Comparative cellular analysis of motor cortex in human, marmoset and mouse

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

Cardiac remodeling in fish: Strategies to maintain heart function during temperature change

Rainbow trout remain active in waters that seasonally change between 4°C and 20°C. To explore how these fish are able to maintain cardiac function over this temperature range we characterized changes in cardiac morphology, contractile function, and the expression of contractile proteins in trout following acclimation to 4°C (cold), 12°C (control), and 17°C (warm). The relative ventricular mass (RVM) of the cold acclimated male fish was significantly greater than that of males in the control group. In addition, the compact myocardium of the cold acclimated male hearts was thinner compared to controls while the amount of spongy myocardium was found to have increased. Cold acclimation also caused an increase in connective tissue content, as well as muscle bundle area in the spongy myocardium of the male fish. Conversely, warm acclimation of male fish caused an increase in the thickness of the compact myocardium and a decrease in the amount of spongy myocardium. There was also a decrease in connective tissue content in both myocardial layers. In contrast, there was no change in the RVM or connective tissue content in the hearts of female trout with warm or cold acclimation. Cold acclimation also caused a 50% increase in the maximal rate of cardiac AM Mg(2+)-ATPase but did not influence the Ca(2+) sensitivity of this enzyme. To identify a mechanism for this change we utilized two-dimensional difference gel electrophoresis to characterize changes in the cardiac contractile proteins. Cold acclimation caused subtle changes in the phosphorylation state of the slow skeletal isoform of troponin T found in the heart, as well as of myosin binding protein C. These results demonstrate that acclimation of trout to warm and cold temperatures has opposing effects on cardiac morphology and tissue composition and that this results in distinct warm and cold cardiac phenotypes

The Dynamic Nature of Hypertrophic and Fibrotic Remodeling of the Fish Ventricle.

Chronic pressure or volume overload can cause the vertebrate heart to remodel. The hearts of fish remodel in response to seasonal temperature change. Here we focus on the passive properties of the fish heart. Building upon our previous work on thermal-remodelling of the rainbow trout ventricle, we hypothesized that chronic cooling would initiate a fibrotic cardiac remodelling, with increased myocardial stiffness, similar to that seen with pathological hypertrophy in mammals. We hypothesized that, in contrast to pathological hypertrophy in mammals, the remodelling response in fish would be plastic and the opposite response would occur following chronic warming. Rainbow trout held at 10 °C (control group) were chronically (> 8 weeks) exposed to cooling (5 °C) or warming (18 °C). Chronic cold induced hypertrophy in the highly trabeculated inner layer of the fish heart, with a 41 % increase in myocyte bundle cross-sectional area, and an up-regulation of hypertrophic markers. Cold acclimation also increased collagen deposition by 1.7-fold and caused an up-regulation of collagen promoting genes. In contrast, chronic warming reduced myocyte bundle cross-sectional area, expression of hypertrophic markers and collagen deposition. Functionally, the cold-induced fibrosis and hypertrophy were associated with increased passive stiffness of the whole ventricle and with increased micromechanical stiffness of tissue sections. The opposite occurred with chronic warming. These findings suggest chronic cooling in the trout heart invokes a hypertrophic phenotype with increased cardiac stiffness and fibrosis that are associated with pathological hypertrophy in the mammalian heart. The loss of collagen and increased compliance following warming is particularly interesting as it suggests fibrosis may oscillate seasonally in the fish heart, revealing a more dynamic nature than the fibrosis associated with dysfunction in mammals

Masson's trichrome stained sections of ventricular compact layer from thermally acclimated rainbow trout.

<p><b>(</b>A) cold, 4°C, (B) control, 12°C, (C) warm, 17°C where pink/ purple is muscle, blue is connective tissue and white or very pale pink is “extra bundular” space.</p

2D-DIGE analysis of cardiac contractile proteins from thermally acclimated trout.

<p>Thermal acclimation did not cause a change in the isoform expression of the identified contractile proteins in the trout ventricle. There was also no detected change in phosphorylation state of any of these proteins between experimental groups. (A) Representative 2D-DIGE analysis of cardiac proteomic changes with thermal acclimation. Proteins extracted from warm and cold acclimated trout were labeled with Cy-3 (green) and Cy-5 (red), respectively. The proteins were focused in the first dimension with an 18-cm, pH range 3–10NL, IPG strip. Superimposed Cy3/ Cy5 image is shown. Protein spots identified: cMyBP-C, myomesin, cardiac troponin T (cTnT), slow skeletal TnT (ssTnt), actin, tropomyosin (Tm) and regulatory light chains (RLC) are indicated. The phosphorylation state of (B) RLC, (C) ssTnT, (D) Tm, (E) cMyBP-C or (F) myomesin were not significantly affected by thermal acclimation. The most basic spot identified in a string of proteins is labeled spot 1. (White bars – cold acclimated, grey bars –control and black bars – warm acclimated).</p

The effect of acclimation temperature on ventricle size.

<p>Cold (4°C) acclimated male rainbow trout had larger ventricle to body mass ratios compared to warm acclimated (17°C) male trout but not control (12°C) trout. Furthermore, cold acclimated male trout had larger ventricle to body mass ratios compared to female trout. Trout were acclimated for a minimum of 2 months, <i>N</i> = 15. Relative ventricular mass: ((heart mass / body mass) * 100). Values are mean ± SEM. Brackets, if present indicate significant differences between sexes at the same acclimation temperature. Different letters above the bars indicate significant difference between acclimation groups. Different letters within the bars for male fish indicate significant differences between acclimation temperatures (p<0.05).</p

Effect of acclimation temperature and assay temperature on the function of actomyosin Mg²⁺-ATPase in rainbow trout ventricle.

<p>Note: Data are mean ± SEM. Values with the same symbol in the same column are significantly different from each other (p<0.05).</p