Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies

Electrophysiological recordings from the Drosophila giant fiber system (GFS).

INTRODUCTION: The giant fiber system (GFS) of Drosophila is a well-characterized neuronal circuit that mediates the escape response in the fly. It is one of the few adult neural circuits from which electrophysiological recordings can be made routinely. This protocol describes a simple procedure for stimulating the giant fiber neurons directly in the brain of the adult fly and obtaining recordings from the output muscles of the GFS: the tergotrochanteral "jump" muscle (TTM) and the large indirect flight muscles (dorsal longitudinal muscles, or DLMs). It is a relatively noninvasive method that allows the investigator to stimulate the giant fibers in the brain and assay the function of several central synapses within this neural circuit by recording from the thoracic musculature

New tools for targeted disruption of cholinergic synaptic transmission in Drosophila melanogaster.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. The α7 subtype of nAChRs is involved in neurological pathologies such as Parkinson's disease, Alzheimer's disease, addiction, epilepsy and autism spectrum disorders. The Drosophila melanogaster α7 (Dα7) has the closest sequence homology to the vertebrate α7 subunit and it can form homopentameric receptors just as the vertebrate counterpart. The Dα7 subunits are essential for the function of the Giant Fiber circuit, which mediates the escape response of the fly. To further characterize the receptor function, we generated different missense mutations in the Dα7 nAChR's ligand binding domain. We characterized the effects of targeted expression of two UAS-constructs carrying a single mutation, D197A and Y195T, as well as a UAS-construct carrying a triple D77T, L117Q, I196P mutation in a Dα7 null mutant and in a wild type background. Expression of the triple mutation was able to restore the function of the circuit in Dα7 null mutants and had no disruptive effects when expressed in wild type. In contrast, both single mutations severely disrupted the synaptic transmission of Dα7-dependent but not glutamatergic or gap junction dependent synapses in wild type background, and did not or only partially rescued the synaptic defects of the null mutant. These observations are consistent with the formation of hybrid receptors, consisting of D197A or Y195T subunits and wild type Dα7 subunits, in which the binding of acetylcholine or acetylcholine-induced conformational changes of the Dα7 receptor are altered and causes inhibition of cholinergic responses. Thus targeted expression of D197A or Y195T can be used to selectively disrupt synaptic transmission of Dα7-dependent synapses in neuronal circuits. Hence, these constructs can be used as tools to study learning and memory or addiction associated behaviors by allowing the manipulation of neuronal processing in the circuits without affecting other cellular signaling

Differential effects of human L1CAM mutations on complementing guidance and synaptic defects in Drosophila melanogaster.

A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin-moesin-radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis

Making an escape: development and function of the Drosophila giant fibre system

Flies escape danger by jumping into the air and flying away. The giant fibre system (GFS) is the neural circuit that mediates this simple behavioural response to visual stimuli. The sensory signal is received by the giant fibre and relayed to the leg and wing muscle motorneurons. Many of the neurons in the Drosophila GFS are uniquely identifiable and amenable to cell biological, electrophysiological and genetic studies. Here we review the anatomy and development of this system and highlight its utility for studying many aspects of nervous system biology ranging from neural development and synaptic plasticity to the aetiology of neural disorder

In vivo and in vitro testing of native α-conotoxins from the injected venom of Conus purpurascens

α-Conotoxins inhibit nicotinic acetylcholine receptors (nAChRs) and are used as probes to study cholinergic pathways in vertebrates. Model organisms, such as Drosophila melanogaster, express nAChRs in their CNS that are suitable to investigate the neuropharmacology of α-conotoxins in vivo. Here we report the paired nanoinjection of native α-conotoxin PIA and two novel α-conotoxins, PIC and PIC[O7], from the injected venom of Conus purpurascens and electrophysiological recordings of their effects on the giant fiber system (GFS) of D. melanogaster and heterologously expressed nAChRs in Xenopus oocytes. α-PIA caused disruption of the function of giant fiber dorsal longitudinal muscle (GF-DLM) pathway by inhibiting the Dα7 nAChR a homolog to the vertebrate α7 nAChR, whereas PIC and PIC[O7] did not. PIC and PIC[O7] reversibly inhibited ACh-evoked currents mediated by vertebrate rodent (r)α1β1δγ, rα1β1δε and human (h)α3β2, but not hα7 nAChR subtypes expressed in Xenopus oocytes with the following selectivity: rα1β1δε \u3e rα1β1δγ ≈ hα3β2 \u3e \u3e hα7. Our study emphasizes the importance of loop size and α-conotoxin sequence specificity for receptor binding. These studies can be used for the evaluation of the neuropharmacology of novel α-conotoxins that can be utilized as molecular probes for diseases such as, Alzheimer\u27s, Parkinson\u27s, and cancer

Functional characterization of GF-TTMn synaptic defects of mutant L1CAM protein expressions in <i>nrg¹⁴</i>;P[nrg180^ΔFIGQY] background.

<p>(A) Schematic of the GF to TTM circuit in Drosophila. For electrophysiological recordings, tungsten electrodes were inserted into the brain for the GF stimulation. The output of the neuronal circuit was recorded from the TTM muscles with glass electrodes. Sample electrophysiological recordings from GF-TTM pathway. In positive control animals (<i>nrg¹⁴</i>/>;P[nrg180^ΔFIGQY]/OK307,UAS-L1) the GF-TTM pathway was able to follow reliably at a one-to-one ratio when the GFs were stimulated in the brain with ten stimuli given at 100 Hz and the Response Latency was 0.87 ms (dashed grey line). In animals, which express L1-C264Y and L1-H210Q protein in a <i>nrg¹⁴</i>/>;P[nrg180^ΔFIGQY] background, only few or no responses (asterisks) could be recorded when the GFs were stimulated in the brain. The Response Latency of all responses was increased. (B) Scatter plots of Following Frequencies. Following Frequencies were only significantly increased (** = p<0.001, * = p value ≤0.05, Mann-Whitney Rank Sum Test) in <i>nrg¹⁴</i>;P[nrg180^ΔFIGQY] in animals that expressed UAS-L1, UAS-L1-E309K, UAS-L1-L120V and UAS-L1-4A driven by OK307. (C) Scatter plots of Response Latencies. In some animals no responses could be recorded from the TTM when the GF was stimulated in the brain (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076974#pone-0076974-t001" target="_blank">Table 1</a>) and thus were not included in the scatter plot. However, in most responding animals that had a decreased ability to follow at a one-to-one ratio at 100 Hz, the Response Latency was increased indicating a reduction in synaptic strength for the GF to TTMn connection.</p

Anatomical characterization of GF guidance defects of mutant L1CAM protein expressions in <i>nrg⁸⁴⁹</i> background.

<p>(A) Schematic of the giant fiber (GF) to TTM (Tergo-trochanteral Muscle) circuit in Drosophila. The GF soma in the brain extends an axon into the second neuromere of the VNC, where it synapses with the Tergo-trochanteral motor neuron (TTMn), which itself innervates the TTM. Rectangle highlights the brain region depicted in B-F. (B) GF anatomy in the brain of a wild-type control fly. Scale bar represents 50 µm. (C) In a <i>nrg⁸⁴⁹</i> animal two GFs were seen to stall in the suboesophageal ganglion. (D) Expression of wild-type human L1CAM in the GF in the <i>nrg⁸⁴⁹</i> background rescued the axonal guidance defect. Expression of L1-Y1070C (E) or L1-H210Q (F) in the <i>nrg⁸⁴⁹</i> background did not rescue or only partially rescued the axonal guidance phenotype. (G) Bar graph shows the quantification of the axonal guidance defects. The rescue of the <i>nrg⁸⁴⁹</i> guidance phenotype by expression of UAS-nrg¹⁸⁰, UAS-L1 and the various mutant L1-constructs represented in percent values. The significant differences (Chi-square analysis, p≤0.05) between Nrg¹⁸⁰, L1, L1-L120V and L1-E309K expression in the <i>nrg⁸⁴⁹</i> background and negative control flies (<i>nrg⁸⁴⁹</i>) are indicated by asterisks.</p

L1-type CAM mutations and their expression in the Drosophila nervous system.

<p>(A) Domain structure of L1CAM showing the locations (asterisks) of different pathological mutations. Mutations L120V, R184Q, H210Q, C264Y, and E309K are in the extracellular Immunoglobulin (Ig) like domains, while Y1070C is in the fifth Fibronectin domain. In the L1-4A protein four amino acids in juxtamembrane region (<u>K</u>GG<u>KY</u>S<u>V</u>) are replaced by alanine residues. The L1-1180 protein lacks part of its C-terminus including the FIGQY motif. The domain structure of Neuroglian (Nrg) indicates the site of the S213L mutation in the second Ig like domain of the <i>nrg⁸⁴⁹</i> protein. The genomic pacman P[nrg180^ΔFIGQY] rescue construct in the <i>nrg¹⁴</i> null mutant background expresses neuronal Nrg¹⁸⁰ that lacks the FIGQY motif. (B) Western blots of pupal stages (in %) and adult (1 and 8 days) flies expressing human UAS-human L1CAM with the OK307 Gal4-driver. Antibodies against the intracellular domain of L1CAM detected 200 kDa and 65 kDa bands. Proteolytic L1CAM cleavage increases with the maturation of the fly nervous system during pupal development and reaches its maximum in the adult. (C) Western blot of transgenic expression of wild-type and mutant UAS-L1 constructs in the wild-type background with the OK307 Gal-4 driver. With an antibody against the intracellular domain of L1CAM a 200 kDa band and 65 kDa band was detected for all L1CAM-constructs except for UAS-L1-C264Y. Only the full length 200 kDa L1CAM form was detected for this construct. No L1CAM protein was detectable in OK307 flies, which served as a negative control. Anti-actin labeling was used as a loading control.</p