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Thermosensory processing in the Drosophila brain

Abstract

In Drosophila, just as in vertebrates, changes in external temperature are encoded by bidirectional opponent thermoreceptor cells: some cells are excited by warming and inhibited by cooling, whereas others are excited by cooling and inhibited by warming1,2. The central circuits that process these signals are not understood. In Drosophila, a specific brain region receives input from thermoreceptor cells2,3. Here we show that distinct genetically identified projection neurons (PNs) in this brain region are excited by cooling, warming, or both. The PNs excited by cooling receive mainly feed-forward excitation from cool thermoreceptors. In contrast, the PNs excited by warming (‘warm-PNs’) receive both excitation from warm thermoreceptors and crossover inhibition from cool thermoreceptors through inhibitory interneurons. Notably, this crossover inhibition elicits warming-evoked excitation, because warming suppresses tonic activity in cool thermoreceptors. This in turn disinhibits warm-PNs and sums with feed-forward excitation evoked by warming. Crossover inhibition could cancel non-thermal activity (noise) that is positively correlated among warm and cool thermoreceptor cells, while reinforcing thermal activity which is anti-correlated. Our results show how central circuits can combine signals from bidirectional opponent neurons to construct sensitive and robust neural codes.

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Figure 1: Projection neurons excited by cooling.
Figure 2: Projection neurons excited specifically by warming.
Figure 3: Projection neurons excited by both warming and cooling.
Figure 4: Inhibitory local neurons responding to thermal stimuli.

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Acknowledgements

We are grateful to C. -H. Lee for the gift of LexAOp-HA-Ort flies, P. Garrity for Gr28b.d-Gal4, and A. Rajan for Gad1-Gal4 (line 2B). A. DiAntonio kindly supplied the anti-dVGluT antibody. We thank the Wilson laboratory, A. Samuel, P. Garrity, L. Griffith and D. Ginty for comments on the manuscript. D. Rogulja’s laboratory and M. Y. Wong shared reagents and expertise. A portion of this work was supported by NIH grant R01 DC008174. W.W.L. is supported by an HHMI International Research Fellowship and a Presidential Scholarship from the MD–PhD Program at Harvard Medical School. R.I.W. is a Howard Hughes Investigator.

Author information

Authors and Affiliations

Authors

Contributions

W.W.L. and R.I.W. conceived the experiments. O.M. and W.W.L. made the thermal stimulation device. W.W.L. performed the experiments. W.W.L. and R.I.W. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Rachel I. Wilson.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Jet micro-thermode schematic.

a, Schematic showing the major components of the jet micro-thermode. A pressurized reservoir forces saline through the inner chamber of a heat exchanger. The saline is then expelled as a jet through a 160 µm nozzle. This jet can be quickly turned on and off via a two-way solenoid valve (the ‘fast’ valve). The outer chamber of the heat exchanger is fed by two pumps which circulate water from a hot and a cold water bath. Two three-way solenoid valves control the flow of water into the exchanger. A third valve (not shown) serves to return the outflow of the heat exchanger to the same bath that is feeding it. b, c, In hot mode and cold mode the heat exchanger is heated or cooled by the continual circulation of water from one of the two baths through the outer chamber. d, In the no-flow configuration, circulation in the outer chamber is stopped. This keeps the heat exchanger at a fairly constant temperature, stabilized by the thermal mass of the water in the outer chamber.

Extended Data Figure 2 Morphologies of neurons in the proximal antennal protocerebrum.

Images are z projections of coronal sections (dorsal is up). The soma remains sealed to the patch pipette at the end of each recording, and so the soma and the proximal part of the primary neurite are not present in the reconstructed morphologies; they are indicated schematically (soma with a black ball and primary neurite with a thick black line). Morphologies shown here were representative of all filled examples of the same neuron type (except as noted below). Regions are colour-coded according to the key in f. a, A fast-cool-PN. These neurons have dendrites in the proximal antennal protocerebrum and axons projecting to two regions of the lateral protocerebrum (posterior lateral protocerebrum and posterior slope). Some fast-cool-PNs arbourized unilaterally (rather than bilaterally) in the proximal antennal protocerebrum; we pooled data from both morphologies because their physiological properties were indistinguishable. b, A slow-cool-PN. These neurons have dendrites in the ipsilateral proximal antennal protocerebrum and axons projecting to a subregion of the calyx of the mushroom body. This morphology has been described previously36 (that study called this neuron a ‘transverse antennal lobe projection neuron’). c, A warm-PN. These neurons have dendrites in the ipsilateral proximal antennal protocerebrum and axons projecting to two regions of the lateral protocerebrum (posterior lateral protocerebrum and posterior slope). d, A warm–cool-PN. These neurons have dendrites in both the ipsi- and contralateral proximal antennal protocerebrum, as well as the ventral-posterior margin of the antennal lobe. Their axons project to two regions of the lateral protocerebrum (posterior lateral protocerebrum and posterior slope). e, Two GABAergic LNs. All LNs we encountered in this study were bilateral. We define an LN here as a neuron that does not extend processes into any region except the proximal antennal protocerebrum (and the midline commissure that links the right and left copies of this brain region). Most LNs arbourized broadly within the posterior antennal protocerebrum and so are well-positioned to mediate cross-talk between warm and cool pathways. f, Locations of colour-coded brain regions in a coronal section through the brain.

Extended Data Figure 3 Adaptation to small temperature steps.

ad, Sensitivity to small temperature steps, shown as the mean change in firing rate versus the change in temperature, averaged across experiments, ±s.e.m. (n = 5–9). Data in red are measured during a late period after stimulus onset (from 4 to 5 s after valve opening). Data in black are measured during an early period after stimulus onset (from 100 to 400 ms after valve opening, except for the warm–cool-PNs, where it was from 100 to 200 ms). Data in black are reproduced from Fig. 1c (a, fast-cool-PNs), Fig. 1g (b, slow-cool-PNs), Fig. 2g (c, warm-PNs) and Extended Data Fig. 8 (d, warm–cool-PNs). Note that slow-cool-PNs show little adaptation to large temperature steps (Fig. 1g). However, their responses to small steps adapt strongly, like those of the other PN types.

Extended Data Figure 4 Innervation patterns in the proximal antennal protocerebrum.

Images are single coronal confocal sections through the proximal antennal protocerebrum, except where noted below. Magenta is neuropil (nc82 immunofluorescence). The approximate boundary of the proximal antennal protocerebrum is indicated in white outline. a, Axon terminals of cold peripheral neurons and warm peripheral neurons. GFP expression is driven by specific Gal4 lines (GMR79C04-Gal4 for the cool cells, Gr28b.d-Gal4 for the warm cells). As noted previously2, the cool peripheral cells project to the lateral part of this brain region, whereas the warm peripheral cells project more medially. Neuropils surrounding the proximal antennal protocerebrum are labelled as LP (lateral protocerebrum), LAL (lateral accessory lobe) and SOG (subesophageal ganglion). The schematic on the right shows a coronal section of the whole brain with the location of the proximal antennal protocerebrum indicated by white outlines. b, Dendrites of three types of projection neurons. Each cell was filled with biocytin and visualized using a fluorescent streptavidin conjugate. The dendrites of the fast-cool-PNs reside in the lateral part of the proximal antennal protocerebrum. The same is true of the dendrites of the slow-cool-PNs (data not shown). The dendrites of the warm-PNs overlap with the axons of both warm and cool peripheral neurons. The dendrites of warm–cool-PNs are sparser than those of other PN types, so in a single section only scattered fragments of dendrite are visible; therefore a z projection through the entire proximal antennal protocerebrum is shown in the image below.

Extended Data Figure 5 Circuit contributions to cool-PN responses.

ad, Fast-cool-PNs. a, Mean firing rate for the fast-cool-PNs, ± s.e.m. Reproduced from Fig. 1b. b, Mean firing rate with synaptic inhibition blocked with picrotoxin and CGP54626 (n = 4–5). c, Mean firing rate in the Gr28b.d mutant (n = 5–10). d, Mean firing rate in the Gr28b.d mutant with inhibition blocked (n = 4–6). eh, Slow-cool-PNs. e, Mean firing rate for the slow-cool-PNs ±s.e.m. Reproduced from Fig. 1f. f, Mean firing rate with synaptic inhibition blocked with picrotoxin and CGP54626 (n = 4–5). g, Mean firing rate in the Gr28b.d mutant (n = 4–5). h, Mean firing rate in the Gr28b.d mutant with inhibition blocked (n = 4). In the Gr28b.d mutant, note that blocking inhibition produces only modest disinhibition in the cool-PNs. This contrasts with our results in the warm-PNs and warm–cool-PNs, where blocking inhibition in the mutant abolished all excitation evoked by the preferred stimulus for these neurons (Fig. 2 and Fig. 3). Genotypes are: ad, Gr28bMB03888;GMR95C02-Gal4,pJFRC2-10XUAS-IVS-mCD8::GFP and eh, Gr28bMB03888;GMR67D03-Gal4,pJFRC2-10XUAS-IVS-mCD8::GFP.

Extended Data Figure 6 Validation of the LexA line for warm peripheral cells and the Gal4 line for cool peripheral cells.

There are six thermoreceptor cells at the base of the arista, comprising three warm cells and three cool cells2. Gr28b.d-Gal4 labels the three warm cells3. a, b, Validation of the Gr28b.d-LexA line for warm peripheral cells. a, We generated a LexA line using a Gr28b.d promoter fragment. Here we show that Gr28b.d-LexA labels three cells at the base of the arista. This image is a z projection of a confocal stack through the base of the arista. Genotype is Gr28b.d-LexA/+;26XLexAop2-mCD8::GFP/+. b, Axon terminals in the brain of peripheral neurons labelled by Gr28b.d-LexA (left) and Gr28b.d-Gal4 (right), both crossed with appropriate CD8::GFP reporter lines. As expected, the medial portion of the proximal antennal protocerebrum (outlined in white) is labelled with GFP. Images are single coronal confocal sections through the proximal antennal protocerebrum. Magenta is neuropil (nc82 immunofluorescence). Image on the right is reproduced from Extended Data Fig. 4. c, d, Validation of the GMR79C04-Gal4 line for cool peripheral cells. c, GMR79C04-Gal4 labels three cells at the base of the arista. Combining this driver with Gr28b.d-Gal4 labels six cells, indicating that these drivers label mutually exclusive populations of aristal neurons. These images show z projections of confocal stacks through the base of the arista. Genotypes are Gr28b.d-Gal4/+;UAS-nls-GFP/+ (left), UAS-nls-GFP/+;GMR79C04-Gal4/+ (middle), Gr28b.d-Gal4/+;UAS-nls-GFP/GMR79C04-Gal4 (right). d, Calcium imaging of neurons at the base of the arista shows that Gr28b.d-Gal4 labels cells that are excited by warming and inhibited by cooling, whereas GMR79C04-Gal4 labels cells that are excited by cooling and inhibited by warming. Shown here are representative experiments using the ‘large fast step’ stimuli. Genotypes are 20xUAS-GCaMP3/+;Gr28b.d-Gal4/+ (warm cell imaging) and 20XUAS-GCaMP3/+;;GMR79C04-Gal4/+ (cool cell imaging). Note that Fig. 4g shows data for all experiments using ‘small fast step’ stimuli.

Extended Data Figure 7 Histamine injection controls.

In these experiments the LexA driver (Gr28b.d-LexA) was omitted, but otherwise the experimental protocol was the same as in Fig. 2e, f and Fig. 3e–g. After histamine was injected into the antenna, there was little effect on the thermal responses of warm-PNs or warm-cool-PNs. a, Recordings from warm-PNs (n = 5). Genotype is LexAOp-HA-Ort/+;GMR95C02-Gal4,pJFRC2-10XUAS-IVS-mCD8::GFP. b, Recordings from warm–cool-PNs (n = 4). Genotype is LexAOp-HA-Ort/+;GMR54A03-Gal4,pJFRC2-10XUAS-IVS-mCD8::GFP.

Extended Data Figure 8 Sensitivity to small temperature steps in warm–cool-PNs.

Sensitivity to small temperature steps, shown as the mean change in firing rate versus the change in temperature, averaged across experiments, ±s.e.m. (n = 5–9). Firing rate changes were averaged over a window from 100 ms to 200 ms after valve opening. Sensitivity was not significantly different in wild type versus the Gr28b.d mutant (unpaired t-tests with iterative Bonferroni corrections, see Methods). Blocking inhibition in these cells produced oscillatory activity that precluded analysis of responses to small steps with inhibition blocked. Genotypes are: GMR54A03-Gal4,pJFRC2-10XUAS-IVS-mCD8::GFP (wild type) and Gr28bMB03888;GMR54A03-Gal4,pJFRC2-10XUAS-IVS-mCD8::GFP (Gr28b.d−/−).

Extended Data Figure 9 Validating the Gad1-Gal4 line as a marker of GABAergic local neurons in the posterior antennal protocerebrum.

In pilot studies, we found that all the local neurons we encountered in the proximal antennal protocerebrum had somata within a distinctive cluster ventral to the antennal lobes. We therefore targeted our electrodes to this cluster when we used a Gad1-Gal4 line26 to drive CD8::GFP expression in putative GABAergic neurons. In order to determine if the CD8::GFP-expressing somata in this cluster are indeed GABAergic, we performed dual immunofluorescence confocal microscopy with anti-CD8 and anti-GABA antibodies. This representative image shows that almost all CD8::GFP-expressing somata in this region are GABA-immunopositive (90% overall), although not all GABAergic somata express CD8::GFP. This image is a single coronal section through the cluster of somata.

Extended Data Figure 10 Additional examples of LNs in the proximal antennal protocerebrum.

For each LN, the sequence of panels is analogous to that of Fig. 4: raw traces (a, e), peri-stimulus time histograms (b, f) and sensitivity plots (c, g). The morphologies of these cells (d and h) are displayed as in Extended Data Fig. 2, with a red outline indicating the boundary of the proximal antennal protocerebrum. ad, A GABAergic LN. This LN is excited by warming and is inhibited by cooling. This cell was recorded in the genotype in pJFRC7-20XUAS-IVS-mCD8::GFP/+;Gad1-Gal4/+. eh, A glutamatergic LN. This LN is excited by cooling and is inhibited by warming, and its properties are similar to those of all the glutamatergic LNs we recorded from. In total we recorded from six glutamatergic LNs, three in the genotype GMR52G03-Gal4,pJFRC2-10XUAS-IVS-mCD8::GFP and three in the genotype GMR91H10-Gal4,pJFRC2-10XUAS-IVS-mCD8::GFP.

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Liu, W., Mazor, O. & Wilson, R. Thermosensory processing in the Drosophila brain. Nature 519, 353–357 (2015). https://doi.org/10.1038/nature14170

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