Cell-type-specific densities in mouse somatosensory cortex derived from scRNA-seq and in situ RNA hybridization.

Cells in the mammalian cerebral cortex exhibit layer-dependent patterns in their distribution. Classical methods of determining cell type distributions typically employ a painstaking process of large-scale sampling and characterization of cellular composition. We found that by combining in situ hybridization (ISH) images with cell-type-specific transcriptomes, position-dependent cortical composition in P56 mouse could be estimated in the somatosensory cortex. The method uses ISH images from the Allen Institute for Brain Science. There are two novel aspects of the methodology. First, it is not necessary to select a subset of genes that are particular for a cell type of interest, nor is it necessary to only use ISH images with low variability among samples. Second, the method also compensated for differences in soma size and incompleteness of the transcriptomes. The soma size compensation is particularly important in order to obtain quantitative estimates since relying on bulk expression alone would overestimate the contribution of larger cells. Predicted distributions of broader classes of cell types agreed with literature distributions. The primary result is that there is a high degree of substructure in the distribution of transcriptomic types beyond the resolution of layers. Furthermore, transcriptomic cell types each exhibited characteristic soma size distributions. Results suggest that the method could also be employed to assign transcriptomic cell types to well-aligned image sets in the entire brain.

Mapping of morpho-electric features to molecular identity of cortical inhibitory neurons.

Knowledge of the cell-type-specific composition of the brain is useful in order to understand the role of each cell type as part of the network. Here, we estimated the composition of the whole cortex in terms of well characterized morphological and electrophysiological inhibitory neuron types (me-types). We derived probabilistic me-type densities from an existing atlas of molecularly defined cell-type densities in the mouse cortex. We used a well-established me-type classification from rat somatosensory cortex to populate the cortex. These me-types were well characterized morphologically and electrophysiologically but they lacked molecular marker identity labels. To extrapolate this missing information, we employed an additional dataset from the Allen Institute for Brain Science containing molecular identity as well as morphological and electrophysiological data for mouse cortical neurons. We first built a latent space based on a number of comparable morphological and electrical features common to both data sources. We then identified 19 morpho-electrical clusters that merged neurons from both datasets while being molecularly homogeneous. The resulting clusters best mirror the molecular identity classification solely using available morpho-electrical features. Finally, we stochastically assigned a molecular identity to a me-type neuron based on the latent space cluster it was assigned to. The resulting mapping was used to derive inhibitory me-types densities in the cortex.

Desmosomal connectomics of all somatic muscles in an annelid larva.

Cells form networks in animal tissues through synaptic, chemical, and adhesive links. Invertebrate muscle cells often connect to other cells through desmosomes, adhesive junctions anchored by intermediate filaments. To study desmosomal networks, we skeletonised 853 muscle cells and their desmosomal partners in volume electron microscopy data covering an entire larva of the annelid Platynereis. Muscle cells adhere to each other, to epithelial, glial, ciliated, and bristle-producing cells and to the basal lamina, forming a desmosomal connectome of over 2000 cells. The aciculae - chitin rods that form an endoskeleton in the segmental appendages - are highly connected hubs in this network. This agrees with the many degrees of freedom of their movement, as revealed by video microscopy. Mapping motoneuron synapses to the desmosomal connectome allowed us to infer the extent of tissue influenced by motoneurons. Our work shows how cellular-level maps of synaptic and adherent force networks can elucidate body mechanics.

A method to estimate the cellular composition of the mouse brain from heterogeneous datasets.

The mouse brain contains a rich diversity of inhibitory neuron types that have been characterized by their patterns of gene expression. However, it is still unclear how these cell types are distributed across the mouse brain. We developed a computational method to estimate the densities of different inhibitory neuron types across the mouse brain. Our method allows the unbiased integration of diverse and disparate datasets into one framework to predict inhibitory neuron densities for uncharted brain regions. We constrained our estimates based on previously computed brain-wide neuron densities, gene expression data from in situ hybridization image stacks together with a wide range of values reported in the literature. Using constrained optimization, we derived coherent estimates of cell densities for the different inhibitory neuron types. We estimate that 20.3% of all neurons in the mouse brain are inhibitory. Among all inhibitory neurons, 18% predominantly express parvalbumin (PV), 16% express somatostatin (SST), 3% express vasoactive intestinal peptide (VIP), and the remainder 63% belong to the residual GABAergic population. We find that our density estimations improve as more literature values are integrated. Our pipeline is extensible, allowing new cell types or data to be integrated as they become available. The data, algorithms, software, and results of our pipeline are publicly available and update the Blue Brain Cell Atlas. This work therefore leverages the research community to collectively converge on the numbers of each cell type in each brain region.

Neural circuitry of a polycystin-mediated hydrodynamic startle response for predator avoidance.

Startle responses triggered by aversive stimuli including predators are widespread across animals. These coordinated whole-body actions require the rapid and simultaneous activation of a large number of muscles. Here we study a startle response in a planktonic larva to understand the whole-body circuit implementation of the behaviour. Upon encountering water vibrations, larvae of the annelid Platynereis close their locomotor cilia and simultaneously raise the parapodia. The response is mediated by collar receptor neurons expressing the polycystins PKD1-1 and PKD2-1. CRISPR-generated PKD1-1 and PKD2-1 mutant larvae do not startle and fall prey to a copepod predator at a higher rate. Reconstruction of the whole-body connectome of the collar-receptor-cell circuitry revealed converging feedforward circuits to the ciliary bands and muscles. The wiring diagram suggests circuit mechanisms for the intersegmental and left-right coordination of the response. Our results reveal how polycystin-mediated mechanosensation can trigger a coordinated whole-body effector response involved in predator avoidance.

Ciliary and rhabdomeric photoreceptor-cell circuits form a spectral depth gauge in marine zooplankton.

Ciliary and rhabdomeric photoreceptor cells represent two main lines of photoreceptor-cell evolution in animals. The two cell types coexist in some animals, however how these cells functionally integrate is unknown. We used connectomics to map synaptic paths between ciliary and rhabdomeric photoreceptors in the planktonic larva of the annelid Platynereis and found that ciliary photoreceptors are presynaptic to the rhabdomeric circuit. The behaviors mediated by the ciliary and rhabdomeric cells also interact hierarchically. The ciliary photoreceptors are UV-sensitive and mediate downward swimming in non-directional UV light, a behavior absent in ciliary-opsin knockout larvae. UV avoidance overrides positive phototaxis mediated by the rhabdomeric eyes such that vertical swimming direction is determined by the ratio of blue/UV light. Since this ratio increases with depth, Platynereis larvae may use it as a depth gauge during vertical migration. Our results revealed a functional integration of ciliary and rhabdomeric photoreceptor cells in a zooplankton larva.

Synaptic and peptidergic connectome of a neurosecretory center in the annelid brain.

Neurosecretory centers in animal brains use peptidergic signaling to influence physiology and behavior. Understanding neurosecretory center function requires mapping cell types, synapses, and peptidergic networks. Here we use transmission electron microscopy and gene expression mapping to analyze the synaptic and peptidergic connectome of an entire neurosecretory center. We reconstructed 78 neurosecretory neurons and mapped their synaptic connectivity in the brain of larval Platynereis dumerilii, a marine annelid. These neurons form an anterior neurosecretory center expressing many neuropeptides, including hypothalamic peptide orthologs and their receptors. Analysis of peptide-receptor pairs in spatially mapped single-cell transcriptome data revealed sparsely connected networks linking specific neuronal subsets. We experimentally analyzed one peptide-receptor pair and found that a neuropeptide can couple neurosecretory and synaptic brain signaling. Our study uncovered extensive networks of peptidergic signaling within a neurosecretory center and its connection to the synaptic brain.

Ciliomotor circuitry underlying whole-body coordination of ciliary activity in the Platynereis larva.

Ciliated surfaces harbouring synchronously beating cilia can generate fluid flow or drive locomotion. In ciliary swimmers, ciliary beating, arrests, and changes in beat frequency are often coordinated across extended or discontinuous surfaces. To understand how such coordination is achieved, we studied the ciliated larvae of Platynereis dumerilii, a marine annelid. Platynereis larvae have segmental multiciliated cells that regularly display spontaneous coordinated ciliary arrests. We used whole-body connectomics, activity imaging, transgenesis, and neuron ablation to characterize the ciliomotor circuitry. We identified cholinergic, serotonergic, and catecholaminergic ciliomotor neurons. The synchronous rhythmic activation of cholinergic cells drives the coordinated arrests of all cilia. The serotonergic cells are active when cilia are beating. Serotonin inhibits the cholinergic rhythm, and increases ciliary beat frequency. Based on their connectivity and alternating activity, the catecholaminergic cells may generate the rhythm. The ciliomotor circuitry thus constitutes a stop-and-go pacemaker system for the whole-body coordination of ciliary locomotion.

A serial multiplex immunogold labeling method for identifying peptidergic neurons in connectomes.

Electron microscopy-based connectomics aims to comprehensively map synaptic connections in neural tissue. However, current approaches are limited in their capacity to directly assign molecular identities to neurons. Here, we use serial multiplex immunogold labeling (siGOLD) and serial-section transmission electron microscopy (ssTEM) to identify multiple peptidergic neurons in a connectome. The high immunogenicity of neuropeptides and their broad distribution along axons, allowed us to identify distinct neurons by immunolabeling small subsets of sections within larger series. We demonstrate the scalability of siGOLD by using 11 neuropeptide antibodies on a full-body larval ssTEM dataset of the annelid Platynereis. We also reconstruct a peptidergic circuitry comprising the sensory nuchal organs, found by siGOLD to express pigment-dispersing factor, a circadian neuropeptide. Our approach enables the direct overlaying of chemical neuromodulatory maps onto synaptic connectomic maps in the study of nervous systems.

Spectral Tuning of Phototaxis by a Go-Opsin in the Rhabdomeric Eyes of Platynereis.

Phototaxis is characteristic of the pelagic larval stage of most bottom-dwelling marine invertebrates. Larval phototaxis is mediated by simple eyes that can express various types of light-sensitive G-protein-coupled receptors known as opsins. Since opsins diversified early during metazoan evolution in the marine environment, understanding underwater light detection could elucidate this diversification. Opsins have been classified into three major families, the r-opsins, the c-opsins, and the Go/RGR opsins, a family uniting Go-opsins, retinochromes, RGR opsins, and neuropsins. The Go-opsins form an ancient and poorly characterized group retained only in marine invertebrate genomes. Here, we characterize a Go-opsin from the marine annelid Platynereis dumerilii. We found Go-opsin1 coexpressed with two r-opsins in depolarizing rhabdomeric photoreceptor cells in the pigmented eyes of Platynereis larvae. We purified recombinant Go-opsin1 and found that it absorbs in the blue-cyan range of the light spectrum. To characterize the function of Go-opsin1, we generated a Go-opsin1 knockout Platynereis line by zinc-finger-nuclease-mediated genome engineering. Go-opsin1 knockout larvae were phototactic but showed reduced efficiency of phototaxis to wavelengths matching the in vitro Go-opsin1 spectrum. Our results highlight spectral tuning of phototaxis as a potential mechanism contributing to opsin diversity.

Inter-individual stereotypy of the Platynereis larval visual connectome.

Developmental programs have the fidelity to form neural circuits with the same structure and function among individuals of the same species. It is less well understood, however, to what extent entire neural circuits of different individuals are similar. Previously, we reported the neuronal connectome of the visual eye circuit from the head of a Platynereis dumerilii larva (Randel et al., 2014). We now report a full-body serial section transmission electron microscopy (ssTEM) dataset of another larva of the same age, for which we describe the connectome of the visual eyes and the larval eyespots. Anatomical comparisons and quantitative analyses of the two circuits reveal a high inter-individual stereotypy of the cell complement, neuronal projections, and synaptic connectivity, including the left-right asymmetry in the connectivity of some neurons. Our work shows the extent to which the eye circuitry in Platynereis larvae is hard-wired.

Neuronal connectome of a sensory-motor circuit for visual navigation.

Animals use spatial differences in environmental light levels for visual navigation; however, how light inputs are translated into coordinated motor outputs remains poorly understood. Here we reconstruct the neuronal connectome of a four-eye visual circuit in the larva of the annelid Platynereis using serial-section transmission electron microscopy. In this 71-neuron circuit, photoreceptors connect via three layers of interneurons to motorneurons, which innervate trunk muscles. By combining eye ablations with behavioral experiments, we show that the circuit compares light on either side of the body and stimulates body bending upon left-right light imbalance during visual phototaxis. We also identified an interneuron motif that enhances sensitivity to different light intensity contrasts. The Platynereis eye circuit has the hallmarks of a visual system, including spatial light detection and contrast modulation, illustrating how image-forming eyes may have evolved via intermediate stages contrasting only a light and a dark field during a simple visual task.

Whole-body gene expression pattern registration in Platynereis larvae.

BACKGROUND: Digital anatomical atlases are increasingly used in order to depict different gene expression patterns and neuronal morphologies within a standardized reference template. In evo-devo, a discipline in which the comparison of gene expression patterns is a widely used approach, such standardized anatomical atlases would allow a more rigorous assessment of the conservation of and changes in gene expression patterns during micro- and macroevolutionary time scales. Due to its small size and invariant early development, the annelid Platynereis dumerilii is particularly well suited for such studies. Recently a reference template with registered gene expression patterns has been generated for the anterior part (episphere) of the Platynereis trochophore larva and used for the detailed study of neuronal development. RESULTS: Here we introduce and evaluate a method for whole-body gene expression pattern registration for Platynereis trochophore and nectochaete larvae based on whole-mount in situ hybridization, confocal microscopy, and image registration. We achieved high-resolution whole-body scanning using the mounting medium 2,2'-thiodiethanol (TDE), which allows the matching of the refractive index of the sample to that of glass and immersion oil thereby reducing spherical aberration and improving depth penetration. This approach allowed us to scan entire whole-mount larvae stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) in situ hybridization and counterstained fluorescently with an acetylated-tubulin antibody and the nuclear stain 4'6-diamidino-2-phenylindole (DAPI). Due to the submicron isotropic voxel size whole-mount larvae could be scanned in any orientation. Based on the whole-body scans, we generated four different reference templates by the iterative registration and averaging of 40 individual image stacks using either the acetylated-tubulin or the nuclear-stain signal for each developmental stage. We then registered to these templates the expression patterns of cell-type specific genes. In order to evaluate the gene expression pattern registration, we analyzed the absolute deviation of cell-center positions. Both the acetylated-tubulin- and the nuclear-stain-based templates allowed near-cellular-resolution gene expression registration. Nuclear-stain-based templates often performed significantly better than acetylated-tubulin-based templates. We provide detailed guidelines and scripts for the use and further expansion of the Platynereis gene expression atlas. CONCLUSIONS: We established whole-body reference templates for the generation of gene expression atlases for Platynereis trochophore and nectochaete larvae. We anticipate that nuclear-staining-based image registration will be applicable for whole-body alignment of the embryonic and larval stages of other organisms in a similar size range.