Substance P-immunoreactive Boutons Research Articles

Neurokinin-1 receptor localisation in guinea pig autonomic ganglia

We have used multiple-labelling immunohistochemistry and confocal microscopy to determine the distribution of immunoreactivity to the tachykinin neurokinin-1 (NK(1)) receptors in guinea pig sympathetic ganglia. Although nerve fibres containing immunoreactivity to substance P were common in all ganglia except the superior cervical ganglia, most neurons expressing NK(1) receptor immunoreactivity were not closely surrounded by pericellular baskets of substance P-immunoreactive boutons. Conversely, many neurons surrounded by baskets of substance P-immunoreactive boutons lacked NK(1) immunoreactivity. In the coeliac and inferior mesenteric ganglia, NK(1) receptor expression was restricted almost entirely to noradrenergic neurons that contained somatostatin immunoreactivity and projected to the enteric plexuses. In the lumbar chain and paracervical ganglia, NK(1) immunoreactivity was expressed by nonnoradrenergic vasodilator neurons containing immunoreactivity to vasoactive intestinal peptide. Taken together, our results show that sympathetic neurons in different functional pathways express NK(1) receptor immunoreactivity. However, the neurons that could respond to endogenously released substance P through NK(1) receptors may be distant from presynaptic release sites. These observations suggest that, in sympathetic ganglia, substance P may modulate ganglionic transmission through heterosynaptic actions on NK(1) receptors.

Substance P-immunoreactive boutons closely appose inspiratory protruder hypoglossal motoneurons in the cat

In anesthetized cats, we recorded intracellularly from 26 hypoglossal motoneurons which were antidromically activated following electrical stimulation of either the medial or lateral branches of the hypoglossal nerve. Twenty-one of these neurons were protruder motoneurons 6 of which had inspiratory activity. Three of the protruder motoneurons with inspiratory activity were filled with Neurobiotin and found to be closely apposed to substance P-like immunoreactive nerve terminals.

Ectopic substance P-immunoreactive boutons are preferentially presynaptic to neurokinin-1 receptor immunoreactive dendrites in the spinal white matter of transgenic mice

A recent immunocytochemical study has shown that substance P (SP) preferentially innervates targets expressing the neurokinin-1 receptor (NK-1r) in the superficial spinal dorsal horn of the rat. Based on these findings, we decided to further investigate the relationship between SP and the NK-1r in a transgenic mouse model in which SP fibres are ectopically located. Double-labelling immunocytochemistry at both the light and electron microscopic levels was performed to study the association between SP and the NK-1r in the spinal white matter of both control and transgenic mice. Light microscopy revealed NK-1r-immunoreactive (IR) dendrites in the white matter of the dorsolateral funiculus in both control and transgenic mice. In transgenic mice, but not in controls, SP-IR fibres were observed in close proximity to the NK-1r-IR dendrites in the white matter. At the ultrastructural level, SP-IR boutons were apposed to NK-1r-IR dendrites in the dorsolateral funiculus of transgenic mice, and a synapse was frequently observed as well. These results indicate that, even in conditions in which SP fibres are ectopically located, they still preferentially innervate targets expressing the NK-1r.

Preferential synaptic relationships between substance P-immunoreactive boutons and neurokinin 1 receptor sites in the rat spinal cord.

Substance P plays an important role in the transmission of pain-related information in the dorsal horn of the spinal cord. Recent immunocytochemical studies have shown a mismatch between the distribution of substance P and its receptor in the superficial laminae of the dorsal horn. Because such a mismatch was not observed by using classical radioligand binding studies, we decided to investigate further the issue of the relationship between substance P and its receptor by using an antibody raised against a portion of the carboxyl terminal of the neurokinin 1 receptor and a bispecific monoclonal antibodies against substance P and horseradish peroxidase. Light microscopy revealed a good correlation between the distributions of substance P and the neurokinin 1 receptor, both being localized with highest densities in lamina I and outer lamina II of the spinal dorsal horn. An ultrastructural double-labeling study, combining preembedding immunogold with enzyme-based immunocytochemistry, showed that most neurokinin 1 receptor immunoreactive dendrites were apposed by substance P containing boutons. A detailed quantitative analysis revealed that neurokinin 1 receptor immunoreactive dendrites received more appositions and synapses from substance P immunoreactive terminals than those not expressing the neurokinin 1 receptor. Such preferential innervation by substance P occurred in all superficial dorsal horn laminae even though neurokinin 1 receptor immunoreactive dendrites were a minority of the total number of dendritic profiles in the above laminae. These results suggest that, contrary to the belief that neuropeptides act in a diffuse manner at a considerable distance from their sites of release, substance P should act on profiles expressing the neurokinin 1 receptor at a short distance from its site of release.

Cells in Laminae III and IV of the Rat Spinal Cord that Possess the Neurokinin-1 Receptor and Have Dorsally Directed Dendrites Receive a Major Synaptic Input from Tachykinin-Containing Primary Afferents

Many neurons with cell bodies in laminae III or IV of the spinal dorsal horn possess the neurokinin 1 receptor and have dorsal dendrites that arborize in the superficial dorsal horn. We have performed a confocal microscopic study to determine whether these cells receive inputs from substance P-containing primary afferents. All neurons of this type received contacts from substance P-immunoreactive axons, and in most cases the contacts onto dorsal dendrites were very numerous. A great majority (90-100%) of substance P-immunoreactive varicosities in contact with these cells were also immunoreactive with antibody to calcitonin gene-related peptide, indicating that they were of primary afferent origin. The density of contacts from substance P-immunoreactive varicosities onto these cells was significantly higher than that seen on cholinergic neurons in lamina III (which do not possess the receptor). Electron microscopy revealed that synapses were present at points of contact between substance P-immunoreactive boutons and dorsal dendrites of cells with the neurokinin 1 receptor. Some cells of this type belong to the spinothalamic tract, and we therefore examined neurons with cell bodies in laminae III or IV that possessed the neurokinin 1 receptor and were labeled retrogradely after thalamic injection of cholera toxin B subunit. These cells also received contacts from substance P-immunoreactive axons on their dorsal dendrites. The results of this study indicate that neurons of this type are a major target for substance P-containing primary afferents.

Substance P and enkephalin immunoreactivities in axonal boutons presynaptic to physiologically identified dorsal horn neurons. An ultrastructural multiple-labelling study in the cat

A combination of intracellular electrophysiological recording and injection of horseradish peroxidase with ultrastructural immunocytochemistry was used to investigate the synaptic interplay between substance P- and enkephalin-immunoreactive axonal boutons and three types of functionally characterized dorsal horn neurons in the cat spinal cord. The dorsal horn neurons were classified as nociceptive specific, wide dynamic range and non-nociceptive based on their responses to innocuous and noxious stimuli. Most of the nociceptive neurons (either nociceptive specific or wide dynamic range) contained enkephalin immunoreactivity, but none of the non-nociceptive neurons were positive for enkephalin. Three types of immunoreactive boutons were found in contact with the functionally characterized dorsal horn neurons. These boutons were positive for either substance P, enkephalin, or substance P+enkephalin. Quantitative analysis revealed that the percentages of substance P-immunoreactive boutons apposed to the cell bodies, proximal dendrites and distal dendrites of nociceptive neurons were significantly higher than those of non-nociceptive neurons. Furthermore, the percentages of substance P+enkephalin-immunoreactive axonal boutons apposed to the distal dendrites of nociceptive neurons were significantly higher than those of non-nociceptive neurons and the percentages of enkephalin-immunoreactive boutons apposed to the cell bodies and proximal dendrites of nociceptive neurons were significantly higher than in non-nociceptive neurons. Finally, neither enkephalin-immunoreactive nor substance P+enkephalin-immunoreactive boutons were ever seen presynaptic to substance P-immunoreactive boutons. These results provide evidence of an anatomical substrate within the dorsal horn for the interaction of substance P-mediated with enkephalin-mediated mechanisms. The data support the idea that the modulation of nociceptive input in the dorsal horn by enkephalinergic neurons occurs mainly via a postsynaptic mechanism, and thus suggest that dorsal horn enkephalinergic neurons participate in a local inhibitory feedback loop in a distinct pathway from the previously postulated opioid-mediated depression of substance P release from primary afferent terminals.

Quantitative analysis of substance P-immunoreactive boutons on physiologically characterized dorsal horn neurons in the cat lumbar spinal cord.

A quantitative analysis of substance P (SP)-immunoreactive (IR) terminals contacting physiologically characterized dorsal horn neurons was performed. Three types of neuron were studied: nociceptive specific (NS) from lamina I (n = 3), wide dynamic range (WDR) from laminae II-IV (n = 3), and nonnociceptive (NN) from lamina IV (n = 3). The nociceptive response of focus was a slow, prolonged depolarization to noxious stimuli, because this response was previously shown to be blocked by selective neurokinin-1 (NK-1) receptor antagonists. Ultrastructural immunocytochemistry was used to quantify the relative number of SP-IR boutons apposed to the intracellularly labeled cell per unit of length (density). Densities of the total population (SP immunoreactive+nonimmunoreactive) of apposed boutons were similar in all three regions (cell body, proximal and distal dendrites) for the three functional types of neuron. NS neurons received a significantly higher density of appositions from SP-IR boutons than NN cells in all three regions. However, compared to WDR cells, NS cells possessed a significantly higher density of appositions from SP-IR boutons only in the cell body and proximal dendrites. WDR cells had a higher density of appositions from SP-IR boutons than NN cells, but only in the proximal and distal dendrites. On average, 33.5% of the SP-IR boutons apposed to the cells displayed a synaptic contact. Finally, 30-45% of the SP-IR boutons apposed to the cells colocalized calcitonin gene-related protein (CGRP) immunoreactivity, indicating their primary sensory origin. The data indicate a direct correlation between the amount of SP-IR input and the nociceptive nature of the cells and suggest that SP acts on NK-1 receptors at a short distance from its release site.

Distribution of substance P-immunoreactive neurons and fibers in the monkey hippocampal formation.

Substance P containing neurons was visualized by immunocytochemistry in the monkey hippocampus, subicular complex, and entorhinal cortex. Immunoreactive neurons were found solely in the hilar region of the dentate gyrus, and in strata oriens and pyramidale of Ammon's horn. In the subicular complex, immunoreactive neurons were located in those layers which were close to the alveus, whereas in the entorhinal cortex most of the substance P-positive neurons appeared in the second and third layers above the lamina dissecans. The majority of substance P-containing neurons were large multipolar cells, but small bipolar and multipolar cells also occurred in Ammon's horn, subiculum and entorhinal cortex. Dendrites of immunoreactive cells were smooth and displayed a few small, faintly stained spines which were hard to identify in the light microscopic preparations, but were visible with electron microscopy. Substance P-positive dendrites were exclusively found in the hilar region and never observed in the upper two-thirds of the molecular layer of the dentate gyrus. Moreover, immunoreactive dendrites rarely penetrated the stratum lacunosum-moleculare of Ammon's horn. In the electron microscopic preparations, somal and dendritic features of substance P-positive neurons were similar to those observed for GABAergic local circuit neurons. Axons of the substance P-immunoreactive local circuit neurons were thin and richly arborized in the upper two-thirds of the molecular layer of the dentate gyrus, in the stratum lacunosum-moleculare of Ammon's horn as well as in the subpial layers of the subicular complex and entorhinal cortex. Their terminals formed exclusively symmetric synapses with dendrites and spines. However, substance P-immunoreactive boutons were not found to make symmetric, axosomatic synapses on the granule cells of the dentate gyrus and very few were present on the pyramidal neurons of Ammon's horn, subicular complex, and entorhinal cortex. Hippocampal neurons, which were immunoreactive for substance P, also contained the neuropeptide somatostatin. However, not all of the somatostatin-containing neurons were substance P-immunoreactive. Thus, substance P-positive neurons are a subpopulation of somatostatin immunoreactive, GABAergic neurons. In conclusion, substance P-immunoreactive neurons are ideally suited for feed-back dendritic inhibition which may control the effectiveness of the main excitatory cortical input to the granule cells of the dentate gyrus and pyramidal neurons of the Ammon's horn.

Enkephalin-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal innervation of the ventrolateral dendritic bundle in the cat sacral spinal cord: An ultrastructural study

The distribution and synaptic arrangement of thyrotropin-releasing hormone-, substance P- and enkephalin-immunoreactive axonal boutons have been studied in the ventrolateral nucleus (Onuf's nucleus) of the upper sacral spinal cord segments in the cat. For this purpose, the peroxidase-antiperoxidase immunohistochemical technique was used. Immunoreactive axonal boutons were traced in complete series of sections in order to reveal synaptic contacts with the bundled dendrites of the ventrolateral nucleus. As judged from the cross-sectional diameter of the postsynaptic dendrites, the distribution of immunoreactive boutons was non-random. Enkephalin-immunoreactive axonal boutons, presumed to be mostly of segmental origin, displayed a rather restricted distribution to mainly (> 80%) medium-to-large dendrites. Thyrotropin-releasing hormone-immunoreactive boutons, that derive from supraspinal levels, were also found to impinge on medium-to-large dendrites (> 80%), indicating a proximal location within the dendritic trees. The skewness toward large postsynaptic dendrites was even more marked for thyrotropin-releasing hormone- than for enkephalin-immunoreactive boutons. Substance P-immunoreactive boutons, that are of either supraspinal or spinal origin, showed a more even distribution throughout the dendritic trees, including both thin distal branches and thick proximal dendrites. In view of the well-known fact that virtually all thyrotropin-releasing hormone-immunoreactive boutons in the ventral horn cocontain substance P (and serotonin) it was assumed that substance P-immunoreactive boutons in synaptic contact with the finest-calibre dendrites as well as most of those with a very proximal juxtasomatic location on the dendritic trees were of segmental origin, while those impinging on medium-to-large dendrites could be of either spinal or supraspinal origin. Fine-calibre dendrites (< 1 μm) represent about 25% of the dendritic branches in the ventrolateral nucleus, but receive, with the exception of substance P (8%), very little (< 3%) peptidergic or GABAergic (Ramírez-León and Ulfhake, 1993) input, although the degree of dendritic membrane covering by bouton profiles in the ventrolateral nucleus does not seem to vary much with the calibre of the postsynaptic dendrite (Ramírez-León and Ulfhake, 1993). Both substance P- and enkephalin-immunoreactive axonal boutons established synaptic contact with more than one dendrite. Furthermore, one and the same bouton could be found in contact with two dendrites that were coupled to each other by a dendro-dendritic contact of desmosomal or puncta adherentia type. This synaptic arrangement was, however, not seen among thyrotropin-releasing hormone-immunoreactive boutons, indicating that these axonal boutons act on a single postsynaptic element, while inputs intrinsic to the spinal cord can show a divergence also at the terminal level.

Substance P-containing hypothalamic afferents to the monkey hippocampus: an immunocytochemical, tracing, and coexistence study.

In order to identify the synaptic connections of substance P-containing afferents within the hypothalamo-hippocampal projection of the monkey, we performed a combined light and electron microscopic, immunocytochemical study, made lesions of the fimbriafornix, and employed retrograde tracing using WGA-HRP. Furthermore, coexistence studies for substance P and GAD were performed to identify the putative transmitters of these hypothalamic projection neurons. A plexus of large substance P-immunoreactive terminals was identified in both the innermost portion of the molecular layer and in CA2. Axon terminals in both plexuses established exclusively asymmetric synapses with spines and dendritic shafts. Substance P-immunoreactive boutons were degenerating 5 days after lesioning, and had disappeared 10 days after ipsilateral fimbria-fornix transection. Thus, these terminals were of extrinsic origin. In contrast, immunoreactive fibers in the outer third of the dentate molecular layer remained unaffected by the lesion. Retrograde tracing combined with immunostaining for substance P revealed the parent cell bodies of the extrinsic substance P-containing afferents in the supramammillary nucleus. Colocalization studies employing a consecutive semi-thin sections technique indicate that these large substance P-containing projection neurons lack GABA as an inhibitory transmitter. These results suggest that hypothalamic afferents of the monkey hippocampus contain substance P. Because these afferents lack GABA as an inhibitory transmitter and establish exclusively asymmetric synapses, this projection may excite hippocampal target neurons.

Substance P immunoreactive boutons form synapses with feline sympathetic preganglionic neurons.

In this study, the relationship between substance P-immunoreactive boutons and antidromically activated sympathetic preganglionic neurons was examined by light and electron microscopy. Sympathetic preganglionic neurons in the T2-T4 spinal segments of the cat were identified by intracellular recording and antidromic activation from the corresponding white ramus. Neurons were filled with lucifer yellow and then stained to reveal, simultaneously, substance P and lucifer yellow immunoreactivity. All of the neurons examined with the light microscope (n = 13) received appositions from substance P-immunoreactive boutons. Appositions were found on all parts of the neuron, including the somata, dendrites, and axon initial segment. In most cases (11/13) few close appositions were seen; however, two neurons received large numbers of appositions from substance P-immunoreactive boutons. On one neuron, 16 substance P-immunoreactive varicosities that were identified as being closely apposed at the light microscope level were serially sectioned and examined with the electron microscope. Of these 16 varicosities, eight either directly contacted the neuron or formed morphologically identifiable synapses. The remaining eight varicosities were separated from the neuron by thin glial processes. Two other sympathetic preganglionic neurons that were examined ultrastructurally also received substance P-immunoreactive synapses and close contacts. These findings suggest that substance P-containing nerve fibres could affect all sympathetic preganglionic neurons but are likely to be important in regulating the activity of only a small proportion of these neurons.

The GABA and substance P input to dopaminergic neurones in the substantia nigra of the rat

In order to examine the synaptic input to dopaminergic neurones in the substantia nigra from GABAergic terminals and terminals that contain substance P, double and triple immunocytochemical studies were carried out at the light and electron microscopic levels in the rat In a first series of experiments sections of the substantia nigra were incubated to reveal axon terminals containing either substance P or glutamate decarboxylase and then incubated to reveal dopaminergic neurones using tyrosine hydroxylase immunocytochemistry. Examination of this material in the light microscope revealed that the many substance P- and glutamate decarboxylase-immunoreactive boutons were associated with the dopaminergic cells. In the electron microscope it was found that the perikarya and dendrites of the dopaminergic neurones received symmetrical synaptic input from terminals that displayed immunoreactivity for substance P or glutamate decarboxylase. A small proportion of the substance P-positive boutons formed asymmetrical synapses. In a second series of experiments sections of the substantia nigra were processed by the pre-embedding immunocytochemical technique for tyrosine hydroxylase and then the post-embedding immunogold technique for gamma-aminobutyric acid (GABA). Examination in the electron microscope revealed that the tyrosine hydroxylase-positive neurones received symmetrical synaptic input from many GABA-positive terminals. Quantitative analyses demonstrated that a minimum of 50–70% of all boutons afferent to the dopaminergic neurones display glutamate decarboxylase or GABA immunoreactivity. Triple immunocytochemical studies i.e. pre-embedding immunocytochemistry for tyrosine hydroxylase and substance P, combined with post-embedding immunogold staining for GABA, revealed that some of the substance P-immunoreactive boutons that were in contact with the dopaminergic neurones also displayed GABA immunoreactivity. In a third series of experiments the combination of anterograde transport of lectin-conjugated horseradish peroxidase or biocytin with post-embedding GABA immunocytochemistry demonstrated that at least one of the sources of GABA-containing terminals in the substantia nigra is the striatum. The results of the present study: (1) demonstrate that dopaminergic neurones in the substantia nigra receive symmetrical synaptic input from GABAergic and substance P-containing terminals, (2) show that a proportion of these terminals contain both substance P and GABA and (3) suggest that the major synaptic input to dopaminergic neurones is from GABAergic terminals and that a part of this innervation is derived from the striatum.

Cholinergic neurons of the chicken ciliary ganglion contain somatostatin

Somatostatin immunoreactivity was studied in the avian ciliary ganglion by immunocyto-chemistry and radioimmunoassay. Immunoreactivity was localized to small diameter cell bodies of neurons from embryos, newly-hatched and adult preparations. Immunostaining of ganglia with a mixture of antisera to substance P and monoclonal antibody to somatostatin indicated that a number of somatostatin-immunoreactive neurons were surrounded by substance P-immunoreactive boutons, which characteristically terminate on choroidal neurons. Staining with a mixture of antisera to choline acetyltransferase and antibody to somatostatin showed that the somatostatin-immunoreactive neurons were less intensely-stained for choline acetyltransferase than were the neurons lacking somatostatin immunoreactivity. Bundles of nerve fibers showing somatostatin and choline acetyltransferase immuno-reactivity were found in the choroid layers of the eye. Radioimmunoassay indicated the presence of somatostatin immunoreactivity in both chick and quail ganglia; the somatostatin immunoreactivity eluted from high pressure liquid chromatography in the same positions as authentic somatostatin 14 and 28. These results show that somatostatin is contained in cholinergic choroidal neurons in the chick and quail ciliary ganglion. Gray B. and Pilar G. [(1987) Soc. Neurosci. Abstr. 13, 790] have recently reported that somatostatin inhibits the release of acetylcholine from a preparation of smooth muscle from the choroid coat.

The postsynaptic targets of substance P-immunoreactive terminals in the rat neostriatum with particular reference to identified spiny striatonigral neurons

Substance P-immunoreactive boutons were examined in the electron microscope in sections of the rat neostriatum that contained retrogradely labelled striatonigral neurons and/or Golgi-impregnated medium-size densely spiny neurons. The post-synaptic targets of the immunoreactive boutons were characterized on the basis of ultrastructural features, their projection to the substantia nigra and/or their somato-dendritic morphology. Substance P-immunoreactive axonal boutons formed symmetrical synaptic specializations. Of a total of 233 randomly identified synaptic boutons 72.5% made contact with dendritic shafts, 15% with dendritic spines and 10.7% with perikarya. The ultrastructural characteristics of some of the postsynaptic neuronal perikarya were consistent with their identification as striatal interneurons. Similarly, the observation of some of the substance P-containing terminals in contact with spines, spine-bearing dendritic shafts and perikarya with the ultrastructural characteristics of medium-size densely spiny neurons suggested that one of the targets of substance P-positive terminals are striatal projection neurons. Direct evidence for this was obtained in sections from rats that had received injections of horseradish peroxidase conjugated with wheatgerm agglutinin in the substantia nigra. The perikarya of retrogradely labeled striatonigral neurons were found to receive symmetrical synaptic input from substance P-positive boutons. Ultrastructural analysis of Golgi-impregnated medium-size densely spiny neurons, some of which were also retrogradely labeled from the substantia nigra, demonstrated directly that this class of neuron was postsynaptic to the substance P-immunoreactive boutons. The combination of Golgi-impregnation with substance P-immunocytochemistry made it possible to study the pattern or topography of the substance P-positive input to medium size densely spiny neurons. The substance P-containing boutons made contact predominantly with perikarya and dendritic shafts. This pattern of input is markedly different from that of other identified inputs to medium-size densely spiny neurons.

An ultrastructural study of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal boutons in the motor nucleus of spinal cord segments L7-S1 in the adult cat

The distribution and fine structure of 5-hydroxytryptamine-, thyrotropin-releasing hormoneand substance P-immunoreactive synaptic boutons and varicosities were studied in the motor nucleus of the spinal cord segments L7-S1 in the cat, using the peroxidase-antiperoxidase immunohistochemical technique and analysis of ultrathin serial sections. The 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive boutons had a similar ultrastructural appearance as judged from serial section analysis. The boutons could be classified into two types on the basis of their vesicular content, with one type containing a large number of small agranular vesicles together with only a few, if any large granular vesicles, while the other type contained a large number of large granular vesicles in addition to small agranular vesicles. The vesicles were spherical or spherical-to-pleomorphic. Postsynaptic dense bodies (Taxi bodies) were occasionally observed in relation to all three types of immunoreactive boutons, which almost invariably formed synaptic junctions with dendrites. Judged by the calibre of the postsynaptic dendrites, the boutons were preferentially distributed to the proximal dendritic domains of motoneurons. In one case, a substance P-immunoreactive bouton formed an axosomatic synaptic contact. In addition to synaptic boutons, 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal varicosities containing a large number of large granular and small agranular vesicles but lacking any form of conventional synaptic contact were observed. Such varicosities were either directly apposing surrounding neuronal elements or separated from the neurons by thin glial processes. The origin of the immunoreactive boutons was not traced, but it was thought likely that the main source of the boutons was neurons with their cell bodies located in the medullary raphe nuclei.

Substance P-Containing terminals in synaptic contact with cholinergic neurons in the neostriatum and basal forebrain: a double immunocytochemical study in the rat

Antibodies against substance P and choline acetyltransferase (ChAT) have been used in a sequential double-immunocytochemical ultrastructural study of the rat forebrain. The peroxidase—anti-peroxidase procedure was used for both antigens, however, two different substrates for the peroxidase reactions were used. The substance --immunoreactive sites were first localized using 3,3′-diamino-benzidine as the substrate, then the ChAT-immunoreactive sites were localized using benzidine dihydrochloride. The reaction product formed by the two substrates was distinguishable in both the light and electron microscopes. Using this procedure, the cell bodies and proximal dendrites of identified cholinergic neurons in the neostriatum were found to receive symmetrical synaptic input from substance P-immunoreactive boutons. A similar pattern of substance P-immunoreactive synaptic input was observed onto magnocellular basal forebrain cholinergic neurons in the ventral pallidum and ventromedial globus pallidus. In both the striatum and basal forebrain substance P-immunoreactive boutons were also seen in contact with structures that did not display ChAT immunoreactivity.