Can We Explain Thousands of Molecularly Identified Mouse Neuronal Types? From Knowing to Understanding.

At the end of 2023, the Whole Mouse Brain Atlas was announced, revealing that there are about 5300 molecularly defined neuronal types in the mouse brain. We ask whether brain models exist that contemplate how this is possible. The conventional columnar model, implicitly used by the authors of the Atlas, is incapable of doing so with only 20 brain columns (5 brain vesicles with 4 columns each). We argue that the definition of some 1250 distinct progenitor microzones, each producing at least 4-5 neuronal types over time, may be sufficient. Presently, this is nearly achieved by the prosomeric model amplified by the secondary dorsoventral and anteroposterior microzonation of progenitor areas, plus the clonal variation in cell types produced, on average, by each of them.

Multimodal cortical neuronal cell type classification.

Since more than a century, neuroscientists have distinguished excitatory (glutamatergic) neurons with long-distance projections from inhibitory (GABAergic) neurons with local projections and established layer-dependent schemes for the ~ 80% excitatory (principal) cells as well as the ~ 20% inhibitory neurons. Whereas, in the early days, mainly morphological criteria were used to define cell types, later supplemented by electrophysiological and neurochemical properties, nowadays. single-cell transcriptomics is the method of choice for cell type classification. Bringing recent insight together, we conclude that despite all established layer- and area-dependent differences, there is a set of reliably identifiable cortical cell types that were named (among others) intratelencephalic (IT), extratelencephalic (ET), and corticothalamic (CT) for the excitatory cells, which altogether comprise ~ 56 transcriptomic cell types (t-types). By the same means, inhibitory neurons were subdivided into parvalbumin (PV), somatostatin (SST), vasoactive intestinal polypeptide (VIP), and "other (i.e. Lamp5/Sncg)" subpopulations, which altogether comprise ~ 60 t-types. The coming years will show which t-types actually translate into "real" cell types that show a common set of multimodal features, including not only transcriptome but also physiology and morphology as well as connectivity and ultimately function. Only with the better knowledge of clear-cut cell types and experimental access to them, we will be able to reveal their specific functions, a task which turned out to be difficult in a part of the brain being so much specialized for cognition as the cerebral cortex.

Neuroarchitecture of the central complex in the Madeira cockroach Rhyparobia maderae: Pontine and columnar neuronal cell types.

Insects have evolved remarkable abilities to navigate over short distances and during long-range seasonal migrations. The central complex (CX) is a navigation center in the insect brain that controls spatial orientation and directed locomotion. It is composed of the protocerebral bridge (PB), the upper (CBU) and lower (CBL) division of the central body, and a pair of noduli. While most of its functional organization and involvement in head-direction coding has been obtained from work on flies, bees, and locusts that largely rely on vision for navigation, little contribution has been provided by work on nocturnal species. To close this gap, we have investigated the columnar organization of the CX in the cockroach Rhyparobia maderae. Rhyparobia maderae is a highly agile nocturnal insect that relies largely but not exclusively on antennal information for navigation. A particular feature of the cockroach CX is an organization of the CBU and CBL into interleaved series of eight and nine columns. Single-cell tracer injections combined with imaging and 3D analysis revealed five systems of pontine neurons connecting columns along the vertical and horizontal axis and 18 systems of columnar neurons with topographically organized projection patterns. Among these are six types of neurons with no correspondence in other species. Many neurons send processes into the anterior lip, a brain area highly reduced in bees and unknown in flies. While sharing many features with the CX in other species, the cockroach CX shows some unique attributes that may be related to the ecological niche of this insect.

Human cortex development is shaped by molecular and cellular brain systems.

Human brain morphology undergoes complex changes over the lifespan. Despite recent progress in tracking brain development via normative models, current knowledge of underlying biological mechanisms is highly limited. We demonstrate that human cerebral cortex development unfolds along patterns of molecular and cellular brain organization, traceable from population-level to individual developmental trajectories. During childhood and adolescence, cortex-wide spatial distributions of dopaminergic receptors, inhibitory neurons, glial cell populations, and brain-metabolic features explain up to 50% of variance associated with regional cortical thickness trajectories. Adult cortical change patterns are best explained by cholinergic and glutamatergic neurotransmission. These relationships are supported by developmental gene expression trajectories and translate to longitudinal data from over 8,000 adolescents, explaining up to 59% of developmental change at population- and 18% at single-subject level. Integrating multilevel brain atlases with normative modeling and population neuroimaging provides a biologically meaningful path to understand typical and atypical brain development in living humans.

Effects of central nervous system electrical stimulation on non-neuronal cells.

Over the past few decades, much progress has been made in the clinical use of electrical stimulation of the central nervous system (CNS) to treat an ever-growing number of conditions from Parkinson's disease (PD) to epilepsy as well as for sensory restoration and many other applications. However, little is known about the effects of microstimulation at the cellular level. Most of the existing research focuses on the effects of electrical stimulation on neurons. Other cells of the CNS such as microglia, astrocytes, oligodendrocytes, and vascular endothelial cells have been understudied in terms of their response to stimulation. The varied and critical functions of these cell types are now beginning to be better understood, and their vital roles in brain function in both health and disease are becoming better appreciated. To shed light on the importance of the way electrical stimulation as distinct from device implantation impacts non-neuronal cell types, this review will first summarize common stimulation modalities from the perspective of device design and stimulation parameters and how these different parameters have an impact on the physiological response. Following this, what is known about the responses of different cell types to different stimulation modalities will be summarized, drawing on findings from both clinical studies as well as clinically relevant animal models and in vitro systems.

Virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq) defines projection neurons involved in sensorimotor integration.

Behavior arises from concerted activity throughout the brain. Consequently, a major focus of modern neuroscience is defining the physiology and behavioral roles of projection neurons linking different brain areas. Single-cell RNA sequencing has facilitated these efforts by revealing molecular determinants of cellular physiology and markers that enable genetically targeted perturbations such as optogenetics, but existing methods for sequencing defined projection populations are low throughput, painstaking, and costly. We developed a straightforward, multiplexed approach, virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq). VECTORseq repurposes commercial retrogradely infecting viruses typically used to express functional transgenes (e.g., recombinases and fluorescent proteins) by treating viral transgene mRNA as barcodes within single-cell datasets. VECTORseq is compatible with different viral families, resolves multiple populations with different projection targets in one sequencing run, and identifies cortical and subcortical excitatory and inhibitory projection populations. Our study provides a roadmap for high-throughput identification of neuronal subtypes based on connectivity.

The development and function of neuronal subtypes processing color and skylight polarization in the optic lobes of Drosophila melanogaster.

The retinal mosaics of many insects contain different ommatidial subtypes harboring photoreceptors that are both molecularly and morphologically specialized for comparing between different wavelengths versus detecting the orientation of skylight polarization. The neural circuits underlying these different inputs and the characterization of their specific cellular elements are the subject of intense research. Here we review recent progress on the description of both assembly and function of color and skylight polarization circuitry, by focusing on two cell types located in the distal portion of the medulla neuropil of the fruit fly Drosophila melanogaster's optic lobes, called Dm8 and Dm9. In the main part of the retina, Dm8 cells fall into two molecularly distinct subtypes whose center becomes specifically connected to either one of randomly distributed 'pale' or 'yellow' R7 photoreceptor fates during development. Only in the 'dorsal rim area' (DRA), both polarization-sensitive R7 and R8 photoreceptors are connected to different Dm8-like cell types, called Dm-DRA1 and Dm-DRA2, respectively. An additional layer of interommatidial integration is introduced by Dm9 cells, which receive input from multiple neighboring R7 and R8 cells, as well as providing feedback synapses back into these photoreceptors. As a result, the response properties of color-sensitive photoreceptor terminals are sculpted towards being both maximally decorrelated, as well as harboring several levels of opponency (both columnar as well as intercolumnar). In the DRA, individual Dm9 cells appear to mix both polarization and color signals, thereby potentially serving as the first level of integration of different celestial stimuli. The molecular mechanisms underlying the establishment of these synaptic connections are beginning to be revealed, by using a combination of live imaging, developmental genetic studies, and cell type-specific transcriptomics.

Reconstruction of 1,000 Projection Neurons Reveals New Cell Types and Organization of Long-Range Connectivity in the Mouse Brain.

Neuronal cell types are the nodes of neural circuits that determine the flow of information within the brain. Neuronal morphology, especially the shape of the axonal arbor, provides an essential descriptor of cell type and reveals how individual neurons route their output across the brain. Despite the importance of morphology, few projection neurons in the mouse brain have been reconstructed in their entirety. Here we present a robust and efficient platform for imaging and reconstructing complete neuronal morphologies, including axonal arbors that span substantial portions of the brain. We used this platform to reconstruct more than 1,000 projection neurons in the motor cortex, thalamus, subiculum, and hypothalamus. Together, the reconstructed neurons constitute more than 85 meters of axonal length and are available in a searchable online database. Axonal shapes revealed previously unknown subtypes of projection neurons and suggest organizational principles of long-range connectivity.

Brainstem Circuits Controlling Action Diversification.

Neuronal circuits that regulate movement are distributed throughout the nervous system. The brainstem is an important interface between upper motor centers involved in action planning and circuits in the spinal cord ultimately leading to execution of body movements. Here we focus on recent work using genetic and viral entry points to reveal the identity of functionally dedicated and frequently spatially intermingled brainstem populations essential for action diversification, a general principle conserved throughout evolution. Brainstem circuits with distinct organization and function control skilled forelimb behavior, orofacial movements, and locomotion. They convey regulatory parameters to motor output structures and collaborate in the construction of complex natural motor behaviors. Functionally tuned brainstem neurons for different actions serve as important integrators of synaptic inputs from upstream centers, including the basal ganglia and cortex, to regulate and modulate behavioral function in different contexts.

Neural Circuits for Goal-Directed Sensorimotor Transformations.

Precisely wired neuronal circuits process sensory information in a learning- and context-dependent manner in order to govern behavior. Simple sensory decision-making tasks in rodents are now beginning to reveal the contributions of distinct cell types and brain regions participating in the conversion of sensory information into learned goal-directed motor output. Task learning is accompanied by target-specific routing of sensory information to specific downstream cortical regions, with higher-order cortical regions such as the posterior parietal cortex, medial prefrontal cortex, and hippocampus appearing to play important roles in learning- and context-dependent processing of sensory input. An important challenge for future research is to connect cell-type-specific activity in these brain regions with motor neurons responsible for action initiation.

Molecular codes for cell type specification in Brn3 retinal ganglion cells.

Visual information is conveyed from the eye to the brain by distinct types of retinal ganglion cells (RGCs). It is largely unknown how RGCs acquire their defining morphological and physiological features and connect to upstream and downstream synaptic partners. The three Brn3/Pou4f transcription factors (TFs) participate in a combinatorial code for RGC type specification, but their exact molecular roles are still unclear. We use deep sequencing to define (i) transcriptomes of Brn3a- and/or Brn3b-positive RGCs, (ii) Brn3a- and/or Brn3b-dependent RGC transcripts, and (iii) transcriptomes of retinorecipient areas of the brain at developmental stages relevant for axon guidance, dendrite formation, and synaptogenesis. We reveal a combinatorial code of TFs, cell surface molecules, and determinants of neuronal morphology that is differentially expressed in specific RGC populations and selectively regulated by Brn3a and/or Brn3b. This comprehensive molecular code provides a basis for understanding neuronal cell type specification in RGCs.

Anatomical characterization of Cre driver mice for neural circuit mapping and manipulation.

Significant advances in circuit-level analyses of the brain require tools that allow for labeling, modulation of gene expression, and monitoring and manipulation of cellular activity in specific cell types and/or anatomical regions. Large-scale projects and individual laboratories have produced hundreds of gene-specific promoter-driven Cre mouse lines invaluable for enabling genetic access to subpopulations of cells in the brain. However, the potential utility of each line may not be fully realized without systematic whole brain characterization of transgene expression patterns. We established a high-throughput in situ hybridization (ISH), imaging and data processing pipeline to describe whole brain gene expression patterns in Cre driver mice. Currently, anatomical data from over 100 Cre driver lines are publicly available via the Allen Institute's Transgenic Characterization database, which can be used to assist researchers in choosing the appropriate Cre drivers for functional, molecular, or connectional studies of different regions and/or cell types in the brain.

A formal ontology of subcellular neuroanatomy.

The complexity of the nervous system requires high-resolution microscopy to resolve the detailed 3D structure of nerve cells and supracellular domains. The analysis of such imaging data to extract cellular surfaces and cell components often requires the combination of expert human knowledge with carefully engineered software tools. In an effort to make better tools to assist humans in this endeavor, create a more accessible and permanent record of their data, and to aid the process of constructing complex and detailed computational models, we have created a core of formalized knowledge about the structure of the nervous system and have integrated that core into several software applications. In this paper, we describe the structure and content of a formal ontology whose scope is the subcellular anatomy of the nervous system (SAO), covering nerve cells, their parts, and interactions between these parts. Many applications of this ontology to image annotation, content-based retrieval of structural data, and integration of shared data across scales and researchers are also described.

Genetic regulation of mec-3 gene expression implicated in the specification of the mechanosensory neuron cell types in Caenorhabditis elegans.

The mec-3 gene, a member of the LIM-homeodomain transcription factors, is required for touch receptor, FLP and PVD neurons to differentiate in the nematode Caenorhabditis elegans. Stably integrated transgenic strains with mec-3-lacZ fusion were generated by irradiating UV light to an unstable transgenic strain with the extrachromosomal DNA. Expression patterns of the mec-3-lacZ fusion were examined in mutant backgrounds (lin-4, lin-14, egl-44, egl-46 and sem-4 genes) which alter touch receptor-specific gene expression. In the lin-4 mutant background, ectopic mec-3-lacZ positive AVM/PVM-like cells were observed in 9% of the animals. By contrast, in the lin-14 mutant background, mec-3-lacZ staining in AVM/PVM cells was lost in 86% of the animals. In the egl-44 and egl-46 mutant backgrounds, expression pattern was the same as wild-type animals. In the sem-4 mutant background, more than half of the animals (54-69%) had ectopic staining cells in the tail in addition to the wild-type staining pattern. The modes of action of these genetically interacting genes in the differentiation of mechanosensory neurons are proposed.