InteractiveFly: GeneBrief

shaking B: Biological Overview | References


Gene name - shaking B

Synonyms -

Cytological map position - 19E2-19E3

Function - transmembrane gap junction protein

Keywords - gap junction protein, innexin, motor neuron pattern regulation, giant fiber system, Johnston's organ neurons, optic lobe

Symbol - shakB

FlyBase ID: FBgn0085387

Genetic map position - chrX:20,771,265-20,826,456

NCBI classification - Innexin

Cellular location - transmembrane



NCBI link: EntrezGene
BIOLOGICAL OVERVIEW

This study used the peristaltic crawling of Drosophila larvae as a model to study how motor patterns are regulated by central circuits. An experimental system was constructed that allows simultaneous application of optogenetics and calcium imaging to the isolated ventral nerve cord (VNC). Next, the effects of manipulating local activity of motor neurons (MNs) on fictive locomotion were observed as waves of MN activity propagating along neuromeres. Optical inhibition of MNs with halorhodopsin3 (NpHR3) in a middle segment (A4, A5 or A6), but not other segments, dramatically decreases the frequency of the motor waves. Conversely, local activation of MNs with channelrhodopsin2 (ChR2) in a posterior segment (A6 or A7) increases the frequency of the motor waves. Since peripheral nerves mediating sensory feedback are severed in the VNC preparation, these results indicate that MNs send signals to the central circuits to regulate motor pattern generation. These results also indicate segmental specificity in the roles of MNs in motor control. The effects of the local MN activity manipulation are lost in shakB2 or ogre2, gap-junction mutations in Drosophila, or upon acute application of the gap junction blocker CBX, implicating electrical synapses in the signaling from MNs. Cell-type specific RNAi suggests shakB and ogre function in MNs and interneurons, respectively, during the signaling. These results not only reveal an unexpected role for MNs in motor pattern regulation but also introduce a powerful experimental system that enables examination of the input-output relationship among the component neurons in this system (Matsunaga, 2017).

Animal movement is accomplished by spatially and temporally coordinated contraction of various muscles throughout the body. It is generally thought that a neuronal network composed of premotor interneurons generates a motor pattern, and this network sequentially activates different classes of motor neurons (MNs). In this view, MNs play only passive roles in pattern generation, relaying the information they receive from upstream interneuronal networks to muscles. By contrast, there is some evidence that MNs themselves contribute to the motor pattern generation. In the crustacean stomatogastric ganglion and in leech swimming circuits, MNs are part of the pattern-forming network. In mammalian spinal cords, MNs send a collateral to innervate Renshaw cells, which in turn convey feedback signals to MNs. However, whether and how MNs regulate motor pattern generation during animal movements remains largely unexplored (Matsunaga, 2017).

Larval Drosophila is emerging as an excellent model system for studying motor pattern generation since one can apply powerful genetic tools including a large collection of Gal4-drivers to study the function of individual component neurons in a numerically simple nervous system. Furthermore, previous development of a platform for electronic microscope (EM) image data reconstruction of the entire nervous system of the larval CNS now allows mapping of the circuit structure that mediates specific behaviors. The larval ventral nerve cord (VNC) consists of three thoracic neuromeres (T1, T2, and T3) and eight abdominal neuromeres (A1-A8). Larval peristaltic crawling is accomplished by successive bilateral muscle contraction that propagates from tail to head. Muscle contraction in each segment is in turn regulated by sequential activation of MNs in the corresponding neuromere of the VNC. Although recent studies have begun to identify several types of premotor interneurons that regulate aspects of movement such as the speed of locomotion and left-right or intersegmental coordination, how a motor pattern is generated by the neural circuits remains largely unknown (Matsunaga, 2017).

In a previous study, halorhodopsin (NpHR) was used to locally and transiently inhibit MN activity in one or a few segments; local activity perturbation was found to halt the propagation of the peristaltic wave at the site of manipulation (Inada, 2011). This suggests that MNs are part of the neural circuits that generate the peristaltic wave. However, how information is retrogradely transmitted from MNs to the central circuits remained unknown. Furthermore, since muscle contraction was usedt as a measure of the motor outputs, changes in the activity dynamics in the CNS could not be studied. That study was extended by constructing a new experimental system in which the effects of local optogenetic manipulation of MNs on global motor activity could be studied in the VNC. Optical inhibition of MNs in a middle segment (A4, A5, or A6) decreased the motor frequency. Conversely, photoactivation of MNs in a posterior segment (A6 or A7) increased the frequency of the motor wave. These results indicate that the local activity level of MNs impacts the global outputs of the motor circuits in a segment-specific manner. It was also show that gap junctions are involved in this process. While this manuscript was in preparation, a study in zebrafish reported that motor neurons retrogradely influence the activity level of the premotor V2a interneurons via gap junctions and regulate motor generation (Song, 2016). Thus, regulation by gap junction-mediated retrograde MN signaling appears to be a common mechanism of motor control (Matsunaga, 2017).

In a previous study (Inada, 2011), MN activity was locally and transiently inhibited in one or a few segments during peristalsis of dissected larvae, and activity manipulation was shown to halt peristalsis. This indicates that MN activity is required for the motor activity wave to propagate along the VNC and suggests the presence of retrograde signaling from MNs to the central circuits. However, since dissected larvae were used, the possibility that the signaling was instead mediated via the sensory feedback of muscular contraction could not be excluded. Furthermore, the mechanism of the retrograde signaling remained unknown (Matsunaga, 2017).

A new experimental system was built that allow studying the direct causal relationship between the manipulation of MN activity and changes in neural dynamics in the motor circuits with superior spatial and temporal resolution. Optical perturbations were applied for a longer period and in a more systematic manner than in the previous study and their effects on the global circuit activity was analyzed. Using the new experimental system, the previous study was extended by showing that (1) MN outputs within the CNS, not mediated by sensory feedback, are critical for motor wave regulation, (2) there is segmental difference in the role of the MN outputs, and (3) the MN signaling is mediated by gap junctions (Matsunaga, 2017).

This study shows that manipulation of motor neuronal activity in just one segment robustly affects the output of the entire motor network in Drosophila larvae. Optical inhibition or activation of MNs in a single segment decreased or increased, respectively, the calcium level of MNs in distant neuromeres. Furthermore, these perturbations strongly affected the frequency of motor waves. Thus, changes in MN activity in one segment affect the activity level and wave generation of the entire motor system (Matsunaga, 2017).

It should be noted that in the isolated VNC preparation used in this study, peripheral nerves with motor activity output and sensory feedback input were severed. Local changes in MN activity therefore influenced the activity of distant MNs through intersegmental neural connections within the CNS, not via sensory feedback. Thus, the results establish the presence of retrograde signaling from MNs that is critical for motor pattern regulation. The identity of the synaptic connections mediating the signaling is currently unknown. They could be direct MN-MN connections, or they may also involve coupling between MNs and interneurons (Matsunaga, 2017).

Electrical synapses are commonly found in the nervous systems of vertebrates and invertebrates. In particular, electrical coupling mediated by gap junctions has been implicated in motor pattern control in various systems. This study has showed that gap junctions are involved in the retrograde MN signaling controlling motor wave frequency in Drosophila larvae. Local photomanipulation of MNs that would normally increase or decrease wave frequency had no effect in shakB2 and ogre2 mutants. This suggests that electrical synapses including ShakB and Ogre mediate the MN signaling controlling motor frequency. In contrast, CBX administration but not shakB2 or ogre2 mutation abolished the calcium level changes of distant MNs induced by the activity manipulation, suggesting that innexins other than those deleted in shakB2 or ogre2 mediate this aspect of motoneuronal communication (eight innexin genes are present in the Drosophila genome). It should also be noted that wave generation normally occurred in the isolated VNCs of shakB2 and ogre2 mutants. There was also no obvious abnormality in the locomotion of the shakB2 or ogre2 larvae. These observations suggest that ShakB and Ogre-mediated MN signaling is part of redundant pathway(s) regulating motor waves. Only upon optical perturbation are the role of MN signaling in wave generation and involvement of ShakB and Ogre manifested (Matsunaga, 2017).

Previous work has reported the existence of electrical coupling between MNs and the premotor excitatory V2a interneurons, a neuronal class that provides a major drive for MNs during locomotion in zebrafish (Song, 2016). Hyperpolarizing or depolarizing MNs decreased or increased the firing activity of V2a interneurons. Furthermore, selective inhibition of MNs during locomotion interrupted the recruitment of V2a interneurons and decreased the frequency of locomotion. Thus, control of locomotor circuits by gap junction-mediated retrograde MN signaling may be an evolutionarily conserved mechanism used in both invertebrates and vertebrates (Matsunaga, 2017).

An interesting feature of MN signaling revealed in this study is segment specificity. On the one hand, inhibition of MNs in A4, A5, or A6, but not other segments, reduced the motor wave frequency. On the other hand, activation of MNs in A6 or A7, but not other segments, increased the frequency of the wave. This segmental discord with regard to the MN signaling may contribute to the regulation of the wave initiation. How can gap junctions selectively mediate one type of activity change but not another? For example, how can the decline but not the elevation in activity level of MNs in the A4 or A5 segment affect the wave frequency? One possibility is the involvement of rectifying electrical synapses. Rectifying electrical synapses have been found in both vertebrates and invertebrates and can mediate unidirectional synaptic transmission in a voltage-dependent manner. Rectifying electrical synapses are often composed of a heteromeric assembly of gap junction proteins on each side of the apposing neurons. A role of ShakB in rectification has been shown in the giant fiber system of adult Drosophila. Two splicing forms of ShakB, ShakB(N) and ShakB(N+16), are expressed in the presynaptic and postsynaptic sites of the giant synapse, respectively. When expressed in neighboring oocytes, these two ShakB variants form heterotypic channels that are asymmetrically gated by voltage. Since RNAi knockdown experiments showed that shakB, but not ogre, is required in MNs to mediate the retrograde signaling, an interesting possibility is the involvement of heterotypic channels composed of ShakB in MNs and Ogre in interneurons. Future studies are necessary first to identify the target neurons that receive the retrograde MN signaling and then to study whether the relevant electrical synapses are indeed rectified. Revealing the information flow mediating the MN retrograde signaling will provide valuable insights on how intersegmentally coordinated motor patterns are generated in this and other systems. The experimental system established in this study can also be applied more generally to study the input-output relationship among the component neurons in this system. While the GAL4/UAS system alone was sufficient to express both GCaMP/RGECO and NpHR/ChR2 in MNs in this study, the introduction of another expression system such as the LexA system will allow expression of GCaMP/RGECO and NpHR/ChR2 in different classes of neurons, including interneurons. This will allow study of the the influence of the optogenetic manipulation of one class of neurons on the activity of others. The functional analyses may also be combined with the circuit diagram elucidated by ongoing EM reconstruction. It is anticipated that such systematic analyses will elucidate fundamental mechanisms of how central circuits coordinate intersegmental movements (Matsunaga, 2017).

Shaking B mediates synaptic coupling between auditory sensory neurons and the giant fiber of Drosophila melanogaster

The Johnston's Organ neurons (JONs) form chemical and electrical synapses onto the giant fiber neuron (GF), as part of the neuronal circuit that mediates the GF escape response in Drosophila. This study examined which of the 8 Drosophila innexins (invertebrate gap junction proteins) mediates the electrical connection at this synapse. The GF is known to express Shaking B (ShakB), specifically the ShakBN+16 isoform only, at its output synapses in the thorax. The shakB2 mutation disrupts these GF outputs and also abolishes JON-GF synaptic transmission. The amplitude of the compound action potential recorded in response to sound from the base of the antenna (sound-evoked potential, or SEP) was reduced by RNAi of the innexins Ogre, Inx3, Inx6 and, to a lesser extent Inx2, suggesting that they could be required in JONs for proper development, excitability, or synchronization of action potentials. The strength of the JON-GF connection itself was reduced to background levels only by RNAi of shakB, not of the other seven innexins. ShakB knockdown prevented Neurobiotin coupling between GF and JONs and removed the plaques of ShakB protein immunoreactivity that are present at the region of contact. Specific shakB RNAi lines that are predicted to target the ShakBL or ShakBN isoforms alone did not reduce the synaptic strength, implying that it is ShakBN+16 that is required in the presynaptic neurons. It was also suggested that gap junction proteins may have an instructive role in synaptic target choice (Pezier, 2016).

Reduced insulin signaling maintains electrical transmission in a neural circuit in aging flies

Lowered insulin/insulin-like growth factor (IGF) signaling (IIS) can extend healthy lifespan in worms, flies, and mice, but it can also have adverse effects (the 'insulin paradox'). Chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber system (GFS), a simple escape response neuronal circuit, by increasing targeting of the gap junctional protein innexin shaking-B to gap junctions (GJs). Endosomal recycling of GJs was also stimulated in cultured human cells when IIS was reduced. Furthermore, increasing the activity of the recycling small guanosine triphosphatases (GTPases) Rab4 or Rab11 was sufficient to maintain GJs upon elevated IIS in cultured human cells and in flies, and to rescue age-related loss of GJs and of GFS function. Lowered IIS thus elevates endosomal recycling of GJs in neurons and other cell types, pointing to a cellular mechanism for therapeutic intervention into aging-related neuronal disorders (Augustin, 2017).

Netrin and frazzled regulate presynaptic gap junctions at a Drosophila giant synapse

Netrin and its receptor, Frazzled, dictate the strength of synaptic connections in the giant fiber system (GFS) of Drosophila melanogaster by regulating gap junction localization in the presynaptic terminal. In Netrin mutant animals, the synaptic coupling between a giant interneuron and the 'jump' motor neuron was weakened and dye coupling between these two neurons was severely compromised or absent. In cases in which Netrin mutants displayed apparently normal synaptic anatomy, half of the specimens exhibited physiologically defective synapses and dye coupling between the giant fiber (GF) and the motor neuron was reduced or eliminated, suggesting that gap junctions were disrupted in the Netrin mutants. When the gap junctions were examined with antibodies to Shaking-B (ShakB) Innexin, they were significantly decreased or absent in the presynaptic terminal of the mutant GF. Frazzled loss of function mutants exhibited similar defects in synaptic transmission, dye coupling, and gap junction localization. These data are the first to show that Netrin and Frazzled regulate the placement of gap junctions presynaptically at a synapse (Orr, 2014).

The results show that Netrin-Frazzled signaling is specifically responsible for localizing gap junctions presynaptically at the GF-TTMn synapse. In the absence of Netrin, the gap junctions are not assembled in the presynaptic terminal and dye coupling is weak or absent in otherwise anatomically normal synapses. Similarly, Frazzled LOF mutants disrupted gap junctions and synaptic transmission. Finally, presynaptic expression of the dominant-negative Frazzled construct that is missing the intracellular domain also disrupts gap junction assembly, dye coupling, and synaptic transmission. In Netrin LOF mutants, axonal pathfinding is normal because the GF always projects into the target region and occasionally branches ectopically in the target region. However, dendritic path finding is dependent on Netrin-Frazzled signaling. In Netrin LOF mutants, the TTMn dendrite that normally projects toward the midline is often missing, as observed in other motor neurons. Finally, Netrin-Frazzled signaling is implicated in target selection, because GFs that reach the target area often do not build synapses, as seen in other model systems (Orr, 2014).

It is hypothesize that the physiological defect seen in Netrin and frazzled mutants arises from a reduction in trans-synaptic coupling between presynaptic and postsynaptic Innexins. Similar phenotypes, long latency, and lack of dye coupling have been observed in the shakB2 mutant, which lacks gap junctions at the GF-TTMn synapse (Phelan, 1996; Phelan, 2008; Allen, 2006). The data suggest that when presynaptic and postsynaptic cells make contact, Netrin-Frazzled signaling is instructive for presynaptic localization of Innexins in the GF terminal to form trans-synaptic gap junctions (Orr, 2014)

The frazzled dominant-negative construct supports the hypothesis that Netrin-Frazzled signaling is instructive in GF-TTMn synaptogenesis and function. Expression of fraC presynaptically disrupts the circuit by interrupting wild-type Netrin-Frazzled signaling. This is demonstrated through disruption of GF-TTMn synaptogenesis and the absence of gap junctions in the presynaptic terminal. However, the expression of UAS-fraC postsynaptically did not disrupt function, but did disrupt the morphology of the postsynaptic neuron. Postsynaptic expression of UAS-fraC disrupted dendritic maturation, resulting in medial dendrite pruning defects and lateral dendrite extension defects. The fraC experiments are interpreted as providing some evidence for Frazzled's cell autonomous role in building this giant synapse. More direct evidence would require rescue experiments. Unfortunately, the relevant genes are located very close to one another, making it difficult to obtain the appropriate recombination event. Future experiments will use recently acquired GAL4 drivers on the third chromosome to clarify this issue. The Frazzled RNAi experiments were uninformative, possibly because RNAi is not a strong enough disruption of frazzled to cause effects in the GFS. In brief, the cell autonomous function of Frazzled warrants further investigation (Orr, 2014)

When UAS-fraC was expressed in the embryo in the Netrin LOF background, it revealed that the disruption of commissures was Netrin dependent. The interaction experiment (NetAΔBΔ/+; A307/+; UAS-fraC/+) revealed a different mechanism by which the dominant-negative fraC obstructed synaptogenesis. In a heterozygous Netrin LOF background, the mutant version of Frazzled was expressed, further knocking down Netrin-Frazzled signaling to disrupt synaptogenesis. The results suggested that fraC was acting as a Netrin sink by binding to secreted Netrin, limiting the amount of Netrin that could bind to wild-type Frazzled receptors (Orr, 2014)

The chemical synaptic component of the GF-TTMn synapse was also observed in the Net LOF mutants using antibodies against the presynaptic density protein bruchpilot (T-bars) with anti-NC82 staining. However, the bruchpilot labeling was not informative. No further effort were made because the cholinergic component has no effect on synaptic circuit function in the adult (Orr, 2014)

In contrast to the GF-TTMn synapse, the GF-PSI synapse is unaffected by the absence of Netrin, Frazzled, or the expression of the dominant-negative Frazzled dominant-negative. This shows that the GF-TTMn synapse specifically is dependent on Netrin-Frazzled signaling for function. This mechanism for gap junction insertion is so specific that neighboring electrical synapses that share the same presynaptic terminal (GF) use different mechanisms for gap junction localization (Orr, 2014)

Netrin regulates Innexins in the GF presynaptic terminal from an external source. Netrin is secreted from two known sources, the midline glia and the postsynaptic target TTMn. A model is proposed for Netrin localization and function in which Netrin is captured on the surface of one neuron (TTMn) by Frazzled and is then presented to Frazzled receptors on another neuron (GF) to transmit signaling. During development, the TTMn extends its medial dendrite toward a source of Netrin, the midline glia. After the TTMn dendrite has grown into the synaptic area by 9% of PD, both the midline glia and TTMn are labeled with Netrin. It is hypothesized that this is important in the induction of synaptic maturation of this synapse (Orr, 2014)

Rescuing Netrin LOF mutants by expressing a secreted form of Netrin specifically in either TTMn or midline glia supports the model that Netrin is presented to the GF to promote synapse formation. The secreted Netrin rescue experiments were effective because Netrin could localize where it would normally as long as it was secreted by a nearby endogenous source. This could explain why it was possibleto rescue the Netrin LOF mutants in a non-cell-autonomous fashion by expressing secreted Netrin in either midline glia or the TTMn independently. Postsynaptic expression of the Frazzled dominant-negative also supports the presentation model. When two copies of Frazzled lacking its intracellular domain were expressed on the TTMn, Netrin could bind to the mutant Frazzled, be presented to the GF, and support normal synaptic function regardless of disrupted intracellular signaling in the TTMn by the deletion of the intracellular domain (Orr, 2014)

In contrast, expressing membrane-tethered UAS-NetBCD8-TM on either the midline glia or TTMn failed to rescue function of the circuit because localization and secretion of Netrin was disrupted. When attempts were made to rescue the Netrin LOF mutants by expressing membrane-tethered NetrinB postsynaptically, the defects were enhanced and the medial dendrite did not extend to the midline in 90% of specimens. However, in the tethered NetB mutant (NetAΔBmyc-TM/>), tethered NetrinB was expressed in both of its endogenous sources, midline glia and TTMn, and the synapse functioned normally. While being expressed under its endogenous promoter, tethered Netrin supported normal synaptogenesis. It is possible that, through the endogenous expression pattern, cells not identified in this study could contribute to the normal phenotype seen in the mutants in a nonlocal manner. However, it was hypothesize that the tethered NetrinB mutant does not behave in a predictable way. It is suggested that this protein is not as tightly membrane bound as the UAS-NetBCD8-TM protein product due to the added extracellular myc domains in the tethered mutant. The tethered mutant's additional myc domains may account for differences in phenotypes due to increased protein flexibility or possible cleavage and secretion from the cell of origin. Considering this, non-cell-autonomous expression of a secreted Netrin rescued Netrin LOF defects, whereas expression of the tethered version using the same GAL4 drivers could not rescue the defects. This is recognized as evidence for the importance of Netrin secretion in GFS synaptogenesis (Orr, 2014)

Molecular mechanism of rectification at identified electrical synapses in the Drosophila giant fiber system

Electrical synapses are neuronal gap junctions that mediate fast transmission in many neural circuits. The structural proteins of gap junctions are the products of two multigene families. Connexins are unique to chordates; innexins/pannexins encode gap-junction proteins in prechordates and chordates. A concentric array of six protein subunits constitutes a hemichannel; electrical synapses result from the docking of hemichannels in pre- and postsynaptic neurons. Some electrical synapses are bidirectional; others are rectifying junctions that preferentially transmit depolarizing current anterogradely. The phenomenon of rectification was first described five decades ago, but the molecular mechanism has not been elucidated. This study demonstrates that putative rectifying electrical synapses in the Drosophila Giant Fiber System are assembled from two products of the innexin gene shaking-B. Shaking-B(Neural+16) is required presynaptically in the Giant Fiber to couple this cell to its postsynaptic targets that express Shaking-B(Lethal). When expressed in vitro in neighboring cells, Shaking-B(Neural+16) and Shaking-B(Lethal) form heterotypic channels that are asymmetrically gated by voltage and exhibit classical rectification. These data provide the most definitive evidence to date that rectification is achieved by differential regulation of the pre- and postsynaptic elements of structurally asymmetric junctions (Phelan, 2008).

The shaking-B (shakB) gene gives rise to several partially identical transcripts, which translate into three distinct proteins: Shaking-B(Neural) (ShakB[N]), Shaking-B(Neural+16) (ShakB[N+16]), and Shaking-B(Lethal) (ShakB[L]). ShakB(N) was originally implicated in synaptic connectivity in the Giant Fiber System (GFS). The mutation shakB2, believed to lie in an exon unique to the shakB(n) transcript, was associated with loss of electrical and dye coupling and gap-junction morphology at GFS synapses. ShakB(N+16) subsequently was found to be partially encoded on this exon; thus, shakB2 disrupts the function of ShakB(N) and ShakB(N+16). To determine directly which of these proteins is required at electrical synapses in the GFS, attempts were made to rescue the mutant phenotype by cell-specific expression of the individual transcripts under GAL4-UAS control. The GAL4 lines A307, which expresses strongly in the Giant Fibers (GFs) and giant commissural interneurons (GCIs) and weakly in the tergotrochanteral muscle motorneurons (TTMns) and peripherally synapsing interneurons (PSIs), which expresses in the GFs, but not in its pre- or postsynaptic partners, were used to direct expression of UAS-shakB(n+16) or UAS-shakB(n) to neurons of the GFS in a shakB2 mutant background (Phelan, 2008).

Gap-junction function in the GFS was examined by monitoring the cell-cell transfer of the fluorescent dye Lucifer Yellow injected into one of the GF axons. In wild-type flies, the dye diffused from the injected GF into the GCIs in the brain, the TTMn, PSI, and other unidentified neurons in the thoracic ganglion. Dye coupling was never observed in shakB2. A307-directed expression of shakB(n+16) in these mutants rescued coupling between the GF and GCIs and between the GF and TTMn; rescue of GF-PSI coupling was observed less frequently, consistent with the comparatively lower levels of expressed protein at these junctions. Expression of shakB(n+16) under the control of c17 did not rescue GF-GCI coupling in shakB2. In the thoracic ganglion, coupling between the GF and PSI was rescued; however, GF-TTMn dye coupling was not convincingly rescued. These data are consistent with the finding that ShakB protein was more concentrated at the GF-PSI contacts than at the GF-TTMn contacts in these flies. In ~24% of preparations, very faint fluorescence was observed in a cell in the approximate position of the TTMn. It was reasoned, therefore, that there was weak rescue at both synapses but that the dye dissipated in TTMn, which is a much larger cell than PSI. To confirm whether this was the case, an alternative method was used to assess GFS synaptic function (Phelan, 2008).

Electrophysiological recordings were made from the tergotrochanteral (TTM) and dorsal longitudinal (DLM) muscles in response to GF stimulation. Using this approach, rescue of the GF-TTM pathway was observed when shakB(n+16) was expressed in shakB2 mutants with either c17 or A307. The level of rescue obtained was slightly higher with A307 but, in both cases, was manifest as a dramatic increase in the number of flies responding, a slight (although not statistically significant) reduction in response latency, and a significant improvement in the response to repetitive stimulation at 100 Hz, indicative of more stable synapses (Phelan, 2008).

Expression of shakB(n) in shakB2 mutants with the stronger GAL4 driver A307 failed to restore dye coupling or synaptic activity in the GFS, although the protein was clearly localized to sites of synaptic contact (Phelan, 2008).

The phenotypic rescue studies demonstrate that shakB(n+16) is required for electrical transmission in the GFS and that shakB(n), despite sharing 96% amino acid identity, cannot functionally substitute (Phelan, 2008).

The use of two GAL4 lines, with different patterns of expression in the GFS, to drive shakB(n+16) allowed determination of which cells of the escape circuit require this protein. Robust rescue of the GF-GCI synapses was observed with A307, which expresses strongly in both of these neurons, but not with c17, which expresses in the GFs only. This suggests that the protein is normally required in both cells. Synapses between the GF and its thoracic ganglion targets TTMn and PSI were rescued when shakB(n+16) was expressed with either A307 or c17. In principle, rescue with A307 could be due to formation of ShakB(N+16) homotypic channels because this GAL driver expresses in the TTMn and PSI as well as in the GF. In practice, postsynaptic expression of the transgene is unlikely to have contributed significantly to synaptic connectivity because the rescue lines are heterozygous for the GAL4 construct, and A307 expression (e.g., of reporter genes) in TTMn and PSI is weak even in the homozygous state. Rescue with c17, which does not express in TTMn or PSI, confirms that coupling between the GF and these cells is not dependent on the presence of shakB(n+16) postsynaptically (Phelan, 2008).

It is concluded that the synapses between the GF and GCIs, which are believed to synchronize activity of right and left GFs, are homotypic gap junctions with both pre- and postsynaptic hemichannels composed of ShakB(N+16). The GF-TTMn and GF-PSI synapses are heterotypic junctions in which presynaptic ShakB(N+16) interacts with a different innexin in the postsynaptic neurons. Consistent with these data, shakB(n+16) has been shown by RNA in situ hybridization to be expressed in the wild-type GFs and in the presumptive GCIs; it is the only shakB transcript detectable in these neurons and is not present at detectable levels in the TTMn or PSI. Two lines of evidence indicate that these cells express shakB(l). First, a GAL4 construct containing the shakB(l) promoter, which is distinct from the shakB(n+16) promoter, drives reporter gene expression in TTMn and PSI (but not in the GFs). Second, ShakB immunoreactivity persists in shakB2 mutants at the midline in the region where the PSI contacts the tip of the TTMn medial dendrite and the GF; given the specificity of the antibody, this must represent ShakB(L) protein. A caveat is that shak-B(l) RNA is not detectable in the TTMn or PSI by in situ hybridization, presumably because expression levels are below the sensitivity of the technique (Phelan, 2008).

Since the pioneering work of Furshpan and Potter, several studies have examined the mechanism of rectification at the crayfish giant motor synapse, the classical rectifying electrical synapse, where transmission can be recorded by inserting electrodes into the pre- and postsynaptic axons. Ideally, one would like to apply the same approach in the Drosophila GFS in order directly to correlate synaptic physiology and molecular genetics. As yet, at least, this is not technically possible because the fruit fly neurons are much smaller in size and less accessible than their crayfish counterparts. To determine, therefore, whether molecular asymmetry at the GFS synapses might underlie electrical rectification, a synapse was 'modeled' in paired Xenopus oocytes (Phelan, 2008).

shakB(n+16) and shakB(l) RNAs were transcribed in vitro and microinjected into connexin-depleted Xenopus oocytes. Both RNAs are efficiently translated by oocytes. The ability of the expressed proteins to form channels was assessed by dual voltage clamp electrophysiology of cell pairs in which one cell expressed ShakB(N+16) and the other ShakB(L) (heterotypic) or both cells of a pair expressed the same protein (homotypic). In heterotypic configuration, channels were reliably induced at RNA levels of 0.1-0.5 ng; the voltage sensitivity of these channels differed significantly from that of homotypic channels composed of either protein (Phelan, 2008).

A striking asymmetry was observed in the response of ShakB(N+16)/ShakB(L) heterotypic channels to transjunctional voltage (Vj). Depolarizing Vj steps applied to the ShakB(N+16)-expressing cell induced large junctional currents (Ijs) that tended to increase over time for Vjs up to 40 mV. For higher Vjs, current increased to its maximum level and then declined slightly. By contrast, when the ShakB(L)-expressing cell was subjected to depolarizing Vjs, induced currents were of low magnitude and showed a voltage-dependent decrease over time. Hyperpolarizing Vjs applied to either cell elicited responses opposite to those observed on depolarization. Junctional currents induced by the application of negative Vjs to the ShakB(N+16)-expressing cell were of low magnitude and decreased in a time- and voltage-dependent manner. Large currents were induced when hyperpolarizing Vjs were applied to the ShakB(L)-expressing cell. These Ijs increased over time to a steady-state level for Vjs up to 40 mV; for higher Vjs, maximum Ij was followed by a slight decline (Phelan, 2008).

The relationship between junctional conductance (Gj) and transjunctional voltage for ShakB(N+16)/ShakB(L) heterotypic channels was examined. Conductance was low and declined with increasing Vj (up to 40-50 mV) for relative negativity of the ShakB(N+16)-expressing cell. Instantaneous Gj, measured 5 ms after the imposition of Vj steps, was not significantly different than steady-state Gj, indicating that the junctions approximated their lowest conductance state, which was always > 0, within 5 ms. The residual conductance presumably represents a small population (~15%-~20%) of voltage-insensitive channels. Instantaneous and steady-state Gj increased in a sigmoidal fashion as the cell expressing ShakB(N+16) was depolarized or the ShakB(L)-expressing cell was hyperpolarized relative to its heterotypic partner. Steady-state data fitted well to a Boltzmann equation, suggesting a single transition from closed to open states. The calculated parameters were essentially the same irrespective of which cell of the pair was subjected to voltage steps (Phelan, 2008).

The asymmetry of the voltage response of heterotypic channels contrasts with a symmetrical response of homotypic channels to applied voltage. ShakB(N+16) channels, which were generally of low conductance, showed no voltage sensitivity. Steady-state and instantaneous Gj were constant with increasing Vj < 40 mV. For higher Vjs, there was a slight but nonsignificant rise in steady-state Gj. Steady-state Gj of ShakB(L) homotypic channels declined with increasing Vjs (≥30 mV) of either polarity (Phelan, 2008).

GFS heterotypic synapses modeled in Xenopus oocytes exhibited classical rectification. As observed at the crayfish giant synapse, depolarizations were preferentially transmitted in one direction only—in this case from the ShakB(N+16)-expressing cell (representing the presynaptic GF) to the ShakB(L) -- expressing cell (representing the postsynaptic TTMn or PSI) -- whereas hyperpolarizing signals passed preferentially in the opposite direction. Apart from this physiological asymmetry, notable features of the crayfish synaptic gap junctions are insensitivity to transmembrane voltage, very rapid responses to changes in transjunctional voltage, and steep rectification. With respect to the first of these, the data are consistent; conductance of ShakB(N+16)/ShakB(L) junctions was not significantly influenced by transmembrane voltage, and, hence, the observed voltage responses reflect Vj dependence only. The initial response to voltage was rapid, occurring within milliseconds of the imposition of Vj steps. At 5 ms (the earliest time at which instantaneous conductance could be reliably measured), the majority of voltage-sensitive channels had closed in response to hyperpolarizing Vjs applied to the ShakB(N+16)-expressing cell or depolarizing Vjs applied to the ShakB(L) cell. The rise to maximum Gj upon depolarization of the ShakB(N+16) cell, or hyperpolarization of the ShakB(L) cell, occurred more slowly so that, at 5 ms, Gj had only attained ~50%-~60% of its maximum value. These timescales are somewhat slower than those reported for the crayfish giant synapse, where junctional currents typically reached their steady-state levels within 1 ms of the application of Vj steps of ≤ 30 mV. ShakB heterotypic junctions showed steep rectification, albeit not as steep as that observed at the crayfish synapse. The Gjmin/Gjmax ratio was 0.21 when the ShakB(N+16) and ShakB(L) cells, respectively, were stepped) as compared to a corresponding ratio of the order of 0.05 for the crayfish giant synapse stepped over a similar Vj range (Phelan, 2008).

Models of the crayfish giant motor synapse propose a structurally asymmetric junction in which one of the two apposed hemichannels contains a fast voltage-dependent gate. Qualitatively at least, the data presented in this study for the Drosophila GFS synapses are entirely consistent with this model. Given that ShakB(N+16) homotypic channels show little voltage sensitivity, the likely location of the voltage gate is postsynaptically in the ShakB(L) hemichannel. Assuming the crayfish synapses are composed of crustacean orthologs of ShakB, the quantitative differences between in situ- and in vitro-expressed junctions may be due to differences in the numbers and/or spatial arrangement of the channels in neurons and oocytes that might influence the kinetics of the voltage response (Phelan, 2008).

This study has combined in vivo and in vitro approaches to characterize the molecular mechanism of transmission at putative rectifying electrical synapses in the Drosophila GFS. Studies in flies demonstrate that ShakB(N+16) in the presynaptic GF is necessary and sufficient to couple this cell to its postsynaptic targets TTMn and PSI, which express ShakB(L). Xenopus oocyte pairs in which ShakB(N+16) is expressed in one cell and ShakB(L) in the adjacent one form heterotypic channels that are asymmetrically gated by transjunctional voltage. Relative positivity of the ShakB(N+16)-expressing cell, or relative negativity of the ShakB(L)-expressing cell, leads to large junctional conductances and vice versa. Taken together, these data strongly support the hypothesis that differential voltage gating of structurally asymmetric gap junctions underlies rectification at arthropod electrical synapses (Phelan, 2008).

The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter

The largest central synapse in adult Drosophila is a mixed electro-chemical synapse whose gap junctions require the product of the shaking-B (shak-B) gene. Shak-B(2) mutant flies lack gap junctions at this synapse, which is between the giant fibre (GF) and the tergotrochanteral motor neuron (TTMn), but it still exhibits a long latency response upon GF stimulation. This study targeted the expression of the light chain of tetanus toxin to the GF, to block chemical transmission, in shak-B(2) flies. The long latency response in the tergotrochanteral muscle (TTM) was abolished indicating that the chemical component of the synapse mediates this response. Attenuation of GAL4-mediated labelling by a cha-GAL80 transgene, reveals the GF to be cholinergic. A temperature-sensitive allele of the choline acetyltransferase gene (chats2)) was used to block cholinergic synapses in adult flies and this also abolished the long latency response in shak-B2 flies. Taken together the data provide evidence that both components of this mixed synapse are functional and that the chemical neurotransmitter between the GF and the TTMn is acetylcholine. These findings show that the two components of this synapse can be separated to allow further studies into the mechanisms by which mixed synapses are built and function (Allen, 2007).

Gap junction proteins are not interchangeable in development of neural function in the Drosophila visual system

Gap junctions (GJs) are composed of proteins from two distinct families. In vertebrates, GJs are composed of connexins; a connexin hexamer on one cell lines up with a hexamer on an apposing cell to form the intercellular channel. In invertebrates, GJs are composed of an unrelated protein family, the innexins. Different connexins have distinct properties that make them largely non-interchangeable in the animal. Innexins are also a large family with high sequence homology, and some functional differences have been reported. The biological implication of innexin differences, such as their ability to substitute for one another in the animal, has not been explored. Recently, it has been shown that GJ proteins are necessary for the development of normal neural transmission in the Drosophila visual system. Mutations in either of two Drosophila GJ genes (innexins), shakB and ogre, lead to a loss of transients in the electroretinogram (ERG), which is indicative of a failure of the lamina to respond to retinal cell depolarization. Ogre is required presynaptically and shakB(N) postsynaptically. Both act during development. This study asked if innexins are interchangeable in their role of promoting normal neural development in flies. Specifically, several innexins were tested for their ability to rescue shakB2 and ogre mutant ERGs; by and large innexins were found to be noninterchangeable. The protein regions required for this specificity were mapped by making molecular chimeras between shakBN and ogre and testing their ability to rescue both mutants. Each chimera rescued either shakB or ogre but never both. Sequences in the first half of each protein are necessary for functional specificity. Potentially crucial residues include a small number in the intracellular loop as well as a short stretch just N-terminal to the second transmembrane domain. Temporary GJs, possibly between the retina and lamina, may play a role in final target selection and/or chemical synapse formation in the Drosophila visual system. In that case, specificity in GJ formation or function could contribute, directly or indirectly, to chemical synaptic specificity by regulating which neurons couple and what signals they exchange. Cells may couple only if their innexins can mate with each other. The partially overlapping expression patterns of several innexins make this 'mix and match' model of GJ formation a possibility (Curtin, 2002a).

Gap junction proteins expressed during development are required for adult neural function in the Drosophila optic lamina

Evidence is provided that gap junction proteins, expressed during development, are necessary for the formation of normally functioning connections in the Drosophila optic lamina. Flies with mutations in the gap junction genes (innexins), shakingB, and ogre have normal photoreceptor potentials but a defective response of the postsynaptic cells in the optic lamina. This is indicated by a reduction in, or absence of, transients in the electroretinogram. Ogre is required in the presynaptic retinal photoreceptors. ShakingB(N) is, at a minimum, required in postsynaptic lamina neurons. Transgenic expression of the appropriate innexins during pupal development (but not later) rescues connection defects. Transient gap junctions have been observed to precede chemical synapse formation and have been hypothesized to play a role in connectivity and synaptogenesis; however, no causal role has been demonstrated. This study shows that developmental gap junction genes can be required for normally functioning neural connections to form (Curtin, 2002b).

Null mutation in shaking-B eliminates electrical, but not chemical, synapses in the Drosophila giant fiber system: a structural study

Mutations in the Drosophila shaking-B gene perturb synaptic transmission and dye coupling in the giant fiber escape system. The GAL4 upstream activation sequence system was used to express a neuronal-synaptobrevin-green fluorescent protein (nsyb-GFP) construct in the giant fibers (GFs); nsyb-GFP was localized where the GFs contact the peripherally synapsing interneurons (PSIs) and the tergotrochanteral motorneurons (TTMns). Antibody to Shaking-B protein stained plaque-like structures in the same regions of the GFs, although not all plaques colocalized with nsyb-GFP. Electron microscopy showed that the GF-TTMn and GF-PSI contacts contained many chemical synaptic release sites. These sites were interposed with extensive regions of close membrane apposition, with faint cross striations and a single-layered array of 41-nm vesicles on the GF side of the apposition. These contacts appeared similar to rectifying electrical synapses in the crayfish and were eliminated in shaking-B2 mutants. At mutant GF-TTMn and GF-PSI contacts, chemical synapses and small regions of close membrane apposition, more similar to vertebrate gap junctions, were not affected. Gap junctions with more vertebrate-like separation of membranes were abundant between peripheral perineurial glial processes; these were unaffected in the mutants (Blagburn, 1999).

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, it was expressed in paired Xenopus oocytes. This study shows that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. This is the first structural component of an invertebrate gap junction to be characterized (Phelan, 1998).


Functions of Shaking B orthologs in other species

Motor neurons control locomotor circuit function retrogradely via gap junctions

Motor neurons are the final stage of neural processing for the execution of motor behaviours. Traditionally, motor neurons have been viewed as the 'final common pathway', serving as passive recipients merely conveying to the muscles the final motor program generated by upstream interneuron circuits. This study reveals an unforeseen role of motor neurons in controlling the locomotor circuit function via gap junctions in zebrafish. These gap junctions mediate a retrograde analogue propagation of voltage fluctuations from motor neurons to control the synaptic release and recruitment of the upstream V2a interneurons that drive locomotion. Selective inhibition of motor neurons during ongoing locomotion de-recruits V2a interneurons and strongly influences locomotor circuit function. Rather than acting as separate units, gap junctions unite motor neurons and V2a interneurons into functional ensembles endowed with a retrograde analogue computation essential for locomotor rhythm generation. These results show that motor neurons are not a passive recipient of motor commands but an integral component of the neural circuits responsible for motor behaviour (Song, 2016).


REFERENCES

Search PubMed for articles about Drosophila Shaking B

Allen, M. J. and Murphey, R. K. (2007). The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter. Eur J Neurosci 26(2): 439-445. PubMed ID: 17650116

Augustin, H., McGourty, K., Allen, M. J., Madem, S. K., Adcott, J., Kerr, F., Wong, C. T., Vincent, A., Godenschwege, T., Boucrot, E. and Partridge, L. (2017). Reduced insulin signaling maintains electrical transmission in a neural circuit in aging flies. PLoS Biol 15(9): e2001655. PubMed ID: 28902870

Blagburn, J. M., Alexopoulos, H., Davies, J. A. and Bacon, J. P. (1999). Null mutation in shaking-B eliminates electrical, but not chemical, synapses in the Drosophila giant fiber system: a structural study. J Comp Neurol 404(4): 449-458. PubMed ID: 9987990

Curtin, K. D., Zhang, Z. and Wyman, R. J. (2002a). Gap junction proteins are not interchangeable in development of neural function in the Drosophila visual system. J Cell Sci 115: 3379-3388. PubMed ID: 12154069

Curtin, K. D., Zhang, Z. and Wyman, R. J. (2002b). Gap junction proteins expressed during development are required for adult neural function in the Drosophila optic lamina. J Neurosci 22: 7088-7096. PubMed ID: 12177205

Inada, K., Kohsaka, H., Takasu, E., Matsunaga, T. and Nose, A. (2011). Optical dissection of neural circuits responsible for Drosophila larval locomotion with halorhodopsin. PLoS One 6(12): e29019. PubMed ID: 22216159

Matsunaga, T., Kohsaka, H. and Nose, A. (2017). Gap junction-mediated signaling from motor neurons regulates motor generation in the central circuits of larval Drosophila. J Neurosci 37(8):2045-2060. PubMed ID: 28115483

Orr, B. O., Borgen, M. A., Caruccio, P. M. and Murphey, R. K. (2014). Netrin and frazzled regulate presynaptic gap junctions at a Drosophila giant synapse. J Neurosci 34: 5416-5430. PubMed ID: 24741033

Pezier, A. P., Jezzini, S. H., Bacon, J. P. and Blagburn, J. M. (2016). Shaking B mediates synaptic coupling between auditory sensory neurons and the giant fiber of Drosophila melanogaster. PLoS One 11: e0152211. PubMed ID: 27043822

Phelan, P., Stebbings, L. A., Baines, R. A., Bacon, J. P., Davies, J. A. and Ford, C. (1998). Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes. Nature 391(6663): 181-184. PubMed ID: 9428764

Phelan, P., Goulding, L. A., Tam, J. L., Allen, M. J., Dawber, R. J., Davies, J. A. and Bacon, J. P. (2008). Molecular mechanism of rectification at identified electrical synapses in the Drosophila giant fiber system. Curr Biol 18(24): 1955-1960. PubMed ID: 19084406

Song, J., Ampatzis, K., Bjornfors, E. R. and El Manira, A. (2016). Motor neurons control locomotor circuit function retrogradely via gap junctions. Nature 529(7586): 399-402. PubMed ID: 26760208


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date revised: 5 November 2017

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