SUMMARY 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 sensing of nociceptive heat. We identify two new protein isoforms of dTRPA1named 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 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.

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 [1]. Paradoxically, female Drosophila have been found to avoid oviposition on sugar which contrasts with known Drosophila feeding preferences [1]. 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.

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.

Chloride influx through GABA-gated Cl À channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl À gradient established in part by K þ -Cl À cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na þ -driven Cl À -HCO 3 À exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K þ -Cl À cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl À extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl À gradient such that Cl À 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 À gradient. Our results show that Na þ -driven Cl À -HCO 3 À 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.