Retinal neural transmission represents a key function of the eye. Identifying the molecular components of this vital process is helped by studies of selected human genetic eye disorders. For example, mutations in the calcium channel subunit gene CACNA1F cause incomplete X-linked congenital stationary night blindness (CSNB2 or iCSNB), a human retinal disorder with abnormal electrophysiological response and visual impairments consistent with a retinal neurotransmission defect. To understand the subcellular basis of this retinal disorder, we generated a mouse with a loss-of-function mutation by inserting a self-excising Cre-lox-neo cassette into exon 7 of the murine orthologue, Cacna1f. Electroretinography of the mutant mouse revealed a scotopic a-wave of marginally reduced amplitude compared with the wild-type mouse and absence of the post-receptoral b-wave and oscillatory potentials. Cone ERG responses together with visual evoked potentials and multi-unit activity in the superior colliculus were also absent. Calcium imaging in Fluo-4 loaded retinal slices depolarized with KCl showed 90% less peak signal in the photoreceptor synapses of the Cacna1f mutant than in wild-type mice. The absence of post-receptoral ERG responses and the diminished photoreceptor calcium signals are consistent with a loss of Ca((2+)) channel function in photoreceptors. Immunocytochemistry showed no detectable Ca(v)1.4 protein in the outer plexiform layer of Cacna1f-mutant mice, profound loss of photoreceptor synapses, and abnormal dendritic sprouting of second-order neurons in the photoreceptor layer. Together, these findings in the Cacna1f-mutant mouse reveal that the Ca(v)1.4 calcium channel is vital for the functional assembly and/or maintenance and synaptic functions of photoreceptor ribbon synapses. Moreover, the outcome of this study provides critical clues to the pathophysiology of the human retinal channelopathy of X-linked incomplete CSNB.

The dendritic patterning of retinal horizontal cells has been shown to be specified by the cone photoreceptor afferents. The present investigation has addressed whether this specification is due to visually dependent synaptic transmission in the outer plexiform layer or to some other early, pre-visual, neural activity. Individually labeled horizontal cells from dark-reared mice, as well as from mice carrying a mutation in the Cacna1f gene, which encodes the pore-forming calcium channel subunit Ca(v)1.4, were assessed for various morphological features. The dark-reared mice showed no alteration in any of these features, despite showing a compromised maximal voltage response in the electroretinograms. The retinas of Cacna1f mutant mice, by contrast, showed conspicuous morphological changes that mimicked the effects observed previously in coneless transgenic mice. These changes were present as early as postnatal day 10, when the shape and density of the cone pedicles appeared normal. Ultrastructurally, however, the pedicles at this early stage, as well as in maturity, lacked synaptic ribbons and the invaginations associated with postsynaptic processes. These results suggest a role for this calcium channel subunit in ribbon assembly in addition to its role in modulating calcium influx and glutamate release. Together, they suggest a complex cascade of interactions between developing cone pedicles and horizontal cell dendrites involving early spontaneous activity, dendritic attraction, ribbon assembly, and pedicle invagination.

The Dutch-German Mennonites are a religious isolate with foundational roots in the 16th century. A tradition of endogamy, large families, detailed genealogical records, and a unique disease history all contribute to making this a valuable population for genetic studies. Such studies in the Dutch-German Mennonite population have already contributed to the identification of the causative genes in several conditions such as the incomplete form of X-linked congenital stationary night blindness (CSNB2; previously iCSNB) and hypophosphatasia (HOPS), as well as the discovery of founder mutations within established disease genes (MYBPC1, CYP17alpha). The Dutch-German Mennonite population provides a strong resource for gene discovery and could lead to the identification of additional disease genes with relevance to the general population. In addition, further research developments should enhance delivery of clinical genetic services to this unique community. In the current review we discuss 31 genetic conditions, including 17 with identified gene mutations, within the Dutch-German Mennonite population.

Studies of public views on stem cell research have traditionally focused on human embryonic stem cells. With more recent scientific research on developing other stem cell sources, a series of focus group studies was undertaken with Canadian adults to examine their views on different stem cell sources (adult, umbilical cord blood, human embryonic stem cells, somatic cell nuclear transfer or SCNT, and interspecies nuclear transfer, or iSCNT). Views on three different policy models--a permissive, middle-of-the-road and restrictive policy approach--were also explored. Participants were recruited from several different social groups including patients, young adults, seniors, members of two ethnic communities, and a mixed group of adults. Participants were generally supportive of the use of adult stem cell sources. While there was also majority support for the use of hESC and SCNT, this was conditional on strict regulatory oversight. There was also majority support for a permissive policy which allows research on hESC and SCNT. General themes that cut across different groups included the potential cost of new technologies to the health care system, issues around who would gain access to these technologies, and trust in the scientific establishment and regulatory systems. A diversity of viewpoints was found as participants justified their positions on stem cell sources and policy approaches, showing more complexity and nuance than has been generally portrayed.

Ca(v)1.4 plays a key role in photoreceptor synaptogenesis and synaptic function in mouse retina. Cacna1f ( G305X ) is a true knockout model for human CSNB2, with prominent defects in cone and rod function. Cacna1f ( nob2 ) is an incomplete knockout model for CSNB2, because alternative splicing in an ETn element leads to some full-length Ca(v)1.4 protein, and some cones surviving to drive photopic visual responses.

The pedicles of cone photoreceptors, labeled with an antibody to mouse cone arrestin (blue), stratify in the outer plexiform layer, where they form synapses with the dendrites of horizontal and bipolar cells. Those synaptic sites are evidenced by the co‐localization of the synaptic ribbon protein, piccolo (red), with the cone arrestin labeling. The remaining red profiles in the outer plexiform layer indicate the sites of the rod spherules. An antibody to cytochrome oxidase (green) labels the mitochondrion‐rich inner segments of all photoreceptors and yields punctate peri‐nuclear labeling within the outer nuclear layer. Many of these features of the outer retina are altered in the Cacna1f‐mutant retina, expressing a defective calcium channel subunit that prevents normal neurotransmission in the outer plexiform layer. J. Comp. Neurol. 506:745–758, 2007. © 2007 Wiley‐Liss, Inc.

The pedicles of cone photoreceptors, labeled with an antibody to mouse cone arrestin (blue), stratify in the outer plexiform layer, where they form synapses with the dendrites of horizontal and bipolar cells. Those synaptic sites are evidenced by the co‐localization of the synaptic ribbon protein, piccolo (red), with the cone arrestin labeling. The remaining red profiles in the outer plexiform layer indicate the sites of the rod spherules. An antibody to cytochrome oxidase (green) labels the mitochondrion‐rich inner segments of all photoreceptors and yields punctate peri‐nuclear labeling within the outer nuclear layer. Many of these features of the outer retina are altered in the Cacna1f‐mutant retina, expressing a defective calcium channel subunit that prevents normal neurotransmission in the outer plexiform layer. J. Comp. Neurol. 506:745–758, 2007. © 2007 Wiley‐Liss, Inc.

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 scd-2 (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. SCD-2 ubiquitination is currently being tested. We have shown that loss of scd-2 will partially rescue the synapse defects of fsn-1 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.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-CreER T2 ; 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 ge...