5. The NK3 receptor immunoreactivity was found in the glia limitans at the pial surface, where astrocytes and beaded fibres were intensely stained. Dots of NK3 receptor immunoreactivity were scattered across all cortical layers. In the white matter, astrocytes and beaded fibres displayed NK3 receptor immunoreactivity, particularly in areas surrounding blood vessels.

Localization of tachykinin receptors and Fos-like immunoreactivity induced by substance P in guinea-pig brain.

1. In the present study, a comparison was made between the distribution of tachykinin NK1 and NK3 receptor immunoreactivity and the distribution of Fos-like immunoreactivity induced by the tachykinin agonist substance P (SP) in the guinea-pig brain. 2. In agreement with results from previous studies in rat brain, NK1 receptor-immunoreactive neurons were found to be widely distributed throughout the brain in the striatum and in diencephalic and mesencephalic structures, while NK3 receptor-immunoreactive neurons were mainly in telencephalic structures. Considerable overlap was observed between NK1 and NK3 receptor distributions. 3. Substance P induced Fos-like immunoreactivity (Fos-LI) in extensive areas of the guinea-pig brain. The induction of Fos-LI was markedly inhibited in many areas by pretreatment with the NK1 receptor antagonist SR 140333. The NK3 receptor antagonist SR 142801 reduced Fos-LI staining in fewer areas, although a reduction was observed in the cortex, striatum and hypothalamus. 4. In general, tachykinin receptors were located at sites corresponding to areas of functional activation by SP, as shown by Fos-LI. These results extend previous studies by adding a functional dimension to tachykinin receptor localization studies.

The substance P antagonist L-760,735 inhibits stress-induced NK1 receptor internalisation in the basolateral amygdala

The distribution of NK1 receptor immunoreactivity in the amygdaloid complex, induction of NK1 receptor endocytosis in the amygdala following immobilisation stress, and the ability of pretreatment with the substance P antagonist L-760,735 or imipramine to block this response were examined in gerbils, a species with human-like NK1 receptor pharmacology. Highest levels of immunolabelling were observed in the anterior, amygdalo-hippocampal and medial nuclei. Less dense labelling was observed in the basolateral nucleus, where it was possible to clearly visualise the distal dendrites of NK1 immunoreactive neurones and quantify the effect of immobilisation stress on NK1 receptor endocytosis morphology, a marker of local substance P release. Immobilisation for 1 h caused an approximately 60% increase in the number of dendritic processes undergoing NK1 receptor endocytosis in the basolateral amygdala that was inhibited by acute pretreatment of animals with L-760,735 (3 mg/kg), but not by imipramine (10 mg/kg). These findings are consistent with other evidence that the amygdala represents a possible site of action for the antidepressant and anxiolytic efficacy of substance P antagonists.

NK-1 Receptor Immunoreactivity in Distinct Morphological Types of Lamina I Neurons of the Primate Spinal Cord

In cat and monkey, lamina I cells can be classified into three basic morphological types (fusiform, pyramidal, and multipolar), and recent intracellular labeling evidence in the cat indicates that fusiform and multipolar lamina I cells are two different types of nociceptive cells, whereas pyramidal cells are innocuous thermoreceptive-specific. Because earlier observations indicated that only nociceptive dorsal horn neurons respond to substance P (SP), we examined which morphological types of lamina I neurons express receptors for SP (NK-1r). We categorized NK-1r-immunoreactive (IR) lamina I neurons in serial horizontal sections from the cervical and lumbar enlargements of four monkeys. Consistent results were obtained by two independent teams of observers. Nearly all NK-1r-IR cells were fusiform (42%) or multipolar (43%), but only 6% were pyramidal (with 9% unclassified). We obtained similar findings in three monkeys in which we used double-labeling immunocytochemistry to identify NK-1r-IR and spinothalamic lamina I neurons retrogradely labeled with cholera toxin subunit b from the thalamus; most NK-1r-IR lamina I spinothalamic neurons were fusiform (48%) or multipolar (33%), and only 10% were pyramidal. In contrast, most (approximately 75%) pyramidal and some (approximately 25%) fusiform and multipolar lamina I spinothalamic neurons did not display NK-1r immunoreactivity. These data indicate that most fusiform and multipolar lamina I neurons in the monkey can express NK-1r, consistent with the idea that both types are nociceptive, whereas only a small proportion of lamina I pyramidal cells express this receptor, consistent with the previous finding that they are non-nociceptive. However, these findings also indicate that not all nociceptive lamina I neurons express receptors for SP.

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.

Characterisation of substance P-induced endocytosis of NK1 receptors on enteric neurons.

Immunoreactivity for NK1 receptors is confined to specific nerve cell bodies in the guinea-pigileum, including inhibitory motor neurons and secretomotor neurons. In the present work, endocytosis of NK1 receptors in these enteric neurons was studied following addition of substance P (SP) to isolated ileum. NK1 receptors were localised with antibodies against the C-terminus of this receptor. Some preparations were incubated with SP tagged with the fluorescent label, Cy3.18, so that the fate of SP bound to receptors could be followed. Preparations were analysed by confocal microscopy. In tissue that was incubated at 4 degrees C in the absence of SP, most NK1 receptor immunoreactivity (IR) was confined to surface membranes of nerve cells. At 37 degrees C in the presence of 10(-7) M SP (plus 3 x 10(-7)M tetrodotoxin to prevent indirect activation via other neurons) the neuronal NK1 receptor was rapidly internalised. After 5 min, NK1 receptor IR was partially internalised, at 20 min NK1 receptor IR was throughout the cytoplasm and in perinuclear aggregates and at 30 min it was again at the cell surface. SP-induced NK1 receptor endocytosis was inhibited by the specific NK1 receptor antagonist, SR140333. Cy3-SP was colocalised with NK1 receptor IR and was internalised with the NK1 receptor. These results show that enteric neurons exhibit authentic NK1 receptors that are rapidly internalised when exposed to their preferred ligand.