B. Lindh

Coexistence of peptides with classical neurotransmitters

In the present article the fact is emphasized that neuropeptides often are located in the same neurons as classical transmitters such as acetylcholine, 5-hydroxy-tryptamine, catecholamines, γ-aminobutyric acid (GABA) etc. This raises the possibility that neurons produce, store and release more than the one messenger molecule. The exact functional role of such coesisting peptides is often difficult to evaluate, especially in the central nervous system. In the periphery some studies indicate apparently meaningful interactions of different types with the classical transmitter, but other types of actions including trophic effects have been observed. More recently it has been shown that some neurons contain more than one classical transmitter, e.g. 5-HT plus GABA, further underlining the view that transfer of information across synapses may be more compex than perhaps hitherto assumed.

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.

NPY-, galanin-, VIP/PHI-, CGRP- and substance P-immunoreactive neuronal subpopulations in cat autonomic and sensory ganglia and their projections

SummaryThe neuronal subpopulations in the cat stellate, lower lumbar and sacral sympathetic ganglia were studied with regard to the cellular distribution of immunoreactivity to tyrosine hydroxylase (TH), acetylcholinesterase (AChE) and various neuronal peptides. Coexistence of neuropeptide Y (NPY)- and galanin (GAL)-like immunoreactivity (LI) was found in a high proportion of the neuronal cell bodies; these cells also contained immunoreactivity to TH, confirming their presumably noradrenergic nature. Some TH- and GAL-immunoreactive principal ganglion cells lacked NPY-LI. Two populations (scattered and clustered) of vasoactive intestinal polypeptide (VIP)- and peptide histidine isoleucine (PHI)-positive cell bodies were found in the sympathetic ganglia studied. The scattered VIP/PHI neurons also contained AChE-LI, calcitonin gene-related peptide (CGRP)-and, following culture, substance P (SP)-LI. The clustered type only contained AChE-LI. In the submandibular and sphenopalatine ganglia, neurons were AChE- and VIP/ PHI-immunoreactive but lacked CGRP- and SP-LI. Many GAL- and occasional TH-positive neurons were found in these ganglia. In the spinal ganglia, single NPY-immunoreactive sensory neuronal cells were observed, in addition to CGRP- and SP-positive neurons. The present results show that there are at least two populations of sympathetic cholinergic neurons in the cat. Retrograde tracing experiments indicate that the scattered type of cholinergic neurons contains four vasodilator peptides (VIP, PHI, CGRP, SP) and provides an important input to sweat glands, whereas the clustered type (containing VIP and PHI) mainly innervates blood vessels in muscles.

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.

Distribution and origin of peptide-containing nerve fibres in the rat and human mammary gland

The structures in the mammary gland involved in milk ejection have been investigated with regard to their relation to different types of peptidergic nerve fibres and their origin. Lactating rats were studied with immunohistochemistry focusing on the nipple, the parenchyma, the mammary blood vessels and the mammary nerve. The human mammary gland was also analysed. In the mammary gland from rat and human, nerve endings in the subepidermis, around smooth muscle cells in the nipple, in the connective tissue surrounding lactiferous ducts and alveoli in the nipple and in the parenchyma of the mammary gland showed immunoreactivity for calcitonin gene-related peptide, substance P, vasoactive intestinal polypeptide, peptide histidine isoleucine, neuropeptide Y, galanin and tyrosine hydroxylase, whereas dynorphin-positive nerve fibres could not be detected. The mammary nerve contained calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y and tyrosine hydroxylase immunoreactivities; the adventitia of the mammary artery contained nerve fibres immunoreactive for neuropeptide Y and tyrosine hydroxylase, while vasoactive intestinal polypeptide-, peptide histidine isoleucine-, calcitonin gene-related peptide- and substance P-positive fibres were found in the tissue surrounding the artery. The wall of the mammary vein had nerve terminals immunoreactive for neuropeptide Y, tyrosine hydroxylase, calcitonin gene-related peptide and substance P. With the help of retrograde tracing using wheat germ agglutinin in combination with immunohistochemistry, projections of calcitonin gene-related peptide-immunoreactive cells in the dorsal root ganglia to the nipple were established. Neurons in the sympathetic stellate ganglion containing neuropeptide Y and tyrosine hydroxylase also projected to the mammary gland. Moreover retrogradely-labelled cells were found in the nodose ganglion, and they were vasoactive intestinal polypeptide-immunoreactive. These results demonstrate a rich distribution of different types of nerve fibres in structures of the mammary gland related to milk ejection. These nerve fibres and their peptides may be involved in the local control of milk ejection.

Substance P mRNA is present in a population of CGRP-immunoreactive cholinergic postganglionic sympathetic neurons of the cat: evidence from combined in situ hybridization and immunohistochemistry

A synthetic oligonucleotide probe, complementary to a sequence of the rat beta-preprotachykinin mRNA coding for part of the mature substance P (SP), was used to localize, by in situ hybridization, SP mRNA to individual paravertebral sympathetic ganglion cells of the cat. Subsequent immunohistochemical analysis revealed that most of these neurons contained immunoreactivity to calcitonin gene-related peptide (CGRP), suggesting that they belong to the cholinergic cell population of the scattered type. These cells contain, in addition to SP and CGRP, vasoactive intestinal polypeptide and peptide histidine isoleucine.

Simultaneous immunohistochemical demonstration of intra-axonally transported markers and neuropeptides in the peripheral nervous system of the guinea pig

SummaryProjections and peptide neurotransmitter/neuromodulator content of autonomic and visceral afferent neurons of the guinea pig were studied after application of the subunit B of cholera toxin (CTB) with or without horseradish peroxidase (HRP) as retrograde and anterograde tracers and subsequent immunohistochemical processing for double staining using antibodies raised to CTB, HRP and various neuropeptides. The results demonstrate that substance P (SP)- and calcitonin gene-related peptide (CGRP)-containing dorsal root ganglion cells project to the pylorus as well as to the celiac superior mesenteric and stellate ganglia as demonstrated with both retrograde and anterograde transport methodology. Binding studies revealed that a small number of the CTB-binding dorsal root ganglion cells contains immunoreactivity to SP and CGRP. The majority of the CTB-binding cells is SP- and CGRP-negative and terminate in the deeper parts of the dorsal horn. After injection of CTB conjugated to HRP (B-HRP) into the nodose ganglion, both motor and sensory elements were labeled in the medulla oblongata. Some of the CTB labeled vagal sensory nerve fibers in the nucleus tractus solitarii (NTS) were also found to contain immunoreactivity to SP or CGRP. The tracer was also transported through the peripheral branch of the nodose ganglion cells and labeled terminals in the esophagus.

The expression of different cytochemical markers in normal and axotomised dorsal root ganglion cells projecting to the nucleus gracilis in the adult rat

Rat lumbar dorsal root ganglion neurones projecting to the nucleus gracilis in the brainstem were retrogradely labelled with Fluoro-Gold and analysed immunocytochemically for their expression of substance P-, calcitonin gene-related peptide-, galanin-, galanin message-associated peptide-, neuropeptide Y-, nitric oxide synthase- and carbonic anhydrase-like immunoreactivity as well as affinity to Griffonia (bandeiraea) simplicifolia lectin I — isolectin B4, RT97 and to choleragenoid. The analysis was made both in uninjured rats and in rats which had been subjected to unilateral sciatic nerve transection and partial resection 3 weeks earlier. The data showed that 6% of the L4 and L5 lumbar dorsal root ganglion cells that projected to the nucleus gracilis showed substance P-like immunoreactivity. Following nerve injury, none of the nucleus gracilis-projecting dorsal root ganglion cells showed substance P-like immunoreactivity. Nineteen per cent of the investigated cell population showed calcitonin gene-related peptide-like immunoreactivity in uninjured rats, but no nucleus gracilisprojecting calcitonin gene-related peptide-positive cells were found after nerve injury. Galanin- and galanin message-associated peptide-like immunoreactivity were found in 2% and 3%, respectively, of the Fluoro-Gold-labelled cell population normally and in 22% and 14%, respectively, after injury. No neuropeptide Y-positive cells were found in the Fluoro-Gold-labelled cell population normally, but after nerve injury, 96% of this population became neuropeptide Y-positive. Nitric oxide synthaselike immunoreactivity was found in 2% of the Fluoro-Gold-labelled cells normally and in 10% after injury. Two per cent of the Fluoro-Gold-labelled cells in the normal cases were stained by Griffonia (bandeiraea) simplicifolia lectin I — isolectin B4. After injury, however, no such double labelling was found. Thirty-four per cent of the Fluoro-Gold-labelled cell population was carbonic anhydrase positive normally, and 42% after injury. Seventy-five per cent of the Fluoro-Gold-labelled cells showed RT97 immunoreactivity normally and 12% after injury. Choleragenoid-like immunoreactivity was found in 99% of the Fluoro-Gold-labelled dorsal root ganglion cells normally and 81% after injury.Immunohistochemical visualisation of choleragenoid transganglionically transported from the injured sciatic nerve combined with neuropeptide Y immunocytochemistry showed that primary afferent fibres and terminals in the nucleus gracilis contain neuropeptide Y following peripheral nerve transection. Taken together, the results indicate that peripherally axotomised nucleus gracilis-projecting neurones undergo marked alterations in their cytochemical characteristics, which may be significant for the structural and functional plasticity of this system after injury.

WGA-HRP and choleragenoid-HRP as anterogradely transported tracers in vagal visceral afferents and binding of WGA and choleragenoid to nodose ganglion neurons in rodents.

The axonal and terminal labelling pattern in the brain stem resulting from the injection of horseradish peroxidase (HRP) conjugate of wheat germ agglutinin (WGA) or choleragenoid into the nodose ganglion of guinea pigs was examined. In addition, the binding profiles of WGA and choleragenoid in the nodose ganglion of guinea pig and rat were examined. The results show that WGA-HRP and choleragenoid-HRP (B-HRP) produce almost identical distribution of axonal and terminal labelling, the difference being some contralateral fibre labelling present only with B-HRP. However, WGA-HRP shows the strongest labelling at short survival times, whereas B-HRP requires longer postoperative survival times to reach maximum labelling intensity. All nodose ganglion neurons appear to bind WGA as well as choleragenoid although to a varying degree. The results of this and previous studies support the view that visceral sensory ganglion cells and the large light subpopulation of somatic dorsal root ganglion cells both bind choleragenoid, whereas the small dark somatic cells show affinity for WGA but rarely for choleragenoid.

Effects of preganglionic sympathectomy on peptides in the rat superior cervical ganglion.

A novel method to selectively lesion preganglionic sympathetic neurones has been combined with immunohistochemistry to study the expression of peptides in the rat superior cervical ganglion (SCG). Thus, systemic administration of monoclonal antibodies against acetylcholinesterase (AChE) caused a marked reduction in the number of enkephalin (ENK)-positive fibres and a total disappearance of fibres immunoreactive for calcitonin gene-related peptide (CGRP) and AChE in the SCG. A marked increase in the number of galanin/galanin message-associated peptide (GAL/GMAP)-immunoreactive cell bodies was also observed. The present results indicate that probably all CGRP and most ENK containing fibres in the rat SCG are of preganglionic origin and that peptides not normally expressed in SCG neurones, e.g. GAL and GMAP, can be upregulated after deafferentation.