Ch. Heym

Correlation of neuronal size and peptide immunoreactivity in the guinea-pig trigeminal ganglion.

SummaryFrequency and size of guinea-pig trigeminal neurones immunoreactive with antisera to α-neo-endorphin(α-neo-END), dynorphin A-(DYN), vasoactive intestinal polypeptide-(VIP), somatostatin-(SOM), and substance P-(SP) are reported. Co-localisation of the various peptides to the same ganglion cells was investigated immunocytochemically in consecutive 7-μm thick paraffin sections. According to their size, all peptide-immunoreactive neurones belong to the class of “small” ganglion cells. Within this cell group, SP-immunoreactive neurones appear to be the largest, followed by SOM-, VIP-, α-neo-END- and DYN-immunoreactive ganglion cells. The observed differences in size are statistically significant with the exception of that between α-neo-END and DYN. This finding correlates well with the observed co-occurrence of the two immunoreactive peptides. All α-neo-END-immunoreactive perikarya are also reactive to VIP antisera. These neurones are significantly smaller than those containing VIP-immunoreactivity exclusively. Ganglion cells displaying co-existence of α-neoEND- and SP-immunoreactivity or VIP- and SP-immunoreactivity are found too infrequently to allow morphometric analysis. Some non-immunoreactive ganglion cells are shown to be approached by dense baskets of VIP-, α-neo-END- or SP-immunoreactive varicose fibres, indicating the presence of intraganglionic modulation sites. The combination of immunohistochemistry and morphometry presented in this study allows the differentiation of diverse populations of primary afferent neurones exhibiting peptide immunoreactivity, most likely reflecting their involvement in different central and peripheral reflex arcs and sensory modalities.

Dopamine-β-hydroxylase-, neurotensin-, substance P-, vasoactive intestinal polypeptide- and enkephalin-immunohistochemistry of paravertebral and prevertebral ganglia in the cat

SummaryPara and prevertebral ganglia of the cat were investigated for immunoreactivity (IR) against neurotensin (NT), vasoactive intestinal polypeptide (VIP), substance P (SP) and enkephalin (ENK). Dopamine-β-hydroxylase- (DBH)-IR was studied in consecutive sections to correlate the distribution of noradrenergic/adrenergic neurons with that of peptidergic nerve fibres and cells.In paravertebral (cervical and thoracic) ganglia, NT-IR or ENK-IR nerve fibres were seen in areas in which DBH-IR fibre networks also occurred. NT-IR varicosities were often in close contact with perikarya of principal ganglionic cells on which DBH-IR varicosities also terminated. Such an association was rarely seen between ENK-IR and DBH-IR fibre baskets. NT-IR and ENK-IR fibre baskets were not found to occur around the same principal ganglionic cell. The distribution of VIP-IR and SP-IR nerve fibres did not coincide with that of DBH-IR fibres.In prevertebral ganglia (celiac-superior mesenteric and inferior mesenteric) DBH-IR or VIP-IR varicosities surrounded the majority of principal ganglionic neurons. ENK-IR or SP-IR fibres were closely associated with only a minority of the neurons; NT-IR networks were rather sparse. Some principal neurons were approached by DBH-IR fibres and by different peptide-IR fibres.In paravertebral ganglia some principal ganglionic cells contained VIP-IR, a few of which were also surrounded by NT-IR varicosities. VIP-IR perikarya in prevertebral ganglia were extremely rare. No NT-IR, SP-IR or ENK-IR principal ganglionic cells were found.Glomus-like paraganglionic cell clusters in paravertebral and prevertebral ganglia exhibited DBH-IR cell bodies. Moreover, the clusters also contained ENK-IR or SP-IR cells. NT-IR varicosities were observed adjacent to clustered paraganglionic cells. Only few singly located paraganglionic cells were NT-IR or ENK-IR.The differential distribution of peptide-IR nerve endings in the investigated ganglia suggests a regulation of impulse transmission that seems to be related to the target organs.

Neuronal pathways in the guinea-pig lumbar sympathetic ganglia as revealed by immunohistochemistry.

SummaryTyrosine hydroxylase (TH)- and peptide-immunoreactivity of postganglionic neurons and of nerve fibres in guinea pig lumbar paravertebral sympathetic ganglia 2–4 after transection of the communicating rami and the visceral branches, respectively, were investigated by single-and double-labelling techniques. Six subpopulations of postganglionic neurons were discriminated immunohistochemically: two cell types, which were immunoreactive to only one of the applied antisera — TH, and vasoactive intestinal polypeptide (VIP); and four cell types in which immunoreactivity was colocalized — TH/neuropeptide Y (NPY), NPY/VIP, dynorphin/α-neoendorphin and dynorphin (α-neoendorphin)/NPY. Small intensely fluorescent (SIF) cells dependent on their location exhibited differential immunobehaviour to NPY-/dynorphin-(α-neoendorphin-) and TH-antisera. Immunoreactivity to substance P (SP), calcitonin gene-related peptide (CGRP), met-enkephalin-arg-phe (MEAP) and leu-enkephalin was present in nerve fibres but not in postganglionic neurons with frequent colocalization of SP/CGRP- and MEAP/leu-enkephalin- and, sometimes leu-enkephalin/SP- and dynorphin/SP-immunoreactivity. TH-immunoreactive intraganglionic nerve fibres were numerically more increased after cutting the visceral branches, than after transection of the communicating rami. Vice versa, NPY-, VIP-, dynorphin- and α-neoendorphin-immunoreactive nerve fibres were particularly increased in number after cutting the communicating rami. Many but not all of the nerve fibres exhibited colocalization of two of these peptides. SP-, CGRP-, and enkephalin-immunoreactive nerve fibres were not visibly affected by cutting the visceral branches but virtually disappeared after lesioning the communicating rami.

Immunohistochemical evidence for extrinsic and intrinsic opioid systems in the guinea pig superior cervical ganglion

SummaryImmunohistochemical localization of the opioid peptides α-neo-endorphin (α-neo-END), dynorphin A (DYN) and leu-enkephalin (leu-ENK) in the guinea pig superior cervical ganglion (SCG) was studied following central denervation, peripheral axotomy, and after application of the depleting drug reserpine and of the neurotoxin 6-hydroxydopamine. The paraganglionic cells of the SCG are shown to form an intrinsic opioid — (α-neo-END, DYN, leu-ENK) — immunoreactive system being not visibly responsive to the experimental procedures. Leu-ENK-immunoreactive fibres ascend in the preganglionic trunk and supply fibre baskets to defined clusters of postganglionic neurones. Principal ganglion cells of the SCG containing α-neo-END-and DYN-immunoreactivity project to extraganglionic targets via the postganglionic nerves. These findings are indicative of a sympathetic α-neo-END-ergic and DYN-ergic innervation of effector organs. They also point to a modulatory function of opioids on neuronal activity in a paravertebral ganglion.

Immunohistochemistry of biogenic polypeptides in nerve cells and fibres of the guinea pig inferior mesenteric ganglion after perturbations

SummaryImmunohistochemistry of peptide-and dopamine-β-hydroxylase-(DBH)-containing varicose nerve fibres and ganglion cells, respectively, in the guinea pig inferior mesenteric ganglion was investigated followinga) transsection of mesenteric (colonic) branches,b) transsection of central (lumbar splanchnic, intermesenteric and hypogastric) branches, andc) transplantation into the spleen.The findings indicate that pathways of different opioid peptides are not identical Met-enkephalin-and met-enkephalin-arg-phe-(cleavage products from pre-proenkephalin) containing fibres course in central branches to make contact in the inferior mesenteric ganglion. Dynorphin-and α-neo-endorphin-(deriving from pre-prodynorphin) containing fibers as well as leu-enkephalin-(included in the dynorphin sequence) fibres appear to rise not only from central and from enteric somata, but also from intraganglionic noradrenergic neurons. Similar pathways seem to be used by VIP-and by neurotensin-immunoreactive cell bodies are rare. Practically all substance P-and most CGRP-immunoreactive fibres enter the ganglion via central branches and, to a large extent, traverse it, but some CGRP-immunoreactive influx appears to come from the intestine. The origin of intraganglionic substance P-and CGRP-immunoreactive fibres after ganglion transplantation remained unidentified. Somatostatin-and neuropeptide Y-immunoreactive fibres predominantly have an intraganglionic origin as have DBH-immunoreactive noradrenergic fibres. The demonstrated alterations in neuropeptide immunoreactivity of intraganglionic and periganglionic nerve fibres following the applied transsection procedures contribute to the present knowledge on origin and destination of peptidergic transmitter segments in the guinea pig inferior mesenteric ganglion. Moreover, the present study provides evidence that intrinsic participitation in intraganglionic fibre supply is more extensive than hitherto believed.

Simultaneous demonstration of catecholamines and phenylethanolamine-n-methyltransferase in the same tissue section by means of formaldehyde-induced fluorescence (FIF) and tetramethyl-rhodamine-isothiocyanate (MRITC) immunofluorescence

Abstract A combined method is presented for the simultaneous demonstration of catecholamines through formaldehyde-induced fluorescence and immunohistochemical identification of phenylethanolamine-N-methyltransferase (PNMT) by indirect immunofluorescence with tetramethylrhodamine-isothiocyanatelabeled antisera. As a result of the application of this method to small intensely fluorescent (SIF) cells in the rat superior cervical ganglion, a minor portion of fluorescence-histochemically identified SIF cells exhibited PNMT-like immunoreactivity. This observation suggests the presence of epinephrine in these cells.

Immunohistochemistry of the guinea-pig trachea using an anti-idiotypic antibody recognizing substance P receptors

SummaryThe airways receive a dense innervation from sensory neurons containing substance P (SP). An anti-SP anti-idiotypic antibody (anti-Id ab) recognizing SP receptors was previously characterized pharmacologically and proved to be useful in immunohistochemistry of the central nervous system. This antibody was used to localize SP binding sites in the guinea-pig trachea by immunohistochemistry. Immunolabelling was considered as specific when it could be prevented by a) preabsorption of the anti-Id ab with a C-terminal specific monoclonal anti-SP antibody, and b) preincubation of the tissue sections with either of the tachykinins, substance P and neurokinin A, in the presence of the inhibitor of neutral endopeptidase, phosphoramidon, and addition of these compounds into the antibody incubation medium. Moreover, immunofluorescence was absent when the acetone-fixed of fresh frozen sections were exposed to the detergent Tween 20 prior to immunohistochemistry, which points to a membrane localization of the detected tissue antigen, as expected for SP receptors. Compared with previous reports on autoradiographic localization of SP receptors in the guinea-pig trachea, the present immunohistochemical approach proved to be superior in enabling discrimination of labelled elements: Trachealis muscle, cylindrical epithelial cells and some roundish, singly lying cells in the epithelium and subepithelial lamina propria displayed specific immunofluorescence. These morphological findings match well with the known pharmacological actions of SP on the guinea-pig trachea.

Immunohistochemical localization of neuropeptide Y in the guinea pig medulla oblongata

SummaryThe immunohistochemical localization of neuropeptide Y (NPY) was correlated with those of dopamine-β-hydroxylase (DBH) and vasoactive intestinal polypeptide (VIP) by mapping serial 7 μm paraffin sections at three levels of the guina pig lower brainstem: a) area postrema, b) dorsal motor nucleus of the vagus, and c) nucleus prepositus of the hypoglossal nerve. Based on differences in transmitter expression, three populations of NPY-immunoreactive (IR) neurons were distinguished: NPY-IR catecholaminergic cells (NPY/CA), NPY-IR VIP-ergic cells (NPY/VIP), and NPY-IR cells which were not reactive to either DBH or VIP. Within these populations, size differences among neurons in characteristic locations allowed differentiation among the following subpopulations: NPY/CA neurons in the lateral reticular nucleus — magnocellular part (mean neuronal size 538 μm2) and parvocellular part (318 μm2)-, in the vagus-solitarius complex (433 μm2), and in the dorsal strip (348 μm2); NPY/VIP neurons in the vagus-solitarius complex (368 μm2) and in the nucleus ovalis (236 μm2). Apart from scattered NPY-IR cell bodies in the regions listed above, NPY-IR cell bodies in the lateral portion of the nucleus solitarius and in the caudal part of the spinal nucleus of the trigeminal nerve did not exhibit IR to either DBH or VIP. NPY-IR neurons in the area postrema occurred too infrequently for co-localization studies. The differential distribution of heterogeneous NPY-IR cell subpopulations may reflect the involvement of NPY in a variety of neuronal functions.

Biochemical analysis of catecholamines in small intensely fluorescent (SIF) cell clusters of the rat superior cervical ganglion

The microlaser technique of isolating small cell clusters has been applied to groups of small intensely fluorescent (SIF) cells in rat superior cervical ganglion. Alternate cryostat sections were either incubated in glyoxylic acid monohydrate or freeze-dried. SIF cell clusters, recognized by glyoxylic acid-induced fluorescence, were re-identified in the consecutive freeze-dried section through dark-field microscopy. Three clusters were dissected with a BTG microlaser unit and collected for biochemical assay. The catecholamine content of the specimens was measured by gas chromatography/mass fragmentography, using 3 deuterated catecholamines as an internal standard and calibration curves of each catecholamine. The findings indicate the presence of these 3 catecholamines in rat SIF cell clusters in a varying amount: in probes, each consisting of 5 cell clusters, the content of norepinephrine averaged approximately 7.3 pmol, of epinephrine below 1 pmol, and of dopamine from below 1 pmol to 14.6 pmol.

Effects of Guanethidine on Paraganglionic Cells in the Superior Cervical Ganglion of the Rat

SummaryParaganglionic cells in the rat superior cervical ganglion were investigated by fluorescence and electron microscopy following treatment with guanethidine for 5–30 days. Control animals received saline and guanidine.Fluorescence cytophotometric measurements revealed a general decrease in the catecholamine content of paraganglionic cells in guanethidine-treated animals. However a few cell clusters showed focal increases. Similarly by electron microscopy there was a general decrease in the number of dense core vesicles in the majority of paraganglionic cells with a minority of cell clusters showing increases.—guanethidine—as well as guanidine—treated animals showed non-specific cytological alterations such as mitochondrial swelling and increase of cytoplasmic glycogen. However no changes of catecholamine contents and of dense core vesicles were noted in control animals.These results confirm the conception that in rat paraganglionic cells the dense core vesicles are the main storage site of catecholamines. The marked difference in the response of some cell clusters to the experimental treatment can be considered as evidence of functional heterogeneity of this cell population in the rat superior cervical ganglion.