Probes for PIEZO1

As we age, our bones undergo a process of loss, often accompanied by muscle weakness and reduced physical activity. This is exacerbated by decreased responsiveness to mechanical stimulation in aged skeleton, leading to the hypothesis that decreased mechanical stimulation plays an important role in age-related bone loss. Piezo1, a mechanosensitive ion channel, is critical for bone homeostasis and mechanotransduction. Here, we observed a decrease in Piezo1 expression with age in both murine and human cortical bone. Furthermore, loss of Piezo1 in osteoblasts and osteocytes resulted in an increase in age-associated cortical bone loss compared to control mice. The loss of cortical bone was due to an expansion of the endosteal perimeter resulting from increased endocortical resorption. In addition, expression of Tnfrsf11b, encoding anti-osteoclastogenic protein OPG, decreases with Piezo1 in vitro and in vivo in bone cells, suggesting that Piezo1 suppresses osteoclast formation by promoting Tnfrsf11b expression. Our results highlight the importance of Piezo1-mediated mechanical signaling in protecting against age-associated cortical bone loss by inhibiting bone resorption in mice.

Spatial gene expression, achieved classically through in situ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for visualization of multiple mRNAs at single-cell resolution and greatly expand the ability to link gene expression to tissue topography, yet there are challenges in efficient quantification and analysis of these high-dimensional datasets. We have therefore developed the single-cell automated multiplex pipeline for RNA (SCAMPR), facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low- and high-abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on gene expression in vagal neuron subtypes.

Sleep disturbances are strongly associated with cardiovascular diseases. Baroreflex, a basic cardiovascular regulation mechanism, is modulated by sleep-wake states. Here, we show that neurons at key stages of baroreflex pathways also promote sleep. Using activity-dependent genetic labeling, we tagged neurons in the nucleus of the solitary tract (NST) activated by blood pressure elevation and confirmed their barosensitivity with optrode recording and calcium imaging. Chemogenetic or optogenetic activation of these neurons promoted non-REM sleep in addition to decreasing blood pressure and heart rate. GABAergic neurons in the caudal ventrolateral medulla (CVLM)-a downstream target of the NST for vasomotor baroreflex-also promote non-REM sleep, partly by inhibiting the sympathoexcitatory and wake-promoting adrenergic neurons in the rostral ventrolateral medulla (RVLM). Cholinergic neurons in the nucleus ambiguous-a target of the NST for cardiac baroreflex-promoted non-REM sleep as well. Thus, key components of the cardiovascular baroreflex circuit are also integral to sleep-wake brain-state regulation.