Ontogenetic development of the guinea pig uterine innervation. An immunohistochemical study of different neuronal markers, neuropeptides and S-100 protein

The ontogenetic development of the guinea pig uterine autonomic innervation was studied immunohistochemically using neurofibrillary protein (NF) and neuron specific enolase (NSE) as general neuronal markers, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) as specific markers for adrenergic innervation and S-100 protein as marker for Schwann cell structure and/or function. In addition, comparisons were made of the development of the different populations of peptide-containing nerves. The structure and time of appearance were similar for nerves with NF-, NSE-, TH- and DBH-immunoreactivities, which were first present in the organ periphery as coarse nerve trunks, then extending centrally and branching into non-varicose nerves. From these, varicose nerves developed first in relation to vessels and then in association with the myometrial smooth musculature. Development was completed earlier in the cervix than in the uterine horns suggesting differences in local environment. In comparison, S-100 nerve-immunoreactivity appeared later but attained complete development more rapidly than axonal structures. Neuropeptide Y-immunoreactive nerves showed a similar developmental pattern to presumed adrenergic nerves, further verifying the assumption of intraneuronal localization of NPY in uterine adrenergic nerves. Other peptide-containing nerves were developed later probably reflecting differences in neuronal growth properties.     

S-100-immunoreactive nerves in the guinea-pig uterus with reference to ultrastructural correlations: Effects of chemical sympathectomy and pregnancy

Summary In the guinea-pig uterus, by use of an indirect immunofluorescence method, S-100 immunoreactivity was found to be restricted to nerves that corresponded in number, distribution and type to adrenergic axons and preterminals. With advancing pregnancy S-100 immunoreactivity completely disappeared in uterine tissue adjacent to a fetus, in parallel with an ultrastructural degeneration of the adrenergic innervation. In the cervix and the uterine horn devoid of a fetus, however, the number and distribution of S-100-immunoreactive nerves was seemingly unchanged and no ultrastructural changes were found in adrenergic nerves. In contrast, chemical sympathectomy produced by 6-hydroxydopamine did not change S-100 immunoreactivity of uterine nerves. These findings suggest that there are differences in the denervation effected by chemical and by pregnancy-induced sympathectomy. The latter probably represents a special type of adrenergic denervation by inducing a degeneration of Schwann cells in addition to destruction of neuronal structures. This may explain the differences in the speed of regeneration of uterine adrenergic nerves following the two types of denervation.     

Organization of the guinea-pig uterine innervation. Distribution of immunoreactivities for different neuronal markers. Effects of chemical- and pregnancy-induced sympathectomy

Summary The structural organization of the guinea-pig uterine innervation was investigated by an immunofluorescence method using neurofibrillary protein (NF) and neuron-specific enolase (NSE) as general neuronal markers. NF- and NSE-immunoreactive nerve trunks and non-varicose nerves formed continuous networks similar to nerves with analogue morphology and with immunoreactivities for tyrosine hydroxylase (TH; adrenergic nerves) and neuropeptide Y (NPY). NF- and NSE-immunoreactive non-varicose nerves occurred in the myometrium and along vessels, where TH- and NPY-immunoreactive varicose nerves were also comparatively frequent. After chemical sympathectomy all TH- and NPY-immunoreactive varicose nerves and most NF- and NSE-immunoreactive non-varicose nerves disappeared, suggesting colocalization of TH, NPY, NF and NSE immunoreactivities. During pregnancy all NF-, NSE-, TH- and NPY-immunoreactive nerve structures disappeared in the foetus-bearing uterine horns whereas in the cervix and non-foetus-bearing uterine horns only the myometrial TH- and NPY-immunoreactive varicose nerves disappeared. After parturition there was a complete structural restoration of all types of immunoreactive nerves in previously non-foetus-related tissue. The reinnervation of this tissue followed a similar time-course to that after chemical sympathectomy. In contrast, the reinnervation of previously foetus-related tissue was much slower and incomplete.In conclusion, the whole autonomic uterine innervation undergoes overt structural changes during pregnancy and these changes are related to the foetus-bearing regions.     

Immunohistochemical demonstration of VIP, [Met5]- and [Leu5]-enkephalin immunoreactive nerve fibres in the human prostate and seminal vesicles

Summary The distribution of nerves containing immunoreactivity for the VIP and enkephalins has been demonstrated in the human prostate and seminal vesicles using the immunoperoxidase bridge. VIP-containing nerves were detected in both organs studied mainly in association with the epithelium, while nerves containing ELI seemed to be related to smooth muscle. Compared with the distribution of adrenergic and cholinergic nerves in the prostate marked differences in the density of the innervation were detected. The possible nature of these peptide-containing nerves is discussed.     

Effects of pregnancy on the extrinsic innervation of the guinea pig uterus. A histochemical, immunohistochemical and ultrastructural study

Summary The extrinsic innervation of the guinea pig uterus was studied by immunohistochemical, ultrastructural and enzyme histochemical methods.The extrinsic innervation was organized in two major ways. One consisted of nerve trunks and non-varicose nerve fibres running in the suspensory ligament, and the other of a plexus of varicose nerve fibres surrounding vessels, and non-vessel-related non-varicose nerve fibres in the mesouterus. The use of different neuronal and Schwann cell markers showed that the extrinsic innervation was predominantly adrenergic and contained only few peptidergic nerves. Acetylcholinesterase-positive (cholinergic) nerves were only found around the uterine artery.In late pregnancy, the extrinsic nerves of the mesouterus adjacent to foetus-containing uterine horns underwent pronounced degenerative changes comprising both Schwann cell and axonal structures. In comparison, no changes were found in extrinsic nerves of mesouteri adjacent to non-foetus-bearing uterine horns or in extrinsic nerves in the suspensory ligaments. Further, chemical sympathectomy produced axonal degeneration but no changes in the Schwann cells.In conclusion, the pregnancy-induced nerve degeneration is of a very special type different from that following chemical sympathectomy and represents a local phenomenon related to the conceptus. Hypothetically, this could be of importance for counteracting disturbances in placental blood flow.     

Studies on the acetylcholinesterase (ache)-positive and -negative autonomic axons supplying smooth muscle in the normal and 6-hydroxydopamine (6-OHDA)treated rat iris

Summary The adrenergic and cholinergic innervation to the rat iris has been studied at a light and electron microscopic level. Catecholamine fluorescence histochemistry showed adrenergic nerves to be present in both the dilatator and the constrictor pupillae regions. At a fine structural level the terminal innervation of the iris was studied and criteria for the differentiation between presumptive adrenergic and presumptive cholinergic axon terminals were examined. To aid this examination presumptive adrenergic axons were either labelled with the false adrenergic transmitter, 5-hydroxydopamine, or chemical sympathectomy performed using 6-hydroxydopamine. The value of using acetylcholinesterase staining as a marker for cholinergic nerve terminals was also studied.Results showed a mixed adrenergic/cholinergic innervation to the dilatator pupillae. In the constrictor pupillae an exclusively cholinergic innervation was found although adrenergic and cholinergic nerves were found supplying the blood vessels and at the dilatator-constrictor interface. These findings are discussed with regard to innervation-function relationships in the iris.     

Protein gene product 9.5 (PGP 9.5)

Summary The guinea pig uterus is supplied by different populations of nerves which can be demonstrated by specific immunocytochemical and histochemical techniques. So far, there has been no single marker displaying entire peripheral innervation patterns. Recently, protein gene product (PGP) 9.5, a cytoplasmic protein in neurons and neuroendocrine cells, was found to visualize both different populations and subtypes of nerves. This prompted the present study of using PGP 9.5 for visualization of the whole uterine innervation. This was performed by the indirect immunofluorescence method using antiserum to PGP 9.5 raised in rabbits.PGP-immunoreactivity was present in all neuronal parts of the extrinsic and intrinsic uterine innervation, including different subpopulations of nerves. This was verified by chemical sympathectomy and sensory denervation with 6-hydroxydopamine and capsaicin-treatment respectively, and double immunostaining.By term a disappearance of uterine PGP-nerve-immunoreactivity was observed which was almost complete in fetus-bearing uterine tissue and further strengthens previous assumptions of a general, pregnancy-induced uterine neuronal degeneration.The developmental time-course and morphology of PGP-immunoreactive nerve structures was similar to that for other neuronal markers and support the suggestion of PGP-immunoreactivity as a general marker for the entire uterine innervation, and suggests that the presence of PGP 9.5-immunoreactivity may coincide with functional maturation of uterine innervation.     

The innervation of the hepatic portal vein in the rabbit: Ultrastructural evidence against “purinergic” neurotransmission

Summary The relative density of adrenergic and non-adrenergic nerves in the hepatic portal vein of the rabbit has been determined ultrastructurally. Adrenergic nerves were visualised with the modified chromaffin procedure of Tranzer and Richards (1976). Nearly equal numbers of adrenergic and non-adrenergic nerve profiles were found, indicating a much greater density of innervation by non-adrenergic nerves than that described by Burnstock et al. (1979) using light microscopic histochemical methods. These results imply that part of the argument used by Burnstock et al. (1979) to support purinergic transmission in rabbit portal vein is probably invalid.     

Functional and histochemical characterization of a uterine adrenergic denervation process in pregnant rats

The time course of pregnancy-induced changes in the contractile responses of isolated uterine rings and sympathetic innervation pattern were studied using electric field stimulation and histofluorescence techniques, respectively, in intact and 6-hydroxydopamine-treated rats. Neurally mediated contractions elicited by field stimulation (0.6 msec, 1-70 Hz, 40 V) were measured in uterine preparations obtained from nonpregnant, 6-hydroxydopamine-treated and 5-, 10-, 15-, 18-, and 22-day (term) pregnant rats. At all frequencies, the amplitudes of contractions were highest in nonpregnant uteri. Stimulation at 1-2.5 Hz evoked contractions in 10-day pregnant uteri but failed to cause contractions on Day 5 and from Day 15 onward. In uterine preparations obtained from term and from 6-hydroxydopamine-treated rats, contractions could not be evoked by stimulation at 1-20 Hz. Fluorescence histochemistry of uterine adrenergic nerves revealed rich perivascular and myometrial innervation in nonpregnant and in pregnant rats through Day 10. Degeneration and loss of adrenergic nerve fibers was apparent by Day 15, and fluorescent myometrial and perivascular nerves were practically absent by Day 22. These findings demonstrate a progressive, frequency-related reduction of nerve-mediated uterine contractions beginning in midterm pregnancy, in parallel with a gradual loss of adrenergic nerve fibers. Pregnancy-induced nerve degeneration may promote the development of nonsynaptic alpha-adrenergic uterine contractile activity towards term. The reduced responsiveness of uterine smooth muscle to electric field stimulation in early pregnancy appears to be unrelated to alterations in uterine innervation but may be related to changes associated with implantation.     

Noradrenergic and cholinergic nerves in the uterus of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus,change with reproductive cycle

The pattern of uterine innervation by noradrenergic (NA) and acetylcholinesterase-positive (AChE) nerves in different reproductive stages of the adult Japanese long-fingered bats were investigated histochemically and immunohistochemically. In the non-pregnant bat, the uterine horn was supplied with abundant NA and AChE nerves. These two types of nerves were closely associated with the uterine arteries and myometrial smooth muscles. In the pregnant bat, NA and AChE nerves supplying the uterus did not degenerate much during hibernating period, but reduced markedly after arousal. In the postpartum bat, the density of nerves recovered progressively. The significant change in the innervation pattern of uterine NA and AChE nerves in the pregnant bats under and after hibernation, and in the postpartum bat must be considered in relation to the adrenergic and cholinergic controlling mechanisms on the uterine function that is matched for the unique reproductive cycle of this bat.     

Cholinergic mechanisms in pial vessels

Summary Plexuses of cholinergic nerve terminals were demonstrated (acetylcholinesterase staining) in pial arteries (down to a diameter of about 15) at the base of the brain and on the brain convexities of mice, rats, rabbits, hamsters, guinea-pigs, and cats. The pial veins were less well supplied than the arteries. Consecutive formaldehyde gas treatment (to visualize adrenergic nerves) and acetylcholinesterase staining revealed that the adrenergic and cholinergic plexuses followed each other closely, the axon terminals running together in the same Schwann cell strands. This was confirmed by electron microscopy after KMnO₄ fixation or 5-hydroxydopamine treatment. The varicosities of cholinergic and adrenergic axons were sometimes seen as close as 250 Å. In the neuro-effector area, the terminals of both nerve types (naked or surrounded by an incomplete Schwann cell covering) approached the smooth muscle cells as close as 800–1100 Å, and they were separated from the latter only by the fused neuronal and muscular basement membranes. In this area axo-axonal contacts were observed. The adrenergic, but not the cholinergic, nerves disappeared after bilateral removal of the superior cervical sympathetic ganglia. Isolated cat middle cerebral artery contracted strongly with acetylcholine, and the effect was inhibited by atropine.With regard to the cholinergic neural control of the intracranial arteries, it may have particular functional implications: (1) that these vessels do have a cholinergic parasympathetic innervation in contrast to most other vascular systems, for example, in the mesenterium, (2) that this cholinergic nerve supply was found to be about equally prominent as the adrenergic (sympathetic) innervation which, in some pial vessels, is even better developed than in the mesenteric arteries, and (3) that the adrenergic and cholinergic systems in the intracranial arteries may interact, even at the level of the neuro-muscular contacts, a complex situation which may be partly responsible for the previous difficulties in defining the autonomic neural influence on the brain circulation.Part of the findings were reported at Journées Internationales de Circulation Cérébrale, Toulouse, April 21–22, 1972.     

Lack of correlation between ultrastructural and pharmacological types of non-adrenergic autonomic nerves

Summary In order to test the premise that non-adrenergic, non-cholinergic (NANC) autonomic nerves have a distinctive ultrastructural appearance, clearly different from that of cholinergic nerves, a detailed quantitative ultrastructural analysis has been made of the non-adrenergic innervation of 15 tissues thought from pharmacological evidence to be innervated by NANC nerves (rat and rabbit anococcygeus muscles; rabbit hepatic portal vein; extrinsically denervated toad lung); cholinergic nerves (atria of rat, rabbit, guinea-pig and toad); or both (guinea-pig cervical and thoracic trachealis muscle; rabbit rectococcygeus muscle; urinary bladder of rat, rabbit, guinea-pig and toad) in addition to their adrenergic supply. Following fixation with a modified chromaffin procedure allowing identification of adrenergic nerves, large, randomly selected samples of non-adrenergic nerve profiles from each tissue were analysed with respect to numbers, relative proportions, and size frequency distributions of different vesicle classes within the profiles. The neuromuscular relationships within each tissue were also analysed. On the basis of these analyses, it is clear that there are no consistent ultrastructural differences between cholinergic and NANC autonomic nerves: neither proportions nor sizes of the vesicles provide any clue as to the transmitter used by a particular nerve. The great majority of nerve profiles, whether cholinergic or NANC, contain predominantly small clear synaptic vesicles. Large filled peptidergic vesicles are no more common in most NANC nerves than in most cholinergic ones. It is concluded, on ultrastructural grounds, that the primary transmitter in these NANC autonomie nerves is most likely to be stored in and released from the small clear vesicles.     

Chemical sympathectomy induced by 5.6-dihydroxytryptamine

Summary The effect of different doses of 5.6-dihydroxytryptamine—a serotonin analogue which produces a degeneration of serotonin containing nerve terminals in the rat brain—on the noradrenaline (NA) content and—storage sites of peripheral sympathetic nerves in the mouse and rat heart, spleen, rectum and vas deferens has been investigated by fluorescence—, electron microscopical and chemical methods. Moderate doses of 5.6-dihydroxytryptamine (5.6-DHT) (10–45 mg/kg ip.) cause a temporary, reversible displacement of noradrenaline from the adrenergic nerves concomitant with a significant increase in the number and opacity of small and especially large granular vesicles. The recovery of the neuronal NA concentration is, however, retarded after doses higher than 45 mg/kg (60 or 100 mg/kg ip.); a partial degeneration of varicose NA terminals is verified fluorescence- and electron microscopically. A combined treatment of animals with tyrosine hydroxylase inhibitors (-methyl-paratyrosine or -propyl-dopacetamide) and 5.6-DHT, in some instances also followed by reserpine, potentiates the destructive properties of 5.6-DHT; a similar potentiation is accomplished by reserpine posttreatment or by an additional pretreatment of animals with reserpine and nialamide.The results suggest that 5.6-DHT when given in moderate doses (up to 45 mg/kg) may be handled by sympathetic adrenergic nerves like a false neurotransmitter which displaces noradrenaline from the stores, but that it causes a chemical degeneration of noradrenaline containing nerve terminals when applied either in single high doses (60 or 100 mg/kg ip.), or when administered in moderate non-degenerative doses together with drugs that impair the neuronal inactivation mechanisms for 5.6-DHT (granular uptake and storage mechanism and/or monoamine oxidase activity) and thus provoke a temporary increase in the amount of free 5.6-DHT in the neuron's cytoplasm.The molar efficiency of 5.6-DHT in causing a chemical sympathectomy is clearly inferior to that caused by 6-hydroxydopamine. The differences are probably mainly due to differences in the affinity of both drugs to the amine uptake system located at the cell membrane and the membrane of the intraneuronal storage vesicles of the adrenergic nerve terminals.Supported by grants from the Deutsche Forschungsgemeinschaft.     

Changes in the levels of neural cell specific proteins in the developing rat brain

The levels of S-100 protein (S-100) and neuron-specific enolase (NSE) in the developing rat brain were determined by a sensitive enzyme immunoassay and the results were compared with those obtained by other methods. Changes with development in the levels of S-100, NSE, and 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase), biochemical markers for astroglia, neurons and oligodendroglia respectively, were determined in various brain regions including the cerebral hemisphere (CH), brain stem (BS) and cerebellum (Ce). The peak increments of S-100, NSE, and CNPase activity were reached later than that of the brain weight in all of the regions. The ratios of S-100/NSE and CNPase/NSE rose during the 21 days after birth in the CH and BS; the S-100/NSE ratio in the CH began to decrease from the 21st day, whereas the CNPase/NSE ratio continued to rise even after the 30th day, suggesting different maturation periods of the different glial cells. In the Ce, the change of these ratios showed a pattern different from those in the other regions. In the CH of rats with experimental microencephaly induced by methylazoxymethanol (MAM), the ratios were almost normal, in spite of the reduction of the brain weight to about 50% of the control.Dedicated to Professor Yasuzo Tsukada.     

Catecholaminergic nerves in the embryonic chick ovary: co-localization with β2-adrenoceptor-bearing steroidogenic cells

Summary The present study investigates the innervation of the embryonic chick ovary with regard to (i) development and compartmentalization of catecholaminergic nerves, and (ii) presence of adrenoceptors on steroidogenic target cells of catecholaminergic nerve terminals. Catecholaminergic nerve fibers visualized by glyoxylic acid-induced histofluorescence first appeared at embryonic day (E) 13. From E15 through E21 the density of fluorescent aminergic nerves increased markedly in parallel with the concentration of catecholamines and numbers of nerve bundles and single axons seen at the electron-microscopic level. Catecholaminergic nerves were confined to the ovarian medulla and closely associated with interstitial cells. Nerve terminals approached interstitial cells up to a distance of 20 nm and, in their majority, exhibited uptake of the false adrenergic transmitter 5-hydroxydopamine. Although adrenaline amounted to 14% of the total catecholamine content at E21, adrenaline immunoreactivity was only detected in adrenal chromaffin cells, but not in nerve fibers or cell bodies within the ovary. Interstitial cells structurally matured between E15 and E21 as documented by an increase of smooth endoplasmic reticulum and tubular mitochondria. Monoclonal antibodies mAB 120 and BRK 2 raised against avian ₁ and mammalian ₂-adrenergic receptors revealed the presence of ₂-adrenoceptor-like immunoreactivity on the surface of interstitial cells, but not on any other cell type.The results are consistent with the notion of a dense adrenergic innervation of the embryonic chick ovarian medulla and its steroidogenic interstitial cells, and suggest the chick ovary as an excellent model for elucidating the functional role of a neural input to steroidogenic cells during development.     

The distribution and colocalization of neuropeptides and catecholamines in nerves supplying the gall bladder of the toad,Bufo marinus

The indirect immunofluorescence technique was used to determine the distribution of peptide-containing axons in the gall bladder of the cane toad, Bufo marinus. In addition, the adrenergic innervation of the gall bladder was examined by use of immunoreactivity to the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and glyoxylic acid-induced fluorescence. On the basis of peptide coexistence, two intrinsic populations of neurones and their projecting fibres could be distinguished substance P neurones and vasoactive intestine peptide neurones. Neither of these two types of neurones contained any other colocalized neuropeptides. Four populations of nerve fibres arising from cell bodies outside the gall bladder were identified: nerves containing colocalized galanin, somatostatin and vasoactive intestinal peptide; nerves containing colocalized calcitonin gene-related peptide and substance P; adrenergic nerves containing neuropeptide Y; and nerves containing only adrenaline.     

Uterine adrenergic and cholinesterase-positive nerves and myometrial catecholamine concentrations during pregnancy in sheep

Uterine adrenergic and cholinesterase (AChE)-positive innervation of the sheep uterus during anestrus and at 4 stages of pregnancy were examined by histochemical methods. In addition, uterine and cervical myometrium concentrations of norepinephrine (NE) and dopamine (DA) were determined using high-performance liquid chromatography. During anestrus, adrenergic and AChE-positive nerve fibers in the uterine myometrium and endometrium were primarily associated with the vasculature. Innervation of myometrial smooth muscle was almost exclusively by adrenergic fibers. In the endometrium, fibers of both types were observed closely associated with endometrial glands, and adrenergic fibers were observed in the connective tissue beneath the luminal epithelium. Density of uterine innervation decreased by day 65 of pregnancy with an additional decrease by day 105. Myometrial NE concentrations were higher in the cervix than the uterus. Uterine NE concentrations generally were not affected by pregnancy. Although cervical NE per gram of tissue decreased during pregnancy, this effect of pregnancy was not detected when NE was expressed per microgram of DNA. Myometrial DA concentrations were higher in uterine segments than in the cervix. DA concentrations decreased during pregnancy in all tissues except the posterior uterine segment. The DA to NE ratio in the uterus was greater than that for the cervix and was not generally affected by the stage of pregnancy. These results demonstrate that cholinergic and adrenergic nerves supply the sheep uterus. Decreasing fiber density during pregnancy suggests that a majority of the innervation to the sheep uterus is supplied by 'short' nerve fibers whose activity is regulated by steroids of pregnancy. The possible role of DA as a neurotransmitter in the sheep uterus is discussed.     

Cholinergic nerves in the human liver

Summary The cholinergic innervation of the human liver was studied. Slices (150–200m thick) of human liver and of the greater hepatic blood vessels (hepatic artery and vein, portal vein) were incubated in a solution of 6-hydroxydopamine (6-HDA) in order to obtain a selective degeneration of adrenergic nerves. Controls were prepared from samples incubated with buffer alone. The slices were cut on a cryostat into 15–20m thick sections and processed for the histochemical detection of cholinesterases.Cholinergic nerve fibres innervate the extra hepatic and the intrahepatic branches of the hepatic artery, the portal vein as well as the hepatic vein. Fewer cholinergic fibres innervate the hepatocytes and the hepatic sinusoids. The 6-HDA treatment does not seem to alter the pattern of the cholinergic innervation of the liver. The findings indicate the presence of a cholinergic parasympathetic innervation in the human liver.     

Immunohistochemical studies of neurochemical markers in normal human buccal mucosa

The content of various substances, such as regulatory peptides, hormones and structural proteins, was investigated in normal buccal mucosa using indirect immunofluorescence. Thin nerve fibres, which from a morphological point of view were most probably sensory, showed immunoreactivity for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide K (NPK) and neurokinin A (NKA). Also galanin (GAL), -melanocyte stimulating hormone (-MSH) and somatostatin (SOM) stained thin fibres were found in the propria, which were, however, few in number and the -MSH staining was weak. CGRP, vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine amide (PHI) and neuropeptide Y (NPY) immunoreactive nerve fibres were observed in close connection to blood vessels. SOM positive cells with processes were found, mostly scattered, in the connective tissue. A population of cells within the epithelium also showed somatostatin immunoreactivity. Protein S-100 (S-100) stained distinct populations of cells at two separate locations. In the propria, cells with one or two slender processes were seen, being mostly single but sometimes forming groups. In the epithelium, dendritic cells with many processes with or without spines were observed, mainly located to the basal layer of the lamina epithelialis. Single nerve fibres and nerve bundles were also stained. Neurofilament (NF) positive fibres, singly and in bundles, as well as endorgan-like structures were seen. Neuron-specific enolase (NSE) and protein gene product 9.5 (PGP 9.5) both stained the same structures, namely single fibres, nerve bundles, nerves surrounding vessels and innervating muscles and glands (if present in the section), as well as Merkel cells. Also with these two markers endorgan-like structures were seen. No clear innervation of the epithelium could be observed with the markers used. No methionine-enkephalin (ENK) or synaptophysin (SYN) immunoreactive material was found.     

Effects of surgical denervations on the autonomic nerves in parotid glands of dogs

Summary The innervation of the dog's parotid has been studied by cholinesterase staining and catecholamine fluorescence. In normal glands cholinergic and adrenergic nerves are plentiful around acini, muscular blood vessels, and to a lesser extent striated ducts. The main ducts, although surrounded by many cholinesterase-positive nerves, are associated with few adrenergic nerves. Severance of the classical parasympathetic post-ganglionic nerve to the gland, the auriculo-temporal, caused a moderate loss of cholinesterase-positive nerves. When this procedure was combined with section of the nerves on the internal maxillary artery there was a greater loss. Fewest cholinesterase-positive nerves remained when, in addition to these two procedures, the facial nerve was cut. These findings support the concept that all three sets of nerves contain some post-ganglionic parasympathetic fibres for the dog's parotid. The source of the remaining nerves is unknown. Preganglionic parasympathetic denervation by section of the tympanic branch of the glossopharyngeal nerve did not reduce the number of cholinesterase-positive nerves. None of these parasympathetic denervations caused reduction of adrenergic nerves, indicating that they do not travel to the gland with the parasympathetic nerves. After superior cervical ganglionectomy a few scattered fluorescent nerves remained in the gland; their origin is unknown.