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R A Baines

Publications and source records attributed to R A Baines.

15 recordsLinked to original sources
PubMed2001

Altered electrical properties in Drosophila neurons developing without synaptic transmission.

We examine the role of synaptic activity in the development of identified Drosophila embryonic motorneurons. Synaptic activity was blocked by both pan-neuronal expression of tetanus toxin light chain (TeTxLC) and by reduction of acetylcholine (ACh) using a temperature-sensitive allele of choline acetyltransferase (Cha(ts2)). In the absence of synaptic activity, aCC and RP2 motorneurons develop with an apparently normal morphology and retain their capacity to form synapses. However, blockade of synaptic transmission results in significant changes in the electrical phenotype of these neurons. Specifically, increases are seen in both voltage-gated inward Na(+) and voltage-gated outward K(+) currents. Voltage-gated Ca(2+) currents do not change. The changes in conductances appear to promote neuron excitability. In the absence of synaptic activity, the number of action potentials fired by a depolarizing ramp (-60 to +60 mV) is increased and, in addition, the amplitude of the initial action potential fired is also significantly larger. Silencing synaptic input to just aCC, without affecting inputs to other neurons, demonstrates that the capability to respond to changing levels of synaptic excitation is intrinsic to these neurons. The alteration to electrical properties are not permanent, being reversed by restoration of normal synaptic function. Whereas our data suggest that synaptic activity makes little or no contribution to the initial formation of embryonic neural circuits, the electrical development of neurons that constitute these circuits seems to depend on a process that requires synaptic activity.

Acetylcholine↗
PubMed2001

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

Gap-Junctional communication between developing Drosophila muscles is essential for their normal development.

Recent experiments have demonstrated that a family of proteins, known as the innexins, are structural components of invertebrate gap junctions. The shaking-B (shak-B) locus of Drosophila encodes two members of this emerging family, Shak-B(lethal) and Shak-B(neural). This study focuses on the role of Shak-B gap junctions in the development of embryonic and larval muscle. During embryogenesis, shak-B transcripts are expressed in a subset of the somatic muscles; expression is strong in ventral oblique muscles (VO4-6) but only weak in ventral longitudinals (VL3 and 4). Carboxyfluorescein injected into VO4 of wild-type early stage 16 embryos spreads, via gap junctions, to label adjacent muscles, including VL3 and 4. In shak-B2 embryos (in which the shak-B(neural) function is disrupted), dye injected into VO4 fails to spread into other muscles. In the first instar larva, when dye coupling between muscles is no longer present, another effect of the shak-B2 mutation is revealed by whole-cell voltage clamp. In a calcium-free saline, only two voltage-activated potassium currents are present in wild-type muscles; a fast IA and a slow IK current. In shak-B2 larvae, these two currents are significantly reduced in magnitude in VO4 and 5, but remain normal in VL3. Expression of shak-B(neural) in a shak-B2 background fully rescues both dye coupling in embryonic muscle and whole-cell currents in first instar VO4 and 5. Our observations show that Shak-B(neural) is one of a set of embryonic gap-junction proteins, and that it is required for the normal temporal development of potassium currents in some larval muscles.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1996

Selective activity of a proctolin analogue reveals the existence of two receptor subtypes.

1. The neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) both potentiates neurally evoked contractions and causes contractures of insect skeletal muscle. In the hindleg extensor tibiae muscle of the locust, Schistocerca gregaria, the proctolin analogue [Afb (p-NO2)2]-proctolin is also able to potentiate neurally evoked contractions but is approximately 1,000-fold less effective in evoking contractures. 2. Proctolin and [Afb (p-NO2)2]-proctolin are equipotent in their ability to elevate the second-messenger inositol trisphosphate in isolated extensor tibiae muscle fiber membranes. 3. [Afb (p-NO2)2]-proctolin is approximately 1,000-fold less effective than proctolin in reducing the resting potassium conductance (GK) in extensor tibiae fibers. 4. We conclude that the action of proctolin on the extensor tibiae muscle is mediated by at least two receptor subtypes and that [Afb (p-NO2)2]-proctolin acts selectively on the receptor that potentiates neurally evoked contractions.

Animals↗
PubMed1995

Analysis of the peptide content of the locust vasopressin-like immunoreactive (VPLI) neurons.

Isolated cell bodies of the locust vasopressin-like immunoreactive (VPLI) neurons, analyzed by HPLC separation and radioimmune assay, contain three arginine vasopressin-like peptides: a previously identified monomer (Fl, Cys-Leu-Ile-Thr-Asn-Cys-Pro-Arg-Gly-NH2) and its antiparallel homodimer (F2), but also the previously unreported parallel homodimer (PDm). VPLI neuron activity significantly reduces the level of cAMP in the CNS. Of the three synthetic peptides, only the monomer (F1, 10(-8) and 10(-6) M) is capable of inhibiting a forskolin-stimulated increase in cAMP in isolated neural membranes. The antiparallel (F2) and parallel dimers (PDm) of this peptide have no effect on this second messenger.

Amino Acid Sequence↗
PubMed1994

Pharmacological analysis of the cholinergic input to the locust VPLI neuron from an extraocular photoreceptor system.

1. The brain of the locust contains an extraocular photoreceptor (EOP), which provides the major synaptic excitation to the vasopressin-like immunoreactive (VPLI) interneuron of the suboesophageal ganglion. Although the precise location of the EOP remains unknown, its activity can be determined indirectly by intracellular recording from the VPLI neuron. The excitatory drive to the VPLI neuron occurs only in darkness and is absent in the light. 2. The EOP is preferentially sensitive to light of wavelength 494 +/- 7 (SD) nm (blue-green) and has an absorption spectrum characteristic of a rhodopsin-like photopigment. 3. In the presence of high divalent saline (20 mM Ca2+ and Mg2+), the VPLI neuron receives excitatory input in the light. This indicates that the excitatory input to the VPLI neuron is from a tonically active descending input, which normally is inhibited by the light-induced activation of the presynaptic EOP. 4. Stimulation of the connectives while recording the resultant excitatory postsynaptic potential (EPSP) evoked in VPLI shows that the descending input projects beyond the suboesophageal ganglion, extending as far as the metathoracic ganglion. 5. Pharmacological analysis shows that the descending input to the VPLI neuron is cholinergic: acetylcholine (ACh) strongly depolarizes the neuron and eserine, an ACh esterase inhibitor, markedly potentiates the synaptic excitation of the VPLI neuron. 6. Nicotinic and muscarinic receptor antagonists show that the excitation of VPLI consists of two pharmacologically discrete components. Nicotinic ACh receptors mediate a fast depolarization, whereas muscarinic ACh receptors evoke a more sustained depolarization. Accordingly, both a fast and slow depolarization can be evoked selectively in VPLI by direct application of either nicotine or muscarine. 7. Voltage-clamp analysis shows that the fast EPSP evoked current is similar to that produced by nicotine in that it decreases linearly with membrane depolarization. The current associated with the sustained depolarization is similar to that evoked by muscarine, increasing nonlinearly with membrane depolarization. 8. Activity of the descending input, or application of muscarine, lowers the spike-initiation threshold of the VPLI neuron, thereby increasing its excitability. 9. It is concluded that the presence of two ACh receptor subtypes act synergistically to allow continuous activity of the VPLI neuron for sustained periods (i.e., throughout the hours of darkness).

Acetylcholine↗
PubMed1992

Comparative studies on the mode of action of proctolin and phorbol-12,13-dibutyrate in their ability to contract the locust mandibular closer muscle.

The role of proctolin has been further investigated in the locust (Locusta migratoria) mandibular closer muscles. Radioactive calcium uptake measurements were made using protease-dissociated muscle cells. Both the phorbol ester, phorbol-12,13-dibutyrate, and proctolin produce tonic contractions which are associated with the influx of extracellular calcium. The thresholds for proctolin and the phorbol ester to contract the muscle were 1-10 nM and 10-100nM, respectively, while their respective thresholds for evoking measurable calcium influx into the muscle cells were 0.1-1 nM for proctolin, and 0.1-1 pM for phorbol-12,13-dibutyrate. The effect of phorbol-12,13-dibutyrate is blocked by a number of protein kinase inhibitors (at a concentration of 0.1 mM), suggesting that an activation of a protein kinase can lead to calcium influx. These inhibitors, however, do not block the effect of proctolin, indicating that these two compounds work through different pathways, possibly converging on the same final target. In light of this finding, a number of other compounds have been tested to try to ascertain how proctolin mediates an increased calcium influx.

Amino Acid Sequence↗
PubMed1991

Pharmacological characterization of a 5-hydroxytryptamine-sensitive receptor/adenylate cyclase complex in the mandibular closer muscles of the cricket, Gryllus domestica.

The mandibular closer muscles of the cricket, Gryllus domestica, contain a 5-hydroxytryptamine (5-HT)-sensitive receptor that is coupled to adenylate cyclase. A structure-activity study of the 5-HT molecule indicates that the integrity of the ethylamine sidegroup and the presence of a negatively charged moiety at the 5 position (-OH, -OCH3) are essential for activity. A pharmacological profile is presented for this receptor. The receptor differs from any reported mammalian 5-HT receptor in that none of the mammalian agonists tested were effective. However, the mammalian antagonists for 5-HT receptors, spiperone, mianserin, and ketanserin as well as the anti-histaminic cyproheptadine were all effective antagonists in this preparation. Preliminary analysis of antagonism, particularly by spiperone, shows that these antagonists are probably acting non-competitively. On the basis of the pharmacological data, and comparisons with other insect systems, the 5-HT receptor present in the cricket mandibular muscles has been tentatively classified as 5-HT2-like.

Adenylyl Cyclases↗
PubMed1990

Proctolin in the innervation of the locust mandibular closer muscle modulates contractions through the elevation of inositol trisphosphate.

Extracts of the locust (Locusta migratoria) mandibular closer muscle separated on reverse-phase HPLC and tested for bio-activity on the locust oviduct contain a bio-active substance that coelutes with authentic proctolin. Furthermore, the effect on oviduct contractions of this compound is indistinguishable from that of authentic proctolin. Antiserum to proctolin stains numerous axons with beaded endings that run along the fibres of the closer muscles and, in addition, the antiserum stains a number of cell bodies in the suboesophageal ganglion, some of which have axons in the mandibular nerve that innervates the mandibular musculature. The function of proctolin appears to be modulatory as its presence significantly increases the amplitude of neurally evoked contractions of the closer muscle. This effect can be mimicked by the addition of inositol 1,4,5-trisphosphate (IP3) to preparations in which the muscles have been permeabilized with dimethyl sulfoxide. The involvement of this second messenger is further implicated as we also show that proctolin produces a large, significant increase in the IP3 content of homogenized muscle.

Animals↗
PubMed1990

Serotoninergic innervation of the locust mandibular closer muscle modulates contractions through the elevation of cyclic adenosine monophosphate.

The mandibular closer muscles of the locust receive innervation that is immunoreactive for the putative transmitter 5-hydroxytryptamine (5-HT). Cobalt-labelling suggests that the origin of this innervation is a group of cells located anteriorly in the suboesophageal ganglion. Bath application of 5-HT while the muscles are active produces marked changes in the contractions, increasing their amplitude, rate of contraction, and rate of relaxation. Incubation of isolated muscles with 5-HT shows that this amine elevates the levels of the cyclic nucleotide cyclic adenosine monophosphate (cAMP). In addition compounds that artificially elevate the levels of cAMP in the muscle--3-isobutyl-1-methylxanthine (IBMX), forskolin, and the cAMP analogue 8-(4-chlorophenylthio) cAMP--mimic the actions of 5-HT, whereas a potent inhibitor of insect adenylate cyclase, adenosine, considerably delays the onset of the effects produced by 5-HT. The effects observed with 5-HT in the mandibular muscle are similar to those of octopamine in the locust extensor tibiae muscle, and it is possible that this is an analogous modulatory system.

Animals↗