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Larva Glial Cells and Axon Tracts

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Volker Hartenstein

Molecular, Cellular and Developmental Biology Department, University of California, Los Angelos, CA

Correspondence: Volker Hartenstein volkerh@mcdb.ucla.edu


Introduction
Methods
Insect Brain Structure
Drosophila Larval Brain
Insect Glial Cells
Models

Introduction

In this database you will find a series of models of the embryonic brain at different stages. For brief a orientation: Drosophila embryogenesis lasts approximately 22 hours. The progenitor cells of the brain and ventral core, called neuroblasts, are born between 3.5 and 6.5 hrs (stages 9-11) of development. These cells proliferate between 4 and about 12 hours (stage 10-15) to produce neurons. Differentiation of the first neurons and glial cells starts at around 8 hrs (stage 12). These neurons form pioneer tracts followed by later differentiating axons. The first set of models visualize the pattern of pioneer tracts in the brain and ventral cord at different stages. Stages are: 12 (approximately 7-9hrs); 13 (9-10.5hrs), 14 (10.5-11.5hrs), 15 (11.5-13hrs), 16 (13-16hrs).

The second set of models illustrates the pattern of glial cells at these stages.

Generation of 3D digital models

Staged Drosophila embryos labeled with anti-FasII and anti-Repo antibodies were viewed as wholemounts under Nomarski optics (Zeiss Axiophot, 40X immersion oil lens). While focussing through the embryos at increments of 2mm, digitized images were taken with a Sony 3CCD camera (Sony Instruments). Digitized images ( "raw section files") were imported into Adobe Photoshop (Adobe). Bezier curves ("paths") were drawn manually around the labeled structures which were to be included in the model. The paths obtained from each section file ("section path files") were imported into the RayDream Designer (Metacreations). Since the section path files were taken from focal planes of one and the same embryo, there was no need for alignment of different sections. In RayDream Designer, section path files were stacked at the proper intervals. A "skin" is synthesized by triangulation. Models were then exported to VRML format. Lighting, camera angle, transparency, reflection and triangle reduction were adjusted in CosmoWorlds (SGI).

Background

1. Insect Brain Structure

Neurons of the insect form a multitude of functionally specialized tracts and neuropile compartments. A considerable diversity in the size and pattern of neuropile compartments has been documented among different insect groups (Hanström, 1928; Bullock and Horridge, 1965). However, it is possible to identify a groundplan of several prominent brain structures common to most insects. Along the anterior-posterior axis (neuraxis), the insect brain is divided into the supraesophageal ganglion, comprising the protocerebrum, deuterocerebrum, tritocerebrum, and the subesophageal ganglion, which arises by the fusion of three segmental ganglia (labium, maxilla, mandible). Sensory and motor nerves of the subesophageal ganglion and the tritocerebrum supply the innervation of the mouthparts; the tritocerebrum also sends axons into a group of peripheral ganglia, the stomatogastric ganglia, which controls feeding behavior. The deuterocerebrum receives the antennal nerve and represents the olfactory center of the brain; the so called posterior slope of the deuterocerebrum (Strausfeld, 1976), a major "output region" of the insect brain, contains numerous interneurons which project onto the motoneurons of the ventral nerve cord. The protocerebrum dominates the brain in size and complexity. Among the conserved elements of the protocerebrum are the mushroom bodies (corpora pedunculata) and their major afferent tract (antenno-glomerular tract), the central complex, pars intercerebralis, optic tubercles and optic lobes. The spatial relationship and interconnectivity between these protocerebral compartments is schematically shown in Fig.1.

The mushroom body consists of a plate of cell bodies (Kenyon cells) located in the dorsal protocerebrum. Kenyon cell axons form a characteristic branched structure (calyx, peduncle, a, b, and g lobes). Afferent axons from the olfactory lobe and various other sources, among them the optic lobe, form contacts with Kenyon cell axons in the calyx neuropile. Medially and anterior of the mushroom bodies is the pars intercerebralis, which in all insects contains neurosecretory cells projecting their axons towards the corpora cardiaca, neuro-endocrine organs involved in molting, cardiovascular and metabolic functions. Axons to and from the pars intercerebralis, interconnecting this brain region with the ventral nerve cord and basal brain regions, form the crossed median bundle. The central complex, an unpaired structure implicated in flight control (Ilius et al., 1994) and built of several distinct neuropile compartments (Power, 1943; Strausfeld, 1976) lies between the calices of the mushroom bodies. The only anatomically defined source of input to the central complex is the ventral body, a spherical neuropile compartment at the base of the protocerebrum, between antennal neuropile and lobes of the mushroom body (Power, 1943; Hanesch et al., 1989). All structures mentioned so far form the so called "midbrain", which is flanked on either side by the optic lobe. The two distal neuropiles of the optic lobe, lamina and medulla, receive the highly ordered axons of the compound eyes. Lamina and medulla project to the inner optic neuropile (lobula complex), which in turn sends axons to its contralateral counterpart, as well as the lateral neuropiles of the midbrain (anterior and posterior optic tubercles; Strausfeld, 1976; Meinertzhagen and Hanson, 1993).

2. Drosophila Larval Brain

The central nervous system of dipteran larvae consists of the paired supraesophageal ganglion (frequently called "brain" in the recent literature) and the ventral nerve cord (Fig.1). The subesophageal ganglion is included in the brain only in the adult fly; during larval stages, it forms the anterior part of the ventral nerve cord. Both supraesophageal ganglion and ventral nerve cord have an outer layer of neuronal and glial cell bodies (cortex) and a central neuropile. In the ventral nerve cord of a mature embryo, the neuropile is formed by a longitudinal component, the connective, and segmentally reiterated pairs of commissures. [Strictly speaking, the term connective denominates bundles of axons that interconnect the neuropiles of neighboring ganglia. In Dipterans, the CNS has condensed to such a degree that one cannot distinguish between areas of neuropile (characterized by terminal axonal branches and synapses) and connectives interconnecting neuropile areas; instead, long range (longitudinal) axonal tracts and terminal branches with synapses are intermingled and are jointly referred to as connective.] At the level of the commissures, nerve roots carrying axons to and from the periphery enter the connectives. In thoracic and abdominal neuromeres, two roots join to form the so called intersegmental nerve, a single root forms the segmental nerve (Fig.2A). The neuropile of the subesophageal ganglion closely resembles that one of the thoracic and abdominal neuromeres Fig.2B). The labial neuromere possesses a pair of commissures and two nerves that show serial homology with the ISN and SN of thoracic and abdominal neuromeres. The posterior labial nerve (corresponding to the SN in view of the fact that it has a single root entering the neuropile at the level of the posterior labial commissure) is called the lateropharyngeal nerve (Schmidt-Ott et al., 1994) and conducts afferent and efferent axons to the pharynx. The anterior, ISN nerve of the labial neuromere (labial nerve) enters the neuropile via two roots; this nerve mainly carries sensory axons from the numerous labial sensilla and some efferent axons to the ventral pharyngeal musculature. The maxillary neuromere has a pair of commissures and gives rise to the maxillary nerve that enters the connective at the level of the anterior maxillary commissure; this nerve carries axons from the maxillary sensory complex and is serially homologous with the segmental nerve of abdominal and thoracic segments (see below). There are no further peripheral nerves entering the subesophageal ganglion anterior to the maxillary nerve. The subesophageal commissure, a massive fibre bundle crossing the midline at the boundary between subesophageal and supraesophageal ganglion, carries axons of mainly tritocerebral origin (see below), although some axons of neurons located in the rudimentary mandibular neuromere also contributes to this commissure (see below).

The neuropile of the larval supraesophageal ganglion has three major components, a vertical component (cervical connective; for this and other previously identified structures, the nomenclature used in the classical literature (e.g., Hertweck, 1931; Bullock and Horridge, 1965) will be used), a transversal component (supraesophageal commissure) and a horizontal component which will be called "protocerebral connective" in the following (Fig.2C-E). Forming the anterior continuation of the connective of the ventral cord, the cervical connective is a thick axon bundle that curves arond the foregut. At the basal part of the brain hemisphere, corresponding to the tritocerebrum, axons of the subesophageal (tritocerebral) commissure and the frontal connective branch off the cervical connective (Hartenstein et al., 1994). The frontal connective carries peripheral axons to and from the stomatogastric nervous system. Further dorsally, the point of entry of the antennal nerve into the cervical connective defines the position of the deuterocerebrum (Tissot et al., NEED DATE). After passing the foregut, the cervical connective branches into the massive supraesophageal commissure and the protocerebral connective; these structures constitute the neuropile of the embryonic protocerebrum. Two peripheral nerves enter the protocerebrum: the optic nerve (or Bolwig's nerve), carrying sensory axons from the larval photoreceptors (Steller et al., 1987), and the nerve to the corpora cardiaca (Hartenstein et al., 1994).

3. Insect Glial Cells

The following classes of glial cells have been distinguished. There are glia cells that ensheath ganglia and nerve tracts ("surface glia"), that surround individual or groups of somata ("cortex glia"), and that form sheaths around the entire neuropile, as well as individual axon bundles ("neuropile glia"). The surface glia cells, called sub-perineurial glia cells in insects, form the blood-brain barrier (Lane, 1984; Edwards et al., 1993). At its external surface, the sub-perineurial glial sheath is surrounded by a capsule of mesodermally derived connective tissue. In annelids this capsule consists of fibroblasts and muscle cells; it forms trabecles penetrating into the ganglia. A similar capsule of mesodermally derived cells exists in insects where it is called perineurium. The perineurium has been excluded from the glia cell population by several authors, based on the fact that it does not directly contact any neurons (Hoyle, 1986), and that it is derived from the mesoderm, whereas all other glia cells are ectodermal in origin (Scharrer, 1939). The fact that molecular markers such as Repo are not expressed in the perineurial cells also points at their non-glial nature. The cortex glia-cells are associated with neuronal cell bodies; they form trophospongia (Hoyle et al., 1986). Hoyle (1986), on morphological and topological grounds, further subdivides cortex glia into cell body glia, satellite glia, and axon hillock glia.

In the ventral nerve cord of Drosophila embryos, the pattern and origin of glia cells has been studied in great detail (Klaembt and Goodman, 1991; Ito et al., 1994). Similar to what has been established for embryonic neurons, glia cells form an almost invariant, segmentally reiterated pattern. Most glia cells are produced by progenitor cells that give rise to neurons as well, i.e., neuro-glioblasts (Bossing et al., 1996; Schmidt et al., 1997). Only the so called longitudinal glia and midline glia (a subset of neuropile associated glia) are formed by progenitors that do not produce neurons (Jacobs et al., 1989; Klaembt et al., 1991).

Models

Brain Glia Models

Stage 11 (565 KB) (gzipped version - 131 KB)
Stage 12 (861 KB) (gzipped version - 194 KB)
Stage 12pns (689 KB) (gzipped version - 157 KB)
Stage 14 (700 KB) (gzipped version - 154 KB)
Stage 14pns (718 KB) (gzipped version - 161 KB)
Stage 16 (780 KB) (gzipped version - 163 KB)

Axon Tract Models

Stage 12 Lateral (627 KB) (gzipped version - 142 KB)
Stage 14 Lateral (713 KB) (gzipped version - 159 KB)
Stage 16 Lateral (716 KB) (gzipped version - 145 KB)


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