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In the early embryonic grasshopper, two pairs of sibling cells near the apex of the antenna pioneer its dorsal and ventral nerve tracts to the brain. En route, the growth cones of these pioneers contact a so-called base pioneer associated with each tract and which acts as a guidepost cell. Both apical and basal pioneers express stereotypic molecular labels allowing them to be uniquely identified. Although their developmental origins are largely understood, the fates of the respective pioneers remain unclear. We therefore employed the established cell death markers acridine orange and TUNEL to determine whether the apical and basal pioneers undergo apoptosis during embryogenesis. Our data reveal that the apical pioneers maintain a consistent molecular profile from their birth up to mid-embryogenesis, at which point the initial antennal nerve tracts to the brain have been established. Shortly after this the apical pioneers undergo apoptosis. Death occurs at a developmental stage similar to that reported elsewhere for pioneers in a leg – an homologous appendage. Base pioneers, by contrast, progressively change their molecular profile and can no longer be unequivocally identified after mid-embryogenesis. At no stage up to then do they exhibit death labels. If they persist, the base pioneers must be assumed to adopt a new role in the developing antennal nervous system.

Neuropils in the cerebral midline of Panarthropoda exhibit a wide spectrum of neuroarchitectures – from rudimentary to highly elaborated – and which at first sight defy a unifying neuroarchitectural principle. Developmental approaches have shown that in model arthropods such as insects, conserved cellular and molecular mechanisms first establish a simple axon scaffold in the brain. However, to be adapted for adult life, this immature ground plan is transformed by a developmental process – known in the grasshopper as “fascicle switching” – in which subsets of neurons systematically redirect their growth cones at stereotypic locations across the brain midline. A topographic system of choice points along the transverse brain axis where axons decussate features in all panarthropods studied even though different modes of neurogenesis and varying degrees of neuropilar elaboration are involved. This suggests that the molecular mechanisms regulating choice point selection may be conserved. In combination with recent cladistic interpretations of arthropod phylogeny based on nuclear protein-coding sequences the data argue for this topographic decussation as having evolved early and being a conserved feature of the Panarthropoda. Differences in elaboration likely reflect both the extent to which neuropilar reorganization has progressed during development and the lifestyle of the individual organism.

The neurons of the insect brain derive from neuroblasts which delaminate from the neuroectoderm at stereotypic locations during early embryogenesis. In both grasshopper and Drosophila, each developing neuroblast acquires an intrinsic capacity for neuronal proliferation in a cell autonomous manner and generates a specific lineage of neural progeny which is nearly invariant and unique. Maps revealing numbers and distributions of brain neuroblasts now exist for various species, and in both grasshopper and Drosophila four putatively homologous neuroblasts have been identified whose progeny direct axons to the protocerebral bridge and then to the central body via an equivalent set of tracts. Lineage analysis in the grasshopper nervous system reveals that the progeny of a neuroblast maintain their topological position within the lineage throughout embryogenesis. We have taken advantage of this to study the pioneering of the so-called w, x, y, z tracts, to show how fascicle switching generates central body neuroarchitecture, and to evaluate the roles of so-called intermediate progenitors as well as programmed cell death in shaping lineage structure. The novel form of neurogenesis involving intermediate progenitors has been demonstrated in grasshopper, Drosophila and mammalian cortical development and may represent a general strategy for increasing brain size and complexity. An analysis of gap junctional communication involving serotonergic cells reveals an intrinsic cellular organization which may relate to the presence of such transient progenitors in central complex lineages.

We have investigated the ontogenetic basis of locustatachykinin-like expression in a group of cells located in the pars intercerebralis of the grasshopper midbrain. These cells project fibers to the protocerebral bridge and the central body via a characteristic set of fiber bundles called the w, x, y, z tracts. Lineage analyses associate the immunoreactive cells with one of four neuroblasts (termed W, X, Y, Z) in each protocerebral hemisphere of the early embryo. Locustatachykinin is a ubiquitous myotropic peptide among the insects and its expression in the pars intercerebralis begins at approximately 60-65% of embryogenesis. This coincides with the appearance of the columnar neuroarchitecture characteristic of the central body. The number of immunoreactive cells in a given lineage is initially small, increases significantly in later embryogenesis, and attains the adult situation (about 7% of a lineage) in the first larval instar after hatching. Although each neuroblast generates progeny displaying a spectrum of cell body sizes, there is a clear morphological gradient, which reflects birth order within the lineage. Locustatachykinin expressing cells are located stereotypically at or near the tip of their lineage, which an age profile reveals places them amongst the first born progeny of their respective neuroblasts. Although these neuroblasts begin to generate progeny at approximately 25-27% of embryogenesis, their daughter cells remain quiescent with respect to locustatachykinin expression for over 30% of embryogenesis.

The grasshopper antenna is an articulated appendage associated with the deutocerebral segment of the head. In the early embryo, the meristal annuli of the antenna represent segment borders and are also the site of differentiation of pioneer cells which found the dorsal and ventral peripheral nerve tracts to the brain. We report here on another set of cells which appear earlier than the pioneers during development and are later found arrayed along these tracts at the border of epithelium and lumen. These so-called nerve tract associated cells differ morphologically from pioneers in that they are bipolar, have shorter processes, and are not segmentally organized in the antenna. Nerve tract associated cells do not express horseradish peroxidase and so are not classical neurons. They do not express antigens such as repo and annulin which are associated with glia cells in the nervous system. Nerve tract associated cells do, however, express the mesodermal/mesectodermal cell surface marker Mes-3 and putatively derive from the antennal coelom and then migrate to the epithelium/lumen border. Intracellular recordings show that such nerve tract associated cells have resting potentials similar to those of pioneer cells and can be dye coupled to the pioneers. Similar cell types are present in the maxilla, a serially homologous appendage on the head. The nerve tract associated cells are organized into a cellular scaffold which we speculate may be relevant to the navigation of pioneer and sensory axons in the early embryonic antennal nervous system.

The central complex is a brain specific structure involved in multimodal information processing and in coordinating motor behaviour. It possesses a highly organized neuroarchitecture, which is remarkably conserved across insect species. A prominent feature of this neuroarchitecture is the stereotypic projection of axons from clusters of neurons in the pars intercerebralis to the central body via the so-called w, x, y and z tracts. Despite extensive analyses of this neuroarchitecture in adults, little is known about its ontogeny in any insect. In this paper we use the expression pattern of the segment polarity gene engrailed to identify those neuroblasts belonging to the protocerebrum of the early embryonic brain of the grasshopper Schistocerca gregaria. We present a new map for this brain region in which the 95 protocerebral neuroblasts in each hemisphere are organized into seven rows, as they are in the neuromeres of the ventral nerve cord. We then identify a subset of four of these neuroblasts as being the progenitor cells for four clusters of neurons, some of whose axons we show project via discrete tracts (w, x, y, z) into the central complex. These tracts begin to form prior to 39% of embryogenesis. We show further, that the cells from one of these clusters (the Z cluster) are organized according to age, and direct axons topologically according to age into the appropriate z tract. This pattern is repeated in each of the other three clusters, thus establishing a clonally based modular system of fibre tracts consistent with the model proposed for this brain region in the adult.

In this paper, we propose an ontogeny for previously identified cells from the median domain in the midline of the embryonic brain of the grasshopper Schistocerca gregaria. The so-called lateral cells (LCs) are characteristically located laterally within the median domain at its border with the protocerebral hemispheres. The LC occurs singly and can be identified in the early embryo on the basis of their expression of the cell surface lipocalin Lazarillo. Using immunocytochemical, dye injection, electron microscopical and histological methods, we show that these LC are neurons and derive as postmitotic cells directly from the epithelium of the median domain. Further, they and the other identified cells of the median domain such as the protocerebral commissure pioneers (PCP), co-express the Mes-3 antigen, consistent with a derivation from the mesectodermal germ layer of the embryo. Subsequent to axogenesis, electron microscopy reveals that these Mes-3-expressing LC fasciculate with the co-expressing PCPs within the developing protocerebral commissure. We present a model for the origin of all these cells based on histological data and bromodeoxyuridine incorporation. The model suggests a delamination of cells from the mesectoderm followed by a migration to their ultimate sites within the median domain.

The primary axon scaffold of the insect brain is established early in embryogenesis and comprises a preoral protocerebral commissure, a postoral tritocerebral commissure and longitudinal fiber pathways linking the two. In both grasshopper and fly its form is approximately orthogonal and is centered around the stomodeum. We show how pioneer fibers from the protocerebrum and tritocerebrum cross the brain midline directly via their respective commissures. The deutocerebrum, however, lacks its own commissure and we describe how deutocerebral pioneers circumnavigate the gut to cross the midline either via the protocerebral commissure or the tritocerebral commissure. In contrast to all other commissures of the central nervous system, the protocerebral commissure persists, albeit in reduced form, in the commissureless mutation in the fly. Besides the com gene, a further, as yet unidentified, mechanism must regulate this commissure. The formation of the tritocerebral commissure involves labial, a member of the Hox gene group. Genetic rescue experiments in labial mutants reveal that the formation of this commissure can be rescued by all other Hox genes except Abdominal-B. However, only in the labial and Deformed null mutants are the commissures associated with the respective expression domains (tritocerebral, mandibular, respectively) absent. This suggests that the molecular mechanisms regulating postoral brain commissure formation are distinct from those in the neuromeres of the ventral nerve cord.

Our analysis of head segmentation in the locust embryo reveals that the labrum is not apical as often interpreted but constitutes the topologically fused appendicular pair of appendages of the third head metamere. Using molecular, immunocytochemical and retrograde axonal staining methods we show that this metamere, the intercalary segment, is innervated by the third brain neuromere-the tritocerebrum. Evidence for the appendicular nature of the labrum is firstly, the presence of an engrailed stripe within its posterior epithelium as is typical of all appendages in the early embryo. Secondly, the labrum is innervated by a segmental nerve originating from the third brain neuromere (the tritocerebrum). Immunocytochemical staining with Lazarillo and horseradish peroxidase antibodies reveal that sensory neurons on the labrum contribute to the segmental (tritocerebral) nerve via the labral nerve in the same way as for the appendages immediately anterior (antenna) and posterior (mandible) on the head. All but one of the adult and embryonic motoneurons innervating the muscles of the labrum have their cell bodies and dendrites located completely within the tritocerebral neuromere and putatively derive from engrailed expressing tritocerebral neuroblasts. Molecular evidence (repo) suggests the labrum is not only appendicular but also articulated, comprising two jointed elements homologous to the coxa and trochanter of the leg.

The expression pattern of the segment polarity gene engrailed was studied at the single cell level in the protocerebrum of the early embryonic brain of the grasshopper Schistocerca gregaria, the neuromere containing the secondary headspot cells. The engrailed protein is first expressed in the protocerebrum at about 22% of embryogenesis by a group of identified neuroblasts bordering the antennal lobe. The number of immunoreactive neuroblasts increases up to 26% of embryogenesis and then rapidly declines so that by 30% only the three most posterior remain immunoreactive. These three neuroblasts become incorporated into the developing antennal lobe of the deutocerebrum. Subsequently, there is a progressive re-expression of the engrailed protein in the protocerebrum by the so-called six secondary headspot cells. These are the first born sibling progeny of three identified protocerebral neuroblasts which themselves expressed the engrailed protein prior to generating their lineages, and so represents a reacquisition of engrailed expression within identified clones. The secondary headspot cells are neurons which direct axonal processes into the developing optic tract and so contribute to the primary axon scaffold of the brain. From our analysis of their ontogeny, we conclude that the secondary headspot cells do not represent a segmental border in the brain.

The expression pattern of the engrailed protein was studied in neuroblasts which delaminate at the border of the protocerebrum and antennal lobe of the deutocerebrum in the early embryonic brain of the grasshopper. The antennal lobe is a complex structure comprising both glomerular and non-glomerular components, a cellular organization which distinguishes it from the striate-like neuropil comprising the remainder of the deutocerebrum. Early in embryogenesis engrailed expression in the protocerebrum is restricted to a compact block of neuroblasts located at its interface with the antennal lobe. Subsequently engrailed expression in these cells disappears in a stepwise manner from anterior to posterior so that by 37% of embryogenesis only a single row of three engrailed positive neuroblasts and their progeny remains. Contemporaneously engrailed expression reappears in a group of more anterior progeny deriving from neuroblasts which are no longer immunoreactive. The three remaining engrailed positive neuroblasts then become separated from their non-immunoreactive neighbours by an invagination of the perineurium called the lateral cleft and come to lie completely within the developing antennal lobe. These cells then direct columns of immunoreactive progeny centrifugally towards the centre of the lobe. Such a protocerebral contribution to the antennal lobe suggests that the evolution and ontogeny of this brain region need to be reconsidered.

The groundplan of longitudinal fiber tracts and commissures comprising the thoracic CNS is remarkably similar throughout a diverse group of holo- and hemimetabolous insects. Among tympanate insects, the axons of tympanal (auditory) receptors project to equivalent tracts and association areas in the central nervous system, even when the ears are located on different parts of the body. In segments without tympanal receptors, mechanoreceptive pleural chordotonal afferents are found to project to these neuropilar areas. Consistent with this, the tympanal receptors of the grasshopper have been shown to be serially homologous with those of the pleural chordotonal organs arrayed along the body wall. In atympanate insects, mechanosensitive receptors project to the equivalent neuropilar areas, via the same routes, as do tympanal receptors in insects with ears. Again, the auditory receptors of tympanate insects such as the grasshopper have been shown to be homologous with the chordotonal receptors of atympanate insects such as Drosophila. Identified first-order interneurons which have their dendrites in areas of neuropil occupied by tympanal afferents have been shown to be serially homologous along the CNS of the grasshopper. Putative homologs to these interneurons have also been identified in a range of other tympanate and atympanate insects. It is proposed that the tympanal receptors are a special part of a general chordotonal system in which homologous peripheral receptors project to sets of homologous interneurons.

The terminal ganglion of the CNS of the locust contains a bilateral pair of Giant Interneurone 1's (GIN 1's). GIN 1 has a lateral cell body, an axon which leaves the ganglion anteriorly via the contralateral connective, and an extensively branched, posteriorly directed dendrite. The response of a GIN 1 to wind stimuli directed at the cerci, or electrical stimulation of the cercal nerve, consists of a depolarization supporting spikes. Wind-sensitive filiform afferents on the cerci send axons via the cercal nerve into the terminal ganglion where they evoke short-latency EPSPs in GIN 1. The input from a number of filiform hairs on a cercus converges onto GIN 1. The response of GIN 1 to a single electrical stimulus applied to the cercal nerve consists of: an initial depolarization and spike, evoked by input from filiform afferents; and a delayed, sometimes suprathreshold, depolarization which is evoked by input from an interneurone. Simultaneous intracellular recordings and stainings reveal this delayed input to GIN 1 originates from a spiking local interneurone. The magnitude of the delayed depolarization in GIN 1 depends on the degree to which the local neurone is hyperpolarized by its own presynaptic input. The response of GIN 1 to cercal stimulation is therefore determined by parallel inputs, both of which are activated by cercal hairs.

An identified interneurone (SA3) of the suboesophageal ganglion has an axon in both circumoesophageal connectives ascending to the brain. This is a novel morphology for a cell in the auditory system of the locust. The neurone is also novel for its physiological responses to sound in that it displays side-dependent response decrement (habituation). Responses to a tone directed at one ear decrement without affecting responses from the other ear. The responses on the decremented side recover when the opposite ear is stimulated. The decrement is the result of a diminishing amplitude of the compound excitatory post-synaptic potential but no inhibition is seen. Response decrement does not occur if the stimulus frequency is varied.

Three qualities of sound—the directional, the temporal and the spectral—are important for intraspecific communication in Orthoptera. The neural mechanisms employed by identified interneurones for encoding these sound qualities are illustrated by examples of physiological processes found at different levels of the CNS. Discussed are: (1) the creation of directional information by local interneurones in the thorax, and the use of time-intensity trading in sound location; (2) mechanisms for encoding the temporal parameters of sound by interneurones ascending to the brain; (3) frequency-dependent neural filtering of auditory information by local interneurones.

Two types of auditory interneurone which ascend from the prothoracic ganglion to the brain in the cricket Gryllus bimaculatus (De Geer) are described. Intracellular recordings were made from the axons of the neurones in the brain under closed-field stimulus conditions and the recorded cells then stained with either cobalt or Lucifer Yellow. Both neurone types—the Plurisegmental ascending low frequency neurone 1 (PALF1), and the Plurisegmental ascending high frequency neurone 1 (PAHF1)—show response characteristics which would make them well suited to encoding the conspecific calling and courtship songs respectively. Further, the projection areas of both neurone types in the brain overlap those of previously identified intraganglionic interneurones, particularly in the anterior-ventral protocerebrum, and it is suggested that an auditory neuropile may exist in this region.