Károly Elekes

Aminergic neurons in the brain of blowflies and Drosophila: dopamine- and tyrosine hydroxylase-immunoreactive neurons and their relationship with putative histaminergic neurons

SummaryThe distribution and morphology of neurons reacting with antisera against dopamine (DA), tyrosine hydroxylase (TH) and histamine (HA) were analyzed in the blowflies Calliphora erythrocephala and Phormia terraenovae. TH-immunoreactive (THIR) and HA-immunoreactive (HAIR) neurons were also mapped in the fruitfly Drosophila melanogaster. The antisera against DA and TH specifically labeled the same neurons in the blowflies. About 300 neurons displayed DA immunoreactivity (DAIR) and THIR in the brain and subesophageal ganglion of the blowflies. Most of these neurons were located in bilateral clusters; some were distributed as bilateral pairs, and two ventral unpaired median (VUM) neurons were seen in the subesophageal ganglion. Immunoreactive processes were found in all compartments of the mushroom bodies except the calyces, in all divisions of the central body complex, in the medulla, lobula and lobula plate of the optic lobe, and in non-glomerular neuropil of protocerebrum, tritocerebrum and the subesophageal ganglion. No DA or TH immunoreactivity was seen in the antennal lobes. In Drosophila, neurons homologous to the blowfly neurons were detected with the TH antiserum. In Phormia and Drosophila, 18 HA-immunoreactive neurons were located in the protocerebrum and 2 in the subesophageal ganglion. The HAIR neurons arborized extensively, but except for processes in the lobula, all HAIR processes were seen in non-glomerular neuropil. The deuto- and tritocerebrum was devoid of HAIR processes. Double labeling experiments demonstrated that TH and HA immunoreactivity was not colocalized in any neuron. In some regions there wasm however, substantial superposition between the two systems. The morphology of the extensively arborizing aminergic neurons described suggests that they have modulatory functions in the brain and subesophageal ganglion.

serotonergic terminals in the neural sheath of the blowfly nervous system: Electron microscopical immunocytochemistry and 5,7-Dihydroxytryptamine labelling

With serotonin immunocytochemistry we have demonstrated an extensive plexus of immunoreactive varicose fibres in the neural sheath of the nervous system of the blowfly, Calliphora. These fibres are located in the neural sheath of the following regions: the maxillary-labial and labrofrontal nerves of the cerebral ganglia, the cervical connective, the dorsal surface of the thoracicoabdominal ganglia, two pairs of prothoracic nerves and the median abdominal nerve. We identified the serotonin-immunoreactive neural processes in the electron microscope by means of the peroxidase-antiperoxidase method. Immunoreactivity was seen in large granular vesicles (ca 100 nm), on membranes of smaller (ca 60 nm) and larger (ca 100 nm) agranular vesicles, along the inner surface of the axolemma, along neurotubules and outer membranes of mitochondria. By conventional electron microscopy we found numerous varicose neural processes in the neural sheath of some of the above regions. These varicosities are of at least two types. One type corresponds to the serotonin-immunoreactive profiles. A second type contains large granular vesicles (ca 200 nm) of variable electron density. 5,7-Dihydroxytryptamine injected into the head capsule labelled varicosities in the neural sheath, corresponding to the ones identified with serotonin immunocytochemistry. The electron-dense labelling was seen in flattened vesicles within these varicosities. We propose that the serotonin-immunoreactive fibers in the neural sheath constitute neurohemal regions for the release of serotonin into the circulation. The finding of another morphological type of varicose fibers in the neural sheath suggests the presence of further putative neurohormones in these regions.

A comparison of four techniques for mapping the distribution of serotonin and serotonin-containing neurons in fixed and living ganglia of the snail,Lymnaea

SummaryThe distribution of serotonin and serotonin-containing neurons was studied in the ganglia of the CNS of the snailLymnaea stagnalis. Results of the application of three different labelling techniques on wholemount preparations were compared with each other and with the serotonin content of the ganglia, measured by high-performance liquid chromatography. Serotonin immunocytochemistry resulted in the highest number of labelled neurons, but the more recently developedin vivo method of 5,6- or 5,7-dihydroxytryptamine-induced pigmentation also proved to be a reliable technique for the visualization of serotonin-containing cell bodies. In comparison with these two techniques, the glyoxylic acid fluorescence method appeared to be less sensitive. The distribution and number of serotonin-containing neurons and biochemically measured serotonin in specific ganglia showed a close correlation. By combining the results of the three labelling techniques, a detailed map of serotonin-containing neurons was constructed, and this was compared with maps of identified neurons prepared from earlier electrophysiological studies. Previously described serotonergic neurons were consistently found, as well as several new serotonin-containirig cell types in the cerebral, visceral and parietal ganglia. A network of serotonin-containing inter- and intraganglionic axon tracts, and thin serotonergic fibres in the perineurium were also demonstrated. Thisin vivo andin vitro identification of serotonin-containing neurons will facilitate further neurophysiological analysis of serotonergic neural mechanisms inLymnaea.

Development of catecholaminergic neurons in the pond snail, Lymnaea stagnalis: I. Embryonic development of dopamine-containing neurons and dopamine-dependent behaviors

The embryonic development of the catecholaminergic system of the pond snail, Lymnaea stagnalis, was investigated by using chromatographic and histochemical methods. High performance liquid chromatography suggested that dopamine was the only catecholamine present in significant concentrations throughout the embryonic development of Lymnaea. Dopamine first became detectable at about embryonic stage (E) 15 (15% of embryonic development) and then increased in amount during early development to reach about 120–140 fmol per animal by around E40. Dopamine content remained stable during mid‐embryogenesis (E40–65), increased slowing for the next couple of days, and then increased rapidly to culminate at about 400 fmol per animal by hatching. The detection of aldehyde‐ and glyoxylate‐induced fluorescence and of tyrosine hydroxylaselike immunoreactivity indicated that the first catecholaminergic cells appeared in the late trochophore or early veliger stage of embryonic development (E32–35). The paired perikarya of these transient apical catecholaminergic (TAC) neurons were located beneath the apical plate, remained outside of the central ganglia during embryogenesis, and no longer contained detectable catecholamines close to hatching. TAC neurons bore cilia on the ends of short processes that penetrated the overlying epithelium; their long processes branched repeatedly under the ciliated apical plate. Several smaller catecholaminergic cells first appeared in the anterior margin of the foot at a stage when the embryos began to metamorphose from the veliger form (E55). Similar bipolar cells later appeared in the tentacle and lips. The axons of all of these small peripheral cells projected centrally and terminated within the neuropil of different central ganglia. Central catecholaminergic neurons, including RPeD1, differentiated only after metamorphosis was complete (E75). Development of locomotor, respiratory, and feeding behaviors correlated with maturation of catecholaminergic neurons, as indicated by histology and chromatography. J. Comp. Neurol. 404:285–296, 1999.

Distribution of FMRFamide-like immunoreactive neurons in the central nervous system of the snail Helix pomatia

SummaryThe distribution of FMRFamide-like immunoreactive (FLI) neurons and their morphological characteristics have been investigated in the central nervous system of the snail, Helix pomatia L. Approximately phageal ganglion complex. More than 50% of the FLI neurons were located in the cerebral ganglia. The FLI neurons could be divided into four groups according to size: (i) giant neurons (over 100 μm); (ii) large neurons (80–100 μm); (iii) medium-sized neurons (40–70 μm); (iv) small neurons (12–30 μm). They were distributed i) in groups or clusters, typical of small neurons and ii) in solitary form or in groups comprising 2–3 cells, typical of large and giant neurons. Giant and large neurons revealed only limited arborizations in the neuropil, but rich branching towards and in the peripheral nerves. Some of the small neurons had extensive arborizations of varicose fibers in the neuropil. They may therefore play some role in integratory processes. Varicose FLI fibers were visualized in the cell body layer of the different ganglia, and in the neural sheath of both the ganglia and the peripheral nerves. We propose a multifunctional involvement of FLI neurons and FMRFamide-like neuropeptides in the Helix nervous system: (i) a synaptic or modulatory role in axo-axonic interactions in the neuropil; (ii) a direct influence on neuronal cell bodies in the cortical layer, (iii) innervation of different peripheral organs; and (iv) remote neurohormonal control of peripheral events through the neural sheath.

Evidence that locustatachykinin I is involved in release of adipokinetic hormone from locust corpora cardiaca

The glandular cells of the corpus cardiacum of the locust Locusta migratoria, known to synthesize and release adipokinetic hormones (AKH), are contacted by axons immunoreactive to an antiserum raised against the locust neuropeptide locustatachykinin I (LomTK I). Electron-microscopical immunocytochemistry reveals LomTK immunoreactive axon terminals, containing granular vesicles, in close contact with the glandular cells cells. Release of AKH I from isolated corpora cardiaca of the locust has been monitored in an in vitro system where the amount of AKH I released into the incubation saline is determined by reversed phase high performance liquid chromatography with fluorometric detection. We could show that LomTK I induces release of AKH from corpora cardiaca in a dose-dependent manner when tested in a range of 10-200 microM. This is thus the first clear demonstration of a substance inducing release of AKH, correlated with the presence of the substance in fibers innervating the AKH-synthesizing glandular cells, in the insect corpora cardiaca.

Dopamine-like immunoreactivity in the bee brain

SummaryThe distribution of dopamine-like immunoreactive neurons is described for the brain of the bee, Apis mellifera L., following the application of a pre-embedding technique on Vibratome sections. Immunoreactive somata are grouped into seven clusters, mainly situated in the protocerebrum. Immunoreactive interneurons have been detected in the different neuropilar compartments, except for the optic lobe neuropils. Strong immunoreactivity is found in the upper division of the central body, in parts of the stalk and in the α-lobe layers of the mushroom bodies. A dense network of many immunoreactive fibres surrounds the mushroom bodies and the central body. It forms a number of interhemispheric commissures/chiasmata, projecting partly into the contralateral mushroom body and central body. The lateral protocerebral neuropil contains some large wide-field-neurons. The antennal-lobe glomeruli receive fine projections of multiglomerular dopamine-like immunoreactive interneurons.

Ultrastructural demonstration of serotonin-immunoreactivity in the nervous system of an insect (Calliphora erythrocephala)

Serotonin (5-HT) immunocytochemistry, was performed on the whole dissected nervous system of the blowfly. Employing the peroxidase-antiperoxidase technique and osmium postfixation, it was possible to examine 5-HT-immunoreactive neuronal elements first light microscopically in 25 microns sections, and, after re-embedding, to analyze the same sections electron microscopically in ultrathin sections. We describe the ultrastructure of 5-HT-positive terminals in the neural sheath of peripheral nerves and in the optic lobes. The immunoreactivity was observed in large (100 nm) granular vesicles, on membranes of clear vesicles, along neurotubules, and along the internal surface of the plasmalemma.

Development of catecholaminergic neurons in the pond snail, Lymnaea stagnalis: II. Postembryonic development of central and peripheral cells

Catecholamines have long been thought to play important roles in different mollusc neural functions. The present study used glyoxylate‐ and aldehyde‐induced histofluorescence to identify central and peripheral catecholaminergic neurons in the snail Lymnaea stagnalis. The majority of these cells were also found to react to antibodies raised against tyrosine hydroxylase. A minority of the catecholaminergic neurons, however, exhibited no such immunoreactivity. The number of central catecholaminergic neurons nearly doubled (from about 45 to about 80 cells) during the first 2–3 days of postembryonic development. Thereafter, catecholaminergic neurons again doubled in number and generally grew by about 100–200% in soma diameter as the snails grew by 1,000% in overall linear measurements. In contrast to the relatively meager addition of central catecholaminergic neurons, several thousand catecholaminergic somata were added to different peripheral tissues during postembryonic development. These small, centrally projecting neurons were particularly concentrated in the lips, esophagus, anterior margin of the foot, and different regions of the male and female reproductive tracts. Chromatographic analyses indicated that dopamine was the major catecholamine present in the central ganglia, foot, and esophagus, although detectable levels of norepinephrine (approximately 20% of dopamine levels) were also found in the ganglia. The total content but not the concentration of dopamine increased within the tissue samples during postembryonic development. The companion study (Voronezhskaya et al. [1999] J. Comp. Neurol. 404:285–296) and the present study furnish a complete description of central and peripheral catecholaminergic neurons from their first appearance in early embryonic development to adulthood. J. Comp. Neurol. 404:297–309, 1999.

Distribution of serotonin-containing neurons in the central nervous system of the snail Helix pomatia

SummaryThe distribution of serotonin (5HT)-containing neurons in the central nervous system of the snail Helix pomatia has been determined in whole-mount preparations by use of immunocytochemical and in vivo 5,6-dihydroxy-tryptamine labelling. 5HT-immunoreactive neuronal somata occur in all but the buccal and pleural ganglia. Immunoreactive fibres are present throughout the central nervous system. The 5HT-immunoreactive neuronal somata characteristically appear in groups, located mainly in the cerebral, pedal, visceral and right parietal ganglia. The majority of 5HT-immunoreactive neurons is located in the pedal ganglia. Additionally a dense network of 5HT-immunoreactive varicose fibres is found in the neural sheath of the central nervous system including all the nerves and ganglia. The number and distribution of 5HT-immunoreactive neurons correlates with that demonstrated by 5,6-dihydroxytryptamine labelling method.