Ultrastructural localization of substance P immunoreactivity in the ventral horn of the rat spinal cord

Summary At the light microscope level, the minute concentrations of substance P (SP) in rat spinal ventral horn can be visualized best by amplification with the double bridge PAP method of Vacca et al. (1975; 1980) in 5 m paraffin tissue sections. Morphologically, the immunoreactive sites resemble punctate bodies. They occur in close apposition with the large ventral horn cells and their associated neuronal processes. By the Sternberger PAP procedure, we now describe these punctate bodies at the electron microscope level. Ultrastructurally, they appear as tiny boutons (terminal and preterminal) and small unmyelinated processes. The boutons and processes typically contain one to several immunolabeled dense core vesicles among many immunolabeled clear vesicles. They range in size near the limit of resolution of the light microscope (LM), thereby justifying further the use of LM amplification staining by the double bridge method. The immunolabeled boutons often synapse with large smooth dendrites (which may originate from motoneurons) by asymmetrical or symmetrical synaptic densities. Their synaptic densities appear immunostained as well. The data support the view that the electrophysiological action of SP in the ventral horn occurs in part by synaptic action along the processes of the ventral horn cells. Other mechanisms of action are considered for the peptide as well. Additional types of membrane specializations (synaptoid junctions) and SP neural circuits are described below.The work, presented at the Histochemical Society's 29 Annual Meeting in Vancouver, B.C. April 1–2, 1978, was partially supported by CCHD 10-12-04-3600-67 (LLV)     

Ultrastructural localization of immunolabeled substance P and methionine-enkephalin-octapeptide in the surface layer of the dorsal horn of rat spinal cord

Summary A preembedding dual immunolabeling technique and electron microscopy were utilized to demonstrate the localization of immunoreactive substance P and methionine-enkephalin-octapeptide (Enk-8) in ultrathin sections of the surface layer (laminae I and II) of rat spinal dorsal horn. The immunoreaction of Enk-8 was visualized as goldtoned silver particles and that of substance P as diaminobenzidine reaction products. Axonal terminals with immunoreactive substance P, and also unlabeled axonal terminals, formed synaptic junctions with the perikarya and dendritic processes of Enk-8-containing neurons. Dendritic profiles immunolabeled for substance P were synaptically linked with unlabeled axons but not with Enk-8-positive ones. Furthermore, it was found that Enk-8 axons and substance P axons terminated synaptically in juxtaposition to one another on the same immunonegative dendrites. Among the Enk-8-containing neurons axonal profiles also appeared to be synaptically associated with immunoreactive Enk-8 dendritic processes.     

Intracellular pH regulation in ventral horn neurones cultured from embryonic rat spinal cord

Intracellular pH was measured with the pH-sensitive fluorescent probe BCECF in spinal cord neurones cultured from rat embryos. At an external pH of 7.3, the average steady-state pHi was 7.18 +/- 0.03 (SEM, n = 97) and 7.02 +/- 0.01 (n = 221) in HEPES-buffered and in bicarbonate-buffered medium, respectively. In both external media, pHi was strongly dependent on external pH (pHe). In HEPES-buffered medium, pHi recovery following an acid load induced by transient application of ammonium required external Na+ and was inhibited by amiloride, indicating the presence of a Na+/H+ exchange. Na(+)- and HCO3(-)-dependent, DIDS-sensitive alkalinizing mechanisms also contributed to pHi regulation in CO2/bicarbonate-buffered medium. The presence of an electrogenic Na(+)-HCO3- cotransporter was confirmed by the alkalinizing effect of KCl application. The fact that pHi is lower in CO2/bicarbonate- than in HEPES-buffered medium and the alkalinization observed upon suppression of external Cl- suggest that the acidifying Cl-/HCO3- transporter plays an important role in defining pHi.     

Intracellular pH regulation in ventral horn neurones cultured from embryonic rat spinal cord

Summary

We have isolated and characterized a cDNA from the marine sponge Geodia cydonlum coding for a new member of the tyrosine protein kinase (TK) family. The cDNA encodes a protein of M_r = 68 710, termed GCTK, which is homologous to class II receptor tyrosine kinases (RTKs). GCTK contains conserved amino acids (aa) characteristic of all protein kinases, and the sequences DLATRN and PIRWMATE which are highly specific for TKs. Furthermore, the sequence N-L-Y-x(3)-Y-Y-R Is highly homologous to the sequence D-[LIV]-Y-x(3)-Y-Y-R found only in class II RTKs. The sponge TK, when compared with mammalian class II RTKs, shows maximum 31% homology in the TK domain indicating that this the oldest member of class II RTK started to diverge from the common ancestral protein kinase 650 million years ago. Using GCTK as a probe we identified three mRNA signals ranging from 2μ6 to 0μ6 kb. Kinase activity was localized only in the cell membranes from G. cydonium (M_r = 65 000), and was not detected in the cytosol of this organism. Antibodies raised against a synthetic peptide, corresponding to the aa residues within the catalytic domain of the sponge TK, recognized strongly two proteins of M_r = 65 000; these proteins, present in membrane fractions, also bound to the anti-phosphotyrosine antibody. These data suggest that the TK cloned from the sponge is a membrane-associated 65 kDa protein. Moreover these results demonstrate that RTKs are present from the lowest group of multicellular eukaryotes, sponges, to mammals, and may suggest that RTKs are involved in a signal transduction pathway.     

Ultrastructural localization of uteroglobin immunoreactivity in rabbit lung and endometrium,and rat ventral prostate

Summary Recent biochemical studies have demonstrated amino acid sequence homologies between uteroglobin from rabbit endometrium and prostatic binding protein from rat ventral prostate. We have studied the ultrastructural distribution of uteroglobin-immunoreactive material in rabbit lung and endometrium and rat ventral prostate using an uteroglobin antibody raised in guinea pigs. Secretory granules of bronchiolar Clara cells, endometrial non-ciliated cells and rat prostate secretory cells gave a positive immunoreaction when this antibody was used. The results indicate a close relationship of immunoreactive epitopes of proteins present in those secretory cells. The functional properties of these proteins (glycoproteins, steroid binding, androgen-dependent secretion) suggest a close functional relationship, for instance a surface action such as coating, capping, masking or lubrication.Supported by grants Au 48/7-8 and Ki 154/9-3 from the Deutsche Forschungsgemeinschaft     

A modified peroxidase--antiperoxidase procedure for improved localization of tissue antigens: localization of substance P in rat spinal cord

A procedure is presented which modifies the Sternberger peroxidase--antiperoxidase (PAP) technique in order to visualize additional amounts of immunodeposits representing the antigen substance (SP) in 5-micrometer paraffin tissue sections of rat spinal cord. For increased sensitivity, the new procedure utilizes a "double bridge" and diaminobenzidine in low pH buffer. The modifications have made possible the visualization of immunoreactive beaded processes and punctate bodies, which were then traced to determine patterns of SP circuitry. Using the modified PAP procedure, the greatest number of immunoreactive processes appeared in the dorsal horn, where some punctate bodies and varicose processes could be seen adjacent to the myelinated afferent fiber bundles that penetrate the substantia gelatinosa as dorsal root collaterals. Additional immunoreactive processes and punctate bodies coursed through the myelinated afferent fiber bundles that penetrate the dorsolateral white matter, and extend into the intermediolateral gray region. Substance P was also identified within immunoreactive processes found in Rexed's laminae V and VI, as well as the central canal region, the dorsal gray commissure, and the ventral gray and white commissures. Since the modifications improved the visualization of SP-containing processes in sparsely populated regions of the spinal cord, especially the ventral horn, they may be useful in demonstrating other antigens that normally occur in small quantities within tissues.     

N-terminally extended substance P is released together with substance P from rat spinal cord.

The release of different forms of substance P-like immunoreactivity (SP-LI) from superfused slices of rat spinal cord was studied. The released SP-LI was characterized by reverse-phase high-performance liquid chromatography and radioimmunoassay with two antisera directed to the C- and N-terminal parts of SP, respectively. The SP-LI detected in the superfusates with the C-terminally directed antiserum was found to consist of (undeca) SP, SP-sulfoxide and a late eluting component which was not detectable with the N-terminally directed antiserum. This component was also found in neutral extracts of the spinal cord. Upon trypsin digestion, it produced SP-LI detectable with both C- and N-terminally directed antiserum which also coeluted with SP. From these results we conclude that this form of SP-LI most likely corresponds to an N-terminally extended form of SP. An increase of the potassium concentration in the superfusion fluid from 5 to 50 mM evoked an increased overflow of both SP and the N-terminally extended SP. The present results indicate that N-terminally extended SP is released by a calcium-dependent mechanism together with SP from terminals in the spinal cord in response to potassium stimulation.     

Immunocytochemical study of substance P containing nerve terminals in rat spinal cord

Summary The subcellular localization of substance P (SP) in the dorsal horn of the rat spinal cord was studied using the unlabelled antibody procedure of Sternberger with different fixatives (4% paraformaldehyde alone or with varying amounts of glutaraldehyde), buffer systems for the immunohistochemical incubations, and the presence or absence of the detergent, Trition X-100. Hand-sliced tissues were compared with Vibratome sections, and showed adequate results which are described below. Labelled terminals of two types could be seen in all samples incubated with anti-SP sera. The two types of positive terminals can be described as those which contained mostly immunoreactive clear vesicles, and those which contained both immunoreactive clear and dense core vesicles. Brief fixation during pressure perfusion with increased concentrations of glutaraldehyde (up to 2%) improved the tissue preservation and, as a result, the intensity and definition of the SP immunoreaction products. The use of Tris or phosphate buffer for the immunohistochemical incubations maintained the intensity of staining in well-fixed tissues. However, Tris incubations contributed to a diffusion of immunoreaction products and increased the number of broken membranes in the labelled processes as well as those of myelin. These phenomena were not observed in phosphate buffer, which preserved the tissue better than Tris. Like Tris, pretreatment with the detergent Triton X-100 (TX) contributed further to the diffusion of the immunoreaction products, and increased the number of broken membranes. For example,without TX, the outer membrane and envelope of the mitochondria became intensely and clearly labelled when phosphate buffer was used for incubations;with TX pretreatment, the staining was far more diffuse, and the intensity of staining became reduced such that only the mitochondrial outer surface appeared somewhat immunopositive. Using phosphate buffer alone, we observed well-defined immunoreaction products around the microtubules of many-containing processes. This finding was less clear in other preparations, especially those pretreatedwith TX. We therefore submit that the conditions of tissue fixation and incubation may influence greatly the data amassed by the technique of immunocytochemistry. Results must be evaluated in view of the methods chosed for each immunocytochemical study. Optimal technical conditions encourage new morphological findings, as shown below concerning the circuitry of SP neurons in the dorsal horn.This work was initiated while Dr. K. Kakudo was a postdoctoral Fellow in Anatomic Pathology, Medical College of Georgia, from Osaka University Medical School, JapanPresently at the Department of Anatomy, Tulane Medical School, New Orleans, Louisiana, USAThe work, presented at the Annual Meeting of the 31st Annual Meeting of the Histochemical Society held in New Orleans, April 11–15, 1980, was partially supported by BRSG 10-16-04-3611-23, CCHD10-12-04-3600-00 (LLV) and the Department of Pathology (address reprint requests to LLV)     

Effects of substance P analogues on spinal dorsal horn neurons

The effects of iontophoretically applied (D-Pro2, D-Phe7, D-Trp9)-SP and (D-Pro2, D-Trp7,9)-SP on the spontaneous and evoked activity of functionally identified cat spinal dorsal horn neurons have been investigated in vivo by means of extracellular single unit recording technique. In addition, the rat spinal cord slice preparation has been used to study the actions of (D-Pro2, D-Trp7,9)-SP and (D-Arg1, D-Pro2, D-Trp7,9, Leu11)-SP on the resting membrane potential of dorsal horn neurons and also on their responses to dorsal root stimulation and exogenous SP application. We have observed that both (D-Pro2, D-Phe7, D-Trp9)-SP and (D-Pro2, D-Trp7,9)-SP produced an excitation of about 15% of all neurons tested and had a weak antagonistic effect against SP in the cat spinal cord. (D-Pro2, D-Trp7,9)-SP suppressed the SP-induced excitation in 63% of examined cells. In addition, depression of the glutamate-induced excitation and spontaneous activity was evident in 10% and 19% of the cat dorsal horn neurons tested, respectively. In the spinal cord slice preparation (D-Arg1, D-Pro2, D-Trp7,9, Leu11)-SP proved to be a more potent antagonist of the SP-induced depolarization and the dorsal root-elicited slow depolarization, if compared with (D-Pro2, D-Trp7,9)-SP.     

Sciatic nerve transection decrease substance P immunoreactivity in the lumbosacral spinal cord of the frog (Rana catesbeiana)

Using immunohistochemistry and optical densitometry, substance P (SP) was investigated in the lumbar spinal cord of the frog Rana catesbeiana after sciatic nerve transection. In control animals, there was a high density of SP fibers in the Lissauer's tract and in the mediolateral band of the dorsal gray matter. Other SP immunoreactive fibers were observed in the dorsal part of the lateral funiculus and in the ventral horn. No SP label was found in any cell bodies. After axotomy, SP immunoreactive fibers decreased in the Lissauer's tract on the same side of the lesion. The other regions remained labeled. The changes were observed at 3 days following axonal injury and persisted at 5, 8 and 15 days. At 20 days, there was no significant difference between the axotomized side and the control one, thus indicating a recovery of the SP expression. These results indicate that the frog may be used as a model to study the effects of peripheral axotomy, contributing to elucidate the SP actions in the pain neuropath.     

Opioid modulation of capsaicin-evoked release of substance P from rat spinal cord in vivo

Capsaicin has been shown to evoke the release of substance P (SP) from small diameter primary afferent fibers. Using an in vivo perfusion of the rat spinal cord, this study examined the pharmacology of opioid receptor systems which modulate the capsaicin-evoked release of SP. The addition of capsaicin (200 μM) to the perfusate raised SP-like immunoreactivity (SP-LI) from resting levels of 31±5 to 74±14 pg/ml or an increase of 139% above the baseline. Using high pressure liquid chromatography (HPLC) the identity of the released SP-LI was determined to coelute primarily with authentic SP or the oxidized form of SP. Opioid receptor agonists were added to the perfusate and their ability to inhibit capsaicin-evoked release of SP-LI was assessed. Morphine (10–100 μM), DAGO (1–100 μM), DPLPE (10–100 μM), but not U50488H (100 μM) produced a dose-dependent reduction in the capsaicin-evoked release of SP-LI. Pretreatment with the opioid receptor antagonist naloxone (1 mg/kg, IP) had no effect on the basal or capsaicin-evoked release of SP-LI. Naloxone pretreatment was able to antagonize completely the opioid-produced inhibition of capsaicin-evoked SP-LI release. These data indicate that the release of SP from primary afferent fibers can be modulated by the activation of mu or delta but not kappa opioid receptors. Further, these data support the hypothesis that spinally administered mu and delta opioid agonists may produce their antinociceptive effect through the presynaptic inhibition of neuropeptide release from small diameter primary afferent fibers.     

Immunocytochemical study of the relations of acetylcholinesterase,enkephalin-, substance P-, choline acetyltransferase- and calcitonin gene-related peptide-immunoreactive structures in the ventral horn of rat spinal cord

Summary Calcitonin gene-related peptide (CGRP)-like immunoreactivity was localized immunocytochemically in the large motoneurons in the ventral horn of rat spinal cord. Using fluorescence double-labelling substance P (SP)-immunoreactive nerve fibres were found to surround both the CGRP-positive and negative motoneurons, whereas enkephalin (ENK)-immunoreactive fibres surrounded mainly CGRP-negative cells. All CGRP-like immunoreactive motoneurons were also choline acetyltransferase (ChAT)- and acetylcholinesterase (AChE)-positive. On the other hand a large population of ChAT- and AChE-positive motoneurons were devoid of CGRP-immunoreactivity. It is probable that CGRP/ChAT/AChE-positive cells surrounded by SP-positive fibres have different functions in motoric nervous system than the CGRP-negative ChAT/AChE-positive cells, which are surrounded by ENK-immunoreactive fibres.     

Neuronal organization of the medial part of the ventral horn of the cat spinal cord

An electron-microscopic study was made of the normal structure of the medial part of the ventral horn (Rexed's laminae VII and VIII) in the cervical portion of the cat's spinal cord, the region where fibers of reticulospinal and vestibulospinal tracts terminate. Neurons of this region can be divided on the basis of the density of their cytoplasmic matrix into "light" and "dark," the dark being much more numerous in this area (26% of the total number counted) than in other parts of the gray matter of the spinal cord. The mean diameter of the soma of the dark cells is smaller than that of the light cells, and it usually is 15–20 µ. Dendrites of the neurons can also be subdivided into "light" and "dark" respectively. The surface of the former is comparatively simple in shape with a small number of appendages and spine-like structures. On the surface of the dark dendrites there are many projections and irregularly shaped lacunae. The glial cells and their processes often completely cover the surface of the soma of the small neurons, and synaptic endings are found on it only where the dendrites leave the soma. Analysis of 1000 randomly chosen synaptic endings showed that 76.1% of them form axo-dendritic synapses, 14.2% axo-somatic, and 9.7% axo-axonal synapses. Of the total number of endings 50.9% contain spherical and 40.9% flattened synaptic vesicles. Some synaptic endings contain special structures under the postsynaptic membrane and have osmiophilic synaptic vesicles. The possible functional role of the pattern of neuronal organization revealed in this region is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 4, No. 2, pp. 176–183, March–April, 1972.     

Ultrastructural study of neurotensin immunoreactivity in the superficial laminae of the dorsal horn of the rat

Neurotensin immunoreactivity was identified in cell bodies, dendrites, spines, axons, terminals and varicosities in superficial laminae of rat spinal cord with the electron microscope. Unlabeled terminals synapsed with neurotensin-immunoreactive cell bodies, dendrites and spines. Presynaptic terminals contained round or pleomorphic vesicles and generally made symmetrical contacts with medium-sized neurotensin-containing dendrites in outer lamina II, and asymmetrical or symmetrical contacts with large and small dendrites and spines in inner lamina II. Neurotensin immunoreactive axons were unmyelinated, and their terminals were presynaptic to unlabeled dendrites and spines in laminae I and II. Terminals contained small, round, clear vesciles (31 nm) and occasional large granular vesicles (78 nm). Contacts in outer lamina II were evenly distributed among dendrites of various sizes and spines, whereas the majority of labeled terminals in inner lamina II made contacts onto small dendrites and spines. These findings indicate that neurotensin effects in rat spinal cord are mediated by axodendritic synapses, and that neurotensin cells at the inner and outer borders of lamina II contact dendrites of efferent neurons or other interneurons in the dorsal horn.     

Projections of afferent ventral root fibers on dorsal horn neurons in the cat spinal cord

Mechanisms of the effect of stimulation of afferent fibers in ventral roots on dorsal horn interneurons were investigated in experiments on anesthetized cats. Dorsal horn interneurons on which such fibers project were shown to exist. In particular, some dorsal horn interneurons can exert an inhibitory influence on effects of dorsal root fiber activation.Institute of Physiology, Academy of Sciences of the Kazakh SSR, Alma-Ata. Translated from Neirofiziologiya, Vol. 17, No. 3, pp. 300–305, May–June, 1985.