Ascertaining cells' synaptic connections and RNA expression simultaneously with barcoded rabies virus libraries.

Brain function depends on synaptic connections between specific neuron types, yet systematic descriptions of synaptic networks and their molecular properties are not readily available. Here, we introduce SBARRO (Synaptic Barcode Analysis by Retrograde Rabies ReadOut), a method that uses single-cell RNA sequencing to reveal directional, monosynaptic relationships based on the paths of a barcoded rabies virus from its "starter" postsynaptic cell to that cell's presynaptic partners. Thousands of these partner relationships can be ascertained in a single experiment, alongside genome-wide RNAs. We use SBARRO to describe synaptic networks formed by diverse mouse brain cell types in vitro, finding that different cell types have presynaptic networks with differences in average size and cell type composition. Patterns of RNA expression suggest that functioning synapses are critical for rabies virus uptake. By tracking individual rabies clones across cells, SBARRO offers new opportunities to map the synaptic organization of neural circuits.

Bombesin-like peptide recruits disinhibitory cortical circuits and enhances fear memories.

Disinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory, and excitatory neurons suggests that each circuit motif may be controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a genetically encoded GRP sensor, optogenetic anterograde stimulation, and trans-synaptic tracing, we reveal that GRP regulates VIP cells most likely via extrasynaptic diffusion from several local and long-range sources. In vivo photometry and CRISPR-Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.

Molecular and anatomical organization of the dorsal raphe nucleus.

The dorsal raphe nucleus (DRN) is an important source of neuromodulators and has been implicated in a wide variety of behavioral and neurological disorders. The DRN is subdivided into distinct anatomical subregions comprised of multiple cell types, and its complex cellular organization has impeded efforts to investigate the distinct circuit and behavioral functions of its subdomains. Here we used single-cell RNA sequencing, in situ hybridization, anatomical tracing, and spatial correlation analysis to map the transcriptional and spatial profiles of cells from the mouse DRN. Our analysis of 39,411 single-cell transcriptomes revealed at least 18 distinct neuron subtypes and 5 serotonergic neuron subtypes with distinct molecular and anatomical properties, including a serotonergic neuron subtype that preferentially innervates the basal ganglia. Our study lays out the molecular organization of distinct serotonergic and non-serotonergic subsystems, and will facilitate the design of strategies for further dissection of the DRN and its diverse functions.

Malaria in pregnancy alters l-arginine bioavailability and placental vascular development.

Reducing adverse birth outcomes due to malaria in pregnancy (MIP) is a global health priority. However, there are few safe and effective interventions. l-Arginine is an essential amino acid in pregnancy and an immediate precursor in the biosynthesis of nitric oxide (NO), but there are limited data on the impact of MIP on NO biogenesis. We hypothesized that hypoarginemia contributes to the pathophysiology of MIP and that l-arginine supplementation would improve birth outcomes. In a prospective study of pregnant Malawian women, we show that MIP was associated with lower concentrations of l-arginine and higher concentrations of endogenous inhibitors of NO biosynthesis, asymmetric and symmetric dimethylarginine, which were associated with adverse birth outcomes. In a model of experimental MIP, l-arginine supplementation in dams improved birth outcomes (decreased stillbirth and increased birth weight) compared with controls. The mechanism of action was via normalized angiogenic pathways and enhanced placental vascular development, as visualized by placental microcomputerized tomography imaging. These data define a role for dysregulation of NO biosynthetic pathways in the pathogenesis of MIP and support the evaluation of interventions to enhance l-arginine bioavailability as strategies to improve birth outcomes.

Genetically Distinct Parallel Pathways in the Entopeduncular Nucleus for Limbic and Sensorimotor Output of the Basal Ganglia.

The basal ganglia (BG) integrate inputs from diverse sensorimotor, limbic, and associative regions to guide action-selection and goal-directed behaviors. The entopeduncular nucleus (EP) is a major BG output nucleus and has been suggested to channel signals from distinct BG nuclei to target regions involved in diverse functions. Here we use single-cell transcriptional and molecular analyses to demonstrate that the EP contains at least three classes of projection neurons-glutamate/GABA co-releasing somatostatin neurons, glutamatergic parvalbumin neurons, and GABAergic parvalbumin neurons. These classes comprise functionally and anatomically distinct output pathways that differentially affect EP target regions, such as the lateral habenula (LHb) and thalamus. Furthermore, LHb- and thalamic-projecting EP neurons are differentially innervated by subclasses of striatal and pallidal neurons. Therefore, we identify previously unknown subdivisions within the EP and reveal the existence of cascading, molecularly distinct projections through striatum and globus pallidus to EP targets within epithalamus and thalamus.