No abstract

Superconducting nanostrip photon detectors have been used as single-photon detectors, which can discriminate only photons’ presence or absence. It has recently been found that they can discriminate the number of photons by analyzing the output signal waveform, and they are expected to be used in various fields, especially in optical-quantum-information processing. Here, we improve the photon-number-resolving performance for light with a high-average photon number by pattern matching of the output signal waveform. Furthermore, we estimate the positive-operator-valued measure of the detector by a quantum detector tomography. The result shows that the device has photon-number-resolving performance up to five photons without any multiplexing or arraying, indicating that it is useful as a photon-number-resolving detector.

SummaryThe highly specific and complex connectivity between neurons is the hallmark of nervous systems, but techniques for identifying, imaging, and manipulating synaptically-connected networks of neurons are limited. Monosynaptic tracing, or the gated replication and spread of a deletion-mutant rabies virus to label neurons directly connected to a targeted population of starting neurons1, is the most widely-used technique for mapping neural circuitry, but the rapid cytotoxicity of first-generation rabies viral vectors has restricted its use almost entirely to anatomical applications. We recently introduced double-deletion-mutant second-generation rabies viral vectors, showing that they have little or no detectable toxicity and are efficient means of retrogradely targeting neurons projecting to an injection site2, but they have not previously been shown to be capable of gated replication in vivo, the basis of monosynaptic tracing. Here we present a complete second-generation system for labeling direct inputs to genetically-targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Spread of the viruses requires complementation of both of the deleted viral genes in trans in the starting postsynaptic cells; suppressing the expression of these viral genes following an initial period of viral replication, using the Tet-Off system, reduces toxicity to the starting cells without decreasing the efficiency of viral spread. Using longitudinal two- photon imaging of live monosynaptic tracing in visual cortex, we found that 94.4% of all labeled cells, and an estimated 92.3% of starting cells, survived for the full twelve-week course of imaging. Two-photon imaging of calcium responses in labeled networks of neurons in vivo over ten weeks showed that labeled neurons’ visual response properties remained stable for as long as we followed them. This nontoxic labeling of inputs to genetically-targeted neurons in vivo is a long-held goal in neuroscience, with transformative applications including nonperturbative transcriptomic and epigenomic profiling, long-term functional imaging and behavioral studies, and optogenetic and chemogenetic manipulation of synaptically-connected neuronal networks over the lifetimes of experimental animals.

10A recent article in Cell reported a new form of modified rabies virus that was apparently capable 11 of labeling neurons "without adverse effects on neuronal physiology and circuit function". These 12 "self-inactivating" rabies ("SiR") viruses differed from the widely-used first-generation deletion-13 mutant (∆G) rabies viruses only by the addition of a destabilization domain to the viral 14 nucleoprotein. However, we observed that the transsynaptic tracing results from that article were 15 inconsistent with the logic described in it, and we hypothesized that the viruses used were actually 16 mutants that had lost the intended modification to the nucleoprotein. We obtained samples of two 17 SiR viruses from the authors and show here that, in both "SiR-CRE" and "SiR-FLPo", the great 18 majority of viral particles were indeed mutants that had lost the intended modification and were 19 therefore just first-generation, ∆G rabies viruses. We also found that SiR-CRE killed 70% of 20 infected neurons in vivo within two weeks. We have shown elsewhere that a ∆G rabies virus 21 encoding Cre can leave a large percentage of labeled neurons alive; we presume that Ciabatti et 22 al. found such remaining neurons at long survival times and mistakenly concluded that they had 23 developed a nontoxic version of rabies virus. Here we have analyzed only the two samples that 24 were sent to MIT by Ciabatti et al., and these may not be from the same batches that were used 25 for their paper. However, 1) both of the two viruses that we analyzed had independently lost the 26 intended modification, 2) the mutations in the two samples were genetically quite distinct from 27 each other yet in both cases caused the same result: total or near-total loss of the C-terminal 28 modification, and 3) the mutations that we found in these two virus samples perfectly explain the 29 otherwise-paradoxical transsynaptic tracing results from Ciabatti et al.'s paper. We suggest that 30 the SiR strategy, or any other such attempt to attenuate a virus by addition rather than deletion, 31 is an inherently unstable approach that can easily be evaded by mutation, as it was in this case. 33 39Its core principles are 1) the selective infection of the targeted neuron group with a recombinant 40 rabies virus with one deleted gene (which in all work published to date is the "G" gene encoding 41 its envelope glycoprotein), and 2) the in vivo complementation of the deletion by expression of 42 the deleted gene in trans in the targeted starting neurons. With all its gene products present in 43 the starting cells, the virus can fully replicate within them and spreads, as wild-type rabies virus 44 does, to the cells directly presynaptic to the initially infected neurons. Unlike wild-type rabies virus, 45 however, once inside the presynaptic cells, the deletion-mutant ("∆G", denoting the deletion of G) 46 is unable to produce its glycoprotein and is therefore unable to spread beyond these secondarily 47 infected cells, resulting in labeling of just the neurons in the in...

Pathway-specific gene delivery is requisite for understanding complex neuronal systems in which neurons that project to different target regions are locally intermingled. However, conventional genetic tools cannot achieve simultaneous, independent gene delivery into multiple target cells with high efficiency and low cross-reactivity. In this study, we systematically screened all receptor-envelope pairs resulting from the combination of four avian sarcoma leukosis virus (ASLV) envelopes (EnvA, EnvB, EnvC, and EnvE) and five engineered avianderived receptors (TVA950, TVB S3 , TVC, TVB T , and DR-46TVB) in vitro. Four of the 20 pairs exhibited both high infection rates (TVA-EnvA, 99.6%; TVB S3 -EnvB, 97.7%; TVC-EnvC, 98.2%; and DR-46TVB-EnvE, 98.8%) and low cross-reactivity (<2.5%). Next, we tested these four receptor-envelope pairs in vivo in a pathway-specific gene-transfer method. Neurons projecting into a limited somatosensory area were labeled with each receptor by retrograde gene transfer. Three of the four pairs exhibited selective transduction into thalamocortical neurons expressing the paired receptor (>98%), with no observed crossreaction. Finally, by expressing three receptor types in a single animal, we achieved pathway-specific, differential fluorescent labeling of three thalamic neuronal populations, each projecting into different somatosensory areas. Thus, we identified three orthogonal pairs from the list of ASLV subgroups and established a new vector system that provides a simultaneous, independent, and highly specific genetic tool for transferring genes into multiple target cells in vivo. Our approach is broadly applicable to pathway-specific labeling and functional analysis of diverse neuronal systems. avian sarcoma leukosis virus | pseudotyped lentiviral vector | pathway-specific gene transfer

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 the development of a noncytotoxic version—a major advance—based on attenuating the rabies virus by the 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 paper's 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 3 wk postinjection. We conclude that the new approach is not robust but that it could become a viable technique given further optimization and validation.

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 achieve circuit mapping 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 RNAseq, and 4,130 retrogradely labeled cells and 2,914 transsynaptically labeled cells in situ. We determined the transcriptomic identities of rabies virus-infected cells robustly using both single-cell RNA-seq and in situ sequencing. We then 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 thus complements existing sequencing-based neuroanatomical techniques and provides a potential path for mapping synaptic connectivity of neuronal types at scale.

During sensory deprivation, the barrel cortex undergoes expansion of a functional column representing spared inputs (spared column), into the neighboring deprived columns (representing deprived inputs) which are in turn shrunk. As a result, the neurons in a deprived column simultaneously increase and decrease their responses to spared and deprived inputs, respectively. Previous studies revealed that dendritic spines are remodeled during this barrel map plasticity. Because cofilin1, a predominant regulator of actin filament turnover, governs both the expansion and shrinkage of the dendritic spine structure in vitro, it hypothetically regulates both responses in barrel map plasticity. However, this hypothesis remains untested. Using lentiviral vectors, we knocked down cofilin1 locally within layer 2/3 neurons in a deprived column. Cofilin1-knocked-down neurons were optogenetically labeled using channelrhodopsin-2, and electrophysiological recordings were targeted to these knocked-down neurons. We showed that cofilin1 knockdown impaired response increases to spared inputs but preserved response decreases to deprived inputs, indicating that cofilin1 dependency is dissociated in these two types of barrel map plasticity. To explore the structural basis of this dissociation, we then analyzed spine densities on deprived column dendritic branches, which were supposed to receive dense horizontal transcolumnar projections from the spared column. We found that spine number increased in a cofilin1-dependent manner selectively in the distal part of the supragranular layer, where most of the transcolumnar projections existed. Our findings suggest that cofilin1-mediated actin dynamics regulate functional map plasticity in an input-specific manner through the dendritic spine remodeling that occurs in the horizontal transcolumnar circuits. These new mechanistic insights into transcolumnar plasticity in adult rats may have a general significance for understanding reorganization of neocortical circuits that have more sophisticated columnar organization than the rodent neocortex, such as the primate neocortex.