Catecholaminergic and peptidergic nerve components of intramural ganglia in the rat heart

Summary Immunohistochemical properties of the terminal nerve network in the rat heart were assessed by use of the elution-restaining method. The colocalization of the enzymes involved in catecholamine synthesis (tyrosine hydroxylase — TH, dopamine--hydroxylase — DBH) as well as the respective distributions of the neuropeptides associated with the adrenergic nervous system (neuropeptide tyrosine — NPY, C-terminal flanking peptide of neuropeptide Y — C-PON) were studied in series of serial sections throughout the interatrial septum and the atrioventricular junction. Our data suggest that ganglion cells of sulcus terminalis as well as the epicardial ganglia enclosed between the superior vena cava and ascending aorta are VIP- and TH-negative, but neuropeptide Y- and DBH-immunoreactive. They give rise to three intraseptal nerves directed towards the specialised structures of the atrioventricular junction. These nerve fascicles contain abundant, thick TH-immunoreactive nerve fibres and scarce, thin NPY- and DBH-immunoreactive fibres. The cell bodies of the intramural ganglion cells localized between the right and left branches of the bundle of His (Moravec and Moravec 1984) are strongly TH- and DBH-immunoreactive. They are innervated by thick nerve fibres having the same immunohistochemical properties (NPY- and DBH-immunoreactivities) as those of a subpopulation of the epicardial ganglion cells and seem to supply some of the TH-immunoreactive nerve fibres directed via the intraseptal nerves to the epicardial ganglia. The existence of a multicomponent nerve network, characterized by a reciprocal innervation of the sinus node and atrioventricular node areas, is suggested by our immunohistochemical data.     

Kinetic characterization of GABA-transaminase from cultured neurons and astrocytes

The enzymatic mechanism and the kinetic parameters of GABA-transaminase extracted from cultured mouse cerebral cortex neurons and astrocytes were studied. Neuronal as well as astrocytic GABA-transaminase obeyed a bi bi ping-pong reaction mechanism. The estimated K_m-values for -ketoglutarate and GABA were significantly lower for astroglial GABA-transaminase compared to the neuronal enzyme suggesting a possible existence of cell specific isozymes of GABA-transaminase. The observed enzymatic mechanism and the magnitude of the estimated kinetic parameters imply that GABA-transaminase synthesized in the two types of cultured neural cells is mechanistically and kinetically equivalent to the enzyme synthesized in the brainin vivo.     

Energy metabolism in glutamatergic neurons,GABAergic neurons and astrocytes in primary cultures

Several aspects of energy metabolism (glucose utilization, lactate production,¹⁴CO₂ production from labeled glucose, glutamate or pyruvate, oxygen consumption and contents of ATP and phosphocreatine) were measured in cerebellar granule cells (glutamatergic) in primary cultures and compared with corresponding data for cerebral cortical neurons (mainly GABA-ergic) and astrocytes. Cerebellar granule cells and astrocytes were metabolically more active than cerebral cortical neurons. Glutamate which is utilized as a major metabolic fuel as astrocytes and, to a lesser extent, in cerebral cortical neurons, was virtually not oxidized in cerebellar granule cells.Special Issue dedicated to Prof. Holger Hydén.     

Distribution of 5-HT (serotonin) immunoreactivity in the central nervous system of the inshore hagfish,Eptatretus burgeri

Summary Immunohistochemical techniques were used to study the distribution of serotonin in the central nervous system of the hagfish,Eptatretus burgeri, in order to produce a detailed map of serotonin-containing structures. In the hypothalamus, many serotonin-containing neurons contacted the cerebrospinal fluid. Most of the serotonin-containing cell bodies were located in the raphe region, where they were compactly distributed at the level of the nucleus motorius tegmenti pars anterior but more diffusely distributed at the level of the nucleus motorius tegmenti pars posterior. Serotonin-containing cell bodies and varicose fibers were widely distributed throughout the brain and upper spinal cord segments, but the distribution density was not even. On the basis of its abundance, serotonin can be judged to have an important function in the control of the hagfish central nervous system. From a phylogenetic point of view, serotonin-containing neurons in the raphe region appear to be a common property of all classes of vertebrates studied except the lampreys, whereas serotonin-containing cerebrospinal fluid-contacting neurons may be considered to be a primitive condition in all nonmammalian vertebrates.     

Expression of Microtubule-Associated Proteins During the Early Stages of Neurite Extension by Brain Neurons Cultured in a Defined Medium

Immunoblotting analysis was used to identify the microtubule-associated proteins (MAPs) present in cultures of mouse brain neurons. Polyclonal antibodies were raised against the two main adult brain MAPs, i.e., MAP2 (300 kDa) and tau (60-70 kDa). Whatever the stage of the culture, which was performed in a defined medium (3 or 6 days), the anti-MAP2 serum detected several high-molecular-weight components (including MAP2) and an entity with 62-65 kDa. Anti-tau revealed essentially a major peak of 48 kDa (young tau) but also slightly cross-reacted with the 62-65 kDa entity. During the culture period (0-6 days) the cells developed progressively a dense neuritic network; the concentration of the different MAPs increased in parallel but at different rates depending on the different species. The increase in concentration of the high-molecular-weight components occurred before that of 48-kDa tau. This suggests that high-molecular-weight MAPs and 48-kDa tau might be involved respectively in the initiation and elongation of neurites. In contrast, and since the main developmental changes in tau composition seen in vivo did not occur during the time course of the culture, this transition might be related to later events of neuronal differentiation.     

An immunocytochemical study of the optic ganglia of the crayfish Astacus leptodactylus (Nordmann 1842) with antisera against biologically active peptides of vertebrates and invertebrates

Summary The peptidergic system in the optic ganglia of Astacus leptodactylus is characterized by the immunocytochemical application of 15 antisera raised against biologically active peptides of vertebrates and invertebrates. Positive reactions were found with anti-FMRFamide, antiMSH, anti-vasotocin, anti-gastrin, anti-CCK, anti-oxytocin, anti-secretin, anti-glucagon and anti-GIP. Based on immunochemical reaction and localization it is possible to distinguish 30 cell groups. Only part of these cell groups is found in the known classical neurosecretory cell regions. This observation demonstrates a more extensive peptidergic system than formerly recognized. The morphology of this peptidergic system suggests that one part is neurohormonal and the other part neurotransmitter-like or neuromodulatory.     

Cross-linked collagen surface for cell culture that is stable,uniform, and optically superior to conventional surfaces

Summary A new type of collagen surface for use with cultures of peripheral nervous system cells is described. Collagen is derivatized to plastic culture dishes by a cross-linking reagent, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate (carbodiimide), to form a uniform and durable surface for cell attachment and growth that allows dry storage, long-term culture, and improved microscopy. Surfaces of collagen derivatized to plastic were compared to surfaces of adsorbed or ammonia-polymerized collagen in terms of collagen binding and detachment, growth by dorsal root ganglion cells, and electron microscopy appearances. Derivatized collagen surfaces retained more collagen and showed much less evidence of degradation and cellular damage over periods of many weeks than did conventional adsorbed surfaces. Long-term survival of cells on derivatized collagen was far superior to that on the other surfaces, with almost 90% of cultures still viable after 10 wk. Transmission electron microscopy showed an organized layer of single fibrils that supported cell growth well, and scanning electron microscopy demonstrated an increased uniformity of derivatized collagen surfaces compared to ammoniated collagen surfaces. Applications for this improved substrate surface are discussed. This work was supported by the Leopold Schepp Foundation, the Dysautonomia Foundation, National Institutes of Health Grants NS14768 and NS11237, and Institutional Core Grant HD06276.     

Actions of Excitatory Amino Acids on Somatostatin Release from Cortical Neurons in Primary Cultures

L-Glutamate, N-methyl-D-aspartic acid (NMDA), quisqualate, and kainate were found to increase endogenous somatostatin release from primary cultures of rat cortical neurons in a dose-dependent manner. The rank order of potency calculated from the dose-response curves was quisqualate greater than glutamate = NMDA greater than kainate, with EC50 values of 0.4, 20, and 40 microM, respectively. Alanine, glutamine, and glycine did not modify the release of somatostatin. The stimulation of somatostatin release elicited by L-glutamate was Ca2+ dependent, was decreased by Mg2+, and was blocked by DL-amino-5-phosphonovaleric acid (APV) and thienylphencyclidine (TCP), two specific antagonists of NMDA receptors. The NMDA stimulatory effect was strongly inhibited by APV in a competitive manner (IC50 = 50 microM) and by TCP in a noncompetitive manner (IC50 = 90 nM). The release of somatostatin induced by the excitatory amino acid agonists was not blocked by tetrodotoxin (1 microM), a result suggesting that tetrodotoxin-sensitive, sodium-dependent action potentials are not involved in the effect. Somatostatin release in response to NMDA was potentiated by glycine, but the inhibitory strychnine-sensitive glycine receptor did not appear to be involved. Our data suggest that glutamate exerts its stimulatory action on somatostatin release essentially through an NMDA receptor subtype.     

Pedal serotonergic neurons modulate the synaptic input of withdrawal interneurons ofHelix

A group of serotonergic cells, located in the pedal ganglia ofHelix lucorum, modulates synaptic responses of neurons involved in withdrawal behavior. Extracellular or intracellular stimulation of these serotonergic cells leads to facilitation of spike responses to noxious stimuli in the putative command neurons for withdrawal behavior. Noxious tactile stimuli elicit an increase in background spiking frequency in the modulatory neurons and a corresponding increase in stimulus-evoked spike responses in withdrawal interneurons. The serotonergic neurons have processes in the neuropil of the parieto-visceral ganglia complex, consistent with their putative role in modulating the activity of giant parietal interneurons, which send processes to the same neuropil and to the pedal ganglia. The serotonergic cells respond to noxious tactile and chemical stimuli. Although the group as a whole respond to noxious stimuli applied to any part of the body, most cells respond more to ipsilateral than contralateral stimulation, and exhibit differences in receptive areas. Intracellular investigation revealed electrical coupling between serotonergic neurons which could underlie the recruitment of members of the group not responding to a given noxious stimulus.     

Regulation of choline acetyltransferase/iacZ fusion gene expression in putative cholinergic neurons of drosophila melanogaster

We have analyzed the distribution of putative cholinergic neurons in whole-mount preparations of adult Drosophila melanogaster. Putative cholinergic neurons were visualized by X-gal staining of P-element transformed flies carrying a fusion gene consisting of 5′ flanking DNA from the choline acetyltransferase (ChAT) gene and a lacZ reporter gene. We have previously demonstrated that cryostat sections of transgenic flies carrying 7.4 kb of ChAT 5′ flanking DNA show reporter gene expression in a pattern essentially similar to the known distribution of ChAT protein. Whole-mount staining of these same flies by X-gal should thus represent the overall distribution of ChAT-positive neurons. Extensive staining was observed in the cephalic, thoracic, and stomodeal ganglia, primary sensory neurons in antenna, maxillary palps, labial palps, leg, wing, and male genitalia. Primary sensory neurons associated with photoreceptors and tactile receptors were not stained. We also examined the effects of partial deletions of the 7.4 kb fragment on reporter gene expression. Deletion of the 7.4 kb fragment to 1.2 kb resulted in a dramatic reduction of X-gal staining in the peripheral nervous system (PNS). This indicates that important regulatory elements for ChAT expression in the PNS exist in the distal region of the 7.4 kb fragment. The distal parts of the 7.4 kb fragment, when fused to a basal heterologous promoter, can independently confer gene expression in subsets of putative cholinergic neurons. With these constructs, however, strong ectopic expression was also observed in several non-neuronal tissues. © 1995 John Wiley & Sons, Inc.