Immunohistochemical evidence for the presence of somatostatin-containing sensory nerve fibres in the human skin

Somatostatin-like immunoreactivity has been found to occur in nerve terminals and fibres of the normal human skin using immunohistochemistry. The immunoreactivity seemed predominantly to be associated with sensory nerves. Thus, nerve fibres displaying weak or moderately strong somatostatin immunofluorescence were observed as free nerve endings of the epidermis and as nerve fibres in the dermis. The immunoreactive nerves were found in low numbers in skin from arm, back and chest, however, only single fibres were seen in sensory specialized areas, such as fingertips. Very few fibres were found close to e.g. blood vessels and sweat glands. Somatostatin, generally believed to be a strongly inhibitory substance, should also be considered when examining the role of different sensory chemical messenger candidates in the human skin.     

Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat

The localization of two small peptides, somatostatin and substance P, has been studied with the indirect immunofluorescence technique. Both peptides were present in small neuronal cell bodies in spinal ganglia, in fibers in the dorsal horn of the spinal cord and in fibers in the intestinal wall. By comparing consecutive sections incubated with antisera to somastostatin and to substance P respectively, it was established that somatostatin, or somatostatin-like immunoreactivity and substance P, or substance P-like immunoreactivity are present in different cells. This is possibly indicated also by a somewhat differential distribution of the immunoreactive fibers in the dorsal horn: the highest concentration of somatostatin-positive fibers was observed in lamina II, whereas abundant substance P-positive fibers were present also in lamina I. Furthermore, numerous substance P-, but no somatostatin-positive fibers, were found around the central canal and in the ventral horns. In the intestinal wall more substance P-positive than somatostatin-positive fibers were seen.The present results indicate that two subpopulations of primary sensory neurons exist, one containing somatostatin, or somatostatin-like immunoreactivity, and the other containing substance P, or substance P-like immunoreactivity.     

Immunohistochemical evidence for an ACTH-like substance in hypothalamic LH-RH neurons

A protein immunochemically related to ACTH was detected in guinea pig hypothalamus using antibodies against synthetic 17–39 ACTH. The strict comparison of both anti-17–39 ACTH and anti-LH—RH stainings enabled to conclude that this substance is localized in LH—RH neurons. The presence of a 17–39 ACTH-like substance in hypothalamic neurons agrees with other investigations which indicate that the hypophysis is probably not the sole source of the brain ACTH-like material.     

Immunohistochemical localization of neurocalcin in human sensory neurons and mechanoreceptors

The localization of neurocalcin in the developing and adult human peripheral nervous system (dorsal root and sympathetic ganglia (DRG, SG), and enteric nervous system (ENS)) was investigated using immunohistochemistry. A subpopulation of large-sized neurons in DRG of 9 and 12 weeks old embryos showed immunoreactivity (IR), whereas the sympathetic ganglia or enteric neurons did not. In adults, neurocalcin IR was restricted to a subpopulation of large (13%) and intermediate (15%) sized neurons in DRG. The protein was also found in muscular (67%) and cutaneous (12%) nerve fibers, as well as in the axons supplying muscular (muscle spindles, Golgi's tendon organs, and perimysial Pacinian corpuscles) and cutaneous (Meissner's but not Pacinian corpuscles) mechanoreceptors, as well as motor end-plates. Present results demonstrate that neurocalcin in both developing and adult humans can be used as a specific marker for a subpopulation of sensory neurons coupled to proprioception and touch, and for axons of motoneurons forming motor end-plates.     

Immunohistochemical evidence for a new group of catecholamine-containing neurons in the basal forebrain of the monkey

Using a specific antibody to the catecholamine (CA) synthesizing enzyme, tyrosine hydroxylase (TH), in combination with the avidin-biotin-peroxidase complex method, we have found evidence for the existence of a new CA-containing cell group extending from the orbitofrontal cortex through the olfactory and pyriform cortices in the brain of two species of monkey. The TH-positive perikarya, which are 4000-5000 in number, are situated within the outer layers of these cortices and also within the olfactory tubercle and horizontal limbs of the diagonal band of Broca. They have small (10-20 microns) somata of round or oval shape. A majority are bipolar with long, slender dendrites but some are small, multipolar with widely branching dendrites. The shape and laminar distribution of these TH-positive neurons suggest that they may serve functions as cortical interneurons.     

Immunohistochemical evidence for the presence of oxytocin in the opossum corpus luteum.

Corpora lutea from opossums late in pregnancy were examined by immunohistochemistry for the presence of oxytocin. Oxytocin-immunoreactivity was observed in all corpora lutea examined but not elsewhere in ovarian tissue. The immunoreactive staining observed was confined primarily to the perinuclear cytoplasm of reactive luteal cells. Not all luteal cells showed oxytocin-immunoreactivity. The immunohistochemical localization of oxytocin in the pregnant opossum corpus luteum demonstrates for the first time this peptide in a metatherian ovary. Its presence in this primitive species suggests that oxytocin has a fundamental role in the physiology of the mammalian ovary.     

Immunohistochemical evidence for the vascular origin of primary adrenal pseudocysts

Pseudocysts are the most common nonfunctioning cystic adrenal lesions associated with symptoms. We have studied tissue from two patients with large adrenal pseudocysts in whom no antecedent cause could be documented. Immunohistochemical examination of these formaldehyde-fixed paraffin-embedded tissues with antibodies directed against the major proteins of the basement membrane (laminin and type IV collagen) revealed intense linear staining surrounding the cystic spaces and at the compressed adrenal cortical/pseudocyst interface. These findings lead us to conclude that these lesions are vascular in nature and, therefore, closely related to the previously recognized adrenal cysts of endothelial origin.     

Coexistence of peptide immunoreactivity in sensory neurons of the cat

The coexistence of the neuropeptides substance P, cholecystokinin, somatostatin and vasoactive intestinal polypeptide in cat sensory neurons has been examined using peroxidase-anti-peroxidase immunocytochemistry. Attempts were also made to locate cells containing bombesin, neurotensin, [Met]enkephalin and [Leu]enkephalin but no immunoreactivity was found when antisera to these peptides was used. Cells in the dorsal root ganglia were studied by cutting 5 microns serial wax sections or 15 microns cryostat sections. Coexistence was established by applying the antiserum to each peptide to serially adjacent 5 microns sections and establishing the presence of peptide-like immunoreactivity in each of 4 different sections through a single cell. Results showed that the distribution and combinations of coexistence of these neuropeptides in the cat is extremely complex; three and sometimes all four antisera showing immunoreactivity with a single cell. About 21% of all ganglion cells contained some immunoreactivity but there were certainly some small cells which did not contain any immunoreactivity. The coexistence of these peptides differed markedly from that previously reported in the rat suggesting that interspecific differences in the neuropeptide content of cells might be much greater than they are for classical neurotransmitters. The results are discussed in relation to the possible role of neuropeptides and the regulation of their production by sensory neurons.     

Further evidence of bilateral cervical projection from the snout primary sensory neurons by HRP method

This report is concerned with a counter-proof of the bilateral pathway of the transganglionic degeneration fibers projecting to the cervical segment following the transection of the infraorbital nerve of the rat and mouse.     

Direct evidence for presynaptic inhibitory mechanisms in crayfish sensory afferents.

1. The central control of sensory inputs from a proprioceptor [chordotonal organ (CO)] in the second joint [coxo-basipodite (CB)] of the fifth leg was studied in crayfish in vitro preparations (Fig. 1A). Simultaneous intracellular recordings from CBCO terminals (CBT) and postsynaptic motoneurons (MNs) were performed along with micropipette pressure ejection or bath application of gamma-aminobutyric acid (GABA), to study the presynaptic mechanisms at work in the CBT (Fig. 1B). 2. Two intracellular recordings were used to show that the spikes never overshoot, and that the more central the recording site within the neuropile, the smaller the spikes (Fig. 2). Only electrotonic conduction occurs, therefore, in the sensory afferents within the ganglion. 3. Pressure ejection of GABA close to the recording site of CBTs in the ganglion (Fig. 3A) gave rise to a membrane depolarization, the reversal potential of which was about -25 mV (Fig. 7), as well as to an increase in the membrane conductance (Fig. 3C) and a decrease in the orthodromic spike amplitude; moreover, it did not elicit either hyperpolarization, or any change in the membrane conductance of the postsynaptic MN (Fig. 3B), which indicates that pressure ejection of GABA affected only a restricted area around the CBT and not the postsynaptic MNs. 4. In CBT, spontaneous primary afferent depolarizations (PADs) occurred irregularly when the activity of the preparation was not rhythmic (Fig. 4A), and in bursts when the preparation displayed fictive locomotion (Fig. 4B). In the latter case, antidromic spikes were sometimes superimposed on PADs (Fig. 4D). The amplitude of the PADs was reduced when picrotoxin (PTX), a GABA antagonist, was applied (Fig. 5), which suggests that GABA may be involved in spontaneous PADs. The reversal potential of PADs was about -25 mV (Figs. 6 and 7). 5. During simultaneous recordings from a CBT and a monosynaptically related MN, GABA applied by pressure ejection close to the CBT (Fig. 8A) completely suppressed the excitatory postsynaptic potentials (EPSPs) elicited by CBT spikes in the MN (Fig. 8, B and D). This was due to a presynaptic mechanism because no change in the membrane potential or membrane conductance was observed in the MN (Fig. 8C) and most of the CBTs associated with a given MN were affected (Fig. 9). 6. Simultaneously recording from a CBT and a monosynaptically related MN demonstrated that, during bouts of PADs, the spike amplitude decreased in proportion to the PAD amplitude (Fig. 10A).(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunohistochemical evidence of substance P-like immunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervous system.

With the indirect immunofluorescence technique of Coons and collaborators a possible coexistence of 5-hydroxytryptamine (5-HT) and substance P in neurons of the lower medulla oblongata was explored. Antisera to 5-HT and to dopadecarboxylase (aromaticl-aminoacid decarboxylase), an enzyme probably present in immunologically indistinguishable forms both in catecholamine and 5-HT neurons, were used as markers for 5-HT neurons and an antiserum raised to synthetic substance P conjugated with bovine serum albumin for substance P-containing neurons. Five or 10 μm thick, consecutive sections were stained with the three antisera. Numerous cell somata in nucleus raphe magnus, nucleus raphe obscurus, nucleus raphe pallidus, pars α of the nucleus reticularis gigantocellularis and nucleus interfascicularis hypoglossi contained both substance P-like immunoreactivity and 5-HT (and dopadecarboxylase) immunoreactive material. After intraventricular or intracisternal injections of 5,6- or 5,7-dihydroxytryptamine, two neurotoxins assumed to cause degeneration mainly of 5-HT neurons, enlarged substance P and 5-HT (and dopadecarboxylase) positive fibres were seen in, around and lateral to the olivary complex. Furthermore, in these rats both substance P and 5-HT positive nerve terminals in the ventral horns of the spinal cord disappeared.These findings indicate that substance P and 5-HT may coexist not only in some cell bodies but also in axons and nerve endings. The latter conclusion must, however, remain tentative since the neurotoxins may cause unspecific damage and thus not only damage 5-HT (and postulated ‘SP-5-HT’) neurons.In further experiments reserpine was used, a drug known to deplete monoamines by affecting their storage sites. With a high dose of reserpine a marked depletion of 5-HT was obtained both in nerve terminals and cell bodies whereas substance P immunoreactive material seemed unaffected. Possible interpretations of these findings is that substance P and 5-HT have different storage sites within the neuron, or that reserpine selectively causes loss of 5-HT and not substance P from the same storage site.     

Pharmacological evidence that somatostatin activates the m-current in hippocampal pyramidal neurons

In the in vitro hippocampal slice somatostatin has been shown to cause a direct hyperpolarization of CA 1 pyramidal neurons by increasing a potassium conductance which is resistant to blockade by tetraethylammonium 4-aminopyridine, or cesium ions. Results reported here demonstrate that this somatostatin-induced hyperpolarization is blocked by 1 mM barium and 5 x 10(-5) M carbachol, with the action of carbachol being reversed by atropine. Barium and carbachol both inactivate the m-current and these results suggest that somatostatin may exert its hyperpolarizing action on CA1 pyramidal cells by activation of the m-current.     

Immunocytochemical evidence for the localization of the GM1 ganglioside in carbonic anhydrase-containing and RT 97-immunoreactive rat primary sensory neurons

Summary Localization of GM1 ganglioside, the receptor for cholera toxin, and choleragenoid, which is the binding subunit of cholera toxin, was studied in the rat L5 dorsal root ganglion. Sections were incubated with choleragenoid and treated immunocytochemically. Choleragenoid-like immunoreactive cells were then examined for possible co-localization with carbonic anhydrase-like, RT 97 (antibody to neurofilament proteins), substance P-like, somatostatin-like and calcitonin gene-related peptide-like immunoreactivity and fluoride-resistant acid phosphatase (FRAP) activity, using adjacent sections. A subpopulation of dorsal root ganglion neurons exhibited choleragenoid-like immunoreactivity. The majority of these were medium-sized and large neurons. The strongest immunoreactivity was found in the area of the plasma membrane, but strong reactivity was also seen in the cytoplasm. The majority of the choleragenoid-like immunoreactive cells showed carbonic anhydrase-like and RT 97 immunoreactivity. Cells showing co-localization of choleragenoid-like and neuropeptide-like immunoreactivity or activity for FRAP were rarely observed. Our results suggest that the GM1 receptor is localized primarily on carbonic anhydrase-containing and RT 97-immunoreactive primary sensory neurons.The main part of this study was carried out in the laboratory of Dr Lana R. Skirboll, Clinical Neuroscience Branch, NIMH, National Institutes of Health, Bethesda, MD 20892, USA.     

Immunohistochemical evidence for the presence of neuropeptides in the hypothalamic suprachiasmatic nucleus of ground squirrels

The cytoarchitecture and immunocytochemical distribution of neuropeptides (corticotropin-releasing factor, CRF; neuropeptide Y, NPY; oxytocin, OXY; vasopressin, VP; and vasoactive intestinal polypeptide, VIP) were studied in the hypothalamic suprachiasmatic nuclei (SCN) in male and female ground squirrels of two species (Spermophilus tridecemlineatus and S. richardsonii). Immunoreactive (IR) perikarya were found in sections incubated with VP or VIP antisera. VP-IR cell bodies were seen in the dorsal and medial parts of the nucleus in colchicine-treated animals. IR fibers were distributed throughout the SCN. In the ventral part of the nucleus, VIP-IR cells were seen in untreated animals and were more pronounced in colchicine-treated animals. VIP-IR fibers and terminals form a dense plexus throughout the nucleus. Furthermore, NPY-IR terminals and fibers with multiple varicosities, but no IR perikarya, were present in the suprachiasmatic nuclei. Within the borders of the SCN, no cell bodies or fibers were stained with CRF or OXY antisera in any animal.     

Immunohistochemical evidences for localization and production of D-serine in some neurons in the rat brain

D-Amino acids are thought not to occur in mammalian tissues. However, previous studies reported D-serine was present only in astrocytes in the rat brain. In the present study, it was indicated by a highly sensitive immunocytochemical method with a D-serine specific antibody that D-serine was contained not only in astrocytes but also in some neurons, such as pyramidal neurons in the cerebral cortex, and neurons in the nucleus of the trapezoid body. Some amacrine cells also showed strong immunoreactivity for D-serine in the eyes which were injected with colchicine into the corpus vitreum.     

Immunohistochemical and neurochemical evidence for the presence of serotonin in the adrenal medulla of the rat

Immunohistochemical and biochemical techniques were used to look for serotonin in the adrenal medulla of the rat. Using antibodies to serotonin, noradrenaline and adrenaline, it could be shown that the adrenaline-storing cells are highly immunoreactive for serotonin. Noradrenaline-storing cells were not stained even after administration of the precursors l-tryptophan and 5-hydroxytryptophan, or of serotonin itself. Specificity of the immune reaction was studied by both absorption and inhibition experiments. Chemical assays showed that rat adrenals contain significant amounts of serotonin (1.4 ± 0.11 μg/g wet weight) which is about 0.4% of the adrenaline levels. Serotonin could be reduced to about 10% of control by a high dose of reserpine. From differential and sucrose gradient centrifugation experiments it was concluded that serotonin is probably stored in granules also containing adrenaline. Administration of 5-hydroxytryptophan led to a marked increase of the serotonin level, preferentially in the granular fraction. This increase could be blocked almost completely by a decar☐ylase inhibitor. Serotonin administration did not result in a statistically significant increase of the serotonin concentration. Serotonin levels were not changed either after administration of l-tryptophan or the tryptophan hydroxylase inhibitor H22/54. These results indicate that there is no significant synthesis of serotonin from l-tryptophan.

It is suggested that the serotonin present in the adrenaline-storing cells is derived from circulating serotonin and/or 5-hydroxytryptophan. Serotonin taken up directly from the circulation or formed by decar☐ylation from 5-hydroxytryptophan is subsequently incorporated in the chromaffan granules.     

Morphological evidence of sensory neurons in the root of the rat tongue

In our previous studies, a large number of substance P (SP)-immunoreactive (IR) nerve fibers were detected in the rat tongue and their number increased after inflammation, suggesting that these fibers might be involved in the axon reflex. Therefore, in this study, we have examined the different neuropeptide-containing nerve elements by light, electron, and confocal laser microscopy. SP, vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) IR varicose fibers were numerous compared with other ones. Small groups of ganglia with perikarya IR for SP, VIP, NPY, galanin, and somatostatin were observed. The SP-IR nerve cell bodies were mainly located in the tunica propria just below the epithelial lining. Double-labeling immunohistochemistry showed that the intrinsic SP-IR neurons did not colocalize VIP. The SP containing nerve terminals were observed in and below the epithelium as well as in very close contact to or making real synapses with other neurons in the intralingual ganglion. Our data confirmed the possibility of intrinsic sensory neurons, which might be the afferent branch of the intralingual reflex arch, while the VIP- and NPY-IR neurons located in the salivary glands, around the blood vessels, and in the muscle layer might constitute the efferent site of this reflex.     

Lectin binding: a genuine marker for transganglionic regulation of human primary sensory neurons

Medium-sized human dorsal root ganglion cells and their central terminals in the upper dorsal horn exert preferential affinity for Ulex europaeus I lectin. Lectin binding of glycoconjugates outlines lamina III (+IIi) and disappears after peripheral neurotomy, which renders it a useful marker to study transganglionic regulation in man.