Morphology Analysis of Descending Tracts in the Mouse Spinal Cord Using Tissue Clearing, Tissue Expansion and Tiling Light Sheet Microscopy Techniques

doi:10.21203/rs.3.rs-3089483/v1

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Morphology Analysis of Descending Tracts in the Mouse Spinal Cord Using Tissue Clearing, Tissue Expansion and Tiling Light Sheet Microscopy Techniques

https://doi.org/10.21203/rs.3.rs-3089483/v1

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published 30 Sep, 2023

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Descending tracts are the pathways to send motor signals from the brain to spinal cord playing an important role in movement coordination. However, there are rare reports to show the full view of the tracts with a long span projection and variety axon morphologies from 3D perspective. Herein, five less-studied descending tracts in the mouse spinal cord were analyzed that originate from midbrain, cerebellum or brainstem individually by the tissue clearing method in combination with tiling light sheet microscopy. By tracing dozens of axons in spinal cord, we reported several novel findings of the different tracts: the collateral “sister” branches occurred only in the axons originating from the cerebellospinal tracts among these five tracts; the axons from superior colliculus terminated only at upper cervical but with abundant branches in the brainstem; the SP5 (spinal trigeminal nucleus) axons crossed the midline of spinal cord to contralateral while the MdV (medullary reticular formation, ventral part) axons had plenty of branches in both cervical and lumbar segments. The characteristics of these branch features in different tracts hinted the function of specific descending pathway. Furthermore, we preliminarily investigated the synaptic connection between the tracts and motor neurons in the spinal cord through hydrogel-based tissue expansion and found several synaptic connections between MdV axons and motor neurons. The present study, for the first time, described the full-view of the projection patterns and axon morphologies of five descending tracts in the mouse spinal cord under 3D view, which was essential for better understanding the function of these descending tracts. Meanwhile, we developed a new method for future study of descending tracts by three-dimensional imaging.

Biological sciences/Biological techniques

Biological sciences/Neuroscience

Movement initiation and accomplishment depend on the function of descending tracts that acts directly on the spinal cord, which originate from diverse brain areas, including the cerebral cortex ^1,2, midbrain ³, brainstem ⁴ and cerebellum ^5–7. These pathways in mammals play a vital role in the initiation of movements of the limbs and trunk, including grasping, walking and posture maintenance. In the past century, by using retrograde tracing methods (eg. adeno-associated virus or dyes), researchers have identified more than 20 brain centers that gave rise to the descending tracts reaching spinal cord.^8,9 Among them, the dominant tracts mainly arose from the cerebral cortex ^1,2,10,11, red nucleus ^12,13, brainstem reticular formation ^14,15 and the vestibular nuclei ¹⁶. Apart from these well-known centers, there are some other nuclei that have been identified, including the medial and interposed cerebellar nuclei (Med, IntP)^5,17, the superior colliculus^18,19 and the spinal trigeminal nucleus^20,21. They could also project to the spinal cord.

The existing evidence has provided a convenient projection map for researchers to study the function of the descending tracts, but there are still many problems worth considering due to technical limitations. For example, the descending tracts generally have a long span, from brain to the spinal cord, whereas the projection map published in the past studies were almost always obtained in 2D tissue slices, through which the information and knowledge were fragmented and couldn’t give a full-view of the real projection pattern. Moreover, if the 2D tissue slice was used to study the long-span tracts, more tissue slices should be prepared and a lot of images should be taken one by one, which is laborious and time consuming. Furthermore, it was hard to study the connectome of descending tracts in spinal cord using 2D image. In the past several years, the development and maturity of tissue clearing methods ^22,23 and light sheet microscopy imaging system¹⁹, has made it possible to obtain 3D information of intact large tissue samples within a short time. Nowadays, according to the different principles of tissue transparency, three major approaches are currently prevalent: hydrophobic²⁴, hydrophilic²⁵, and hydrogel-based methods^26,27. CUBIC²⁸, DISCO²⁹ and MAP³⁰ are three the most representative methods. In general, hydrophobic tissue-clearing methods often provide transparency of an intact specimen quickly, while hydrophilic methods perform better in preserving the emission of fluorescent proteins. Meanwhile, hydrogel-based methods can expand the tissues, which further increases both the transparency and effective resolution³¹. Thus, in order to know more details and connectome information about the axons of descending neurons in spinal cord, the hydrophilic and hydrogel-based methods were used in this study to obtain the intact 3D cleared tissue. In our previous study, we have reported optimized tissue clearing and tissue expansion methods, called eCubic and CMAP (Clearing and Magnification Analysis of Proteome) respectively, for the large sample clearing and expansion by modifying the CUBIC and MAP methods³². Our previous data have shown that the tissues treated by CMAP had higher fluorescence preservation and mechanical strength.

Therefore, in this study, by combing the hydrophilic tissue clearing method and tiling light sheet microscopy system, we studied five descending tracts in the spinal cord individually originating from cerebellum, brainstem, and midbrain. Firstly, we clarified the patterns of tracts projection in the spinal cord under high magnification. Next, through sparse labelling neurons, we traced dozens of single axons of each tract individually, and further analyzed the distribution and intensity of axon branches in different areas of spinal cord. Finally, by using the CMAP method, we also detected direct connections between the projection axons and the motor neurons in spinal cord ventral horn. Overall, these data provide an accessible resource to understand the descending tracts in depth and contribute new methods to the study of the descending tracts.

Distribution of descending neurons in the brain

Movement is controlled by the descending tracts that project to the spinal cord from different brain motor areas, including the cortex, red nucleus, brainstem, and cerebellum⁷. To verify the specific distribution of descending neurons in the brain, AAV2-Retro-EGFP was injected bilaterally to cervical spinal cord to label the neurons. Next, we performed the tissue clearing followed with 3D imaging of the brain after four weeks (Fig. 1A-C). Consistent with previous studies^17,33, retro-labeled neurons were observed within the cortex, red nucleus, vestibular nucleus and reticular formation by analyzing four brains (Fig. 1D-K), thus affirming the reliability of this viral tool for labeling descending neurons. Apart from those well-known descending tracts, we also observed retro-labelled neurons within the spinal trigeminal nucleus (SP5, Fig. 1F, M), medullary ventral (MdV) reticular formation (Fig. 1F, M) and two subregions in deep cerebellum nuclei, namely IntA/P and Med (Fig. 1F, L). Through fasciculus, the IntA/P and Med trajectory extended to the contralateral cerebellum and then descended to the spinal cord (Fig. 1L). In the previous study, we have studied the projection patterns of three dominant descending tracts, containing the cortex, red nucleus and reticular formation ³⁴. In this study, we will focus on these less-studied descending pathways to analyze the projection pattern and axon features in the spinal cord using high magnification 3D imaging microscopy.

Axon projection pattern of Med neurons in spinal cord

Cerebellospinal neurons, consisting of Med neurons and IntA/P neurons, play an essential role in motor commands, like forelimb performance and skilled locomotor learning ^17,35,36. In order to study the projection pattern of Med neurons in spinal cord, the Med neurons were sparsely labelled with the mixture virus of AAV2/9-hSyn-cre and AAV-hSyn-DIO-tomato in ratio of 1:2. Our data showed that all of the axons terminated at contralateral side of the cervical and upper thoracic (T1-T2) region of spinal cord (Fig. 1A-D, Supplementary video 1). From a horizontal view, the propriety of axons extended along the middle ventral with abundant collateral branches, with only few axons projected along the lateral ventral region of spinal cord (Fig. 2E, F). From a coronal view, it can be seen that axon can extend to the edge of the ventral of both upper cervical and lower cervical segments (Fig G, H). After tracing the axons manually, two main types of axon branch patterns were detected: branches generated only at upper cervical segment (Fig. 2J-M), and those generated at both upper and lower cervical segments (Fig. 2N-Q). We analyzed the branches at the both areas, finding that the intensity of lower cervical is higher than that at upper cervical (Fig. 2R). Furthermore, in the traced axons, we found out that many collateral branches can further generate sister branches (Fig. 2T), i.e., the collateral branch divided into two branches immediately at the branch node. And both sister branches could be extended far away, which was obviously different from the single collateral branch (Fig. 2S). Moreover, the axons containing collateral “sister” branches were all of this type (Fig. 2M-Q). This phenomenon was hard to detect under 2D imaging system. Finally, we counted the number of axons with collateral “sister” branches in the all-traced axons, and found that the percentage of this type of axon was up to 75%. As a medium for information transfer, axons are responsible for transmitting information from the soma to the axon terminals and further to the next level of neurons. Therefore, we speculated that the “sister” branches may enlarge the dominant area, which is conductive to information transfer.

Axon projection pattern of IntA/P neurons in spinal cord

Anterior interpositus nucleus (IntA) and posterior interpositus nucleus (IntP) were very close to each other in neuroanatomy, so we studied the both areas together to investigate the projection pattern (Fig. 3A, B). Similar to the Med, the most axons from the IntA/P crossed to contra-cerebellum and descended to the ventral of spinal cord with the axons terminated at cervical segment (Fig. 3C, D). The most axons extended along ventral part, while few axons were distributed at the lateral side of cervical segment (Fig. 3C, D). From a coronal view, the terminations of axon branches were accumulated in the laminae VII and VIII of both upper and lower cervical segments (Fig. 3E- H). After the axons were traced individually, we found out that the branches were evenly distributed in the upper cervical or lower cervical segment (Fig. 3I-Q, W). Meanwhile, as in Med, we also found many “sister” branches in IntA/P, and the percentage of “sister” branches was about 70%(Fig. 3X), which was close to the percentage in Med.

To sum up, both Med and IntA/P neuron axons had close projection pattern. Their axon termination was distributed at the same area (laminae VII and VIII), and they had approximately the close percentage of “sister” branches to enlarge the area to transmit information. However, the Med had a higher percentage in upper cervical segment while IntA/P had a close percentage in different areas. Next, we explored whether the cerebellospinal tracts had direct connection with motor neurons. To answer the question, we injected the same virus into IntA/P on ChAT-eGFP mice, and perfused the mouse after 4–6 weeks. Next, we combined tissue expansion with tiling light sheet microscopy to image the intact cervical segment of the spinal cord (Fig. 3R). After enlarging the tissue 3–4 times by CMAP, we found out that the axon termination didn’t occur in the area of motor neurons accumulated in both upper and lower cervical regions (Fig. 3S-V), which hinted no direct connection of them with motor neurons.

Axon projection pattern of SP5 neurons in spinal cord

SP5 (spinal trigeminal nucleus) is a small motor center in the brainstem. Prior study has reported that labeled neurons in SP5 can be detected by injecting the rabies into the mouse biceps ³⁷. However, they didn’t deeply observe the pattern of SP5 axons in the spinal cord. In this study, by injecting anterograde virus into the SP5 and observing the axon branches in the spinal cord, we found most axons terminated at cervical or upper thoracic segment (Fig. 4A-C). We analyzed the axons number bilaterally, discovering that almost 80% of axons projected to ipsilateral cervical region, while ~ 20% of neurons terminated at contralateral cervical region (Fig. 4E). We observed the branches of axons in the coronal view and found that the branches were distributed at dorsolateral cervical segment. Unexpectedly, some branches crossed the midline and terminated at contralateral spinal cord (Fig. 4D). Similarly, we traced the axons in the spinal cord manually to detect the projection patterns in the cervical segment. We observed two different types of projection patterns: terminated at upper cervical (C1-C4, Fig. 4I-L) and lower cervical regions (C5-C8, Fig. 4M-R). In the pattern of upper cervical projection, most axons possessed few branches (Fig. 4I, K, O) and only few axons possessed several branches (Fig. 4J). By contrast, in the pattern of lower cervical projection, most axons possessed several branches and only few axons possessed few branches (Fig. 4P). Meanwhile, we traced some interesting axons which could cross the midline and get extended from ipsilateral to contralateral (Fig. 4L-M). In addition, we also observed an interesting axon in the lower cervical projection pattern, which originated from lateral upper cervical segment, but turned to middle in the lower cervical segment and then extended lots of branches (Fig. 4N). According to the traced results, we finally counted the intensity of axons in upper and lower cervical segments, finding that the branch intensity was higher in lower cervical segment while the branches in upper cervical had comparable intensity to that in all the projection areas (Fig. 4F).

Axon projection pattern of MdV neurons in spinal cord

The medullary reticular formation ventral part (MdV) is a key brainstem area specifically connected to forelimb-innervating spinal motor neurons ³⁷. A previous study has shown glutamatergic premotor neurons in MdV received synaptic input from key upper motor centers and were recruited during motor tasks³⁷, but up to now there are no reports to study the axon output of the MdV neurons systemically. In order to have a broader understanding of axons output of MdV neurons, we injected anterograde virus in MdV area (Fig. 5A-B) and studied the axon projection pattern of MdV neurons in the whole spinal cord (Fig. 5C). Firstly, through the whole spinal cord view and the coronal view, it could be seen that the labelled axons were mainly distributed at dorsolateral and ventral parts of spinal cord (Fig. 5C-E). Next, in order to study the projection pattern of MdV neurons in spinal cord, the MdV neurons were sparsely labelled with mixture virus of the AAV-Cre and AAV-DIO-tdTomato viruses at a ratio of 1:3 − 1:5. After the axons were traced individually, several different projection patterns were detected. In general, according to the distance of axon projection on the spinal cord, there are three projection patterns: terminated at the cervical (Fig. 5N-P), upper thoracic (Fig. 5M) and lumbar and sacral (Fig. 5G-L). The properties of axon branch properties in different projection patterns were observed and found something interesting. For example, for those axons that terminated only in cervical or upper thoracic regions, axon branches accumulated in cervical region (Fig. 5M-P). While, for the axons that terminated in lumbar and sacral regions, the distribution of axon branches was slightly more complicated. For instance, some axon branches were only distributed in lumbar region (Fig. 5J), some were distributed in both thoracic and lumbar regions (Fig. 5I, K) and some were distributed in both cervical and lumbar regions (Fig. 5H, L). These projection branch patterns may suggest the role of MdV neurons in motor control. We have also analyzed the branch intensity in cervical and lumbar regions, and the intensity was higher in cervical region than in lumbar region (Fig. 5F). Furthermore, to detect whether the MdV neurons can directly drive the motor neuron, the AAV-cre and AAV-DIO-tdToamto mixture virus was injected into MdV of ChAT-eGFP mouse. In combination with the tissue expansion and tiling light sheet microscope imaging system, we detected whether the axons had synaptic connection with motor neurons. The lower cervical and lumbar region were expanded individually (Fig. 5Q, 5U). Our results showed abundant synaptic connections between MdV axons and motor neurons in both cervical and lumbar regions, which indicates that the MdV axons could drive the motor neurons directly (Fig. 5Q-Z, Supplementary video 2).

Tectospinal tract in spinal cord

The tectospinal tract in mammals arises from neurons in the superior colliculus and projects to the spinal cord^18,19,38. It is primarily involved in the control of head and neck movements. In our studies, we detected rare neurons in superior colliculus through retrograde injection into spinal cord (Fig. 1E), which was consistent with the published finding that rare neurons project to spinal cord from superior colliculus⁸. By observing the projection patterns of superior colliculus neurons, we found that majority axon fibers crossed in the dorsal tegmental decussation and got extended along the ventral part of brainstem, but only rare axons entered to the spinal cord (Fig. 6A-B). Next, we traced and observed the axons that projected to the spinal cord. Our results showed that all the axons along the ventral column of spinal cord were extended to the contralateral-upper cervical (Fig. 6C-F). Meanwhile, we found the axon branches could be extended to the dorsal area of spinal cord (Fig. 6F). We have also accessed the branch numbers and found that in all the traced axons, only few branches (less than 3 branches) were detected (Fig. 6G-Q), which was in sharp contrast with the large number of branches in brainstem (Fig. 6B). This phenotype may be related to the function of superior colliculus, which mainly participated in the movement of head and neck. Also, the branches of axon were extended to the middle and dorsal area of spinal cord, and they were unlikely to connect with the motor neurons in ventral horn of spinal cord directly.

Spatial distribution of MdV neurons in brain

When the axons of MdV neurons were traced, it was observed that the labelled axons projected to cervical or lumbar region, which made us to be interested in how those neurons with different projection patterns are distributed within the brain. Thus, we labelled the neurons with the injection of AAV2-retro-EGFP in cervical spinal cord and injection of AAV2-retro-tdTomato in lumbar spinal cord, respectively (Fig. 7A). Whole CNS was imaged using tiling light sheet microscopy after being delipidated by eCUBIC (Fig. 7B). We found that the GFP positive neurons and tdTomato positive neurons in the motor cortex and red nucleus had specific spatial distribution (Fig. 7C,7D, Supplementary video 3), which was consistent with prior studies^{17 9,33}. Next, we focused on the spatial distribution of MdV neurons and found there was no obvious spatial distribution like that in motor cortex or red nucleus (Fig. 7E). However, we observed that many neurons in MdV were either GFP or tdTamato positive or GFP and tdTamato double positive (Fig. 7F), which inversely demonstrates our findings that MdV neurons have complex branching patterns in different segments of the spinal cord. It is further implied that our tracing results were reliable (Fig. 6G-P, Fig. 7G).

Descending pathways carry motor commands to the spinal cord, linking motor plan to task execution. In the past few decades, researchers used retrograded virus and dye to label the tracts and study the morphology and distribution in the spinal cord under the context of 2D sectioning. However, this system was laborious and caused the information to be fragmented. Furthermore, it was inaccessible to analyze the single, intact axon morphology in detail, which corresponds with function of the tracts. In the present study, we offered a new method to study the descending tracts comprehensively, through the virus labelling, tissue clearing and light sheet microscopy in combination. We demonstrated that by using this method, it was possible to analyze distribution and morphology of the tracts from 3D image under high magnification. Firstly, we injected retrograded virus into spinal cord to detect the nuclei projecting to spinal cord directly by means of intact brain imaging. Then we focused on five pathways and further analyzed distributions of these pathways and axon morphologies in the spinal cord under high magnification. Furthermore, we analyzed whether the tracts had direct connection with motor neurons in the ventral horn of spinal cord by using CMAP.

Classical anatomical studies have shown that the majority cerebellospinal neurons (from Med and IntA/P) projected to contralateral spinal cord, and the termination of axons was distributed in laminae VII and VIII of cervical spinal cord ^5,17,39. Consistent with the prior description, our data showed that both Med and IntA/P pathways projected to contralateral cervical segment of spinal cord along the mid-ventral surface. And the tracing results, for the first time, showed that the IntA/P and Med neurons had high ratio of “sister” branches, which was difficult to detect under 2D system. The branch number of axons was related to the number of next layer neurons, who received upper command information. The Med axon branches had higher intensity in upper cervical region while IntA/P had higher intensity in lower cervical region which may imply that the information flow area and target were varied between two pathways. However, the reason for the difference requires further functional analysis between the both pathways. Next, we have imaged intact cervical region of spinal cord after injecting the anterograde virus in ChAT-eGFP mouse. No direct synaptic connection was detected between the descending axons and the motor neurons neither in upper cervical nor in lower cervical segment, which was consistent with the previous study¹⁷.

In our study, we found the majority SP5 axons terminated at cervical region and only few axons were extended to thoracic spinal cord in mouse which was consistent with the findings in human brain²¹. Previous study showed that SP5 neurons can be labeled by injecting the monosynaptic rabies virus in forelimb muscle which implied that the SP5 was associated with forelimb control. This might explain why majority labelled axons in our study terminated in the lower cervical segment and have a high density of axon branches. In this study, we also observed several axon branches in upper cervical region, which may hint the role of SP5 in face and head control. Surprisingly, we firstly traced several SP5 axons that could cross the midline to the contralateral in the spinal cord, which reflected the complexity and diversity of the SP5 axon morphology and distribution, although the function of the different types of axons was not yet clear.

In 2014, Maria et al. reported that MdV neurons was important for skilled forelimb motor behavior ³⁷. However, in their published data, we also observed MdV axons projected into the lumbar region, which may hint its function in hindlimb motor control. In our study, we imaged the whole spinal cord, and observed that the MdV axon mainly projected to the cervical and lumbar region, and some of them even projected to the sacral region. And the single axon tracing results showed that most of the axon branches were distributed in either cervical or lumbar region, which may indicate that MdV played a specific role in forelimb and hindlimb control. While the higher intensity of branching distribution in cervical segment than lumbar segment may suggest the different dominate degree of MdV neurons in forelimb and hindlimb motor control. In Maria’s study, they only reported the synaptic connection between the MdV axons and the motor neuron in cervical region³⁷, however, in this study, we found that the MdV axons had synaptic connections with motor neurons in both cervical and lumbar regions.

Finally, we visualized the relative spatial distribution of neurons in brain that project to cervical and lumbar spinal cord combined AAV2-retro virus from 3D view. Consistent with previous studies, the descending neurons from motor cortex and red nucleus had apparent spatial distribution which controlled the forelimb and hindlimb^17,33, while, there was no obvious spatial distribution of MdV neurons. Surprisingly, we found that several neurons were both GFP positive and tdTmato positive in the ventral part of MdV, which inversely demonstrated our findings that MdV neurons had complex branching patterns in different segments of the spinal cord. Above all, these results indicated that our tracing results were reliable.

Overall, in this study, combining the tissue clearing, tissue expansion and tilling light sheet microscopy imaging system, we firstly reported the full-view of the projection patterns and axon morphologies of five descending tracts in the mouse spinal cord under 3D view. Our results both validated the reported data and uncovered some new phenomena that can’t be observed at 2D level. Although it looked available to understand the descending tracts more sufficient and comprehensively through the 3D imaging system, it is not yet possible to image the entire brain at high magnification. Even so, the present and future information from 3D view will give researchers revelation on functional speculation.

Animals and virus

All the experiments were carried out in accordance with the ARRIVE guidelines (https://arriveguidelines.org). This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Westlake University, Hangzhou, China. All the methods were performed in accordance with relevant guidelines and regulation. All mice were housed on a 12hr/12hr light dark cycle with standard chow. ChAT-GFP mouse line was maintained on a background of C57BL/6J, which was kindly provided by Dr. Liang Wang from Zhejiang University. In this study, 20 adult male mice were used. Four mice were used to detect the descending neurons by injecting retrograded virus in cervical region of spinal cord while three mice were used to study the relative spatial distribution of descending neurons by injecting retrograded virus in cervical and lumbar regions, respectively. Two to four mice were analyzed in each tract (Med: 2; IntA/P: 3, SP5: 2; MdV: 4; superior colliculus: 2). All AAV viruses were purchased from the Brain VTA (Brain VTA Co., Ltd., Wuhan, China). Viral particles were injected at a titer of 1-5E12 genome copies per mL.

Surgery and virus injection

Spinal cord injection: Mice were anesthetized by intraperitoneal injection of amobarbital sodium. The injection process was performed according to a previously described method⁴⁰. Briefly, a small incision was made in the skin and the underlying musculature and adipose tissues were teased to expose the vertebral column. Then, the tissue joining the dorsal processes of consecutive vertebrae was removed. After cleaned the vertebral surfaces with fine tweezers, vertebral was gently separated to reveal the dorsal surface of the spinal cord. Stereotaxic apparatus (RWD Technology Corp., Ltd) was used here to confirm the injection depth. AAV(2/retro)-hSyn-EGFP or AAV(2/retro)-hSyn- tdTomato was pressure injected through a pulled glass needle at a depth of 0.8-1 mm from the dorsal surface and 1uL of viral particles was released to 3 points of each region at a rate of 50 nl/min. Following injections, the muscles were sutured with absorbable sutures followed by sewing up the skin with vetbond (3M). In addition, mice received intradermal injection of meloxicam SR for analgesia, and then were placedon heating pad until it woke up. At last, the mice were returned to their home cages. Cervical region (C5-C8) and lumbar region (L2-L4) injections were performed in line with the same protocol.

Virus injection for mouse brain: Stereotaxic apparatus (RWD Technology Corp., Ltd) was used to fix mouse brain during the virus injection. Virus solution was pressure injected through a pulled glass needle. Mixed virus solution used in this experiment, rAAV2-hSyn-cre and rAAV2-DIO-tdTomato mixed with the ratio of 1:1 to 1: 5 to inject to WT mice or ChAT-GFP mice. Craniotomies were made above the corresponding target areas (Med are AP: -6.00, ML: -0.3, DV: -3.0; IntA/P are AP: -6.20, ML: -1.8, DV: -3.0; Superior colliculus are AP: -4.50, ML: -0.5, DV: -1.2; SP5 are AP: -6.00, ML: -1.5, DV: -4.0 from pia; MdV are AP: -7.20, ML: -0.5, DV: -3.2 from pia) and the viral particles were released at a rate of 10 nL/min, 100-200 nL per site. Following injections, the skin incision was closed using vetbond and returned to their homecages.

Histology and Immunofluorescence

Anesthetized mice were perfused with PBS followed by cold 4% paraformaldehyde (PFA). The whole CNS was harvested and post-fixed in cold 4% PFA overnight with gentle shaking. The coat of spinal cord was peeled softly with precision tweezers after washing several times with PBS. To verify the injection site, the brain was sliced into 50 μm with vibratome and incubated in DAPI (1:5000). The slices with fluorescence were checked under confocal microscope (Zeiss LSM 980). The spinal cord with desired injection site was reserved fortissue clearing and tissue expansion experiment.

Tissue clearing and tissue expansion

The tissue was treated with eCubic and CMAP as previously reported³². Preparation of cleared spinal cord and brain(eCubic): The tissue was immersed in the delipidation solution(15 wt% urea,10% wt% N-butyldiethanolamine,10 wt% Triton X-100 and 65 wt% ddH₂O) at 37℃ for 3-5 days with gently shaking until the tissue became transparent. The delipidation solution was replaced every 24 hours. Following that, the delipidated tissue was transferred to RI matching buffer(25 wt% urea, 22.5 wt% sucrose, 22.5 wt% antipyrine,10 wt% triethanolamine) at 25℃ for 2 days with daily change. Finally, the tissue was embedded with 2% agarose gel made with the RI matching buffer. The embedded tissue was amounted on the sample holder and imaged in silicone oil with tiling light sheet microscopy.

Preparation of expanded spinal cord samples (CMAP): After clearing the sample with the procedure as described above, the tissue was washed with 0.01 M PBS for 2-3 hours at room temperature with gently shaking. Next, the spinal cord was incubated with monomer solution (30% Acrylamide, 0.075% N,N-Dimethylacrylamide, 10% Sodium acrylate,0.5% 2,2’-Azobis dihydrochloride in 0.01M PBS) at 4℃ for 2 days with gently shaking. After that, the spinal cord was polymerized and embedded by using ultraviolet on ice. Finally, the polymerized sample was placed in filtered DI water at room temperature for 2 days for subsequent expansion with daily DI water change. The expanded sample was glued on an iron sheet, mounted on the sample holder and imaged using DI water as the imaging buffer.

Imaging and statical analysis

The imaging system has been reported in previous studies^34,41,42. The microscope used a fluorescence macro zoom microscope (Olympus MVX10) with Olympus MVPLAPO 1X when the whole brain was imaged. Images were captured under 6.3X magnification. The Nikon Plan Apo 10X Glyc was performed when we imaged whole spinal cord. In addition, the aperture was changed to Olympus XLPLN 10X SV MP when the tissue expansion samples were imaged. All pictures were aligned semi-automatically using the Amira software. Axon tracing was performed by using the Amira filament tracing package manually. the axons were traced from trunk to the furthest branch terminal. The cell segmentation was performed by using the spot function of Imaris 9.3. The intensity of collateral branches was analyzed using GraphPad Prism 9. All data are presented as mean ± s.e.m. The statistical analysis was performed using unpaired Student’s t-tests.

CMAP

Clearing and Magnification Analysis of Proteome

Primary motor cortex

Secondary motor cortex

Secondary somatosensory cortex

Red nucleus

Gigantocellular reticular nucleus

Vestibular nucleus

Superior colliculus

Med

Medial cerebellar nucleus

IntA/P

Interposed cerebellar nucleus, anterior part/ posterior part

MdV

Medullary reticular nucleus ventral part

Sp5

Spinal trigeminal nucleus.

Contra

Contralateral

Ipsi

Ipsilateral

1st cervical segment

4th cervical segment

5th cervical segment

8th cervical segment

1st thoracic segment

2nd thoracic segment

Acknowledgements

We thank Dr. Xuzhao Li and Xiaoxuan Zhang for their help on manuscript revision and helpful discussion. We thank the Dr. Liang Wang for kindly providing ChAT-eGFP mouse line from Zhejiang University. We thank the advanced biomedical technology core facility, the laboratory animal resource center and high-performance computing center for facility support and technical assistance. L.G. acknowledges support from Westlake University, the Westlake Education Foundation, the National Natural Science Foundation of China (32150015) and the Zhejiang Province Natural Science Foundation (LR20C070002).

Contributions

L.G. conceived the project, designed the experiments, and supervised the research; J.X. and R.F. prepared tissue clearing samples, Y.C. and J.X. performed the tissue expansion samples. J.X. analyzed the data; J.X. wrote the manuscript. All authors read and approved the final manuscript. J.X. and R.F. made an equal contribution to this work. All authors approved the final manuscript.

Competing interests

The authors declare no competing interests.

Data availability

The data is available upon request from the corresponding author.

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No competing interests reported.

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Morphology Analysis of Descending Tracts in the Mouse Spinal Cord Using Tissue Clearing, Tissue Expansion and Tiling Light Sheet Microscopy Techniques

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Abstract

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Introduction

Results

Distribution of descending neurons in the brain

Axon projection pattern of Med neurons in spinal cord

Axon projection pattern of IntA/P neurons in spinal cord

Axon projection pattern of SP5 neurons in spinal cord

Axon projection pattern of MdV neurons in spinal cord

Tectospinal tract in spinal cord

Spatial distribution of MdV neurons in brain

Discussion

Methods and materials

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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Journal Publication

Version 1