Isabelle Cournil

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

SummaryThe 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.

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.

Gamma‐Aminobutyric Acid‐Inhibitory Motor Innervation of Leg Muscles of the Shore Crab

The inhibitory motor innervation of a crustacean leg was studied in the crab, Carcinus maenas. In in vitro preparations of the central nervous system and the proximal leg nerves, motor nerve recordings demonstrate the presence of a single common inhibitory motor neuron which elicits picrotoxin‐sensitive inhibitory junction potentials in a distal leg muscle, the accessory flexor. This inhibitor is the common inhibitor (CI). Immunohistochemical detection of the inhibitory motor neuron neurotransmitter, gamma‐aminobutyric acid (GABA), allows us to identify three immunoreactive motor neuron axons in sections of the distal leg nerves and of proximal leg nerves. One corresponds to the CI whereas the other two are the specific inhibitors, one to the stretcher and one to the opener muscles. After nickel chloride backfills of the CI in proximal leg nerves, GABA immunodetection fails and thus confirms that CI is the single inhibitor having branches in proximal leg nerves.

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.

Lobster Stomatogastric GABA System

GABA immunodetection on paraffin serial sections coupled with (1) Lucifer yellow intracellular staining to obtain double labelling (2) nickel chloride axonal backfilling to suppress GABA immunoreactivity and (3) electrophysiological techniques, has allowed identification of all 8 GABAergic neurones that project to the lobster stomatogastric ganglion (STG). It will now be possible to examine whether these neurones containing the same neurotransmitter, constitute a functionally discrete input system.

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.

A Method to Study Projections of Gabaergic Neurons Using Deletion of the Gaba Immunostaining by Metallic Ions

GABAergic transmission plays an important role in the function of both the vertebrate and invertebrate nervous system. However, the physiological effects of discrete GABAergic neurons on neural circuits are not so well defined. Immunocytochemistry has been used to localize GABA in neural somata, fibers and terminals, and this maps its distribution throughout whole nervous systems. However, to understand how a particular GABAergic neuron or group of neurons influences a target neuron or neuronal network, it is necessary to devise anatomical methods that allow the identification of the appropriate GABA containing neurons and enable their characterization in electrophysiological studies. It is difficult to use the simple observation of immunostained preparations to resolve the projections of individual axons when they pass through regions of ganglionic neuropil, where labelled axon terminals interfere with the staining of fibers, or through the junction between nerves where axons may ramify into several branches. To overcome these limitations, we present here an original method that is specific to GABAergic neuronal systems.

Characterization of FMRF-Amide-Like Peptides in the Locust, Schistocerca Gregaria

FMRFamide was the first peptide isolated and characterized in a growing interphyletic family of neuropeptides (see Greenberg et al., 1988). In the locust, Schistocerca gregaria, the distribution of FMRFamide-like immunoreactivity has been described using immunocytochemistry and radioimmunoassay, and suggests both neurohormonal and neuromodulatory roles for peptides from this family (see Evans and Myers, 1986; Robb and Evans, submitted).