Localization of neurokinin 1 receptor (NK1R) immunoreactivity in rat esophagus

The aim of the present immunohistochemical study was to investigate the localization of neurokinin 1 receptor (NK1R) in rat esophagus and examine the relationship between NK1Rs and intrinsic cholinergic, nitrergic, or substance P (SP) neurons. NK1R immunoreactivity (IR) was observed on the nerve cell bodies in the myenteric ganglia throughout the esophagus, but not on striated muscles and smooth muscle cells of the muscularis mucosae. The frequency of occurrence of NK1R neurons was highest in the cervical esophagus and lowest in the lower thoracic esophagus. Considerable immunoreactivity was seen on the nerve cell surfaces and was also present in the cytoplasm of cell somas and in the initial part of the axons, but not in any other nerve fibers or terminals. Dogiel type I-like morphology was observed in some of the NK1R neurons; however, the majority exhibited polymorphic morphology. Double immunolabeling indicated that a majority (77%) of the NK1R neurons were immunoreactive for choline acetyltransferase (ChAT), while a minority (23%) were immunoreactive for nitric oxide synthase (NOS)-IR. Most of the NK1R neurons (92%) were innervated by the SP nerve fibers. Triple immunolabeling indicated that 70% of the NK1R neurons were associated with intrinsic SP nerve fibers (without CGRP-IR), 59% were associated with extrinsic SP nerve fibers (with CGRP-IR), and 35% were associated with both intrinsic and extrinsic SP nerve fibers. These results suggest that SP/tachykinin released from the SP nerve fibers of intrinsic and/or extrinsic origin activates the predominantly intrinsic cholinergic neurons via NK1Rs to influence neuronal transmission or motility in rat esophagus.     

Postnatal development of NK1, NK2, and NK3 neurokinin receptors expression in the rat retina.

The biological effects of tachykinins are mediated by three distinct receptors, the neurokinin 1 receptor (NK1-R), NK2-R, and NK3-R. There is no information available concerning the development of these receptors in the retina. In the present study, we investigated the localization of tachykinin receptors, using antisera directed against NK1-R, NK2-R, and NK3-R in the adult and developing rat retinas. Numerous NK1-R immunoreactive (NK1-R IR) cells were already observed in the proximal part of the neuroblastic layer in the retina at postnatal day 5 (P5). The distribution and intensity of NK1-R IR cells and processes in the inner nuclear layer (INL) and inner plexiform layer (IPL) at P10 were similar to those of adult retina. Most NK1-R IR cells located in the proximal part of INL, which were morphologically amacrine cells. In the contrast to the early expression of NK1-R IR cells, no NK3-R IR structures existed in the neuronal elements of the retina until P10. NK3-R IR processes were first detected in the outer plexiform layer (OPL) at P10. At P15, NK3-R IR somata were slightly stained in the distal and middle parts of the INL, and NK3-R IR processes were present in the OPL and the upper part of the IPL. During P15-P30, the number of NK3-R IR somata located in the INL remarkably increased. These NK3-R IR cells were morphologically bipolar and amacrine cells. This study provides differential cellular distribution of NK1-R IR cells and NK3-R IR cells in the INL of the rat retina. Our findings suggest that NK1-R and NK3-R are involved in different visual circuits and retinal maturation, and NK3-R may play previously unknown important roles in the visual processes of the rat.     

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.     

Tachykinin receptor (NK(1), NK(2), NK(3)) binding sites in the rat caudal brainstem following neonatal capsaicin administration

Binding of [125I]-Bolton-Hunter substance P ([125I]-BHSP), [125I]-neurokinin A and [3H]-senktide to tachykinin NK(1), NK(2) and NK(3) receptors, respectively, was examined in caudal brainstem sections of 10-week-old rats pretreated as neonates (P2) with capsaicin (50 mg/kg, s.c.) or vehicle. [125I]-BHSP binding was localised to the nucleus of the solitary tract (NTS), hypoglossal nucleus and inferior olivary complex, whereas [125I]-neurokinin A and [3H]-senktide binding were confined to the NTS. The distribution and density of binding sites were similar in vehicle- and capsaicin-pretreated rats, suggesting that sensory neuron ablation by neonatal capsaicin does not affect tachykinin receptor numbers in the rat caudal brainstem.     

Distribution of immunoreactivity for the NK1 receptor on different subpopulations of sympathetic preganglionic neurons in the rat

The distribuktion of immunoreactivity to the recptor for substance P, the neurokinin 1 (NK1) receptor, was ezamined in preganglionic sympathetic neurons of the rat by usingimmunohistochemistry and retroggrade neuronal tracing abour one-third of all sympathetic preganglionic neurons were NK1 receptor immunoreactiv, and most of the NK1 receptor immunoractive neurons were also nitric oxide synthase immunoreactive the proportions ofsympathetic preganglionic neurons projecting to the superior and inferior mesenteric ganglia, addrenal glnd, and lumbar sympathetic hain which werre nK1 rdeptor-immunoreactive were determined. Most (89%) of the preganglionic neurons projecting to the addrenal glands were NK1 rceptor immunoreactive few (17%) of the preganglionic neurons projecting to the L5 sympathetic hain ganglion were immunoreactive for the receptor, while preganglionic neurons projecting tothe prevertebral ganglia were NK1 receptor immunoractive at intermediate frequencies (61–64%). Thus, substance P acting on NK1 receptors in likely to be importnt inthe pregnglionic pathways to the addrenal medulla and viscera via the preveertebral ganglia, but the NK1 receptor withthe enzyme nitric oxide synthase was also examined. The co-localisation of NK1 receptor-immunoreactive neurons were also nitric oxide synthase immunoreactiv. Thus NK1 receptors occur onpreganglionic neurons over many spinal segments and in a range of prganglionic pathways, as well as in a range of combinations with nitric oxide shnthase. The heterogeneity ofjpreganglionic neurons showing NK1 receeptor immunoreactivity may reglect the involvement of NK1-mediated trnsmkssion in a variety of functional pathways, most notably thepreganglionic projections to the adrenal medulla and to the viscera. © 1996 Wiley-Liss, Inc.     

Basic FGF-like immunoreactivity in the developing and adult rat brainstem

Although a variety of in vitro and in vivo actions of basic fibroblast growth factor (bFGF) on neuronal cells have been documented, the physiological role of this protein in the nervous system is still contested. Since the distribution of a molecule in the nervous system may provide cues for an understanding of its possible roles, we have begun to study its cellular localization in the central and peripheral nervous system using immunocytochemistry with an anti-bFGF-specific antibody. Here we provide an account on the distribution of bFGF-like immunoreactivity (bFGF-IR) in the brainstem of the developing and adult rat. Basic FGF-IR was found to be widely distributed in motor and sensory nuclei. In all nuclei examined, only subpopulations of neurons were stained. Different staining patterns were found. For example, in the red nucleus weakly or unstained perikarya were surrounded by numerous immunoreactive fibers, often in close contact with the neuronal surface. In the reticular formation and facial nerve, many neuronal cell bodies showed a strong IR that extended into the processes. Glial cells were consistently unstained. During early postnatal development changes of the distribution of bFGF IR were found. From this wide distribution pattern of bFGF-IR, we conclude that bFGF may have more general and, possibly, diverse functions rather than a restricted role for a particular subset of neurons. Variations in the staining pattern of nerve cell bodies in a single nucleus may suggest a function related to neuronal activity.     

The murine neurokinin NK1 receptor gene contributes to the adult hypoxic facilitation of ventilation

Substance P and neurokinin-1 receptors (NK1) modulate the respiratory activity and are expressed early during development. We tested the hypothesis that NK1 receptors are involved in prenatal development of the respiratory network by comparing the resting respiratory activity and the respiratory response to hypoxia of control mice and mutant mice lacking the NK1 receptor (NK1-/-). In vitro and in vivo experiments were conducted on neonatal, young and adult mice from wild-type and NK1-/- strains. In the wild strain, immunohistological, pharmacological and electrophysiological studies showed that NK1 receptors were expressed within medullary respiratory areas prior to birth and that their activation at birth modulated central respiratory activity and the membrane properties of phrenic motoneurons. Both the membrane properties of phrenic motoneurons and the respiratory activity generated in vitro by brainstem-spinal cord preparation from NK1-/- neonate mice were similar to that from the wild strain. In addition, in vivo ventilation recordings by plethysmography did not reveal interstrain differences in resting breathing parameters. The facilitation of ventilation by short-lasting hypoxia was similar in wild and NK1-/- neonates but was significantly weaker in adult NK1-/- mice. Results demonstrate that NK1 receptors do appear to be necessary for a normal respiratory response to short-lasting hypoxia in the adult. However, NK1 receptors are not obligatory for the prenatal development of the respiratory network, for the production of the rhythm, or for the regulation of breathing by short-lasting hypoxia in neonates.     

Distinct distributions of two bombesin receptor subtypes in the rat central nervous system.

We have previously demonstrated the presence of two distinct bombesin receptor subtypes in the rat CNS and distinguished them as bombesin/gastrin-releasing peptide (BBS/GRP) and neuromedin B (NMB)-preferring binding sites. In the present study, we conducted a complete evaluation of the distribution of these binding sites throughout the rat brain using in vitro receptor autoradiography. The BBS/GRP-preferring binding sites were characterized as those that bound 125I-(Tyr4)BBS but not 125I-(D-Tyr0)NMB. At these sites 125I-(Tyr4)BBS binding was inhibited in the presence of 100 nM BBS but not by the same concentration of NMB. In contrast, NMB-preferring sites bound both radioligands and binding at these sites was inhibited in the presence of 100 nM NMB. Our results indicate that the distributions of BBS/GRP and NMB-preferring binding sites are widespread and distinct at all levels of the rat brain suggesting these peptides mediate separate functions in the rat central nervous system.     

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. J. Comp. Neurol. 381:439-448, 1997. © 1997 Wiley-Liss, Inc.     

Increased tachykinin NK(1) receptor immunoreactivity in the prefrontal cortex in schizophrenia.

BACKGROUND: Changes in levels of substance P and substance P-binding sites have been implicated in schizophrenia. However, no studies have used receptor-specific antibodies to directly investigate the substance P (neurokinin 1) receptor in schizophrenia. METHODS: We used an antibody directed against the human neurokinin-1 receptor to compare the distribution of neurokinin-1 receptors in the prefrontal cortices from six subjects with schizophrenia and six control subjects, matched for age, gender, and postmortem interval. RESULTS: In control tissue, dots of neurokinin-1 receptor immunoreactivity were observed in layer I to upper/mid layer III only. In contrast, dots of neurokinin-1 receptor immunoreactivity were observed in all layers of the prefrontal cortex in subjects with schizophrenia, and the density of dots was significantly greater than in control subjects. CONCLUSIONS: This is the first report of increased neurokinin-1 receptor immunoreactivity in the prefrontal cortex in subjects with schizophrenia. These changes may have implications for understanding the pathophysiology of the prefrontal cortex in schizophrenia and for the treatment of this disorder.     

Glutamic acid decarboxylase-like immunoreactivity in brainstem auditory nuclei of the rat

The distribution of GABA-producing neurons in the brainstem auditory nuclei of the rat was investigated immunohistochemically by using an antibody to glutamic acid decarboxylase (GAD). In the cochlear nuclei, GAD immunoreactive neurons are present only in the superficial granular and molecular layers, whereas terminals are found in all subdivisions of the nuclei and are particularly dense surrounding large spherical cells and one type of stellate cell. In the superior olivary complex, GAD immunoreactive neurons are located in the lateral olivary nucleus and throughout the periolivary region. Immunoreactive terminals are distributed along dendrites of principal cells of the medial and lateral olivary nuclei and are clustered around somata of globular neurons of the nucleus of the trapezoid body. An extremely dense band of immunoreactive somata and terminals is present along the ventral edge of the olivary complex. The ventral, intermediate, and dorsal nuclei of the lateral lemniscus contain small fusiform GAD-immunoreactive neurons and a moderately dense plexus of immunoreactive terminals. The inferior colliculus contains a large population of GAD-immunoreactive perikarya and an extremely dense accumulation of immunoreactive terminals in the central, dorsomedial, and external nuclei. These observations indicate that GABA systems are involved in function at all levels of the brainstem auditory pathway.     

Localisation of NK1 receptor immunoreactivity to neurons and interstitial cells of the guinea-pig gastrointestinal tract

Tachykinins, including substance P, neurokinin A, and neuropeptides K and γ, are expressed widely in the peripheral nervous system where they affect smooth muscle contraction, exocrine gland secretion, vascular permeability, and neurotransmission. Substance P, the preferred ligand for the NK1 receptor, is found in high concentrations in the enteric nervous system. In the present study, the localisation and distribution of the NK1 receptor was studied throughout the gastrointestinal tract of the guinea-pig by using a polyclonal antiserum raised against the C-terminal 15 amino acids of the NK1 receptor. Co-localisation with other neuronal markers was examined in the ileum. Nerve cell bodies reactive for the NK1 receptor were found in the myenteric plexus of all regions and the submucous plexus of the small and large intestines. In the small intestine, the interstitial cells of Cajal were also immunoreactive. Immunoreactivity was largely confined to cell surfaces. Almost all immunoreactive myenteric nerve cells had Dogiel type I morphology, and most of these were immunoreactive for nitric oxide synthase, a transmitter of inhibitory neurons to the muscle and of descending interneurons. Neuropeptide Y-containing secretomotor neurons in the submucous and myenteric plexuses also exhibited NK1 receptor immunoreactivity. NK1 receptors were present on a minority of tachykinin immunoreactive neurons of submucous ganglia. The results suggest that receptors on the longitudinal muscle might not be conventional NK1 receptors, that excitation of the circular muscle of the ileum is indirect, perhaps via the interstitial cells of Cajal, and that enteric inhibitory neurons may be excited via NK1 receptors. © 1996 Wiley-Liss, Inc.     

Distribution of Fos-like immunoreactivity within the rat brain following intraventricular injection of the selective NK(3) receptor agonist senktide

Neurokinin B (NKB) is one member of an evolutionarily conserved family of neuropeptides, the tachykinins. Preferential binding of NKB to endogenous NK(3) receptors affects a variety of biological and physiological processes, including endocrine secretions, sensory transmission, and fluid and electrolyte homeostasis. In light of its widespread biological actions, immunohistochemical detection of the c-Fos protein product was used to study the distribution of neuronal activation in the rat brain caused by intraventricular (icv) injections of the selective NK(3) receptor agonist (succinyl-[Asp(6), N-Me-Phe(8)] substance P [6-11]), senktide. Quantitative analysis revealed that treatment with isotonic saline or 200 ng senktide resulted in the differential expression of Fos-like immunoreactivity (FLI) throughout the brain. Senktide induced the highest number of FLI neurons in the lateral septum, bed nucleus of the stria terminalis, amygdala, paraventricular nucleus of the hypothalamus, median preoptic nucleus, organum vasculosum of the lamina terminalis, supraoptic nucleus, periaqueductal gray, and medial nucleus of the solitary tract compared to isotonic saline controls. Additional regions that contained elevated FLI following icv injection of senktide, relative to saline injection, included the cerebral cortex, lateral hypothalamic nucleus, suprachiasmatic nucleus, ventral tegmental area, substantia nigra, inferior colliculus, locus coeruleus, zona incerta, and arcuate nucleus. Our data indicate that activation of NK(3) receptors induces the expression of FLI within circumscribed regions of the rat brain. This pattern of neuronal activation overlaps with nuclei known to regulate homeostatic processes, such as endocrine secretion, cardiovascular function, salt intake, and nociception.     

Reduction in choline acetyltransferase immunoreactivity but not muscarinic-m2 receptor immunoreactivity in the brainstem of SIDS infants

The cholinergic neurotransmitter system is vital for several brainstem functions including cardiorespiratory control and central chemosensitivity. This study has examined aspects of the cholinergic neurotransmitter system in the brainstem of sudden infant death syndrome (SIDS) and control infants. The cellular localisation and the optical density of the immunoreactivity of the cholinergic enzyme choline acetyltransferase (CHAT-IR) and the muscarinic acetylcholine receptor m2 (m2-IR) in the medulla was described in 14 SIDS and 9 control cases. There was a reduction in the number of CHAT-IR neurons in the hypoglossal nucleus (control: 71.2+/-8.3% vs SIDS: 46.1+/-5.3%) and the dorsal motor nucleus of the vagus (DMV) (control: 77.2+/-5.0% vs SIDS: 52.5+/-7.4%) and reduced optical density of CHAT-IR in the hypoglossal nucleus (control: 0.20+/-0.01 vs SIDS; 0.14+/-0.02) in SIDS infants. In contrast there were no changes in the optical density of m2-IR in the hypoglossal nucleus, the DMV, or the arcuate nucleus. Hypoplasia of the arcuate nucleus was observed in one SIDS infant. These results suggest that there is a specific defect in some cholinergic motor neurons in the medulla of SIDS infants. This could lead to abnormal control of cardiovascular and respiratory function and airway patency and may be one of the contributing factors in the etiology of SIDS.     

Generation of a transgenic model to address regulation and function of the human neurokinin 1 receptor (NK1R)

We have generated mouse transgenic lines using yeast artificial chromosome (YAC) technology which demonstrate expression from the human NK1 receptor (NK1R) locus. We introduced a 380 kb fragment encompassing the human NK1R gene and flanking regions which we hoped would recapitulate the expected endogenous expression of the human gene. To visualise this expression the NK1 locus co-expresses the green fluorescence protein gene (GFP) under the control of an internal ribosome entry site (IRES) sequence. We have generated five mouse lines that express the human NK1 receptor gene with and without the marker gene. All the lines incorporating the marker gene appear to exhibit the same expression pattern in analysis of selected anatomical regions throughout the mouse. The lack of a human specific NK1R antibody determined that we could not distinguish between expression of the transgene and endogenous NK1R. Our analysis has shown transgene expression in brain areas known to express NK1R in human such as the hippocampus and caudate putamen. The majority of these cells were also positive for GFP fluorescence. These transgenic lines may prove a good pre-clinical model as drugs can be addressed against both the human receptor and modulators of its expression in vivo.     

Distribution of galanin receptor 1 immunoreactivity in the rat stomach and small intestine

Galanin affects gastrointestinal functions by activating different G protein-coupled receptors. Here, we identified the sites of expression of the galanin receptor 1 (GAL-R1) subtype in the rat stomach and small intestine by using immunohistochemistry with an antibody raised to the third intracellular loop of rat GAL-R1 (GAL-R1(Y225-238)) and confocal microscopy. Antibody specificity was confirmed by (1) the detection of a band at approximately 70 kDa in Western blot of membranes from GAL-R1 transfected cells, (2) the cell surface staining of GAL-R1 transfected cells, which was not detected in control cells, and (3) the abolition of Western signal and tissue immunostaining by preadsorbing the antibody with the peptide used for immunization. GAL-R1 immunoreactivity was localized to the cell surface of enterochromaffin-like cells, and of myenteric and submucous neurons, and to fibers distributed to the plexuses, interconnecting strands, muscle layers, vasculature, and mucosa. A dense network of GAL-R1 immunoreactivity was observed in the deep muscular plexus in very close association with interstitial cells of Cajal visualized by c-kit immunostaining. In the ileum, 81.6% of GAL-R1 myenteric neurons and 70.7% of GAL-R1 submucosal neurons were substance P immunoreactive. Vasoactive intestinal polypeptide immunoreactivity was found in 48.3% of GAL-R1 submucosal neurons, but not in GAL-R1 myenteric neurons. These findings support the hypothesis that GAL-R1 mediates galanin actions on gastrointestinal motility and secretion by modulating the release of other neurotransmitters and contributes to galanin-induced inhibition of gastric acid secretion by means of the suppression of endogenous histamine release.     

Post-natal development of type 1 cannabinoid receptor immunoreactivity in the rat hippocampus

Type 1 cannabinoid receptors, selectively located on axon terminals of GABAergic interneurons in the hippocampus, are known to be involved in endocannabinoid-mediated retrograde synaptic signalling. The question arises whether type 1 cannabinoid receptors appear on these axons during early post-natal life, when GABAergic transmission is still depolarizing, and whether there are any developmental changes in the cellular or subcellular expression pattern. Here we demonstrate, using single and double immunocytochemical methods at the light and electron microscopic levels, that type 1 cannabinoid receptors are expressed only on the membrane of axon terminals and pre-terminal axons but not on the soma-dendritic membrane at all examined timepoints between post-natal days 0 and 20, similar to the adult distribution. All type 1 cannabinoid receptor-positive boutons formed symmetric synapses. Granular labelling in the somata was already strong at post-natal day 0 and corresponded to multivesicular bodies, lysosomes, Golgi apparatus and rough endoplasmic reticulum. The type 1 cannabinoid receptor-positive axons were shown to originate largely from cholecystokinin-immunoreactive basket and bistratified neurons throughout the hippocampus (90% of all type 1 cannabinoid receptor-containing cells) and dentate gyrus (70% of all type 1 cannabinoid receptor-containing cells). The remaining cells have not been identified but probably belong to the somatostatin- and/or neuropeptide Y-containing subsets, as cholecystokinin-negative, type 1 cannabinoid receptor-positive axons have been observed in strata moleculare and lacunosum-moleculare of the dentate gyrus and CA1-3, respectively, where these neurons are known to arborize. No cell types were found that expressed type 1 cannabinoid receptors transiently at some developmental stage. We conclude that the cellular and subcellular pattern of type 1 cannabinoid receptor expression during early post-natal life is similar to the adult pattern and type 1 cannabinoid receptors are expressed on the cholecystokinin-containing axons as soon as synapse formation begins. This suggests that retrograde synaptic signalling by endocannabinoids is required for the normal operation of GABAergic neurotransmission even before it becomes hyperpolarizing.     

Morphology and neurokinin 1 receptor expression of spinothalamic lamina I neurons in the rat spinal cord

Distinct morphological types of spinothalamic tract (STT) lamina I (LI) neurons have been identified in the cat and monkey spinal dorsal horn. Because these morphological types appear to differ in functional properties and receptor expression, we examined their distribution in the rat to test how their identification relates to earlier classification schemes. LI STT cells were retrogradely labeled with cholera toxin subunit b (CTb). Three types were recognized on the basis of cell body shape and proximal dendrites in the horizontal plane: fusiform, multipolar, and pyramidal. The relative distribution of these types was: 43, 26, and 28%, respectively, similar to that observed in the cat and monkey. 3D reconstructions were used to view each cell in all three major projection planes: horizontal, parasagittal, and transverse. Most LI STT neurons appeared fusiform in the parasagittal plane even though they belonged to different types based on their appearance in the horizontal plane, except in the most lateral portion of the dorsal horn, where LI curves ventrally. The proportion of STT neurons within LI was quantified by using the optical dissector method. To label all LI neurons, we used an anti-neuron-specific nuclear protein (NeuN) antibody. We found that approximately 9% of LI neurons projected to the thalamus. We also investigated neurokinin 1 receptor (NK-1r) expression in LI STT neurons. As in the monkey, most pyramidal STT neurons did not express NK-1r. These results provide further evidence that distinct morphological types of neurons differ in phenotype but not in their projection pattern.     

Recognition of neurokinin 1 receptor (NK1-R): an antibody to a peptide sequence from the third extracellular region binds to brain NK1-R

Substance P (SP) can produce cytokine-like responses by astrocytes and mononuclear cells. In an effort to identify neurokinin-1-receptors (NK1-R), an antibody to NK1-R was generated by using a linear peptide sequence from the deduced third extracellular region (ECR) corresponding to the seven transmembrane rat brain NK1-R. The ECR-3 peptide was coupled to keyhole-limpet hemocyanin and the antisera produced in rabbits was purified by binding to a peptide-affinity matrix. The specificity for the anti-peptide antibody was shown by its reactivity to the ECR-3 peptide by ELISA. The anti-ECR-3 peptide antibody could detect, by Western blot analysis of SDS-PAGE-separated rat brain membranes, a single band with an apparent molecular weight (MW) of 53–54 kDa. An affinity matrix made from the anti-ECR-3 antibody was used to isolate NK1-R from rat brain membranes which exhibited two products on SDS-PAGE with apparent MW of 54 and 44 kDa. The C6 astrocytes were shown to express NK1-R as determined by [¹²⁵I]Bolten-Hunter SP binding to intact cells with a K_d = 0.32 nM. These C6 cells did not co-express either NK2-R or NK3-R when analyzed at the mRNA level. The anti-ECR-3 peptide antibody could inhibit [¹²⁵I]Bolten-Hunter SP binding to intact C6 astrocytes and CHO cells expressing NK1-R by greater than 95% when compared to normal rabbit IgG which failed to inhibit radiolabeled SP binding. Thus, an antibody which recognizes surface determinants to the NK1-R could be generated upon immunization with an NK1-R peptide.     

NGF receptor immunoreactivity in aged rat brain

The cellular distribution of nerve growth factor (NGF) receptor (NGFR) immunoreactivity in 3 cholinergic nuclei (medial septal nucleus, nucleus of the diagonal band and nucleus basalis magnocellularis) of the aged rat brain was compared to that of young-adult animals. In young-adult rats, NGFR immunoreactivity was strong in the neuronal body and in the whole dendritic tree. In aged animals, NGFR immunoreactivity was weak in both cell body and dendrites and was practically absent in the dendrite's distal portion. The loss of dendritic NGFR may play a critical role in the decline of neuronal function in the aging brain.