Immunohistochemical localization of serotonin and choline acetyltransferase in sensory neurones of the locust.

Sensory neuronal cell bodies in the leg of locust, Schistocerca gregaria, were visualized with antibodies to locust choline acetyltransferase and with antibodies to serotonin by the avidin-biotin peroxidase technique. Two groups of sensory cells react with the antibody to choline acetyltransferase: One group is associated with external mechanoreceptors (i.e., hair-plate hairs and campaniform sensilla) and the other with internal proprioceptors (i.e., chordotonal organs and multiterminal receptors). Sensory cells which react with the antibody to serotonin are associated only with internal proprioceptors being found in both chordotonal organs and multiterminal receptors. In the metathoracic femoral chordotonal organ indirect evidence suggests that some sensory cells are reactive to both antibodies. Some multiterminal receptors react with anti-choline-acetyltransferase, while others react with antiserotonin. These results support the conclusion that most insect sensory neurones are cholinergic but some are serotoninergic.

The locust wing hinge stretch receptors. I. Primary sensory neurones with enormous central arborizations.

In locusts a single-celled stretch receptor (SR) neurone at the base of each wing monitors wing elevation and contributes to the control of the flight motor output. The central projections of these neurones are very complex but consistent in detail in the three species studied (Chortoicetes, Locusta and Schistocerca). The hindwing SR projects to the second and third thoracic ganglia, the forewing SR to the first, second and third thoracic ganglia. Both send fine axons into the abdominal connective. Within the ganglia each SR forms an extensive arborization, entirely ipsilateral and mainly in the dorsal neuropile, divided into medial, mediolateral, and lateral branches. The projections of the two ipsilateral SR neurones overlap almost completely in the second and third ganglia. There are recurrent loops between branches of a single neurone both within and between ganglia. Light microscope analysis shows apparent contacts between the SR aneurones and flight motor neurones and other wing sensory afferents, as well as long interneurones, other motor neurones and two types of multiaxonal neurones of unknown function. There are three groups of contacts between each SR and a flight motor neurone: laterally on the main branches, medially with the terminal twigs; and in the anterior dorso-medial glomerulus, where the inter ganglionic recurrent branch also terminates. All contacts are ipsilateral except for those with the contralateral branches of the dorsal longitudinal muscle motor neurones. We suggest that the SR neurones are multifunctional. Differential information transfer could result both from the spatial distribution of synaptic connections with the motor neurones and from filtering caused by low safety factors at branch junctions. Information in the lateral branching could be used for general excitation and control of firing frequency of the motor neurones; that in the medial branch for wing control and co-ordination.

The locust wing hinge stretch receptors. II. Variation, alternative pathways and "mistakes" in the central arborizations.

The central arborizations of the stretch receptor (SR) neurones are very consistent from one individual to another. Superimposition of normalized neurones from eight individuals of Locusta show very little variation even in the detailed branching pattern. There is, however, a commonly found alternative course for the main medial branch in the metathoracic ganglion. Rare, radical departures from the normal branching pattern are termed "mistakes." Only three have been found, all in the forewing SR projection, one with an extra branch and two with missing branches. Terminals of twigs in the alternative region of the medial branch occupy consistent positions in the neuropile, although these are reached by different routes. Mistakes have terminals in areas normally containing SR endings. Both these findings suggest that there may be labelled sites in the neuropile which the growing tips of the SR neurones seek out.

Serotoninergic innervation of the locust mandibular closer muscle modulates contractions through the elevation of cyclic adenosine monophosphate.

The mandibular closer muscles of the locust receive innervation that is immunoreactive for the putative transmitter 5-hydroxytryptamine (5-HT). Cobalt-labelling suggests that the origin of this innervation is a group of cells located anteriorly in the suboesophageal ganglion. Bath application of 5-HT while the muscles are active produces marked changes in the contractions, increasing their amplitude, rate of contraction, and rate of relaxation. Incubation of isolated muscles with 5-HT shows that this amine elevates the levels of the cyclic nucleotide cyclic adenosine monophosphate (cAMP). In addition compounds that artificially elevate the levels of cAMP in the muscle--3-isobutyl-1-methylxanthine (IBMX), forskolin, and the cAMP analogue 8-(4-chlorophenylthio) cAMP--mimic the actions of 5-HT, whereas a potent inhibitor of insect adenylate cyclase, adenosine, considerably delays the onset of the effects produced by 5-HT. The effects observed with 5-HT in the mandibular muscle are similar to those of octopamine in the locust extensor tibiae muscle, and it is possible that this is an analogous modulatory system.

Synaptic connections between the hindwing stretch receptor and flight motor neurones in the locust revealed by double cobalt labelling for electron microscopy.

Synaptic interactions between sensory and motor neurones in the locust flight system have been investigated by using intracellular labelling with cobalt and nickel for electron microscopy. Simultaneous axonal filling of two neurones with different concentrations of metal ions produces differential labelling, so that contacts between them in the central nervous system can be recognized. We have investigated the connectivity of the hindwing stretch receptor neurone (SR) with a direct hindwing depressor motor neurone (MN 127) known from physiological experiments to receive monosynaptic input from the SR, and an indirect hindwing depressor motor neurone (MN 112/1), for which no monosynaptic connection with the SR has been reported. We have found no direct synapses between the SR and MN 112/1, although some of their branches lie close together in the neuropile. We have, however, found some evidence for polysynaptic connections between them. There are many synapses of conventional dyadic morphology from both the lateral and mediolateral branches of the SR to MN 127; the medial branch was not examined. Those from the lateral branch contact the motor neurone on branches close to the neuropilar segment, while those from the mediolateral branch contact long, thin distal twigs. We estimate that there are about 600 anatomical synapses between these two neurones. Our results suggest that a large number of widely distributed anatomical synapses constitute the physiological synaptic connection between the SR and MN 127. The dyadic arrangement of these synapses provides an anatomical correlate for the physiologically established divergence of SR outputs onto interneurones and motor neurones.

Identifiable neurons in the locust central nervous system that react with antibodies to serotonin.

A detailed account is given of a number of neurons in the locust central nervous system that react with antibody raised to serotonin-albumin complex. The antibody was applied to a series of frozen sections of locust ganglia and visualized by using the peroxidase immunohistochemical procedure. The neurons described include certain afferents and their related neuropiles, a small number of efferents and several systems of interneurons, some of which are segmentally repeated, some run from the brain through the whole nerve cord, while others are confined to the brain. It has been possible to identify many of the neurons from previous descriptions obtained from cobalt, Golgi, and osmium ethyl gallate methods.

Morphology of the vasopressin-like immunoreactive (VPLI) neurons in many species of grasshopper.

It has previously been shown that the pair of vasopressin-like immunoreactive (VPLI) neurons of the locust, Locusta migratoria, have cell bodies on the ventral midline of the suboesophageal ganglion and extensive arborisations in all ganglia of the central nervous system. In the present study, we have stained vasopressin-like immunoreactive neurons in 16 additional species of grasshopper, and consistently find this pair of extensive neurons: we assume these to be interspecies homologues. However, the anatomy of these neurons falls into two morphological types: the first, typified by Schistocerca gregaria, has most of its processes distributed in dorsal and lateral neuropil of all ganglia; the second, typified by Locusta migratoria, is equally extensive in its arborisation, but the distribution of branches is shifted peripherally into the optic lobes and the proximal portions of peripheral nerves. It has been suggested that the peripheral fibres in Locusta migratoria are neurohaemal organs for the release of a vasopressin-like diuretic peptide. Our sample of 17 Acridoid species has deliberately selected animals from very different habitats, but our extensive survey of VPLI anatomy shows that peripheral fibres are only present in species from the subfamily Oedipodinae (of which Locusta migratoria is a member) and that no peripheral fibres are present in any of the species from the 4 other subfamilies of the Acridoidea that we have examined. The presence of peripheral fibres is therefore determined by phylogeny and not by habitat. The absence of peripheral VPLI fibres in most grasshopper species examined in this study probably means that the release of putative diuretic hormone from VPLI to control water homeostasis cannot be a conserved function of this ubiquitous neuron. In contrast, the extensive central arborisations and rare antigenicity, which are highly conserved features of the VPLI neuron in all those grasshoppers we have examined, suggests that any conserved role is more likely to be central. A central role for the VPLI neuron has yet to be determined.

Electron microscopy of serotonin-immunoreactive neuron branches and terminals in the locust central nervous system.

Using a pre-embedding peroxidase technique, we have investigated the ultrastructure of elements that react with antibodies to 5-hydroxytryptamine in the central nervous system of the locust. Reactive neuron profiles are widespread, and contain a variety of vesicle types: small lucent vesicles, some of which are stained only on their outer membranes while others are stained internally, and large dense granules that again differ in the staining properties of their cores. Some of the reactive profiles contain synaptic specializations, while others receive synaptic inputs from unlabelled elements. The heterogeneity of reactive profiles makes it impossible to define ultrastructural characteristics of putative serotonergic terminals that might be generally applicable. The differential reactivity both of small vesicles and of large dense granules may indicate functional differences within these categories.

Morphology, ultrastructure and synapse distribution of putative serotonergic salivary neurons in the locust.

Neurons innervating the locust salivary glands have been investigated using the electron microscope. We have examined the projections in the suboesophageal ganglion of two identified salivary neurons by differential double labelling with cobalt ions, followed by silver intensification. Numerous synaptic inputs occur on the arborizations of the salivary neurons, particularly on fine branches and on small spines arising from larger branches. Although a few instances occur of common input to both salivary neurons from a single presynaptic element, many of the appositions between branches seen in the light microscope do not represent functional connections. A few structures resembling presynaptic dense bodies have been observed in salivary neuron profiles, but with few synaptic vesicles. Large dense granules are present in some labelled profiles, but not in the vicinity of synapse-like membrane specializations. We have also examined unidentified neuron profiles within the acini of the salivary glands, which contain large dense granules and small vesicles. There is good evidence that these unidentified terminals correspond to the suboesophageal salivary neurons. The central arborizations of the salivary neurons appear to serve largely as sites of synaptic input, whereas the peripheral terminals are likely sites of transmitter release. The results are considered in the context of the known immunoreactivity of the salivary neurons with antibodies to 5-hydroxytryptamine, and compared with analogous systems in other insect groups.

Methods for imaging labeled neurons together with neuropil features in Drosophila.

We describe staining protocols for serial semithin sections of Drosophila central ganglia that allow visualization of gene expression in particular neurons with counterstaining to display the ganglion architecture. Green fluorescent protein (GFP), expressed in a subset of sensory neurons from a selected enhancer trap line, is visualized by conventional immunohistochemistry with a peroxidase-linked antibody, and neural architecture is revealed by reduced silver staining. This makes visible in histological sections the same GFP-labeled cells seen with confocal microscopy, but with the especial advantage that neuropil structures are also revealed at the level of individual cells and neuron processes. Not only does this allow the physical relationships among intracellularly labeled neurons to be determined by reference to specific features in the neuropil but it also enables a function to be ascribed provisionally to particular regions of neuropil. These methods have particular utility for mapping morphological information on specific neurons in the context of central nervous system architecture, both in adult Drosophila and during development.

Immunohistochemical localization of choline acetyltransferase in the central nervous system of the locust.

Immunohistochemistry of the locust central nervous system with antibody to choline acetyltransferase (ChAT) purified from the same species shows: first, there are relatively few immunoreactive cell bodies in the CNS; and second, sensory neuropiles, such as the ventral association centre and the ventral VAC (vVAC), the anterior ring tract, the tritocerebrum and the antennal lobe, are immunoreactive. That ChAT is contained in sensory neurones is suggested by immunoreactivity found in peripheral neurone cell bodies. These results indicate that acetylcholine serves primarily as a sensory transmitter in the locust.

Some insect sensory neurones contain 5-hydroxytryptamine.

Immunocytochemistry of the locust central nervous system shows that most segmental nerves, in particular those of the legs, contain afferent fibres that react with antibody to 5-hydroxytryptamine (5-HT). Adsorption controls indicate that the antigen is 5-HT or a closely related compound. This is supported by the finding of significant amounts of 5-HT in leg nerves using reverse phase high-performance liquid chromatography (HPLC) and electrochemical detection. On the other hand 5-HT was not detectable in locust antennal and cercal nerves with either immunocytochemistry or with HPLC. These results strongly support that some populations of sensory neurones in the locust contain 5-HT.

Confocal quality imaging of afferent neurons from semi-thin sections of Drosophila ganglia.

The aim of this study was to develop protocols for computer imaging of the thoraco-abdominal ganglion of Drosophila from serial semi-thin sections, in which specific neurons were stained and related to neuropilar structures. The central projections of a subset of transgenically labelled sensory neurons were revealed by immunohistochemistry, while Nomarski optics were used to show motor neuron targets in the neuropil. Digital photomicrographs of each section were aligned and the resultant image stacks rendered into three-dimensional (3D) images that can be rotated in real time. The result is a detailed, in-depth visualization of labelled neurons at a resolution comparable with that in confocal reconstructions, which also allows investigation of their relationships with other components of the neuropil.

Celloidin-wax sandwich microtomy: a novel and rapid method for producing serial semithin sections.

A method is described that allows rapid and reliable serial sectioning down to thicknesses of 1 micron. The tissue is first embedded in celloidin and then in wax and trimmed so that the block is sandwiched between two layers of wax. This combines the virtues of both media. The celloidin gives greater support to tissue than wax and enables the cutting of semithin sections. The wax allows ribbons of serial sections to be produced as in conventional wax microtomy. This makes it easy to produce serial semithin sections as a matter of routine.

Innervation of dipteran eclosion muscles: ultrastructure, immunohistochemistry, physiology and death.

The thoracic eclosion muscles of flies die by cytotoxic attack under neural control. We have investigated the innervation, ultrastructure and immunohistochemistry of the ventral eclosion muscle of Glossina. Two neurons located in the thoracic ganglion innervate this muscle. One of these is immunoreactive for serotonin and does not provide motor innervation. It appears to terminate near the attachment of an immunocyte involved in the dismantling of the muscle. The neuromuscular junction has features that distinguish it from any other chemical junction. A narrow, 3 nm gap separates pre- and post-synaptic membranes and this apparently acts to limit diffusion into and out of the junction. The immunocyte may use neuromuscular innervation as a path-finder to all muscle fibres and may even receive direct input from this source. Neuromuscular transmission is probably chemical as decreasing temperature results in decreasing amplitude of the (graded) muscle potential.

Spatial distribution of synapses onto thoracic motor neurones in locusts.

Aggregates of synaptic vesicles, stained black by the zinc iodideosmium procedure, can be visualised with the light microscope in 1 mum plastic sections. This allows the main branches of a neurone to be reconstructed relatively rapidly and the associated vesicle aggregates to be plotted. By resectioning, the identity of the vesicle aggregates has been confirmed with the electron microscope. Two flight motor neurones in the mesothoracic ganglion of the locust have been examined. One is identified as a dorsal longitudinal muscle motor neurone (muscle 112) and the other is probably a subalar neurone (muscle 99). Both have a large density of vesicle aggregates on the neuropilar segment, the widest part of the main neuronal axis, but few on the neurite within 250 mum of the cell body. The larger branches arising from the neuropilar segment tend to have a lower density of aggregates than fine branches, which suggests that synapses to the branches may occur mainly on the distal twigs. These results are an important preliminary step in determining the integrative functions of such neurons and have immediate implications in the interpretation of microelectrode recordings.