Substance P-like Immunoreactive Fibers Research Articles

Involvement of Dorsal Column Nucleus Neurons in Nociceptive Transmission in Aged Rats

To clarify the functional role of the dorsal column nucleus (DCN) in nociception in rats with advancing age, single neuronal activity and substance P-like immunoreactivity (SP-LI) of the gracile nucleus (GN) were studied in aged rats (29 to 34 mo old) and adult rats (9 to 12 mo old). A total of 122 neurons [aged: 34 wide-dynamic-range (WDR), two nociceptive-specific (NS), and 32 low-threshold mechanical (LTM) neurons; adult: 22 WDR and 32 LTM neurons] were recorded from GN. For WDR neurons, the latency to antidromic activation of the ventral posterior lateral nucleus of the thalamus showed no difference between the aged and adult rats. Sciatic nerve stimulation with C-fiber intensity induced responses of GN with significantly longer latency in aged rats than in adults, whereas there was no difference in the response latency to A-fiber intensity stimulation. Background activity and afterdischarges were significantly higher in the aged rats than those in the adult rats. Responses to noxious mechanical and thermal stimuli were significantly greater in the aged rats during application of graded stimuli. There were no significant differences in responses to nonnoxious mechanical stimulus, mechanical response threshold, and the size of the receptive fields between neurons in the aged and adult rats. The area occupied by SP-LI fibers in the GN and the size of SP-LI dorsal root ganglia neurons were significantly larger in aged rats than in adults. The present findings suggest that the hyperexcitability of GN neurons could be involved in abnormal noxious pain sensations with advancing age.

Organization of the ferret lateral cervical nucleus and cervicothalamic tract

To elucidate the organization of the ferret spinocervicothalamic pathway (SCTP), we examined the lateral cervical nucleus (LCN) and the termination of the cervicothalamic tract (CTT) in this species. In thionin-stained sections, the ferret LCN appeared as an easily delineated column of cells in the dorsolateral funiculus from about mid-C3 to the rostral end of C1, with most cells located in the C1 and C2 segments. In transverse sections, the LCN was elongated along a dorsolateral to ventromedial axis and in the rostral half of C2 and caudal half of C1 continuous with the neck of the dorsal horn. The number of ferret LCN cells was estimated to 2,500-3,700, with an average of 3,340. Substance P-like immunoreactive fibers located preferentially in the ventromedial part of the LCN, whereas serotonin-like immunoreactive fibers were found throughout the nucleus. Anterograde transport of wheat germ agglutinin-horseradish peroxidase conjugate and biotinylated dextran amine demonstrated that the ferret CTT terminates extensively in the peripheral parts of the ventral posterior lateral nucleus. Sparser termination was evident in the ventral posterior inferior nucleus, in the medial nucleus of the posterior complex, and in the medial part of the magnocellular medial geniculate nucleus. Thus, although the LCN is significantly smaller in ferrets than in cats and raccoons, the organization of the LCN and of the cervicothalamic tract is closely similar in the three species. These findings indicate a conserved general organization of the SCTP among carnivores.

Calcitonin gene-related peptide- and substance P-like immunoreactive fibers in the spermatic nerve and testis of the dog

In order to determine if calcitonin gene-related peptide (CGRP) and substance P (SP) coexist in peripheral spermatic nerve fibers, we carried out a double-staining immunofluorescence study using confocal microscopy and fluorescence microscopy. CGRP- and SP-like immunoreactivity (LI) coexisted in the spermatic nerve trunk and in the single fibers running along the surface of the testis. The great majority of the SP-containing fibers also held CGRP-LI, although some fibers contained CGRP-LI without SP-LI. These observations are consistent with previous observations on testicular dorsal root ganglion neurons. Additionally, we carried out an immunogold silver staining for CGRP and found CGRP-containing nerve bundles, single nerve fibers and their nerve terminals. Some CGRP-containing nerve terminals were located very superficially in the tunica albuginea (<5 μm from the surface).

Effect of celiac ganglionectomy on tachykinin innervation, receptor distribution and intestinal responses in the rat

Substance P (SP) is an important neurotransmitter in the control of intestinal motility and is found in both the enteric and sympathetic nervous systems. This study examined the effect of celiac ganglionectomy on (1) mechanical properties of the circular muscles of the duodenum, ileum and proximal colon, (2) circular muscle responses to SP and neurokinin A, (3) distribution of substance P-like immunoreactive nerves, and 4) the distribution of neurokinin 1 and neurokinin 2 receptors. Celiac ganglionectomy resulted in an effective sympathectomy as evidenced by a marked decrease in norepinephrine content and tyrosine hydroxylase staining in the duodenum, ileum and proximal colon. The in vitro length/tension characteristics of the circular muscle of the duodenum, ileum and colon were unchanged after ganglionectomy. In all regions of the gut studied, substance P and neurokinin A caused dose-dependent contractions that were unaltered by celiac ganglionectomy. Immunohistochemistry revealed moderate substance P-like immunoreactive fibers in the myenteric plexus, submucosal plexus and circular muscle of the ileum, while in the colon, substance P-like immunoreactivity was intense in the myenteric plexus, and moderate in the circular muscle. In vitro autoradiography showed minimal binding of SP (NK1 receptor) or neurokinin A (NK2 receptor) in the ileum and significantly greater binding in the circular muscle layer of the colon. Celiac ganglionectomy did not affect substance P-like immunoreactivity, or NK1 or NK2 receptor binding. A greater contractile response to neurokinins was seen in the colon than in the duodenum or ileum, which paralleled the receptor density. The studies demonstrate that surgical celiac ganglionectomy, unlike chemical sympathectomy, does not affect the substance P innervation, receptor density or physiological responses of the intestine. The greater contractile response of the colon than the ileum parallels the greater receptor density rather than the peptide content as determined by immunhistochemistry.

Central origins of substance P-like immunoreactive fibers and terminals in the spinal trigeminal caudal subnucleus in the rat

After trigeminal rhizotomy, some substance P-like immunoreactive (SP-LI) fibers and terminals in the spinal trigeminal caudal subnucleus (Vc), specially in its superficial laminae (laminae I and II), still remained in the rat. Employing a combination of Fluoro-Gold retrograde tracing and immunofluorescence histochemical staining for SP, we found that the main central origins of these SP-LI fibers and terminals were midbrain periaqueductal gray (PAG), nucleus raphe magnus (NRM) and other raphe nuclei, and nucleus reticularis gigantocellularis pars α; all of them are important structures of the endogenous pain control system. The present results provided morphological evidence for PAG or NRM stimulation could inhibit neuronal activities in the Vc evoked by orofacial nociceptive stimulation and also suggested that SP might be an important neurotransmitter or neuromodulator for endogenous pain control system.

610 Simultaneous determination of CCK-LI, dopamine and serotonin levels in the dialysates of rat frontal cortex after treatment with veratrine, NMDA and scopolamine

By using immunocytochemical method, the prenatal ontogeny of substance P-like immunoreactivity (SP-LI) was demonstrated in the dorsal motor nucleus of the vagus nerve (nX) and the nucleus tractus solitarii (nTS) of the human fetus at fetal age (menstruation age) of 11.5 weeks to 40 weeks. The time of initial appearance of SP-LI in the human brainstem nTS was between the fetal age 11.5 weeks and 16 weeks. At fetal age 16 weeks, the nTS showed moderate density of SP-LI fibers and terminals in subnucleus dorsalis of the nTS and nX. While the fetus grew, the density of SP-LI in the human fetus brainstem nTS and nX increased gradually from fetal age 16 weeks to 40 weeks. According to the Nissl staining, at fetal age 23 weeks, the nTS of human fetus can be subdivided into dorsal, medial, dorsolateral, ventrolateral and ventral gelatinosus subnuclei. The cytoarchitectonic subdivisions of human fetus nTS is in good agreement with the results obtained by immunocytochemical staining. These findings indicated that substance P (SP) might play an important role in the development of human brainstem nX, nTS, their related cranial nerves, and in their functional establishment during the pranatal period.

Tachykinin-related neuropeptides in the central nervous system of the snail Helix pomatia: an immunocytochemical study

The distribution of neurons reacting with an antibody raised against an insect neuropeptide, locustatachykinin I, was investigated in the CNS of the snail Helix pomatia. The localization of the neurons was compared with that of the substance P-like immunoreactive (SPLI) neurons in the different ganglia. Altogether, there are ∼ 800–1000 locustatachykinin-like immunoreactive (LomTKLI) neurons in the Helix CNS, occurring with an overwhelming dominancy (83.5%) in the cerebral ganglia. Within the cerebral ganglia, the majority of LomTKLI neurons were localized in the procerebrum. The number of SPLI neurons was high; ∼ 2000 SPLI nerve cells were found in the Helix CNS. The majority (44.5%) of SPLI neurons was also found in the cerebral ganglia and they were also concentrated in the procerebrum. The neuropils of all ganglia were densely innervated by both LomTKLI and SPLI fibers except the medullary mass of the procerebrum where only SPLI elements form an extremely dense innervation. In addition to the neuropil processes, LomTKLI neurons sent axon processes to the peripheral nerves. SPLI fibers also formed a dense network of varicose fibers in the connective tissue sheath around the ganglia where they innervated the blood vessel walls too. Immunolabeling on alternating cryostat sections revealed that LomTKLI and SPLI neurons are localized near each other in most cases; co-localization of the two immunoreactive materials could be seen in a very small number of neurons of the pedal and pleural ganglia. The present results show that the Helix CNS possesses distinct neuronal populations using different tachykinin-related peptides. It is suggested that the differential distribution of these neuropeptides also implies a diversity in their central and peripheral functions.

Distribution of substance P‐like immunoreactivity in the brain of the elasmobranch Scyliorhinus canicula

Immunohistochemical methods were used to study the distribution of substance P in the brain of the small-spotted dogfish (Scyliorhinus canicula). Substance P-like immunoreactive (SP-IR) cell bodies and fibers were widely distributed. In the telencephalon, sparse populations of SP-IR neurons are present in the olfactory bulbs, pallium, and subpallium. In the subpallium numerous SP-IR boutons form unusual coats ("pericellular appositions") on SP-immunonegative neurons. In the diencephalon numerous SP-IR cerebrospinal fluid-contacting neurons are present in the preoptic recess organ and organon vasculosum hypothalami. Numerous SP-IR fibers also run in the hypothalamus, although no immunoreactivity was observed in the habenulo-interpeduncular system. A terminal field of SP-IR fibers is present in the median eminence. In the mesencephalic tegmentum, SP-IR neurons were observed in the Edinger-Westphal nucleus. SP-IR fibers are present at high density in the basal tegmentum, forming a conspicuous tract. In the hindbrain, numerous SP-IR fibers were observed in the isthmal region, the trigeminal descending root, the visceral sensory area and commissural nucleus, and the visceromotor column. SP-IR fibers occur at high density in the substantia gelatinosa of the rostral spinal cord.

The development of non-retinal afferent projections to the frog optic tectum and the substance P immunoreactivity of tectal connections

Accessibility in early development and the presence of a retinotopic map have made the amphibian optic tectum a popular system for exploration of the role of synaptic function in central map refinement. Although a great deal is known about the development of retinal innervation of the tectum, little information exists about the development of non-retinal tectal inputs. Since these other afferent systems may contribute to the synaptic drive of developing tectal cells and thereby be involved in the activity-dependent refinement of the retinotectal map, we sought to determine whether these inputs are present at the early tadpole stages when the first retinal axons refine their synaptic order within the tectal neuropil. Rhodamine-labelled latex beads, retrogradely transported from injection sites in the optic tecta, were used to identify tectal afferent projections. Projection patterns in very young tadpoles were identical to those found in juvenile frogs and heavily labelled regions included areas of the posteroventral tegmental field, the posterior tuberculum, the ventromedial thalamic nucleus, the ventral part of the ventrolateral thalamic nucleus, the suprachiasmatic nucleus and discrete regions within the central and anterior thalamic nuclei. Ipsilateral nucleus isthmi cells were also labelled, indicating the existence of an isthmo-tectal projection in even the youngest animals examined. Additionally, substance P-like immunoreactive tecto-isthmal fibers were traced from the optic tectum to the nucleus isthmi. The presence of these connections suggests that feedback from the nucleus isthmi and/or input from the other brain areas projecting to the tectum may play a role in modulating the cellular mechanisms that underlie the formation of the visual map.

Prenatal ontogeny of substance P-like immunoreactivity in the nucleus tractus solitarii and dorsal motor nucleus of the vagus nerve of the human fetus: an immunocytochemical study

By using immunocytochemical method, the prenatal ontogeny of substance P-like immunoreactivity (SP-LI) was demonstrated in the dorsal motor nucleus of the vagus nerve (nX) and the nucleus tractus solitarii (nTS) of the human fetus at fetal age (menstruation age) of 11.5 weeks to 40 weeks. The time of initial appearance of SP-LI in the human brainstem nTS was between the fetal age 11.5 weeks and 16 weeks. At fetal age 16 weeks, the nTS showed moderate density of SP-LI fibers and terminals in subnucleus dorsalis of the nTS and nX. While the fetus grew, the density of SP-LI in the human fetus brainstem nTS and nX increased gradually from fetal age 16 weeks to 40 weeks. According to the Nissl staining, at fetal age 23 weeks, the nTS of human fetus can be subdivided into dorsal, medial, dorsolateral, ventrolateral and ventral gelatinosus subnuclei. The cytoarchitectonic subdivisions of human fetus nTS is in good agreement with the results obtained by immunocytochemical staining. These findings indicated that substance P (SP) might play an important role in the development of human brainstem nX, nTS, their related cranial nerves, and in their functional establishment during the pranatal period.

Prenatal ontogeny of substance P-like immunoreactivity in the parabrachial nucleus of the human fetus — an immunocytochemical study

Using an immunocytochemical method, the prenatal ontogeny of substance P-like immunoreactivity (SP-LI) was demonstrated in the parabrachial nucleus (PB) of human fetus, fetal age (menstruation age) 11.5 to 40 weeks. The time of initial appearance of SP-LI in the human brainstem PB was between fetal age 11.5 weeks and 16 weeks. While the fetus grew, the density of SP-LI fibers and terminals in the human fetus brainstem PB increased constantly from fetal age 16 weeks to 40 weeks. These findings indicate that substance P (SP) might play an important role in the human parabrachial nucleus development and in its functional establishment during the prenatal period.

The development of substance P-like immunoreactivity in the goldfish brain.

The development of substance P-like immunoreactivity (SPLI) in the goldfish brain was studied by means of the indirect peroxidase-antiperoxidase technique and an antibody to substance P. By 80 h after fertilization, the first SPLI-cell bodies appear in the ventricular zone of the future diencephalon and the first SPLI-fibers appear in the olfactory bulbs. Two days after hatching (which occurs at 100 h after fertilization), SPLI fibers connecting the olfactory bulbs and hypothalamus are seen. In the optic tectum SPLI-fibers appear for the first time 5 days after hatching. In the brain stem, SPLI-cell bodies appear in juvenile animals 40 days after hatching. The highest number and intensity of SPLI-cell bodies and fibers are found in the area postrema. SPLI-cell bodies are also seen in the gustatory nucleus, nucleus ambiguous, reticular formation of the medulla, dorsal motor nucleus of the vagus and commissural nucleus of Cajal. The significant information gained from the present study is: 1. The rostro-caudal sequence in which the SPLI appears in the developing nuclei of the goldfish brain 2. The reduction of SPLI-cell bodies in some nuclei with age Thus, in the brain stem, SPLI-cell bodies that were labeled in juvenile goldfish were not seen in adults. This might be due to changes in the rate of axonal transport, changes of the SP phenotype during development or cell death. The developmental sequence and relative timing in which SPLI-cell bodies appear in the goldfish, rat and mice are similar.

Neuropeptide Y- and substance P-like immunoreactive nerve fibers in the rat dura mater encephali

Density and pattern of nerve fibers with neuropeptide Y-like immunoreactivity (NPY-LI) and substance P-like immunoreactivity (SP-LI) in the rat dura mater encephali were investigated by light and electron microscopy using whole-mount preparations. NPY-LI fibers are observed throughout the encephalic dura mater. A remarkable net of NPY-LI nerve fibers is located in the walls of the sagittal and transverse sinuses. Beyond that NPY-LI network, distinct NPY-LI nerve fibers or plexus occur in the rostral falx, parietal dura mater of the olfactory bulb, supratentorial dura mater, parietal dura mater of the cerebellum, tentorium cerebelli and the ventral dura mater. Electron microscopic studies reveal that NPY-LI is exclusively located in unmyelinated axons of small and large nerve fiber bundles, with or without a perineural sheath. Immunopositive C-fibers are predominantly associated with the vascular bed. SP-LI nerve fibers have a moderate and more uniform distribution in the encephalic dura mater. A distinct plexus of SP-LI fibers follows the branches of the middle meningeal artery and the adjacent dura mater. SP-LI fibers are most prominent in the parietal dura mater of the cerebellum. Fine beaded SP-LI fibers, arising from larger SP-LI fiber bundles, are observed in close association to the capillary bed. SP-LI axons are all unmyelinated. They are found in larger nerve fiber bundles with a perineural sheath or in Schwann cells lacking any perineural sheath. The function of NPY-LI and SP-LI nerve fibers in the rat dura mater is discussed in relation to their topography, density and termination.

Sensory-motor neuromodulation of sympathetic vasoconstriction in the rabbit central ear artery

Histochemical and pharmacological studies were performed on the rabbit central ear artery. In perivascular nerves, positive immunoreactivity for calcitonin gene-related peptide and substance P was demonstrated. Calcitonin gene-related peptide-like immunoreactivity was also found to be colocalised with substance P-like immunoreactivity in a subpopulation of perivascular nerves. In vitro incubation with 6-hydroxydopamine did not alter the intensity and/or density of either the calcitonin gene-related peptide- or substance P-like immunoreactive fibres, whereas incubation with capsaicin significantly reduced both. In pharmacological studies, calcitonin gene-related peptide reduced the vasoconstrictor responses to exogenous noradrenaline and α,β-methylene ATP and to electrical field stimulation in a concentration-dependent manner. In segments of the central ear artery preconstricted with noradrenaline, relaxation mediated by calcitonin gene-related peptide was endothelium-independent. These results shed new light on the innervation and nervous control of the rabbit central ear artery previously thought to be exclusively under sympathetic (adrenergic and purinergic) control. Further, the results suggest that calcitonin gene-related peptide localised in sensory nerves in the rabbit central ear artery may act as an inhibitory modulator of excitatory sympathetic vascular neurotransmission.

Appearance of substance P-like immunoreactive neurons in the rat superior colliculus after neonatal eye enucleation

The possibility that eye enucleation within one day after birth has an effect on the postnatal development of substance P (SP)-like-immunoreactive (SP-I) structures in the superior colliculus (SC) was investigated in the rat. Results were compared with those in animals enucleated at postnatal day 15. All the animals were allowed to survive until postnatal day 90, after which changes in SP-I neurons and fibers were identified immunohistochemically. In colchicine-treated rats, the most remarkable changes occurred in SP-I neurons following eye enucleation at birth; large numbers of SP-I neurons appeared in the ventral part of the stratum griseum superficiale (SGS), stratum opticum (SO) and stratum griseum intermediale (SGI) of the deafferentated SC. SP-I neurons did not appear in these layers, when deafferentation of the SC was carried out in rats at postnatal day 15. These findings suggest strongly that eye enucleation at birth affects the production of SP of neurons in the ventral part of the SGS, SO and SGI at the deafferentated SC. The appearance of SP-I neurons in the neonatal eye enucleation may be due, at least partially, to reorganization of another neuronal system in the SC.

Distribution of substance P-like immunoreactive fibres and terminals in the medulla oblongata of the human infant

This study provides a detailed report of the distribution and density of substance P-like immunoreactive fibres and terminals within the human infant medulla. Seven brains with no signs of macroscopic alteration fixed usually within 24–48 h after death were used. Free floating transverse sections (50–60 μm) were then immunostained with a monoclonal antibody against substance P using the avidin-biotin-peroxidase technique. Morphologically three types of substance P-like immunoreactive fibres were observed: those with small varicosities of < 1 μm, those with medium varicosities of 1–2 μm and those with large varicosities of 2–4 μm. Very dense substance P-like immunoreactivity was present within the spinal trigeminal nucleus, parts of the gracile and cuneate fasciculi and the paracommissural subnucleus of the solitary nucleus; dense substance P-like immunoreactivity was present within the dorsal nucleus of the vagus nerve, commissural, medial, dorsal, dorsolateral ventral and ventrolateral subnuclei of the solitary nuclear complex, parasolitary nucleus, raphe obscurus, inferior olivary complex (medial, dorsal, dorsomedial nuclei) and ventrolateral part of the dorsal reticular nucleus; moderate substance P-like immunoreactivity was present within the gelatinosus nucleus of the solitary tract, lateral reticular nucleus proper, intermediate reticular zone and parvocellular reticular nucleus; sparse substance P-like immunoreactivity was present within the hypoglossal nucleus, retroambigual nucleus and much of the reticular formation (dorsal, parvocellular, ventral gigantocellular, dorsal paragigantocellular nuclei); and very sparse substance P-like immunoreactivity was present within the nucleus ambiguus, medial vestibular nucleus and parts of the reticular formation (ventral, medial, gigantocellular, ventral gigantocellular, dorsal paragigantocellular nuclei). Substance P-like immunoreactivity was absent in the area postrema, intercalated nucleus, gracile and cuneate nuclei, in the epiolivary, subtrigeminal and parvocellular divisions of the lateral reticular nucleus, spinal vestibular nucleus, and in the solitary and pyramidal tracts. In several regions the substance P-like immunoreactive fibres formed distinct pericellular arrays around the somata and dendrites of neurons (hypoglossal nucleus, dorsal nucleus of the vagus nerve, retroambigual nucleus, intermediate reticular zone). The results indicate the high specificity of the localization of substance P in various structures of the brainstem and underline the presumed significance of this peptide in autonomic and sensorimotor functions of the brain.

Convergence of serotonin-, enkephalin- and substance P-like immunoreactive afferent fibers onto cat medullary dorsal horn projection neurons: a triple immunocytochemical staining technique combined with the retrograde HRP-tracing method

Convergence of serotonin (5-HT)-, enkephalin (ENK)-, and substance P (SP)-like immunoreactive (LI) afferent fibers onto cat medullary dorsal horn projection neurons was examined by retrogradely labeling trigeminothalamic neurons and immunocytochemically labeling 5-HT, ENK and SP fibers in the same tissue section using distinct chromogens. Convergence of 5-HT-, ENK- and SP-LI fibers upon single projection neurons was often observed in laminae I and V.

Peptidergic regulation of secretory activity in amphibian olfactory mucosa: Immunohistochemistry, neural stimulation, and pharmacology

The role of substance P in the regulation of secretion from sustentacular cells, Bowman's glands and deep glands in the amphibian olfactory mucosa was investigated using immunohistochemical, electrophysiological, and pharmacological methods. Substance P-like immunoreactive varicose fibers extended through the olfactory epithelium, terminating at or near the surface. In addition, immunoreactive varicose fibers innervated Bowman's glands, deep glands, and blood vessels in the lamina propria. Innervation of Bowman's gland was sparse, with fibers terminating on basal acinar cell membranes; deep gland innervation was abundant, with fibers often extending between acinar cells almost to the lumen. Stimulation of the ophthalmic branch of the trigeminal nerve resulted in slow potentials recorded at the surface of the olfactory epithelium. When the olfactory mucosae from trigeminal-stimulated animals were examined histologically, morphological signs of secretory activity were observed, suggesting that substance P was released from the trigeminal nerve terminals. Topical application of 10(-5) to 10(-3) mol substance P resulted in morphological signs of secretion that were very similar to those seen as a result of trigeminal stimulation. Thus, substance P released from trigeminal fibers may modulate secretory activity within the olfactory mucosa.

Convergence of serotonin-, enkephalin- and substance P-like immunoreactive afferent fibers on single pudendal motoneurons in Onuf's nucleus of the cat: a light microscope study combining the triple immunocytochemical staining technique with the retrograde HRP-tracing method

Convergence of serotonin (5-HT)-, enkephalin (ENK)-, and substance P (SP)-like immunoreactive (LI) afferent fibers on single pudendal motoneurons within Onuf's nucleus was demonstrated: pudendal motoneurons were retrogradely labeled with horseradish peroxidase applied to the pudendal nerve. Subsequently, 5-HT-LI fibers were stained by the immunoperoxidase method, and then ENK- and SP-LI fibers were stained by the double immunofluoresence method.

Substance P-like immunoreactive fibres in the ventromedial mesencephalic tegmentum of rat

The distribution and morphology of the substance P-like immunoreactive (SP-IR) fibres and terminals in the rat ventromedial mesencephalic tegmentum (VMT) were studied using qualitative and quantitative immunohistochemical methods at light and electron microscopic levels. All five component nuclei of the VMT were examined and the size, number and density of immunoreactive terminals determined. The SP-IR fibres were distributed heterogeneously within the VMT. Under the electron microscope, SP-IR axon terminals contained both clear and densecored vesicles and made both symmetrical and asymmetrical synapses. The ultrastructure of the SP-IR terminals appeared to differ between nuclei. Small, clear vesicle terminals made symmetrical synaptic junctions with small calibre dendrites in the paranigral nucleus while large, clear and dense-cored vesicle terminals made asymmetrical junctions with somata and large calibre dendrites in the interfascicular nucleus. Quantitative differences between the VMT nuclei were also seen in the density of SP-IR terminals, the paranigral nucleus contained the highest density and the rostral linear nucleus the lowest. A comparison between the number of SP-IR terminals and the total number of axon terminals in the VMT reveals that the majority of all terminals in the paranigral nucleus were SP-IR, as well as the majority of axosomatic synapses in the interfascicular nucleus. These regional differences in the SP-IR innervation suggest that substance P and related peptides may perform several specific functions within the VMT and therefore have a more variable influence on this region than was previously thought.