Marianne Schultzberg

Distribution of peptide- and catecholamine-containing neurons in the gastro-intestinal tract of rat and guinea-pig: Immunohistochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrin/cholecystokinin, neurotensin and dopamine β-hydroxylase

Abstract The distribution of peptide-containing neurons in the oesophagus, stomach and small and large intestine of the rat and the guinea-pig has been studied with the indirect immunofluorescence technique of Coons & Co-workers (1958) using antisera to substance P, vasoactive intestinal polypeptide (VIP), enkephalin, somatostatin, gastrin and neurotensin. (The gastrin antiserum is to the C-terminal portion and consequently reacts also with cholecystokinin (CCK)-like peptides.) For comparison, the noradrenergic innervation was visualized with antiserum to dopamine β-hydroxylase. For improved visualization of peptide-containing cell bodies, a mitotic inhibitor (colchicine or vinblastine) was applied locally on the different parts of the gastro-intestinal tract of several animals. Substance P-, VIP-, enkephalin- and somatostatin-like immunoreactivity was observed in all parts of the gastro-intestinal tract studied. Gastrin/CCK had a more limited distribution, especially in the guinea-pig and neurotensin was seen only in certain regions and layers of the rat gastro-intestinal tract. Immunoreactivity to all peptides except neurotensin was observed both in cell bodies and fibres; immunoreactivity to neurotensin has so far only been seen in nerve fibres. Substance P and enkephalin immunoreactive cells were often numerous in the myenteric plexus, whereas VIP and somatostatin immunoreactive cells were preferentially located in the submucous plexus. Some VIP immunoreactive cells were observed in the lamina propria. Large numbers of especially substance P-, VIP- and enkephalin-containing fibres were often seen in the circular muscle layer and in the two ganglionic plexuses. Substance P immunoreactive fibres formed the densest network in the ganglionic plexuses, whereas VIP immunoreactive fibres constituted the most impressive network in the lamina propria and often extended into the most superficial parts of the mucosa. Enkephalin immunoreactive structures were mainly confined to the circular and longitudinal muscle layers and the myenteric plexus. Somatostatin immunoreactive fibres were mainly found in the ganglionic plexuses. Peptide-containing fibres, particularly these containing substance P and VIP were often seen along blood vessels, but never with such a density as the noradrenergic (dopamine β-hydroxylase immunoreactive) fibres. No somatostatin or neurotensin immunoreactive fibres were observed in relation to clearly identifiable blood vessels. The possible coexistence of two peptides in one neuron was studied. For this part of the study the proximal colon and five antisera, namely substance P, VIP, enkephalin. somatostatin and gastrin/CCK antisera were selected. Evidence was obtained for the occurrence of a somatostatin-like and a gastrin/ CCK-like peptide in the same neurons. This may indicate a common precursor for the two peptides in these particular neurons. Each of the substance P-, VIP- and enkephalin-like peptides. on the other hand, seem to be present in different neuronal populations, which were themselves distinct from the somatostatin-gastrin/CCK immunoreactive neurons. In addition, somatostatin immunoreactive neurons different from the gastrin/CCK immunoreactive ones seem to exist. The gastrin/CCK immunoreactive fibres around blood vessels may represent a further, separate population of fibres, since no somatostatin immunoreactive fibres were seen at this location. The findings indicate the existence of numerous subpopulations of enteric neurons, each characterized by its content of a certain peptide (or peptides). The axons of most of these neurons probably terminate in the wall of the gastro-intestinal tract, but some seem to project to other organs. In addition, some peptide-containing fibres in the gastro-intestinal wall may have an extrinsic origin. The relationship between these peptide-containing neurons and the cholinergic enteric neurons and any of the other non-cholinergic. non-adrenergic inhibitory and excitatory neurons present in the enteric nervous system is not known. It is, however, noteworthy that a somatostatin-like peptide seems to be present in noradrenergic neurons of prevertebral ganglia that project to the intestine. The possibility must be kept in mind that one or more of the peptides in the gut could be localized in neurons that contain other potential transmitters, e.g. acetylcholine. The wide variety of pharmacological actions of these neuronal peptides on smooth muscle and neurons in the gut and on its blood vessels raises the possibility that some of them may be neurotransmitters.

Occurrence of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in certain cholinergic neurons of the cat: Evidence from combined immunohistochemistry and acetylcholinesterase staining

Abstract Postganglionic sympathetic and parasympathetic neurons of the cat were studied with the indirect immunofluorescenee technique using antiserum to vasoactive intestinal polypeptide (VIP) and with acetylcholinesterase staining. In the stellate and in L7 and S1 sympathetic ganglia some of the principal ganglion cells (10–15% of the total) contained both VIP-like immunorcactivity and intense acetylcholinesterase activity, suggesting the presence of a VIP-like peptide in a population of sympathetic cholinergic neurons. This was corroborated by (1) overlapping accumulations of VIP immunoreactivity and acetylcholinesterase staining around a ligation of the sciatic nerve, (2) very similar distribution patterns of VIP immunoreactive and acetylcholinesterase-containing fibres around sweat glands of the food pad and around some blood vessels in several tissues, and (3) the disappearance of both types of staining of fibres around sweat glands and skeletal muscle vessels after lumbo-sacral sympathectomy. In other sympathetic ganglia, as well as in the parasympathetic pelvic ganglion plexus and in some ganglia of the bladder wall, VIP immunoreactivity was also found in neurons rich in acetylcholinesterase. On the other hand, it should be emphasized that the occurrence of a VIP-like peptide in presumably cholinergic neurons does not seem to be a general phenomenon. No marked overlap was observed between VIP immunoreactive and acetylcholinesterase-rich cell bodies in most ganglia in the urinary bladder wall and nerve terminals of the smooth muscle layer. Furthermore, no obvious correlation in the number of VIP immunoreactive and acetylcholinesterase-containing fibres were seen in certain other nerves such as the spinal ventral roots, the splanchnic nerve and the perivascular superior mesenteric nerves. Thus, three groups of peripheral autonomic neurons have been defined in the present study: neurons rich in acetylcholinesterase that also contain a VIP-like peptide; VIP immunoreactive neurons; and neurons rich in acetylcholinesterase. The functional significance of a VIP-like peptide in probable cholinergic neurons is at present unclear. Preliminary data indicate that, whereas acetylcholine directly stimulates sweat secretion, VIP may cooperate in the production of sweat by increasing local blood flow. Thus, VIP, released together with acetylcholine, may be responsible for the atropine-resistant vasodilatation in sweat glands and other exocrine glands.

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.

Substance P- and CGRP-immunoreactive nerves in bone

The present study demonstrates the occurrence of substance P (SP)- and calcitonin gene related peptide (CGRP)-immunoreactive nerve fibres in bone, bone marrow, periosteum, synovial membrane and soft tissues adjacent to the bone. The distribution pattern of the two types of nerves was similar, although the CGRP-positive fibres generally were more numerous. Both types of nerves were particularly abundant near the epiphyseal plate, in the bone marrow of patella and epiphyses, and in the periosteum. Many SP- and CGRP-immunoreactive fibres were also observed around blood vessels.

Cytokine regulation of neuronal survival

Abstract: Interleukin‐1 is a cytokine involved in the immune response to infection and inflammation as well as a growth promotor for several cell types. Interleukin‐1‐like immunoreactive material has been found in the nervous system. We now show that antisera, which blocked the T‐cell proliferative effects of interleukin‐1α, decreased neuronal cell counts (to 40% of control) in dissociated spinal cord cultures derived from fetal mice. This neuronal loss was prevented by addition of interleukin‐1α, and to a lesser extent by interleukin‐1β. Exogenous interleukin‐1α increased the survival of neurons when added to cultures in which the electrical activity was blocked with tetrodotoxin, whereas no such cytokine‐related increase in neuronal survival was observed in electrically active cultures. The antiserum‐induced death could also be prevented by cotreatment of the cultures with 0.1 nM vasoactive intestinal peptide, a substance that induces the secretion of neuronal trophic factors from nonneuronal spinal cord cells and thereby increases neuronal survival in electrically inactive cultures. These studies indicate that the cytokine interleukin‐1, or an immunologically cross‐reactive protein, can increase neuronal survival.

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.

Neuropeptide Y-, tyrosine hydroxylase- and vasoactive intestinal polypeptide-immunoreactive nerves in bone and surrounding tissues

Nerve fibres immunoreactive to neuropeptide Y (NPY), tyrosine hydroxylase (TH) and vasoactive intestinal polypeptide (VIP) were demonstrated in rat bone and adjacent tissues. The distribution of NPY- and TH-positive fibres differed from that of VIP-positive fibres. NPY- and TH-immunoreactive fibres were almost exclusively found close to or within the blood vessel walls, mostly in the vicinity of the epiphyseal plate, but also in the Volkmann canals. VIP-positive fibres were predominantly present in the epiphysis and periosteum and only occasionally around blood vessels. This study demonstrates that bone and surrounding tissues have a supply of both noradrenergic and peptide-containing nerves. The differential distribution of these nerves may reflect specific roles in the local regulation of bone physiology, such as blood flow, bone formation or resorption.

VIP-, enkephalin-, substance P- and somatostatin-like immunoreactivity in neurons intrinsic to the intestine: immunohistochemical evidence from organotypic tissue cultures

Small intestine from 18-day fetal mice grown for 3 weeks in organotypic tissue culture was found to contain numerous VIP, enkephalin, substance P and some somatostatin immunoreactive nerve fibers. Since these cultures should be devoid of all afferent or other extrinsic neuronal inputs, it is concluded that there are VIP, enkephalin, substance P and somatostatin containing neurons intrinsic to the intestinal wall. However, all 4 peptides may also be present in neurons originating outside the gastrointestinal tract as well as in the intrinsic neurons.

Blood-brain barrier alterations in ageing and dementia

The current pathogenic scenarios of different types of dementia are based on a number of common mechanisms of neurodegeneration, such as accumulation of abnormal proteins (within or outside cells), mitochondrial dysfunction and oxidative stress, calcium homeostasis dysregulation, early synaptic disconnection and late apoptotic cell death. Ageing itself is associated with mild cognitive deterioration, probably due to subtle multifactorial changes resulting in a global decrease of a functional brain reserve. Increased age is a risk factor for neurodegeneration and key pathological features of dementia can also be found in aged brains. One of the underexplored brain structures in ageing and dementia is the blood-brain barrier (BBB), a complex cellular gate which regulates tightly the transport of molecules into and from the central nervous system. Disruption of this barrier is now increasingly documented not only in brain vascular disease but also in ageing and neurodegenerative disorders. To date, such evidence points mainly at an association between various dementia forms and disruption of the BBB. But, in reviewing such results, and taking into account the exquisite sensitivity of neuronal function to the composition of the interstitial brain fluid (IBF), which is regulated by the BBB, we would like to propose the existence of a possible causal link between alterations of BBB and conditions associated with cognitive decline.

Coexistence of cholecystokinin- and substance P-like peptides in neurons of the dorsal root ganglia of the rat

Using the indirect immunohistochemical technique with antisera to cholecystokinin and to substance P, the spinal dorsal horn and dorsal root ganglia of normal and colchicine-treated rats were studied. In the spinal cord a similar distribution of substance P- and cholecystokinin-positive networks in the superficial layers of the dorsal horn was observed. In the dorsal root ganglia several cholecystokinin and substance P immunoreactive cell bodies were seen in colchicine-treated rats. After elution and restaining for substance P, of sections previously stained for cholecystokinin, it was found that all cholecystokinin-positive cells also contained substance P-like immunoreactivity and vice versa.