Christine Heym

The sensory and sympathetic innervation of guinea-pig lung and trachea as studied by retrograde neuronal tracing and double-labelling immunohistochemistry

The sympathetic and sensory innervation of guinea-pig trachea and lung were studied by means of retrograde neuronal tracing using fluorescent dyes, and double-labelling immunofluorescence. Sympathetic neurons supplying the lung were located in stellate ganglia and in thoracic sympathetic chain ganglia T2-T4; those supplying the trachea resided in the superior cervical and stellate ganglia. Retrogradely labelled sympathetic neurons were usually immunoreactive to tyrosine hydroxylase; the majority also contained neuropeptide Y immunoreactivity. However, a small number were non-catecholaminergic (i.e. tyrosine hydroxylase negative), but neuropeptide Y immunoreactive. Within the airways, tyrosine hydroxylase/neuropeptide Y-immunoreactive axons were found in the smooth muscle layer, around blood vessels including the pulmonary artery and vein, and to a lesser extent in the lamina propria. Periarterial axons contained in addition dynorphin immunoreactivity. Sensory neurons supplying the lung were located in jugular and nodose vagal ganglia as well as in upper thoracic dorsal root ganglia; those supplying the trachea were most frequently found bilaterally in the nodose ganglia and less frequently in the jugular ganglia. A spinal origin of tracheal sensory fibres could not be consistently demonstrated. With regard to their immunoreactivity to peptides, three types of sensory neurons projecting to the airways could be distinguished: (i) substance P/dynorphin immunoreactive; (ii) substance P immunoreactive but dynorphin negative; and (iii) negative to all peptides tested. Substance P-immunoreactive neurons innervating the airways invariably contained immunoreactivity to neurokinin A and calcitonin gene-related peptide. Retrogradely labelled neurons located in the nodose ganglia belonged almost exclusively (greater than or equal to 99%) to the peptide-negative group, whereas the three neuron types each represented about one-third of retrogradely labelled neurons in jugular and dorsal root ganglia. Within the airways, axons immunoreactive to substance P/neurokinin A and substance P/calcitonin gene-related peptide were distributed within the respiratory epithelium of trachea and large bronchi, in the lamina propria and smooth muscle from the trachea down to the smallest bronchioli (highest density at the bronchial level), in the alveolar walls, around systemic and pulmonary blood vessels, and within airway ganglia. Those axons also containing dynorphin immunoreactivity were restricted to the lamina propria and smooth muscle. The origin of nerve fibres immunoreactive for vasoactive intestinal polypeptide, of which a part were also neuropeptide Y immunoreactive, could not be determined by retrograde tracing experiments. Vasoactive intestinal polypeptide-immunoreactive fibres terminating within airway ganglia may be of preganglionic parasympathetic origin, whereas others (e.g. those found in smooth muscle) may arise from intrinsic ganglia.(ABSTRACT TRUNCATED AT 400 WORDS)

Nitric oxide synthase in cardiac nerve fibers and neurons of rat and guinea pig heart.

Participation of nitric oxide (NO) in the autonomic innervation of rat and guinea pig hearts was investigated by applying the NADPH diaphorase technique and immunohistochemistry with NO synthase antiserum. We present evidence that NO synthase is localized in cardiac ganglion cells and nerve fibers innervating the sinuatrial and atrioventricular nodes, the myocardium, local neurons, coronary arteries, and pulmonary vessels, suggesting an involvement of NO in neurogenic heart rate regulation, myocardial cell function, neuronal transmission in cardiac ganglia, and coronary as well as pulmonary vasodilation.

Neuropeptide distribution in the cervico-thoracic paravertebral ganglia of the cat with particular reference to calcitonin gene-related peptide immunoreactivity

SummaryParaffin sections of cervical and upper thoracic paravertebral ganglia of the cat were investigated by immunohistochemistry using antisera directed against calcitonin gene-related peptide (CGRP). The relationships of CGRP-immunoreactive structures to those exhibiting immunoreactivity to antisera against other regulatory peptides and dopamine-β-hydroxylase (DBH), respectively, were studied in consecutive sections. Singly scattered CGRP-immunoreactive neuronal perikarya were observed in the superior and middle cervical ganglia as well as in the stellate ganglion. These neurons also displayed immunoreactivity to vasoactive intestinal polypeptide (VIP), and some additionally exhibited faint substance-P immunoreactivity. DBH- and neuropeptide Y-immunoreactive ganglion cells were not identical with CGRP-immunoreactive neuronal cell bodies.According to the immunoreactive properties of varicosities, which abut on CGRP/VIP-immunoreactive perikarya, three types of CGRP/VIP-immunoreactive ganglion cells could be distinguished: (1) CGRP/VIP-immunoreactive neurons being surrounded by somatostatin-immunoreactive nerve fibers, (2) neurons being approached by both DBH- and met-enkephalin-immunoreactive varicosities, and (3) neurons receiving both DBH- and neurotensin-immunoreactive fibers. The stellate and upper thoracic ganglia harbored clusters of intensely VIP-immunoreactive somata, which lacked CGRP-immunoreactivity. Fine somatostatin-immunoreactive and coarse CGRP-immunoreactive fibers were distributed within these clusters, whereas patches of neurotensin-immunoreactive fibers were complementarily arranged. At all segmental levels investigated, a few postganglionic neurons were approached by both CGRP-immunoreactive and substance P-immunoreactive varicosities, but lacked a VIP-immunoreactive innervation. Therefore, CGRP/substance P-immunoreactive fiber baskets appeared rather to be of extraganglionic origin than to emerge from intraganglionic CGRP/VIP/SP neurons. CGRP-immunoreactive cell bodies or fibers were absent in clusters of small paraganglionic cells, but some of the solitary paraganglionic cells displayed CGRP-immunoreactivity. Our findings establish the presence of CGRP-immunoreactivity in a population of sympathetic neurons in the cat. A highly differentiated, segment-dependent organizational pattern of neuropeptides in cervico-thoracic paravertebral ganglia was demonstrated.

Techniques in neuroanatomical research

I General Research Methods in Neuroanatomy.- 1 General Methods in Light Microscopy of the Nervous System.- 2 General Methods in Transmission Electron Microscopy of the Nervous System..- 3 Freeze-Etching in Neuroanatomy..- 4 General Methods in Scanning Electron Microscopy of the Nervous System.- 5 Lesion Methods in Neurobiology..- 6 General Methods for Characterization of Brain Regions..- II Light Microscopical Research Methods in Neuroanatomy.- 7 Enzyme Histochemistry of Nervous Tissue..- 8 The Golgi Methods..- 9 Fluorescence Histochemistry of Biogenic Monoamines..- 10 Immunohistochemistry and Immunocytochemistry of Nervous Tissue..- 11 Identification of Single Neurons by Intracellular Application of Tracers..- 12 Light Microscopical Autoradiography of Nervous Tissue..- 13 Brain Localization of Hormones and Drugs by Thaw-mount Autoradiography, Combined Autoradiography-Formaldehyde Induced Fluorescence, and Combined Autoradiography-Immunohistochemistry..- 14 Combined Immunocytochemistry and Autoradiography: In Vivo Injections of Monoclonal Antibodies and Radioactive Amines (Substance P and 3H-Serotonin).- III Electron Microscopical Research Methods in Neuroanatomy.- 15 Quick-Freezing Methods in Neuroanatomy..- 16 Ultrastructural Histochemistry of Nervous Tissue..- 17 The Zinc Iodide-Osmium Tetroxide (ZIO) Method..- 18 Combined Freeze-Fracturing and Autoradiography Techniques: Freeze-Fracture Autoradiography..- IV Investigation of Living Nervous Tissue.- 19 Extracellular Marking and Retrograde Labelling of Neurons..- 20 Cell, Tissue, and Organ Culture in Neuroanatomy..

Localization, regulation and functions of neurotransmitters and neuromodulators in cervical sympathetic ganglia.

Cervical sympathetic ganglia represent a suitable model for studying the establishment and plasticity of neurochemical organization in the nervous system since sympathetic postganglionic neurons: (1) express several neuromediators, i.e., short acting transmitters, neuropeptide modulators and radicals, in different combinations; (2) receive synaptic input from a limited number of morphologically and neurochemically well‐defined neuron populations in the central and peripheral nervous systems (anterograde influence on phenotype); (3) can be classified morphologically and neurochemically by the target they innervate (retrograde influence on phenotype); (4) regenerate readily, making it possible to study changes in neuromediator content after axonal lesion and their possible influence on peripheral nerve regeneration; (5) can be maintained in vitro in order to investigate effects of soluble factors as well as of membrane bound molecules on neuromediator expression; and (6) are easily accessible. Acetylcholine and noradrenaline, as well as neuropeptides and the recently discovered radical, nitric oxide, are discussed with respect to their localization and possible functions in the mammalian superior cervical and cervicothoracic (stellate) paravertebral ganglia. Furthermore, mechanisms regulating transmitter synthesis in sympathetic neurons in vivo and in vitro, such as soluble factors, cell contact or electrical activity, are summarized, since modulation of transmitter synthesis, release and metabolism plays a key role in the neuronal response to environmental influences.

The origin of ovarian neuropeptide Y (NPY)-immunoreactive nerve fibres from the inferior mesenteric ganglion in the pig.

SummaryApplying a double-immunofluorescence technique, the porcine ovary is demonstrated to receive two populations of NPY-immunoreactive nerve fibres originating from the inferior mesenteric ganglion: one with colocalized tyrosine hydroxylase and supplying predominantly the ovarian vasculature, and a second, solely NPY-immunoreactive and almost exclusively associated with growing follicles. A third group of tyrosine hydroxylase-and dopamine-β-hydroxylase-positive, but NPY-negative nerve fibres is associated with ovarian blood vessels and, to a minor extent, with ovarian follicles. As revealed by retrograde tracing, the vast majority of postganglionic neurons projecting to the ovary is located in a discrete area of the ganglion, suggesting a somatotopic organization of the porcine inferior mesenteric ganglion. Moreover, the finding indicate that three subpopulations of postganglionic sympathetic neurons with different chemical codes supply different target components of the porcine ovary. The physiological relevance of the described neurons in the nervous control of ovarian functions remains to be elucidated.

Immunohistochemical demonstration of the synthesis enzyme for nitric oxide and of comediators in neurons and chromaffin cells of the human adrenal medulla.

Within the human adrenal medulla immunoreactivity for the nitric oxide (NO)-generating enzyme nitric oxide synthase (NOS) was demonstrated in neurons, nerve fibres and chromaffin cells. Correlation of NOS-immunoreactivity with immunostaining for the peptides neuropeptide Y, somatostatin, substance P or vasoactive intestinal polypetide and for the catecholamine synthesis-enzyme tyrosine hydroxylase, respectively, in nerve cell bodies revealed colocalization of NOS only with substance P. Sparse intramedullary NOS-immunoreactive varicose nerve fibres associated with blood vessels or with chromaffin tissue were devoid of immunoreactivities for tyrosine hydroxylase or for the investigated peptides. Small NOS-immunolabeled cells belonged to the catecholamine-containing chromaffin cell population and costored VIP, but were distinct from the somatostatin- or neuropeptide Y- immunostained chromaffin subpopulations. The localization of NOS in distinct structural components of the human adrenal medulla indicates that NO is produced in different cell types and may reflect a differential role of this messenger system in autonomic control of adrenal gland function.

Substance P-immunoreactive nerve fibers in tracheobronchial lymph nodes of the guinea pig: Origin, ultrastructure and coexistence with other peptides ☆

Double-labeling immunofluorescence of guinea pig tracheobronchial lymph nodes revealed complete coincidence of SP and CGRP immunoreactivities in perivascular nerves and axons of the medullary lymphatic tissue. Additional dynorphin A or cholecystokinin immunoreactivity was seen only in some of the medullary fibers. Ultrastructurally, all SP-immunoreactive axons were unmyelinated and displayed vesicle-containing varicosities. Retrograde neuronal tracing combined with immunohistochemistry revealed a sensory origin from dorsal root ganglia of SP/CGRP-immunoreactive fibers ramifying within paratracheal lymph nodes, and an additional neuronal population being devoid of SP/CGRP immunoreactivity. The findings provide evidence for several types of sensory nerve fibers innervating lymph nodes.

Vasoactive intestinal peptide but not galanin promotes survival of neonatal rat sympathetic neurons and neurite outgrowth of PC12 cells

The synthesis of the neuropeptide galanin (GAL) is greatly enhanced after axonal lesion in different neuron populations of the peripheral and central nervous system. In sympathetic ganglia, GAL-immunoreactive nerve fiber baskets have been found surrounding postganglionic neurons after axotomy. Until now, it is unclear if GAL may be involved in neuronal survival or regeneration as suggested for vasoactive intestinal peptide (VIP) that is also upregulated after nerve lesion. We have, therefore, studied the effects of GAL on survival of sympathetic neurons dissociated from newborn rat superior cervical ganglia and on neurite outgrowth of PC12 cells. These effects were compared to those elicited by VIP. Whereas VIP promoted survival of about 10% of sympathetic neurons 2 days after nerve growth factor deprivation and induced neurite outgrowth of PC12 cells already at 6 h after addition of the peptide, GAL had no effect in either of these culture systems. While the induction of VIP may be beneficial for axotomized neurons, the functional significance of increased GAL levels remains to be established.

Ultrastructure of calcitonin gene-related peptide-and substance P-like immunoreactive nerve fibres in the carotid body and carotid sinus of the guinea pig

SummaryPrevious studies have demonstrated that substance P-(SP) and calcitonin gene-related peptide-like immunoreactivities (CGRP-LI) coexist in sensory nerve fibres in the guinea-pig carotid body and carotid sinus. In the present study the ultrastructure of these nerve fibres was investigated by means of single-and double-labelling immunocytochemistry. In both, carotid body and carotid sinus immunoreactive fibres were unmyelinated axons of small dianeter (0.12–0.56 μm). At the subcellular level, SP-and CGRP-LI were colocalized in intra-axonal dense core vesicles, suggesting corelease and simultaneous action of these two compounds. SP/CGRP-LI nerve fibres within the carotid body were mainly found in the interparenchymal connective tissue, but also occurred in relationship to blood vesslesl and nests of glomus cells. Neither in the carotid body not in the carotid sinus, SP/CGRP-LI axons corresponded to the large terminals which are generally considered to represent the main chemoreceptor and baroreceptor endings, respectively. Thus, SP/CGRP-LI fibres either belong to the chemo-and baroreceptors of the C-fibre class or constitute a fibre population not directly involved in conduction of baro-and chemoreflexes.