For many years this laboratory has been involved in two main programs of research, both devoted to structural organisation of the nervous system from a developmental and evolutionary perspective, using accessible nervous systems offered by certain invertebrate species. Each program exploits the characteristics of its particular nervous system. Our two favourites are: the visual system of the fly, and the larval nervous system of the ascidian, or sea-squirt. Both offer morphological determinacy, having fixed numbers of cells that occupy fixed locations relative to each other, and can thus be repeatedly recognised in different animals. Other preparations have included the slit sensilla of spiders and the central neuropiles of the mushroom body and related regions of the Drosophila brain.
Our work on these projects is enabled by the skilled support of Jane Anne Horne in computer and imaging methods, Harjit Seyan in micro- and histological methods, Zhiyuan Lu in serial-EM and 3-D reconstruction methods, Rita Kostyleva in quantitative EM of the lamina, and Dorota Tarnogorska in lab support and fly care. We also have bright young undergraduate students.
- How nerve cells establish, maintain and modify their synaptic connections. We have examined the highly determinate photoreceptor synapses of the first neuropile, or lamina, of the optic lobe behind the fly's compound eye. Our background knowledge of these has proved useful in studies on various mutants of genes for synaptic proteins, in past collaborations with Tom Schwarz (Children’s Hospital, Boston, MA), Harold Atwood (University of Toronto) and Robin Hiesinger (UT Southwestern Medical Center, Dallas, TX), and for our current study on the role of the cell adhesion molecules Dscam in photoreceptor synaptogenesis (Larry Zipursky, UCLA, CA). We have also identified the range of targets that can support the development of photoreceptor synapses in ectopic locations: either when ectopic eyes send their axons into the central brain (a collaboration with Patrick Callaerts, in Leuven, Belgium); or when runt is over-expressed in photoreceptors, in a project conducted by Tara Edwards, for her Ph.D. Birgit Greiner (Dr. phil., University of Lund, Sweden), who held an Erwin-Schrödinger Stipendium of the FWF and was an honorary Dalhousie Killam Fellow, with an undergraduate student Nancy Butcher, has examined the ultrastructure of synaptic macromolecules using EM tomography, in a project conducted in collaboration with the lab of Jack McMahan (Stanford Medical School, CA). Various studies also indicate considerable plasticity amongst these synapses. A postdoctoral fellow, Yoshitaka Hamanaka (Ph.D., Osaka City University, Japan) is examining the expression of gene products involved in synaptic function, plasticity and synaptogenesis, using immuno-EM methods. Claudia Groh (Dr. rer. nat., Würzburg, Germany), a visiting postdoctoral fellow of the DAAD, is examining F-actin mobilisation of dendritic spines in lamina neurons in response to changes in visual exposure. These studies are all supported by the National Eye Institute of NIH.
- The synaptic circuits of the fly’s visual system. Postdoctoral fellow, Shin-ya Takemura (Ph.D., Yokahama City University, Japan), has examaned the synaptic circuits of the second neuropile, or medulla, using serial-EM methods. Neuronal profiles are labelled by targeted expression of EM markers using Gal4 lines generated in collaborating laboratories, Chi-Hon Lee (NIH, Bethesda, MD) and others. This work is continuing partly as a visiting scientist at the Janelia Farm Research Campus of HHMI, in collaboration with Mitya Chklovskii. These studies are also supported by the National Eye Institute of NIH.
- The synaptic circuits of the mushroom bodies in the fly’s brain. In collaboration with Kouji Yasuyama, a sometime visitor from Kawasaki Medical School in Okayama, Japan, and Friedrich-Wilhelm Schürmann (Georg-August-University Göttingen, Germany) we have undertaken an analysis of the synaptic circuits of the microglomeruli of the mushroom body calyx, a high-order integrative centre required for olfactory learning and memory, and the upstream inputs to the lateral horn. Continuing this work, Claudia Groh is examining volumetric changes that result from dendritic plasticity among the cells of the calyx, in response to olfactory conditioning and is further examining synaptic microcircuits using serial-EM reconstructions of calycal microglomerul. These studies are supported by NSERC.
- We have a longstanding collaboration with the laboratory of Prof. Elzbieta Pyza, a visiting scientist from the Jagiellonian University in Kraków, Poland, who has examined circadian fluctuations in the number of photoreceptor synaptic contacts, and in the size of the lamina cells that form these, as well as in the action of neuromodulators in bringing about these changes. We are examining the role of a major circadian peptide, pigment dispersing factor (PDF). In collaboration with Paul Taghert (Washington University, St. Louis, MO), Yoshi Hamanaka is examining the effects of transforming photoreceptors with the dimmed gene for peptidergic neurons, in attempts to insert a secretory pathway for the neuropeptides, FMRF and PDF. In collaboration with Kouji Yasuyama, we are examining the PDF-immunoreactive innervation of the fly protocerebrum and mushroom body calyx. Finally, in collaboration with Yasuyuki Shimohigashi (Kyushu University, Fukuoka, Japan) and Miki Shimohigashi (Fukuoka University, Japan), we also plan to examine the actions of endocrine disruptors, particularly bisphenol A, at nuclear receptors (transcription factors) during neuronal development.
- Janusz Borycz (Ph.D., Institute of Pharmacology, Polish Academy of Science, Kraków), a Research Associate and former NATO Postdoctoral Fellow, developed HPLC methods to determine histamine, a neurotransmitter of photoreceptors in the fly, and has examined the metabolism of histamine via a β-alanyl metabolite called carcinine. Various mutants of histamine metabolism, in particular two mutants tan and ebony of the metabolic pathway that shuttles histamine and its metabolite between photoreceptor terminals and surrounding glia, reveal these pathways in Drosophila. Recent work is devoted to measuring the release of histamine from the fly’s eye, using microdialysis. In related studies, and in collaboration with David Krantz (UCLA School of Medicine, Los Angeles, CA) and Bernd Hovemann (Ruhr University of Bochum, Germany), Jolanta Borycz, a postdoctoral fellow, is studying the expression and action of the vesicle transporter VMAT, and ABC transporter genes white, brown and scarlet, in the visual system. She has developed microdissection, fractionation and centrifugation methods for this work. Finally, a graduate student Tara Edwards is working to establish the nature of the transport mechanisms in a histamine and carcinine shuttle pathway between photoreceptor terminals and surrounding glial cells. These studies have been supported by CIHR and NIH.
- We have also examined the neuroembryogenesis of the ascidian larva, and the normal structure of its simple nervous system. The latter is remarkable both as the evolutionary herald of the chordate dorsal nerve cord, and because it has only about 300 cells, some of which at least are uniquely identifiable. A recent graduate student Janice Imai has identified many of the cell types in the nervous system, using transient transfection with GFP. This work is being continued by Kerrianne Ryan, a graduate student in the lab who is examining the synaptic circuits from serial-section EM, with the aim of identifying the central pattern generator for larval swimming. These studies are supported by NSERC. .