Richard A. Baines

Calcium Influx via TRP Channels Is Required to Maintain PIP2 Levels in Drosophila Photoreceptors

The trp (transient receptor potential) gene encodes a Ca2+ channel responsible for the major component of the phospholipase C (PLC) mediated light response in Drosophila. In trp mutants, maintained light leads to response decay and temporary total loss of sensitivity (inactivation). Using genetically targeted PIP2-sensitive inward rectifier channels (Kir2.1) as biosensors, we provide evidence that trp decay reflects depletion of PIP2. Two independent mutations in the PIP2 recycling pathway (rdgB and cds) prevented recovery from inactivation. Abolishing Ca2+ influx in wild-type photoreceptors mimicked inactivation, while raising Ca2+ by blocking Na+/Ca2+ exchange prevented inactivation in trp. The results suggest that Ca2+ influx prevents PIP2 depletion by inhibiting PLC activity and facilitating PIP2 recycling. Without this feedback one photon appears sufficient to deplete the phosphoinositide pool of approximately 4 microvilli.

Electrophysiological development of central neurons in the Drosophila embryo.

In this study, we describe the development of electrical properties of Drosophila embryonic central neurons in vivo. Using whole-cell voltage clamp, we describe the onset of expression of specific voltage- and ligand-gated ionic currents and the first appearance of endogenous and synaptic activity. The first currents occur during midembryogenesis [late stage 16, 13–14 hr after egg laying (AEL)] and consist of a delayed outward potassium current (IK) and an acetylcholine-gated inward cation current (IACh). As development proceeds, other voltage-activated currents arise sequentially. An inward calcium current (ICa) is first observed at 15 hr AEL, an inward sodium current (INa) at 16 hr AEL, and a rapidly inactivating outward potassium current (IA) at 17 hr AEL. The inward calcium current is composed of at least two individual and separable components that exhibit small temporal differences in their development. Endogenous activity is first apparent at 15 hr AEL and consists of small events (peak amplitude, 5 pA) that probably result from the random opening of relatively few numbers of ion channels. At 16 hr AEL, discrete (10–15 msec duration) currents that exhibit larger amplitude (25 pA maximum) and rapid activation but slower inactivation first appear. We identify these latter currents as EPSCs, an indication that functional synaptic transmission is occurring. In the neurons from which we record, action potentials first occur at 17 hr AEL. This study is the first to record from Drosophila embryonic central neurons in vivo and makes possible future work to define the factors that shape the electrical properties of neurons during development.

Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes

In most multicellular organisms direct cell–cell communication is mediated by the intercellular channels of gap junctions. These channels allow the exchange of ions and molecules that are believed to be essential for cell signalling during development and in some differentiated tissues. Proteins called connexins, which are products of a multigene family, are the structural components of vertebrate gap junctions,. Surprisingly, molecular homologues of the connexins have not been described in any invertebrate. A separate gene family, which includes the Drosophila genes shaking-B and l(1)ogre, and the Caenorhabditis elegans genes unc-7 and eat-5, encodes transmembrane proteins with a predicted structure similar to that of the connexins. shaking-B and eat-5 are required for the formation of functional gap junctions,. To test directly whether Shaking-B is a channel protein, we expressed it in paired Xenopus oocytes. Here we show that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. To our knowledge, this is the first structural component of an invertebrate gap junction to be characterized.

Regulation of Neuronal Excitability through Pumilio-Dependent Control of a Sodium Channel Gene

Dynamic changes in synaptic connectivity and strength, which occur during both embryonic development and learning, have the tendency to destabilize neural circuits. To overcome this, neurons have developed a diversity of homeostatic mechanisms to maintain firing within physiologically defined limits. In this study, we show that activity-dependent control of mRNA for a specific voltage-gated Na+ channel [encoded by paralytic (para)] contributes to the regulation of membrane excitability in Drosophila motoneurons. Quantification of para mRNA, by real-time reverse-transcription PCR, shows that levels are significantly decreased in CNSs in which synaptic excitation is elevated, whereas, conversely, they are significantly increased when synaptic vesicle release is blocked. Quantification of mRNA encoding the translational repressor pumilio (pum) reveals a reciprocal regulation to that seen for para. Pumilio is sufficient to influence para mRNA. Thus, para mRNA is significantly elevated in a loss-of-function allele of pum (pumbemused), whereas expression of a full-length pum transgene is sufficient to reduce para mRNA. In the absence of pum, increased synaptic excitation fails to reduce para mRNA, showing that Pum is also necessary for activity-dependent regulation of para mRNA. Analysis of voltage-gated Na+ current (INa) mediated by para in two identified motoneurons (termed aCC and RP2) reveals that removal of pum is sufficient to increase one of two separable INa components (persistent INa), whereas overexpression of a pum transgene is sufficient to suppress both components (transient and persistent). We show, through use of anemone toxin (ATX II), that alteration in persistent INa is sufficient to regulate membrane excitability in these two motoneurons.

The roles of the dystrophin-associated glycoprotein complex at the synapse.

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene and is characterized by progressive muscle wasting. A number of Duchenne patients also present with mental retardation. The dystrophin protein is part of the highly conserved dystrophin-associated glycoprotein complex (DGC) which accumulates at the neuromuscular junction (NMJ) and at a variety of synapses in the peripheral and central nervous systems. Many years of research into the roles of the DGC in muscle have revealed its structural function in stabilizing the sarcolemma. In addition, the DGC also acts as a scaffold for various signaling pathways. Here, we discuss recent advances in understanding DGC roles in the nervous system, gained from studies in both vertebrate and invertebrate model systems. From these studies, it has become clear that the DGC is important for the maturation of neurotransmitter receptor complexes and for the regulation of neurotransmitter release at the NMJ and central synapses. Furthermore, roles for the DGC have been established in consolidation of long-term spatial and recognition memory. The challenges ahead include the integration of the behavioral and mechanistic studies and the use of this information to identify therapeutic targets.

Pumilio Binds para mRNA and Requires Nanos and Brat to Regulate Sodium Current in Drosophila Motoneurons

Homeostatic regulation of ionic currents is of paramount importance during periods of synaptic growth or remodeling. Our previous work has identified the translational repressor Pumilio (Pum) as a regulator of sodium current (I Na) and excitability in Drosophila motoneurons. In this current study, we show that Pum is able to bind directly the mRNA encoding the Drosophila voltage-gated sodium channel paralytic (para). We identify a putative binding site for Pum in the 3′ end of the para open reading frame (ORF). Characterization of the mechanism of action of Pum, using whole-cell patch clamp and real-time reverse transcription-PCR, reveals that the full-length protein is required for translational repression of para mRNA. Additionally, the cofactor Nanos is essential for Pum-dependent para repression, whereas the requirement for Brain Tumor (Brat) is cell type specific. Thus, Pum-dependent regulation of I Na in motoneurons requires both Nanos and Brat, whereas regulation in other neuronal types seemingly requires only Nanos but not Brat. We also show that Pum is able to reduce the level of nanos mRNA and as such identify a potential negative-feedback mechanism to protect neurons from overactivity of Pum. Finally, we show coupling between I Na (para) and I K (Shal) such that Pum-mediated change in para results in a compensatory change in Shal. The identification of para as a direct target of Pum represents the first ion channel to be translationally regulated by this repressor and the location of the binding motif is the first example in an ORF rather than in the canonical 3′-untranslated region of target transcripts.

Postsynaptic expression of tetanus toxin light chain blocks synaptogenesis in Drosophila.

During the development of the nervous system embryonic neurons are incorporated into neural networks that underlie behaviour. For example, during embryogenesis in Drosophila, motor neurons in every body segment are wired into the circuitry that drives the simple peristaltic locomotion of the larva. Very little is known about the way in which the necessary central synapses are formed in such a network or how their properties are controlled. One possibility is that presynaptic and postsynaptic elements form relatively independently of each other. Alternatively, there might be an interaction between presynaptic and postsynaptic neurons that allows for adjustment and plasticity in the embryonic network. Here we have addressed this issue by analysing the role of synaptic transmission in the formation of synaptic inputs onto identified motorneurons as the locomotor circuitry is assembled in the Drosophila embryo. We targeted the expression of tetanus toxin light chain (TeTxLC) to single identified neurons using the GAL4 system. TeTxLC prevents the evoked release of neurotransmitter by enzymatically cleaving the synaptic-vesicle-associated protein neuronal-Synaptobrevin (n-Syb) [1]. Unexpectedly, we found that the cells that expressed TeTxLC, which were themselves incapable of evoked release, showed a dramatic reduction in synaptic input. We detected this reduction both electrophysiologically and ultrastructurally.

Alternative splicing in the voltage-gated sodium channel DmNav regulates activation, inactivation, and persistent current.

Diversity in neuronal signaling is a product not only of differential gene expression, but also of alternative splicing. However, although recognized, the precise contribution of alternative splicing in ion channel transcripts to channel kinetics remains poorly understood. Invertebrates, with their smaller genomes, offer attractive models to examine the contribution of splicing to neuronal function. In this study we report the sequencing and biophysical characterization of alternative splice variants of the sole voltage-gated Na+ gene (DmNav, paralytic), in late-stage embryos of Drosophila melanogaster. We identify 27 unique splice variants, based on the presence of 15 alternative exons. Heterologous expression, in Xenopus oocytes, shows that alternative exons j, e, and f primarily influence activation kinetics: when present, exon f confers a hyperpolarizing shift in half-activation voltage (V1/2), whereas j and e result in a depolarizing shift. The presence of exon h is sufficient to produce a depolarizing shift in the V1/2 of steady-state inactivation. The magnitude of the persistent Na+ current, but not the fast-inactivating current, in both oocytes and Drosophila motoneurons in vivo is directly influenced by the presence of either one of a pair of mutually exclusive, membrane-spanning exons, termed k and L. Transcripts containing k have significantly smaller persistent currents compared with those containing L. Finally, we show that transcripts lacking all cytoplasmic alternatively spliced exons still produce functional channels, indicating that splicing may influence channel kinetics not only through change to protein structure, but also by allowing differential modification (i.e., phosphorylation, binding of cofactors, etc.). Our results provide a functional basis for understanding how alternative splicing of a voltage-gated Na+ channel results in diversity in neuronal signaling.

Miniature Neurotransmission Regulates Drosophila Synaptic Structural Maturation

Summary Miniature neurotransmission is the transsynaptic process where single synaptic vesicles spontaneously released from presynaptic neurons induce miniature postsynaptic potentials. Since their discovery over 60 years ago, miniature events have been found at every chemical synapse studied. However, the in vivo necessity for these small-amplitude events has remained enigmatic. Here, we show that miniature neurotransmission is required for the normal structural maturation of Drosophila glutamatergic synapses in a developmental role that is not shared by evoked neurotransmission. Conversely, we find that increasing miniature events is sufficient to induce synaptic terminal growth. We show that miniature neurotransmission acts locally at terminals to regulate synapse maturation via a Trio guanine nucleotide exchange factor (GEF) and Rac1 GTPase molecular signaling pathway. Our results establish that miniature neurotransmission, a universal but often-overlooked feature of synapses, has unique and essential functions in vivo.

Synaptic Strengthening Mediated by Bone Morphogenetic Protein-Dependent Retrograde Signaling in the Drosophila CNS

Retrograde signaling is an essential component of synaptic development and physiology. Previous studies show that bone morphogenetic protein (BMP)-dependent retrograde signaling is required for the proper development of the neuromuscular junction (NMJ) in Drosophila. These studies, moreover, raised the significant possibility that the development of central motor circuitry might similarly be reliant on such signaling. To test this hypothesis, retrograde signaling between postsynaptic motoneurons and their presynaptic interneurons is examined. Postsynaptic expression of an adenylate cyclase encoded by rutabaga (rut), is sufficient to strengthen synaptic transmission at these identified central synapses. Results are presented to show that the underlying mechanism is dependent on BMP retrograde signaling. Thus, presynaptic expression of an activated TGF-β receptor, thickvien (tkv), or postsynaptic expression of a TGF-β ligand, glass-bottom boat (gbb), is sufficient to phenocopy strengthening of synaptic transmission. In the absence of gbb, endogenous synaptic transmission is significantly weakened and, moreover, postsynaptic overexpression of rut is unable to potentiate synaptic function. Potentiation of presynaptic neurotransmitter release, mediated by increased postsynaptic expression of gbb, is dependent on normal cholinergic activity, indicative that either the secretion of this retrograde signal, or its transduction, is activity dependent. Thus, in addition to the development of the NMJ and expression of myoactive FMRFamide-like peptides in specific central neurons, the results of the present study indicate that this retrograde signaling cascade also integrates the development and function of central motor circuitry that controls movement in Drosophila larvae.