(A) Survival rates of fish as a function of time after different treatments. The number of fish in each treatment group is given in the legend. Larvae were injected at 5 dpf.

(A) Survival rates of fish as a function of time after different treatments. The number of fish in each treatment group is given in the legend. Larvae were injected at 5 dpf.

(A) Comparison of swimming speed, vertical position in the water column (Z position), and social distance in groups of five fish at room temperature (RT; 25 – 26 °C) and at 36 °C. Fish were kept at the indicated temperature for 15 min. Each datapoint represents the mean value during a one-minute time window (10 time windows in total). Box plots show median and 25th and 75th percentiles, circles and error bars indicate mean and s.d. over time windows. Swimming trajectories were analyzed in two dimensions using DeepLabCut. Z position is normalized to the distance between the bottom of the tank (0) and the water surface (1). Social distance is the mean distance between all pairs of fish in the group. (Swimming speed: p<0.001, Z position: p<0.001, p=0.53, Wilcoxon rank sum test). (B) Swimming speed of adult fish kept individually in 1 L tanks measured at 27 °C. Fish were either kept at 27 °C or at 36 °C for 7 days prior to measurements and received different treatments (no injection, buffer injection, or HSV1 injection). Plot symbols represent data from individual fish; box plots show median and 25th and 75th percentiles, circles and error bars indicate mean and s.d. over individual fish. N: number of fish. No significant differences were observed (p>0.54; ANOVA test). (C) Sequence of virus injections, temperature changes and associative conditioning to assess effects of experimental manipulations on discrimination learning. (D) Learning index (behavioral discrimination score) on the last day of training. Plot symbols represent data from individual fish; box plots show median and 25th and 75th percentiles, circles and error bars indicate mean and s.d. over individual fish. N: number of fish. Control fish (group 1) were not injected and kept at standard laboratory temperature. The experimental group (group 2) was injected with HSV1[LTCMV:jGCaMP7b], an amplicon type of HSV1 with an insert encoding the calcium indicator GCamp7b (Dana et al., 2019) under the control of a non-specific promoter for long-term expression (LTCMV), and subsequently kept at 36 °C for 2 days before training (A). Performance was not significantly different between groups (p=0.76, Wilcoxon rank sum test).

(A) Expression of DsRed (magenta) 6 days after injection of HSV1[LTCMV:DsRed] into the OB (arrow) of an adult Tg[vglut1:GFP] fish kept at 26 °C (maximum projection of confocal stack). Boxed areas (OB and Dp) are enlarged below. The number of DsRed-expressing neurons is low compared to DsRed expression at 36 °C (Figure 1). (B) DsRed expression in the dorsal telencephalon at different timepoints after injection of HSV1[LTCMV:DsRed] into the ipsilateral OB. Fish were kept at 36 °C. Black dots represent data from individual fish, box plot indicates median and 25th and 75th percentiles, circles and error bars indicate mean and s.d. over individual fish. N: number of fish.

(A) Expression of GFP 48 hr after injection of HSV1[LTCMV:GFP] into the optic tectum of zebrafish larvae (3 dpf; maximum intensity projection of confocal stack). Larvae were kept after the injection at 28.5, 32, or 35 °C. (B) Expression of GFP 48 hr after injection of HSV1[LTCMV:GFP] into the optic tectum of a larva at 5 dpf (maximum intensity projection of confocal stack). The larva was kept after the injection at 35 °C. (C) Expression of GFP 48 hr after injection of HSV1[LTCMV:GFP] into the optic tectum of a larva at 14 dpf (maximum intensity projection of confocal stack). The larva was kept after the injection at 35 °C. (D) Expression of GFP 48 hr after injection of HSV1[LTCMV:GFP] into trunk muscles at 7 dpf (maximum intensity projection of confocal stack). The larva was kept after the injection at 35 °C. Note retrograde labeling of motor neurons (M.N.). (E) Expression of GFP 48 hr after injection of HSV1[UAS:GFP] into the hindbrain of a Tg[gad1b:Gal4; gad1b:DsRed] larva at 7 dpf (maximum intensity projection of confocal stack). The larva was kept after the injection at 35 °C. Note co-localization of DsRed and GFP in hindbrain and cerebellum.

(A) Injection of HSV1[UAS:TVA-mCherry] into the telencephalon of adult wildtype fish. No expression of TVA-mCherry was detectable (granular particles are autofluorescent endogenous objects). (B) Coronal section through the telencephalon at the level of Dp after injection of HSV1[UAS:TVA-mCherry] into Tg[gad1b:Gal4; gad1b:GFP] double transgenic fish. The injection was targeted to a volume around Dp. mCherry was expressed predominantly in a cluster of GFP-positive neurons associated with Dp. Note long-range projections of mCherry-expressing neurons to multiple telencephalic areas. (C) Enlargements of boxed region in (a). Arrowheads indicate GFP+/mCherry +neurons.

(A) Injection of HSV1[UAS:TVA-mCherry] into the telencephalon of adult wildtype fish. No expression of TVA-mCherry was detectable (granular particles are autofluorescent endogenous objects). (B) Coronal section through the telencephalon at the level of Dp after injection of HSV1[UAS:TVA-mCherry] into Tg[gad1b:Gal4; gad1b:GFP] double transgenic fish. The injection was targeted to a volume around Dp. mCherry was expressed predominantly in a cluster of GFP-positive neurons associated with Dp. Note long-range projections of mCherry-expressing neurons to multiple telencephalic areas. (C) Enlargements of boxed region in (a). Arrowheads indicate GFP+/mCherry +neurons.

(A) Expression of GFP and mCherry after injection of HSV1[UAS:GFP & UAS:TVA-mCherry] into the cerebellum of Tg[gad1b:Gal4] fish. In this virus, two expression constructs, UAS:GFP and UAS:TVA-mCherry, are packaged into the same virus particles. Expression is observed in Purkinje neurons and in putative Golgi cells. Note high rate of co-expression of GFP and mCherry. ML: molecular layer; PL: Purkinje layer; GL: granular layer. (B) Expression of GFP and mCherry in the Purkinje layer after co-injection of two independent viruses (HSV1[UAS:GFP] and HSV1[UAS:TVA-mCherry]) into the cerebellum of Tg[gad1b:Gal4] fish. Note that the rate of co-expression was high even though GFP and mCherry were delivered by separate viruses. Note also that the overall expression was sparse, implying that co-expression was unlikely to occur by chance. (C) Percentage of GFP and mCherry-expressing neurons among all fluorescent neurons. Filled circles represent data from individual fish, box plot indicates median and the 25th and 75th percentiles, and open circles indicate mean over individual fish. N: number of fish.

(A) Schematic: injection of HSV1[LTCMV:Gal4] into the OB and subsequent co-injection of HSV1[UAS:GCaMP6f] and HSV1[UAS:Chrimson-tdTomato] into the dorsal telencephalon of wildtype fish.(B) Simultaneously recorded calcium transients evoked by optical stimulation of different light intensity (vertical lines) in three example neurons. (C) Mean change GCaMP6f evoked by optical stimulation of different light intensity (11–18 light stimuli at each intensity). Left: GCaMP6f ⁺/tdTomato^- neurons (N=8 out of 21 GCaMP6f⁺ neurons). Right: GCaMP6f ⁺/tdTomato⁺ neurons (N=13 out 21 GCaMP6f⁺ neurons). Shading shows SEM. Responses to light stimulation were seen in all individual tdTomato⁺ neurons (N=13; not shown).

(A) Top: Expression of GFP in the cerebellum after injection of HSV1[UAS:GFP] into the cerebellum of Tg[gad1b:Gal4] fish. Bottom: Expression of TeNT-GFP after injection of HSV1[UAS:TeNT-GFP] into the cerebellum of Tg[gad1b:Gal4] fish. (B) Examples of swimming trajectories (15 min) of individual Tg[gad1b:Gal4] fish that received injections of HSV1[UAS:GFP] (left) or HSV1[UAS:TeNT-GFP] into the cerebellum. Note that the fish injected with HSV1[UAS:TeNT-GFP] covered less territory, showed fewer long straight swims, and showed a tendency to stay lower in the water column. (C) Mean swimming speed and 2D space occupancy of Tg[gad1b:Gal4] fish that received injections of HSV1[UAS:GFP] (left) or HSV1[UAS:TeNT-GFP] into the cerebellum. Plot symbols represent data from individual fish; box plots show median and 25th and 75th percentiles, circles and error bars indicate mean and s.d. over individual fish. N: number of fish. p=0.03 for swimming speed, p=0.0012 for space occupancy, Wilcoxon rank sum test.

(A) Absence of expression after injection of EnvA-RVΔG-GFP into the telencephalon of adult wildtype fish.(B) Absence of expression after injection of EnvA-RVΔG-GFP into the optic tectum of wildtype fish at 7 dpf.

(A) Absence of expression after injection of EnvA-RVΔG-GFP into the telencephalon of adult wildtype fish.(B) Absence of expression after injection of EnvA-RVΔG-GFP into the optic tectum of wildtype fish at 7 dpf.

(A) Experimental scheme: Rabies virus (EnvA-RVΔG-GFP) was injected into the telencephalon of transgenic fish expressing TVA-mCherry in GABAergic neurons (Tg[gad1b:Gal4; UAS:TVA-mCherry]). (B) Expression of TVA-mCherry and GFP when fish were kept at 26 °C for 6 days after injection. Note almost complete absence of GFP expression.(C) Expression of TVA-mCherry and GFP when fish were kept at 36 °C for 6 days after injection. Note strong GFP expression.(D) Expression of TVA-mCherry and GFP six days after injection when the housing temperature was increased from 26 °C to 36 °C 3 days after injection. GFP expression was weak and sparse.

(A) Schematic: injection of EnvA-RVΔG-GFP into the OB of adult Tg[gad1b:Gal4;UAS:TVA-mCherry] fish. (B) Example of FACS analysis of GFP and mCherry expression. Boxes depict cells selected as mCherry+/GFP+ (EnvA-RVΔG-GFP infected gad1b neurons), mCherry+/GFP- (non-infected gad1b neurons), mCherry-/GFP- (negative control containing other OB cells). gad1b is one of two isoforms of gad1 that are expressed differentially in GABAergic neurons. (C) Expression of marker genes (x-axis) in infected gad1b neurons (mCherry+/GFP+; green), non-infected gad1b neurons (mCherry+/GFP-; magenta), and other OB cells (mCherry-/GFP-; black). Cells classified as gad1b-positive by fluorescence markers, but not other cells, expressed gad1b but not gad1a, the other gad1 isoform. Expression of fluorescent marker genes followed the detection of fluorescent markers by FACS. The neuronal marker elav3 was present in all three pools. Plot symbols represent data from individual samples; box plots show median and 25^th and 75^th percentiles, circles and error bars indicate mean and s.d. over individual samples (N=8 samples). (D) Expression of negative markers for GABAergic neurons. The selected marker genes (slc17a6a, slc17a6b, tbx21, lhx2b, and lhx9) should be expressed in mitral cells of the OB and other excitatory neurons but not in GABAergic neurons. Consistent with this expectation, expression of all negative markers was low or absent in pools of gad1b cells selected by FACS (N=8 samples).

Expression of GFP in the cerebellum after sequential injection of (1) HSV1[UAS:TVA-mCherry] and HSV1[UAS:zoSADG] and (2) EnvA-RVΔG-GFP into the cerebellum of Tg[gad1b:Gal4] fish. Outlined regions are enlarged. Only few labeled neurons were detected. Left: EnvA-RVΔG-GFP was injected 2 days after HSV1 injection. Right: EnvA-RVΔG-GFP was injected 4 days after HSV1 injection.

Expression of GFP in the cerebellum after sequential injection of (1) HSV1[UAS:TVA-mCherry] and HSV1[UAS:zoSADG] and (2) EnvA-RVΔG-GFP into the cerebellum of Tg[gad1b:Gal4] fish. Outlined regions are enlarged. Only few labeled neurons were detected. Left: EnvA-RVΔG-GFP was injected 2 days after HSV1 injection. Right: EnvA-RVΔG-GFP was injected 4 days after HSV1 injection.

(A) Co-injection of EnvA-RVΔG-GFP and HSV1[UAS:TVA-mCherry] into Dp of Tg[vglut1:Gal4] fish in the absence of glycoprotein. Coronal section through the injected telencephalic hemisphere at the level of Dp. Area outlined by dashed rectangle is enlarged below and red channel is enhanced. Co-expression of GFP (green) and mCherry (magenta) indicates starter cells. (B) Same as in (A) but with trans-complementation of zoSADG in starter neurons by co-injection of HSV1[UAS:zoSADG]. Left: coronal section through the injected telencephalic hemisphere. Right: coronal section through the ipsilateral olfactory bulb. Expression of GFP only (green) indicates transneuronally labeled neurons.

(A) Coronal cross sections through the OB and anterior telencephalon from Tg[vglut2a:RFP; vglut1:GFP] double transgenic fish. Note that vglut2a (magenta) is expressed by axons of olfactory sensory neurons innervating glomeruli in the OB and by a subset of mitral cells, while expression of vglut1 (green) in the OB is weak or absent. Dotted lines outline OBs. (B) More posterior coronal cross sections through the telencephalon of the same fish at the level of Dp. Note that expression of vglut2a and vglut1 in the telencephalon are largely complementary. Neurons in Dp express primarily vglut1. Dotted areas indicate the dorsal lateral telencephalic area (Dl) and Dp.

(A) Expression of GFP (green) and TVA-mCherry (red) 6 days after injection of EnvA-RVΔG-GFP into the spinal cord of Tg[gad1b:Gal4;UAS:TVA-mCherry] fish at 7 dpf. Boxed region is enlarged on the right. (B) Same after co-injection of EnvA-RVΔG-GFP and HSV1[UAS:zoSADG] into the spinal cord of Tg[gad1b:Gal4;UAS:TVA-mCherry] fish at 7 dpf.