Substance P-immunoreactive Terminals Research Articles

Connections between EM2- and SP-containing terminals and GABAergic neurons in the mouse spinal dorsal horn

Endomorphin-2 (EM2) demonstrates a potent antinociceptive effect in pain modulation. To investigate the potential interactions of EM2- and substance P (SP)-containing primary afferents and γ-amino butyric acid (GABA)-containing interneurons in lamina II in nociceptive transmission, connections between EM2- and SP-containing terminals and GABAergic neurons in the spinal dorsal horn were studied. Double-immunofluorescent labeling showed that approximately 62.3 % of EM2-immunoreactive neurons exhibited SP-immunostaining, and 76.9 % of SP-immunoreactive neurons demonstrated EM2-immunoreactivities in the dorsal root ganglion (DRG). Dense double-labeled EM2- and SP-immunoreactivities were mainly observed in lamina II of the lumbar dorsal horn. Furthermore, triple-immunofluorescent labeling results revealed that EM2 and SP double-labeled terminals overlapped with GABAergic neurons. Immuno-electron microscopy confirmed that the EM2- or SP-immunoreactive terminals formed synapses with GABA-immunoreactive dendrites in lamina II of the lumbar dorsal horn. During noxious information transmission induced by formalin plantar injection, GABAergic neurons expressing FOS in their nuclei were contacted with EM2- or SP-immunoreactive terminals. These results suggest that the interactions between EM2- and SP-containing terminals and GABAergic interneurons in the lamina II influence pain transmission and modulation in the spinal dorsal horn.

Relationships between neurokinin receptor-expressing interstitial cells of Cajal and tachykininergic nerves in the gut.

The so-called interstitial cells of Cajal (ICC) are distributed throughout the muscle coat of the alimentary tract with characteristic intramual location and species-variations in structure and staining. Several ICC sub-types have been identified: ICC-DMP, ICC-MP, ICC-IM, ICC-SM. Gut motility is regulated by ICC and each sub-type is responsible for the electrical activities typical of each gut region and/or muscle layer. The interstitial position of the ICC between nerve endings contain tachykinins. Three distinct tachykinin receptors (NK1r, NK2r and NK3r) have been demonstrated by molecular biology. Each of them binds with different affinities to a series of tachykinins (SP, NKA and NKB). In the ileum, SP-immunoreactive (SP-IR) nerve fibers form a rich plexus at the deep muscular plexus (DMP), distributed around SP-negative cells, and ICC-DMP intensely express the SP-preffered receptor NK1r; conversely a faint NK1r-IR is detected on the ICC-MP and mainly after receptor internalization was induced by agonists. ICC-IM are never stained in laboratory mammals, while those of the human, antrum are NK1r-IR. RT-PCR conducted on isolated idleal ICC-MP and gastric ICC-IM showed that these cells express NK1r and NK3r. Colonic ICC, except those in humans, do not express NK1r-IR, at least in resting conditions. Outside the gut, NK1r-IR cells were seen in the arterial wall and exocrine pancreas. In the mouse gut only, NK1r-IR is present in non-neuronal cells located within the intestinal villi, so-called myoid cells, which are c-kit-negative and α-smooth muscle actin-positive. Immunohistochemistry and functional studies confirmed that ICC receive input from SP-IR terminals, with differences between ICC sub-types. In the rat, very early after birth, NK1r is expressed by the ICC-DMP and SP by the related nerve varicosities. Studies on pathological conditions are few and those on mutant strains practically absent. It has only been reported that in the inflamed ileum of rats the NK1r-IR ICC-DMP disappear and that at the peak of inflammatory conditions ICC-MP are NK1r-IR. In ileum of mice with a mutation in the W locus, ICC-DMP were seen to express c-kit-IR but not NK1-IR, and SP-IR innervation seems unchanged. In summary, there are distinct ICC populations, each of them under a different tachkininergic control and, likely, having different functions. Further studies are recommended at the aim of understanding ICC involvement in modulating/transmitting tachykininergic inputs.

μ-Opioid Receptors Often Colocalize with the Substance P Receptor (NK1) in the Trigeminal Dorsal Horn

Substance P (SP) is a peptide that is present in unmyelinated primary afferents to the dorsal horn and is released in response to painful or noxious stimuli. Opiates active at the mu-opiate receptor (MOR) produce antinociception, in part, through modulation of responses to SP. MOR ligands may either inhibit the release of SP or reduce the excitatory responses of second-order neurons to SP. We examined potential functional sites for interactions between SP and MOR with dual electron microscopic immmunocytochemical localization of the SP receptor (NK1) and MOR in rat trigeminal dorsal horn. We also examined the relationship between SP-containing profiles and NK1-bearing profiles. We found that 56% of SP-immunoreactive terminals contact NK1 dendrites, whereas 34% of NK1-immunoreactive dendrites receive SP afferents. This result indicates that there is not a significant mismatch between sites of SP release and available NK1 receptors, although receptive neurons may contain receptors at sites distant from the peptide release site. With regard to opioid receptors, we found that many MOR-immunoreactive dendrites also contain NK1 (32%), whereas a smaller proportion of NK1-immunoreactive dendrites contain MOR (17%). Few NK1 dendrites (2%) were contacted by MOR-immunoreactive afferents. These results provide the first direct evidence that MORs are on the same neurons as NK1 receptors, suggesting that MOR ligands directly modulate SP-induced nociceptive responses primarily at postsynaptic sites, rather than through inhibition of SP release from primary afferents. This colocalization of NK1 and MORs has significant implications for the development of pain therapies targeted at these nociceptive neurons.

Immunohistochemical localization of substance P and substance P receptor (NK1) in the olivary pretectal nucleus of the rat.

The olivary pretectal nucleus (OPN) is the first central nucleus in the pupillary light reflex arc (PLR). Substance P (SP) is a neuropeptide present in the OPN. The present immunohistochemical study, performed at the ultrastructural level, aimed to determine the synaptic localization of SP and SP receptor in the OPN. Three types of SP-positive terminals were found. The most abundant type was of retinal origin, characterized by electron-lucent mitochondria and round vesicles, organized in glomerular structures, making asymmetric synaptic contacts with dendrites, and profiles containing pleomorphic vesicles, also making synaptic contacts with dendrites. The second type of SP-immunoreactive terminal contained electron-dense mitochondria and pleomorphic vesicles. This type made symmetric synaptic contacts and may originate from the ventral part of the lateral geniculate nucleus. The third type of SP-immunoreactive terminals contained electron-dense mitochondria, clear round vesicles, and made an asymmetric synaptic contact. This type originates from the contralateral OPN. SP receptors of the NK1 subtype were revealed to be on dendrites and were part of the glomerular-like arrangement. On account of the present observations, it can be concluded that retinal projections to the OPN use SP as a neuromodulator and synapse on NK1 receptor-containing dendrites of large neurons projecting to the Edinger-Westphal nucleus. Since SP also modulates the parasympathetic component of the PLR, we postulate that SP plays a modulating role in all components of the PLR.

Dual ultrastructural localization of mu-opiate receptors and substance p in the dorsal horn.

Opiates active at the mu-opiate receptor (MOR) produce antinociception, in part, through actions involving substance P (SP), a peptide present in both unmyelinated primary afferents and interneurons within the dorsal horn. We examined potential functional sites for interactions between SP and MOR by using dual electron microscopic immunocytochemical localization of antisera against SP and a sequence-specific antipeptide antibody against MOR in rat cervical spinal dorsal horn. The distribution was compared with that of the functionally analogous dorsal horn of the trigeminal nucleus caudalis. Many of the SP-immunoreactive terminals in the dorsal horn contacted dendrites that contain MOR (53% in trigeminal; 70% in cervical spinal cord). Conversely, within the cervical spinal dorsal horn 79% of the MOR-labeled dendrites that received any afferent input were contacted by at least one SP-containing axon or terminal. Although SP-immunoreactive dendrites were rare, many of these (48%) contained MOR, suggesting that the activity of SP-containing spinal interneurons may be regulated by MOR ligands. A few SP-labeled terminals also contained MOR (12% in trigeminal; 6% in cervical spinal cord). These data support the idea that MOR ligands produce antinociception primarily through modulation of postsynaptic second-order nociceptive neurons in the dorsal horns of spinal cord and spinal trigeminal nuclei, some of which contain SP. They also suggest, however, that in each region, MOR agonists can act presynaptically to control the release of SP and/or glutamate from afferent terminals. The post- and presynaptic MOR sites are likely to account for the potency of MOR agonists as analgesics.

Ultrastructure of substance P-immunoreactive terminals and their relation to vascular smooth muscle cells of rat small mesenteric arteries

Mesenteric arteries of the rat are surrounded by a plexus of primary afferent nerve terminals which contain both substance P (SP) and calcitonin gene-related peptide (CGRP). The ultrastructural arrangement of the innervation was studied in second-order branches of the rat mesenteric artery using immunohistochemical labelling with antibodies against SP. The structure and distribution of SP-immunoreactive (SP+) and SP-negative (SP−, i.e., virtually all noradrenergic) axons and their terminals within the adventitia of the artery have been determined. Sixteen percent of axons and 22% of varicosities in the perivascular plexus were SP+. Most of the SP+ varicosities lay between 0.4 and 2 μm from the smooth muscle cells, whereas most SP− varicosities lay much closer to the vessel (i.e., <1 μm). SP+ varicosities typically contained the same number and size of small synaptic vesicles and mitochondria as SP− varicosities, but there were more large dense-cored vesicles in the SP+ varicosities. Unlike SP− varicosities, the peptidergic varicosities did not show clustering of synaptic vesicles toward one part of the axon membrane, and none of them formed junctions with the smooth muscle cells. Close relationships between SP+ and SP− varicosities lacked any detectable structural specialization. The arrangement of SP+ (primary afferent) terminals and their association with vascular smooth muscle cells indicates that peptide released from afferent terminals must diffuse further than noradrenaline from sympathetic terminals to reach the vascular smooth muscle. J. Comp. Neurol. 416:277–290, 2000. © 2000 Wiley-Liss, Inc.

Ultrastructure of substance P-immunoreactive terminals and their relation to vascular smooth muscle cells of rat small mesenteric arteries

Mesenteric arteries of the rat are surrounded by a plexus of primary afferent nerve terminals which contain both substance P (SP) and calcitonin gene-related peptide (CGRP). The ultrastructural arrangement of the innervation was studied in second-order branches of the rat mesenteric artery using immunohistochemical labelling with antibodies against SP. The structure and distribution of SP-immunoreactive (SP+) and SP-negative (SP-, i.e., virtually all noradrenergic) axons and their terminals within the adventitia of the artery have been determined. Sixteen percent of axons and 22% of varicosities in the perivascular plexus were SP+. Most of the SP+ varicosities lay between 0.4 and 2 microm from the smooth muscle cells, whereas most SP- varicosities lay much closer to the vessel (i.e., <1 microm). SP+ varicosities typically contained the same number and size of small synaptic vesicles and mitochondria as SP- varicosities, but there were more large dense-cored vesicles in the SP+ varicosities. Unlike SP- varicosities, the peptidergic varicosities did not show clustering of synaptic vesicles toward one part of the axon membrane, and none of them formed junctions with the smooth muscle cells. Close relationships between SP+ and SP- varicosities lacked any detectable structural specialization. The arrangement of SP+ (primary afferent) terminals and their association with vascular smooth muscle cells indicates that peptide released from afferent terminals must diffuse further than noradrenaline from sympathetic terminals to reach the vascular smooth muscle.

Ultrastructural evidence that substance P neurons form synapses with noradrenergic neurons in the A7 catecholamine cell group that modulate nociception

Potent antinociception can be produced by electrical stimulation of spinally projecting noradrenergic neurons in the A7 catecholamine cell group and this effect is blocked by intrathecal injection of α 2-adrenoceptor antagonists. Microinjection of substance P near A7 neurons also produces antinociception that is blocked by intrathecal injection of α 2-adrenoceptor antagonists. These observations suggest that substance P produces antinociception by activating noradrenergic A7 neurons. However, it is not known whether this effect of substance P is produced by a direct or an indirect action on A7 neurons. Although light microscopic studies have demonstrated the existence of both substance P-containing axon terminals and neurokinin-1 receptors in the region of the A7 cell group, it is not known whether substance P terminals form synapses with noradrenergic A7 neurons. These experiments used double-labeling immunocytochemical methods and electron microscopic analysis to determine whether substance P-containing axons form synapses with noradrenergic neurons in the A7 cell group. Pre-embedding immunocytochemistry, combined with light and electron microscopic analysis, was used to provide ultrastructural evidence for synaptic connections between substance P-immunoreactive terminals labeled with immunoperoxidase and tyrosine hydroxylase-immunoreactive A7 neurons labeled with silver-enhanced immunogold. Tyrosine hydroxylase labeling was found in perikarya and dendrites in the A7 region, and substance P labeling was found in axons and synaptic terminals. Substance P-labeled terminals formed asymmetric synapses with tyrosine hydroxylase-labeled dendrites, but only a few of these were present on tyrosine hydroxylase-labeled somata. Substance P-labeled terminals also formed asymmetric synapses with unlabeled dendrites, and many unlabeled terminals formed both symmetric and asymmetric synapses with tyrosine hydroxylase-labeled dendrites. These results demonstrate that substance P neurons form a significant number of synapses with the dendrites of noradrenergic A7 neurons and support the conclusion that microinjection of substance P in the A7 cell group produces antinociception by direct activation of spinally projecting noradrenergic neurons.

Control of negative inotropic vagal preganglionic neurons in the dog: synaptic interactions with substance P afferent terminals in the nucleus ambiguus?

Previous research from this laboratory has shown that substance P-immunoreactive (SP) terminals synapse upon negative chronotropic vagal preganglionic neurons (VPNs), but not upon negative dromotropic VPNs, of the ventrolateral nucleus ambiguus (NA-VL). Moreover, SP agonists injected into NA-VL cause bradycardia without decreasing AV conduction. In the current study, we have: (1) defined the electron microscopic characteristics of the SP neurons of NA-VL in dog; and (2) tested the hypothesis that SP nerve terminals synapse upon negative inotropic VPNs of NA-VL, retrogradely labeled from the cranial medial ventricular (CMV) ganglion. Numerous SP terminals and a few SP neurons were observed in the vicinity of retrogradely labeled neurons. SP terminals were observed forming synapses with unlabeled dendrites and with SP dendrites, but never with the retrogradely labeled neurons. Together, these results and earlier findings suggest that SP agonists may be able to induce bradycardia without decreasing AV conduction or ventricular contractility.

Synaptic Connections Between Spiny Neurons of the Direct and Indirect Pathways in the Neostriatum of the Rat: Evidence from Dopamine Receptor and Neuropeptide Immunostaining

The flow of cortical information through the basal ganglia occurs through the so-called 'direct pathway' and 'indirect pathways'. The object of the present work was to attempt to determine whether spiny neurons in the neostriatum that give rise to the direct pathway (i.e. the striatonigral/entopeduncular pathway) and those giving rise to the indirect pathways (i.e. striatopallidal pathway) are synaptically interconnected. The approach was to carry out double immunocytochemistry at the electron microscopic level using antibodies against peptides or dopamine receptor subtypes that are selectively associated with the neurons that give rise to the direct (substance P or D1 receptors) and indirect pathways (enkephalin or D2 receptors). Sections of perfuse-fixed rat neostriatum were immunostained to reveal both substance P immunoreactivity and D2 receptor immunoreactivity or enkephalin and D1 receptor immunoreactivity, respectively. Double peroxidase methods were employed using different chromogens that were distinguishable at both the light and electron microscopic levels. In the electron microscope substance P-immunoreactive terminals were seen in synaptic contact with dendritic structures that displayed immunoreactivity for D2 receptor. Similarly, enkephalin-immunoreactive terminals were seen in synaptic contact with D1-immunoreactive dendritic structures. Thus, axon collaterals of neurons giving rise to the direct pathway form synaptic contacts with neurons that give rise to the indirect pathway and axon collaterals of neurons giving rise to the indirect pathway form synaptic contact with neurons that give rise to the direct pathway. These results indicate that the two pathways of information flow through the basal ganglia are synaptically linked at the level of the neostriatum. spiny neurons, direct pathway,indirect pathways, rat neostriatum

Substance P innervation of neurons projecting to the paraventricular hypothalamic nucleus in the rat nucleus tractus solitarius

After injection of WGA-HRP-colloidal gold in the rat paraventricular nucleus (PVN), retrogradely labeled neurons were found mainly in the medial and commissural subnuclei of the nucleus tractus solitarius (NTS) around 0.5 mm caudal to the obex which is closely related to cardiovascular function. Electron microscopic immunohistochemistry in these areas demonstrated synaptic contacts between retrogradely labeled neurons and substance P-immunoreactive terminals. Innervation of NTS-PVN projection systems by substance P is suggested.

Evidence for a preferential loss of enkephalin immunoreactivity in the external globus pallidus in low grade Huntington's disease using high resolution image analysis

Previous studies have shown that in advanced cases of Huntington's disease, enkephalin-immunoreactive striatal projections to the external globus pallidus may be more affected than substance P-containing striatal projections to the inner segment of the pallidum [Reiner A. et al. (1988) Proc. natn. Acad. Sci. U.S.A. 85, 5733-5737]. Other immunohistochemical [Ferrante R. J. et al. (1990) Soc. Neurosci. Abstr. 16, 1120] and neurochemical observations [Storey E. and Beal M.F. (1993) Brain 116, 1201-1222] suggest no difference in the loss of these peptide-containing pathways in Huntington's disease. In view of the potential significance of this issue for understanding the neuropathological process in Huntington's disease, we examined the globus pallidus in control and Huntington's disease brains, using a quantitative approach which involved high resolution image analysis of 7 microns frozen sections to determine the overall density of peptide-immunoreactive terminals. Results showed that in the controls there was no significant difference between the density of enkephalin- and substance P-immunoreactive terminals in the external and internal globus pallidus, respectively. In all Huntington's disease brains, including grade 1 cases, enkephalin-immunoreactive terminals in the external globus pallidus were significantly reduced compared to substance P-positive boutons in the internal segment of the adjacent section. In comparison to controls, enkephalin immunoreactivity in all Huntington's disease cases was significantly lower; substance P-immunoreactive terminals in the internal globus pallidus were significantly lower than controls in some of the grade 2 cases and in the grade 3 cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptic morphology of substance P terminals on catecholamine neurons in the commissural subnucleus of the nucleus tractus solitarii in the rat.

The ultrastructure of substance P-containing nerve terminals synapsing on catecholamine neurons in the rat commissural subnucleus of the nucleus tractus solitarii (NTScom) was studied using a double immunocytochemical labeling technique. Although there were numerous tyrosine hydroxylase-immunoreactive (TH-I) somata present, substance P immunoreactive (SP-I) cell bodies were only occasionally found in the NTScom. At the light microscopic level, many SP-I terminals were seen closely associated with TH-I dendrites and somata. At the electron microscopic level, SP-I terminals synapsing on TH-I structures were also readily encountered. SP-I terminals contained small, clear, and predominantly spherical vesicles (32 +/- 4 nm diameter), as well as large dense-cored vesicles approximately 100 nm in diameter. Postsynaptic TH-I dendritic profiles of various calibers and somata were encountered. These postsynaptic TH-I structures often showed postsynaptic densities. The morphological features of the SP-TH synapses in the present study, that is, the size of synaptic vesicles and the presence of postsynaptic densities, are quite different from those of central carotid sinus afferent synapses reported in our previous study [Chen et al. (1992), J. Neurocytol., 21:137-147]. Therefore, most of the SP terminals of the SP-TH synapses in the NTScom appear not to originate from the carotid sinus afferents. SP-I second-order neurons of the carotid sinus afferent pathway [Chen et al. (1991), J. Auton. Nerv. Syst., 33:97-98] may be one of the possible sources of such terminals.

Substance P nerve terminals synapse upon negative chronotropic vagal motoneurons

Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic ganglia on the surface of the heart which are capable of selective control of sino-atrial rate, atrio-ventricular conduction, and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates heart rate (the SA ganglion). Medullary tissues were processed for the histochemical visualization of retrogradely labeled neurons and for the immunohistochemical detection of the neurotransmitter substance P (SP) by dual labeling light and electron microscopic methods. Negative chronotropic retrogradely labeled cells were found in a long slender column in the ventrolateral nucleus ambiguus (NA-VL) which enlarged somewhat at the level of the area postrema. These cells were found bilaterally, but they were asymmetrically distributed. Half the animals showed a pronounced right side predominance in retrograde labeling, while the other half of the animals showed a lesser left side predominance. These observations may help to explain some of the controversy in the literature concerning the relative influence of the right and left vagus nerves on sinus rate. Ultrastructural examination demonstrated axo-somatic and axo-dendritic contacts between SP nerve terminals and retrogradely labeled negative chronotropic NA-VL neurons. SP immunoreactivity was often associated with large dense-core vesicles in terminals forming either symmetric or asymmetric synapses. These observations provide a potential anatomical substrate for the centrally mediated bradycardia elicited by microinjections of SP into the NA. SP immunoreactive terminals were also observed to make axo-somatic, axo-dendritic, and axo-axonic synapses with unlabeled neurons in NA-VL. These data suggest that SP may also modulate the activity of other vagal preganglionic neurons.

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.

Sprouting of remaining substance P-immunoreactive fibers in the monkey dentate gyrus following denervation from its substance P-containing hypothalamic afferents.

This study analyzed the response of intrinsic substance P-immunoreactive fibers in the monkey dentate gyrus to disruption of the supramammillo-hippocampal projection. This projection normally forms a thin plexus of large, substance P-immunoreactive terminals in the innermost portion of the dentate molecular layer and establishes exclusively asymmetric synapses with dendritic shafts and spines of dentate neurons. Conversely, substance P-containing terminals have never been observed in synaptic contact with granule cell bodies. Ten days after ipsilateral fimbria-fornix transection, the prominent band of large immunostained axons in the inner molecular layer of the ipsilateral fascia dentata disappeared. Four and five weeks following transection, however, some small, substance P-containing terminals were observed in the innermost portion of the dentate molecular layer and the granule cell layer. These terminals established exclusively symmetric synapses with the somata and proximal dendritic shafts of granule cells. These results suggest that, following transection of the hypothalamo-hippocampal fiber tract, presumptive intrinsic substance P-containing axons are capable of sprouting into the granule cell layer and the former termination field of the hypothalamic fibers. The symmetric synapses established with granule cell bodies and their proximal dendrites might indicate a shift from an extrinsic excitation to an intrinsic inhibition of granule cells following disruption of substance P-containing hypothalamic afferents.

Synaptic connections of central carotid sinus afferents in the nucleus of the tractus solitarius of the rat. II. Connections with substance P-immunoreactive neurons.

A combined transganglionic transport and immunocytochemical technique was used to study the synaptic morphology of central carotid sinus afferents and substance P-immunoreactive neurons in the commissural subnucleus of the nucleus of the tractus solitarius in rats. A large population of substance P-immunoreactive neurons (88.32%) were seen in close association with central carotid sinus afferents by light microscopy. However, many labelled central carotid sinus afferents appeared not associated with substance P-immunoreactive neurons in the nucleus of the tractus solitarius. Substance P-immunoreactive neurons were spindle, pear, or oval-shaped with a short axis ranging from 5 to 11 microns. Their long axis was oriented predominantly in a lateral-medial direction along the path of the central carotid sinus afferents from the solitary tract to the midline. Synaptic contacts between central carotid sinus afferents and substance P-structures, including dendritic profiles of different calibers and somas, were readily found by electron microscopy. Many central carotid sinus afferents were also found in synaptic contact with non-immunoreactive dendrites and somas. Appositions between central carotid sinus afferents and unlabelled axon terminals were common, but only in a few cases were morphological manifestations of synapses revealed. In the latter, the substance P-immunoreactive terminals appeared mostly presynaptic but postsynaptic ones were also encountered. Our data provide the evidence that some of the substance P-immunoreactive cells in the nucleus of the tractus solitarius are 2nd order neurons of the carotid sinus afferent pathway. The possibility that some of the substance P-immunoreactive neurons in the nucleus of the tractus solitarius may be interneurons and mediate carotid sinus afferent inputs to catecholaminergic neurons in the nucleus of the tractus solitarius is considered. Our findings also provide an anatomical substrate for a possible presynaptic modulatory role of central carotid sinus afferents on the inputs from other brain centers to the substance P-neurons in the nucleus of the tractus solitarius.

Substance P synaptic interactions with GABAergic and dopaminergic neurons in rat substantia nigra: An ultrastructural doublelabeling immunocytochemical study

The distribution of substance P (SP), tyrosine hydroxylase (TH), and glutamic acid decarboxylase (GAD) immunoreactivity in the substantia nigra of the rat was studied by means of an ultrastructural double-labeling immunocytochemical method. Direct synaptic contact between SP-immunoreactive terminals and GAD-positive nigral neurons was more often observed in the pars lateralis than the pars reticularis and was rarely observed in the pars compacta. Substance P-positive terminals also formed synapses with cell bodies and dendrites of TH-positive, dopaminergic neurons in the pars compacta and pars reticulata. Multiple SP-immunoreactive terminals were often observed with symmetrical and, less frequently, asymmetrical synapses on individual TH-containing dendrites. Evidence of SP-containing terminals contacting both GABAergic and dopaminergic neurons in the substantia nigra suggests a direct excitatory action upon nigral projection neurons.

Ultrastructural characterization of substance-P-immunoreactive synaptic terminals in the cat's normal and rhizotomized trigeminal subnucleus caudalis.

Deafferenting injuries often cause transient or permanent physiological alterations within the central projection field of affected primary afferent fibers. Aberrant sensory perceptions, dysesthesias, and hyperalgesias represent the clinical sequelae of such injuries; however, the results of experimental deafferentations have been subject to a variety of interpretations (Rodin and Kruger, 1984b). Neurochemical studies show an increased sensitivity of partially deafferented neurons to substance P (SP). Our previous studies (Hoffmann et al., 1991) documented, primarily at the light-microscopic level, a moderate transient loss of SP-immunoreactive (SPIR) boutons in the trigeminal subnucleus caudalis (Vc)--a loss that seemed to preferentially affect the slightly larger, possibly complex boutons with multiple contacts. However, despite the elimination of the trigeminal input, the larger boutons reappeared. In the present study, therefore, we examined Vc using electron-microscopic immunocytochemistry, in order to document these changes over time and to clarify the structure and relationships of this population of boutons. SPIR boutons occurred in lamina I and II degrees of the substantia gelatinosa of Vc, ranged in size from 1 to 5 microns in diameter, and displayed mixed populations of clear and dense-core vesicles. Most formed single or multiple axodendritic junctions, but a significant number engaged in axoaxonic contacts with both SPIR-labeled and unlabeled terminals. A small number appeared to be the central element of a typical glomerulus, particularly in lamina II degrees. Three to seven days following an ipsilateral retrogasserian rhizotomy, synaptic degeneration was evident in the substantia gelatinosa and often involved glomerular terminals. However, most of these were SPIR-negative and occurred primarily in lamina II degrees. Those SPIR boutons that displayed degenerative features often made single or multiple axodendritic contacts, and in some instances were scalloped. By 30 days, most remaining SPIR boutons were small, with a lower incidence of contacts; however, some of these were axoaxonic. In addition, many SPIR terminals were only very lightly stained--a feature not encountered to such an extent in the contralateral Vc. At 45 days, complex SPIR boutons were again evident in the field, and some showed densely packed vesicles. An increased incidence of clusters of two to four SPIR axoaxonic contacts was also observed. Finally, almost all SPIR boutons encountered at this stage were intensely stained. It is suggested that these alterations represent a compensatory neuroplastic response on the part of overlapping cervical and cranial primary afferents to the partial deafferentation resulting from the interruption of the trigeminal root.(ABSTRACT TRUNCATED AT 400 WORDS)

Plant‐Derived Neurotoxic Amino Acids (β‐N‐Oxalylamino‐l‐Alanine and β‐N‐Methylamino‐l‐Alanine): Effects on Central Monoamine Neurons

In the present study the subacute effects of beta-N-oxalylamino-L-alanine (BOAA) and beta-N-methylamino-L-alanine (BMAA) on CNS monoamine neurons in rats were investigated following intracisternal injections or local intracerebral administration into substantia nigra. In vitro effects of BOAA and BMAA on high-affinity synaptosomal uptake of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) were also examined. Intracisternal administration of BMAA decreased NA levels in hypothalamus, whereas no effects were seen on DA or 5-HT levels. Following intranigral injections of BOAA, NA levels tended to decrease in several regions, whereas the DA levels and the levels of DA metabolites were unaffected in all regions analyzed. Loss of tyrosine hydroxylase (TH) immunoreactivity in the intranigral injection sites and the presence of TH-immunoreactive pyknotic neurons near the borders of the injection sites were observed following both BOAA and BMAA treatments. Furthermore, substance P-immunoreactive terminals in substantia nigra pars reticulata were also found to have disappeared within the lesioned area following either BOAA or BMAA injections. Incubations with both BOAA and BMAA (10(-5) M) reduced high-affinity [3H]NA uptake in cortical synaptosomes to 69% and 41% of controls, respectively, whereas the striatal high-affinity [3H]DA uptake and the cortical high-affinity [3H]5-HT uptake were unaffected by BOAA or BMAA. The results demonstrate that both BOAA and BMAA can affect central monoamine neurons, although the potency and specificity of these substances on monoamine neurons when administered acutely into cerebral tissue or liquor cerebri seem to be low. However, the in vitro studies indicate selective effects of both compounds on NA neurons in synaptosomal preparations.