Interneurones subserving ocelli in two species of trichopterous insects: morphology and central projections

Summary The central projections of ocellar interneurones in two species of trichopterous insects Agrypnia varia F. and Limnephilus flavicornis F. were analysed by use of cobalt iontophoresis. The interneurones were classified into three groups: large-, medium- and small-caliber neurones based on the diameters of the axons. Seven large-diameter neurones project from each lateral ocellus into the central nervous system. Of these, four neurones terminate in the posterior slope (three ipsilateral and one contralateral). Three neurones possess branches in the contralateral posterior slope and proceed down the cervical connective into the thoracic ganglia. Medium-sized neurones connect the neuropiles of the three ocelli to each other. Small-diameter neurones contact the contralateral lobula and medulla of the optic lobes and connect the three ocellar neuropiles. Large-diameter neurones of the median ocellus were found to terminate bilaterally or ipsilaterally in the posterior slope. In the posterior slope four different subregions can be recognised: (1) the dorso-lateral, (2) the ventro-lateral, (3) the lateral, into which large-diameter interneurones of the lateral ocelli send branches, and (4) the medial, innervated by interneurones of the median ocellus. Interneurones of the median ocellus send branches into the lateral region as well.     

Central projections of first-order ocellar interneurons in two orthopteroid insects Acheta domesticus and Periplaneta americana

Summary The central projections of ocellar first-order interneurons in the cricket, Acheta domesticus, and the cockroach, Periplaneta americana, were examined in silver-intensified cobalt preparations. Ten morphologically different types of ocellar interneurons among a total of 44 are recognized in the cricket, and five different types among a total of 26 in the cockroach, indicating that these species have simpler ocellar systems than those described previously in locusts. Ocellar interneurons arborize in the following regions of neuropil in both the cricket and cockroach: the ocellar foci of the posterior protocerebrum, the posterior deutocerebrum, the protocerebral bridge, the ocellar synaptic plexus, ocellar nerves and tracts, and the lobula and medulla of the optic lobes. Ocellar first-order interneurons thus project predominantly to sites where they are likely to synapse with other ocellar and optic-lobe interneurons.     

Central projections of first-order ocellar interneurons in the bug Triatoma infestans (Heteroptera: Reduviidae)

The projections of first-order ocellar interneurons were analyzed in the hematophagous bug Triatoma infestans by cobalt filling. The axons run between the calyces of the mushroom bodies and dorsal of the central body to different regions of the brain and the subesophageal and thoracic ganglia. The interneurons can be grouped into large L cells and small S cells. The L cells have cell bodies ranging from 11.5 to 25 μm and axons ranging from 8 to 25 μm diameter (measured in the ocellar nerve); the S cells have smaller cell bodies of 9 μm or less and axon diameters less than 5 μm. The projections of ten L cells are described in detail; they project to the protocerebral posterior slope (PS), the other ocellus (O), the optic neuropile, and the subesophageal, pro-, meso-, and metathoracic ganglia, either to ipsi- (PS I, II), or contra- (PS IV, V), or bilateral areas. In this case projections occur to the same areas (PSO, PS III) or different areas at each side (PSOE; E = eye). Large-descending (LD) first-order interneurons project to the contralateral posterior slope of the protocerebrum, the deutocerebrum, and subesophageal, pro-, mesa-, and metathoracic areas (LD I-III). Cell bodies are located in the dorsal protocerebral lobes and pars intercerebralis, except the PS II neuron and three LD cells, which are located in the ipsilateral posterior protocerebrum. This is the first report about ocellar pathways in Hemiptera. Their adaptive function is discussed with reference to the bugs' behavior as Chagas disease vectors. © 1996 Wiley-Liss, Inc.     

Ocellar projections within the central nervous system of the worker honey bee,Apis mellifera

Summary The projections of ocellar fibres within the brain and thorax of the honey bee, Apis mellifera, were established using a modified cobalt sulphide technique, supplemented by serial sectioning of the brain for the light microscope.The results are: 5 large fibres in each lateral nerve and 12 in the median nerve have wide-field terminal arborisations in ocellar association areas on either side of the posterior slope area. 9 medium-sized fibres in each lateral nerve and 12 in the median nerve form a second ocellar association area on each side of the perioesophageal foramen. A group of fine fibres, stained via the ocellar nerves, arborise just below and anterior to the protocerebral bridge. 10 medium-sized fibres run from the level of the ocellar nerve tracts to the first and second thoracic ganglia, branching in a number of discrete areas within each ganglion. These fibres also form a restricted ocellar association area within the suboesophageal ganglion. A few fibres run between the higher-order optic centres and the ocellar tract. The large- and mediumsized fibres give off short, stout spines from their axons within the ocellar tracts.     

Lateral ocellar nerve projections in the dragonfly brain

Summary The central projections of the lateral ocellar neurons of the dragonfly were examined using whole nerve cobalt iontophoresis, supplemented by sectioning of the nerve and brain for inspection in the light and electron microscopes. At E.M. level the presence of cobalt in filled axon profiles and cell bodies was confirmed by analysis of X-ray energy spectra in the microscope.The pathways, cell body sites and terminal arborizations of four large (7–25 m diameter) lateral ocellar neurons are described. Two of these fibers arborize in the ipsilateral posterior neuropil of the protocerebrum and two cross the brain and arborize in the contralateral posterior neuropil. Within each half of the posterior neuropil, two spatially separated regions of ocellar input have been identified. These regions receive median ocellar input plus input from either the ipsi- or contralateral ocellus, but not both. The arborizations of the contralateral fibers are more extensive than those of the ipsilateral fibers.One of the contralateral neurons crosses the brain in the region of the protocerebral bridge giving off a collateral in that region before descending to the posterior neuropil. This collateral arborizes almost immediately in a region receiving input from arborizations of a number of small ocellar neurons (those less than 5 m in diameter) from the ipsilateral ocellar nerve, together with small neurons from the median ocellar nerve, forming a region in each half of the brain which receives input from all three ocelli. The small lateral ocellar neurons associated with these arborizations have cell bodies adjacent to the lateral ocellar tracts.This work was supported in part by National Institute of Health Grants 2 RO1 EY-00777 and 1 KO4 EY-00040     

Lobula plate and ocellar interneurons converge onto a cluster of descending neurons leading to neck and leg motor neuropil in Calliphora erythrocephala

Summary In the fly, Calliphora erythrocephala, a cluster of three Y-shaped descending neurons (DNOVS 1–3) receives ocellar interneuron and vertical cell (VS4–9) terminals. Synaptic connections to one of them (DNOVS 1) are described. In addition, three types of small lobula plate vertical cell (sVS) and one type of contralateral horizontal neuron (Hc) terminate at DNOVS 1, as do two forms of ascending neurons derived from thoracic ganglia. A contralateral neuron, with terminals in the opposite lobula plate, arises at the DNOVS cluster and is thought to provide heterolateral interaction between the VS4–9 output of one side to the VS4–9 dendrites of the other. DNOVS 2 and 3 extend through pro-, meso-, and metathoracic ganglia, branching ipsilaterally within their tract and into the inner margin of leg motor neuropil of each ganglion. DNOVS 1 terminates as a stubby ending in the dorsal prothoracic ganglion onto the main dendritic trunks of neck muscle motor neurons. Convergence of VS and ocellar interneurons to DNOVS 1 comprises a second pathway from the visual system to the neck motor, the other being carried by motor neurons arising in the brain. Their significance for saccadic head movement and the stabilization of the retinal image is discussed.     

Mapping of serotonin-immunoreactive neurons in the larval nervous system of the flies Calliphora erythrocephala and Sarcophaga bullata

Summary Serotonin-immunoreactive (5-HTi) neurons were mapped in the larval central nervous system (CNS) of the dipterous flies Calliphora erythrocephala and Sarcophaga bullata. Immunocytochemistry was performed on cryostat sections, paraffin sections, and on the entire CNS (whole mounts).The CNS of larvae displays 96–98 5-HTi cell bodies. The location of the cell bodies within the segmental cerebral and ventral ganglia is consistent among individuals. The pattern of immunoreactive fibers in tracts and within neuropil regions of the CNS was resolved in detail. Some 5-HTi neurons in the CNS possess axons that run through peripheral nerves (antenno-labro-frontal nerves).The suboesophagealand thoracico-abdominal ganglia of the adult blowflies were studied for a comparison with the larval ventral ganglia. In the thoracico-abdominal ganglia of adults the same number of 5-HTi cell bodies was found as in the larvae except in the metathoracic ganglion, which in the adult contains two cell bodies less than in the larva. The immunoreactive processes within the neuropil of the adult thoracico-abdominal ganglia form more elaborate patterns than those of the larvae, but the basic organization of major fiber tracts was similar in larval and adult ganglia. Some aspects of postembryonic development are discussed in relation to the transformation of the distribution of 5-HTi neurons and their processes into the adult pattern.     

The distribution of neurones immunoreactive for β-tyrosine hydroxylase,dopamine and serotonin in the ventral nerve cord of the cricket,Gryllus bimaculatus

The cellular localization of the biogenic amines dopamine and serotonin was investigated in the ventral nerve cord of the cricket, Gryllus bimaculatus, using antisera raised against dopamine, -tyrosine hydroxylase and serotonin. Dopamine-(n<-70) and serotonin-immunoreactive (n<-120) neurones showed a segmental arrangement in the ventral nerve cord. Some neuromeres, however, did not contain dopamine-immunoreactive cell bodies. The small number of stained cells allowed complete identification of brain and thoracic cells, including intersegmentally projecting axons and terminal arborizations. Dopamine-like immunostaining was found primarily in plurisegmental interneurones with axons descending to the soma-ipsilateral hemispheres of the thoracic and abdominal ganglia. In contrast, serotonin-immunostaining occurred predominantly in interneurones projecting via soma-contralaterally ascending axons to the thorax and brain. In addition, serotonin-immunoreactivity was also present in efferent cells and afferent elements. Serotonin-immunoreactive, but no dopamine-immunoreactive, varicose fibres were observed on the surface of some peripheral nerves. Varicose endings of both dopamine-and serotonin-immunoreactive neurones occurred in each neuromere and showed overlapping neuropilar projections in dorsal and medial regions of the thoracic ganglia. Ventral associative neuropiles lacked dopamine-like immunostaining but were innervated by serotonin-immunoreactive elements. A colocalization of the two amines was not observed. The topographic representation of neurone types immunoreactive for serotonin and dopamine is discussed with respect to possible modulatory functions of these biogenic amines in the central nervous system of the cricket.     

Anatomy of the ocellar interneurons of acridid grasshoppers

Summary The anatomy of the small ocellar interneurons in the brain of the acridid grasshopper Schistocerca vaga was revealed by cobalt-filling the three ocellar nerves and subsequent reconstructions from silver-intensified (Timm's method) serial sections.In total, 61 small ocellar interneurons were repeatedly identified with arborizations in many areas of the brain and optic lobe, including in particular the posterior neuropil, ocellar tracts, protocerebral bridge, lobula, ventral bridge and tritocerebral crotch, calyces, and antenno-glomerular tracts.Each ocellar nerve contains the axons of small cells that arborize in the other two ocellar tracts; these tracts are sites of ocellar integration. Direct interactions between the ocelli and compound eyes are suggested by the projections of small ocellar interneurons into the proximal lobula. Small cell arborizations from all three ocelli are distributed across much of the protocerebral bridge, implying a role for the bridge as an ocellar neuropil within the brain.Four of the small interneurons could be seen in whole-mount preparations and are demonstrated to be identical in five species of acridid grasshoppers of two different subfamilies: Schistocerca vaga, S. gregaria, Gastrimargus africanus, Trimerotropis pallidipennis, and Arphia conspersa.     

GABA and glutamate-like immunoreactivity at synapses received by dorsal unpaired median neurones in the abdominal nerve cord of the locust

Dorsal unpaired median (DUM) neurones in the abdominal ganglia of the locust were impaled with microelectrodes and some were injected intracellularly with horseradish peroxidase so that their synapses could be identified in the electron microscope. Simultaneous recordings from DUM neurones in different abdominal ganglia revealed that they received common postsynaptic potentials from descending interneurones. Post-embedding immunocytochemistry using antibodies against GABA and glutamate was carried out on ganglia containing HRP-stained neurones. GABA-like immunoreactivity was found in 39% (n=82) of processes presynaptic to abdominal DUM neurones and glutamate-like immunoreactivity in 21% (n=42) of presynaptic processes. Output synapses from the DUM neurites were rarely observed within the neuropile. Structures resembling presynaptic dense bars but not associated with synaptic vesicles, were seen in some large diameter neurites.     

Direct excitation of nonspiking local interneurones by exteroceptors underlies tactile reflexes in the locust

Summary Tactile stimulation of a leg of the locustSchistocerca gregaria can lead to specific reflex movements of that leg. At the same time nonspiking interneurones that are presynaptic to the participating motor neurones are excited or inhibited, suggesting that they are directly involved in these reflexes. The afferent pathways mediating these effects have been examined by recording from individual afferents and nonspiking interneurones.Afferent spikes fromtrichoid orcampaniform sensilla on specific regions of a leg evoke chemically-mediated EPSPs with a constant central latency of about 1.5 ms in certain nonspiking interneurones. The branches of an interneurone and the afferents from which it receives inputs overlap in the neuropil of the ganglion.No afferents have been found to evoke IPSPs directly in the nonspiking interneurones. Instead the inhibition is caused by a population of spiking local interneurones that are themselves excited directly by the afferents, and whose spikes evoke IPSPs in certain nonspiking interneurones.The tactile reflexes can involve movements about one or more joints of the leg, and these coordinated responses are explained by the participation of specific nonspiking interneurones that distribute the sensory inputs to the appropriate sets of motor neurones. For example, when hairs on the dorsal surface of a tarsus are touched, the tarsus is levated. This reflex involves nonspiking local interneurones which are excited directly by these hair afferents and which make direct excitatory connections with the single levator tarsi motor neurone.     

Serotonin-like immunoreactivity in the central nervous system and gonad of the scallop,Patinopecten yessoensis

Summary The localization of neurons containing serotonin in the central nervous system and the gonad of the scallop, Patinopecten yessoensis, was examined immunohistochemically. In the central nervous system a large number of immunoreactive perikarya were observed in the following regions: a part of the anterior lobe of the cerebral ganglion; the posterior lobe of the cerebral ganglion; the pedal ganglion; and the accessory ganglion. No immunoreactive perikarya were found in the visceral ganglion. Numerous immunoreactive fibers were revealed in the neuropil of all central ganglia. In the gonadal region immunoreactive fibers were distributed around the gonoduct and along the germinal epithelium.This work was supported by a grant from the Ministry of Education, Science and Culture, Japan     

Location and major postembryonic changes of identified 5-HT-immunoreactive neurones in the terminal ganglion of a cricket (Acheta domesticus)

Summary In the terminal ganglion of the cricket, Acheta domesticus, the somata of certain interneurones and efferent neurones consistently react to 5-HT immunohistochemistry. There are serially homologous pairs of bilateral interneurones seen in the neuromeres of the 7th to the 10th segment and hindgut neurones with their somata located at the posterior median end of the ganglion. In adult crickets, pairs of large efferent neurones with lateral somata supply specific genital muscles in the 8th and the 9th segment of females. In males, only one pair of these efferent neurones supplies genital muscles of the 9th segment only. These identified 5-HT-immunoreactive neurones are not detected in larval crickets before development of the genital apparatus.     

Leu-callatostatin gene expression in the blowflies Calliphora vomitoria and Lucilia cuprina studied by in situ hybridisation: comparison with Leu-callatostatin confocal laser scanning immunocytochemistry

In situ hybridisation studies using a digoxigenin-labelled DNA probe encoding the Leu-callatostatin prohormone of the blowflies Calliphora vomitoria and Lucilia cuprina have revealed a variety of neurones in the brain and thoracico-abdominal ganglion, peripheral neurosecretory neurones, and endocrine cells of the midgut. With two exceptions, the hybridising cells are the same as those previously identified in immunocytochemical studies of sections and whole-mounts using Leu-callatostatin COOH-terminal-specific antisera. Within the brain and suboesophageal ganglion, there is a variety of neurones ranging from a single pair of large cells situated in the dorsal protocerebrum, to the several pairs of neurones in the tritocerebrum, some of which, in immunocytochemical preparations, can be seen to project via axons in the cervical connective to the thoracico-abdominal ganglion. In the medulla of the optic lobes, numerous small interneurones hybridise with the probe, as do clusters of similar-sized neurones close to the roots of the ocellar nerves. These results indicate that the Leu-callatostatin neuropeptides of the brain play a variety of roles in neurotransmission and neuromodulation. There are only three pairs of Leu-callatostatin-immunoreactive neurones in the thoracico-abdominal ganglion, at least two pairs of which project axons along the median abdominal nerve to provide extensive innervation of the hindgut. The Leu-callatostatin peripheral neurosecretory cells are located in close association with both nerve and muscle fibres in the thorax. In addition to neuronal Leu-callatostatin, the presence of the peptide and its mRNA has been demonstrated in endocrine cells in the posterior part of the midgut. These observations provide an example of a named brain/gut peptide in an insect.     

Intracellular stainings of the large ocellar second order neurons in the cockroach

Summary The large ocellar second order neurons (L-neurons) in the cockroach,Periplaneta americana have been studied physiologically by intracellular recordings and morphologically by intracellular and whole nerve cobalt stainings. All the recorded L-neurons showed similar light responses, i.e., light on-hyperpolarization and a small number of off-spikes. All the stained L-neurons had an ocellar arborization covering the whole region of the ocellar neuropile and an central arborization in the region posterior to the protocerebral bridge.     

Serotonin-immunoreactive and dopamine-immunoreactive neurones in the terminal ganglion of the cricket,Acheta domestica: Light- and electron-microscopic immunocytochemistry

Summary The distribution and ultrastructure of serotonin- and dopamine-immunoreactive (5-HTi and DAi) neurones have been investigated in the terminal ganglion of the cricket, Acheta domestica, using a pre-embedding chopper technique. Special attention has been paid to the immunoreactive structures in the neuropil. 5-HTi structures are extensively distributed and densely packed throughout the 5 neuromeres of the terminal ganglion and originate from several interneurones and efferent neurones. In contrast, DAi fibres are distributed sparsely although they extend to all neuromeres of the ganglion and originate from 6 interneurons only. For both 5-HTi and DAi neurones characteristic axonal projections and branching patterns can be distinguished. The 5-HTi axons exhibit rich varicose arborizations, whereas DAi neurones possess fewer varicosities in the neuropil. Electron microscopy shows that 5-HTi varicosities contain small ( 60 nm) and large ( 100 nm) agranular vesicles, and large ( 100 nm) granular vesicles, whereas in DAi varicosities small ( 60 nm) agranular and large ( 100 nm) granular vesicles are seen. Both 5-HTi and DAi varicosities form synaptic contacts. We conclude that both serotonin and dopamine may be used as neurotransmitters in the terminal ganglion of the cricket.Fellow of the Alexander von Humboldt-Stiftung     

Tyrosine hydroxylase in the cerebral ganglia of the American cockroach (Periplaneta americana L.): an immunohistochemical study

We have investigated the distribution of tyrosine-hydroxylase-like immunoreactivity in the cerebral ganglia of the American cockroach, Periplaneta americana. Groups of tyrosine-hydroxylase-immunoreactive cell bodies occur in various parts of the three regions of the cerebral ganglia. In the protocerebrum, single large neurons or small groups of neurons are located in the lateral neuropil, adjacent to the calyces, and in the dorsal portion of the pars intercerebralis. Small scattered cell bodies are found in the outer layers of the optic lobe, and clusters of larger cell bodies can be found in the deutocerebrum, medial and lateral to the antennal glomeruli. Thick bundles of tyrosine-hydroxylase-positive nerve fibers traverse the neuropil in the proto- and deutocerebrum and innervate the glomerular and the nonglomerular neuropil with fine varicose terminals. Dense terminal patterns are present in the medulla and lobula of the optic lobe, the pars intercerebralis, the medial tritocerebrum, and the area surrounding the antennal glomeruli, the central body and the mushroom bodies. The pattern of tyrosine-hydroxylase-like immunoreactivity is similar to that previously described for catecholaminergic neurons, but it is distinctly different from the distribution of histaminergic and serotonergic neurons.     

Parallel effects of joint receptors on motor neurones and intersegmental interneurones in the locust

Summary At the distal end of a mesothoracic tibia of the locust,Schistocerca gregaria, is a chordotonal organ which monitors the position and movement of the tarsus relative to the tibia. It contains approximately 35 receptors that variously encode different spatial and temporal parameters (position, velocity and direction of movement). Some excite intersegmental interneurones that respond phasically or tonically, with directional sensitivity to active or imposed movements of the tarsus. Some of these interneurones are also excited by intrinsic movements of the tarsal segments. Others, besides being excited by tarsal proprioceptive inputs, are also excited by exteroreceptors on the tarsus.When stimulated mechanically or electrically, chordotonal afferents evoke excitatory postsynaptic potentials with a central latency of between 0.9 and 1.4 ms simultaneously in the intersegmental interneurones and in tarsal motor neurones. The central arborizations of the afferents, the intersegmental interneurones and the tarsal motor neurones overlap in certain neuropilar regions of the mesothoracic ganglion. Other afferents cause an inhibition of the motor neurones, with a longer and non-consistent latency suggesting the involvement of other intercalated interneurones.These results indicate that proprioceptive inputs from the tarsal joint receptors are transmitted in parallel and monosynaptically to tarsal motor neurones and to the intersegmental interneurones.     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

Localisation of substance P-, somatostatin-, vasoactive intestinal polypeptide and met-enkephalin-immunoreactive nerves in the peripheral and central nervous systems of the leech (Hirudo medicinalis)

Summary The distribution of substance P (SP)-, somatostatin (SOM)-, vasoactive intestinal polypeptide (VIP)- and met-enkephalin (mENK)-immunoreactive nerve fibres and cell bodies has been studied in the gastrointestinal tract, lateral blood vessel (heart) and segmental ganglia of the leech (Hirudo medicinalis). In the crop and intestine, there was a sparse distribution of VIP-, SP-, SOM- and mENK-immunoreactive nerves, while in the intestine, a dense network of SP-, a moderate network of SOM-, and a sparse distribution of mENK- and VIP-immunoreactive nerve fibres was seen. SP-, SOM- and VIP-immunoreactive nerve cell bodies were found in all the gut regions studied, the greatest number being in the intestine. No mENK-containing cell bodies were seen in any region of the gastrointestinal tract. The heart contained a few SP-, SOM-, and VIP-immunoreactive nerve fibres, but no nerve cell bodies were found. Immunoreactive nerve cell bodies were also present in the segmentai ganglia. A typical midbody ganglion contained up to seven pairs of SP-containing neurones, four pairs of SOM-containing neurones, two pairs of VIP-containing neurones and one to three pairs of mENK-immunoreactive nerve cell bodies. The lateral pair of large SOM-immunoreactive nerve cell bodies is of similar size and correct position to the lateral N cells. One of the pairs of large SP-immunoreactive nerve cell bodies is probably identical to the Leydig cells. A tentative identification of other immunofluorescent nerve cells is attempted. Immunoreactive nerve fibres to all four peptides were distributed throughout the neuropil, those to SP being the most numerous.