Gαq and Phospholipase Cß signaling regulate nociceptor sensitivity in Drosophila melanogaster larvae.

Drosophila melanogaster larvae detect noxious thermal and mechanical stimuli in their environment using polymodal nociceptor neurons whose dendrites tile the larval body wall. Activation of these nociceptors by potentially tissue-damaging stimuli elicits a stereotyped escape locomotion response. The cellular and molecular mechanisms that regulate nociceptor function are increasingly well understood, but gaps remain in our knowledge of the broad mechanisms that control nociceptor sensitivity. In this study, we use cell-specific knockdown and overexpression to show that nociceptor sensitivity to noxious thermal and mechanical stimuli is correlated with levels of Gαq and phospholipase Cß signaling. Genetic manipulation of these signaling mechanisms does not result in changes in nociceptor morphology, suggesting that changes in nociceptor function do not arise from changes in nociceptor development, but instead from changes in nociceptor activity. These results demonstrate roles for Gαq and phospholipase Cß signaling in facilitating the basal sensitivity of the larval nociceptors to noxious thermal and mechanical stimuli and suggest future studies to investigate how these signaling mechanisms may participate in neuromodulation of sensory function.

An evolutionarily conserved switch in response to GABA affects development and behavior of the locomotor circuit of Caenorhabditis elegans.

The neurotransmitter gamma-aminobutyric acid (GABA) is depolarizing in the developing vertebrate brain, but in older animals switches to hyperpolarizing and becomes the major inhibitory neurotransmitter in adults. We discovered a similar developmental switch in GABA response in Caenorhabditis elegans and have genetically analyzed its mechanism and function in a well-defined circuit. Worm GABA neurons innervate body wall muscles to control locomotion. Activation of GABAA receptors with their agonist muscimol in newly hatched first larval (L1) stage animals excites muscle contraction and thus is depolarizing. At the mid-L1 stage, as the GABAergic neurons rewire onto their mature muscle targets, muscimol shifts to relaxing muscles and thus has switched to hyperpolarizing. This muscimol response switch depends on chloride transporters in the muscles analogous to those that control GABA response in mammalian neurons: the chloride accumulator sodium-potassium-chloride-cotransporter-1 (NKCC-1) is required for the early depolarizing muscimol response, while the two chloride extruders potassium-chloride-cotransporter-2 (KCC-2) and anion-bicarbonate-transporter-1 (ABTS-1) are required for the later hyperpolarizing response. Using mutations that disrupt GABA signaling, we found that neural circuit development still proceeds to completion but with an ∼6-hr delay. Using optogenetic activation of GABAergic neurons, we found that endogenous GABAA signaling in early L1 animals, although presumably depolarizing, does not cause an excitatory response. Thus a developmental depolarizing-to-hyperpolarizing shift is an ancient conserved feature of GABA signaling, but existing theories for why this shift occurs appear inadequate to explain its function upon rigorous genetic analysis of a well-defined neural circuit.

Thermotaxis, circadian rhythms, and TRP channels in Drosophila.

The fruit fly Drosophila melanogaster is a poikilothermic organism that must detect and respond to both fine and coarse changes in environmental temperature in order maintain optimal body temperature, synchronize behavior to daily temperature fluctuations, and to avoid potentially injurious environmental hazards. Members of the Transient Receptor Potential (TRP) family of cation channels are well known for their activation by changes in temperature and their essential roles in sensory transduction in both invertebrates and vertebrates. The Drosophila genome encodes 13 TRP channels, and several of these have key sensory transduction and modulatory functions in allowing larval and adult flies to make fine temperature discriminations to attain optimal body temperature, detect and avoid large environmental temperature fluctuations, and make rapid escape responses to acutely noxious stimuli. Drosophila use multiple, redundant signaling pathways and neural circuits to execute these behaviors in response to both increases and decreases in temperature of varying magnitudes and time scales. A plethora of powerful molecular and genetic tools and the fly's simple, well-characterized nervous system have given Drosophila neurobiologists a powerful platform to study the cellular and molecular mechanisms of TRP channel function and how these mechanisms are conserved in vertebrates, as well as how these channels function within sensorimotor circuits to generate both simple and complex thermosensory behaviors.

Egg laying decisions in Drosophila are consistent with foraging costs of larval progeny.

Decision-making is defined as selection amongst options based on their utility, in a flexible and context-dependent manner. Oviposition site selection by the female fly, Drosophila melanogaster, has been suggested to be a simple and genetically tractable model for understanding the biological mechanisms that implement decisions. Paradoxically, female Drosophila have been found to avoid oviposition on sugar which contrasts with known Drosophila feeding preferences. Here we demonstrate that female Drosophila prefer egg laying on sugar, but this preference is sensitive to the size of the egg laying substrate. With larger experimental substrates, females preferred to lay eggs directly on sugar containing media over other (plain, bitter or salty) media. This was in contrast to smaller substrates with closely spaced choices where females preferred non-sweetened media. We show that in small egg laying chambers newly hatched first instar larvae are able to migrate along a diffusion gradient to the sugar side. In contrast, in contexts where females preferred egg laying directly on sugar, larvae were unable to migrate to find the sucrose if released on the sugar free side of the chamber. Thus, where larval foraging costs are high, female Drosophila choose to lay their eggs directly upon the nutritious sugar substrate. Our results offer a powerful model for female decision-making.

Thermosensory and nonthermosensory isoforms of Drosophila melanogaster TRPA1 reveal heat-sensor domains of a thermoTRP Channel.

Specialized somatosensory neurons detect temperatures ranging from pleasantly cool or warm to burning hot and painful (nociceptive). The precise temperature ranges sensed by thermally sensitive neurons is determined by tissue-specific expression of ion channels of the transient receptor potential(TRP) family.We show here that in Drosophila, TRPA1 is required for the sensing of nociceptive heat. We identify two previously unidentified protein isoforms of dTRPA1, named dTRPA1-C and dTRPA1-D, that explain this requirement. A dTRPA1-C/D reporter was exclusively expressed in nociceptors, and dTRPA1-C rescued thermal nociception phenotypes when restored to mutant nociceptors. However,surprisingly, we find that dTRPA1-C is not a direct heat sensor. Alternative splicing generates at least four isoforms of dTRPA1. Our analysis of these isoforms reveals a 37-amino-acid-long intracellular region (encoded by a single exon) that is critical for dTRPA1 temperature responses. The identification of these amino acids opens the door to a biophysical understanding of a molecular thermosensor.

Two types of chloride transporters are required for GABA(A) receptor-mediated inhibition in C. elegans.

Chloride influx through GABA-gated Cl(-) channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl(-) gradient established in part by K(+)-Cl(-) cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na(+)-driven Cl(-)-HCO(3)(-) exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K(+)-Cl(-) cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl(-) extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl(-) gradient such that Cl(-) flow through GABA-gated channels is reversed and excites rather than inhibits cells. Neuronal expression of both transporters is upregulated during synapse development, and ABTS-1 expression further increases in KCC-2 mutants, suggesting regulation of these transporters is coordinated to control the cellular Cl(-) gradient. Our results show that Na(+)-driven Cl(-)-HCO(3)(-) exchangers function with KCCs in generating the cellular chloride gradient and suggest a mechanism for the close tie between pH and excitability in the brain.

The potassium chloride cotransporter KCC-2 coordinates development of inhibitory neurotransmission and synapse structure in Caenorhabditis elegans.

Chloride influx through GABA-gated chloride channels, the primary mechanism by which neural activity is inhibited in the adult mammalian brain, depends on chloride gradients established by the potassium chloride cotransporter KCC2. We used a genetic screen to identify genes important for inhibition of the hermaphrodite-specific motor neurons (HSNs) that stimulate Caenorhabditis elegans egg-laying behavior and discovered mutations in a potassium chloride cotransporter, kcc-2. Functional analysis indicates that, like mammalian KCCs, C. elegans KCC-2 transports chloride, is activated by hypotonic conditions, and is inhibited by the loop diuretic furosemide. KCC-2 appears to establish chloride gradients required for the inhibitory effects of GABA-gated and serotonin-gated chloride channels on C. elegans behavior. In the absence of KCC-2, chloride gradients appear to be altered in neurons and muscles such that normally inhibitory signals become excitatory. kcc-2 is transcriptionally upregulated in the HSN neurons during synapse development. Loss of KCC-2 produces a decrease in the synaptic vesicle population within mature HSN synapses, which apparently compensates for a lack of HSN inhibition, resulting in normal egg-laying behavior. Thus, KCC-2 coordinates the development of inhibitory neurotransmission with synapse maturation to produce mature neural circuits with appropriate activity levels.

Photostimulation alters c-Fos expression in the dorsal raphe nucleus.

Retinal afferents to the dorsal raphe nucleus (DRN) have been described in a number of species, including Mongolian gerbils, but functional correlates of this optic pathway are unknown at present. To determine whether temporally modulated photostimulation can affect c-Fos expression in the gerbil DRN, quantitative analysis of c-Fos-immunoreactive (c-Fos-ir) neurons was conducted following 60-min exposure to pulsed (2 Hz) photostimulation at selected times over the 12:12 h light/dark cycle. For comparison, c-Fos expression was also analyzed in the subnuclei of the lateral geniculate complex and in the suprachiasmatic nucleus (SCN). In the DRN, a substantial reduction was observed in the number of c-Fos immunoreactive (c-Fos-ir) neurons during the light period and early dark period in photostimulated vs. control animals. Similar results were obtained in the intergeniculate leaflet (IGL) and ventral lateral geniculate (VLG). However, no significant changes were observed in the number of c-Fos-ir neurons in the dorsal lateral geniculate nucleus or suprachiasmatic nucleus (SCN) following photostimulation, except for an increase in the middle of the dark period. These findings indicate that photic stimulation can lead to a suppression or down-regulation of c-Fos expression in the DRN that is probably mediated via the direct retinal pathway to the DRN in this species. The similarity between c-Fos expression profiles in the DRN and IGL/VGL suggest that efferent projections from the DRN may modulate c-Fos expression to visual stimulation in these subnuclei of the lateral geniculate complex.