Further immunofluorescence-microscopic evidence for myosin in various peripheral nerves

Summary An indirect immunofluorescence microscopic technique using antibodies from rabbits against highly purified myosin from chicken gizzard was applied to various peripheral nerves (cranial nerves V, VII, X). Myosinspecific immunoreactivity was found in the axoplasm, in Schwann cells, in the perineural sheath and in vascular walls.This work was supported by grants from Deutsche Forschungsgemeinschaft (Un 34/4, Dr 91/1, Ste 105/19). Thanks are due to Ursula König, Renate Steffens and Christine Mahlmeister for skilful technical assistance.     

Pancreatic polypeptide-like material in nerves and endocrine cells of the rat

A region-specific antiserum (AbS11) raised against the carboxyl-terminal hexapeptide of pancreatic polypeptide has been employed to measure rat pancreatic polypeptide specifically and to demonstrate apparent immunoreactivity in nerves and in endocrine cells outside the pancreas. The concentration of pancreatic polypeptide in the head of the rat pancreas measured with AbS11 (176 +/- 47 pmol/g) was 750 fold higher than that measured with a conventional anti-bPP antiserum (0.23 +/- 0.08 pmol/g). Column chromatographs of rat pancreatic extracts demonstrated two peaks of immunoreactivity both eluting after the porcine pancreatic polypeptide standard. AbS11 also detected specific immunoreactivity in rat brain (470 fmol/g) which went undetected in convention assays. Although immunohistochemical studies with AbS11 and human pancreatic polypeptide antiserum demonstrated immunoreactivity in the same population of pancreatic endocrine cells, immunoreactive nerve fibres and enteroglucagon cells were only demonstrable with AbS11. These studies demonstrate that the carboxyl terminus of rat pancreatic polypeptide is immunochemically similar to that of higher mammals. Furthermore, neural and extrapancreatic endocrine variants of this peptide share an immunochemical determinant contained within the carboxyl-terminal hexapeptide.     

Stimulation-depletion of serotonin and noradrenaline from vesicles of sympathetic nerves in the pineal gland of the rat

Summary The pineal gland of the rat receives a rich nervous supply originating from the superior cervical ganglia. These fibers contain serotonin in addition to their neurotransmitter, noradrenaline. Cytochemical studies at the ultrastructural level have shown that both amines are present in the cores of the granular vesicles that are characteristic of these nerves. It is presently shown that the bilateral electrical stimulation of the preganglionic fibers innervating the ganglia markedly reduces the number of small sites reacting cytochemically for both noradrenaline and serotonin, these sites corresponding to the cores of small granular vesicles, while the larger reactive sites (cores of large vesicles) remain unaltered. The vesicles are retained in nerve terminals after stimulation, as observed in conventionally processed tissues, although with altered sizes and shapes. Apart from these cytochemical and structural changes, nerve stimulation also reduces the endogenous noradrenaline content of the pineal gland. Thus, both noradrenaline and serotonin are released from their storage sites in pineal sympathetic nerves after electrical stimulation in vivo. This suggests the possibility that several substances with presumed transmitter or modulatory functions might be simultaneously released by nerve impulses from a given nerve terminal.     

Galanin-immunoreactive nerves in the mouse and rat pancreas

Summary Galanin-containing nerve fibers have previously been observed in the human, dog, and pig pancreas. Whether the mouse and rat pancreas also contain galanin nerve fibers has been a matter of debate. Therefore, we examined the distribution of galanin in the mouse and the rat pancreas. Further, the possible localization of galanin to adrenergic nerves was studied using sequential immunostaining for galanin and tyrosine hydroxylase (TH). In the mouse pancreas, numerous galanin-immunoreactive (GIR) nerve fibers occurred around blood vessels. They were less numerous in the exocrine parenchyma and in association with the islets. In contrast, in the rat pancreas, only a few GIR nerves were found. They were located around blood vessels and scattered in the exocrine parenchyma. Occasionally, GIR nerves were also observed in the islets. There was a dense distribution of TH-immunoreactive fibers in both the mouse and the rat pancreas. Sequential immunostaining revealed co-localization of galanin and TH immunoreactivity in nerve fibers in both the mouse and the rat pancreas. Following chemical sympathectomy using 6-hydroxydopamine (6-OHDA), not all GIR nerves disappeared. In the mouse pancreas a remaining population of galanin nerves was found around blood vessels, and occasionally in the islets. In the rat pancreas, a few GIR nerves were seen also after chemical sympathectomy. We conclude that intrapancreatic GIR nerves also occur in the mouse and the rat. These findings suggest that many of the GIR nerves are adrenergic but that non-adrenergic, possibly intrinsic or sensory GIR nerves exist as well in both the mouse and the rat pancreas.     

Further evidence for the existence of B-lymphocyte subpopulations.

We have studied the homing properties of B lymphocytes by using ⁵¹Cr-labeled lymphoid cells obtained from athymic, nu/nu mice, and animals made T-lymphocyte deficient by thymectomy and lethal irradiation followed by reconstitution with syngeneic bone marrow. Comparison was made to the patterns of distribution observed when cell preparations containing normal numbers of T and B lymphocytes were migrated. A small but significant percentage of labeled lymphocytes from lymph nodes, spleen, Peyer's Patches, and bone marrow of T-cell-deficient animals was shown to be lymph node seeking. Secondary transfers of lymph node cells from primary recipients caused enrichment of this lymph node-seeking population. Treatment of T-lymphocyte-deficient lymphoid cell preparations with neuraminidase reduced the percentages of cells homing to the lymph nodes. The data showed that B lymphocytes exhibit unique homing properties when injected into normal recipients. In addition, direct comparison of the homing patterns of B lymphocytes prepared from spleen and lymph nodes of athymic mice revealed differences suggesting that these lymphoid organs contained unique mixtures of at least two different kinds of B cell. The evidence supports the notion that the B-lymphocyte populations contain at least two subpopulations, one of which possesses the ability to home to lymph nodes.     

Ultrastructural identification of non-adrenergic,non-cholinergic nerves in the rat anococcygeus muscle

The innervation of the rat anococcygeus muscle has been investigated ultrastructurally following fixation with a modified chromaffin reaction for the demonstration of biogenic amines (Tranzer and Richards, 1976). Three types of nerve profiles were revealed: (1) 60-70% of the profiles are adrenergic; (2) less than 5% of the profiles appear to be cholinergic; (3) up to 40% of the profiles are distinguished by the presence of a characteristically high proportion of electron-opaque, chromaffin-negative vesicles, 85-110 nm in diameter. This third type of profile was not affected by 6-OHDA, and is considered to represent the non-adrenergic, non-cholinergic inhibitory innervation of this tissue. Because of the morphological similarity of this nerve type, apart from the smaller vesicle size, to classical peptidergic nerve endings, they have been termed "small p-type" (sp-type). These results are discussed in relation to a previous report describing only two types of nerve profiles in this tissue (Gillespie and Lüllmann-Rauch, 1974).     

Plasticity in developing rat uterine sensory nerves: the role of NGF and TrkA

In the present study we investigated the effects of infantile/prepubertal chronic oestrogen treatment, chemical sympathectomy with guanethidine and combined sympathectomy and chronic oestrogen treatment on developing sensory nerves of the rat uterus. Changes in sensory innervation were assessed quantitatively on uterine cryostat tissue sections stained for calcitonin gene-related peptide (CGRP). Uterine levels of NGF protein, using immunohistochemistry and ELISA, and mRNA, using Northern blots and in situ hybridization, were also measured. Finally, levels of TrkA NGF receptor in sensory neurons of T13 and L1 dorsal root ganglia (DRG), which supply the uterus, were assessed using densitometric immunohistochemistry. These studies showed that: (1) chronic oestrogen treatment led to an 83% reduction in the intercept density of CGRP-immunoreactive nerves; (2) sympathectomy had no effect on the density of uterine sensory nerves or on the pattern of oestrogen-induced changes; (3) NGF mRNA and protein increased following sympathectomy or chronic oestrogen treatment; and (4) oestrogen produced increased intensity of labelling (28%) for TrkA receptors in small-diameter sensory neurons, but decreased labelling (13%) in medium-sized neurons, which represent the large majority of the DRG neurons supplying the upper part of the uterine horn. Contrary to expectations, increased levels of NGF after sympathectomy and oestrogen treatment did not lead to increased sensory innervation of the uterus. The possibility that alterations in neuronal levels of TrkA contribute to the lack of response of uterine sensory nerves to the oestrogen-induced increase in NGF levels is discussed.This work was supported by The Wellcome Trust, UK (CRIG Grant 058122/Z/99/Z/JC/KO), and PEDECIBA, Universidad de la República, Montevideo, Uruguay     

Effects of carvacrol on defects of ischemia-reperfusion in the rat liver

Many plants found in nature have been used to treat various illnesses. One such plant is oregano (Kekik in Turkish). Health beneficial effects of carvacrol obtained from oregano oil have been shown scientifically. We have investigated the comparative effects of carvacrol in the liver of rats subjected to ischemia-reperfusion defect, with silymarin. To test the effects we formed four groups using male Wistar albino rats. Group I was control. The other three groups of animals were administered 60 min prior to surgical operation single doses of physiological serum, carvacrol and silymarin, respectively. Group II, III and IV animal were subjected to 45 min long liver ischemia and 60 min reperfusion. Blood and tissue samples were collected for biochemical and histological analysis following the test.AST and ALT values obtained after biochemical analysis of the serums showed statistically significant difference in group II than the other three groups. A statistical evaluation of the serum AST levels among the groups II, III and IV showed that both groups III and IV which had no difference in between were significantly different in a positive way from group II (p<0.001). As to the serum ALT levels, difference between group II and group III (p<0.001) and group II and group IV (p<0.01) was found significant. No statistical difference was observed in groups I, III and IV for GSH, MDA and CAT levels of the liver. A statistical evaluation of the GSH level in group III and group IV was found to be significantly different from group II (p<0.001) without any difference between them. A similar evaluation for MDA and CAT levels among the revealed no difference between group III and group IV, however, group II showed difference with group II and group IV (p<0.05).Histological findings were in harmony with the biochemical results. We conclude that carvacrol protects the liver against defects caused by ischemia and reperfusion, and carvacrol is not hepatotoxic at the applied dosage.     

Morphine sulphate induced histopathological and histochemical changes in the rat liver

In this study, the histopathological and histochemical changes due to chronic usage of morphine sulphate in liver were assessed in rats with both light and electron microscopes. Twenty male albino rats (Rattus norvegicus) (130-150 g) were included and divided into four groups. Normal saline (5 ml) was given orally as placebo in the control group (N = 5). Morphine groups (N = 5) received morphine orally at a single dose of 5 ml/kg/day for 10, 20 and 30 days (groups II, III and IV), respectively. Liver specimens from all groups were evaluated for histopathological and histochemical changes. Light microscopy revealed severe centrilobular congestion, portal fibrosis with bile ductal proliferation and an increased inflammatory infiltration and focal parenchymal necrosis. Histochemical study revealed a progressive depletion of general carbohydrates and an increase in total protein contents. These changes were confirmed at ultrastructural level, including the presence of accumulated lipid in the hepatocytes; deposits of a collagen-like fibrous material were seen in the space of Disse and a reduction in the number of endothelial cell fenestrations. Our findings pointed out the risk of increased lipid fibrosis and hepatic damage due to long-term use of morphine. Although opioids are reported to be effective in pain management, their toxic effects should be kept in mind during chronic usage.     

Sensory collaterals, intramural ganglia and motor nerves in the guinea-pig bladder: evidence for intramural neural circuits

The afferent output from the bladder is important for triggering micturition. This study identifies different types of afferent nerve and explores the connections of their collateral fibres on intramural ganglia and potential ganglionic targets. The experiments were performed on tissues from male guinea-pigs (n=16). Fibres positive for choline acetyl transferase (ChAT⁺) were found to originate close to the urothelium, to transit the sub-urothelial interstitial cell layer and to pass into the lamina propria. A different population of fibres, immunopositive for calcitonin gene-related peptide (CGRP), capsaicin receptors or neurofilament protein (NF), were seen to intertwine with the ChAT⁺ fibres in the lamina propria. The ChAT⁺ fibres did not express NF. Ganglia with ChAT⁺ and NF⁺ neurones were found in the lamina propria and muscle. ChAT⁺ fibres, with pronounced terminal varicosities, were present on the nerve cell bodies. Two types were noted: NF⁺ terminals and those with little or no NF (NF⁻) suggesting that their origins were the ChAT⁺ afferent collaterals and the adjacent ganglia. Fibres containing CGRP or substance P were seen on the ganglionic cells. α1B adrenergic receptors were also found on the neurones indicative of adrenergic synapses. Thus, the ganglia had multiple inputs. Different types of ChAT⁺ nerves were seen in the muscle: NF⁺ and NF⁻. The ChAT⁺/NF⁺ nerves may represent a ganglionic output to the muscle. This complex neuronal network may therefore represent the elements generating and modulating bladder sensations. The role of such a scheme in bladder pathology and the therapeutic sites of action of anticholinergic and sympathomimetic drugs are discussed.We gratefully acknowledge the support of Pfizer. This work was supported by a grant from the Detrol Research Programme.     

Distribution and morphological characteristics of the pit cells in the liver of the rat

Summary Pit cells, on which almost no further contributions have been presented since the first report by Wisse et al. (1976), are described in detail in the rat liver. These cells show several characteristic features: 1) rod-cored vesicles, a new type of vesicular inclusion observed first in our study; 2) electron-dense granules, which we consider to arise from multivesicular bodies by the accumulation of dense material; and 3) well-developed pseudopodia. Although these features clearly differentiate pit cells from conventional lymphocytes, these two cell types display similarities (i) in a number of ultrastructural features, (ii) in the pattern of their intralobular distribution, and (iii) in their presence in the spleen and peripheral blood.     

Occurrence of uranaffin-positive synaptic vesicles in both adrenergic and non-adrenergic nerves of the rat anococcygeus muscle

Summary The uranaffin reaction in rat anococcygeus muscle, which receives a dual innervation of both adrenergic and non-cholinergic, non-adrenergic nerves was examined. Dense reaction product was observed in the vesicular membranes and/or the cores of some synaptic vesicles in the adrenergic nerve terminals. Occasional vesicles were filled up with dense reaction product. In the prominent population of small clear vesicles, however, no dense reaction product was observed. The number of small granular vesicles in the adrenergic nerve terminals was markedly increased after the administration of 5-hydroxydopamine (5-OHDA). These granular vesicles were moderately stained with uranaffin deposit on the cores but their limiting membranes possessed no uranaffin deposit at all.In the non-adrenergic nerve terminals, on the other hand, uranaffin deposit of variable density was observed on the cores of large granular vesicles but never on their limiting membranes or on the small clear vesicles. There was no change in the axon profiles after the administration of 5-OHDA.The possible occurrence of purines in the cores of large granular vesicles in the non-adrenergic nerves is discussed.     

Second-order ocellar neurons in the brain of the honeybee (Apis mellifera)

Summary Electrophoretic injection of Procion Yellow M-R4 into the ocellar tract of the worker bee has revealed the following:Two types of giant axon run from the lateral ocellus to the circumesophageal neuropile, where one branches ipsilaterally and the other contralaterally. A third type comes from the median ocellus and can be traced into the cervical connectives. The largest dendritic complex is in the circumesophageal neuropile; in addition, fiber endings have been demonstrated in the following areas: in the subretinal region, along the optic commissure, in the medulla interna, in the subesophageal ganglion and between the neurosecretory cells of the pars intercerebralis. — The giant fibers are enclosed in a glial sheath.Three types of cell body are described. One is associated with the glia; another, larger cell type comprises giant-axon somata. The third type of cell is small, and cannot yet be identified.Some of the histological results are discussed with respect to the possible function of the ocellus.     

Galanin-immunoreactive nerves in the female rat paracervical ganglion and uterine cervix: distribution and reaction to capsaicin

Summary The presence and distribution of galanin-immunoreactivity was examined in the uterine cervix and paracervical autonomic ganglia of the female rat. Some animals were treated with capsaicin to determine if galanin-immunoreactivity was present in small-diameter primary afferent nerves. Other animals were treated with the noradrenergic neurotoxin 6-hydroxydopamine to ascertain if galanin-immunoreactivity was present in sympathetic noradrenergic nerves. Galanin-immunoreactive nerve fibers were sparse in the cervical myometrium and vasculature, but numerous in the paracervical ganglion where they appeared to innervate principal neurons. Immunoreactivity was also present in dorsal root ganglia, dorsal horn of spinal cord, and inferior mesenteric ganglia. Capsaicin treatment resulted in a marked reduction of galanin-immunoreactivity in the spinal cord dorsal horn, but not in the dorsal root ganglia, paracervical ganglia, or cervix (although there was a substantial reduction of substance P-, neurokinin A-, and calcitonin gene-related peptide-immunoreactivity in the dorsal horn, dorsal root ganglia, and uterine cervix). 6-Hydroxydopamine treatment did not cause any appreciable change in the galanin-immunoreactivity in any tissues. We conclude that galanin-like immunoreactivity is expressed in nerve fibers innervating the paracervical ganglia and uterine cervix of the female rat. This immunoreactivity is probably present in afferent nerves and could play a role in neuroendocrine reflexes and in reproductive function.     

Further evidence for the existence of a membrane potential in Trypanosoma brucei brucei

The distribution of 137Cs+, in the presence of valinomycin, has been used to measure the magnitude of the membrane potential (delta psi) in bloodstream forms of Trypanosoma brucei brucei. Values of the delta psi falling in the range -100 mV to -160 mV were observed and the maintenance of this delta psi was sensitive to certain ionophores and protonophores.     

Effects of capsaicin on reproductive function in the female rat: Role of peptide-containing primary afferent nerves innervating the uterine cervix in the neuroendocrine copulatory response

Summary Nerves immunoreactive for the peptides substance P, neurokinin A, calcitonin gene-related peptide or cholecystokinin-octapeptide innervate the uterine cervix in the rat. Nerve terminals are associated with the myometrial and vascular smooth muscle and are distributed throughout the endocervix. These nerves degenerate following neonatal capsaicin treatment indicating that they are small-diameter, unmyelinated, C-type primary afferent nerves. Adult female rats, treated with capsaicin as neonates, exhibit decreased fertility following mating and diminished sensitivity to the induction of pseudopregnancy following copulomimetic electrical stimulation of the cervix. The results also demonstrate that hypothalamo-adenohypophyseal-ovarian interactions, corpus luteum progesterone secretion and uterine sensitivity to progesterone are normal in capsaicin-treated rats. Taken together, these data suggest that the reproductive dysfunction observed in capsaicin-treated rats is due to destruction of the afferent limb of the neuroendocrine copulatory response that facilitates the luteal progesterone secretion necessary to support pregnancy or pseudopregnancy. Thus, it is concluded that the afferent limb of this neuroendocrine response in the rat consists primarily of unmyelinated, peptide-containing, C-type primary afferent nerves.A preliminary report of this study was presented at the Substance P and Neurokinins Symposium in Montreal, 1986, and published in the symposium proceedings (Traurig et al. 1987)