L.-G. Elfvin

Enkephalin immunoreactive nerve fibres and cell bodies in sympathetic ganglia of the guinea-pig and rat.

The occurrence and distribution of enkephalin-like immunoreactivity was studied by light microscopy, using an indirect fluorescent-labelled antibody technique, in the superior cervical ganglion, the inferior mesenteric ganglion and the coeliac-superior mesenteric ganglion complex of the guinea-pig and rat. In theguinea-pig a very dense network of enkephalin-positive fibres was observed in the inferior mesenteric ganglion and a less dense one in the coeliac-superior mesenteric ganglion complex. In both ganglia some ‘small intensely fluorescent’ cells were immunoreactive. In the superior cervical ganglion only few fluorescent fibres were seen but several ‘small intensely fluorescent’ cells were enkephalin-positive. In therat the inferior and coeliac-superior mesenteric ganglia contained medium-dense networks of enkephalin-positive fibres. An irregularly distributed network of fluorescent fibres was observed in the superior cervical ganglion, where also several principal ganglion cells were enkephalinimmunoreactive, particularly after colchicine treatment. These findings indicate the presence of several peripheral neuron systems containing enkephalin or a similar peptide. Several antisera raised to methionine- and leucine-enkephalin as well as to α- and β-endorphin were used. Some of these antisera were compared by incubating sections of the inferior mesenteric ganglion with increasing dilutions of antiserum as well as with antisera treated with increasing concentrations of methionine- and leucine-enkephalin, respectively. On the basis of these findings the problem of differentiating between methionine- and leucine-enkephalin is discussed.

Chapter 4 Coexistence of neuronal messengers — an overview

Publisher Summary This chapter discusses results demonstrating that neurons often contain more than one chemical compound. The different types of coexistence situations are described, including (1) a classical transmitter and one or more peptides, (2) more than one classical transmitter, and (3) a classical transmitter, a peptide, and adenosine triphosphate (ATP). The functional significance of these histochemical findings is at present difficult to evaluate, but in studies on the peripheral nervous system evidence has been obtained that classical transmitter and peptide are coreleased and interact in a cooperative way on effector cells. In addition to enhancement, there is evidence that other types of interactions may occur—for example, the peptide may inhibit the release of the classical transmitter. Also in the central nervous system (CNS), indirect evidence is present for similar mechanisms—that is, to strengthen transmission at synaptic (or non-synaptic) sites and for the peptide inhibition of release of a classical transmitter. Multiple messengers may provide the means for increasing the capacity for information transfer in the nervous system.

Fluorescence microscopical, immunohistochemical and ultrastructural studies on sympathetic ganglia of the guinea pig, with special reference to the SIF cells and their catecholamine content

The small intensely fluorescent cells—SIF cells—in the inferior mesenteric ganglion of the guinea pig are all strongly fluorescent when studied after preparation with the Falck-Hillarp method for demonstrating catecholamines. By using an immunohistochemical fluorescent method probably all SIF cells could be shown to exhibit strong tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) positive fluorescence, indicating that they store large amounts of noradrenaline. Some SIF cells show the presence of phenylethanolamine-N-methyl transferase (PNMT) indicating that they also store adrenaline. Ultrastructurally the cells are shown to contain two types of large granules. Most cells have highly opaque granules similar in structure to the noradrenaline-storing granule in the adrenal medulla of many mammals. A few cells have less opaque granules similar to adrenaline-storing granules. Some cells contain a mixture of both granule types. The SIF cells have long processes located close to blood capillaries either at the endothelium or at the pericytes. At the pericytes there are accumulations in the cell processes of small dense core vesicles, similar in structure to the presynaptic vesicles observed in adrenergic nerve terminals. The principal ganglion cells contain TH and DBH but not PNMT and thus seem to store only noradrenaline. The SIF cells in the superior cervical ganglion show a high fluorescence intensity after incubation with TH or DBH antiserum but no specific fluorescence was demonstrated after incubation with antiserum towards PNMT. In the superior cervical ganglion of the guinea pig the SIF cells therefore appear to store noradrenaline exclusively. Probably all principal ganglion cells contain TH but not PNMT, whereas the DBH content seems to vary considerably. The possibility that some principal ganglion cells preferentially store dopamine is discussed.

Structural studies on the connectivity of the inferior mesenteric ganglion of the guinea pig

After injection of horseradish peroxidase (HRP) into the inferior mesenteric ganglion (IMG) of the guinea pig, labeled cell bodies were found in the solar plexus ganglia, the ganglia of the pelvic plexus and in the nodose ganglia as well as in the spinal cord and dorsal root ganglia at the T13-L4 levels. By using the fluorescent tracer True blue, labeled cell bodies could also be detected in the myenteric and submucosa ganglia of the distal colon. Application and uptake of HRP by the colonic nerves resulted in labeling of cell bodies in the dorsal root ganglia, the IMG and in the solar plexus ganglion complex indicating that the latter neurons send their axons in the intermesenteric nerve through the IMG to continue in the colonic nerves to the distal colon. When HRP was applied and diffused into the hypogastric nerves labeled cell bodies were seen in the IMG, the dorsal root ganglia and in the preganglionic nuclei of the lumbar spinal cord with 50% of the labeled neurons located dorsal and dorsolateral to the central canal. The finding of such a diversity in the nerve supply to the IMG, supports the view that the ganglion plays an important role in the integration of visceral reflexes.

Distribution and origin of peptide-containing nerve fibers in the celiac superior mesenteric ganglion of the guinea-pig

The origin of the peptidergic nerve fibers and terminals in the celiac superior mesenteric ganglion of the guinea-pig was studied. The distribution of immunoreactivity to enkephalin, substance P, calcitonin gene-related peptide, cholecystokinin, vasoactive intestinal polypeptide/peptide histidine isoleucine, bombesin and dynorphin was analysed in intact animals and in animals subjected to various denervation and ligation procedures. The present results show that each of the connected nerve trunks carries peptidergic pathways and contributes to the peptidergic networks in the celiac superior mesenteric ganglion. Thus, the thoracic splanchnic nerves contain enkephalin-, substance P- and calcitonin gene-related peptide-immunoreactivity of which substance P and calcitonin gene-related peptide coexist in the same nerve fibers. In addition, cholecystokinin-, vasoactive intestinal polypeptide/peptide histidine isoleucine- and dynorphin-immunoreactivity is present in some fibers. All of these immunoreactivities are present in sensory neurons except enkephalin which probably originates in the spinal cord. The mesenteric nerves carry enkephalin-, calcitonin gene-related peptide-, cholecystokinin-, vasoactive intestinal polypeptide/peptide histidine isoleucine-, bombesin- and dynorphin-immunoreactive fibers from the intestine and are the main source for cholecystokinin, vasoactive intestinal polypeptide/peptide histidine isoleucine, bombesin and dynorphin fibers. Double-staining experiments indicate that many of these peptides are synthesized in the same enteric neurons. Also the intermesenteric nerve contains peptide-immunoreactive fibers to the celiac superior mesenteric ganglion from different sources, probably including the distal colon as well as dorsal root ganglia and spinal cord at lower thoracic and lumbar levels. The results are discussed in relation to earlier morphological and physiological studies supporting the view of a role of the celiac superior mesenteric ganglion in local reflex mechanisms involved in regulation of gastrointestinal functions.

Primary sensory afferents in the inferior mesenteric ganglion and related nerves of the guinea pig: An experimental study with anterogradely transported wheat germ agglutinin-horseradish peroxidase conjugate

Peripheral visceral afferents in the guinea pig were labeled by injections of wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the L2 and L3 dorsal root ganglia bilaterally. After anterograde transport of the tracer the following areas were examined for the presence of HRP-labeled fibers: the inferior mesenteric ganglion (IMG), the inferior mesenteric artery (IMA) with surrounding tissue, the hypogastric nerves, parts of the descending and sigmoid colon as well as the urinary bladder. Large numbers of heavily labeled fibers were found in the IMG, in the colonic nerves around the IMA and in the hypogastric nerves. In the IMG, profiles suggestive of being labeled axon terminals were observed. Labeled fibers were observed in the muscle layers of the colon and in the bladder wall. The results show that anterograde tracing with WGA-HRP can be used successfully in analyzing the morphology and structural organization of visceral afferents in the periphery.

Effects of peripheral axotomy on neuropeptides and nitric oxide synthase in dorsal root ganglia and spinal cord of the guinea pig: an immunohistochemical study

The effect of axotomy (3, 10 and 21 days) on the expression of some neuronal markers was analysed in dorsal root ganglia and spinal cord of guinea-pigs using immunohistochemistry. Three weeks following injury, substance P-like immunoreactivity (-LI) was slightly reduced in the DRGs of the ipsilateral side, whereas a marked increase in neuropeptide Y(NPY)-LI could be detected ipsilaterally and a smaller increase contralaterally. NPY-LI was mainly expressed in small, but also some medium-sized and large neuron profiles after axotomy. Galanin-LI showed a moderate bilateral increase. No significant changes could be observed in DRGs for calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide-, peptide histidine isoleucine- or nitric oxide synthase-LIs. In the ventral horn CGRP-LI was slightly increased bilaterally in motoneurons, most pronounced on the injured side. Autotomy behaviour was seen in seven of the nine animals in the twenty-one day group. The present results demonstrate that also in guinea-pigs several peptides undergo distinct changes in their expression after peripheral nerve injury. However, in contrast to rats and monkeys, galanin-LI is only moderately increased in guinea-pigs. Neuropeptide Y showed a dramatic increase mainly in small neurons, in contrast to the upregulation in large neurons in the rat. Thus, distinct species differences exist with regard to the cellular response to nerve injury.

Nitric oxide synthase, choline acetyltransferase, catecholamine enzymes and neuropeptides and their colocalization in the anterior pelvic ganglion, the inferior mesenteric ganglion and the hypogastric nerve of the male guinea pig

By the indirect immunofluorescence method, the distribution of nitric oxide synthase (NOS)-like immunoreactivity (LI) and its possible colocalization with neuropeptide immunoreactivities, with two enzymes for the catecholamine synthesis pathway, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH), as well as the enzyme for the acetylcholine synthesis pathway, choline acetyltransferase (ChAT) were studied in the anterior pelvic ganglion (APG), the inferior mesenteric ganglion (IMG) and the hypogastric nerve in the male guinea pig. The analyses were performed on tissues from intact animals, as well as after compression/ligation or cut of the hypogastric nerve. In some cases the colonic nerves were also cut. Analysis of the APG showed two main neuronal cell populations, one group containing NOS localized in the caudal part of the APG and one TH-positive group lacking NOS in its cranial part. The majority of the NOS-positive neurons contained ChAT-LI. Some NOS-positive cells did not contain detectable ChAT, but all ChAT-positive cells contained NOS. NOS neurons often contained peptides, including vasoactive intestinal peptide (VIP), neuropeptide tyrosine (NPY), somatostatin (SOM) and/or calcitonin gene-related peptide (CGRP). Some NOS cells expressed DBH, but never TH. The second cell group, characterized by absence of NOS, contained TH, mostly DBH and NPY and occasionally SOM and CGRP. Some TH-positive neurons lacked DBH. In the IMG, the NOS-LI was principally in nerve fibers, which were of two types, one consisting of strongly immunoreactive, coarse, varicose fibers with a patchy distribution, the other one forming fine, varicose, weakly immunoreactive fibers with a more general distribution. In the coarse networks, NOS-LI coexisted with VIP- and DYN-LI and the fibers surrounded mainly the SOM-containing noradrenergic principal ganglion cells. A network of ChAT-positive, often NOS-containing nerve fibers, surrounded the principal neurons. Occasional neuronal cell bodies in the IMG contained both NOS- and ChAT-LI. Accumulation of NOS was observed, both caudal and cranial, to a crush of the hypogastric nerve. VIP accumulated mainly on the caudal side and often coexisted with NOS. NPY accumulated on both sides of the crush, but mainly on the cranial side, and ENK was exclusively on the cranial side. Neither peptide coexisted with NOS. Both substance P (SP) and CGRP showed the strongest accumulation on the cranial side, possibly partly colocalized with NOS. It is concluded that the APG in the male guinea-pig consists of two major complementary neuron populations, the cholinergic neurons always containing NOS and the noradrenergic neurons containing TH and DBH. Some NOS neurons lacked ChAT and could represent truly non-adrenergic, non-cholinergic neurons. In addition, there may be a small dopaminergic neuron population, that is containing TH but lacking DBH. The cholinergic NOS neurons contain varying combinations of peptides. The noradrenergic population often contained NPY and occasionally SOM and CGRP. It is suggested that NO may interact with a number of other messenger molecules to play a role both within the APG and IMG and also in the projection areas of the APG.

The distribution of the sympathetic preganglionic neurons projecting onto the stellate ganglion of the guinea pig. A horseradish peroxidase study.

After injection of horseradish peroxidase (HRP) into the stellate ganglion (SG) of the guinea pig, labelled cells were found in the C8-T11 spinal cord segments. The HRP-positive neurons were located in the nucleus intermedio-lateralis pars principalis and funicularis (ILp and ILf) and in the nucleus intercalatus proprius and pars paraependymalis (ICp and ICpe). The ICp and ICpe neurons represented approximately one-fourth of the total number of labelled cells. The ILp and ILf neurons were mainly located in the T2 and T3 segments whereas the ICp and ICpe neurons occurred mainly in the T4 to T6 segments where the ICpe cell group formed a continuous cell column dorsal to the central canal. The possible correlation between the segmental distribution and peripheral sympathetic effects was discussed.

Anterograde transsynaptic transport of WGA-HRP from spinal afferents to postganglionic sympathetic cells of the stellate ganglion of the guinea pig

After injection of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) or choleragenoid conjugated HRP (B-HRP) into lower cervical and upper thoracic dorsal root ganglia (DRG), HRP reaction product was observed in peripheral fibers of spinal afferents and in postganglionic cell bodies of the stellate ganglion (SG) in the guinea pig. After WGA-HRP injection into C8-T3 or T5 DRG, HRP-labelled cells were observed to cluster at the rami within the SG, with peak labelling observed 36 h after injection. SG cell labelling occurred with B-HRP as well, but not with native HRP after injection into thoracic DRG. Injection of this tracer in C8 DRG gave rise to a small number of labelled cells. In contrast to the labelling pattern following thoracic or C8 DRG injections, injection of WGA-HRP or native HRP into C6 DRG, led to random SG cell labelling. We conclude that the anterograde transsynaptic transport, following injection of WGA-HRP into thoracic DRG, provides a method to selectively label a population of postganglionic sympathetic neurons within the SG. A combination of transsynaptic and retrograde transport appears to be responsible for labelling after injection into C8 DRG, whereas labelling after C6 DRG injections seems to be due primarily to retrograde transport.