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A viral toolbox for conditional and transneuronal gene expression in zebrafish

The zebrafish is an important model in systems neuroscience but viral tools to dissect the structure and function of neuronal circuitry are not established. We developed methods for efficient gene transfer and retrograde tracing in adult and larval zebrafish by herpes simplex viruses (HSV1). HSV1 was combined with the Gal4/UAS system to target cell types with high spatial, temporal, and molecular specificity. We also established methods for efficient transneuronal tracing by modified rabies viruses in zebrafish. We demonstrate that HSV1 and rabies viruses can be used to visualize and manipulate genetically or anatomically identified neurons within and across different brain areas of adult and larval zebrafish. An expandable library of viruses is provided to express fluorescent proteins, calcium indicators, optogenetic probes, toxins and other molecular tools. This toolbox creates new opportunities to interrogate neuronal circuits in zebrafish through combinations of genetic and viral approaches.


Introduction 14
The zebrafish is an important vertebrate model in systems neuroscience because its small, optically 15 accessible brain provides unique opportunities to analyze neuronal circuits and behavior 1,2 . Key methods 16 established in zebrafish include large-scale imaging of neuronal population activity, behavioral 17 approaches including virtual realities, and genetic manipulations 1-8 , but efficient methods for viral gene 18 transfer are still lacking. Viral vectors are important experimental tools in mammals because they enable 19 the visualization and manipulation of defined neurons with high spatial, temporal and molecular 20 specificity, and because they can bypass the need to generate transgenic animals 9,10 . Moreover, engineered 21 viral tools that cross synapses allow for the visualization and physiological analysis of synaptically 22 connected neuronal cohorts [11][12][13] . Our goal was to establish similar methods for specific viral gene transfer 23 and transneuronal tracing in zebrafish. 24 25 Results 26

Optimization of the temperature regime for viral gene transfer 27
Previous studies demonstrated that adeno-associated viruses (AAVs), which are widely used for gene 28 transfer in mammals and other amniotes, fail to infect neurons in the zebrafish brain 14 . Infection of 29 zebrafish neurons by other viruses has been reported but efficiency was usually low and viral vectors for 30 conditional gene expression in transgenic fish have not been described. To improve upon these points, we 31 significantly impaired by virus injection or subsequent incubation at temperatures near 37 ºC. 48 To examine the temperature-dependence of HSV1-mediated gene expression we injected adult zebrafish 49 control of a non-specific promoter for long-term expression (LTCMV). Injections were targeted 52 unilaterally into the olfactory bulb (OB) and fish were then maintained at 26 ºC or 36 ºC for 6 days after 53 injection (Fig. 1a). Consistent with previous results 15 , reporter expression (DsRed) was observed in the 54 injected OB (Fig. S1c). Moreover, retrogradely labeled neurons were present in telencephalic areas that 55 project to the OB, most notably in the posterior zone of the dorsal telencephalon (Dp), the homolog of 56 mammalian olfactory cortex (Fig. S1c). After incubation at 36 ºC, substantially more neurons expressed 57 DsRed (Fig. 1b). In addition, retrograde labeling was detected in a small but distinct population of 58 neurons in the dorsal telencephalon that was not seen at 26 ºC (Fig. 1b, c). DsRed expression at 36 ºC was 59 first detected ~12 hours after injection and stable for at least 10 days (Fig. S1d). Moreover, increasing the 60 temperature from 26 ºC to 36 ºC three days after the injection failed to enhance expression (Fig. 1a, c). 61 These results show that adjusting the temperature to that of mammalian hosts can substantially increase 62 the efficiency of HSV1-mediated viral gene transfer in zebrafish. 63 and measurements of odor-evoked activity revealed no signs of toxicity of RVΔG alone, indicating that 293 transneuronally labeled neurons are healthy even though starter neurons disappear. 294 Transneuronal spread of rabies viruses was glycoprotein-dependent and labeled substantial numbers of 295 putative presynaptic neurons from different starter neurons, both in adult zebrafish and in larvae. 296 Convergence ratios were similar to those observed in rodents despite the fact that zebrafish neurons are 297 smaller and probably receive fewer synaptic inputs. These results suggest that the efficiency of 298 transneuronal tracing was in the same range as in rodents although quantitative comparisons are difficult. 299 In the future, further improvements may be achieved by modifications that are currently explored in 300 rodents such as the design of optimized glycoproteins 29 or trans-complementation with a second viral 301 gene 38 . The apparent long-term toxicity of the glycoprotein may be circumvented by transient expression 302 using a negative feedback system 39 . Hence, rabies-based transneuronal tracers are promising tools for the 303 structural and functional dissection of neuronal circuitry in zebrafish. 304 Retrograde tracing of connected neuronal cohorts using rabies viruses has become an important approach 305 to decipher the functional logic of neuronal connectivity in rodents. Our results pave the way for 306 applications of this approach in zebrafish. The small size of zebrafish facilitates the combination of 307 neuronal circuit tracing with other methods such high-resolution imaging of neuronal activity patterns 308 throughout large brain areas. Transneuronal tracing in the anterograde direction from starter neurons that 309 were not genetically defined has previously been achieved using VSVs in larval zebrafish 23,24 . Recently, it 310 has been reported that the cytotoxicity of VSVs used in initial studies can be reduced 25 , suggesting that 311 anterograde transneuronal tracing using VSVs may complement retrograde transneuronal tracing by 312 rabies viruses. Our experiments were designed primarily to explore HSV1 and rabies viruses as tools for 313 structural and functional analyses of neuronal circuits and behavior. Moreover, the ability of HSV1 and 314 rabies viruses, as well as VSVs, to infect larval neurons may be exploited for studies of development, 315 although the temporal resolution will be limited by the time required for viral gene expression (days).

Virus injection 522
Virus injections were performed as described 15 with minor modifications. Adult fish were anesthetized in 523 0.03 % tricaine methanesulfonate (MS-222) and placed under a dissection microscope. MS-222 (0.01 %) was 524 continuously delivered into the mouth through a small cannula. A small craniotomy was made over the dorsal 525 telencephalon near the midline, over the OBs, or over the cerebellum. If multiple viruses were injected in the 526 same region, virus suspensions were mixed. Phenol red (0.05 %) was added to the suspension to visualize the 527 injection. Micropipettes pulled from borosilicate glass were inserted vertically through the craniotomy. The 528 depth of injections was approximately 100 m in the dorsal telencephalon, 50 m in the OB, 100-150 m in 529 the cerebellum, 300-500 m in Dp and 50 -200 m in the tectum. The injected volume was 50 nl -100 nl. 530 After surgery, fish were kept in standard holding tanks at 35 °C -37 °C unless noted otherwise. 531 Larval fish were anesthetized in 0.03 % tricaine methanesulfonate (MS-222) and placed on an agarose plate 532 under a dissection microscope. Micropipettes pulled from borosilicate glass were inserted vertically into the 533 target region (spinal cord, muscle or brain) without a craniotomy. The injected volume was 250 pl -500 pl. 534 After retraction of the pipette, fish were kept in standard tanks in an incubator at 28.5 °C, 32 °C or 35 °C. 535 are expressed prior to the injection of EnvA-RVΔG-GFP, we also performed experiments with two separate 538 injections. However, the number of GFP-positive neurons was not increased when EnvA-RVΔG-GFP 539 was injected two (n = 3 fish) or four (n = 3 fish) days after HSV1[UAS:TVA-mCherry] and 540 HSV1[UAS:zoSADG] (not shown), consistent with observations in rodents 45,46 . We assume that this 541 observation can be explained by at least two factors. First, rabies virus appears to remain close to the 542 injection site for an extended period of time, possibly because it has high affinity for membranes. Second, 543 it is difficult to precisely target the two separate injections at the same neurons. 544 Transneuronal spread of the rabies virus was determined by quantitative analyses of neurons that expressed 545 GFP but not TVA-mCherry. In theory, some of these neurons could be two (or more) synapses away from the 546 starter cell if they target other neurons that received only the glycoprotein but not TVA-mCherry, if these 547 neurons in turn were presynaptic to a starter neuron. However, because labeling was sparse and because the 548 number of neurons receiving G only should be low, the probability of such events should be very low. 549 Moreover, because transneuronal gene expression is observed only after a delay of >6 days, multi-step events 550 should be very rare 10 days post infection. Multi-step events were therefore not taken into account in our 551 quantitative analyses. 552 553

Clearing of brain samples 554
We adapted the original Cubic protocol 47 to small samples such as adult zebrafish brains. After fixation with 555 4 % paraformaldehyde overnight, samples were soaked with reagent 1A (10%w/v Triton, 5wt% NNNN-556 tetrakis (2HP) ethylenediamine and 10 %w/v urea) for 2.5 h at room temperature and for 6 h at 37 °C with 557 mild shaking and multiple solution exchanges. Subsequently, samples were washed in PBS overnight at room 558 temperature. On the next day, samples were treated with reagent 2 (25 % w/v urea, 50 % w/v sucrose and 10 % 559 w/v triethanolamine) for refractive index matching, mounted in glass bottom dishes, and covered by 16 x 16 560 mm cover glasses to avoid drift. Images were acquired using a Zeiss 10x water-immersion objective lens (N.A.

Optogenetics and imaging in vivo 581
Multiphoton calcium imaging and simultaneous ChrimsonR stimulation was performed as described 48 using a 582 modified B-scope (Thorlabs) with a 12 kHz resonant scanner (Cambridge Technology) at excitation 583 wavelengths of 930 nm (GCaMP6f) or 1100 nm (tdTomato) and an average power under the objective of 584 30 mW at 930 nm. Optogenetic stimulation with ChrimsonR was performed using an LED (UHP-T-595, 585 Prizmatix; 595 nm). Light paths for imaging and stimulation were combined using a dichroic mirror (ZT775sp-586 2p, Chroma). Emitted light was split using a second dichroic mirror (F38-555SG, Semrock), band-pass filtered 587 with a 525/50 filter (Semrock) for GCaMP6f imaging and with a 607/70 filter (Semrock) for tdTomato, and 588 focused onto a GaAsP photomultiplier (H7422, Hamamatsu). The signal was amplified (DHPCA-100, Femto), 589 digitized at 800 MHz (NI5772, National Instruments), and band-pass filtered around 80 MHz using a digital 590 Fourier-transform filter implemented on an FPGA (NI5772, National Instruments). LED activation was 591 synchronized to the turnaround phase of the resonant scanner when no data were acquired. Images were 592 acquired at 60 Hz with a resolution of 750 x 400 pixels, corresponding to a field of view of 300 µm x 250 µm. 593 Images were acquired sequentially in four different focal planes by moving the objective (Nikon 16x, 0.8 NA) 594 with a piezo-electric linear actuator (Physik Instrumente; effective frame rate: 15 Hz per plane). Anatomical 595 snapshots were generated by averaging 1000 images in the absence of optogenetic stimulation. 596 Basel-golden fish were head-fixed in a custom chamber and neurons in the dorsal telencephalon were imaged 597 through the intact skull 5 . Optogenetic stimuli (500 ms duration) were applied randomly every 6 -11 s. The 598 average power of each stimulus was chosen at random to be 1.3 mW, 2.4 mW, 4.7 mW, or 8.6 mW under the 599 objective. The total duration of an imaging session was 11 min. 600 Raw images were full frame registered to correct for motion. Regions of interest were manually selected based 601 on neuronal co-expression of GCaMP6f and ChrimsonR-tdTomato (N = 11 neurons). Raw fluorescence traces 602 were calculated as mean of the pixel values in a given region of interest in each imaging frame. Raw traces 603 were then corrected for slow drift in fluorescence using an 8th-percentile filtering with a 15 s window 49  ester (OGB-1; Thermo Fisher Scientific) was injected into the OB as described 18 . Two-photon calcium 616 imaging started >1 hr after dye injection. Odors were prepared and delivered to the nose for 5 s as described 52 . 617 Inter-stimulus intervals (ISIs) were >2 min. 618 Multiphoton calcium imaging was performed using a custom-built microscope with a 20x water immersion 619 objective (NA 1.0; Zeiss) and galvo scanners 50 . Excitation wavelengths were 930 nm (OGB-1) or 1010 nm 620 (mCherry). The average power under the objective was 50 mW at 930 nm and 20 mW at 1010 nm. The emitted 621 light was split by a dichroic mirror (DMSP550L, Thorlabs), band-pass filtered with a 515/30 filter (Chroma) or 622 with a 641/75 filter (Semrock), and collected with a GaAsP photomultiplier (H7422-40MOD or H11706P-40, 623 Hamamatsu). Images were acquired at 8 Hz using Scanimage 5.5-1 (Vidrio Technologies, LLC) 53 with a 624 resolution of 256 x 256 pixels. 625 626

Tissue dissociation and cell sorting 627
EnvA-RVΔG-GFP was injected into one or both OBs of Tg[gad1b:Gal4;UAS:TVA-mCherry] fish. Cells were 628 dissociated and sorted as per previous protocol 54 with modification for fish. Briefly, after 3 -4 days at 36 °C, 629 fish were anesthetized by cooling to 4 °C and decapitated in ACSF supplemented with 50 mM 2-Amino-5-630 phosphonovaleric acid (APV), 20 mM 6,7-Dinitroquinoxaline-2,3-dione (DNQX), 5 mg/ml actinomycin D 631 (ACT-D), tetrodotoxin (TTX) 100 nM and 10 g/l Trehalose. OB samples were taken and harvested and kept in 632 ACSF. After pooling OB samples from 3 -5 fish, samples were treated with pronase-mix (1 g/l protease type 633 xiv and 33 mg/l of collagenase in ACSF) for 10 minutes and the solution was replaced with fresh ACSF 634 containing 1 % FBS. Samples were then triturated gently with small custom-made glass pipettes and stored on 635 ice. DAPI was added to the samples to detect dead cells and cells were sorted based on GFP and mCherry 636 fluorescence using a 70 or 100 m nozzle (BD FACSAria III; BD Biosciences). Sorted cells were kept in a 637 lysate buffer and stored at -80 °C until further processing. 638 639 20

RNA sequencing 640
RNA was purified using a single-cell RNA purification Kit (Norgen). mRNA-seq libraries were generated 641 using the SmartSeq2 approach 55 with the following modifications: For cDNA pre-amplification, up to 10 ng of 642 RNA was used as input and reverse transcription was performed using Superscript IV (Thermo Fisher 643 Scientific -50 °C for 10 min, 80 °C for 10 min). Amplified cDNA (1 ng) was converted to indexed sequencing 644 libraries by tagmentation, using in-house purified Tn5 56 and Illumina Nextera primers. Libraries were 645 sequenced on an Illumina HiSeq2500, as 50 bp single-end reads.

Odor discrimination training and analysis of behavior 668
In the experimental group, Basel-golden fish were injected with HSV1[LTCMV:jGCaMP7b] into both OBs and 669 kept at 36 °C for 2 days prior to behavioral training. Associative conditioning was performed as described 6,18 . 670 Briefly, individual fish were acclimated to the behavioral setup without food for 1 -3 days and subsequently 671 trained to associate one odor stimulus (CS + : alanine) with a food reward, whereas a second odor stimulus (CS − : 672 tryptophan) was not rewarded. Each odor was infused into the tank for 30 s nine times per day in an alternating 673 sequence (inter-trial interval: 20 min) for five consecutive days. A small amount of food was delivered at a 674 specific location at the end of the presentation of the CS + but not the CS -. 3D swimming trajectories were 675 reconstructed from videos acquired by two orthogonal cameras (Logitech HD Pro C920). The following 676 behavioral components were extracted from the trajectories: swimming speed, elevation in water column, 677 presence in the reward zone, surface sampling, distance to the odor inflow and rhythmic circular swimming. 678 The components were combined into a compound score of appetitive behavior as described 6 . The learning 679 index was calculated as the difference between the mean behavioral scores in response to the CS + and CS − 680 during the final day of training (last nine trials with each odor) in each fish.

Data availability 687
Transcriptomic data generated in this study have been deposited at ArrayExpress (accession number: E-688 MTAB-11083). 689 Requests for HSV1 and the vectors shown in Fig. 2a should be addressed to R.L.N. 690 The data that support the findings of this study are available from the corresponding authors upon reasonable 691 request. 692 693