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The mushroom bodies are paired neuropils in the insect brain that act as multimodal sensory integration centers and are involved in learning and memory. Our studies, by using 5-bromo-2-deoxyuridine incorporation and the Feulgen technique, show that immediately before pupation, the brain of the developing honey bee (Apis mellifera) contains approximately 2,000 neuroblasts devoted to the production of the mushroom body intrinsic neurons (Kenyon cells). These neuroblasts are descended from four clusters of 45 or fewer neuroblasts each already present in the newly hatched larva. Subpopulations of Kenyon cells, distinct in cytoarchitecture, position, and immunohistochemical traits, are born at different, but overlapping, periods during the development of the mushroom bodies, with the final complement of these neurons in place by the mid-pupal stage. The mushroom bodies of the adult honey bee have a concentric arrangement of Kenyon cell types, with the outer layers born first and pushed to the periphery by later born neurons that remain nearer the center of proliferation. This concentricity is further reflected in morphologic and immunohistochemical traits of the adult neurons, and is demonstrated clearly by the pattern of expression of Drosophila myocyte enhancer factor 2 (DMEF2)-like immunoreactivity. This is the first comprehensive study of larval and pupal development of the honey bee mushroom bodies. Similarities to patterns of neurogenesis observed in the mushroom bodies of other insects and in the vertebrate cerebral cortex are discussed.

The multicatalytic proteinase complex is a multi-subunit, high molecular weight proteinase present in the nucleus and cytoplasm of eukaryotic cells. This catalytic complex is involved in diverse cellular functions as part of the ubiquitin proteolysis system, including non-lysosomal proteolysis, antigen presentation, cell cycle progression, and cell proliferation, and in the programmed death of intersegmental muscles after adult eclosion in the tobacco hornworm moth, Manduca sexta. We have investigated the distribution of the multicatalytic proteinase complex in the central nervous system of this moth. At all stages of post-embryonic development, most cell types exhibited consistent, low levels of cytoplasmic and nuclear immunoreactivity for the multicatalytic proteinase complex. High levels of cell-specific accumulation of the complex were, however, demonstrated in abdominal neurosecretory cells and in imaginal cells in the larval brain, the larval segmental ganglia, and the developing wing discs. Imaginal cells exhibited intense immunoreactivity for the multicatalytic proteinase complex only until the onset of terminal differentiation. Intersegmental muscles undergoing programmed cell death exhibited intense cytoplasmic immunoreactivity for the multicatalytic proteinase, while persisting flight muscles and dying neurons were characterized by basal levels of staining. These staining patterns suggest that the multicatalytic proteinase of Manduca sexta serves multiple functions and is associated with the period of developmental arrest displayed by imaginal cells prior to metamorphosis. © 1996 Wiley-Liss, Inc.

Selective neuronal death is a normal component of metamorphosis in the moth, Manduca sexta. In particular, the three unfused abdominal ganglia of the ventral nerve cord serve as a useful experimental preparation in which to study the regulation of the molecular mechanisms that mediate programmed cell death. Ubiquitin, a highly conserved 76-amino acid protein found in all eukaryotic cells, has previously been shown to be present in increased amounts in some tissue undergoing programmed cell death (e. g., larval intersegmental muscles inManduca sextamoths, dying cells in developing tunicates), but not in others (T-cells, Drosophila ommatidial cells, cultured sympathethic neurons deprived of nerve growth factor). It has been hypothesized that the need for ubiquitin-dependent proteolysis is increased in dying cells, and that the accumulation of ubiquitin might serve as an early marker for cells commited to die. Immunohistochemical localization of ubiquitin at the light microscopic level in the adbominal gaglia of Manduca sextasuggests that this protein plays a number of important roles in neuronal physiology and may be associated with the death of some neurons in this tissue. The most intense staining of neuronal cytoplasm, however, was found not in dying neurons, but instead in sets of persisting neurons that may serve a primarily neurosecretory or neuromodulatory function. The staining obtained in these cells with antibodies directed against ubiquitin was developmentally regulated. © 1994 Wiley-Lisx, Inc.

The ventral nerve cord of holometabolous insects is reorganized during metamorphosis. A prominent feature of this reorganization is the migration of subsets of thoracic and abdominal larval ganglia to form fused compound ganglia. Studies in the hawkmoth Manduca sexta revealed that pulses of the steroid hormone 20-hydroxyecdysone (20E) regulate ganglionic fusion, but little is known about the cellular mechanisms that make migration and fusion possible. To test the hypothesis that modulation of cell adhesion molecules is an essential component of ventral nerve cord reorganization, we used antibodies selective for either the transmembrane isoform of the cell adhesion receptor fasciclin II (TM-MFas II) or the glycosyl phosphatidylinositol-linked isoform (GPI-MFas II) to study cell adhesion during ganglionic migration and fusion. Our observations show that expression of TM-MFas II is regulated temporally and spatially. GPI-MFas II was expressed on the surface of the segmental ganglia and the transverse nerve, but no evidence was obtained for regulation of GPI-MFas II expression during metamorphosis of the ventral nerve cord. Manipulation of 20E titers revealed that TM-MFas II expression on neurons in migrating ganglia is regulated by hormonal events previously shown to choreograph ganglionic migration and fusion. Injections of actinomycin D (an RNA synthesis inhibitor) or cycloheximide (a protein synthesis inhibitor) blocked ganglionic movement and the concomitant increase in TM-MFas II, suggesting that 20E regulates transcription of TM-MFas II. The few neurons that showed TM-MFas II immunoreactivity independent of endocrine milieu were immunoreactive to an antiserum specific for eclosion hormone (EH), a neuropeptide regulator of molting.

We explored the neural basis of age- and task-related plasticity in circadian patterns of activity in the honeybee. To identify putative circadian pacemakers in the bee brain, we used antibodies against Drosophila melanogaster and Antheraea pernyi PERIOD and an antiserum to crustacean pigment-dispersing hormone (PDH) known to cross-react with insect pigment-dispersing factors (PDFs). In contrast to previous results from Drosophila, PDH and PER immunoreactivity (-ir) were not colocalized in bee neurons. The most intense PER-ir was cytoplasmic, in two groups of large neurons in the protocerebrum. The number of protocerebral PER-ir neurons and PER-ir intensity within individual cells were highest in brains collected during subjective night and higher in old bees than in young bees. These results are consistent with previous analyses of brain per mRNA in honeybees. Nuclear PER-ir was found throughout the brain, including the optic and antennal lobes. A single group of PDH-ir neurons (approximately 20/optic lobe) was consistently and intensely labeled at the medial margin of the medulla, independent of age or time of day. The processes of these neurons extended to specific neuropils in the protocerebrum and the optic lobes but not to the deutocerebrum. The patterns displayed by PER- and PDH-ir do not completely match any patterns previously described. This suggests that, although clock proteins are conserved across insect groups, there is no universal pattern of coexpression that allows ready identification of pacemaker neurons within the insect brain.

In the course of examining afferents to ventromedial hypothalamic (VMH) neurons using horseradish peroxidase (HRP), we needed to know how close to an iontophoretic deposit site neurons could be proved to be retrogradely labeled. In evaluating cells near but clearly outside HRP deposit sites visualized after a 24-hour survival period, for example, neurons which had been filled with HRP by somal or dendritic uptake could not be treated as retrogradely labeled and thus would not add to studies of intrahypothalamic connections. Rats were given standardized iontophoretic applications of HRP into VMH (continuous positive current 0.25 mu amp for 1 minute) and sacrificed after 5 or 15 minutes, 1, 4, 8, 12, or 24 hours in order to examine the pattern of HRP spread. The chromogen was tetramethylbenzidine. The volume of the application site visualized at 24 hours was less than 10% of maximum site size, which occurred at 1 hour. Since the cells located within the maximal spread boundary are candidates for nonretrograde labeling, HRP data on local connections obtained even from small iontophoretic deposits must be evaluated in the light of the demonstrated expansion and subsequent contraction of the application site. These results may also hold implications for the precision with which distant connections can be examined using the HRP retrograde method, as sites that appear discrete when visualized after 24-hour survival may have overlapped at shorter times post-iontophoresis. Incorporation of retrograde tracers into polyacrylamide gels provides an effective alternative to pressure injection or iontophoresis of aqueous tracer solutions. We describe a method for filling micropipettes with HRP-polyacrylamide gel. The pipettes are then implanted into brain sites to provide a confined pool of HRP. With postimplantation survival of 24 hour or longer, this method produces sites comparable in size to iontophoretic sites examined at 24 hours and results in improved retrograde labeling. Some results obtained with this method concerning the afferent connections of the dorsomedial hypothalamus are described.

Fluorescent dye retrograde tracing was combined with steroid hormone autoradiography to study the midbrain projections of the estrogen-concentrating neurons in the preoptic region of the rat brain. Microinjections of the dyes DAPI, true blue, or a mixture of DAPI and primuline were made into the ventral tegmental area and into the midbrain central gray of ovariectomized, adrenalectomized 2-3-month-old female rats; 3 or 4 days later these animals were injected with [3H]estradiol; the brains were then processed for autoradiography. After exposures of from 3 to 12 months, the autoradiograms were developed and examined for reduced silver grains under cell nuclei (indicating binding of [3H]estradiol) and retrogradely transported fluorescent dye in the cytoplasm (indicating an efferent projection to the midbrain). Numerous [3H]estradiol-concentrating neurons in the medial preoptic region were found to send their axons to the medial midbrain. The largest numbers of estrogen target neurons that were afferent to the ventral tegmental area and to the midbrain central gray were found in the medial preoptic nucleus, in the surrounding medial preoptic area, and in the ventral bed nucleus of the stria terminalis. Double-labeled neurons were also identified in the preoptic suprachiasmatic area, in the lateral preoptic area, and in the rostral anterior hypothalamic area. Thus, a subset of the gonadal steroid target cells of the preoptic region have long projections to the medial midbrain, and a subset of the medial preoptic neurons that project to the ventral tegmental area and to the midbrain central gray concentrate estrogen. Behaviors (for example, maternal behavior, male copulatory behavior, and wheel-running) that are regulated by estrogen action in the medial preoptic region may be controlled by the direct estrogen-sensitive pathway to the medial midbrain revealed in this study.