Pattern of expression of engrailed in relation to gamma-aminobutyric acid immunoreactivity in the central nervous system of the adult grasshopper.

Engrailed (En) protein expression in neurons of the mesothoracic and metathoracic ganglia of the adult grasshopper, Schistocerca americana, was examined by immunohistochemistry. Each neuromere had a dorsally located cluster of En-positive neurons within the dorsal unpaired median (DUM) group, comprising one cluster in the mesothoracic ganglion (T2) and four clusters in the metathoracic ganglion, one for each component neuromere (T3, A1, A2, A3). Ventrally, En-positive neurons occurred in the posterior one-third of each neuromere. In T2 and T3, three ventral groups of neurons were labeled bilaterally. In the abdominal neuromeres, many fewer ventral neurons were En-positive. These also were bilaterally symmetrical, but did not occur in patterns that allowed assignment of homology with the T2 and T3 groups. Altogether, En-positive neurons comprised roughly 10% of the ganglionic populations. In the bilateral groups, as in the DUM groups, En expression was restricted to interneurons, consistent with the suggestion that En expression contributes to some aspect of interneuronal phenotype. En-positive neurons in the DUM groups also expressed gamma-aminobutyric acid (GABA) immunoreactivity. Further study showed that all neurons in one En-positive bilateral group and some neurons in another bilateral group were GABA immunoreactive, but that neurons in a third bilateral group were En-positive only. Additionally, several discrete clusters of neurons were GABA-immunoreactive but En-negative. A provisional morphological scheme is presented, which relates the several neuronal clusters to their likely neuroblasts of origin, as a basis for further research into the composition of neuronal lineages.

Engrailed negatively regulates the expression of cell adhesion molecules connectin and neuroglian in embryonic Drosophila nervous system.

Engrailed is expressed in subsets of interneurons that do not express Connectin or appreciable Neuroglian, whereas other neurons that are Engrailed negative strongly express these adhesion molecules. Connectin and Neuroglian expression are virtually eliminated in interneurons when engrailed expression is driven ubiquitously in neurons, and greatly increased when engrailed genes are lacking in mutant embryos. The data suggest that Engrailed is normally a negative regulator of Connectin and neuroglian. These are the first two "effector" genes identified in the nervous system of Drosophila as regulatory targets for Engrailed. We argue that differential Engrailed expression is crucial in determining the pattern of expression of cell adhesion molecules and thus constitutes an important determinant of neuronal shape and perhaps connectivity.

Engrailed protein is expressed in interneurons but not motor neurons of the dorsal unpaired median group in the adult grasshopper.

We report that the homeodomain protein Engrailed (En) is differentially expressed by neuronal type. Expression was examined within identified midline neurons in T3, A1, and A2 neuromeres of the adult grasshopper by using immunohistochemistry. All save a few neurons in the adult dorsal unpaired median (DUM) group arise embryonically from a single precursor, the median neuroblast. DUM neurons are efferent neurons, local interneurons, or intersegmental interneurons, recognizable as such by their distinct morphologies and neurotransmitter phenotypes. We show that interneurons are En-positive, whereas efferents are En-negative. In the T3 DUM group, the 70 or so interneurons contained cytoplasmic immunoreactivity for gamma-aminobutyric acid (GABA) and glutamate decarboxylase. In double-labeling experiments, all GABA-immunoreactive neurons were also En-positive, and all En-positive neurons contained GABA immunoreactivity. In complementary experiments, the 20 or so efferents in the T3 DUM group, which are octopaminergic, were selectively labeled with a histological marker and then processed to reveal En immunoreactivity. No efferents in the group were En-positive. The abdominal DUM groups contain fewer neurons, but the same dichotomy of labeling was found. The En pattern is established during embryogenesis, with the type-specific pattern apparent by stage 90% of development, the earliest stage examined here. The differential expression of En in the embryo and its continued expression in the adult nervous system suggest a role in the development and maintenance of neuronal phenotype. Morphological differences between efferents and interneurons are discussed in light of a hypothesis that En mediates differential expression of cell adhesion or cell-affinity molecules.

Neurons of the median neuroblast lineage of the grasshopper: a population study of the efferent DUM neurons.

A group of lineally related neurons in the grasshopper was studied to determine the number of efferent neurons in the group and their morphological types. The neurons arise from the median neuroblast of the third thoracic neuromere and comprise what is commonly known as the DUM or dorsal unpaired median group. Of some 92 neurons in the group, about 20 are efferent neurons, the remainder being local or intersegmental interneurons. As part of our continuing developmental studies, we wished to identify the efferent neurons within the lineage and to determine their number. Ten efferent DUM neurons had been described in earlier studies, where neurons were stained individually through microelectrodes. The remaining unidentified neurons might be novel types, multiples of known types, or both, possibilities that would not be readily distinguished through further staining of neurons individually. Rather, we used methods of retrograde staining and axon tracing that allowed us to examine the entire group of efferent DUM neurons. Nineteen efferent neurons were identified, comprising two DUM1s, five DUM3s, six DUM3,4s, three DUM3,4,5s, and three DUM5s; neurons were named according to the lateral nerves containing their axons. The efferent neurons were further divided by type according to the distribution of axonal branches in lateral nerves, the course of the primary neurite within the deep or superficial DUM tract, and the diameter of the cell body.

Development of segment specificity in identified lineages of the grasshopper CNS.

The purpose of this study was to determine the factors underlying differences in population size and composition between segmentally homologous neuronal lineages. The segmental median neuroblasts (MNBs) of grasshoppers are identified stem cells that each produce a midline group of neurons. We traced the embryonic development of the group in two disparate segments, counting MNB progeny and profiles of dying cells in fixed and stained preparations of staged embryos. In the metathoracic segment (T3), about 95 MNB progeny survive embryonic development, whereas in the next posterior segment, the first abdominal (A1), only about 60 survive. In T3, the MNB arises at 29% of embryogenesis and dies at 78%, whereas in A1 the MNB arises at 30% and dies at 73%. In T3, the number of MNB progeny initially increases at a steady rate, 10 cells being added per 5% of embryogenesis. Between 70% and 78% growth tapers off; although the T3 MNB continues to divide, cells die at the same time, specifically removing last-born progeny. By contrast, in A1 the MNB progeny increase in two phases, one from 30% to 45% and the other from 60% to 73%, again at the rate of 10 cells per 5%. Between the two phases, the number of A1 progeny is stable. The A1 MNB continues to divide, but cells die at the same time, specifically removing earlier-born progeny. The episodes of cell death in A1 and T3 coincide with embryonic molts, and thus may be hormonally triggered. Cell death is greater in A1 than T3, accounting for most of the difference in population size. The difference in MNB longevity makes a lesser contribution. The present data, together with corollary anatomical data (Thompson and Siegler, 1991), support the hypothesis that progeny fated to become certain neuronal types are selectively removed from the two MNB lineages: intersegmental interneurons from T3 and efferent neurons and local interneurons from A1.

Motor neurons supplying hindwing muscles of a grasshopper: topography and distribution into anatomical groups.

The motor neurons supplying the dorsoventral wing muscles in the metathorax of the grasshopper Schistocerca americana were stained by backfilling muscle-nerves with cobaltous chloride, which was then precipitated and intensified as silver sulfide. Stained motor neurons were examined in wholemounts and in sectioned ganglia. Two unifunctional muscles are innervated by a single motor neuron each, whereas four bifunctional muscles, involved in leg and wing movements, are innervated by two or three motor neurons each. Each of the motor neurons is in one of four identified anatomical groups. All members of a group have primary neurites that enter the ganglion core as part of an anatomically defined bundle and have cell bodies that lie near each other in the ganglion cortex (Siegler and Pousman, J Comp Neurol: 297:298, 1990). The morphology of the neuropilar branches of the motor neurons is correlated with the pairwise topographic arrangement of the target muscles rather than the muscles' functions. Thus, motor neurons that innervate antagonistic pairs of muscles may be more similar in morphology than motor neurons that innervate synergistic muscles. We find no evidence that particular main branches of the different motor neurons are uniquely associated with a particular motor function. Significant differences in the nature and distribution of synaptic inputs to motor neurons of different function would necessarily be expected as a basis for known differences in synaptic connectivity, but may not be apparent at the level of resolution available from light microscope examination.

Anatomy and physiology of spiking local and intersegmental interneurons in the median neuroblast lineage of the grasshopper.

The range of anatomical and physiological properties in the adult progeny of an identified neuroblast was investigated. Some 80-90 adult neurons constitute the dorsal unpaired median (DUM) group of the grasshopper metathoracic ganglion. Within the group are efferent, octopaminergic neurons with large cell bodies and overshooting action potentials. Our objective was to determine the properties of the neurons with small cell bodies that make up the majority of the clone, some 60-70 neurons, about which scant information was available. The small DUM neurons have cell body diameters of 10-20 microns and stain with antibodies to GABA (Thompson and Siegler, '89: Proc. Soc. Neurosci. 15:1296 (abstr.); Witten and Truman, '89: Proc. Soc. Neurosci. 15:365 (abstr.)). By employing intracellular electrophysiological and morphological techniques, we have established that the small DUM neurons are spiking interneurons, expressing passively conducted action potentials in the cell body. They fall into two basic classes: local interneurons with bilateral branches in the auditory neuropiles, and intersegmental interneurons with bilateral branches widespread in the methathoracic ganglion and axons traveling in both anterior connectives. The local interneurons typically respond to sound, whereas the intersegmental interneurons selectively respond to wind on the head or to generalized movements by the animal. Primary neurites of small and large DUM neurons enter the neuropil in a bundle, but the neurites of DUM interneurons are more posterior and have a separate trajectory from those of the efferent DUM neurons once in the ganglion core. A model is presented for the sequential development of efferent, local, and intersegmental DUM neurons from the median neuroblast.

Distribution of motor neurons into anatomical groups in the grasshopper metathoracic ganglion.

Motor neurons of the main muscles of the hind legs and the hind wings of the grasshopper are distributed into eight anatomical groups within each half of a bilaterally symmetrical segmental ganglion. A group contains 5 to 24 identified motor neurons, accounting for 164 (82 pairs) of the some 200 motor neurons within the population. The motor neurons within a given group may contribute axons to more than one of the lateral nerves, and conversely each lateral nerve contains axons arising from motor neurons in separate groups. Groups may include synergistic and antagonistic motor neurons as well as those that have unrelated functions. The motor neurons of a given muscle may occur together in a single group, or separately in two or more groups. The description of groups provides a way of classifying neurons to simplify and organize the large amount of data on the structure and function of individually identified neurons within the ganglion. The organization of the motor neurons into groups may reflect their developmental origin from individual neuroblasts, and the detailed information about the pattern of groups in the adult thus allows specific predictions to be made about the composition of neurons within neuronal lineages.

Motor neurons of grasshopper metathoracic ganglion occur in stereotypic anatomical groups.

Anatomical groups containing identified motor neurons of the main muscles of the legs and the wings are described in a segmental ganglion of the adult grasshopper. The groups occur reproducibly in ganglia of different individuals and are a simplifying and organizing feature of ganglionic morphology. The motor neurons within each group have cell bodies near each other in the cortex of the ganglion and primary neurites that enter the ganglionic core as a discrete bundle. The primary neurite bundles are distinctive in shape and position and have the same composition in every individual, despite variations in the positions of the cell bodies of the contributing motor neurons. The primary neurite bundle of a group is separate from those of other groups and separate from bundles of motor axons that exit or sensory axons that enter the ganglion. Each group of cell bodies in the cortex appears from light microscope examination to be held separately within a glial surround. Areas of glial cell cytoplasm may extend considerably beyond the boundaries of the neuronal cell bodies, to give shape and structural integrity to the cortex. Similarities between the morphology of the adult groups reported here and the descriptions by others of embryonic and larval nervous systems suggest to us that the motor neurons of each group are the progeny of a single neuroblast.

Receptive fields of motor neurons underlying local tactile reflexes in the locust.

The receptive fields of motor neurons to a hind leg were mapped by recording intracellularly from their cell bodies or from the muscle fibers they innervate while stimulating mechanoreceptors on the surface of that leg. Each motor neuron is affected by a specific array of receptors that make up its receptive field. Boundaries along the anteroposterior or dorsoventral axes of the leg divide the receptive fields into excitatory and inhibitory regions. Proximodistal boundaries may correspond to the articulations between parts of the leg. Motor neurons that innervate antagonistic muscles have complementary receptive fields, so that the region that is excitatory for one is inhibitory for the other. The receptive fields of the motor neurons overlap. Tactile stimulation therefore leads to a specific local reflex that involves the coordinated movement of the segments of a leg. Five local reflexes are described, each of which moves the leg away from the site of stimulation. Afferents from the external mechanoreceptors do not synapse directly on the motor neurons, but instead on spiking local interneurons, some of which then synapse directly on motor neurons. These local interneurons have smaller receptive fields delineated by the same boundaries, so that the receptive fields of the motor neurons can be constructed from appropriate combinations of them. It is suggested that receptive fields are organized as "functional maps" that are appropriate for particular behavioral responses rather than solely to preserve or refine spatial information.

The structure of locust nonspiking interneurones in relation to the anatomy of their segmental ganglion.

The morphology of eight nonspiking local interneurones in the metathoracic ganglion of the locust is described in relation to known tracts, commissures, regions of neuropile, and identified motor neurones. They are compared with the spiking local interneurones in the same ganglion. Each nonspiking local interneurone was injected intracellularly with cobalt, following characterization of its physiological effects on identified leg motor neurones. The shapes of the nonspiking interneurones are diverse, although all have processes restricted to one ganglion and lack an axon. Their cell bodies are distributed in the ventral and dorsal cortex of the ganglion. Interneurones with cell bodies in similar places have similar basic structures, with primary neurites in the same commissure or tract, and major branches in the same tracts. The fine branches of all the interneurones have the same texture throughout, and occur in the same lateral region of neuropile, dorsal to the prominent neurite of the fast extensor tibiae motor neurone. Some interneurones have branches that extend both to the midline and to the dorsal boundary of the neuropile, but none have branches in the ventral, medial neuropile. This distribution of branches corresponds with two known features of the physiology of these interneurones: they make what appear physiologically to be direct connections with motor neurones, and have branches in the same region of the neuropile as the motor neurones. They do not appear to receive direct inputs from hair afferents, and they have no branches in the ventral neuropile to which these afferents project.

Organization of receptive fields of spiking local interneurons in the locust with inputs from hair afferents.

The receptive fields of spiking local interneurons in the locust were defined by making intracellular recordings from them while stimulating mechanoreceptors on the surface of a hindleg. All the interneurons tested have their cell bodies near the ventral midline, in the so-called "midline" group. Those described here receive inputs only from external mechanoreceptors; others receive inputs from internal proprioceptors alone or from receptors of both kinds. The receptors on the surface of a hindleg that contribute to the receptive field of an interneuron may be clustered together in a discrete area or be distributed in separate regions that provide either excitation or inhibition. An interneuron may have a receptive field that is wholly excitatory or one with both excitatory and inhibitory regions. Excitatory but not inhibitory effects are mediated by direct connections between afferents and interneurons. The longitudinal boundaries of most receptive fields occur along one of the major axes of the leg. For example, hairs on the anterior half of a leg can be excitatory, while hairs on the posterior half are inhibitory; or those on the dorsal half are excitatory, and those on the ventral half are inhibitory. The proximal-distal boundaries of a receptive field often correspond to the articulations between the segments of a leg, although they may also occur within a segment of a leg where there are no other obvious anatomical discontinuities. The receptive fields of these interneurons often overlap, and an individual afferent from a hair excites more than one interneuron. In this way a particular region on the surface of a hindleg may be mapped onto as many as 12 interneurons. The size of a receptive field is not correlated with its position along the proximal-distal axis of the leg, but the smallest fields occur on the distal tibia or span the femorotibial joint.

The morphology of two groups of spiking local interneurons in the metathoracic ganglion of the locust.

Two bilaterally symmetrical groups of spiking local interneurons are described in a segmental ganglion of the locust. Interneurons in both groups are excited by specific sets of sensory receptors on one leg. The cell bodies of the anterior-lateral group lie amongst approximately 40 small cell bodies at the anterior of the ganglion, close to the lateral edge of an anterior connective. Interneurons in this group have primary neurites in Ventral Commissure I ( VCI ), and dorsoventral processes in the Oblique Tract, which divide the extensive neuropilar branches into distinct ventral and dorsal regions. Cell bodies of the midline group lie amongst a group of approximately 100 small cell bodies near the ventral midline. Interneurons in this group have primary neurites in Ventral Commissure II ( VCII ), and dorsoventral processes in the Perpendicular Tract, which divide the neuropilar branches into dorsal and ventral regions. The ventral branches of interneurons in both groups are numerous and of a uniform texture, whereas the dorsal branches are sparse and varicose. The ventral branches project to the same ventral areas of neuropil as the afferents from some hairs on a hind leg. The dorsal branches of a midline interneuron and the branches of a leg motor neuron that it affects project to the same dorsal area of neuropil. Some midline interneurons receive direct inputs from leg hair afferents and make direct connections with leg motor neurons.

The morphological diversity and receptive fields of spiking local interneurons in the locust metathoracic ganglion.

Twenty-one types of spiking local interneurons are described in a segmental ganglion of the locust. All have their cell bodies in a group at the ventral midline of the metathoracic ganglion. The interneurons are characterized by their shape as revealed by intracellular injection of dye, and by their physiology as revealed by intracellular recording. Each interneuron conforms to a basic plan, but the characteristic shape of each is derived from the elaboration of branches in some regions of the neuropil and by their absence in other regions. Some interneurons have ventral branches that extend over most of one-half of the metathoracic neuropil, whilst others have ventral branches restricted to a small region of neuropil. A few interneurons have dorsal branches that enter the first abdominal neuromere . Each type of interneuron is excited by a specific array of mechanoreceptors on the hind leg ipsilateral to its neuropilar branches. Some interneurons have a wide receptive field that encompasses most of the dorsal surfaces of the distal three parts of a leg, whilst others have a field limited to the spurs at the distal end of the tibia. The relationship between the shape of an interneuron and the size or orientation of its receptive field is discussed.

Spiking local interneurons as primary integrators of mechanosensory information in the locust.

A population of spiking local interneurons in the metathoracic ganglion of the locust is vigorously excited by particular sensory stimuli from the hindlegs and participates in local postural reflexes. We examined the inputs from singly innervated mechanoreceptors (hairs and campaniform sensilla) to these spiking local interneurons, to nonspiking local interneurons, and to motor neurons that are also elements of local reflex pathways. Recordings were made intracellularly from the interneurons and motor neurons and extracellularly from afferent fibers. The physiological evidence is consistent with the spiking local interneurons being excited by direct, chemically mediated synaptic inputs from the afferents. Each afferent spike is followed at a constant latency by an excitatory postsynaptic potential (EPSP) in a spiking local interneuron, even at instantaneous frequencies as high as 300 Hz. The estimated synaptic delay is 1.5 ms, similar to that measured at other presumed monosynaptic connections within the same ganglion. Cobalt stains of individual interneurons, and of the central projections of afferent fibers show that both branch within the same ventral region of neuropil. Afferents from several hairs and campaniform sensilla converge on an individual spiking local interneuron. One interneuron is shown to receive inputs from at least seven hairs and four campaniform sensilla, but these represent only a tiny fraction of the total number of such sensilla on a hindleg. Practical limitations to the number of sensilla that can be tested for each interneuron means that the degree of convergence is likely to be considerably underestimated. We found no evidence that nonspiking local interneurons or motor neurons receive direct inputs from the afferents tested. Neurons of both types are, however, affected by stimulation of individual hairs, and the resulting pattern of postsynaptic potentials (PSPs) is similar to the pattern of spikes evoked in the spiking local interneurons. We infer from the evidence presented here and elsewhere (10, 11) that the spiking local interneurons are involved in at least two types of pathways for local interactions: 1) sensory neuron-spiking local interneuron-motor neuron, and 2) sensory neuron-spiking local interneuron-nonspiking local interneuron-motor neuron. We conclude that the spiking local interneurons are major elements in the primary integration of inputs from external receptors on the hindlegs.

Spiking local interneurons mediate local reflexes.

A local spiking interneuron in the locust is excited by particular sensory stimulation of a hind leg and forms an inhibitory connection with one hind leg motor neuron. Its behavioral effect is to mediate a local postural reflex. This interneuron is one of a population of interneurons with similar morphology and physiology that participate in the same local circuits as the better known nonspiking local interneurons.

Octopamine mediated relaxation of maintained and catch tension in locust skeletal muscle.

1. The modulatory actions of an identified octopaminergic neurone (DUMETi) that projects to the extensor-tibiae muscle of the locust hind leg depend upon the frequency of stimulation of the slow motoneurone (SETi) to this muscle. 2. At low frequencies of SETi stimulation (1Hz and below) the predominant modulatory effects are increases in the amplitude and relaxation rate of twitch tension. At higher frequencies, where twitches summate but tetanus is incomplete (up to 20 Hz), the reduction of maintained tension becomes considerably more important. 3. Both octopamine application and DUMETi stimulation reduce the amount of catch tension displayed by the extensor muscle when SETi is fired in a variety of different stimulus patterns. The extensor-tibiae muscle is itself 'pattern sensitive' since is shows a 'positive spacing effect' when SETi is stimulated at an average frequency of 1 Hz. 4. It is suggested that a primary function of DUMETi is to change the response of the muscle from one that favours maintenance of posture to one that favours rapid changes in joint position or force, such as might occur during locomotion.