Fascicle switching generates a chiasmal neuroarchitecture in the embryonic central body of the grasshopper Schistocerca gregaria.

The central body is a prominent neuropilar structure in the midbrain of the grasshopper and is characterized by a fan-shaped array of fiber columns, which are part of a chiasmal system linking anterior and posterior commissures. These columns are established during embryogenesis and comprise axons from cell clusters in the pars intercerebralis, which project to the central body via the so-called w, x, y, z tracts. Up to mid-embryogenesis the primary axon scaffold in both the brain and ventral nerve cord comprises a simple orthogonal arrangement of commissural and longitudinal fiber pathways. No chiasmata are present and this pattern is maintained during subsequent development of the ventral nerve cord. In the midbrain, individual axons entering the commissural system from each of the w, x, y, z tracts after mid-embryogenesis (55%) are seen to systematically de-fasciculate from an anterior commissure and re-fasciculate with another more posterior commissure en route across the midline, a feature we call "fascicle switching". Since the w, x, y, z tracts are bilaterally symmetrical, fascicle switching generates chiasmata at stereotypic locations across the midbrain. Choice points for leaving and entering fascicles mark the anterior and posterior positions of each future column. As the midbrain neuropil expands, the anterior and posterior groups of commissures condense, so that the chiasmata spanning the widening gap between them become progressively more orthogonally oriented. A columnar neuroarchitecture resembling that of the adult central body is already apparent at 70% of embryogenesis.

An ontogenetic analysis of locustatachykinin-like expression in the central complex of the grasshopper Schistocerca gregaria.

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.

Evidence that the primary brain commissure is pioneered by neurons with a peripheral-like ontogeny in the grasshopper Schistocerca gregaria.

The commissures represent a major neuroarchitectural feature of the central nervous system of insects and vertebrates alike. The adult brain of the grasshopper comprises 72 such commissures, the first of which is established in the protocerebral midbrain by three sets of pioneer cells at around 30% of embryogenesis. These pioneers have been individually identified via cellular, molecular and intracellular dye injection techniques. Their ontogenies, however, remain unclear. The progenitor cells of the protocerebral midbrain are shown via Annulin immunocytochemistry to be compartmentalized, belonging either to the protocerebral hemispheres or the so-called median domain. Serial reconstructions based on bromodeoxyuridine incorporation confirm that their lineages do not intermingle. Dye injection into progenitor cells and progeny confirms this compartmentalization, and reveals that none of the pioneers are associated with a lineage of cells deriving from a protocerebral neuroblast or midline precursor. Immunocytochemical data as well as dye injection into identified pioneers over several developmental stages indicate that they differentiate directly from epithelial cells, but not from classical progenitor cells. That the commissural pioneers of the protocerebrum represent modified epithelial cells involves a different ontogeny to that described for pioneers in the ventral nerve cord, but parallels that of pioneer neurons of the peripheral nervous system.

Building the central complex of the grasshopper Schistocerca gregaria: axons pioneering the w, x, y, z tracts project onto the primary commissural fascicle of the brain.

The central complex is a major neuropilar structure in the insect brain whose distinctive, modular, neuroarchitecture in the grasshopper is exemplified by a bilateral set of four fibre bundles called the w, x, y and z tracts. These columns represent the stereotypic projection of axons from the pars intercerebralis into commissures of the central complex. Each column is established separately during early embryogenesis in a clonal manner by the progeny of a subset of four identified protocerebral neuroblasts. We report here that dye injected into identified pioneers of the primary brain commissure between 31 and 37% of embryogenesis couples to cells in the pars intercerebralis which we identify as progeny of the W, X, Y, or Z neuroblasts. These progeny are the oldest within each lineage, and also putatively the first to project an axon into the protocerebral commissure. The axons of pioneers from each tract do not fasciculate with one other prior to entry into the commissure, thereby prefiguring the modular w, x, y, z columns of the adult central complex. Within the commissure, pioneer axons from columnar tracts fasciculate with the growth cones of identified pioneers of the existing primary fascicle and do not pioneer a separate fascicle. The results suggest that neurons pioneering a columnar neuroarchitecture within the embryonic central complex utilize the existing primary commissural scaffold to navigate the brain midline.

Embryonic development of a peripheral nervous system: nerve tract associated cells and pioneer neurons in the antenna of the grasshopper Schistocerca gregaria.

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.

Embryonic development of the sensory innervation of the antenna of the grasshopper Schistocerca gregaria.

The establishment of the sensory nervous system of the antenna of the grasshopper Schistocerca gregaria was examined using immunocytochemical methods and in the light of the appendicular and articulated nature of this structure. The former is demonstrated first by the expression pattern of the segment polarity gene engrailed in the head neuromere innervating the antenna, the deutocerebrum. Engrailed expression is present in identified deutocerebral neuroblasts and, as elsewhere in the body, is continuous with cells of the posterior epithelium of the associated appendage, in this case the antenna. Second, early expression of the glial homeobox gene reversed polarity (repo) in the antenna is by a stereotypic pair of cells at the antenna base, a pattern we show is repeated metamerically for each thoracic appendage of the embryo. Subsequently, three regions of Repo expression (A1, A2, A3) are seen within the antenna, and may represent a preliminary form of articulation. Bromodeoxyuridine incorporation reveals that these regions are sites of intense cell differentiation. Neuron-specific horseradish peroxidase and Lazarillo expression confirm that the pioneers of the ventral and dorsal tracts of the antennal sensory nervous system are amongst these differentiating cells. Sets of pioneers appear simultaneously in several bands and project confluent axons towards the antennal base. We conclude that the sensory nervous system of the antenna is not pioneered from the tip of the antenna alone, but in a stepwise manner by cells from several zones. The early sensory nervous systems of antenna, maxilla and leg therefore follow a similar developmental program consistent with their serially homologous nature.

Embryonic development of the sensory innervation of the clypeo-labral complex: further support for serially homologous appendages in the locust.

The clypeo-labrum, or upper lip, of insects is intimately involved in feeding behavior and is accordingly endowed with a rich sensory apparatus. In the present study we map the temporal appearance of all major clusters of sensory cells on this structure in the locust during the first half of embryogenesis. The identities of these sensory cell clusters were defined according to the origin of the branching point of their axons from the labral sensory nerve as seen at mid-embryogenesis. The first sensory cells to differentiate from the labral epithelium do so at stereotypic sites beginning at around 32% of embryogenesis. Bilaterally symmetrical clusters of differentiated neurons rapidly appear and pioneering of the labral sensory nerve on each side is performed by a specific cell from each cluster. This cell directs its axon anteriorly towards a bilaterally symmetrical pair of cells, the frontal commissure pioneers, on either side of the developing frontal ganglion. The final trajectory of the sensory nerve within the labrum closely matches the pattern of Repo-expressing glial cells. The majority of the sensory cell clusters differentiate during embryogenesis, but the number of sensory cells in some clusters are modified significantly during postembryonic development. Comparing the innervation pattern of the clypeo-labrum with that of other mouthparts and the leg at mid-embryogenesis, we find a striking similarity in organization which we interpret as support for the homologous appendage hypothesis.

Morphological and molecular data argue for the labrum being non-apical, articulated, and the appendage of the intercalary segment in the locust.

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.

Synaptic input from serial chordotonal organs onto segmentally homologous interneurons in the grasshopper Schistocerca gregaria.

We investigated the synaptic inputs from the serially homologous pleural, tympanal and wing-hinge chordotonal organs onto a set of identified homologous interneurons (714, 539, 529) in the ventral nerve cord of the grasshopper Schistocerca gregaria. Cobalt backfills show that afferents from all chordotonal organs project into stereotypic tracts in the central nervous system in which intracellular staining reveals the interneurons to have dendritic arborizations. Neuron 714 was found to receive excitatory bilateral synaptic input from all the serial chordotonal organs tested, from the second thoracic segment down to the seventh abdominal segment. Neuron 531, by contrast, only receives input from the chordotonal afferents on the first abdominal segment; those on the axon side are excitatory, while those on the soma side are inhibitory. The pattern of chordotonal input onto neuron 529 is similar to that seen for neuron 714, with the exception that neuron 529 receives no input from the forewing chordotonal organs. The pattern of afferent connectivities onto neurons 714, 531 and 529 differs with respect to those afferents which synapse directly or indirectly with the respective neuron. The synaptic inputs demonstrate a segmental specialization in the chordotonal system and thereby offer an insight into information processing in a modular sensory system.

Neurogenesis in the median domain of the embryonic brain of the grasshopper Schistocerca gregaria.

Embryonic development in the median domain of the brain of the grasshopper Schistocerca gregaria was investigated with immunohistochemical, histological, and intracellular dye injection techniques. The early head midline is divisible into a dorsal median domain and a ventral median domain based on the orientation of cell somata in each region. At 25% of embryogenesis, a single large midline precursor differentiates in the dorsal median domain and produces a lineage of six neuronal progeny before degenerating. No further precursors arise. In addition, the primary commissure pioneers and a pair of lateral neurons differentiate directly from the ectoderm in this region. Lucifer yellow dye injected into the midline precursor stains only this cell and its progeny. Similarly, there is no dye coupling from the primary commissure pioneers to the midline lineage or to neuroblasts of the brain hemispheres. Neurogenesis in the dorsal median domain therefore proceeds separately within each subset of cells, and is not related to development in the brain hemispheres. Beginning at 42% of embryogenesis, the primary commissure pioneers undergo a morphological transformation and concomittantly express the Term-1 antigen. Expression continues throughout embryogenesis and into the adult, where the midline primary commissure pioneer cells are the only ones labeled by Term-1 in the entire brain. The cellular organization of the dorsal median domain therefore remains remarkably conserved throughout embryogenesis, even as the brain undergoes extensive morphological transformation.

Embryonic development of the pars intercerebralis/central complex of the grasshopper.

We have studied the embryonic development of the pars intercerebralis/central complex in the brain of the grasshopper using immunocytochemical and histochemical techniques. Expression of the cell-surface antigen lachesin reveals that the neuroblasts of the pars intercerebralis first differentiate from the neuroectoderm at around 26% of embryogenesis. Differentiation of medial and lateral neuroblasts occurs first. By the 28% stage a more or less uniform sheet of 20 neuroblasts has formed. As a result of both cell proliferation and cell translocation, the pars intercerebralis proliferative cluster in each hemisphere expands so that at 30% the most medial neuroblasts lie apposed at the midline. We followed the further development of the pars intercerebralis of each brain hemisphere using bromo-deoxy-uridine incorporation and osmium-ethyl-gallate staining. Within the pars intercerebralis itself, the neuroblasts redistribute into discrete subsets. The neuroblasts of each subset generate clusters of progeny which extend in a stereotypic, subset-specific direction in the brain. We have used this feature to identify one subset of four neuroblasts as being the likely progenitor cells for four clusters of embryonic neurons (W, X, Y, Z) which develop at around 55% of embryogenesis. We show that these progeny project axons via four discrete fascicles (w, x, y, z) into the embryonic central complex. At the single cell level, Golgi impregnation reveals that the axons from these neighbouring cell clusters remain discrete, and those from the same cluster tightly fasciculated, as they project into the central complex, consistent with a modular organization for this brain region.

Morphogenetic reorganization of the brain during embryogenesis in the grasshopper.

We have studied the morphogenetic reorganization that occurs in the grasshopper brain during embryogenesis. We find that morphogenetic movements occur at three organizational levels during brain development. First, the entire developing brain changes its orientation with respect to the segmental chain of ventral ganglia. A 90 degrees shift in the attitude of the brain neuraxis occurs during embryogenesis due to a gradual upward movement of the cerebral structures in the head. Second, the clusters of proliferating neuroblasts and progeny that generate the neuroarchitecture of the mature brain move relative to one another and to nonneural structures such as the stomodeum. This is especially pronounced for the pars intercerebralis and for the tritocerebrum, as shown by annulin and engrailed immunoreactivity. Third, individual neuroblasts within a given proliferative cluster undergo positional reorganization during embryogenesis. Identified neuroblasts of the tritocerebrum and the pars intercerebralis are displaced within the brain. We conclude that the transformation of the simple sheet-like structure of the early embryonic brain into the highly differentiated structure of the mature brain involves a series of morphogenetic movements that occur in virtually all parts of the brain.

Parallel processing of afferent input by identified interneurones in the auditory pathway of the noctuid moth Noctua pronuba (L.).

1. Interneurones 501 and 504 are identified sound-sensitive interneurones in the pterothoracic ganglion of the noctuid moth Noctua pronuba. Both neurones receive monosynaptic input from the A1 afferent and experiments with current injection suggest that the synapse is chemical. The EPSPs evoked in either IN 501 or 504 by the A1 afferent do not facilitate. 2. Temporal integration in INs 501 and 504 was compared by presenting the moth with tones at repetition rates found in the search, approach and terminal phases of the echolocating call of a hunting bat. INs 501 and 504 differ in their capacity to resolve stimulus repetition rates because the mean decay times of their compound EPSPs differ by a factor of three, although both interneurones receive monosynaptic input from the A1 afferent. 3. The features extracted from the authentic, prerecorded, call of an echolocating bat at the level of the pterothoracic ganglion were examined by recording sequentially from a range of interneurones in the same preparation. The capacity of INs 501 and 504 to encode the various phases of the call was examined in the light of their measured mean decay times and related to the avoidance behaviour of the insect.

Information processing at a central synapse suggests a noise filter in the auditory pathway of the noctuid moth.

1. The central projections of the A1 afferent were confirmed via intracellular recording and staining with Lucifer Yellow in the pterothoracic ganglion of the noctuid moths, Agrotis infusa and Apamea amputatrix (Fig. 1). Simultaneous recordings of the A1 afferent in the tympanal nerve (extracellularly) and in the pterothoracic ganglion (intracellularly) confirm the identity of the stained receptor as being the A1 cell. 2. The major postsynaptic arborizations of interneurone 501 in the pterothoracic ganglion were also demonstrated via intracellular recording and staining (Fig. 2). Simultaneous recordings of the A1 afferent (extracellularly) and neurone 501 (intracellularly) revealed that each A1 spike evokes a constant short latency EPSP in the interneurone (Fig. 2Bi). Neurone 501 receives only monaural input from the A1 afferent on its soma side as demonstrated by electrical stimulation of each afferent nerve (Fig. 2Bii). EPSPs evoked in neurone 501 by high frequency (100 Hz) electrical stimulation of the afferent nerve did not decrement (Fig. 2Biii). These data are consistent with a monosynaptic input to neurone 501 from the A1 afferent. 3. The response of neurone 501 to a sound stimulus presented at an intensity near the upper limit of its linear response range (30 ms, 16 kHz, 80 dB SPL) was a plateau-like depolarization, with tonic spiking activity which continued beyond the end of the tone. The instantaneous spike frequency of the response was as high as 800 Hz, and was maintained at above 600 Hz for the duration of the tone (Fig. 3). 4. The relationship between the instantaneous spike frequency in the A1 afferent and that recorded simultaneously in neurone 501 is linear over the entire range of A1 spike frequencies evoked by white noise sound stimuli (Fig. 4). Similarly, the relationship between instantaneous spike frequency in the A1 afferent and the mean depolarization evoked in neurone 501 is also linear for all A1 spike frequencies tested (Fig. 5). No summation of EPSPs occurred for A1 spike frequencies below 100 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Presynaptic inhibition of identified wind-sensitive afferents in the cercal system of the locust.

The paired cerci located at the tip of the locust abdomen bear a large number of wind-sensitive filiform hairs, each of which sends an axon via the cercal nerve into the terminal ganglion of the CNS. The filiform afferents fire bursts of action potentials when their hairs are displaced by wind or mechanical stimuli. Filiform axon terminals in the CNS are depolarized concomitantly with the discharge of another type of unit (a primary afferent-depolarizing, or PAD, unit) recorded in the cercal nerve. The instantaneous spike frequency of PAD unit discharges matches the evoked depolarization very closely, and during such depolarizations spike amplitudes in the filiform afferent terminals are reduced by up to 55%. Depolarizing current pulses injected into the axonal terminals of an identified filiform afferent evoke spikes that are blocked by the PAD unit, probably via an intercalated interneuron. The PAD unit makes a monosynaptic connection with only one of the 4 giant interneurons (GIN 2) on each side of the terminal ganglion, and indirect connections with 2 others. Depolarizing current pulses injected into the neuropilar segments of GINs evoke fewer spikes when the PAD unit is active, consistent with the PAD unit's mediation of conductance changes in postsynaptic cells. Iontophoretic injection of Lucifer yellow shows the PAD unit to be an afferent with axon terminals overlapping those of filiform afferents and posteriorly directed branches of interneurons such as GIN 2 in the CNS. Passive movements of a cercus, monitored with a position transducer, show that the PAD unit fires discrete bursts during cercal displacement. The PAD unit most probably has its soma and dendrites in tissue spanning the cercal base. By responding to cercal movements sufficient to also activate filiform hairs, and by mediating conductance changes in both the presynaptic terminals of filiform afferents and the postsynaptic membranes of interneurons, the PAD unit desensitizes a pathway to movement-generated afferent input, and ensures that the locust remains sensitive to external wind stimuli.

Modulation of auditory responsiveness in the locust.

The auditory responsiveness of a number of neurones in the meso- and metathoracic ganglia of the locust, Locusta migratoria, was found to change systematically during concomitant wind stimulation. Changes in responsiveness were of three kinds: a suppression of the response to low frequency sound (5 kHz), but an unchanged or increased response to high frequency (12 kHz) sound; an increased response to all sound; a decrease in the excitatory, and an increase in the inhibitory, components of a response to sound. Suppression of the response to low frequency sound was mediated by wind, rather than by the flight motor. Wind stimulation caused an increase in membrane conductance and concomitant depolarization in recorded neurones. Wind stimulation potentiated the spike response to a given depolarizing current, and the spike response to a high frequency sound, by about the same amount. The strongest wind-related input to interneuron 714 was via the metathoracic N6, which carries the axons of auditory receptors from the ear. The EPSP evoked in central neurones by electrical stimulation of metathoracic N6 was suppressed by wind stimulation, and by low frequency (5 kHz), but not high frequency (10 kHz), sound. This suppression disappeared when N6 was cut distally to the stimulating electrodes. Responses to low frequency (5 kHz), rather than high frequency (12 kHz), sounds could be suppressed by a second low frequency tone with an intensity above 50-55 dB SPL for a 5 kHz suppressing tone. Suppression of the electrically-evoked EPSP in neurone 714 was greatest at those sound frequencies represented maximally in the spectrum of the locust's wingbeat. It is concluded that the acoustic components of a wind stimulus are able to mediate both inhibition and excitation in the auditory pathway. By suppressing the responses to low frequency sounds, wind stimulation would effectively shift the frequency-response characteristics of central auditory neurones during flight.

Heterogeneous properties of segmentally homologous interneurons in the ventral nerve cord of locusts.

The G, B1, and B2 neurons are three prominent interneurons located in adjacent segmental ganglia in the central nervous system of locusts. Previous studies on the adult nervous system have shown that each of these cells has its own distinctive morphology and responsiveness to auditory input. Previous studies on the embryonic nervous system have described the lineage and development of one of these cells, the G neuron, in the mesothoracic (T2) segment. In this paper it is shown that the G, B1, and B2 neurons are segmental homologues in that they arise from equivalent lineages during embryogenesis in the T2, T3, and A1 segments, respectively. Each cell arises (along with its identified sibling neuron) from the division of the second ganglion mother cell of neuroblast 7-4. The segment-specific morphology of the G homologues was determined in the T3 and A1 segments between 60-70% of embryonic development, and their identity was established as the adult B1 and B2 neurons by comparing the distinctive cell-specific features of their morphology between embryo and adult. Although all three neurons display striking morphological differences, they all share certain structural features in common, including the location of their primary axons and neurites in specific tracts in the neuropil. By recording intracellularly from the main neurites of the G, B1, and B2 neurons, clear differences were found in the synaptic inputs each of the neurons receives and the synaptic outputs each makes. For example, G and B2, but not B1, receive direct monosynaptic input from the descending contralateral movement detector (DCMD) interneurons and from auditory afferents; B1, but not B2, connects directly to G; and B2, but not B1 or G, connects directly to flight motoneurons. The main conclusion from these observations is that lineally equivalent neurons in different segments can develop similar primary structures but quite different secondary morphologies and synaptic connections. How these segment-specific differences arise during embryogenesis remains unknown.