of other pathways regulated by fsn-1. We are in the process of identifying other components of signaling pathways through which fsn-1 regulates synapse ...
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Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603
[P65] Identifying synapse formation genes regulated by a presynaptic E3 ubiquitin ligase E. Liao ∗ , W. Hung, M. Zhen Samuel Lunenfeld Research Institute, Canada The neuronal synapse is an asymmetric structure consisting of pre and post-synaptic terminals in direct apposition. Synapse formation is a highly regulated process requiring the interaction of many genetic pathways. To identifying genes that are required for the proper formation of synapses we are using the GABAergic nervous system of C. elegans as our model system. C. elegans is an excellent model system due to its strong genetics and characterized and invariant nervous system. Fluorescently tagged synaptic proteins allow for the visualization of synapses in live animals and thus the identification of synapse mutants through genetic screens. We have identified a protein complex that controls synapse morphology. This SCF ubiquitin ligase complex consists of the F-box protein FSN-1, the RING finger protein RPM-1, Skp1, and Cullin. This complex is required pre-synaptically and is localized to the peri-active zone. We hypothesize that this protein complex controls synapse formation by down regulating synapse-promoting factors through an ubiquitin mediated process. We have identified a possible target or downstream effector of synapse formation in the receptor protein tyrosine kinase scd2 (suppressor of constitutive dauer). We have observed that protein levels of an SCD-2::GFP fusion protein increase in the absence of fsn-1 in vivo. SCD2 ubiquitination is currently being tested. We have shown that loss of scd-2 will partially rescue the synapse defects of fsn1 and rpm-1, and suppression of fsn-1 defects is specific for alleles of scd-2 that are defective in the C-terminal kinase. The incomplete suppression of fsn-1 by scd-2 suggests the existence of other pathways regulated by fsn-1. We are in the process of identifying other components of signaling pathways through which fsn-1 regulates synapse morphology. Keywords: Synapse; Ubiquitin; fsn-1; C. elegans doi:10.1016/j.ijdevneu.2006.09.128 [P67] Cell fate specification and axonogenesis in neurons fate mapped from the embryonic rhombic lip R. Machold ∗ , C. Klein, G. Fishell NYU Medical Center, USA The cerebellum develops from an embryonic primordium within dorsal rhombomere 1 (r1) that contains two germinal zones: the ventricular neuroepithelium and the cerebellar rhombic lip. Specification of neurons in the rhombic lip requires the basic helix–loop–helix (bHLH) transcription factor
Math1, a mouse homolog of Drosophila atonal. Recently, we have fate mapped the cerebellar rhombic lip using an in vivo inducible cre/loxP recombination strategy (Math1-CreERT2 ; R26RstopLacZ) to permanently label cohorts of Math1+ cells at early and late embryonic stages. At stages prior to E12.5, with the exception of the deep cerebellar nuclei, we find that Math1+ rhombic lip neurons migrate out of the cerebellar primordium into the rostral hindbrain to populate specific nuclei that include cholinergic neurons of the mesopontine tegmental system. Interestingly, many of these hindbrain nuclei are components of the auditory and vestibular systems, suggesting that Math1 expression may impart a circuit identity on pools of rhombic lip neural progenitors. We will present new data from a microarray analysis of the Math1 populations generated in the cerebellar rhombic lip between E11.5 and E13.5 we performed to characterize the genes expressed downstream of Math1 that direct the differentiation of these populations. Furthermore, using a recently developed reporter line that expresses membrane targeted GFP, we have begun to characterize the formation of axonal projections between Math1-derived nuclei, as well as other projections to the rostral CNS. We will present this data along with expression data for candidate cell surface molecules that may mediate the specific projection patterns observed in the embryonic development of these neural circuits. Keywords: Cell fate specification; Axonogenesis; Math1; Hindbrain development doi:10.1016/j.ijdevneu.2006.09.129 [P68] Cacna1f-mutant mouse exhibits altered synaptogenesis N. Orton ∗ , W.K. Stell, T. Bech-Hansen University of Calgary, Canada Introduction: Mutations in CACNA1F, which encodes the human Cav 1.4 subunit of an L-type voltage-gated calcium channel, cause incomplete Congenital Stationary Night Blindness (CSNB2). This highly variable X-linked retinal disorder is characterized by night blindness, reduced visual acuity, and an abnormal electrophysiological response in addition to secondary visual impairments. Recently we reported pronounced morphological changes in second-order neurons of the retina of a transgenic model of CSNB2 (Anon., 2005): ON-bipolar and horizontal cell processes extended distally from the outer plexiform layer, deep into the outer nuclear layer (ONL). Key preand post-synaptic components: bassoon, ribeye, and mGluR6 occurred only rarely in their normal location in the OPL or even ectopically in the ONL. This suggested that photoreceptor synapses either fail to develop properly, or degenerate after being formed, in the mutant retina. In the present study we attempted to distinguish between these two alternatives. Methods: We used transmission electron microscopy and immunocytochemistry to study the retinas from a mouse model of CSNB2, G305X, which we generated by targeted disruption
Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603
of the Cacna1f gene. In the studies reported here, we compared synaptic structure in the outer plexiform layer (OPL) of agematched mutant and control animals at postnatal days 10 and 14, the peak period for photoreceptor synaptogenesis in normal mice. Results: Transmission electron microscopy indicated that presynaptic specializations of photoreceptors (cone pedicles, rod spherules, synaptic ribbons) and synaptic contacts between photoreceptors and second-order neurons (synaptic membrane specializations, invagination of postsynaptic processes) were rare, and highly abnormal when present, in the retinas of mutant compared with age-matched control animals. Immunocytochemistry for bassoon and ribeye showed reduced expression of bassoon at P10 and P14 and the presence of ribeye in the ONL at P14. Additionally, staining for PKC␣ and calbindin at P14 showed the extension of bipolar and horizontal-cell processes into the ONL respectively. Discussion: These observations show that normal photoreceptor synapses fail to develop, rather than first develop and subsequently degenerate, in the Cacna1f-mutant retina. Therefore, these results suggest a key role for Cav 1.4 in photoreceptor synaptogenesis in the mouse retina, and potentially provide specific clues to the functional defect of the retina in some CSNB2 patients. Acknowledgement Supported by FFB-Canada and CIHR.
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alterations in amplitude and/or time courses during development, and if so, what are the underlying mechanisms for these changes. In this study, we investigated the developmental properties of AMPA receptor mediated miniature excitatory postsynaptic currents (AMPAR-mEPSCs) in zebrafish M-neurons by means of whole cell patch-clamp recordings. We recorded AMPARmEPSCs in zebrafish ranging in age from 36 h postfertilization (hpf) to 72 hpf. Events were isolated and recorded in the presence of tetrodotoxin (TTX), strychnine and picrotoxin, and were completely blocked by the AMPA-receptor blocker, 6-cyano7-nitroquinoxaline-2,3-dione (CNQX) thereby confirming their identity. AMPAR-mEPSCs showed an increase in frequency during development but no significant change in amplitude. Analysis of the kinetic properties of the mEPSCs demonstrated a developmental speeding in their decay kinetics, but no significant change in their activation kinetics; the 20–80% rise time remained constant. These results indicate a change in the properties of AMPAR-mEPSCs as the M-neuron matures and that the changes predominantly occur between 36 and 48 hpf. Pharmacological and electrophysiological tools are currently being used to assess the possibility of subunit switches in the development of AMPA-containing synapses. Keywords: AMPA receptor; EPSC; Zebrafish maunther neurons; Development doi:10.1016/j.ijdevneu.2006.09.131 [P70]
Keywords: Cacna1f; CSNB; Synaptogenesis; Knockout Reference Anon., 2005. Hum. Mol. Genet. 14, 3035–3046.
Regulation of cortical synapse maturation by the NR2B subunit of the NMDA receptor B. Hall ∗ , B. Ripley, A. Ghosh University of California, San Diego, USA
doi:10.1016/j.ijdevneu.2006.09.130 [P69] AMPA receptor-mediated currents in developing zebrafish mauthner neurons S.A. Patten ∗ , D. Ali University of Alberta, Canada Glutamate, the major excitatory transmitter in the vertebrate CNS, activates two main classes of ionotropic recepors: NMDA and AMPA/kainate. The amplitude and time course of the synaptic currents mediated via these receptors are major determinants of cell excitability and synaptic information efficacy. Therefore, changes in the nature of these currents will impact upon spike output. It is well established that during synaptic development the amplitude and time courses of synaptic currents change, but the mechanisms that underlie these changes for AMPA receptor-mediated currents, remain controversial. We undertook the present study to determine if the AMPA receptor-mediated currents on zebrafish Mauthner neurons (M-neuron), experience
Cortical development involves the formation and maturation of billions of synapses, requiring precise regulation over the expression and insertion of distinct receptor subunits at synaptic sites. Maturation of excitatory synapses is accompanied by a change in the contribution of NMDA receptor subunits from predominantly NR2B to NR2A as well as increased incorporation of AMPA receptors. While these changes appear to be ubiquitous at excitatory cortical synapses it is unclear what role the NR2B subunit might play in directly orchestrating this maturation. Whole-cell recordings revealed that spontaneous synaptic activity in E18 derived cortical cultures arises after ≈7 days in vitro (DIV). In 2B null cultures both frequency and amplitude of AMPA-mediated synaptic currents were enhanced compared with littermate controls. Enrichment of synaptic AMPARs was not accompanied by an increase in surface expression of AMPAR subunits (GluR1/GluR2) as assessed by immunohistochemical staining, however the percentage of synapses containing GluR1 or GluR2 was ≈2 fold higher than controls. Co-culturing wildtype and 2B null neurons demonstrated that
