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 4130 retrogradely labeled cells and 2914 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.

In the brain, messages are relayed from one cell to the next through intricate networks of axons and dendrites that physically interact at junctions known as synapses. Mapping out this synaptic connectivity ­ that is, exactly which neurons are connected via synapses ­ remains a major challenge. Monosynaptic tracing is a powerful approach that allows neuroscientists to explore neural networks by harnessing viruses which spread between neurons via synapses, in particular the rabies virus. This pathogen travels exclusively from 'postsynaptic' to 'presynaptic' neurons ­ from the cell that receives a message at a synapse, back to the one that sends it. A modified variant of the rabies virus can therefore be used to reveal the presynaptic cells connecting to a population of neurons in which it has been originally introduced. However, this method does not allow scientists to identify the exact postsynaptic neuron that each presynaptic cell is connected to. One way to bypass this issue is to combine monosynaptic tracing with RNA barcoding to create distinct versions of the modified rabies virus, which are then introduced into separate populations of neurons. Tracking the spread of each version allows neuroscientists to spot exactly which presynaptic cells signal to each postsynaptic neuron. So far, this approach has been used to examine synaptic connectivity in neurons grown in the laboratory, but it remains difficult to apply it to neurons in the brain. In response, Zhang, Jin et al. aimed to demonstrate how monosynaptic tracing that relies on barcoded rabies viruses could be used to dissect neural networks in the mouse brain. First, they confirmed that it was possible to accurately detect which version of the virus had spread to presynaptic neurons using both in situ and single-cell RNA sequencing. Next, they described how this information could be analysed to build models of potential neural networks, and what type of additional experiments are required for this work. Finally, they used the approach to identify neurons that tend to connect to the same postsynaptic cells and then investigated what these have in common, showing how the technique enables a finer understanding of neural circuits. Overall, the work by Zhang, Jin et al. provides a comprehensive review of the requirements and limitations associated with monosynaptic tracing experiments based on barcoded rabies viruses, as well as how the approach could be optimized in the future. This information will be of broad interest to scientists interested in mapping neural networks in the brain.

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

Rabies-virus-based 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. Viral spread 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 12-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 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.

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 (ΔG) vectors limits their use to short-term experiments. We recently introduced second-generation, double-deletion-mutant (ΔGL) 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 ΔGL viruses can be used for monosynaptic tracing with far lower cytotoxicity than the first-generation system. Here, we introduce third-generation (ΔL) rabies viral vectors, which appear to be as nontoxic as second-generation ones but have the major advantage of growing to much higher titers, resulting in significantly increased numbers of retrogradely labeled neurons in vivo.

"Self-inactivating" rabies viruses are susceptible to loss of their intended attenuating modification.

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.

Compensatory remodeling of a septo-hippocampal GABAergic network in the triple transgenic Alzheimer's mouse model.

BACKGROUND: Alzheimer's disease (AD) is characterized by a progressive loss of memory that cannot be efficiently managed by currently available AD therapeutics. So far, most treatments for AD that have the potential to improve memory target neural circuits to protect their integrity. However, the vulnerable neural circuits and their dynamic remodeling during AD progression remain largely undefined. METHODS: Circuit-based approaches, including anterograde and retrograde tracing, slice electrophysiology, and fiber photometry, were used to investigate the dynamic structural and functional remodeling of a GABAergic circuit projected from the medial septum (MS) to the dentate gyrus (DG) in 3xTg-AD mice during AD progression. RESULTS: We identified a long-distance GABAergic circuit that couples highly connected MS and DG GABAergic neurons during spatial memory encoding. Furthermore, we found hyperactivity of DG interneurons during early AD, which persisted into late AD stages. Interestingly, MS GABAergic projections developed a series of adaptive strategies to combat DG interneuron hyperactivity. During early-stage AD, MS-DG GABAergic projections exhibit increased inhibitory synaptic strength onto DG interneurons to inhibit their activities. During late-stage AD, MS-DG GABAergic projections form higher anatomical connectivity with DG interneurons and exhibit aberrant outgrowth to increase the inhibition onto DG interneurons. CONCLUSION: We report the structural and functional remodeling of the MS-DG GABAergic circuit during disease progression in 3xTg-AD mice. Dynamic MS-DG GABAergic circuit remodeling represents a compensatory mechanism to combat DG interneuron hyperactivity induced by reduced GABA transmission.

Recombinant Protein Production Using the Baculovirus Expression Vector System (BEVS).

The baculovirus expression vector system (BEVS) is one of the most popular eukaryotic systems for recombinant protein production. The focus of our protein production platform is the provision of recombinant proteins for research use, where generally only small quantities are required, in the range of tens of micrograms to a few hundred milligrams. Here, we present methods that reflect our standard operating procedures and setup to be able to frequently, and often repeatedly, produce many different types of proteins.

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.

Cingulate-motor circuits update rule representations for sequential choice decisions.

Anterior cingulate cortex mediates the flexible updating of an animal's choice responses upon rule changes in the environment. However, how anterior cingulate cortex entrains motor cortex to reorganize rule representations and generate required motor outputs remains unclear. Here, we demonstrate that chemogenetic silencing of the terminal projections of cingulate cortical neurons in secondary motor cortex in the rat disrupts choice performance in trials immediately following rule switches, suggesting that these inputs are necessary to update rule representations for choice decisions stored in the motor cortex. Indeed, the silencing of cingulate cortex decreases rule selectivity of secondary motor cortical neurons. Furthermore, optogenetic silencing of cingulate cortical neurons that is temporally targeted to error trials immediately after rule switches exacerbates errors in the following trials. These results suggest that cingulate cortex monitors behavioral errors and updates rule representations in motor cortex, revealing a critical role for cingulate-motor circuits in adaptive choice behaviors.

Targeting thalamic circuits rescues motor and mood deficits in PD mice.

Although bradykinesia, tremor and rigidity are the hallmark motor defects in patients with Parkinson's disease (PD), patients also experience motor learning impairments and non-motor symptoms such as depression1. The neural circuit basis for these different symptoms of PD are not well understood. Although current treatments are effective for locomotion deficits in PD2,3, therapeutic strategies targeting motor learning deficits and non-motor symptoms are lacking4-6. Here we found that distinct parafascicular (PF) thalamic subpopulations project to caudate putamen (CPu), subthalamic nucleus (STN) and nucleus accumbens (NAc). Whereas PF→CPu and PF→STN circuits are critical for locomotion and motor learning, respectively, inhibition of the PF→NAc circuit induced a depression-like state. Whereas chemogenetically manipulating CPu-projecting PF neurons led to a long-term restoration of locomotion, optogenetic long-term potentiation (LTP) at PF→STN synapses restored motor learning behaviour in an acute mouse model of PD. Furthermore, activation of NAc-projecting PF neurons rescued depression-like phenotypes. Further, we identified nicotinic acetylcholine receptors capable of modulating PF circuits to rescue different PD phenotypes. Thus, targeting PF thalamic circuits may be an effective strategy for treating motor and non-motor deficits in PD.

Brain-wide mapping of inputs to the mouse lateral posterior (LP/Pulvinar) thalamus-anterior cingulate cortex network.

The rodent homolog of the primate pulvinar, the lateral posterior (LP) thalamus, is extensively interconnected with multiple cortical areas. While these cortical interactions can span the entire LP, subdivisions of the LP are characterized by differential connections with specific cortical regions. In particular, the medial LP has reciprocal connections with frontoparietal cortical areas, including the anterior cingulate cortex (ACC). The ACC plays an integral role in top-down sensory processing and attentional regulation, likely exerting some of these functions via the LP. However, little is known about how ACC and LP interact, and about the information potentially integrated in this reciprocal network. Here, we address this gap by employing a projection-specific monosynaptic rabies tracing strategy to delineate brain-wide inputs to bottom-up LP→ACC and top-down ACC→LP neurons. We find that LP→ACC neurons receive inputs from widespread cortical regions, including primary and higher order sensory and motor cortical areas. LP→ACC neurons also receive extensive subcortical inputs, particularly from the intermediate and deep layers of the superior colliculus (SC). Sensory inputs to ACC→LP neurons largely arise from visual cortical areas. In addition, ACC→LP neurons integrate cross-hemispheric prefrontal cortex inputs as well as inputs from higher order medial cortex. Our brain-wide anatomical mapping of inputs to the reciprocal LP-ACC pathways provides a roadmap for understanding how LP and ACC communicate different sources of information to mediate attentional control and visuomotor functions.

Effects of a Resident's Reputation on Laparoscopic Skills Assessment.

OBJECTIVE: To quantify the effect of a resident's reputation on the assessment of their laparoscopic skills. METHODS: Faculty gynecologists were randomized to receive one of three hypothetical resident scenarios: a resident with high, average, or low surgical skills. All participants were then asked to view the same video of a resident performing a laparoscopic salpingo-oophorectomy that differed only by the resident description and provide an assessment using a modified OSATS (Objective Structured Assessment of Technical Skills) and a global assessment scale. RESULTS: From September 6, 2020, to October 20, 2020, a total of 43 faculty gynecologic surgeons were recruited to complete the study. Assessment scores on the modified OSATS (out of 20) and global assessment (out of 5) differed significantly according to resident description, where the high-performing resident scored highest (median scores of 15 and 4, respectively), followed by the average-performing resident (13 and 3), and finally, the low-performing resident (11 and 3) (P=.008 and .043, respectively). CONCLUSION: Faculty assessment of residents in gynecologic surgery is influenced by the assessor's knowledge of the resident's past performance. This knowledge introduces bias that artificially increases scores given to those residents with favorable reputations and decreases scores given to those with reputed surgical skill deficits. These data quantify the effect of such bias in the assessment of residents in the workplace and serve as an impetus to explore systems-level interventions to mitigate bias.

Anterior thalamic dysfunction underlies cognitive deficits in a subset of neuropsychiatric disease models.

Neuropsychiatric disorders are often accompanied by cognitive impairments/intellectual disability (ID). It is not clear whether there are converging mechanisms underlying these debilitating impairments. We found that many autism and schizophrenia risk genes are expressed in the anterodorsal subdivision (AD) of anterior thalamic nuclei, which has reciprocal connectivity with learning and memory structures. CRISPR-Cas9 knockdown of multiple risk genes selectively in AD thalamus led to memory deficits. While the AD is necessary for contextual memory encoding, the neighboring anteroventral subdivision (AV) regulates memory specificity. These distinct functions of AD and AV are mediated through their projections to retrosplenial cortex, using differential mechanisms. Furthermore, knockdown of autism and schizophrenia risk genes PTCHD1, YWHAG, or HERC1 from AD led to neuronal hyperexcitability, and normalization of hyperexcitability rescued memory deficits in these models. This study identifies converging cellular to circuit mechanisms underlying cognitive deficits in a subset of neuropsychiatric disease models.

An amygdala circuit that suppresses social engagement.

Innate social behaviours, such as mating and fighting, are fundamental to animal reproduction and survival1. However, social engagements can also put an individual at risk2. Little is known about the neural mechanisms that enable appropriate risk assessment and the suppression of hazardous social interactions. Here we identify the posteromedial nucleus of the cortical amygdala (COApm) as a locus required for the suppression of male mating when a female mouse is unhealthy. Using anatomical tracing, functional imaging and circuit-level epistatic analyses, we show that suppression of mating with an unhealthy female is mediated by the COApm projections onto the glutamatergic population of the medial amygdalar nucleus (MEA). We further show that the role of the COApm-to-MEA connection in regulating male mating behaviour relies on the neuromodulator thyrotropin-releasing hormone (TRH). TRH is expressed in the COApm, whereas the TRH receptor (TRHR) is found in the postsynaptic MEA glutamatergic neurons. Manipulating neural activity of TRH-expressing neurons in the COApm modulated male mating behaviour. In the MEA, activation of the TRHR pathway by ligand infusion inhibited mating even towards healthy female mice, whereas genetic ablation of TRHR facilitated mating with unhealthy individuals. In summary, we reveal a neural pathway that relies on the neuromodulator TRH to modulate social interactions according to the health status of the reciprocating individual. Individuals must balance the cost of social interactions relative to the benefit, as deficits in the ability to select healthy mates may lead to the spread of disease.

Phenotypic characterization of frontal cortex microglia in a rat model of post-traumatic stress disorder.

INTRODUCTION: Post-traumatic stress disorder (PTSD) is an anxiety disorder induced by psychologically traumatic events. Using a rat model, this study aimed to determine whether psychological trauma alters relative expression between pro-inflammatory and anti-inflammatory markers in microglia. To meet this goal, expression of genes encoding i-NOS, arginase, TNF-α, interleukin-10, CD74, and Mannose Receptor C was analyzed on multiple days following trauma exposure. METHODS: Single-prolonged stress (SPS) was used to model PTSD in male Sprague-Dawley rats. Twenty-four rats (12 Controls and 12 SPS-exposed) were sacrificed on Days 1, 3, and 7 post-SPS. Twenty-four (12 Controls and 12 SPS-exposed) additional rats were exposed to classical fear conditioning on Day 7, and fear extinction on Days 8, 9, 10, 15, 16, and 17. Freezing behavior was measured to assess fear resolution. Microglial isolates were collected from the frontal cortex, and RNA was extracted. Changes in relative expression of target genes were quantified via RT-PCR. RESULTS: SPS rats showed significant decreases in IL-10 and TNF-α expression and increases in the i-NOS:Arginase and TNF-α:IL-10 ratios compared to Controls on Day 1, but not on Day 3 or Day 7 for any of the dependent variables. Day 17 SPS rats showed a significant decrease in IL-10 expression and an increase in the TNF-α:IL-10 ratio, further characterized by a significant inverse relationship between IL-10 expression and fear persistence. CONCLUSION: Psychological trauma impacts the immunological phenotype of microglia of the frontal cortex. Consequently, future studies should further evaluate the mechanistic role of microglia in PTSD pathology.

Predicting military working dog core temperature during exertional heat strain: Validation of a Canine Thermal Model.

Military working dogs (MWDs) operate under a wide range of conditions, including hot environments. Predicting how long a MWD can safely work without overheating is important for both health and performance. A Canine Thermal Model (CTM) was developed to predict core temperature (Tc) of MWDs. The CTM calculates heat storage from the balance of heat production from metabolism and heat exchange with the environment. Inputs to the CTM are: meteorological conditions (ambient temperature, relative humidity, solar radiation and wind speed), physical characteristics of the dog (mass, length), and metabolic activity (MET level, estimated from accelerometer data). The CTM was validated against Tc measured in 23 MWDs during training sessions (11.6 ± 5.0 min (mean ± standard deviation), range 4-26 min) in October (24 °C, 52% RH), March (14 °C, 74% RH), or August (28 °C, 64% RH), and 24 kennel MWDs during a standard exercise walk (11.4 ± 3.3 min, range 5.6-18 min) in July (26 °C, 77% RH). The CTM was considered acceptable if predicted Tc was within ±0.5 °C of measured Tc at the end of exercise. Compared to Tc at the end of training sessions (39.8 ± 0.6 °C, range 38.4-41.1 °C) and exercise walks (40.0 ± 0.7 °C, range 38.9-41.4 °C), the CTM-predicted Tc was within ±0.5 °C for 71 of 84 cases (85%) and 19 of 24 cases (79%), respectively. The mean difference between CTM-predicted and measured final Tc during training was -0.04 ± 0.43 °C, with 80 of 84 cases (95%) within the range of ±2 SD (Bland Altman comparison). During exercise walks the mean difference was -0.15 °C ± 0.57, with 23 of 24 cases (96%) within ±2 SD. These results support the use of the CTM to predict Tc of MWDs for the types of physical activities described above.

Supramammillary nucleus synchronizes with dentate gyrus to regulate spatial memory retrieval through glutamate release.

The supramammillary nucleus (SuM) provides substantial innervation to the dentate gyrus (DG). It remains unknown how the SuM and DG coordinate their activities at the circuit level to regulate spatial memory. Additionally, SuM co-releases GABA and glutamate to the DG, but the relative role of GABA versus glutamate in regulating spatial memory remains unknown. Here we report that SuM-DG Ca2+ activities are highly correlated during spatial memory retrieval as compared to the moderate correlation during memory encoding when mice are performing a location discrimination task. Supporting this evidence, we demonstrate that the activity of SuM neurons or SuM-DG projections is required for spatial memory retrieval. Furthermore, we show that SuM glutamate transmission is necessary for both spatial memory retrieval and highly-correlated SuM-DG activities during spatial memory retrieval. Our studies identify a long-range SuM-DG circuit linking two highly correlated subcortical regions to regulate spatial memory retrieval through SuM glutamate release.

Shared and distinct transcriptomic cell types across neocortical areas.

The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex.

Persistence of maternal antibodies to influenza A virus among captive mallards (Anas platyrhynchos).

Wild waterfowl are maintenance hosts of most influenza A virus (IAV) subtypes and are often the subjects of IAV surveillance and transmission models. While maternal antibodies have been detected in yolks and in nestlings for a variety of wild bird species and pathogens, the persistence of maternal antibodies to IAVs in mallard ducklings (Anas platyrhynchos) has not been previously investigated. Nonetheless, this information is important for a full understanding of IAV transmission dynamics because ducklings protected by maternal antibodies may not be susceptible to infection. In this study, we examined the transfer of IAV-specific maternal antibodies to ducklings. Blood samples were collected approximately every five days from ducklings hatched from hens previously infected with an H6 strain of IAV. Serum samples were tested for antibodies to IAV by an enzyme-linked immunosorbent assay. The median persistence of maternal antibodies in ducklings was 12.5 days (range: 4-33 days) post-hatch. The majority of ducklings (71%) had detectable maternal antibodies from 4 to 17 days post-hatch, while a small subset of individuals (29%) had detectable maternal antibodies for up to 21-33 days post-hatch. Antibody concentrations in hens near the time of egg laying were correlated with maternal antibody concentrations in the initial blood sample collected from ducklings (0-4 days post-hatch). Knowledge of the duration of maternal antibodies in ducklings will aid in the interpretation of IAV serological surveillance results and in the modeling of IAV transmission dynamics in waterfowl.