Inositol 1,4,5,-tris-phosphate receptor

DEVELOPMENTAL BIOLOGY

Embryonic

As seen by northern analysis Ins3PR is present in 0 to 6 hour early embryos, but this expression cannot be detected by in situ hybridization. The first distinct hybridization is seen at approximately 10.5 hours as a segmental pattern in cells of the ventral epidermis lying along the intersegmental furrows and in posterior epidermal cells. In late stage-13 embryos, this expression intensifies and extends to the head region within cells of the gnathal buds, which lie ventral to the stomodeal opening. The spatiotemporal appearance of these cells in the head region suggests that they are likely to be progenitors of anterior sense organs. In stage 14 embryos, the anterior expression extends dorsally to cells in the clypeolabrum, while the lateral epidermal expression decreases to barely detectable levels. By stage 15, all detectable expression is localized to the anterior head region, presumably in primordia of anterior sense organs. As development progresses, some of these sense organs can be identified as two pairs of dorsal structures that lie along the pharynx walls and a more lateroventral pair, which is likely to be the labial organ. Finally, at stage 17, only the labial organ expresses this gene (Hasan, 1992).

Expression of the InsP3R appears in early mesoderm in stage 9 embryos. This expression is enhanced as differentiation of the mesoderm occurs into somatic and visceral layers at stage 10. Maximum expression is observed in both the somatic and visceral mesoderm at stage 13. Two rows of cardioblasts in the dorsal midline also stain strongly at stage 13. Staining of the visceral mesoderm can be observed surrounding the gut but later this disappears. This disappearance of the InsP3R can be roughly correlated with differentiation of the myoblast clusters into muscle fibers in the somatic muscles (Raghu, 1995).

In the cephalic region, anti-InsP3R antibody staining appears in the developing procephalic mesoderm at stage 9 and begins intensifying in stage 10. At stage 13 staining in the pharyngeal muscles is very distinctive in the anterior regions. Other clusters of muscles in the cephalic region also appear to stain strongly at stage 14. No InsP3R protein could be detected in any of the sense organs (Raghu, 1995).

Distribution of Ryanodine and IP₃R receptors and the endoplasmic reticulum Ca²⁺-ATPase

The biochemistry, distribution and phylogeny of Drosophila ryanodine (RyR) and inositol triphosphate (IP₃R) receptors and the endoplasmic reticulum Ca²⁺-ATPase (SERCA) have been characterized by using binding and enzymatic assays, confocal microscopy and amino acid sequence analysis. [³H]-ryanodine binding in total membranes is enhanced by AMP-PCP, caffeine and xanthine, whereas Mg²⁺, Ruthenium Red and dantrolene are inhibitors. [³H]-ryanodine binding showed a bell-shaped curve with increasing free [Ca²⁺], without complete inhibition at millimolar levels of [Ca²⁺]. [³H]-IP₃ binding is inhibited by heparin, 2-APB and xestospongin C. Microsomal Ca²⁺-ATPase activity is inhibited by thapsigargin. Confocal microscopy demonstrates abundant expression of ryanodine and inositol triphosphate receptors and abundant Ca²⁺-ATPase in Drosophila embryos and adults. Ryanodine receptor is expressed mainly in the digestive tract and parts of the nervous system (Vázquez-Martínez, 2003).

To characterize RyR, IP₃R and SERCA protein distribution in fly tissues, fluorescent compounds specific for RyR (TX-R-BODIPY-ryanodine), IP₃R (FL-Heparin), and thapsigargin-sensitive SERCA (FL-BODIPY-thapsigargin) were used. The signal associated with fluorescent ryanodine is present in practically all cells of early embryos. The label localizes mainly to the cytoplasm of cells. The fluorescent ryanodine signal observed in older embryos (stages 15-17) clearly shows RyR at higher concentrations in the digestive tract. The label associated with Drosophila SERCA and IP₃R in early embryos is also present in practically all cells. In late embryos, label is present in nearly all tissues and is distributed more homogeneously than ryanodine signals. Labeling for all three fluorescent compounds is seen in tissues derived from all germinal layers: ectoderm (epidermis), mesoderm (muscle), and endoderm (digestive tract). As seen for the ryanodine receptor, higher magnification views of cells labelled with thapsigargin and heparin also show cytoplasmic staining. Co-localization of these compounds with fluorescent ryanodine illustrates that SERCA and RyR are highly coexpressed in the digestive tract, whereas coexpression of IP₃R and RyR is evenly distributed. Label observed in these experiments is specific, since coincubation with excess ryanodine, heparin or thapsigargin abolishes labeling for ryanodine, for heparin, and for thapsigargin (Vázquez-Martínez, 2003).

Adult tissues were stained with BODIPY TR-X Ryanodine, BODIPY FL-Thapsigargin and FL-Heparin. A generalized RyR expression was observed, with higher levels in the digestive tract, muscle, and adult optic lobe and retina. Label is cytoplasmic, as in embryos. Staining is seen in tissues of ectodermal origin (nervous system, mesodermal origin (indirect flight and leg muscles), and endodermal origin (digestive tract). Staining for heparin was seen also in practically all adult tissues, and more homogeneous in levels than RyR. Most tissues show extensive colocalization of both labels. Colocalization of fluorescent ryanodine with fluorescent thapsigargin was coincidental (Vázquez-Martínez, 2003).

Published in situ hybridization data offer a very restricted expression pattern, with higher levels in late embryos in prospective antenno-maxillary complex (a complex comprising the dorsal and terminal organs) and the labial organ. In contrast, the data reveal widespread expression of the IP₃R protein: consistent with mutant defects, expression of the protein occurs at all stages and tissues. There is also high expression in the digestive tract, again consistent with the requirement for intracellular Ca²⁺ dynamics in visceral muscle function and consistent with immunocytochemistry data. Staining has less marked regional differences than RyR staining. The data support the idea that IP₃R protein, like RyR protein, is also contributed maternally and/or is expressed at levels not readily detectable by in situ hybridization at all stages and tissues. This underscores the value of examining both transcript and protein expression data, although some caution should be exercised, since heparin may label other proteins besides IP₃R protein (Vázquez-Martínez, 2003).

Larval

In both the wing and leg imaginal discs weak, though specific, staining is found associated with myoblasts. In the eye-antennal disc, however, similar staining is found to be associated with the developing photoreceptor neurons that lie just behind the morphogenetic furrow. No specific staining was found to be associated with the antennal portion of the eye-antennal disc. InsP3R staining can be detected in the dorsolongitudinal indirect flight muscles. Compared to the level of myoblast staining in the imaginal discs, these myoblasts stain more strongly. Among the myoblasts seen at this stage, the ones that lie close to the larval templates appear to stain more strongly than the migrating myoblasts. Unlike what is seen in embryonic muscles development, where the InsP3R disappears after myoblast proliferation and fusion is over, in pupal development InsP3R expression is not turned off and the antigen can be seen to be associated with the nuclei of the fused myoblasts (Raghu, 1995).

Neuronal expression of the InsP3R in developing antennae occurs after differentiation in late pupae. At 86 hours after puparium formation, well after specification of neural precursors in the antenna which occurs about 14 hours after puparium formation, InsP3R can be seen in differentiated antennal neurons. Expression is also seen in cells of nonneuronal identity. These cells form antennal muscles (Raghu, 1995).

The appearance of InsP3R in taste neurons of pupal labellar hairs was compared with the ability of the developing labellar hairs to respond to a taste stimulus. Similar to what is seen with the antennal neurons, the taste neurons at the base of the labellar hairs do not express the InsP3R until 86 hours after puparium formation. Mechanosensory response can be detected from 84 hour pupae by bending the shaft of the bristle. Only by 96 hours after puparium formation was the taste response to 0.1 M NaCl fully developed. Thus the appearance of the InsP3R in the taste neurons of the proboscus correlates well with the time when the taste hairs are developing the ability to respond to taste stimuli (Raghu, 1995).

Adult

In the adult, InsP3R mRNA can be detected in the cortex of the central brain and the antenna. Expression is also detectable in eyes and legs (Hasan, 1992).

Strong antennal staining of InsP3R protein is seen in sections through the second and third antennal segment. Staining is also observed at the base of sensory hairs present in the maxillay palp and the proboscus. The maxillary palp acts as a second olfactory organ in Drosophila, while the proboscus is primarily a taste organ. In the proboscus the staining is clearly associated with sensory neurons present at the base of taste hairs. These data suggest that IP3 may act as a second messenger during both olfactory and taste transduction in Drosophila, in a pathway similar to what has been shown to exist in vertebrates. In the brain the antigen is restricted to cell bodies and is probably nuclear-membrane-associated or perinuclear in localization. This is also the case in the eye, where the staining is associated with the nuclei of each photoreceptor cone and pigment cell. Perinuclear/nuclear-membrane staining is also seen in the muscles of the head (Raghu, 1995).

Effects of Mutation or Deletion

Histological examination of various tissues from 4-day-old InsP3R knockout mutant larvae (corresponding to late third larval instar of control larvae) reveals a pronounced defect in the growth of larval tissues and imaginal progenitor cells. For instance, brain neuroblasts and midgut progenitor cells can be recognized but do not undergo any significant proliferation. As a result, the size of the brain corresponds to that of control late-first or early-second instar larvae. Salivary glands and principal midgut epithelial cells also undergo little endoreplication of the DNA. During normal development, larval cells perform up to 10 rounds of endoreplication, resulting in large cells with highly polytenic chromosomes. Nuclei in knockout larvae undergo little endoreplication. InsP3R mutants also show dramatic defects in the development of imaginal discs; they are rudamentary at best, with little if any cell proliferation or differentiation (Acharya, 1997).

The Drosophila light-sensitive channels TRP and TRPL are prototypical members of an ion channel family responsible for a variety of receptor-mediated Ca(2+) influx phenomena, including store-operated calcium influx. While phospholipase Cbeta is essential, downstream events leading to TRP and TRPL activation remain unclear. The role of the InsP₃ receptor (InsP₃R) was examined by generating mosaic eyes homozygous for a deficiency of the only known InsP₃R gene in Drosophila. Absence of gene product was confirmed by RT-PCR, Western analysis, and immunocytochemistry. Mutant photoreceptors undergo late onset retinal degeneration; however, whole-cell recordings from young flies demonstrate that phototransduction is unaffected, since quantum bumps, macroscopic responses in the presence and absence of external Ca(2+), light adaptation, and Ca(2+) release from internal stores are all normal. Using the specific TRP channel blocker La(3+) it was demonstrated that both TRP and TRPL channel functions are unaffected. These results indicate that InsP₃R-mediated store depletion does not underlie TRP and TRPL activation in Drosophila photoreceptors (Raghu, 2000).

The inositol 1,4,5-trisphosphate (IP3) receptor is an intracellular calcium channel that couples cell membrane receptors, via the second messenger IP3, to calcium signal transduction pathways within many types of cells. IP3 receptor function has been implicated in development, but the physiological processes affected by its function have yet to be elucidated. In order to identify these processes, mutants in the IP3 receptor gene (itpr) of Drosophila were generated and their phenotype during development was studied. All itpr mutant alleles are lethal. Lethality occurs primarily during the larval stages and is preceded by delayed molting. Insect molting occurs in response to the periodic release of the steroid hormone ecdysone which, in Drosophila, is synthesized and secreted by the ring gland. The observation of delayed molting in the mutants, coupled with the expression of the IP3 receptor in the larval ring gland led the authors to examine the effect of the itpr alleles on ecdysone levels. On feeding ecdysone to mutant larvae, a partial rescue of the itpr phenotype is observed. In order to assess ecdysone levels at all larval stages, transcripts were examined of an ecdysone-inducible gene, E74; these transcripts are downregulated in larvae expressing each of the itpr alleles. Thus, disruption of the Drosophila IP3 receptor gene leads to lowered levels of ecdysone. Synthesis and release of ecdysone from the ring gland is thought to occur in response to a neurosecretory peptide hormone secreted by the brain. It is proposed that this peptide hormone requires an IP3 signaling pathway for ecdysone synthesis and release in Drosophila and other insects. This signal transduction mechanism that links neuropeptide hormones to steroid hormone secretion might be evolutionarily conserved (Venkatesh, 1997).

A role for inositol 1,4,5-trisphosphate (IP₃) as a second messenger during olfactory transduction has been postulated in both vertebrates and invertebrates. However, given the absence of either suitable pharmacological reagents or mutant alleles specific for the IP₃ signaling pathway, an unequivocal demonstration of IP₃ function in olfaction has not been possible. The role of a well-established cellular target of IP₃, the IP₃ receptor, has been studied in olfactory transduction in Drosophila. For this purpose existing viable combinations of IP₃R mutant alleles, as well as a newly generated set of viable itpr alleles, were examined for olfactory function. In all of the viable allelic combinations primary olfactory responses were found to be normal. However, a subset of itpr alleles (including a null allele) exhibit faster recovery after a strong pulse of odor, indicating that the IP₃R is required for maintenance of olfactory adaptation. Interestingly, this defect in adaptation is dominant for two of the alleles tested, suggesting that the mechanism of adaptation is sensitive to levels of the IP₃R (Despande, 2000).

Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. Using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting have been examined in Drosophila. Previous studies have shown that mutants for the Inositol 1,4,5-trisphosphate receptor gene (Itpr) are larval lethals. In addition, they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Mutants for adenylate cyclase (rut) synergize, during larval molting, with Itpr mutant alleles, indicating that both cAMP and InsP3 signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP3 receptor. Furthermore, these studies predict the existence of feedback inhibition through protein kinase A on the InsP3 receptor by increased levels of 20-hydroxyecdysone (Venkatesh, 2001).

An understanding of the signaling pathways that control insect molting has come primarily from pharmacological and biochemical studies on lepidopterans with similar studies extending to Drosophila. These studies have shown that neural factors, which include the PTTH, stimulate the prothoracic gland (a part of the ring gland in higher Dipterans including Drosophila) to synthesize and secrete ecdysone, which is subsequently converted to its active form of 20-hydroxyecdysone in other tissues. Biochemical and molecular analyses of PTTH isolated from lepidopterans and Drosophila have shown that the peptide hormone is quite different in the two classes of insects, indicating that signaling downstream of PTTH in the prothoracic gland may also differ. In fact, while extracellular calcium is required for secretion of ecdysone in both systems, cAMP has been demonstrated to be a second messenger only in lepidopterans. Molecular identification of other key players, such as the PTTH receptor and the channel for entry of extracellular calcium, has not yet been determined. The first indication that insect larval molting is regulated by InsP₃ signaling came from analysis of Drosophila mutants for the InsP₃ receptor gene. Data presented in this study now implicate, in addition, the cAMP pathway in control of larval molting in Drosophila. Since exogenous 20-hydroxyecdysone can rescue the molting delays caused by disruption of either pathway, it is likely that both pathways control 20-hydroxyecdysone levels during molting. Due to technical difficulties associated with measuring 20-hydroxyecdysone levels in Drosophila larvae, these measurements could not be carried out directly. Instead, transcript levels of an ecdysone-inducible gene, E74, were used as an indirect measure of 20-hydroxyecdysone levels (Venkatesh, 2001).

Interestingly, steroid secretion by the adrenal fasciculata-reticularis cells of mammalian adrenal glands in response to adrenocorticotrophic hormone occurs through the cAMP pathway, while InsP₃-mediated Ca²⁺ release is required for the steroidogenic action of Angiotensin II on adrenal glomerulosa cells. An increase in cytosolic Ca²⁺ levels is thought to affect multiple steps in mammalian steroid biosynthesis, including one crucial step that requires the transfer of endogenous cholesterol from the outer to the inner mitochondrial membrane. The data presented in this study support a similar model in which 20-hydroxyecdysone levels are regulated through activation of both InsP₃ and cAMP signals. The presence of multiple genes encoding adenylate cyclases allows rut mutant alleles to proceed through molting normally. Presumably, however, activity of the alternate adenylate cyclase(s) is dependent on InsP₃ receptor function since removal of the Itpr gene in rut mutant backgrounds leads to phenotypes that are synergistic. Activation of the two second messenger pathways probably occurs in the ring gland via PTTH and other as yet unidentified neural factors. Alternate explanations whereby InsP₃ and/or cAMP signaling are required for PTTH release from neurons or during conversion of 20-hydroxyecdysone precursors to 20-hydroxyecdysone cannot be ruled out at this stage. In either event the two pathways act in parallel to maintain 20-hydroxyecdysone levels perhaps via nonoverlapping downstream targets (Venkatesh, 2001).

Since ecdysone secretion occurs as tightly regulated peaks, preceding each molt, inherent in the system should be a mechanism that inhibits ecdysone secretion once the peak level has been reached. On the basis of data from the UAS-mC* transgene (coding for a dominant active form of PKA), it is suggested that increased levels of 20-hydroxyecdysone in the hemolymph initiate a negative feedback loop that requires PKA activation and inhibition of the InsP₃ receptor. Thus the activated PKA phenotype is not rescued by increased levels of 20-hydroxyecdysone, but is rescued by increased levels of the itpr transgene. Interestingly, the effect of the UAS-mC* transgene on molting is also lost when Itpr gene levels are reduced as in larvae of the genotype UAS-mC*/+; 1664GAL4/itpr^90B0. This observation supports the idea that the Itpr gene is downstream of the UAS-mC* effect, and in addition suggests that the negative feedback is highly sensitive to levels of the Itpr gene. While these results demonstrate interactions between the two signaling pathways, the molecular basis of these interactions is unknown as yet. Since mammalian InsP₃ receptors can be directly phosphorylated by PKA, the possibility exists that a similar mechanism might operate in the negative feedback step predicted from these results. However, both predicted isoforms of the Drosophila InsP₃ receptor, which are present in larval tissues and derive from two known splice variant forms of the itpr cDNA, lack putative PKA phosphorylation sites as determined by Prosite analysis. It is possible that a low-abundance isoform of the InsP₃ receptor exists in specific larval cells that may be directly regulated by PKA. Additionally, there are almost certainly other unidentified players in this system that this study has not revealed. It should be possible to identify some or all of these factors using suitable genetic interaction screens in the future (Venkatesh, 2001).

Genetic dissection of itpr gene function reveals a vital requirement in aminergic cells of Drosophila larvae

Signaling by the second messenger inositol 1,4,5-trisphosphate is thought to affect several developmental and physiological processes. Mutants in the inositol 1,4,5-trisphosphate receptor (itpr) gene of Drosophila exhibit delays in molting while stronger alleles are also larval lethal. In a freshly generated set of EMS alleles for the itpr locus single point mutations in seven mutant chromosomes have been sequenced and identified. The predicted allelic strength of these mutants matches the observed levels of lethality. They range from weak hypomorphs to complete nulls. Interestingly, lethality in three heteroallelic combinations has a component of cold sensitivity. The temporal focus of cold sensitivity lies in the larval stages, predominantly at second instar. Coupled with the observation that an itpr homozygous null allele dies at the second instar stage, it appears that there is a critical period for itpr gene function in second instar larvae. The focus of this critical function is shown to lie in aminergic cells, by rescue with UAS-itpr and DdCGAL4. However, this function does not require synaptic activity, suggesting that InsP₃-mediated Ca²⁺ release regulates the neurohormonal action of serotonin (Joshi, 2004).

To identify the tissue/cells that contribute to lethality observed in itpr mutants, tissue- and cell-specific GAL4 strains were used to drive expression of the UAS-itpr transgene in ug3/sv35 organisms, the majority of which die as second instars. Expression of the InsP₃ receptor in larvae is known to be in the brain and ring gland complex, among other tissues. Consequently, attempts were made to rescue lethality with lines that express in the central nervous system, peripheral nervous system, and ring gland. Complete rescue of second instar lethality was obtained with elavGAL4, including a normal transition from second to third instar larvae. elavGAL4-rescued organisms also pupate but are unable to eclose as adults. Expression of the elav gene is known to occur in all postmitotic neurons. In addition, significant expression of elavGAL4 is seen in cells of the ring gland, including the corpora cardiaca in second and third instar larvae. Several GAL4 lines were tested that were known to express in subsets of larval neurons and expression was looked for in cells of the ring gland. Among the GAL4 lines, which could rescue second to third instar lethality to varying levels, were DdCGAL4 and c929. In the ug3/sv35 strain, 6.3 ± 0.6 third instar larvae were seen at 152-160 hr AEL. GAL4 expression in these strains occurs in all dopamine and serotonin neurons (DdCGAL4) and in all peptidergic neurons (c929). Similar to elavGAL4, both strains also have significant GAL4 expression in the corpora cardiaca region of second and third instar larval ring glands. Since the corpora cardiaca is the only tissue of overlap between DdCGAL4 and C929, the modest rescue observed with c929 is attributed to these cells. The higher level of rescue observed in DdCGAL4-containing organisms indicates that in addition to the corpora cardiaca, dopamine and serotonin neurons in the larval brain also require itpr gene function. Of these two neurotransmitters, serotonin immunoreactivity has also been reported in the corpora cardiaca. This was confirmed by immunostaining second and third instar ring glands with a polyclonal antiserum to serotonin. Taken together, these data point toward a critical requirement for the InsP₃ receptor in serotonin-containing cells of second instar larvae (Joshi, 2004).

To obtain an understanding of the cellular function performed by the InsP₃ receptor in DdCGAL4 positive cells, synaptic function in these cells was inhibited by expression of the UAS-tetanus toxin transgene (UAS-TNT. Expression of tetanus toxin in Drosophila neurons is known to specifically block evoked neurotransmitter release and to reduce the frequency of spontaneous quantal release. DdCGAL4-driven expression of UAS-TNT did not affect larval viability or molting. In contrast, when TNT expression is under control of c929 in peptidergic neurons, there is a significant loss of viability in second instar larvae. Therefore, the TNT transgene used in this work is functional and capable of affecting viability, which is dependent on the neurons where it is expressed. Complete loss of DdCGAL4-expressing cells, however, is critical for larval viability, since expression of a cell death gene (UAS-hid) in these cells resulted in a high level of larval lethality (Joshi, 2004).

Analysis of larval lethality with the newly generated InsP₃ alleles described in this study has provided new insights into InsP₃ receptor function in Drosophila. The observations are consistent with the existence of a physiological process in second instar larvae, which requires a critical level of activity from the zygotically derived InsP₃ receptor, the absence of which leads to lethality. This requirement occurs prior to the InsP₃ receptor's role in regulating larval molting as suggested by the following observation. While feeding of 20-hydroxyecdysone can rescue molting delays in a nonlethal allele, it is not able to rescue lethality of any allelic combination. However, the focus of both these defects in itpr mutants could lie in serotonin cells. This idea is supported by the fact that 5-HT immunoreactive fibers have been seen extending to the prothoracic gland and corpora allata. A role for serotonin in larval molting has been proposed in other insects (Joshi, 2004).

From the rescue profiles obtained with elavGAL4 and DdCGAL4 it is also clear that there is a pupal phase of lethality, which is rescued effectively by UAS-itpr expression in the domains of neurGAL4 and prosGAL4. To understand the cause of pupal lethality, the expression of these strains in pupae needs further investigation (Joshi, 2004).

Both serotonin and dopamine are best known in their roles as neurotransmitters. However, serotonin is also known to have an essential role during gastrulation of Drosophila embryos when it is thought to trigger changes in cell adhesiveness by as-yet-unknown cellular mechanisms. In the context of neurons, serotonin can act as a neurotransmitter or as a neurohormone. On the basis of the results of experiments with expression of UAS-TNT in DdCGAL4-positive cells, it is proposed that serotonin's action as a neurohormone is critical for larval viability. The partial rescue of second instar lethality by UAS-itpr expression in serotonin-positive neurohemal cells of the corpora cardiaca (c929GAL4), supports this idea. Neurohemal cells have no synaptic activity and their function is to secrete either stored or freshly synthesized neurohormones. Serotonin-positive fibers and varicosities extend from the corpora cardiaca to other regions of the ring gland, the aorta wall, and the surface of the gut. The effect of serotonin release from these fibers thus could be on the physiological function of any of these tissues. A direct test of this hypothesis would be to see the effect of inhibiting secretory pathways required for neurohormonal release (unrelated to neurotransmission) in serotonin cells, such as the one described recently in Drosophila neurons (Murthy, 2003). However, at present the possibility that a neurohormone other than serotonin is secreted by DdCGAL4-expressing cells, that might be the critical factor in the observed lethality, cannot be ruled out (Joshi, 2004).

The observations related to cold sensitivity in ug3/ka1091 also support the idea of the InsP₃ receptor's role in regulating neurohormonal release from aminergic cells. Exposure to cold temperatures is known to have an inhibitory effect on secretory pathways described in CHO cells and in chromaffin cells. Moreover, certain exocytosis mutants in yeast are cold sensitive (Joshi, 2004).

Further investigation is required to understand the nature of extracellular signals that activate the InsP₃ receptor in DdCGAL4-positive cells. It is known that several neurosecretory neurons send their processes to the corpora cardiaca. Serotonin and dopamine cells of the larval brain and ventral ganglion are mostly interneurons and probably receive inputs from many types of neurons. A combination of cellular and genetic studies will be required to understand the nature of these signals (Joshi, 2004).

Loss of flight and associated neuronal rhythmicity in Inositol 1,4,5-trisphosphate receptor mutants of Drosophila

Coordinated flight in winged insects requires rhythmic activity of the underlying neural circuit. Drosophila mutants for the inositol 1,4,5-trisphosphate (InsP₃) receptor gene (itpr) are flightless. Electrophysiological recordings from thoracic indirect flight muscles show increased spontaneous firing accompanied by a loss of rhythmic flight activity patterns normally generated in response to a gentle puff of air. In contrast, climbing speed, the jump response, and electrical properties of the giant fiber pathway are normal, indicating that general motor coordination and neuronal excitability are much less sensitive to itpr mutations. All mutant phenotypes are rescued by expression of an itpr⁺ transgene in serotonin and dopamine neurons. Pharmacological and immunohistochemical experiments support the idea that the InsP₃ receptor functions to modulate flight specifically through serotonergic interneurons. InsP₃ receptor action appears to be important for normal development of the flight circuit and its central pattern generator (Banerjee, 2004 ).

This study shows that InsP₃R is required for air puff-induced flight and associated neuronal rhythmicity. This requirement is in the domain of DdCGAL4 (serotonin and dopamine)-positive neurons during the first 48 hr of pupal development. From pharmacological and anatomical experiments, it appears likely that the InsP₃R is required in serotonergic neurons, although a requirement in dopaminergic cells cannot be ruled out at this stage (Banerjee, 2004).

Changes in intracellular calcium levels as a consequence of InsP₃R function are likely to affect diverse aspects of neuronal physiology. The itpr alleles studied here are viable and hence their behavioral and physiological phenotypes probably reflect neuronal functions most sensitive to InsP₃-mediated Ca²⁺ release. Interestingly, the foci of these functions lie in aminergic cells that release the neurotransmitters serotonin or dopamine. Furthermore, despite the fact that the observed phenotypes are behavioral and physiological, a significant proportion of these arise from a developmental requirement for the InsP₃R. Thus, normal formation and functioning of the air puff-stimulated flight circuit in adults requires the itpr gene during its formation and growth in pupae (Banerjee, 2004).

It is known that motoneurons innervating the indirect flight muscles undergo dendritic and axonal remodeling during pupariation. From several studies in both invertebrates and vertebrates, it appears that among the biogenic amines, serotonin can modulate axon outgrowth. In serotonin-positive cerebral giant cells of Lymnaea stagnalis, release of 5-HT autoregulates axon growth by inducing growth cone collapse. The cerebral giant cells form part of the neural circuit that controls rhythmic feeding behavior in these molluscs. In Drosophila, inhibition of serotonin synthesis has been shown to cause excessive branching of serotonergic axon terminals during embryonic and larval development. More recently, serotonin has been shown to affect development of the swimming circuit in zebrafish, and a role for serotonergic interneurons has been described during development of the left-right coordination of rhythmic motor activity in rat spinal cord. Together with these studies, it is reasonable to postulate that itpr mutants affect development of the flight circuit through modulating serotonin release. The observation that the flight deficit (~45%) in DdCGAL4/UAS-TNT organisms (in which tetanus toxin is expressed in the pattern of the the Ddc promoter) is less than that of a majority of the itpr mutants (>80%) suggests that a component of this release is neurohormonal and not entirely because of evoked neuronal activity. A role for the InsP₃R in neurohormonal secretion is also apparent in DdCGAL4-positive neurons and neurohemal cells of second instar Drosophila larvae. These data raise the possibility that release of serotonin is particularly sensitive to intracellular Ca²⁺ levels regulated through the InsP₃R. This idea needs to be tested rigorously by additional experiments (Banerjee, 2004).

Both serotonin and dopamine have been shown to modulate the acute activity of motor circuits. Increased serotonin release has been shown to increase the postsynaptic response of flight motoneurons in locust. Recently, serotonin was shown to alter motoneuron firing in Drosophila larvae in a biphasic manner. The increased frequency of spontaneous firing and the loss of air puff induced flight in itpr mutants suggest that disruption of InsP₃R function could alter normal firing of motor neurons, which innervate the DLMs. The required InsP₃R activity does not reside in the motor neurons but rather in aminergic (possibly serotonergic) neurons innervating and controlling motor neuron activity. One explanation is that inhibitory connections that modulate flight motor neurons have been lost or weakened during development. However, as adult feeding of PCPA results in lowered synthesis of serotonin and resembles the blocking effects of release of serotonin by TNT from these interneurons and also partially phenocopies itpr phenotype, it is possible that the InsP₃R continues to play a central role in flight coordination (Banerjee, 2004).

Anatomical analysis has shown that cell bodies positive for serotonin and DdcGAL4 exist in the thoracic ganglia: no arborization from these cell bodies toward flight motoneurons was detected. Instead, the data suggest that 5-HT-positive varicosities on flight motor neurons arise from serotonin and DdcGAL4-positive axon tracts that descend from the brain. This observation argues for a serotonergic modulation of flight motor neuron function from neurons in the brain. Localization of the InsP₃R to these neurons and their axon tracts was not feasible, because immunohistochemistry with existing antisera to the Drosophila InsP₃R gives a cross-reaction with nonspecific antigen(s) in adult CNS preparations. The existing itprGAL4 line could not be used either, because it represents a subset of the itpr gene expression domain and does not express in the adult nervous system. Very likely, the physiological defects observed are attributable to a combination of inappropriate circuit formation during development and altered neuronal activity in adults. Although development has a significant contribution, the existing tools do not allow investigation of an adult-specific requirement in an unambiguous manner. However, given the perdurance of the InsP₃R in adults (as judged by the expression of a UAS-itprGFP transgene induced in pupae by the same heat shock regime as that for UAS-itpr⁺), a contribution to acute flight signaling remains a distinct possibility (Banerjee, 2004).

Tbese studies show that the InsP₃R is essential for development of the neural circuit that probably functions as the central pattern generator for air puff-induced flight. The function of the InsP₃R is in aminergic cells, indicating that at a cellular level, it functions in the release of serotonin and/or dopamine. Earlier, Ca²⁺ release from vesicular InsP₃Rs in chromaffin cells, acinar cells, and islet cells has been proposed to release Ca²⁺ required for secretion. The profound developmental and functional actions of InsP₃R, through release of biogenic amines, is a novel finding and could have relevance for vertebrate systems, where serotonin has been shown to affect neural circuit development. Significantly, knock-outs for the InsP₃R1 in mice exhibit ataxia and motor discoordination at birth (Banerjee, 2004).

Ectopic expression of a Drosophila InsP(3)R channel mutant has dominant-negative effects in vivo

The inositol 1,4,5-trisphosphate (InsP₃) receptor is a tetrameric intracellular calcium channel. It is an integral component of the InsP₃ signaling pathway in multicellular organisms, where it regulates cellular calcium dynamics in many different contexts. In order to understand how the primary structure of the InsP₃R affects its functional properties, the kinetics of Ca²⁺-release in vitro from single point mutants of the Drosophila InsP₃R have been determined. Among these, the Ka901 mutant in the putative selectivity-filter of the pore is of particular interest. It is non-functional in the homomeric form whereas it forms functional channels (with altered channel properties) when co-expressed with wild-type channels. Due to its changed functional properties the Ka901 mutant protein has dominant-negative effects in vivo. Cells expressing Ka901:WT channels exhibit much higher levels of cytosolic Ca²⁺ upon stimulation as compared with cells over-expressing just the wild-type DmInsP₃R, thus supporting in vitro observations that increased Ca²⁺ release is a property of heteromeric Ka901:WT channels. Furthermore, ectopic expression of the Ka901 mutant channel in aminergic cells of Drosophila alters electrophysiological properties of a flight circuit and results in defective flight behavior (Srikanth, 2005).

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date revised: 15 April 2007

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