Biology subjects

Matsuyama, M.

Publications and source records attributed to Matsuyama, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Characterization of a maintainable human myeloid model of VEXAS syndrome with enhanced TNF-induced cell death and DAMP release

Background: VEXAS syndrome is an adult-onset autoinflammatory disorder caused by somatic UBA1 mutations. UBA1-mutated myeloid cells have been reported to exhibit increased susceptibility to inflammatory cell death; however, the mechanisms underlying this phenotype and the extracellular consequences of enhanced cell death remain incompletely understood. Methods: Disease-associated UBA1 M41 mutations were introduced into U937 cells by CRISPR/Cas9-mediated genome editing. The resulting cells were characterized by genomic, molecular, and functional analyses, with particular focus on UBA1-associated cellular phenotypes, cell death signaling, and extracellular release of damage-associated molecular patterns (DAMPs). Results: Genome editing yielded UBA1-mutated cells that could be maintained under standard culture conditions while recapitulating multiple molecular and cellular features associated with VEXAS syndrome. Long-read sequencing revealed the intended UBA1 M41 mutation on one allele and a CRISPR/Cas9-induced on-target genomic deletion on the other. The mutant cells exhibited reduced UBA1b and increased UBA1c expression, prominent cytoplasmic vacuolization, reduced proliferative capacity, and increased basal cell death. They also showed enhanced susceptibility to TNF-induced cell death under conditions favoring either apoptosis or necroptosis. Mechanistic analyses demonstrated enhanced apoptotic and necroptotic signaling, together with increased basal abundance of RIPK1, RIPK3, and MLKL. Pharmacological inhibition of RIPK1, RIPK3, or MLKL attenuated membrane permeabilization under caspase-inhibited conditions. Enhanced cell death was accompanied by increased extracellular release of ATP, HMGB1, and S100A8/A9. Conclusions: We characterized a maintainable UBA1-mutated human myeloid model that recapitulates multiple molecular and cellular features associated with VEXAS syndrome. Using this model, we showed that UBA1 dysfunction is associated with heightened susceptibility to TNF-dependent apoptotic and necroptotic cell death and increased extracellular release of multiple DAMPs. These findings provide insight into the cellular consequences of UBA1 dysfunction and establish a tractable experimental platform for further mechanistic studies of VEXAS syndrome.

immunology↗

bioRxiv2025

Cellular fitness is determined by ribosomal protein S12-mediated release of a truncated Xrp1

Multicellular tissues require continuous optimization to maintain their structure and function by actively eliminating viable, yet unfit cells via a mechanism known as cell competition. During this process, unfit loser cells commonly upregulate the C/EBP family transcription factor Xrp1, which causes their elimination, indicating that intracellular Xrp1 levels determine cellular fitness. However, despite its central role, the mechanism by which Xrp1 is upregulated in unfit cells remains unknown. Here, we show that Xrp1 is upregulated through a previously unrecognized post-transcriptional regulatory mechanism mediated by the ribosomal protein S12 (RpS12). Surprisingly, Xrp1 mRNA is abundantly expressed even in wild-type cells, yet it is not translated. This is due to an upstream open reading frame (uORF) located in the 5UTR of the initial coding exon of Xrp1 mRNA, which inhibits the translation of the main Xrp1 ORF. Intriguingly, in unfit cells, RpS12 causes splicing-mediated skipping of this initial exon, leading to the use of an alternate start codon that generates a short isoform of Xrp1 protein, causing cell death. Notably, protein structural analysis reveals that RpS12 is highly homologous to that of a spliceosomal component SNU13, suggesting the role of RpS12 in directly regulating alternative splicing of Xrp1 mRNA. Our findings thus provide not only a mechanistic insights into how cellular fitness is determined but also how tissue optimization is achieved.

cell biology↗

bioRxiv2025

Development of a sandwich ELISA for the detection of bovine A1 beta-casein

The genetic variant A2 beta-casein is associated with fewer digestive and absorption issues compared to A1 beta-casein, leading to increased global demand for A2 milk. However, contamination with the A1 variant during collection, transportation, or sterilization of A2 milk poses a risk, necessitating a verification test to ensure A2 milk does not contain A1 beta-casein. We developed an A1-specific monoclonal antibody (mAb) and a general mAb that reacts with both A1 and A2 variants using the iliac lymph node method. A sandwich ELISA was created using the general mAb as the capture antibody and the A1-specific mAb as the detection antibody to identify A1 beta-casein in milk. This ELISA successfully detected A1 beta-casein in raw and pasteurized A2 milk, including ultra-high temperature treated milk. The test identified A1 beta-casein when the A1 spike in A2 milk exceeded 1% in volume, indicating its capability to detect contamination from one A1A1 cow in a herd of one hundred A2A2 cows. The developed A1 beta-casein ELISA is suitable for high-throughput analysis and can be valuable for monitoring A1 beta-casein contamination in commercially produced A2 milk.

zoology↗

bioRxiv2025

Emergence of cell polarity by reciprocal interactions between Wnt and core PCP components

Planar cell polarity (PCP) is established by asymmetric localization of core PCP components in each cell. Since some Wnt proteins can induce PCP in vertebrates, it has been proposed that Wnt concentration gradients provide spatial cues for polarization. Here, however, we present evidence that Wnt11 can regulate PCP in a gradient-independent manner. In the neural plate of Xenopus embryos, endogenous Wnt11 does not form an obvious gradient in the direction of PCP, but is polarized at cell boundaries together with core PCP components. Wnt11 polarization is dependent on core PCP components, while polarization of core PCP components can also be induced by Wnt11, indicating a mutual amplification loop between Wnt11 and core PCP components in PCP formation. Furthermore, Wnt11 and core PCP components compose an intercellular loop to coordinate the direction of polarity. Thus, we propose that local and reciprocal interactions between Wnt11 and core PCP components can generate PCP.

developmental biology↗

bioRxiv2025

Dynamic polarization of heparan sulfate proteoglycans is involved in planar cell polarity and localization of endogenous Wnt11 during vertebrate neural tube formation.

Planar cell polarity (PCP) on a tissue plane is established by asymmetric polarization of core PCP components in individual cells. Recently, we found that Wnt11 organizes PCP in Xenopus embryos by accumulating locally with core PCP components to be polarized, but not by acting as a directional cue forming a global gradient. Since heparan sulfate proteoglycans (HSPGs) associate with Wnt on cell membranes, how HSPGs are involved in Wnt polarization and PCP formation is a central issue. Here, we show that HSPGs are dynamically organized in a polarized manner, as with core PCP components and Wnt11, by associating with them. While HSPGs with HS chains enriched by N-sulfation and N-deacetylation are polarized, depending on Wnt and core PCP components, NDST1, which catalyzes these modifications, is required for Wnt11 polarization and PCP formation. Thus, mutual regulation among HS chains, Wnt11, and PCP components appears to be essential for PCP formation. HighlightsO_LIndst1 is required for Wnt11 localization and proper PCP formation. C_LIO_LINDST1-modified HS chains are polarized in the neural plate during development. C_LIO_LIPolarization of NDST1-modified HS chains depends on PCP formation. C_LIO_LIPositive feedback loop results in polarized distribution of HSPGs, Wnt11, and PCP. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/669988v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1db75adorg.highwire.dtl.DTLVardef@10aec47org.highwire.dtl.DTLVardef@1e50d5org.highwire.dtl.DTLVardef@4cd41c_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2025

Deep Tissue Second-Harmonics of Collagen Fibers in a Transparent Rat Heart from a Myocardial Infarction Model

The combination of tissue-clearing techniques with light-sheet microscopy has enabled detailed visualization of histological changes from the micrometer to millimeter scale, deepening understanding of various disease processes. However, these protocols are not fully optimized for animal species beyond mice or for organs outside the brain. Additionally, the lack of suitable fluorescent probes for target molecules limits their broader application. In this study, we present a protocol for whole-organ clearing of rat hearts in a myocardial infarction model, achieving complete transparency and enabling label-free imaging of collagen fibers in the myocardial wall up to a depth of [~]5 mm using Second Harmonic Generation (SHG) microscopy. For the first time, we successfully compared collagen fiber orientations between infarcted and healthy myocardial regions. Our approach facilitates high-resolution tissue remodeling analysis in cardiovascular research without the need for antibody staining, demonstrating that tissue-clearing techniques are feasible even in animal species with limited available antibodies. Summary statementWe present a novel method for visualizing collagen fibers deep within the heart using a combination of tissue clearing and advanced microscopy, providing valuable data for cardiac research.

biochemistry↗

bioRxiv2024

Novel and Efficient Plasmid Transfection Protocols for Functional Analysis of Genetic Elements in Human Cardiac Fibroblasts

Cardiac fibroblasts, have lower gene transfer efficiency compared to dermal fibroblasts, posing challenges for plasmid-based gene transfer methods. A higher transfer efficiency could enable improved insight into heart pathology and development of novel therapeutic targets. In this study we compared eleven commercially available transfection reagents and eight plasmid purification methods. Finally, we systematically evaluated 150 unique transfection conditions (incubation times, addition of innate immune inhibitors, reagent to plasmid ratios etc) to optimize the methodology. The aim was to develop an optimized plasmid transfection protocol specifically tailored for primary human cardiac fibroblasts with high efficiency and minimal toxicity. Leveraging our newly developed methodology, we could perform comprehensive profiling of nine viral and native enhancer/promoters, revealing regulatory sequences governing classical fibroblast marker (VIM) and resident cardiac fibroblast marker (TCF21) expression. We believe that these findings can help advance many aspects of cardiovascular research. In conclusion, we here report for the first time a plasmid transfection protocol for cardiac fibroblasts with minimal cell toxicity.

molecular biology↗

bioRxiv2023

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing

Mapping the connectivity of diverse neuronal types provides the foundation for understanding the structure and function of neural circuits. High-throughput and low-cost neuroanatomical techniques based on RNA barcode sequencing have the potential to map circuits at cellular resolution and a brain-wide scale, but existing Sindbis virus-based techniques can only map long-range projections using anterograde tracing approaches. Rabies virus can complement anterograde tracing approaches by enabling either retrograde labeling of projection neurons or monosynaptic tracing of direct inputs to genetically targeted postsynaptic neurons. However, barcoded rabies virus has so far been only used to map non-neuronal cellular interactions in vivo and synaptic connectivity of cultured neurons. Here we combine barcoded rabies virus with single-cell and in situ sequencing to perform retrograde labeling and transsynaptic labeling in the mouse brain. We sequenced 96 retrogradely labeled cells and 295 transsynaptically labeled cells using single-cell RNA-seq, and 4,130 retrogradely labeled cells and 2,914 transsynaptically labeled cells in situ. We found that the transcriptomic identities of rabies virus-infected cells can be robustly identified using both single-cell RNA-seq and in situ sequencing. By associating gene expression with connectivity inferred from barcode sequencing, we distinguished long-range projecting cortical cell types from multiple cortical areas and identified cell types with converging or diverging synaptic connectivity. Combining in situ sequencing with barcoded rabies virus complements existing sequencing-based neuroanatomical techniques and provides a potential path for mapping synaptic connectivity of neuronal types at scale.

neuroscience↗

bioRxiv2022

Development of novel cytoprotective small compounds inhibiting mitochondria-dependent apoptosis

We identified cyto-protective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was developed, which is orally bioactive and penetrates the blood-brain/retina barriers. M109S protected retinal cells in the mouse models of Stargardt disease and macular degeneration. M109S directly interacted with Bax and inhibited the conformational change and mitochondrial translocation of Bax. M109S inhibited ABT-737-induced apoptosis both in Bax-only and Bak-only MEFs. M109S also inhibited apoptosis induced by staurosporine (mouse embryonic fibroblasts), etoposide (Neuro2a cells), and obatoclax (ARPE19 cells). M109S is a novel small molecule protecting cells from mitochondria-dependent apoptosis both in vitro and in vivo. M109S has the potential to become a new research tool for studying cell death mechanisms and to develop therapeutics targeting mitochondria-dependent cell death pathway. (128words)

cell biology↗

bioRxiv2022

Functionally conserved Pkd2, mutated in autosomal dominant polycystic kidney disease, localizes to the endoplasmic reticulum and regulates cytoplasmic calcium homeostasis in fission yeast

Mutations in PKD1 or PKD2 genes lead to autosomal dominant polycystic kidney disease (ADPKD) that is the most frequent family inherited renal disorder. These genes encode polycystin-1/PC-1 and polycycstin-2/PC-2, respectively. Although the genetic basis of ADPKD is well established, the crucial functions of polycystins underlying onset and development of cyst formation remain elusive. Fission yeast Schizosaccharomyces pombe has a single polycystin homolog, Pkd2, which is essential for cell growth. In this study, the truncation analyses of Pkd2 reveal that Pkd2 localizes to not only the plasma membrane but also the endoplasmic reticulum (ER) and regulates cytoplasmic calcium signaling in fission yeast. Internal transmembrane domains within Pkd2 are sufficient for these processes. Surprisingly, more than half of Pkd2 is not required for cell viability. Cytoplasmic calcium levels are mainly regulated through C-terminus of Pkd2. Importantly, human Pkd2 also localizes to the ER and furthermore, fully complements the loss of fission yeast Pkd2. As the functions of polycystin-2 are conserved, fission yeast provides a suitable model to study the mechanism of ADPKD as well as polycystins.

cell biology↗

bioRxiv2022

Single-deletion-mutant, third-generation rabies viral vectors allow nontoxic retrograde targeting of projection neurons with greatly increased efficiency

Rabies viral vectors have become important components of the systems neuroscience toolkit, allowing both direct retrograde targeting of projection neurons and monosynaptic tracing of inputs to defined postsynaptic populations, but the rapid cytotoxicity of first-generation ({Delta}G) vectors limits their use to short-term experiments. We recently introduced second-generation, double-deletion-mutant ({Delta}GL) rabies viral vectors, showing that they efficiently retrogradely infect projection neurons and express recombinases effectively but with little to no detectable toxicity; more recently, we have shown that {Delta}GL viruses can be used for monosynaptic tracing with far lower cytotoxicity than the first-generation system. Here we introduce third-generation ({Delta}L) rabies viral vectors, which, like first-generation vectors, have only a single gene deleted from their genomes (in this case the viral polymerase gene L) but which appear to be as nontoxic as second-generation ones, based on in vivo longitudinal structural and functional two-photon imaging and ex vivo electrophysiology. Although third-generation vectors are therefore phenotypically very similar to second-generation ones, we show that they have the major advantage of growing to much higher titers, and this key difference results in significantly increased numbers of retrogradely labeled neurons in vivo. These {Delta}L rabies viral vectors therefore constitute a new state of the art for minimally perturbative, pathway-specific expression of recombinases and transactivators in mammalian neurons selected on the basis of their axonal projections. Because replication of deletion-mutant rabies viruses within complementing cells is precisely the process that underlies monosynaptic tracing, the higher replication efficiency of this new class of rabies viral vectors furthermore suggests the potential to provide the foundation of an improved nontoxic monosynaptic tracing system.

neuroscience↗

bioRxiv2021

Long-term labeling and imaging of synaptically-connected neuronal networks in vivo using nontoxic, double-deletion-mutant rabies viruses

Monosynaptic tracing is a widely-used technique for mapping neural circuitry, but its cytotoxicity has confined it primarily to anatomical applications. Here we present a second-generation system for labeling direct inputs to targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Spread of the viruses requires expression of both deleted viral genes in trans in postsynaptic source cells; suppressing this expression with doxycycline following an initial period of viral replication reduces toxicity to postsynaptic cells. Longitudinal two-photon imaging in vivo indicated that over 90% of both presynaptic and source cells survived for the full twelve-week course of imaging. Ex vivo whole-cell recordings at 5 weeks postinfection showed that the second-generation system perturbs input and source cells much less than does the first-generation system. Finally, two-photon calcium imaging of labeled networks of visual cortex neurons showed that their visual response properties appeared normal for 10 weeks, the longest we followed them.

neuroscience↗

bioRxiv2021

Trim41 is essential for preventing X chromosome chaotic synapsis in male mice

Meiosis is a hallmark event in germ cell development that accompanies sequential chromosome events executed by numerous molecules. Therefore, characterization of these factors is one of the best strategies to clarify the mechanism of meiosis. Here, we report tripartite motif-containing 41 (TRIM41), a ubiquitin ligase E3, as an essential factor for proper meiotic progression and fertility in male mice. Trim41 KO spermatocytes exhibited synaptonemal complex protein 3 (SYCP3) overloading, especially on the X chromosome, showing extensive self-synapsis of X chromosome and non-homologous synapsis between the X chromosome and autosomes. Furthermore, the mutant mice lacking the RING domain of TRIM41, required for the ubiquitin ligase E3 activity, phenocopied Trim41 KO mice. We then examined the behavior of mutant TRIM41 ({Delta}RING-TRIM41) and found that {Delta}RING-TRIM41 accumulated on the chromosome axes with overloaded SYCP3. This result showed that TRIM41 exerts the function on the chromosome axes. In summary, our study revealed that Trim41 is essential for preventing SYCP3 overloading and chaotic synapsis of the X chromosome, suggesting a TRIM41-mediated mechanism for regulating unsyapsed axes during male meiotic progression. Summary statementTrim41-disruption caused abnormal synapsis configuration of the X chromosome and complete infertility in male mice. Thus, TRIM41 prevents the sex chromosome from chaotic synapsis.

developmental biology↗

bioRxiv2021

LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction of structures including dynein arms, radial spokes (RSs), microtubules, and the dynein regulatory complex (DRC). RSs facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 different proteins (RSP1-23), with the roles of RSP13, RSP15, RSP18, RSP19, and RSP21 remained poorly understood. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. Through immunoelectron microscopy, we demonstrate that LRRC23 localizes to the RS complex within murine sperm flagella. We further found that LRRC23 was able to interact with RSHP9 and RSPH3A/B. The knockout of Lrrc23 resulted in RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was unaffected by the loss of this protein. Spermatozoa lacking LRRC23 were unable to efficiently pass through the uterotubal junction and exhibited defective zona penetration. Together these data indicate that LRRC23 is a key regulator underpinning the integrity of RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia. Author summary

cell biology↗

bioRxiv2021

Creation of X-linked Alport Syndrome Rat Model with Col4a5 Deficiency

Alport syndrome is an inherited chronic human kidney disease, characterized by glomerular basement membrane abnormalities. This disease is caused by mutations in COL4A3, COL4A4, or COL4A5 gene. The knockout mice for Col43, Col44, and Col45 are developed and well characterized for the study of Alport syndrome. However, disease progression and effects of pharmacological therapy depend on the genetic variability. This model is reliable only to mice. Therefore in this study, we created a novel Alport syndrome rat model utilizing rGONAD technology. Col45 deficient rats showed hematuria, proteinuria, high levels of BUN, Cre, and then died at 18 to 28 weeks of age (Hemizygous mutant males). Histological and ultrastructural analyses displayed the abnormalities including parietal cell hyperplasia, mesangial sclerosis, and interstitial fibrosis. Then, we demonstrated that 3/4/5 (IV) and 5/5/6 (IV) chains of type IV collagen disrupted in the Col45 deficient rats. Moreover, immunofluorescence analyses revealed that some glomeruli of Col45 mutant rats were found to be disrupted from postnatal day 0. Thus, Col45 mutant rat is a reliable candidate for Alport syndrome model for underlying the mechanism of renal diseases and further identifying potential therapeutic targets for human renal diseases.

bioengineering↗

bioRxiv2021

KCTD19 associates with ZFP541 and HDAC1 and is required for meiotic exit in male mice

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. Immunoprecipitation-mass spectrometry identified zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1) as binding partners of KCTD19, indicating that KCTD19 is involved in chromatin modification. Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 were essential for meiotic exit in male mice. Author summaryMeiosis is a fundamental process that consisting of one round of genomic DNA replication and two rounds of chromosome segregation producing four haploid cells. To properly distribute their genetic material, cells need to undergo complex chromosome events such as a physical linkage of homologous chromosomes (termed synapsis) and meiotic recombination. The molecules involved in these events have not been fully characterized yet, especially in mammals. Using a CRISPR/Cas9-screening system, we identified the potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis in male mice. Further, we identified zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1) as binding partners of KCTD19. By observing meiosis of Zfp541 knockout germ cells, we found that Zfp541 was also essential for meiotic completion. These results show that the KCTD19/ZFP541 complex plays a critical role and is indispensable for male meiosis and fertility.

developmental biology↗

bioRxiv2019

The p21 dependent G2 arrest of the cell cycle in epithelial tubular cells links to the early stage of renal fibrosis

Renal fibrosis is accompanied with the progression of chronic kidney disease (CKD). Despite a number of past and ongoing studies, our understanding of the underlying mechanisms remains elusive. Here we explored the progression of renal fibrosis by using a mouse model, unilateral ureter obstruction (UUO). We found that in the initial stage of the progression where extracellular matrix did not deposit yet, the proximal tubular cells arrested at the G2 of the cell cycle. This G2 arrest was induced prior to activation of both DNA damage checkpoint and Wnt/{beta}-Catenin pathway. Further analyses in vivo and in vitro indicated the cyclin dependent kinase inhibitor p21 is involved in the G2 arrest after the damage. The newly produced monoclonal antibody against p21 revealed that the p21 levels were sharply upregulated in response to the damage during the initial stage, but dropped down toward the later stage. To examine the function of p21 in the progression of renal fibrosis, we constructed the novel p21 deficient mice by i-GONAD. Compared with wild-type mice, p21 deficient mice showed the exacerbation of the fibrosis. Thus we propose that during the initial stage of the fibrosis following the renal damage, tubular cells arrest in the G2 phase depending on p21, thereby safeguarding the kidney functions.

cell biology↗

bioRxiv2019

\"Self-inactivating\" rabies viruses are just first-generation, ΔG rabies viruses

Monosynaptic tracing using rabies virus is an important technique in neuroscience, allowing brain- wide labeling of neurons directly presynaptic to a targeted neuronal population. A 2017 article reported development of a noncytotoxic version - a major advance - based on attenuating the rabies virus by addition of a destabilization domain to the C-terminus of a viral protein. However, this modification did not appear to hinder the ability of the virus to spread between neurons. We analyzed two viruses provided by the authors and show here that both were mutants that had lost the intended modification, explaining the papers paradoxical results. We then made a virus that actually did have the intended modification in at least the majority of virions and found that it did not spread efficiently under the conditions described in the original paper, namely, without an exogenous protease being expressed in order to remove the destabilization domain. We found that it did spread when the protease was supplied, although this also appeared to result in the deaths of most source cells by three weeks postinjection. We conclude that the new approach is not robust but that it could become a viable technique given further optimization and validation. SIGNIFICANCE STATEMENTRabies virus, which spreads between synaptically-connected neurons, has been one of the primary tools used by neuroscientists to reveal the organization of the brain. A new modification to rabies virus was recently reported to allow the mapping of connected neurons without adverse effects on the cells health, unlike earlier versions. Here we show that the conclusions of that study were probably incorrect and based on having used viruses that had lost the intended modification because of mutations. We also show that a rabies virus that does retain the intended modification does not spread efficiently between neurons under the conditions reported previously; however, it does spread between neurons under different conditions, suggesting that the approach may be successful if refined further.

neuroscience↗