Mishra A et al. (2018), The Drosophila Gr28bD product is a non-specific...

XB-ART-54476

Sci Rep 2018 Jan 17;81:901. doi: 10.1038/s41598-017-19065-4.

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The Drosophila Gr28bD product is a non-specific cation channel that can be used as a novel thermogenetic tool.

Mishra A , Salari A , Berigan BR , Miguel KC , Amirshenava M , Robinson A , Zars BC , Lin JL , Milescu LS , Milescu M , Zars T .

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Extrinsic control of single neurons and neuronal populations is a powerful approach for understanding how neural circuits function. Adding new thermogenetic tools to existing optogenetic and other forms of intervention will increase the complexity of questions that can be addressed. A good candidate for developing new thermogenetic tools is the Drosophila gustatory receptor family, which has been implicated in high-temperature avoidance behavior. We examined the five members of the Gr28b gene cluster for temperature-dependent properties via three approaches: biophysical characterization in Xenopus oocytes, functional calcium imaging in Drosophila motor neurons, and behavioral assays in adult Drosophila. Our results show that Gr28bD expression in Xenopus oocytes produces a non-specific cationic current that is activated by elevated temperatures. This current is non-inactivating and non-voltage dependent. When expressed in Drosophila motor neurons, Gr28bD can be used to change the firing pattern of individual cells in a temperature-dependent fashion. Finally, we show that pan-neuronal or motor neuron expression of Gr28bD can be used to alter fruit fly behavior with elevated temperatures. Together, these results validate the potential of the Gr28bD gene as a founding member of a new class of thermogenetic tools.

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Species referenced: Xenopus
Genes referenced: dtl lgals4.2

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	Figure 1. Biophysical properties of Gr28bD in Xenopus oocytes. (A) Temperature response: oocytes injected with Gr28bD cRNA display temperature-activated inward current in response to temperature steps. Control oocytes injected with RNAse-free water exhibit no significant current. (B) Response of Gr28bD current to slow temperature ramps (≈2.5 °C/60 s). (C) Temperature sensitivity: the logarithmic plot of normalized Gr28bD current versus slow ramp temperature exhibits two ranges of temperature sensitivity. Red and blue lines represent linear fits to data in each range (14–19 °C and 21–35 °C). Q10 values were calculated using the equation Q10 = 1010×s, where s is the slope of the linear fit. The average estimates are shown (n = 12). (D) Voltage response: control and Gr28bD currents in response to voltage steps at two different temperatures. Endogenous currents are small at all voltages and temperatures. (E) Current-voltage relationships for GR28bD-expressing and control oocytes at 10 and 35 °C, as obtained with the voltage step protocol in D. Gr28bD current does not show voltage sensitivity at the tested temperatures. The symbols are as in D. (F) Ionic selectivity: Gr28bD currents were obtained as in E, at 35 °C, with Na+ partially replaced by NMDG+ in the external solution (96 mM Na+ : 0 mM NMDG+, 48 : 48, and 2 : 94). The measured reversal potentials were (in mV): −12.0 ± 0.7, −23.7 ± 0.2, and −59.0 ± 0.9, respectively. The holding voltage was −60 mV in all experiments. Data shown in A, B, C, and D are representative examples. In E and F, data points are mean ± SEM (n = 6–8). The currents were obtained under ND96 solution (see Methods), except where otherwise noted.
	Figure 2. Temperature-dependent modulation of cellular activity by Gr28bD. (A) Left panel: dorsal view of adult Drosophila dissected to expose ventral nerve cord motor neurons to saline bath. Right panel: composite image of epifluorescence (green on black) and two-photon 3D reconstruction (gray scale) of abdominal neuromere neurons expressing GCaMP6f. The green trace is an example of single-cell activity, as obtained via epifluorescence from the indicated region of interest. (B) Single-cell fluorescence signals (blue traces) obtained in response to a temperature step (red traces), analyzed by continuous wavelet-transform (spectrogram). Top panel: representative data from control flies expressing only GCaMP6f in motor neurons. Middle and bottom panels: representative data sets illustrating two types of temperature responses in flies expressing GCaMP6f + Gr28bD in motor neurons. (C) Change in average fluorescence between low and high temperature. ΔF/F was calculated as (FH−FL)/FL, where FL and FH are the average fluorescence intensities (in arbitrary units) for a given cell, at low and high temperature. Data points are mean ± SDV (n = 21 cells from five flies for GCaMP6f, and n = 20 cells from six flies for GCaMP6f + Gr28bD). The blue lines represent mean ± SDV for each data set. ΔF/F = −0.1524 ± 0.1358 in control flies and 0.03656 ± 0.10 in GR28bD-expressing flies (unpaired t-test; p-value < 0.00001).
	Figure 3. Temperature-dependent modulation of fly behavior by Gr28bD. (A) Incapacitation by temperature: flies overexpressing GR28b genes had their locomotor activity recorded for 90 seconds at 24–40 °C in two-degree steps. The plot shows the cumulative proportion of flies not incapacitated at each temperature. Most flies with pan-neuronal or motor neuron overexpression of GR28bD were temporarily incapacitated at 34 or 36 °C, respectively. Gr28bB overexpression had an effect at the very high end of the tested temperature range. (B) Control and Gr28bD-overexpressing flies were exposed to 32–40 °C in one-degree steps. Most flies overexpressing Gr28bD pan-neuronally were incapacitated at 33–35 °C. The heterozygous genetic control flies did not show incapacitation over the tested temperatures. Incapacitation is faster and recovery is slower at higher exposure temperatures. (C) Example position traces of a non-incapacitated fly (top panel) and of an incapacitated fly that recovered (bottom trace). The red area represents the exposure to high temperature and the arrows mark the times of incapacitation and recovery. (D) Time to incapacitation at different temperatures: flies overexpressing Gr28bD and flies heterozygous for the GAL4 driver and UAS-Gr28bD construct were tested. Most flies with Gr28bD pan-neuronal overexpression were incapacitated within 10 seconds at 36, 38, and 40 °C, but it took much longer at 34 °C. Few flies from the control genotypes were incapacitated at the temperatures tested. (E) Time to recovery from incapacitation: flies recovered faster when subjected to lower temperature. In A, B, and D, statistical difference between control and experimental genotypes was determined with a Kaplan-Meier test, p < 0.000001 = ****. In each experiment, 60–80 flies of each genotype were tested.

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