Optical inhibition of larval zebrafish behaviour with anion channelrhodopsins

Abstract

Background: Optical silencing of activity provides a way to test the necessity of neurons in behaviour. Two light-gated anion channels, GtACR1 and GtACR2, have recently been shown to potently inhibit activity in cultured mammalian neurons and in Drosophila. Here, we test the usefulness of these channels in larval zebrafish, using spontaneous coiling behaviour as the assay.

Results: When the GtACRs were expressed in spinal neurons of embryonic zebrafish and actuated with blue or green light, spontaneous movement was inhibited. In GtACR1-expressing fish, only 3 μW/mm² of light was sufficient to have an effect; GtACR2, which is poorly trafficked, required slightly stronger illumination. No inhibition was seen in non-expressing siblings. After light offset, the movement of GtACR-expressing fish increased, which suggested that termination of light-induced neural inhibition may lead to activation. Consistent with this, two-photon imaging of spinal neurons showed that blue light inhibited spontaneous activity in spinal neurons of GtACR1-expressing fish, and that the level of intracellular calcium increased following light offset.

Conclusions: These results show that GtACR1 and GtACR2 can be used to optically inhibit neurons in larval zebrafish with high efficiency. The activity elicited at light offset needs to be taken into consideration in experimental design, although this property can provide insight into the effects of transiently stimulating a circuit.

Keywords: Behaviour; Chloride pump; Neural circuit; Optogenetics; Zebrafish.

Fig. 1

Expression of GtACR1 and GtACR2 in transgenic zebrafish. a, b Expression of GtACR1-eYFP in the trunk of 1-day-old GAL4s1020t, UAS:GtACR1-eYFP (a) and GAL4s1020t, UAS:GtACR2-eYFP (b) embryos. Labelled cells are located in the ventral regions of the spinal cord. c, d High magnification view of the trunk of fish expressing GtACR1-eYFP (c) or GtACR2-eYFP (d) in spinal neurons. There is label in the plasma membrane (arrows). For GtACR2, the label is dimmer. Puncta are visible (arrowheads). e An 8-dpf gng8:GAL4, UAS:GtACR1-eYFP larva, showing label in habenula neurons and in axons innervating the interpeduncular nucleus. f Expression of GtACR1-eYFP in olfactory neurons of a 3-day-old fish carrying the GAL4s1011t driver. g, h Forty-eight-hour-old embryos labelled with acridine orange. Dying cells are strongly labelled and appear as bright spots. These are detected in both GtACR1-expressing (g) and non-expressing (h) fish. There is no evidence of cell death in the ventral spinal cord of GtACR1-expressing fish. Anterior is to the left in all panels except f, where anterior is to the top. All panels are lateral views, except e and f, which are dorsal views. Panels a, c, d and f are single planes, while b, e, g and h are maximum projections. OE olfactory epithelium, IPN interpeduncular nucleus, lHb left habenula, rHb right habenula

Fig. 2

Light sensitivity of GtACR1- and GtACR2-expressing fish. a, b Response of ACR1 fish to blue (a) and green (b) light of three different intensities (high, medium and low). For each panel, the top trace shows the movement of each fish in 1 s. This is measured by number of pixels that differ, relative to the cross-sectional area of the chorion. Each line represents one fish. The lower trace shows the mean value and 95% CIs. Controls refer to siblings lacking eYFP expression that were exposed to high intensity light. c, d Effect of blue (c) and green (d) light of different intensities on ACR2-expressing fish. e, f The effect of red light on ACR1- (e) and ACR2- (f) expressing fish. Line colour corresponds to the colour of light used for illumination. For all conditions, the period of illumination lasted from t = 15 s to t = 30 s

Fig. 3

The effect of light and loss of light on spontaneous movement of GtACR1 embryos. a, c, e The amount of movement displayed by individual embryos, in the 5 s before light onset (‘5 s before’), in the first 5 s after light onset (‘First 5 s’) and in the first 5 s after light offset (‘5 s after’). Different intensities of blue (a), green (c) and red (e) light were tested. Controls refer to siblings lacking eYFP expression. The line colour of each plot corresponds to the light colour employed for illumination. b, d, f The mean amount of movement relative to the period before light onset. A negative value indicates inhibition of spontaneous movement, whereas a positive value indicates elevated levels of movement. Mean differences and 95% CIs are reported alongside P values from Wilcoxon tests. Blue (b) and green (d) light both affect spontaneous movement. Red light has a small effect (f)

Fig. 4

The effect of light and loss of light on spontaneous movement of GtACR2 embryos. a, c, e The amount of movement displayed by individual embryos, in the 5 s before light onset (‘5 s before’), in the first 5 s after light onset (‘First 5 s’) and in the first 5 s after light offset (‘5 s after’). Three different intensities of blue (a) and green (c) light and one intensity of red light (e) were tested. The colour of the lines indicates the colour of light used for illumination. Controls for each experiment were siblings not expressing eYFP. b, d, f The mean amount of movement relative to the period before light onset. A negative value indicates inhibition of spontaneous movement, whereas a positive value indicates elevated levels of movement. Mean differences and 95% CIs are reported; all P values are results of Wilcoxon tests. Blue (b) light affected spontaneous movement at medium and high intensities, whereas green light only had a weak effect at high intensity (d). Red light had no effect

Fig. 5

The effect of low intensity amber light on spontaneous movement of NpHR-expressing embryos. a, b Lateral view of a 24-h-old embryo expressing NpHR-mCherry under the 1020 GAL4 driver. a Overview of expression in the spinal cord. b. High magnification view, showing expression of NpHR-mCherry in spinal neurons. c–f Time course of movement of embryos, with (c, e) or without (d, f) halorhodopsin expression, in a 45-s recording with exposure to 15 s of amber light. The period of illumination is indicated by the black bar. c and d show movement of individual embryos, while e and f show mean values and 95% CIs. g, h Average amount of movement in the 5 s before light onset (‘5 s before’), in the first 5 s after light onset (‘First 5 s’) and in the first 5 s after light offset (‘5 s after’), in NpHR-expressing embryos (g) and non-expressing siblings (h). i Difference in movement in the first 5 s after light onset and after light offset, relative to the period before light, in NpHR-expressing and non-expressing embryos. Intensity of amber light used = 17 μW/mm²

Fig. 6

Calcium imaging of spinal neurons in GtACR1 embryos and non-expressing siblings. a Ventral spinal neurons of a 24-h-old 1020:GAL4, UAS:GtACR1-eYFP, elavl3:GCaMP6f embryo. The green arrowhead indicates a neuron with bright membrane label and puncta, suggesting expression of GtACR1-eYFP. The orange arrowhead indicates a neuron without GtACR1-eYFP expression. b Time course of fluorescence intensity in neurons, represented as change relative to minimum fluorescence. There is a reduction in activity during delivery of blue light. c, d Activity in dorsal spinal neurons, which do not express the GAL4 driver, in another 24-h-post-fertilization (hpf) GAL4s1020t, UAS:GtACR1-eYFP, elavl3:GCaMP6f embryo. Spontaneous activity was detected before and after, but not during, the period of blue light delivery. e, f Spontaneous activity in six neurons in a 26-h-old embryo with no GtACR1-eYFP expression. e Increase in fluorescence intensity occurs during the period of illumination with blue light (arrowheads). g, h The response of six spinal neurons in a 4-day-old embryo expressing GtACR1. There is no activity before light in these neurons, but there is a rise in GCaMP6f fluorescence after termination of the blue light. In panels b, d, f and h, the blue shaded region and bar indicate the period in which light was delivered. The colours of the traces represent relative change in fluorescence of the cells indicated by the arrowheads with the corresponding colours in the image on the left