Glucocorticosteroids up-regulate the expression of cholecystokinin mRNA in the rat paraventricular nucleus.

Adrenalectomy abolishes corticosteroid feedback onto the hypothalamic-pituitary-adrenal axis. This results in an increased biosynthetic and secretory activity of corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN), sustained in the absence of hormone replacement. In the PVN, cholecystokinin (CCK) is present both in parvicellular CRH-containing and in magnocellular oxytocin (OXY)-containing neurons. We presently studied the glucocorticoid feedback regulation of the expression of cholecystokinin (CCK) mRNA in rats after: (i) adrenalectomy, (ii) sham surgery or (iii) adrenalectomy with corticosterone replacement. Using 35S-labeled CRH and p-CCK cRNA probes and in situ hybridization, CRH and CCK mRNAs were radiolabeled. The total amount of hybridization labeling (integrated density), was quantified in adjacent series of cryosections regularly spaced throughout the PVN. The OXY mRNA detection served to identify PVN magnocellular areas. Adrenalectomy was shown to induce: (i) a 75% increase in CRH mRNA labeling in the PVN, (ii) a concomitant 43% decrease in CCK mRNA labeling but only in the anterior part of the PVN and occurring both in CCK/CRH area (two thirds of it) and CCK/OXY area (one third of it) and (iii) that they were fully reversed by corticosterone replacement. Thus, glucocorticoids that are well known to negatively feedback on CRH expression in parvicellular PVN neurons are also capable of positively regulating CCK expression in anterior PVN neurons, both in parvicellular and magnocellular areas.

Dopamine in the lobster Homarus gammarus: II. Dopamine-immunoreactive neurons and development of the nervous system.

Dopamine-immunoreactive neurons were revealed in lobster embryos, larvae, and postlarvae, and staining patterns were compared to neuronal labeling in the juvenile lobster nervous system (Cournil et al. [1994] J. Comp. Neurol. 344:455-469). Dopamine immunoreactivity is first detected by midembryonic life in 35-40 neuronal somata located anteriorly in brain and subesophageal ganglion. When the lobsters assume a benthic life during the first postlarval stage, an average of 58 cell bodies are labeled. The acquisition of dopamine in lobster neurons is a protracted event spanning embryonic, larval, and postlarval life and finally reaching the full complement of roughly 100 neurons in juvenile stages. Some of the dopaminergic neurons previously identified in the mature nervous system, such as the paired Br cells, L cells, and mandibular cells, are labeled in embryos and persist throughout development. In contrast, other neurons stain transiently for dopamine during the developmental period, but, by the adult stage, these neurons are no longer immunoreactive. Such transiently labeled neurons project to the foregut, the thoracic dorsal muscles, the neurohormonal pericardial plexus, and the pericardial pouches. It is proposed that these neurons are alive and functioning in adult lobster but that dopamine levels have been abolished, providing that neurotransmitter status is a dynamic, changing process.

Dopamine in the lobster Homarus gammarus. I. Comparative analysis of dopamine and tyrosine hydroxylase immunoreactivities in the nervous system of the juvenile.

As a catecholamine, dopamine belongs to a class of molecules that have multiple transmitter and hormonal functions in vertebrate and invertebrate nervous systems. However, in the lobster, where many central neurons have been identified and the peripheral innervation pattern is well known, the distribution of dopamine-containing neurons has not been examined in detail. Therefore, immunocytochemical methods were used to identify neurons likely to contain dopamine and tyrosine hydroxylase in the central nervous system of the juvenile lobster Homarus gammarus. Approximately 100 neuronal somata stain for the catecholamine and/or its synthetic enzyme in the brain and ventral nerve cord. The systems of neurons labeled with dopamine and tyrosine hydroxylase antibodies have the following characteristics: 1) the two systems are nearly identical; 2) every segmental ganglion contains at least one pair of labeled neurons; 3) the positions and numbers of cell bodies labeled with each antiserum are similar in the various segmental ganglia; 4) six labeled neurons are anatomically identified; two interneurons from the brain project within the ventral cord to reach the last abdominal ganglion, two neurons from the commissural ganglia are presumably neurosecretory neurons, and two anterior unpaired medial abdominal neurons project to the hindgut muscles; and 5) no cell bodies are labeled in the stomatogastric ganglion, but fibers and terminals in the neuropil are stained. The remarkably small numbers of labeled neurons and the presence of very large labeled somata with far-reaching projections are distinctive features consistent with other modulatory aminergic systems in both vertebrates and invertebrates.

A rhythmic modulatory gating system in the stomatogastric nervous system of Homarus gammarus. I. Pyloric-related neurons in the commissural ganglia.

1. Operation of the pyloric neural network in the crustacean stomatogastric ganglion (STG) depends on constant firing of modulatory inputs from anterior ganglia. We have identified two bilaterally symmetrical pairs of these inputs in the commissural ganglia (COGs) of the European rock lobster Homarus gammarus. During operation of the pyloric CPG, they fired in pyloric time, out of phase with the pyloric pacemakers. 2. One of the pair was the commissural pyloric (CP) neuron and the other was homologous to the P neuron described in the spiny lobster Panulirus interruptus. We describe their morphology and location in the COG. The CP neuron projected to the STG via the superior esophageal nerve (son) and the stomatogastric nerve (stn), whereas the P neuron projected via the inferior esophageal nerve (ion) and stn. 3. To determine the total number of commissural neurons projecting to the STG, we used cobalt and Lucifer yellow backfilling from their cut axons in the stn. With the ion cut, there were between 8 to 12 labeled somata in each COG including CP cell body, whereas only 2 somata (including P) were labeled with the son cut. Among these neurons, CP and P appeared to be the only commissural neurons that fired in pyloric time and projected in the STG on the pyloric network. 4. The CP neuron produced monosynaptic excitatory postsynaptic potentials (EPSPs) on the pyloric dilator (PD), lateral pyloric (LP), and inferior cardiac (IC) neurons, whereas the P neuron produced monosynaptic EPSPs on all pyloric motoneurons but IC. The P neuron was gamma-aminobutyric acid immunoreactive, and the P-derived EPSPs in pyloric neurons were reversibly blocked by bicuculline, picrotoxin, and D-tubocurarine. 5. The CP and P neurons were electrically coupled, and modification of membrane potential in either one of them appreciably changed the firing frequency of the coupled neuron. 6. A negative-feedback loop from the pyloric anterior burster (AB) interneuron provoked simultaneous rhythmic inhibitions in the P and CP neurons. Together with the electrical coupling, the rhythmic inhibition contributed to synchronize firing of the two commissural neurons. 7. The following papers in the series of describe the modulatory and rhythmic control exerted by the P and CP neurons over the pyloric pattern generator.

Distribution of modulatory inputs to the stomatogastric ganglion of the crab, Cancer borealis.

The pyloric and gastric mill neural networks in the crustacean stomatogastric ganglion receive modulatory inputs from more anteriorly located ganglia via the stomatogastric nerve. In this study we employed biocytin backfilling and immunostaining, as well as electron microscopy, to determine the origin of these inputs in the crab, Cancer borealis. Fiber counts from electron micrographs of sections through the stomatogastric nerve showed that this nerve contains 55-60 medium to large diameter fibers (1-13 microns). These fibers were individually wrapped by several layers of membrane, presumably glial in origin. There was also a single cluster of jointly wrapped, small diameter (< 1 micron) fibers that may originate from peripheral sensory somata. Biocytin backfills revealed that approximately two thirds of the individually wrapped fibers in this nerve originate from somata in the other three ganglia of the stomatogastric nervous system, including the paired commissural ganglia and the single oesophageal ganglion. There were approximately 20 biocytin-labeled somata in each commissural ganglion and 3 somata in the oesophageal ganglion. An additional ten somata were localized to the stomatogastric ganglion itself. This accounts for nearly all of the medium to large diameter fibers in the stomatogastric nerve. We used double-labeling with backfills and immunocytochemistry to determine that there are two proctolin-immunoreactive neurons and four FMRFamide-like immunoreactive neurons among the biocytin-labeled neurons in each commissural ganglion. Both peptides modulate neural network activity in the stomatogastric ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)

The innervation of the closer muscle of the mesothoracic spiracle of the locust.

The closer muscle of the mesothoracic spiracle of the locust, Schistocerca gregaria is innervated by two excitatory motoneurones and also by processes of a peripherally located neurosecretory cell. Within the muscle, ultrastructural studies show the presence of two types of excitatory nerve terminal which differ in the content of dense cored vesicles and in their distribution. The ventral segment of the muscle is innervated predominantly by terminals with small clear vesicles and only an occasional dense-cored vesicle. The central part of the muscle is innervated predominantly by terminals with small clear vesicles and larger numbers of dense-cored vesicles. The dorsal segment of the muscle is innervated exclusively by a neurosecretory type innervation. The small neurohaemal organ of the median nerve close to the spiracle muscle is immunoreactive to an antibody raised against bovine pancreatic polypeptide but no immunoreactive processes enter the muscle itself. The muscle possesses specific octopaminergic receptors that increase cyclic AMP levels and the possibility that the neurosecretory input to the muscle is provided by either a central or peripheral octopamine containing neurone is discussed.

A method for the determination of projection areas of GABA immunoreactive neurons in the invertebrate nervous system.

Axonal transport of metallic salts (nickel or cobalt chloride) has been widely used for the anatomical mapping of neural pathways. We show here that when nickel is introduced into GABAergic neurons it completely eliminates GABA immunolabelling. We have used this property to determine the axonal projections of GABAergic neurons in the stomatogastric system of Crustacea. For example, following nickel backfills from either cut axons or from terminals, GABA immunostaining labels only those GABA-immunoreactive neurons which had not been retrogradely labelled with nickel and hence did not project in the cut nerve or to the neuropile uptake site. By comparing such immunolabelled preparations with those not pretreated with nickel the projection patterns of all the GABA immunoreactive neurons in a given system can be revealed. This effect of nickel appears to be selective for GABA immunostaining, insofar as it does not interfere with the immunodetection of either the peptide proctolin or a FMRFamide-like peptide. This method may prove to be a useful tool for analyzing GABAergic neuronal pathways in the nervous systems of invertebrates.

Identification of all GABA-immunoreactive neurons projecting to the lobster stomatogastric ganglion.

The stomatogastric ganglion of lobsters (Homarus or Jasus) contains a large number of gamma-aminobutyric acid-immunoreactive processes originating from ten fibres in the single input nerve, the stomatogastric nerve. The cell bodies and axonal pathways of these ten fibres have been identified using gamma-aminobutyric acid immunohistochemistry in combination with Lucifer Yellow staining (double labelling) and nickel chloride backfilling (selective gamma-aminobutyric acid immunoinhibition). It is shown that eight gamma-aminobutyric acid-immunoreactive neurons project to the stomatogastric ganglion: gamma-aminobutyric acid neurons 1 and 2, found posterior to the oesophageal ganglion, entering the stomatogastric nerve via the oesophageal nerve as well as sending an axonal branch into each superior oesophageal nerve; gamma-aminobutyric acid neurons 3 and 4, found anterior to the oesophageal ganglion, each sending an axonal branch into each inferior oesophageal nerve to reach the stomatogastric nerve via the commissural ganglion and the superior oesophageal nerve; and gamma-aminobutyric acid neurons 5 and 6, found in each commissural ganglion, projecting into the stomatogastric nerve via the inferior oesophageal nerve, the oesophageal ganglion and the oesophageal nerve. These gamma-aminobutyric acid-immunoreactive neurons were also characterized by electrophysiological methods coupled with Lucifer Yellow labelling, and their picrotoxin-sensitive effects on several stomatogastric ganglion neurons were demonstrated. The present results provide a firm basis for further studies concerning the physiological significance of one class of neurochemically-defined input neurons to stomatogastric ganglion networks.

Co-localization of FLRF- and vasopressin-like immunoreactivity in a single pair of sexually dimorphic neurones in the nervous system of the locust.

The distribution of Phe-Leu-Arg-Phe (FLRF)-like immunoreactivity is described in the brain and in the ganglia of the ventral nerve cord of the locust Schistocerca gregaria. A single homologous pair of immunoreactive cell bodies occurs ventrally and medially in the suboesophageal ganglion. Each cell sends a process dorsally which bifurcates into anteriorly and posteriorly running neurites. The single anterior neurite passes along the circumoesophageal connectives to the brain where it ascends in a posterior running tract, giving off branches to innervate the tritocerebral neuropile and ending in an extensive network of highly varicose immunoreactive processes in the protocerebral neuropile. No processes are seen in the optic lobes or associated with the structured neuropiles of the muschroom bodies. The single posterior neurite from each cell passes into the suboesophageal-prothoracic connectives. It runs in the lateral dorsal tract of each ganglion in the ventral nerve cord as a highly varicose process and in each ganglion gives rise to an ipsilateral network of varicose processes in the dorsal and lateral neuropiles. In the seventh and terminal abdominal ganglia the innervation pattern exhibits sexual dimorphism. Vasopressin-like immunoreactivity is co-localized in the same pair of suboesophageal neurones and their processes. A similar pair of ventral median neurones stains with both antibodies in the suboesophageal ganglion of another species of locust, Locusta migratoria. Although the basic distribution pattern of immunoreactive processes is similar in both species there are also marked species differences in the pattern.

Suppression of oscillatory activity in crustacean pyloric neurons: implication of GABAergic inputs.

Generation of rhythmic pyloric motor output in the crustacean stomatogastric ganglion results from synaptic connections and cellular properties of a 14-cell network of pyloric neurons. These cellular properties are under the influences of modulatory inputs, which act, for the most part, in an activating mode, i.e., they enhance the bursting properties of the pyloric neurons and/or their ability to express their regenerative properties. Here we attempt to demonstrate that the pyloric motor output is also under the control of suppressive afferent inputs that are able to stop the pyloric rhythm in a long-lasting manner. Immunohistochemistry, using GABA antibodies, indicates that GABAergic-like fibers are present in both the stomatogastric ganglion and its afferent nerve. Bath-applied GABA suppresses spontaneous pyloric rhythmic activity. This is due to an inability of the pyloric pacemakers to express their bursting properties. The suppressive effect of GABA is blocked by picrotoxin and mimicked by muscimol. Isolating the pyloric neurons from all descending spiking influences with tetrodotoxin demonstrates that exogenously applied GABA acts directly on the pyloric neurons. To confirm the existence of a physiological suppressive system for the pyloric motor pattern, we show that the stimulation of an afferent nerve, known to contain GABA-like fibers, also causes the cessation of rhythmic activity and the inability of the pyloric neurons to express their bursting properties.

Coexistence of dopamine and serotonin in an identified neuron of the lobster nervous system.

The combination of several analytical methods, i.e. chemical analysis (high performance liquid chromatography), biochemical analysis (radioimmunoassay) and immunohistochemistry, has shown that a single neuron can contain two 'classical' neurotransmitters.

All-or-none control of the bursting properties of the pacemaker neurons of the lobster pyloric pattern generator.

In Crustacea the central pattern generator for the pyloric motor rhythm (filtration to the midgut) is known to be located within the stomatogastric ganglion (STG); its cycling activity is known to be organized by three endogenous burster neurons acting as pacemakers and driving 11 follower neurons. In Homarus, recordings from the isolated stomatogastric nervous system (Fig. 1) indicate that (1) the pyloric output can be generated only when the STG is afferented (i.e., connected to the more rostral oesophageal and commissural ganglia) (Fig. 2) and (2) the deafferentation of the STG results in a complete loss of the bursting properties of the pacemaker neurons (Fig. 4). Manipulation of the STG inputs responsible for unmasking the properties of the pacemakers strongly suggests that (1) they are not phasic inputs (Fig. 5) and (2) they are long-term acting inputs (Fig. 6). These results provide evidence for a neural all-or-none control of the bursting properties of the pacemaker neurons of a motor pattern generator.

Immunohistochemical evidence for the intracellular formation of basement membrane collagen (type IV) in developing tissues.

Antibodies to type IV collagen obtained from the basement membrane of the mouse EHS tumor were incubated with sections of rat incisor teeth and other tissues for immunostaining by direct or indirect methods. In all locations, the immunostaining was pronounced in basement membranes in which it was restricted to the "basal lamina" layer, from which "bridges" often extended to nearby basal laminae. Usually no immunostaining was detectable in the cells associated with the basement membranes. However, examination of the capillaries at the posterior extremity of the rat incisor tooth, where tissues are at an early stage of development, showed immunostaining not only of the basement membrane, but also of the endothelial cells. The staining was localized in rough endoplasmic reticulum cisternae, some Golgi saccules and their peripheral distensions, and structures believed to be secretory granules. These findings suggest that the synthesis of type IV collagen proceeds along the classical secretory pathways through rough endoplasmic reticulum and Golgi apparatus. At the same time, immunostaining was usually lacking in the cells of the capillaries that had migrated about 2 mm away from the posterior end of the incisor tooth and also in the cells of most other tissues examined, even though the associated basal laminae were reactive. It is, therefore, presumed that the production of type IV collagen may be high in cells at an early stage of development and that any later production and turnover of basement membrane collagen can only be minimal.

Immunohistochemical localization of procollagens. I. Light microscopic distribution of procollagen I, III and IV antigenicity in the rat incisor tooth by the indirect peroxidase-anti-peroxidase method.

Frozen sections of the growing end of the rat incisor tooth were exposed to antisera or affinity prepared antibodies against partially purified type I, II, or IV procollagen in the hope of detecting the location of the corresponding antigens by the peroxidase-anti-peroxidase technique. The distribution of immunostaining was similar with antisera as with purified antibodies of a given type, but differed for each type; that is, predentin, odontoblasts, pulp and periodontal tissue were the sites of type I; blood vessel walls, pulp and periodontal tissue, of type III; and basement membranes, of type IV antigenicity. It was demonstrated, at least in cases of type I and III, that immunostaining detected the corresponding procollagens and related substances, but not the corresponding collagens. The interpretation of these observations is that: 1) odontoblasts elaborate procollagen I for release to predentin and subsequent transformation to dentinal collagen I; 2) pulp and periodontal cells produce procollagens I and III which presumably become collagens I and III respectively, while the adventitial cells of blood vessels give rise to collagen III; and 3) procollagen IV is associated with basement membranes and, occasionally, adjacent cells.

Immunohistochemical localization of procollagens. II. Electron microscopic distribution of procollagen I antigenicity in the odontoblasts and predentin of rat incisor teeth by a direct method using peroxidase linked antibodies.

In an attempt to locate procollagen I in rats odontoblasts, antibodies raised in rabbits were purified by affinity methods and linked to peroxidase. They were then incubated with chopped slices from the growing end of rat incisor teeth. The antibodies binding to the antigens in the slices were visualized by reacting the peroxidase moiety with diaminobenzidine in the presence of hydrogen peroxide. The slices were then embedded in Epon and sectioned for ultrastructural study. Within odontoblasts, the immunostaining indicative of procollagen I antigenicity is moderate in rough endoplasmic reticulum cisternae, strong in spherical and cylindrical Golgi distensions, intense in secretory granules, and variable in lysosomal structures. In predentin, immunostaining is intense close to the odontoblast layer, but decreases gradually in a distal direction. Hence, procollagen I (and/or substances endowed with similar antigenicity such as pro alpha (I) chains and procollagen fragments) is present: 1) along the intracellular pathway of collagen precursors where its concentration gradually increases to reach a maximum in secretory granules; 2) in predentin, into which it is released from the granules for transformation into nonimmunoreactive collagen I; and 3) in lysosomal structures where some of it is hydrolyzed.

Dissociation of aspartate aminotransferase into subunits. Effect of ligands upon this dissociation.

Frontal and zonal analysis of the chromatography of aspartate aminotransferase (EC2.61.1), pig heart cytosolic enzyme, on Bio-Gel P150 shows that holo- and apoenzyme can dissociate at pH 8.3. Ultracentrifugation and fluorescence depolarization confirm this result. Kinetic analysis of the fluorescence depolarization experiments favors a biphasic phenomenon: a few minutes for the faster one and several hours for the slower one. The apparent dissociation constant is 0.8 muM for the apoenzyme and 0.18 muM for the pyridoxal 5'-phosphate form of the holoenzyme. In the presence of sucrose or 0.1 M L-aspartate or a mixture of 70 mM L-glutamate and 2 mM alpha-ketoglutarate, the holoenzyme is dimeric at concentrations higher than 5 nM. The addition of a mixture of the substrates L-glutamate and alpha-ketoglutarate to a monomeric holoenzyme leads to dimerization. The stability of the dimeric form is in the order: holoenzyme + substrates greater than apoenzyme.