Substance P Terminals Research Articles

Effects on ventilation (VE) and neuromodulator concentration of cholinergic receptor blockade at the pre‐Bötzinger Complex (preBötC)

We previously found that microdialysis of the muscarinic acetylcholine receptor antagonist, Atropine (AT, 50mM) in mock cerebrospinal fluid (mCSF) into the Kӧlliker‐Fuse Nucleus (KFN) of awake and sleeping goats (n=6) reduces VE and breathing frequency (f) (J Appl Physiol, 109: 159–170, 2010). In contrast, dialysis of 50mM AT into the preBötC of awake goats (n=6) increased VE and f, and increased the concentration of serotonin (5‐HT) and substance P (SP) in the effluent mCSF (FASEB J, 25: 1074.1, 2011). To gain insight into the mechanism by which cholinergic receptor blockade in the preBötC increases VE and f, we dialyzed in one awake goat, mCSF into the preBötC on one side, and simultaneously dialyzed 5mM or 50mM AT into the preBötC of the contralateral side. 5mM AT did not affect VE, f, or effluent 5‐HT, but minimally increased effluent SP, indicating a dose‐dependent AT effect. In contrast, 50mM AT increased VE, f, and effluent 5‐HT and SP. The increase in 5‐HT and SP was restricted to the side receiving AT. These data suggest that cholinergic receptor blockade in the preBötC affects local, not global, levels of these excitatory neuromodulators, which affect the level of breathing. We hypothesize that AT blocks cholinergic receptors of pre‐synaptic 5‐HT and SP terminals at the preBötC, the effect of which is enhanced release of 5‐HT and SP, which subsequently increases VE and f. Support: NIH HL25739 & Veterans Administration

Substance P, tyrosine hydroxylase and serotonin terminals in the rat caudal nucleus ambiguus

Substance P (SP), tyrosine hydroxylase (TH) and serotonin inputs onto laryngeal motoneurons (LMNs) are known to exist, but the distribution of their terminals in the caudal nucleus ambiguus (NA), remains unclear. Using immunofluorescence and confocal microscopy, we assessed simultaneously the distribution of SP, TH, serotonin and synaptophysin immunoreactive (ir) terminals in the caudal NA. SP, TH and serotonin-ir varicosities were considered to represent immunoreactive synapses if, using confocal microscopy, they were co-localized with the presynaptic protein, synaptophysin. Relative to the total number of synapses, we found only a modest number of SP, TH or serotonin-ir synaptic terminals in the caudal NA. The density of SP-ir synaptic terminals was higher than that of TH-ir and serotonin-ir synaptic terminals. Our results suggest that SP, TH, and serotonin-ir inputs may play only a modest role in regulating the activity of LMN. We conclude that SP, TH and serotonin are not always co-localized in terminals forming inputs with LMN and that they arise from separate subpopulations of neurons.

Trafficking of neurokinin‐1 receptors in serotonin neurons is controlled by substance P within the rat dorsal raphe nucleus

Substance P (SP) modulates serotonin neurotransmission via neurokinin-1 receptors (NK1rs), and exerts regulatory effects on mood through habenular afferents to the dorsal raphe nucleus (DRN). We have previously demonstrated that, in the caudal DRN of rat, some serotonin neurons are endowed with NK1rs that are mostly cytoplasmic, whereas these receptors are mostly membrane bound in non-serotonin neurons. Here, we first examined by double-labeling immunocytochemistry the relationships between SP axon terminals and these two categories of DRN neurons. Almost half of the SP terminals were synaptic and many were in close contact with serotonin dendrites, but never with non-serotonin dendrites. In additional double-immunolabeling experiments, most if not all dendrites bearing membranous NK1rs appeared to be GABAergic. Treatment with the selective neurokinin-1 antagonist RP67580 modified the subcellular distribution of NK1rs in serotonin neurons. At 1 h after administration of a single dose, the receptor distribution was unchanged in both dendritic types but, after daily administration for 7 or 21 days, the plasma membrane and cytoplasmic density of NK1rs were increased in serotonin dendrites, without any change in non-serotonin dendrites. These treatments also increased NK1r gene expression in the caudal DRN. Lastly, a marked increase in the membrane (but not cytoplasmic) density of NK1rs was measured in serotonin dendrites after bilateral habenular lesion. These results suggest that the trafficking of NK1rs represents a cellular mechanism in control of the modulation of serotonin neuron activity by SP in DRN.

An immunohistochemical study on a unique colocalization relationship between substance P and GABA in the central nucleus of amygdala

Substance P (SP) is a neuropeptide contained in axon terminals. Various classical neurotransmitters coexist with SP in mammalian brains, but there has been no information on the colocalizing substances in the central nucleus of amygdala (CeA), where both SP and its specific receptor are highly concentrated. The present study aimed at determining the colocalizing neurotransmitter in SP terminals in CeA by multi-label immunohistochemistry combined with digitized quantitative analysis. Unexpectedly, most of SP-containing boutons did not show immunoreactivities for any of the transmitters or their marker proteins examined (GABA, glycine, glutamate, acetylcholine, serotonin, or dopamine). Electron microscopy demonstrated small clear vesicles in addition to dense core vesicles within SP-positive terminals that formed symmetrical synapses, indicating the presence of some classical neurotransmitter, most likely GABA. Therefore tissues were fixed by zinc–aldehyde to enhance immunoreactivity for a low level of glutamic acid decarboxylase (GAD), the GABA synthetic enzyme. This led to weak but consistent labeling for GAD in the majority of SP-positive boutons in CeA. By contrast, definite GAD-immunoreactivity was confirmed in SP-containing boutons in the substantia nigra pars reticulata even in specimens treated with a conventional fixative, indicating that negligible GAD labeling in CeA is not ascribed to methodological problems such as interference by the presence of SP but actually reflects low GAD content. These data suggest a unique mode of synaptic transmission at amygdalar SP-containing terminals where slowly-acting SP is concentrated but both GABA and its synthetic enzyme are maintained at low levels, possibly underlying long-lasting responses in emotions.

Genioglossal hypoglossal motoneurons contact substance P-like immunoreactive nerve terminals in the cat: a dual labeling electron microscopic study

This study investigated the synaptic interactions between hypoglossal motoneurons that project to the genioglossus muscle and substance P (SP) containing immunoreactive nerve terminals. Cholera toxin B conjugated to horseradish peroxidase (CTB-HRP) was injected into the right half of the genioglossus muscle in four anesthetized cats. Two days later, the animals were perfused with acrolein fixative. Tetramethylbenzidine (TMB) was the chromogen used to detect retrogradely labeled cells containing CTB-HRP. The tissues were then processed for immunocytochemistry using an antiserum raised against SP with diaminobenzidine (DAB) as the chromogen. At the light microscopic level, labeled cells were observed primarily ipsilaterally in ventral and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were observed at the level of the area postrema. At the electron microscopic level, SP-like immunoreactive nerve terminals formed synaptic contacts with retrogradely labeled dendrites and perikarya. Nineteen percent of the terminals that contacted retrogradely labeled cells contained SP. These are the first ultrastructural studies demonstrating synaptic interactions between protruder hypoglossal motoneurons and SP terminals. These studies demonstrate that hypoglossal motoneurons which innervate the major protruder muscle of the tongue, the genioglossus muscle, may be modulated by SP. Thus, SP may play a role in the control of protrusive movements of the tongue acting via neurokinin receptors.

Distribution, immunohistochemical characteristics and nerve pathways of primary sensory neurons supplying the porcine vas deferens.

The present study investigated: (1) the distribution and chemical coding of primary sensory neurons supplying the vas deferens in juvenile pigs by the use of retrograde tracing combined with double-labelling immunofluorescence, (2) nerve pathways from dorsal root ganglia (DRG) to the vas deferens by means of denervation procedures involving transection of the hypogastric or pelvic nerve combined with a retrograde tracing method, and (3) possible interactions of the substance P (SP)/calcitonin gene-related peptide (CGRP)-immunoreactive varicose nerve fibres on vas deferens projecting neurons (VDPN) in the anterior pelvic ganglion (APG). The vast majority of VDPN were found mainly in the lumbar L2, L3 and sacral S2, S3 pairs of DRG and showed a clear ipsilaterally organized projection pattern. Immunohistochemistry revealed that most of these neurons contained SP and/or CGRP, occasionally coexpressed with galanin. Interestingly, pronounced differences in the expression of SP and/or CGRP were observed between the lumbar and sacral VDPN in that most of the lumbar but less than half of the sacral neurons stained for these peptides. Denervation experiments showed that the neurons located within the lumbar DRG project through the ipsilateral hypogastric nerve, whereas those found within the sacral DRG send their processes through the ipsilateral and contralateral pelvic nerve. In the nerve-lesioned animals, especially in those with the hypogastric nerve cut, a dramatic reduction in the number of SP and/or CGRP-containing nerve terminals surrounding the efferent VDPN within the APG was observed. This study has disclosed the distribution and, for the first time, chemical coding and nerve pathways of vas deferens-projecting primary sensory neurons in a mammalian species, the pig. The results obtained also provide some novel information about the possible morphological and functional relationship between vas deferens-projecting primary sensory and pelvic efferent nerve cells.

Influence of passive avoidance learning by substance P in the basolateral amygdala.

Neuropeptide substance P (SP) has reinforcing and memory facilitating effects after its peripheral or central application. Rats self-inject SP into the ventromedial caudate-putamen and SP microinjections into the basal forebrain induce place preference with a simultaneous increase of dopamine level. In the amygdaloid body SP positive neurones and terminals have been identified. The aim of the present study was to examine the possible reinforcing effects of SP in the basolateral amygdala (ABL). CFY male rats were conditioned in two-compartment passive avoidance paradigm and place preference was examined in two-compartment-box and in circular open field. Animals were microinjected bilaterally with 10 ng SP, 100 ng SP or vehicle solution (0.4 microl/side) into the ABL. Results showed that post-shock infusion of 10 ng SP significantly enhanced passive avoidance learning while 100 ng SP was ineffective. In two-compartment-box and in circular open field place preference did not develop after SP treatments, however. Our data are the first to demonstrate that SP in the ABL is involved in learning and memory processes related to aversive situations. Results that SP microinjections were not followed by rewarding-reinforcing consequences in place preference paradigms indicate that the local SP network in the ABL is not involved in neuronal circuitry responsible for addictive behaviour.

Substance P afferent terminals innervate vagal preganglionic neurons projecting to the trachea of the ferret

Airway disorders, such as asthma and chronic obstructive bronchitis, are, in part, due to abnormalities in the nervous control of the airways. However, the ultrastructural circuitry and neurochemical anatomy of afferents modulating the output of airway-related vagal preganglionic neurons (VPNs) in the nucleus ambiguus are poorly understood. We have examined the potential role of substance P (SP) immunoreactive afferents in the regulation of anatomically identified airway VPNs. Cholera toxin b-subunit conjugated to horseradish peroxidase was used as a retrograde cell body tracer to identify the central VPNs innervating the extra-thoracic trachea. Immunocytochemistry was employed to identify SP afferents. The external formation of the nucleus ambiguus was examined by electron microscopy using a simultaneous double labeling method. Cell bodies of tracheal VPNs were 31.7±1.18×23.0±1.3 μm (means±S.E.M.) in size, contained abundant endoplasmic reticulum, had a round nucleus with a prominent nucleolus, no satellite body and displayed somatic and dendritic spines. Somato-somatic appositions, somato-dendritic appositions without intervening glial processes and dendritic “bundling” commonly seen in esophageal motoneurons were not observed. The ultrastructural morphology of tracheal VPNs were also clearly distinguishable from pharyngeal and laryngeal motoneurons in other divisions of the nucleus ambiguus which lack somatic spines. These data are consistent with the hypothesis that differences in the ultrastructure and synaptology of the different divisions of the nucleus ambiguus may be associated with specific physiological functions. The mean size (±S.E.M.) of SP nerve terminals was 1.57±0.06×0.79±0.03 μm. SP terminals formed 17.5% of the axo-dendritic and 15.9% of the axo-somatic synapses which were observed upon retrogradely labeled tracheal VPNs. Synaptic contacts observed were both symmetric and asymmetric. These synaptic interactions define, in part, the neurochemical anatomy of neuronal circuits modulating vagal preganglionic control of tracheal functions.

Synaptic interactions of retrogradely labeled genioglossus motoneurons with substance P-like immunoreactive terminals in the cat: a double labeling immunoelectron microscopic study

The genioglossus muscle (GM) is the principal protruder of the tongue and comprises a major portion of the caudal body of the tongue. The position of the GM is a major determinant of upper airway resistance. Our laboratory is interested in identifying the neurochemical microcircuitry of functionally identified hypoglossal motoneurons. We have found that substance P (SP) makes synaptic contacts with hypoglossal motoneurons that innervate intrinsic tongue muscles [1] in the cat. The purpose of the present study was to investigate whether there are synaptic interactions between motoneurons that innervate the GM and SP-immunoreactive nerve terminals utilizing double labeling immunoelectron microscopy. Cholera toxinB conjugated to horseradish peroxidase (CTB-HRP) was injected into the right GM of three cats. Two days later, the animals were deeply anesthetized and perfused with an acrolein/paraformaldehyde mixture. Cells containing CTB-HRP were labeled with a histochemical reaction utilizing tetramethylbenzidine (TMB) as the chromogen. TMB forms a crystalline reaction product that is very distinct at the electron microscopic level. The tissues were then processed for immunocytochemistry using an antiserum against SP. The chromogen used in this case, diaminobenzidine, yields an amorphous reaction product. At the light microscopic level, retrogradely labeled cells were observed primarily ipsilaterally to the injection site. These neurons were located predominantly in the ventral and ventrolateral portions of the nucleus primarily at the level of the area postrema. At the electron microscopic level, numerous SP-labeled terminals were observed some of which made synaptic contacts with retrogradely labeled GM's. SP terminals formed synapses with retrogradely labeled dendrites and perikarya. SP-labeled terminals also synapsed on unlabeled dendrites. These are the first ultrastructural studies demonstrating synaptic interactions between hypoglossal motoneurons that innervate an extrinsic tongue muscle and SP nerve terminals. These studies demonstrate that motoneurons that innervate the GM are modulated by SP and that SP appears to play a role in the control of tongue movement and GM activity.

Action of substance P (neurokinin-1) receptor activation on rat neostriatal projection neurons.

Substance P (SP) acts as a neurotransmitter in the neostriatum through the axon collaterals of spiny projection neurons. However, possible direct or indirect actions of SP on the neostriatal output neurons have not been described. Targets of SP terminals within the neostriatum include interneurons, spiny neurons, afferent fibers and boutons. SP induces the release of both dopamine (DA) and acetylcholine (ACh). Since some postsynaptic actions of both DA and ACh on spiny neurons are known, we asked if activation of neostriatal NK1-class receptors is able to reproduce them. The SP NK1-receptor agonist, GR73632 (1 microM), had both excitatory and inhibitory actions on virtually all spiny neurons tested at resting potential. The excitatory action was blocked by atropine and coursed with an increase in firing rate and input resistance (R(N)). The inhibitory action was blocked by haloperidol and coursed with a reduction in firing rate and R(N). Therefore, the release of both DA and ACh induced by NK1-receptor activation modulates indirectly the excitability of the projection neurons. SP facilitates the actions of these transmitters on the spiny neuron. A residual excitatory response to the NK1-receptor agonist was observed in 30% of a sample of neurons tested in the presence of both haloperidol and atropine. The increase in R(N) that accompanied this response could be observed in the presence of 1 microM TTX or 100 microM Cd2+, suggesting a direct effect. Double labeling showed that only SP-immunoreactive neurons were facilitated by NK1-receptor activation in these conditions.

Substance P in the hypoglossal nucleus of the rat

The distribution of substance P (SP)-containing synaptic terminals in the hypoglossal nucleus (XII) of adult rats was examined by retrograde peroxidase labelling and immunocytochemistry. From the location of peroxidase injections into the tongue and of labelled neurones in the ventral lamina of XII, motor neurones that supply intrinsic vertical, longitudinal and transverse fibres as well as the extrinsic muscle genioglossus appear to have been labelled. SP-containing terminals were found making contact, and sometimes dual synapses, with unlabelled neuronal dendrites but not with retrogradely labelled somata or dendrites. These findings suggest that SP terminals may contact dendrites of interneurones or of neurones supplying other extrinsic muscles located in the anterior part of the tongue. Dual SP-containing synapses between XII motor neurones may be the means by which tongue muscle fibres are recruited and their function synchronized.

Substance P immunoreactive nerve terminals in the dorsolateral nucleus of the tractus solitarius: roles in the baroreceptor reflex

Physiological and light microscopic evidence suggest that substance P (SP) may be a neurotransmitter contained in first-order sensory baroreceptor afferents; however, ultrastructural support for this hypothesis is lacking. We have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase (HRP). The dorsolateral subnucleus of the nucleus tractus solitarius (dlNTS) was processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical visualization of SP by dual labeling light and electron microscopic methods. Either HRP or SP was readily identified in single-labeled unmyelinated axons, myelinated axons, and nerve terminals in the dlNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the dlNTS, which were simultaneously identified as CSN primary afferents. However, only 15% of CSN terminals in the dlNTS were immunoreactive for SP. Therefore, while the ultrastructural data support the hypothesis that SP immunoreactive first-order neurons are involved in the origination of the baroreceptor reflex, they suggest that only a modest part of the total sensory input conveyed from the carotid sinus baroreceptors to the dlNTS is mediated by SP immunoreactive CSN terminals. Five types of axo–axonic synapses were observed in the dlNTS. SP immunoreactive CSN afferents were very rarely involved in these synapses. Furthermore, SP terminals were never observed to form the presynaptic element in an axo–axonic synapse with a CSN afferent. Therefore, SP does not appear to be involved in the modulation of the baroreceptor reflex in the dlNTS.

Synaptic interactions of retrogradely labeled hypoglossal motoneurons with substance P-like immunoreactive nerve terminals in the cat: a dual-labeling electron microscopic study.

This study has investigated the synaptic interactions between hypoglossal motoneurons and substance P (SP)-immunoreactive terminals. Cholera toxin B conjugated to horseradish peroxidase was injected into the tip of the tongue on the right side of six ketamine-anesthetized cats. Two to five days later, the animals were killed. Cells containing HRP were labeled with a histochemical reaction utilizing tetramethylbenzidine (TMB) as the chromogen. TMB forms crystalline reaction products that are very distinct at the electron microscopic level. The tissues were then processed for immunocytochemistry using an antiserum against SP. The chromogen used in this case, diaminobenzidine, yields amorphous reaction products. At the light microscopic level, labeled cells were observed primarily ipsilaterally in both intermediate and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were seen at the level of obex. At the electron microscopic level, both asymmetric and symmetric synapses were observed. SP-immunoreactive nerve terminals formed asymmetric synapses with labeled dendrites and symmetric synapses with labeled perikarya. SP-labeled terminals also synapsed on unlabeled dendrites and somata. These are the first ultrastructural studies demonstrating synaptic interactions between hypoglossal motoneurons and SP terminals. These studies demonstrate that hypoglossal motoneurons that innervate intrinsic tongue muscles are modulated by SP and that SP may play a role in the control of fine movements of the tongue.

What are the roles of substance p and neurokinin-1 receptors in the control of negative chronotropic or negative dromotropic vagal motoneurons? a physiological and ultrastructural analysis

Recent data indicate that there is a cardiotopic organization of negative chronotropic and negative dromotropic neurons in the nucleus ambiguus (NA). Negative dromotropic neurons are found in the rostral ventrolateral NA (rNA-VL), negative chronotropic neurons are found in the caudal ventrolateral NA (cNA-VL), and both types of neurons are found in an intermediate level of the ventrolateral NA (iNA-VL). Substance P (SP) immunoreactive nerve terminals synapse upon negative chronotropic vagal motoneurons in the iNA-VL, and SP microinjections in the NA cause bradycardia. In the present report we have attempted to: (1) define the type of tachykinin receptor which mediates the negative chronotropic effect of SP microinjections into the iNA-VL; (2) define the physiological effect of microinjections of a selective SP agonist into the rNA-VL on atrioventricular (AV) conduction; and (3) find ultrastructural evidence for synaptic interactions of SP-immunoreactive nerve terminals with negative dromotropic vagal motoneurons in the rNA-VL. Microinjections of the excitatory amino acid glutamate (Glu) into the iNA-VL to activate all local vagal preganglionic neurons caused both bradycardia and a decrease in the rate of AV conduction. Injections of the selective neurokinin-1 (NK-1) receptor agonist drug GR-73632 also caused bradycardia, however the rapid onset of agonist induced desensitization prevented an evaluation of potential effects on AV conduction in the iNA-VL. These data suggest that the SP-induced bradycardia which can be elicited from the NA is mediated, at least in part, by NK-1 receptors. Microinjections of Glu into the rNA-VL caused a decrease in AV conduction without an effect on cardiac rate. On the other hand, GR-73632 microinjections into rNA-VL did not affect AV conduction. Following injections of the β subunit of cholera toxin conjugated to horseradish peroxidase (CTB-HRP) into the left atrial fat pad ganglion which selectively mediates changes in AV conduction, retrogradely labeled neurons were histochemically visualized in the rNA-VL. These tissues were subsequently processed for the simultaneous immunocytochemical visualization of SP, and examined by electron microflapy. Histochemically labeled neurons were large, multipolar, with abundant cytoplasm containing large masses of rough endoplasmic reticulum, and exhibited distinctive dendritic and somatic spines. Unlabeled nerve terminals were noted to form either asymmetric or symmetric synapses with dendrites, dendritic spines, and perikarya of histochemically labeled neurons. SP-immunoreactive nerve terminals were also detected in the rNA-VL. SP terminals typically contained numerous small pleomorphic vesicles, multiple large dense core vesicles, and several mitochondria, and they synapsed upon unlabeled dendritic profiles. A total of 154 SP-immunoreactive nerve terminals were observed on photomicrographs of tissues which also contained histochemically labeled profiles. None made an identifiable synapse with a retrogradely labeled profile on the sections examined. In summary, both physiological and ultrastructural data indicate that SP terminals in the iNA-VL do modify the output of negative chronotropic vagal motoneurons. This effect is mediated by NK-1 receptors. On the other hand both physiological and ultrastructural data indicate that SP terminals in the rNA-VL do not modify the output of negative dromotropic vagal motoneurons. Therefore different mechanisms (neurotransmitters or receptors) mediate the central vagal control of cardiac rate and AV conduction.

Synaptic interactions of substance P immunoreactive nerve terminals in the baro- and chemoreceptor reflexes of the cat

The neurochemical anatomy and synaptic interactions of morphologically identified chemoreceptor or baroreceptor afferents in the nucleus of the solitary tract (NTS) are poorly understood. A substantial body of physiological and light microscopic evidence suggests that substance P (SP) may be a neurotransmitter contained in first order sensory chemo- or baroreceptor afferents, however ultrastructural support for this hypothesis is lacking. In the present report we have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase. Medullary tissues including the commissural NTS (cNTS) were processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical detection of SP by dual labeling light and electron microscopic methods. At the light microscopic level, dense bilateral labeling with TMB was found in the tractus solitarius (TS) and cNTS, caudal to the obex. Rostral to the obex, significant ipsilateral TMB labeling was detected in the dorsal, dorso-lateral, and medial subnuclei of the NTS, as well as in the TS. Significant staining of SP immunoreactive processes was detected in most subnuclei of the NTS. The cNTS was examined by electron microscopy. Either HRP or SP were readily identified in single labeled unmyelinated axons, myelinated axons, and nerve terminals in the cNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the cNTS which were simultaneously identified as CSN primary afferents. These ultrastructural data support the hypothesis that SP immunoreactive first order neurons are involved in the origination of the chemo- and baroreceptor reflexes. Axo-axonic synapses were observed between CSN primary afferent terminals and: (a) unlabeled nerve terminals; (b) other CSN primary afferent terminals; and (c) terminals containing SP. Axo-axonic synapses were also observed between CSN primary afferents which contained SP, and other SP terminals. These observations may mediate the morphological bases for multiple forms of presynaptic inhibition in the cNTS, including those involved in cardiorespiratory integration. In conclusion, our results indicate that SP immunoreactive nerve terminals may be important in both the origination and the modulation of the chemo- and/or baroreceptor reflexes.

Synaptic relationship between substance P and the substance P receptor: light and electron microscopic characterization of the mismatch between neuropeptides and their receptors.

Light microscopic studies have demonstrated significant mismatches in the location of neuropeptides and their respective binding sites in the central nervous system. In the present study we used an antiserum raised against a synthetic peptide corresponding to the carboxyl-terminal tail of the substance P (SP) receptor (SPR) to further explore the relationship between a neuropeptide and its receptor. Light microscopy revealed an excellent correlation between the patterns of SPR immunoreactivity and of 125I-labeled SPR-binding sites in the central nervous system. The SPR appeared to be exclusively expressed by neurons; in fact, the SPR decorates the somatic and dendritic surface of neurons, producing Golgi-like images. Electron microscopic analysis in cortex, striatum, and spinal cord revealed that approximately 70% of the surface membrane of immunoreactive neurons is SPR laden. Simultaneous electron microscopic labeling of SP and SPR demonstrated significant mismatch at the synaptic level. Although some SP terminals contacted SPR-immunoreactive membrane, no more than 15% of the SPR-laden membrane apposed synaptic terminals. These results suggest that in contrast to more "classical" central and peripheral nervous system synapses, wherein the receptor immediately apposes the site of neurotransmitter storage and release, much of the surface of SPR-expressing neurons can be targeted by SP that diffuses a considerable distance from its site of release.

Proliferation of SP- and 5HT-Containing Terminals in Lamina II of Rat Spinal Cord Following Dorsal Rhizotomy: Quantitative EM-Immunocytochemical Studies

The density of substance P (SP) and serotonin (5HT) immunoreactivity in laminae I and II of rat spinal cord changes following dorsal rhizotomy in a manner consistent with sprouting by intrinsic SP and descending 5HT systems (Wang et al. J. Comp. Neurol. 304: 555, 1991). In this study we used quantitative EM-immunocytochemistry to examine whether the increase in substance P and serotonin immunoreactivity seen at the light microscopic level was related to increased numbers of SP- and 5HT-containing terminals in lamina II. Dorsal roots were sectioned and their ganglia removed from L1 to S2 unilaterally in 18 rats. After 3 (acute), 10 (subacute), or 60 (chronic) days, rats were perfused, and the L5 segments of the spinal cord were removed and prepared for electron microscopy. Lumbosacral deafferentation completely and permanently eliminated complex terminals in lamina II at L5 but the number of simple terminals increased by 46% compared to the control side. This result was similar to that shown previously in cat (Murray and Goldberger, J. Neurosci. 6: 3205, 1986) and suggested that intact intrinsic systems sprouted to form new terminals to compensate for the terminals lost by deafferentation. Quantitative electron microscopic immunocytochemistry demonstrated that the number of terminals containing SP in deafferented lamina II decreased by 58% at 3 days postoperatively and then increased by 10 days and recovered to normal levels by 60 days. The loss of SP terminals in the acute group is due to the loss of SP-containing dorsal root afferents, while the recovery in the chronic group suggests replacement of lost terminals by intrinsic SP systems. These results therefore indicate that SP-containing terminals show homotypic sprouting in response to complete dorsal root deafferentation. The number of 5HT-containing terminals in lamina II of spinal cord increased by 56% on the deafferented side in the chronic group. The increase in 5HT-containing terminals indicates that descending 5HT systems undergo heterotypic sprouting in response to dorsal rhizotomy.

Differential effects on substance P immunoreactivity in the rat suprachiasmatic and olivary pretectal nuclei

We examined the effects of constant dark or constant light on substance P (SP) and/or neuropeptide Y (NPY) immunoreactive fibres in the rat suprachiasmatic nucleus (SCN) and olivary pretectal nucleus (OPT) by immunohistochemistry. After constant dark, SP immunoreactive fibres and terminals decreased slightly in the SCN, while they increased markedly in the OPT. After constant light, they increased markedly in the SCN, but were little changed in the OPT. NPY immunoreactive fibres and terminals in the SCN decreased slightly after after constant light, but there were no effects on these fibres after constant dark. These findings suggest that SP immunoreactive fibres are involved in mediating illumination discrimination in the SCN and/or OPT.

Substance P innervation of the rat and cat thalamus. I. Distribution and relation to ascending spinal pathways.

An antiserum for substance P (SP) with minimal cross-reactivity for other tachykinins was employed to map the distribution of SP-positive nerve fibers and terminals in the thalamus of cats and rats with special emphasis on the innervation by these fibers of nuclei related to the somatosensory system. In both species SP innervation is predominantly along the midline, in medial and posterior thalamic regions, and sparser in sensory relays for specific modalities. Among the most densely innervated nuclei are the parafascicular, paraventricular, rhomboid, central medial and parts of mediodorsal, lateral posterior, and ventral lateral geniculate. SP innervation of somatosensory-related nuclei is also evident in central lateral nucleus, posterior complex (PO), and in ventroposterolateral (VPL) nucleus of both cats and rats. In VPL of cats SP fibers and terminals are present along its ventral and lateral border, a paralaminar area in which spinothalamic fibers have been shown to terminate and where neurons responsive to noxious stimuli have been reported. Also in rats the SP innervation of VPL is similar to that of spinothalamic tract fibers. The SP innervation of somatosensory thalamic nuclei may be supplied, at least in part, by spinothalamic afferent as suggested by the depletion of SP after anterolateral chordotomy but not after ablation of the dorsal column nuclei. The presence of SP-positive spinothalamic neurons in the spinal cord is reported in the following paper.

Ultrastructural localization of substance P, met‐enkephalin, and somatostatin immunoreactivity in lamina X of the primate spinal cord

The ultrastructural localization of substance P (SP), met-enkephalin (MENK), and somatostatin (SS) in the lamina X area surrounding the central canal of the macaque monkey was examined by the indirect peroxidase-antiperoxidase method. The most common synaptic terminals in lamina X were simple terminals (S) with small rounded or pleomorphic clear vesicles; one to two dense-core vesicles were occasionally also present. These were found on soma, dendrites, and dendritic spines, in all regions of lamina X. A second class of terminal with round or oval clear vesicles was glomerular (G) in shape, with scalloped edges, and contained many mitochondria. These large terminals had several synaptic contacts onto dendrites, spines, and small terminals and were found mainly in the lateral region. The third class (L) contained small clear vesicles and several vesicles with large, dense cores (100-125 nm), and also contacted dendrites, mainly lateral to the canal. The fourth class of terminal (D) contained small clear vesicles and several vesicles with small, dense cores (75-100 nm); these contacted dendrites and somata in all areas. Very few terminals with flat vesicles were identified. There was an unequal distribution of immunoreactivity among the several terminal classes identified in lamina X. Most SP terminals were S terminals, but SP L terminals were also common; few were D terminals. MENK terminals were usually either S terminals or D terminals; L terminals were rarely MENK positive. SS terminals were commonly D terminals or S terminals; L terminals were also rarely SS positive. Only SP terminals were identified as G terminals. Synaptic targets of SP, MENK, and SS terminals were most commonly dendrites. In addition to unlabelled neurons, peptidergic neurons and their processes were also synaptic targets of terminals containing the same peptide. The distributions of these peptides in primate lamina X differ from that of the same peptides in primate superficial dorsal horn. These differences are important, in consideration of some of the parallels that may be drawn between the lamina X area and the superficial dorsal horn; both areas have high concentrations of the same peptides, receive nociceptive primary afferents, and contain spinothalamic and other projection neurons. Nevertheless, comparison of the distribution of immunoreactivity among terminal classes indicates that neurochemical organization at the ultrastructural level is quite distinct in each of the two areas. This may also reflect other roles of the lamina X area, including its involvement in visceral functions, although it would be expected that this element might be less prominent at the cervical levels we investigated.