Use of a tissue clearing technique combined with retrograde trans-synaptic viral tracing to evaluate changes in mouse retinorecipient brain regions following optic nerve crush

Abstract

Successful establishment of reconnection between retinal ganglion cells and retinorecipient regions in the brain is critical to optic nerve regeneration. However, morphological assessments of retinorecipient regions are limited by the opacity of brain tissue. In this study, we used an innovative tissue cleaning technique combined with retrograde trans-synaptic viral tracing to observe changes in retinorecipient regions connected to retinal ganglion cells in mice after optic nerve injury. Specifically, we performed light-sheet imaging of whole brain tissue after a clearing process. We found that pseudorabies virus 724 (PRV724) mostly infected retinal ganglion cells, and that we could use it to retrogradely trace the retinorecipient regions in whole tissue-cleared brains. Unexpectedly, PRV724-traced neurons were more widely distributed compared with data from previous studies. We found that optic nerve injury could selectively modify projections from retinal ganglion cells in the hypothalamic paraventricular nucleus, intergeniculate leaflet, ventral lateral geniculate nucleus, central amygdala, basolateral amygdala, Edinger-Westphal nucleus, and oculomotor nucleus, but not the superior vestibular nucleus, red nucleus, locus coeruleus, gigantocellular reticular nucleus, or facial nerve nucleus. Our findings demonstrate that the tissue clearing technique, combined with retrograde trans-synaptic viral tracing, can be used to objectively and comprehensively evaluate changes in mouse retinorecipient regions that receive projections from retinal ganglion cells after optic nerve injury. Thus, our approach may be useful for future estimations of optic nerve injury and regeneration.

Introduction

The optic nerve transports visual messages from retinal ganglion cells (RGCs) into the brain. As neurons specific to the central nervous system, RGCs lack the ability to regenerate. Thus, the optic nerve crush (ONC) procedure, in which RGCs are seriously damaged, is widely used to investigate nerve degradation and regeneration in mice (Li et al., 1999; Donahue et al., 2020; Patel et al., 2020). The reformation of eye-to-brain connections is an important histological and functional indicator of successful optic nerve regeneration (Laha et al., 2017; Yang et al., 2020). According to our findings and those of others, the ONC alters synaptic plasticity, astrocyte activity, and the cortical network within the visual cortex contralateral to the ONC side in mice (Groleau et al., 2020; Zhan et al., 2020). However, whether the ONC leads to changes in the retinorecipient regions related to RGCs in the whole mouse brain remains unclear.

Tissue clearing techniques may be a practicable way to scan retinorecipient regions in the brain after ONC. Combined with whole-organ antibody labeling, viral encoding with fluorescent protein tracing, or the use of transgenic animals, the tissue clearing technique is preferable for visualizing the neural circuits and connectome (Tian et al., 2020; Ueda et al., 2020; Lee et al., 2021; Wang et al., 2021; Li et al., 2022). This is preferable to whole brain imaging of the rodent brain, which can require hundreds or even thousands of sections, can take more than a month to complete, is prone to errors associated with misreading the regions on the different planes, and is vulnerable to missing data because of the large amount of information. A previous study indicated that tissue clearing may enable a fast and comprehensive scan of the spinal cord, dorsal root ganglia, and sural nerve in terms of nerve regeneration (Daeschler et al., 2022). Tissue clearing combined with light-sheet imaging can be used to evaluate whole body pancreatic innervation in diabetic mice and patterns of cancer metastasis (Pan et al., 2019; Alvarsson et al., 2020). Thus, this approach has great potential for use in neuroscience and pathology. With large numbers of samples, it will be possible to conduct comprehensive clinical pathological examinations in the future. However, few studies have examined the possibility of applying the tissue clear technique in assessments of central nervous system regeneration.

Two strains of the pseudorabies virus (PRV) are used in laboratory research: PRV-Becker and PRV-Bartha. PRV-Becker is more similar to wild-type PRV, infects RGCs, and is transported both anterogradely and retrogradely (Card et al., 1991). In contrast, PRV-Bartha is used more widely because it comes from a live vaccine and is transported retrogradely (Card et al., 1991; Pickard et al., 2002; Card and Enquist, 2014; Xu et al., 2020). PRV-Bartha has mutations in the Us genes (gE, gI, and Us9). For example, Us9 is a structural protein found in all α-herpesvirus. It appears in infected cell membranes and near the trans-Golgi network, and enables the spread of PRV-Becker infection from axons to cell bodies (Brideau et al., 2000). Hence, PRV-Bartha does not have the ability to infect neurons anterogradely, but it can infect them retrogradely (i.e., from postsynaptic to presynaptic neurons) (Enquist, 2002). Therefore, an isogenic version of PRV-Bartha, encoded with a fluorescence protein such as PRV724, could be used to examine the function and survival of RGCs and to label retinorecipient brain regions.

Here, we administered intravitreal injections of PRV724 to ONC mice, and used the tissue optical clearing technique to analyze changes in retinorecipient brain regions.

Methods

Ethics statement

All experiments were carried out in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (Percie du Sert et al., 2020), the Guide for the Care and Use of Laboratory Animals, and the Association for Research in Vision and Ophthalmology (ARVO) statement on animal use. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Laboratory Animal Center, Zhongshan School of Medicine, Sun Yat-sen University (approval No. 2019-1210; approval date: March 28, 2019).

Experimental animals

We used 3-month-old C57BL/6 or Thy1-yellow fluorescence protein (Thy1-YFP) transgenic mice (RRID: MGI: 3581649) of both sexes, which had been identified by ZYZ and YRH via polymerase chain reaction (PCR) and bred in specific-pathogen-free conditions, for our experiments. We purchased 3-month-old C57BL/6 mice weighing 20–25 g from the Guangdong Medical Laboratory Animal Center in China (license No. SCXK (Yue) 2022-0002), and obtained 3-month-old Thy1-YFP transgenic mice from the Zhongshan School of Medicine, Sun Yat-sen University. The mice were divided randomly into a control and an ONC group. At least three mice per group were sacrificed in each experiment. The specific numbers are given in Figure 1A–C. We used a total of 74 mice, including 55 C57BL/6 mice and 19 Thy1-YFP mice. RGC survival was analyzed in 8 C57BL/6 mice (four males and four females). The residual RGCs fibers and axons in the optic nerves were analyzed in 6 C57BL/6 mice (three males and three females). Retrograde fibers were analyzed in 7 C57BL/6 mice (three males and four females). An intravitreal injection of PRV724 was administered in 38 C57BL/6 mice (19 males and 19 females), and an intravitreal injection of phosphate buffer solution (PBS) was administered in three C57BL/6 mice (one male and two females). Two C57BL/6 mice (two males) died prematurely because of the lethality of PRV724. An intravitreal injection of PRV724 was administered in 16 Thy1-YFP mice (eight males and ten females), and one mouse (female) died prematurely because of the lethality of PRV724. Counts for RGC staining were performed in 6 Thy1-YFP mice (three males and three females). Among these mice, four brains from the above C57BL/6 mice (two males and two females) and nine brains from the above Thy1-YFP mice (four males and five females) were analyzed after tissue clearing. The anesthesia used for all experiments was an intraperitoneal injection of 1% pentobarbital sodium (80 mg/kg; Merck, Frankfurter, Darmstadt, Germany) and topically applied promecaine hydrochloride eyedrops (5 mg/mL; Alcon, Fort Worth, TX, USA).

Figure 1.

Flow charts of experiments.

(A) Schematic figure of the timepoints and the number of mice subjected to ONC surgery and different groups. (B) Schematic figure showing the timepoints of ONC surgery, intravitreal injection of PRV724, and sampling. (C) Schematic figure of the timepoints of ONC surgery, intravitreal injection of PRV724 injection, sampling, tissue clearance time, and imaging times. 3 mo: Three-month old; CTB: cholera toxin B subunit; GAP-43: growth-associated protein-43; NF-L: neurofilament-light chain; ONC: optic nerve crush; PRV: pseudorabies virus; RBPMS: RNA binding protein with multiple splicing.

Identification of Thy1-YFP transgenic mice

A 0.5 cm piece of tail was removed from each mouse using sterilized scissors on ice. The tail pieces were processed using a mouse tail rapid genotype identification kit (Beyotime, Shanghai, China). The PCR amplifier (Bio-rad, Hercules, CA, USA) settings were as follows: 94°C for 3 minutes, 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 27 seconds, 72°C for 10 minutes, and 4°C for storage. The cycle was repeated 30 times. The primers used for genotyping were designed for Thy1-YFP sequences (forward: 5′-ACA GAC ACA CAC CCA GGA CA-3′, reverse: 5′-CGG TGG TGC AGA TGA ACT T-3′). The electrophoresis was run in 2% agarose gel.

ONC surgery

As previously described (Quan et al., 2020; Wu et al., 2020; Zhan et al., 2020), after ensuring adequate anesthesia, the lower bulbar conjunctiva of each mouse was incised at 180°, and the optic nerve of the left eye was carefully exposed without obvious bleeding under a stereo microscope (Phenix, Shangrao, Jiangxi, China). Then, the optic nerve was crushed for 5 seconds using cross-action forceps (Dumont, Montignez, Switzerland), at a position 1 to 2 mm behind the eyeball. Tobramycin and dexamethasone ointment (Alcon) were applied to both eyes to prevent cornea dryness and infection.

Intravitreal injection

Based on previous studies (Quan et al., 2020; Wu et al., 2020), we first ensured adequate anesthesia, and then intravitreally injected 1 µL of cholera toxin B subunit (CTB)-Alexa 488 (BrainVTA, Wuhan, Hubei, China) into the left eye using a 33-gauge needle (Hamilton Company, Reno, NV, USA). To avoid injectant leakage, the needle was kept in the eye for 1 minute and then slowly retracted. Tobramycin and dexamethasone ointment (Alcon) were applied to both eyes to prevent cornea dryness and infection. The samples were collected 3 days after the intravitreal injection of CTB-Alexa 488.

We purchased PRV724, a recombinant virus with retrograde spread and monomeric red fluorescent protein (mRFP) based on PRV-Bartha (Jia et al., 2019), from BrainVTA. We intravitreally injected 2 µL doses containing 1 × 10⁹ or 3 × 10⁹ pfu/mL PRV724, as described above. Because PRV724 is lethal in most rodents (Fu et al., 2021), the whole procedure was performed in a BSL-2 laboratory. To maximize the trans-synaptic retrogradation of PRV724, the mice were closely observed. Once they showed inactivity, slow movements, shaky movements, or tremors (Song et al., 2021), euthanasia was performed. Euthanasia was performed an average of 5–6 days after the procedure, and mice without obvious signs of sickness were sacrificed on the 6^th day.

Histology

The mice were deeply anesthetized and transcardially perfused with 4°C PBS followed by 4% paraformaldehyde (PFA). To obtain flat-mounted retinas, intact retinas were removed from the eyeballs using a stereo microscope (Phenix) and immersed in 4% PFA at 4°C overnight for follow-up experiments. For frozen retinal sections, the eyeballs were immediately enucleated and immersed in 4% PFA overnight, and then dehydrated and embedded in TissueTek OCT compound (SAKURA, Torrance, CA, USA). The 10-µm frozen retina sections were prepared for follow-up experiments. For frozen sections of optic nerves, the intact optic nerves were enucleated from the bottom region of the brain and immersed in 4% PFA overnight. Then, they were dehydrated and embedded in TissueTek OCT compound (SAKURA). We prepared 10-µm vertical and 14-µm transverse frozen sections for follow-up experiments. For whole brain optical clearing, whole brains were carefully removed from the skull using a stereo microscope.

Fluorescence immunohistochemistry

The flat-mounted retinas were blocked with 10% normal goat serum (Boster, Wuhan, China) and 0.01% Triton-X 100 at 4°C overnight and then incubated with primary antibodies against RNA binding protein with multiple splicing (RBPMS; rabbit, 1:100, Proteintech, Rosemont, IL, USA, Cat# 15187-1-AP, RRID: AB_2238431) or neurofilament-light chain (NF-L; rabbit, 1:250, CST, Danvers, MA, USA, Cat# 2837S, RRID: AB_823575) for 2 days at 4°C. After 3 washes with PBS-0.1% Tween 20, the retinas were incubated with Alexa Fluor 488 or 647 goat anti-rabbit IgG secondary antibody (1:500, CST, Cat# 4412S, RRID: AB_1904025 or Cat# 4414S, RRID: AB_10693544) for 1 day in a dark environment at 4°C, and an antifade reagent with diamidino-phenyl-indole (DAPI; F6057, Sigma-Aldrich, St. Louis, MO, USA) was applied. The number of RGCs (RBPMS⁺ cells) in each mouse was counted in three randomly selected places in the central, mid-peripheral, and peripheral retina. The nerve fiber density (NF-L⁺ fibers) in each mouse was measured at three randomly selcted places in the central, mid-peripheral, and peripheral retina using ImageJ 1.52v (National Institutes of Health, Bethesda, MD, USA) (Schneider et al., 2012). Using ImageJ, cells with co-labeling of RBPMS and mRFP were identified as PRV724-infected RGCs.

The frozen sections were blocked with 10% normal goat serum for 2 hours at 26°C and then incubated with primary antibodies against RBPMS (rabbit, 1:200, Proteintech, Cat# 15187-1-AP, RRID: AB_2238431), growth-associated protein-43 (GAP-43; rabbit, 1:200, Sigma-Aldrich, Cat# AB5220, RRID: AB_2107282), or opsin 4 polyclonal antibody (rabbit, 1:100, Invitrogen, Carlsbad, CA, USA, Cat# PA1-781, RRID: AB_2156044) overnight at 4°C. After three washes with PBS-0.1% Tween 20, goat anti-rabbit Alexa Fluor 488 (1:500, CST, Cat#4412S, RRID: AB_1904025) or goat anti-mouse Alexa Fluor 647 (1:500, CST, Cat#4410, RRID: AB_1904023) was applied. The tissue samples were incubated at 26°C in a dark environment for 2 hours, and then coverslip slides were applied with an antifade reagent with DAPI (F6057, Sigma-Aldrich). The RBPMS⁺ cells were identified as RGCs, and the opsin 4⁺ cells were identified as intrinsically photosensitive RGCs (ipRGCs) (Kwong et al., 2010; Mao et al., 2014). Cells co-labeled with RBPMS, opsin 4, and mRFP were identified as ipRGCs infected with PRV724. The axons in the optic nerve were stained with GAP-43.

Tissue optical clearing

Optical clearing of the brains was based on the polyethylene glycol-associated solvent system (Jing et al., 2018) and the kits were purchased from Leads Bio-Technology, Shanghai, China. The brains were immersed in 4% PFA at 4°C for a 48-hour fixation period. After fixation, the brains were transferred to a Quadrol decolorization solution and left for 2 days at 37°C in a 60-r/min shaker. The brains were then immersed in graded series of delipidation solutions at 37°C in a 60-r/min shaker for 2–3 days, and then immersed in a dehydration solution for 2 days followed by a benzyl benzoate-PEGMMA500 clearing medium for at least 1 day. The clearing time depended on the transparency of the tissue, and averaged 6–9 days. The brains were preserved in a clearing medium at room temperature in a dark environment.

Microscopy imaging

Images of non-cleared samples were acquired using a confocal microscope (LSM 780 or LSM 880, Zeiss, Oberkochen, Germany). We used a 10× objective lens (FLUAR, numerical aperture (NA) = 0.5, work distance (WD) = 2 mm), 20× objective lens (PLANAPOCHROMAT, NA = 0.8, WD = 550 µm), and 63× objective lens (PLANAPOCHROMAT, NA = 1.4, WD = 190 µm) for imaging.

Image stacks of cleared samples were acquired with a step size of ~5 µm using a LiTone XL Light-sheet microscope (Light Innovation Technology Ltd., Hong Kong Special Administrative Region, China) equipped with an sCMOS camera (Hamamatsu ORCA-flash 4.0 V3). Samples were illuminated from 4 directions horizontally via thin Line Bessel light-sheets, and imaged with a 4× objective lens (Olympus XLFLUOR4X/340, NA = 0.28) from the bottom. We used Litscan 3.0 to create light-sheet microscope hyper-stacks as 16-bit grayscale TIFF images for each channel separately.

Image processing

We used Zen 2011 SP2 (Version 8.0.0.273, Carl Zeiss GmbH, Oberkochen, Germany) software to collect non-cleared sample data. The RGC counts and fluorescence intensity values were measured using ImageJ.

Data processing, analysis, 3-dimensional (3D) rendering, and video generation for the cleared samples were performed on an HP workstation Z8G4 with a 24 core Xeon processor, 256 GB RAM, and a Nvidia RTX k4000 graphics card, or on an HP workstation Z840 with an 8 core Xeon processor, 196 GB RAM, and an Nvidia Quadro k5000 graphics card. Stitching of the tile scans from the light-sheet microscopy was performed using the Litscan 3.0 normalized cross correlation stitching mode. All raw image data were collected in a lossless TIFF format (8-bit images for confocal microscopy and 16-bit images for light-sheet microscopy). The 16-bit images were converted to 8-bit images using Fiji version 2.5.0 (National Institutes of Health, Bethesda, MD, USA) to enable faster processing with different software. To consolidate the cleared brain regions and the 3D reconstruction, we used the AI open code “BIRDS”, created by Wang et al. (2021). The procedure was performed as described in the tutorial (https://github.com/bleach1by1/BIRDS_plugin) (Wang et al., 2021). The locations of the different regions were based on the Allen Brain Reference Atlases (http://atlas.brain-map.org) for transverse slices, sagittal slices, and coronal slices (Additional Figure 1 ^{(1.8MB, tif)} ). Imaris version 9.0.2 (Bitplane AG, Belfast, UK) was also used in the 3D reconstruction of the cleared brain tissue.

PRV724-labeled neuron counts

Counting was performed for representative images containing each brain region. Two researchers (ZYZ and YRH) independently counted the numbers of PRV724-labeled neurons, and the data were averaged for statistical analysis. The ONC side was identified as the ipsilateral side and the side contralateral to the ONC eye was identified as the contralateral side.

Statistical analysis

Although we used no statistical methods to predetermine sample sizes, our sample sizes are similar to those reported in a previous publication (Ren et al., 2021). The investigators (ZYZ and YRH) evaluated the data individually, and were blinded to each other’s assessments, but not to group membership. We adopted the averages of their evaluations for statistical analysis, which was performed with the mean ± standard error of the mean (SEM) via SPSS 23.0 (IBM SPSS Statistics, Armonk, NY, USA) and Prism GraphPad 7.00 (GraphPad Software Inc., San Diego, CA, USA, www.graphpad.com) software. A two-way analysis of variance with Sidak’s multiple comparisons test was applied. A P value of < 0.05 was considered statistically significant.

Results

Residual RGCs and axons had survived at 4 weeks after ONC

Previous studies have reported that approximately 10% of RGCs had survived at 4 weeks after ONC (Tran et al., 2019). We wanted to determine whether the axons stretching from the RGCs had survived, so we used a pan-RGC marker antibody and an axon marker antibody to stain the RGC and RGC nerve fibers, as well as the axons, after ONC administration in 1 eye of 3-month-old mice. As expected, we found that ONC led to a marked reduction in the number of RGCs (n = 4 eyes per group, Figure 2A upper panel). When compared with the number of RGCs in controls (2314.5 ± 287.9/mm²), the number of RGCs in the central retina 4 weeks after ONC was only 196.0 ± 61.6/mm². Furthermore, 4 weeks after ONC, the numbers of RGCs in the mid-peripheral retina and peripheral retina were 248.0 ± 85.6/mm² and 237.0 ± 86.1/mm², respectively. The survival rates were 8.47%, 9.6%, and 13.9% in the central retina, mid-peripheral retina, and peripheral retina, respectively. To confirm the existence of retrograde nerve fibers, we used the retrograde tracer CTB (Figure 2B lower panel). The results showed that the nerve fibers that remained after ONC included retrograde nerve fibers (control: n = 4 eyes, ONC: n = 3 eyes). Additionally, using an NF-L antibody, we found that 4 weeks after ONC, 74.0%, 48.3%, and 68.3% of the fluorescent signal remained in the central, mid-peripheral, and peripheral retina, respectively (n = 3 eyes per group, Figure 2C). The number of RGCs was significantly reduced (P < 0.0001 for all regions; Figure 2D). The fluorescent signals of CTB-stained nerve fibers were also significantly reduced in the center (P = 0.0019) and mid-peripheral (P = 0.002) retina, respectively (Figure 2E). CTB-stained nerve fibers were less injured in the peripheral retina, and the total numbers of nerve fibers in the central (P = 0.02) and mid-peripheral (P = 0.001) retina 4 weeks after ONC were significantly lower than those in controls (Figure 2F). These data strongly suggest that the nerve fibers in the peripheral retina were less damaged.

Figure 2.

Residual RGCs and axons had survived at 4 weeks after ONC.

(A) The decrease in RGCs after ONC. Dual-color staining of the central, mid-peripheral, and peripheral retinas via an intravitreal injection of CTB (Alexa Fluor 488, green, labeling axons) and RBPMS (Alexa Fluro 647, gray, labeling RGCs) in the control and ONC groups. Scale bars: 50 µm. (B) The decrease in retrograde nerve fibers after ONC. Single channel of CTB staining from A. Scale bars: 50 µm. (C) The decrease in total nerve fibers after ONC. Immunostaining of the central, mid-peripheral, and peripheral retinas with NF-L staining (Alexa Fluro 488, gray, labeling nerve fibers). Scale bars: 50 µm. (D–F) Quantification of the numbers of RGCs (n = 4 eyes per group), CTB fluorescence density (control: n = 4 eyes ONC: n = 3 eyes), and NF-L fluorescence density (n = 3 eyes each group) in the control and ONC groups. (G) The decrease in axons after ONC. Axon immunostaining in the central and peripheral optic nerves via GAP-43 (Alexa Fluor 488, green, labeling axons) and DAPI (blue, labeling nucleus) in the control and ONC groups (n = 3 per group); the central and peripheral regions are further magnified (gray) in the yellow boxes. Scale bars: 50 µm. (H) Quantification of the GAP-43 fluorescence density in the control and ONC groups (n = 3 per group). Data from three independent experiments are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 (two-way analysis of variance followed by Sidak’s multiple comparisons). CTB: Cholera toxin B subunit; DAPI: diamidino-phenyl-indole; GAP-43: growth-associated protein-43; NF-L: neurofilament-light chain; ONC: optic nerve crush; RBPMS: RNA binding protein with multiple splicing; RGCs: retinal ganglion cells.

We used the axon antibody GAP-43 to stain the axons in the optic nerve (n = 3 per group), and found that the fluorescent signals of the axons were significantly reduced after ONC (center: P < 0.0001 and peripheral: 0.0003, respectively) (Figure 2G and H). However, some axons had survived at 4 weeks after ONC. Collectively, these findings show that RGCs and axons survived at 4 weeks after ONC, including retrograde axons.

Retrogradely labeled RGCs in ONC eyes

To investigate the retinorecipient regions connected to RGCs, we used retrograde trans-synaptic virus PRV724 constructed with mRFP, an isogenic version of PRV-Bartha. Three weeks after ONC, PRV724 was intravitreally injected into the ONC eyes of the experimental group or the normal eyes of the control group (D21). After 6–7 days (D27–28), we collected whole-mount retinas or frozen retina sections (Figure 1B). Because 1 × 10⁹ pfu/mL PRV724 did not obviously infect RGCs in the control group (n = 3 eyes; Figure 3A), we used 3 × 109 pfu/mL PRV724 in subsequent studies (Figure 3B). There were no red fluorescent signals in the retinas of the contralateral eye (n = 4 eyes; Figure 3C), which was similar to eyes intravitreally injected with PBS (n = 3 eyes; Figure 3D).

Figure 3.

Retrograde labeling of RGCs in ONC eyes.

(A, B) Comparison of different dose of PRV724 intravitreal injection. 1 × 10⁹ pfu/mL PRV724 infected fewer RGCs than 3 × 10⁹ pfu/mL PRV724. Confocal images of the retina in the control group after intravitreal injection of 1 × 10⁹ pfu/mL (n = 3 eyes) or 3 × 10⁹ pfu/mL PRV724 (red) (n = 3 eyes), co-stained with RBPMS (Alexa Fluor 488, green). (C) The contralateral retinas were not infected with PRV724. Confocal images of the retina contralateral to the PRV724-injected eye (red) (n = 3 eyes), co-stained with RBPMS (Alexa Fluro 488, green). (D) An intravitreal injection of PBS produced no mRFP signals compared with an intravitreal injection of PRV724. Confocal images of retinas from PBS-injected eyes (n = 3 eyes) co-stained with RBPMS (Alexa Fluro 488, green). (E) PRV724 did not infect fewer RGCs in the ONC mice than in the control mice. Confocal images of retinas from ONC mice with PRV724-injected eyes (n = 3 eyes) co-stained with RBPMS (Alexa Fluro 488, green). (F) PRV724 infected a higher ratio of RGCs in the ONC group than in the control group. Co-labeling of RGCs and PRV724 was observed from flat-mounted retinas. Figure shows magnified confocal images of flat-mounted retinas from both control and ONC mice after an intravitreal injection of PRV724 (red) (n = 3 eyes), with RGCs stained with RBPMS (Alexa Flour 488, green). Yellow triangles show the co-labeled RGCs (mRFP+ RGCs). (G) PRV724 infected a greater ratio of RGCs in the ONC group than in the control group. Co-labeling of RGCs and PRV724 was observed from cross-sectioned retinas. Figure shows retinal cross-sections from both control and ONC mice after an intravitreal injection of PRV724 (red) (n = 3 eyes), with RGCs stained with RBPMS (Alexa Flour 488, green) and nuclei stained with DAPI (blue). Red arrows show the co-labeled RGCs. (H) Quantification of the RGCs in the control group and the PRV724-injected control group (n = 3 eyes per group; the number of RGCs in the control group is shown in Figure 1C). (I) Quantification of the RGCs in the ONC group and the PRV724-injected ONC group (n = 3 eyes per group; the number of RGCs in the ONC group is shown in Figure 1C). (J) Quantification of the ratio of mRFP+ RGCs/total RGCs in the control (n = 16 eyes) and PRV724-injected ONC groups (n = 11 eyes). (K) PRV724 did not infect fewer ipRGCs in the ONC group than in the control group. Confocal immunostaining images of cross-sections from wild-type mice in the control group and ONC group after an intravitreal injection of PRV724 (red). The ipRGCs were stained with opsin 4 (Alexa Flour 488, green) and the nuclei were stained with DAPI (blue) (n = 3 eyes per group). Green triangles indicate ipRGCs. Red triangles indicate PRV724-labeled cells. Yellow triangles show the PRV724-infected ipRGCs. Scale bars: 50 µm. Data from 3 independent experiments are presented as the mean ± SEM. *P < 0.05 (two-way analysis of variance followed by Sidak’s multiple comparisons). DAPI: Diamidino-phenyl-indole; ipRGCs: intrinsically photosensitive retinal ganglion cells; mRFP: monomeric red fluorescent protein; ONC: optic nerve crush; PRV: pseudorabies virus; RBPMS: RNA binding protein with multiple splicing; RGCs: retinal ganglion cells.

To verify the infection specificity of PRV724 in RGCs, we used the pan-RGC marker RBPMS antibody to stain the retina. The results showed that PRV724-labeled RGCs were present in both the control and ONC groups (Figure 3B, E, and F), but the retinas of PRV724-injected eyes were distorted and swollen because of PRV toxicity (n = 3 eyes per group; Figure 3G). To determine whether PRV724 reduced the number of RGCs, we counted the number of RGCs in the control and ONC groups using whole-mount retina staining with RBMPS antibody, which is a pan-RGC marker. When compared with the data in Figure 1D, there was no significant loss of RGCs after intravitreal injection of PRV724 over 5–6 days in the control or ONC groups (n = 3 eyes per group, all P > 0.05; Figure 3H, and I). Nevertheless, the results showed that the PRV724 infection was mostly limited in RGCs regardless of ONC administration. Although there were fewer RGCs in ONC eyes, the proportion of infected RGCs appeared to be higher. By counting the number of RGCs with red fluorescence (mRFP+ RGCs) and the total number of RGCs in the infected regions of retinas, we found that the percentage of mRFP+ RGCs/total RGCs in control eyes following a PRV724 intravitreal injection was 5.8 ± 1.2% (n = 16 eyes), while the percentage of mRFP+ RGCs/total RGCs in ONC eyes was 10.9 ± 2.1% (n = 11 eyes). There was a small but significant difference between these 2 groups (P = 0.04, Figure 3J).

A previous study showed that PRV-Bartha exclusively infects ipRGCs (Viney et al., 2007), and among the 46 types of RGCs, 2 types of αRGCs and all 5 types of ipRGC were injury-resistant to ONC (Rheaume et al., 2018; Tran et al., 2019; Yang et al., 2020). ipRGCs accounted for over 82% of the surviving RGCs 60 days after the ONC procedure (Pérez de Sevilla Müller et al., 2014). Hence, we next confirmed whether PRV724 exclusively infected ipRGCs, as reported in a previous study (Viney et al., 2007). We stained the retinal cross-sections with the ipRGC marker opsin 4 antibody, and found that while some PRV724-infected RGCs were stained with opsin 4, other opsin 4-labeled RGCs were not infected with PRV724 in the control group (n = 3 eyes; Figure 3K upper row). PRV724-infected RGCs were also co-stained with opsin 4 in the ONC group (n = 3 eyes; Figure 3K bottom row). However, not all PRV724-labeled cells were co-stained with opsin 4 (Figure 3K middle row). Hence, PRV724 might not exclusively infect ipRGCs. Additionally, when GAP-43 antibody was used to stain the intact optic nerve crossover at the optic chiasm, no red fluorescent signals were observed (n = 3; Additional Figure 2A ^{(5.2MB, tif)} –D ^{(5.2MB, tif)} ). This suggests that PRV724 did not reveal the axons of RGCs. Taken together, these findings confirm that PRV724 can label RGCs in ONC eyes.

Overview of PRV724-traced retinorecipient regions

After confirming our labeling strategy, we sought to better identify the whole brain neurons innervated by the RGCs. Accordingly, 6–7 days after the intravitreal injection of PRV724 in the left eye, we removed the whole brain with or without the eyeballs and optic nerve (Thy1-YFP control mice n = 4, wild-type control mice n = 4). The mouse brains were analyzed using whole brain optical tissue clearing, with a transparency time of 7 days (Figure 1C). As expected, the brains were fully transparent with no visible shrinkage (Figure 4A), and they could be imaged via a light-sheet microscope (Figure 4B and Additional Video 1). The locations of different brain areas were determined following standard procedures, as described in the Methods section (Additional Figure 1A ^{(1.8MB, tif)} –H ^{(1.8MB, tif)} ). To better locate the positions, we used Thy1-YFP mice. We found that PRV724 was able to label the RGCs in C57BL/6 (wild-type) mice as well as in Thy1-YFP mice, regardless of ONC administration (Additional Figure 3A ^{(1.8MB, tif)} ). There was no significant difference in the number of RGCs in the C57BL/6 mice (wild-type) versus Thy1-YFP mice, regardless of ONC administration (n = 3 eyes per group; Additional Figure 3B ^{(2.5MB, tif)} ). Also, the percentage of RFP⁺RGCs/total RGCs in the C57BL/6 mice (wild-type) and Thy1-YFP mice did not differ significantly, regardless of ONC administration (n = 5 eyes per group; Additional Figure 3C ^{(2.5MB, tif)} ).

Figure 4.

Overview of tissue-cleared brain.

(A) Representative images of a brain before clearing (left), after clearing (middle), and after RI matching (right). (B) View from different angles of the 3D cleared brain from a Thy1-YFP mouse. Scale bar: 5 mm in A, 100 µm in B. 3D: Three-dimensional; RI: refractive index; YFP: yellow fluorescent protein.

We focused on retinorecipient nuclei known to receive robust RGC innervation including the suprachiasmatic nucleus (SCN), ventral part of the lateral geniculate complex (LGv), intergeniculate leaflet (IGL), olivary pretectal nucleus (OPN), and superior colliculus (SC) (Hattar et al., 2002, 2006; Beier et al., 2021). However, PRV724 labeling of neurons was inconsistent in the SCN, OPN, and SC (data not shown), apart from the LGv and IGL. Hence, data from the SCN, OPN, and SC were not included in this study. Consistent with previous studies using PRV-Bartha (Card et al., 1991; Smith et al., 2000; Pickard et al., 2002; Smeraski et al., 2004), we observed trans-synaptically labeled neurons in the paraventricular hypothalamic nucleus (PVH), Edinger-Westphal nucleus (EWN), oculomotor nucleus, IGL, LGv, and superior vestibular nucleus (SUV) in both hemispheres (Figure 5A–C). Unexpectedly, PRV724 labeled neurons were also observed in the basolateral amygdala nucleus (BLA), central amygdalar nucleus (CeA), red nucleus (RN), VII nucleus, and gigantocellular reticular nucleus (GRN) in both hemispheres (Figures 5A, C, and D).

Figure 5.

Overview of PRV724-traced retinorecipient regions in control and ONC mice.

(A) There were fewer PRV724-labeled neurons in the BLA, CeA, and PVH of ONC mice versus control mice. The locations of the BLA, CeA, and PVH in the control (left) and ONC (right) mice. (B) There were fewer PRV724-labeled neurons in the IGL, LGv, and EWN in the ONC mice than in the control mice. The locations of the IGL, LGv, oculomotor nucleus, and EWN in the control (left) and ONC (right) mice. (C) The number of PRV724-labeled neurons was unchanged in the LC, SUV, and RN in ONC mice. The locations of the LC, SUV, and RN in the control (left) and ONC (right) mice. (D) The number of PRV724-labeled neurons was unchanged in the VII nucleus and GRN in the ONC mice. The locations of the VII nucleus and GRN in the control (left) and ONC (right) mice. Scale bars: 500 µm. BLA: Basolateral amygdala nucleus; CeA: central amygdalar nucleus; EWN: Edinger-Westphal nucleus; GRN: gigantocellular reticular nucleus; IGL: intergeniculate leaflet of the lateral geniculate complex; LC: locus ceruleus; LGv: ventral part of the lateral geniculate complex; ONC: optic nerve crush. PRV: pseudorabies virus; PVH: paraventricular hypothalamic nucleus; RN: red nucleus; SUV: superior vestibular nucleus.

Common properties of PRV724-traced retinorecipient regions

As shown in Figure 6A, the signals of PRV724-labeled neurons were distributed equally across the ipsilateral and contralateral BLA (P = 8498), while the signals of PRV724-labeled neurons were mostly located in the contralateral CeA (P = 0.0073) and contralateral PVH (P = 0.0164) (n = 4; Figure 6B–D). As shown in Figure 6E, PRV724-labeled neurons in the IGL and LGv were distributed equally across both hemispheres (P > 0.9999 and P = 0.9928), while the signals of PRV724-labeled neurons were mostly located in the contralateral oculomotor nucleus (P = 0.0305) (n = 4; Figure 6F–H). The EWN is located at the midline, so there was no comparison between the ipsilateral and contralateral sides (Figure 6I).

Figure 6.

ONC administration selectively eliminates projections from RGCs in the BLA, CeA, PVH, IGL, LGv, oculomotor nucleus, and EWN.

Light-sheet images of Thy1-YFP mouse brains from the control mice and those subjected to ONC with PRV724 intravitreal injection. (A) The decrease in PRV724-labeled neurons in the BLA, CeA, and PVH after ONC. Magnified images of the BLA, CeA, and PVH. Top panel: control. Bottom panel: ONC. The band at the top of the figure indicates the ipsilateral and contralateral sides of the brain. (B–D) Quantification of PRV724-labeled neurons in the BLA, CeA, and PVH. Black dots: control (n = 4); red squares: ONC (n = 5). (E) The decrease in PRV724-labeled neurons in the IGL, LGv, oculomotor nucleus, and EWN after ONC. Magnified images of the IGL, LGv, oculomotor nucleus, and EWN. Top panel: control. Bottom panel: ONC. Scale bars: 100 µm in A and E. The band at the top of the figure indicates the ipsilateral and contralateral sides of the brain. (F–I) Quantification of PRV724-labeled neurons in the IGL, LGv, oculomotor nucleus, and EWN. Black dots: control (n = 4), red squares: ONC (n = 5). Data from three independent experiments are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way analysis of variance followed by Sidak’s multiple comparisons). BLA: Basolateral amygdala nucleus; CeA: central amygdalar nucleus; EWN: Edinger-Westphal nucleus; IGL: intergeniculate leaflet of the lateral geniculate complex; LGv: ventral part of the lateral geniculate complex; ONC: optic nerve crush; PRV: pseudorabies virus; PVH: paraventricular hypothalamic nucleus; YFP: yellow fluorescent protein.

As shown in Figure 7A, the signals of PRV724-labeled neurons were distributed equally across the ipsilateral and contralateral LC (P = 0.9875), SUV (P = 0.8998), and RN (P = 0.8424) (n = 4; Figure 7B–D). As shown in Figure 7E, PRV724-labeled neurons were distributed equally across the ipsilateral and contralateral VII nucleus (P = 0.9418), as well as the GRN (P = 0.7549) (n = 4; Figure 7F and G). Our results confirmed that PRV724-labeled RGCs send more projections to the contralateral PVH, oculomotor nucleus, and CeA.

Figure 7.

The brain regions that receive projections from RGCs are unchanged after ONC.

Light-sheet images of Thy1-YFP mouse brains from the control mice and those subjected to ONC with PRV724 intravitreal injection. (A) The number of PRV724-labeled neurons did not significantly decrease after ONC in the LC, SUV, and RN. Magnified images of the LC, SUV, and RN. Top panel: control. Bottom panel: ONC. The band at the top of the figure indicates the ipsilateral and contralateral sides of the brain. (B–D) Quantification of PRV724-labeled neurons in the LC, SUV, and RN. Black dots: control (n = 4), red squares: ONC (n = 5). (E) The number of PRV724-labeled neurons did not significantly decrease after ONC in the VII nucleus and GRN. Magnified images of the VII nucleus and GRN are shown. Top panel: control. Bottom panel: ONC. Scale bars: 100 µm in A and E. The band at the top of the figure indicates the ipsilateral and contralateral sides of the brain. (F, G) Quantification of PRV724-labeled neurons in the VII nucleus and GRN. Black dots: control (n = 4), red squares: ONC (n = 5). Data from three independent experiments are presented as the mean ± SEM and were analyzed by a two-way analysis of variance followed by Sidak’s multiple comparisons. GRN: Gigantocellular reticular nucleus; LC: locus ceruleus; ONC: optic nerve crush; PRV: pseudorabies virus; RN: red nucleus; SUV: superior vestibular nucleus; YFP: yellow fluorescent protein.

ONC administration selectively alters PRV724-traced retinorecipient regions

Given that the ONC procedure has a considerable impact not only on the survival of RGCs but also on the function and structure of the brain (Zhan et al., 2020; Zhang et al., 2020), we next examined changes in projections from PRV724-labeled RGCs after ONC using optical tissue clearing (Thy1-YFP ONC mice, n = 5). After ONC, we observed fewer PRV724-labeled neurons in the BLA, CeA, and PVH of both hemispheres (Figure 6A). The distribution of PRV724-labeled neurons was decreased after ONC in the ipsilateral BLA (P = 0.0006) and contralateral BLA (control: n = 4 and ONC: n = 5, P = 0.0210, Figure 6B). Furthermore, the distribution of PRV724-labeled neurons was decreased in the contralateral CeA (P < 0.0001), whereas in the ipsilateral CeA (P = 0.0989), we found no significant difference between the control and ONC group (control: n = 4 and ONC: n = 5; Figure 6C). In the PVH of both hemispheres, the distribution of PRV724-labeled neurons was also decreased after ONC (control: n = 4 and ONC: n = 5, ipsilateral P = 0.0440 and contralateral P < 0.0001; Figure 6D). Moreover, the distribution of PRV724-labeled neurons was less apparent in the IGL, LGv, oculomotor nucleus, and EWN of both hemispheres (Figure 6E). The distribution of PRV724-labeled neurons in the contralateral IGL was significantly reduced (P = 0.0496), and that of the ipsilateral IGL was also reduced but this was not significant (P = 0.0791) (control: n = 4 and ONC: n = 5; Figure 6F). In the LGv, the distribution of PRV724-labeled neurons was less apparent in both hemispheres (control: n = 4 and ONC: n = 5, ipsilateral P = 0.0019 and contralateral P = 0.0009; Figure 6G). In the oculomotor nucleus, the distribution of PRV724-labeled neurons was only reduced on the contralateral side (P = 0.0418)—changes on the ipsilateral side were insignificant (P = 0.8907) (control: n = 4 and ONC: n = 5, Figure 6H). In the EWN, which is a thin structure in the midline of the brain, the distribution was also reduced (control: n = 4 and ONC: n = 5, P = 0.0020; Figure 6I).

ONC administration appeared to have no effect on the distribution of PRV724-labeled neurons in the LC, SUV, RN, VII nucleus, and GRN (Figure 7A and E). The distributions of cells in these areas were not significantly different across hemispheres (control: n = 4 and ONC: n = 5, P = 0.2035 and 0.1751 in the ipsilateral and contralateral LC, 0.3743 and 0.2541 in the ipsilateral and contralateral SUV, 0.9939 and 0.9923 in the ipsilateral and contralateral RN, 0.9563 and 0.5664 in the ipsilateral and contralateral VII nucleus, and 0.6415 and 0.4921 in the ipsilateral and contralateral GRN, respectively, Figure 7B–D, F, and G). Thus, our results indicate that ONC administration selectively altered PRV724-traced retinorecipient regions including the BLA, CeA, PVH, IGL, LGv, oculomotor nucleus, and EWN.

Discussion

The main goals of this study were to identify the retinorecipient regions of RGCs in mice brains, to map their changes after ONC, and to offer a practicable approach for evaluating neural regeneration in future. To ensure that signals continued to be sent from the RGCs after ONC, we first confirmed that ~13.9% of the RGCs survived the procedure, along with retrograde nerve fibers and axons in the optic nerves. Next, to determine whether PRV724, a new isogenic version of PRV-Bartha, had the same neurotropic effect on RGCs, we performed unilateral intravitreal injection of PRV724. The retinal immunofluorescence data showed that PRV724 primarily infected RGCs in both control and ONC mice. We then performed entire brain imaging using a tissue clearing technique to fully scan the retinorecipient regions that received projections from PRV724-labeled RGCs, and investigated the changes in these retinorecipient regions after ONC. Together, our results demonstrate that PRV724-traced retinorecipient regions changed in specific areas after ONC, including the BLA, CeA, PVH, IGL, LGv, oculomotor nucleus, and EWN. Thus, reconnection between RGCs and these areas might be a good measure of optic nerve regeneration.

Only a few RGCs survive after ONC, and approximately 1% of their axons pass through the crush site at the optic nerve (Duan et al., 2015). Our data were consistent with previous findings in that few RGCs survived, and almost half of their nerve fibers had been lost. This was accompanied by a striking decrease in the number of axons in the optic nerves. When studying retinorecipient regions using a retrograde trans-synaptic virus, the integrity of the retrograde nerve fibers should first be confirmed. Previous studies have shown that retrograde tracer CTB can be used to label axons after ONC, and while most retrograde axons are expected to be lost, a few retrograde axons generally survive and pass through the injury site (Duan et al., 2015; Patel et al., 2020; Hilla et al., 2021). Our findings from retinal tissue indicate that retrograde nerve fibers were present after ONC. Together, these data suggest that although ONC seriously damages the optic nerve, a retrograde physiological structure exists. Thus, this model provides an opportunity to study retinorecipient regions using a retrograde trans-synaptic virus.

Recent studies have shown that PRV724 can be used to trace neurons via injection into the anterior chamber of the eyes or brain area (Yang et al., 2021; Zhai et al., 2021). We confirmed that PRV724 could label RGCs, as well as some ipRGCs. Moreover, PRV724 labeled other cells in the retina. Viney et al. observed that during the first wave of infection (0.5–4 days), almost 99% of PRV152 (an isogenic version of PRV constructed with green fluorescence protein)-labeled cells were ipRGCs, while after 5 days, only 50% of PRV-labeled cells were ipRGCs (Viney et al., 2007). One possible explanation is that PRV724 infected Müller cells near the cell bodies of infected RGCs through gap junctions, as described in a previous study (Viney et al., 2007). The extra labeling of other cells in the retina is related to the local circuit of ipRGCs (Viney et al., 2007). However, in the present study we could not confirm that the PRV724-labeled cells were exclusively ipRGCs. Additionally, we found that the number of RGCs in the PRV724-infected retinas did not significantly differ from that in non-infected retinas, despite the fact that PRV724 caused retinal disorder and distortion. This enabled us to rule out the possibility that PRV724 affected RGC survival.

Via intravitreal or intracameral injection of PRV, a previous report found PRV-labeled neurons to be distributed in the SCN, IGL, LGv, and PVH in rodent brains (Smith et al., 2000). Another study in golden hamsters found that PRV-labeled neurons were not only present in the SCN, LGv, IGL, and PVH, but also in the EWN, lateral terminal nucleus, and OPN (Pickard et al., 2002). Furthermore, Smeraski et al. (2004) observed that PRV-labeled neurons were distributed in multiple regions in rat brains, including the principal sensory nucleus of the trigeminal and superior salivatory nucleus. Yang et al. (2021) found that after the injection of PRV into the anterior chamber, neurons in the SCH, PVH, EW, superior salivatory nucleus, and ventral mammillary nucleus were labeled. Previous studies have indicated that the seemingly disparate distributions of PRV-labeled retinorecipient nuclei might be caused by differences in the susceptibility of neurons in different animals (i.e., golden hamster, rat, and mouse) as well as limitations associated with slice analysis. For instance, large datasets such as those containing hundreds or even thousands of rodent brain sections may be susceptible to missing information. Hence, methods that enable viewing of the entire brain are preferable for visualizing retinorecipient regions connected to RGCs. However, deep regions in opaque tissue are impossible to image without slicing. In tissue clearing, the refractive index of the medium is matched to that of scattering particles to produce optical transparency in the tissue (Ueda et al., 2020), and light-sheet microscopes have an extended range of fast imaging options for large samples. Our light-sheet imaging method for the entire brain enabled a more comprehensive assessment of retinorecipient regions connected to RGCs via PRV labeling. Although our results have similar characteristics to previous studies, there are some distinct findings to discuss.

Previous studies on ipRGCs have shown that they send projections to the SCH to control circadian rhythms (Hattar et al., 2002) and to the OPN to participate in the pupil light-reflex (Hattar et al., 2006; Sonoda et al., 2020). However, although there were some PRV724 retinal cells co-labeled with opsin 4, we did not consistently observe PRV724-labeled neurons in these areas in all 9 brains (control: n = 4, ONC: n = 5, data not shown). One study found that the SCH was labeled with PRV only if the OPN was labeled in some mice (Smeraski et al., 2004). This might depend on the number of PRV724-infected ipRGCs in different animals, and so this issue requires further investigation.

The death of most RGCs explained our results in the LGv, IGL, CeA, BLA, PVH, and EWN after ONC. The LGv processes not only visual-related information but also non-visual information (Monavarfeshani et al., 2017). The IGL is another non-visual processor that receives projections mainly from ipRGCs, although it receives a few projections from other types of RGCs (Hattar et al., 2006). The amygdala is a limbic structure that is involved in a variety of functions, including emotion and memory. The CeA is associated with conditional fear, anxiety, and mediates the secretion of corticotrophin-releasing factor (Kalin et al., 2004). The CeA participates in mediating eyeblink conditioning (Lee and Kim, 2004) and receives information about visually conditioned stimuli and projections from the LGv (Halverson and Freeman, 2010; Farley et al., 2018). Hence, the CeA might be strongly related to the processing of visual-related information from RGCs. The BLA has complex connections with multiple areas of the brain (Hintiryan et al., 2021). The most well-studied function of the BLA is fear control (Adhikari et al., 2015). Recent evidence has shown that the response to visual threats is controlled by input from the nucleus reuniens, which projects to the medial prefrontal cortex and then to the BLA, resulting in freezing motions when facing a visual threat (Salay et al., 2018). However, we did not observe PRV724-labeled neuron distributions in either the nucleus reuniens or the medial prefrontal cortex (data not shown). The BLA is directly connected to secondary visual cortical areas such as the anteromedial and anterolateral areas (Hintiryan et al., 2021). The LGv also participates in reacting to visual threats and sends projections to the nucleus reuniens, which has been found to increase freezing motions (Fratzl et al., 2021; Salay and Huberman, 2021). Therefore, the BLA might participate in the processing of visual-related information and indirectly receive projections from the LGv. Previous studies have shown that PRV-labeled RGCs project to the PVH (Smith et al., 2000; Viney et al., 2007; Yang et al., 2021), whereas studies on ipRGCs have not shown that they project to the PVH (Hattar et al., 2002; Ecker et al., 2010; Cui et al., 2015; Sonoda et al., 2020; Beier et al., 2021). The PVH has recently been identified as playing a crucial role in the circadian regulation of weakness, and it receives some inputs from the CeA, SUV, and GRN (Chen et al., 2021). In addition, although ipRGCs play an important role in regulating circadian rhythms, rods also contribute to this function (Altimus et al., 2010). Hence, the PVH might mainly receive visual-related projections from the CeA, and the loss of these projections after ONC could have resulted in a decrease in the number of PRV724-labeled neurons in the PVH. Light stimulation has been found to activate the noradrenergic pathway, of which the LC is a central component (Liu et al., 2017; Szabadi, 2018), and then project to the EWN to control pupil diameter (Nobukawa et al., 2021). Other studies have found that ipRGCs participate in the control of the pupil light-reflex (Hattar et al., 2006; Sonoda et al., 2020). These results suggest that the LC might receive non-visual projections from ipRGCs, and then project these signals to the EWN. Taken together, these data indicate that brain areas including the LGv, IGL, CeA, BLA, PVH, and EWN might receive projections from specific RGCs that died after ONC, whereas the LC receives projections from specific ipRGCs that had survived at 4 weeks after ONC.

We also examined the distribution of PRV724-labeled neurons in regions related to motor function including the SUV, oculomotor nucleus, RN, VII nucleus, and GRN, and found that apart from that in the oculomotor nucleus, these distributions remained unchanged after ONC. The GRN is the main output center for the regulation of locomotor patterns and rhythms (Lemieux and Bretzner, 2019), and the RN and LC might project to the GRN to regulate locomotion (Zörner et al., 2014). It is possible that the SUV and oculomotor nucleus coordinate to mediate vertical eye movements in monkeys (Horn and Straka, 2021). Further research is needed to determine the nature of projections from RGCs to motor-related nuclei, and to understand why the distributions remained unchanged in the SUV, RN, VII nucleus, and GRN despite changing in the oculomotor nucleus after ONC.

The major limitation of this study was that the changes in PRV724-labeled brain regions after ONC were strongly correlated with behaviors and functions such as fear avoidance, circadian rhythms, pupillary reflex, and motor function. Detailed assessments of fear avoidance, sleep, the pupillary reflex, and motor function are needed before and after ONC to uncover the functions of PRV724-labeled RGCs. Another limitation was that we did not definitively determine the types of PRV724-labeled RGCs, although single cell sequencing of PRV724-infected retinas may address this limitation.

In conclusion, we confirmed that a combination of tissue clearing and trans-synaptic viral tracing enabled the objective examination of retinorecipient regions of RGCs after ONC in mice brains. This, our method is applicable to future investigations of optic nerve regeneration.

Additional files:

Additional Figure 1 ^{(1.8MB, tif)} : The location procedures of the brain area.

Additional Figure 1

The location procedures of the brain area.

Light-sheet images from cleared brains were obtained from the Y-axis to the X-axis (transverse). Take the CeA region as an example. (A) Locate the transverse section of the PRV724 (red dots) labeled areas. (B) Compare with the 3D Allen Brain Atlas. (C) Locate the sagittal section of the same PRV724 labeled areas. (D, E) Compare the sagittal section with the 2D and 3D Allen Brain Atlas. (F) Locate the coronal section of the same PRV724 labeled areas. (G, H) Compare the coronal section with the 2D and 3D Allen Brain Atlas. 2D: Two-dimensional; 3D: three-dimensional; CeA: central amygdalar nucleus; PRV: pseudorabies virus.

Additional Figure 2 ^{(5.2MB, tif)} : PRV724 did not label the axons.

Additional Figure 2

PRV724 did not label the axons.

(A) Confocal immunostaining images of the ON and OC of control mice 6 days after PRV724 intravitreal injection, showing axons stained with GAP-43 (Alexa Fluor 488, green) and nuclei stained with DAPI (blue). (B) The regions of OC and left ON of a control mouse are further magnified. (C) Confocal immunostaining images of the ON and OC of an ONC mouse after intravitreal injection of PRV724, showing axons stained with GAP-43 (Alexa Fluor 488, green) and nuclei stained with DAPI (blue). (D) The regions of control OC and left ON of an ONC mice are further magnified. Scale bars: 50 μm. DAPI: Diamidino-phenyl-indole; GAP-43: growth-associated protein-43; OC: optic chiasm; ON: optic nerve; ONC: optic nerve crush; PRV: pseudorabies virus.

Additional Figure 3 ^{(2.5MB, tif)} : Wild-type mice and Thy1-YFP mice shared the same characteristics.

Additional Figure 3

Wild-type mice and Thy1-YFP mice shared the same characteristics.

(A) The number of total RGCs or PRV724-infected RGCs do not differ in WT mice or Thy1-YFP mice before and after ONC. Immunostaining images of the central, mid-peripheral and peripheral retinas with RBPMS (Alexa Fluro 647, gray, labeling RGCs), Thy1-YFP RGCs (green), and PRV724-labeled RGCs (red). In the control and ONC groups of Thy1-YFP mice with PRV724 intravitreal injection. Scale bars: 50 μm. (B) Quantification of the number of RGCs in the WT and Thy1-YFP mice in control and ONC groups (n = 3 eyes per group). The WT data are from Figure 2D. (C) Quantification of the mRFP+ RGC ratio in Thy1-YFP mice compared with that in WT mice in the control and ONC groups. The WT mice data are from Figure 3J (n = 5 eyes per group). Data from 3 independent experiments are presented as the mean ± SEM, and were analyzed by two-way analysis of variance followed by Sidiki’s multiple comparisons. mRFP: Monomeric red fluorescent protein; ns: not significant; ONC: optic nerve crush; PRV: pseudorabies virus; RBPMS: RNA binding protein with multiple splicing; RGCs: retinal ganglion cells; WT: wild-type; YFP: yellow fluorescent protein.

Additional Video 1: The video of tissue-cleared Thy1-YFP mouse brain under light-sheet microscope.