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In JoVE (1)

Other Publications (8)

Articles by Jens Rister in JoVE

 JoVE Neuroscience

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

1Department of Biology, New York University

JoVE 4347

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Other articles by Jens Rister on PubMed

Flies Lacking All Synapsins Are Unexpectedly Healthy but Are Impaired in Complex Behaviour

Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine-regulate neurotransmitter release by phosphorylation-dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock-out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock-out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified 'model' synapse of the larval nerve muscle preparation revealed no difference between wild-type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild-type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.

Differential Potencies of Effector Genes in Adult Drosophila

The GAL4/UAS gene expression system in Drosophila has been crucial in revealing the behavioral significance of neural circuits. Transgene products that block neurotransmitter release and induce cell death have been proved to inhibit neural function powerfully. Here we compare the action of the five effector genes shibire(ts1), Tetanus toxin light chain (TNT), reaper, Diphtheria toxin A-chain (DTA), and inwardly rectifying potassium channel (Kir2.1) and show differences in their efficiency depending on the target cells and the timing of induction. Specifically, effectors blocking neuronal transmission or excitability led to adult-induced paralysis more efficiently than those causing cell ablation. We contrasted these differential potencies in adult to their actions during development. Furthermore, we induced TNT expression in the adult mushroom bodies. In contrast to the successful impairment in short-term olfactory memory by shibire(ts1), adult TNT expression in the same set of cells did not lead to any obvious impairment. Altogether, the efficiency of effector genes depends on properties of the targeted neurons. Thus, we conclude that the selection of the appropriate effector gene is critical for evaluating the function of neural circuits.

Distinct Functions of Neuronal Synaptobrevin in Developing and Mature Fly Photoreceptors

Neuronal synaptobrevin (n-Syb, alias VAMP2), a synaptic vesicle membrane protein with a central role in neurotransmission, is specifically cleaved by the light chain of tetanus neurotoxin (TNT) that is known to reliably block neuroexocytosis. Here, we study fly photoreceptors transmitting continuous, graded signals to first order interneurons in the lamina, and report consequences of targeted expression of TNT in these cells using the UAS/GAL4 driver/effector system. Expressing the toxin throughout photoreceptor development causes developmental, electrophysiological, and behavioral defects. These can be differentiated by confining toxin expression to shorter developmental periods. Applying a method for controlled temporal and spatial TNT expression, we found that in the early pupa it impaired the development of the retina; in the midpupa, during synapse formation TNT caused a severe hypoplasia of the lamina that persisted into adulthood and left the photoreceptor-interneuron synapses of the lamina without function. Finally, during adulthood TNT neither blocks synaptic transmission in photoreceptors nor depletes the cells of n-Syb. Our study suggests a novel, cell type-specific function of n-Syb in synaptogenesis and it distinguishes between two synapse types: TNT resistant and TNT sensitive ones. These results need to be taken into account if TNT is used for neural circuit analysis.

Dissection of the Peripheral Motion Channel in the Visual System of Drosophila Melanogaster

In the eye, visual information is segregated into modalities such as color and motion, these being transferred to the central brain through separate channels. Here, we genetically dissect the achromatic motion channel in the fly Drosophila melanogaster at the level of the first relay station in the brain, the lamina, where it is split into four parallel pathways (L1-L3, amc/T1). The functional relevance of this divergence is little understood. We now show that the two most prominent pathways, L1 and L2, together are necessary and largely sufficient for motion-dependent behavior. At high pattern contrast, the two pathways are redundant. At intermediate contrast, they mediate motion stimuli of opposite polarity, L2 front-to-back, L1 back-to-front motion. At low contrast, L1 and L2 depend upon each other for motion processing. Of the two minor pathways, amc/T1 specifically enhances the L1 pathway at intermediate contrast. L3 appears not to contribute to motion but to orientation behavior.

Distinct Roles for Two Histamine Receptors (hclA and HclB) at the Drosophila Photoreceptor Synapse

Histamine (HA) is the photoreceptor neurotransmitter in arthropods, directly gating chloride channels on large monopolar cells (LMCs), postsynaptic to photoreceptors in the lamina. Two histamine-gated channel genes that could contribute to this channel in Drosophila are hclA (also known as ort) and hclB (also known as hisCl1), both encoding novel members of the Cys-loop receptor superfamily. Drosophila S2 cells transfected with these genes expressed both homomeric and heteromeric histamine-gated chloride channels. The electrophysiological properties of these channels were compared with those from isolated Drosophila LMCs. HCLA homomers had nearly identical HA sensitivity to the native receptors (EC(50) = 25 microM). Single-channel analysis revealed further close similarity in terms of single-channel kinetics and subconductance states ( approximately 25, 40, and 60 pS, the latter strongly voltage dependent). In contrast, HCLB homomers and heteromeric receptors were more sensitive to HA (EC(50) = 14 and 1.2 microM, respectively), with much smaller single-channel conductances ( approximately 4 pS). Null mutations of hclA (ort(US6096)) abolished the synaptic transients in the electroretinograms (ERGs). Surprisingly, the ERG "on" transients in hclB mutants transients were approximately twofold enhanced, whereas intracellular recordings from their LMCs revealed altered responses with slower kinetics. However, HCLB expression within the lamina, assessed by both a GFP (green fluorescent protein) reporter gene strategy and mRNA tagging, was exclusively localized to the glia cells, whereas HCLA expression was confirmed in the LMCs. Our results suggest that the native receptor at the LMC synapse is an HCLA homomer, whereas HCLB signaling via the lamina glia plays a previously unrecognized role in shaping the LMC postsynaptic response.

The Neural Substrate of Spectral Preference in Drosophila

Drosophila vision is mediated by inputs from three types of photoreceptor neurons; R1-R6 mediate achromatic motion detection, while R7 and R8 constitute two chromatic channels. Neural circuits for processing chromatic information are not known. Here, we identified the first-order interneurons downstream of the chromatic channels. Serial EM revealed that small-field projection neurons Tm5 and Tm9 receive direct synaptic input from R7 and R8, respectively, and indirect input from R1-R6, qualifying them to function as color-opponent neurons. Wide-field Dm8 amacrine neurons receive input from 13-16 UV-sensing R7s and provide output to projection neurons. Using a combinatorial expression system to manipulate activity in different neuron subtypes, we determined that Dm8 neurons are necessary and sufficient for flies to exhibit phototaxis toward ultraviolet instead of green light. We propose that Dm8 sacrifices spatial resolution for sensitivity by relaying signals from multiple R7s to projection neurons, which then provide output to higher visual centers.

Deciphering the Genome's Regulatory Code: the Many Languages of DNA

The generation of patterns and the diversity of cell types in a multicellular organism require differential gene regulation. At the heart of this process are enhancers or cis-regulatory modules (CRMs), genomic regions that are bound by transcription factors (TFs) that control spatio-temporal gene expression in developmental networks. To date, only a few CRMs have been studied in detail and the underlying cis-regulatory code is not well understood. Here, we review recent progress on the genome-wide identification of CRMs with chromatin immunoprecipitation of TF-DNA complexes followed by microarrays (ChIP-on-chip). We focus on two computational approaches that have succeeded in predicting the expression pattern driven by a CRM either based on TF binding site preferences and their expression levels, or quantitative analysis of CRM occupancy by key TFs. We also discuss the current limits of these methods and highlight some of the key problems that have to be solved to gain a more complete understanding of the structure and function of CRMs.

The Retinal Mosaics of Opsin Expression in Invertebrates and Vertebrates

Color vision is found in many invertebrate and vertebrate species. It is the ability to discriminate objects based on the wavelength of emitted light independent of intensity. As it requires the comparison of at least two photoreceptor types with different spectral sensitivities, this process is often mediated by a mosaic made of several photoreceptor types. In this review, we summarize the current knowledge about the formation of retinal mosaics and the regulation of photopigment (opsin) expression in the fly, mouse, and human retina. Despite distinct evolutionary origins, as well as major differences in morphology and phototransduction machineries, there are significant similarities in the stepwise cell-fate decisions that lead from progenitor cells to terminally differentiated photoreceptors that express a particular opsin. Common themes include (i) the use of binary transcriptional switches that distinguish classes of photoreceptors, (ii) the use of gradients of signaling molecules for regional specializations, (iii) stochastic choices that pattern the retina, and (iv) the use of permissive factors with multiple roles in different photoreceptor types.

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