Proprioceptors are responsible for the conscious sensation of limb position and movement, muscle tension or force, and balance. Recent evidence suggests that Piezo2 is a low threshold mechanosensory receptor in the peripheral nervous system, acting as a transducer for touch sensation and proprioception. Thus, we characterized proprioceptive neurons in the mesencephalic trigeminal nucleus that are involved in processing proprioceptive information from the face and oral cavity. This is a specific population of neurons that produce rapidly adapting mechanically-activated currents that are fully dependent on Piezo2. As such, we analyzed the deficits in balance and coordination caused by the selective deletion of the channel in proprioceptors (conditional knockout). The data clearly shows that Piezo2 fulfills a critical role in a defined homogeneous population of proprioceptor neurons that innervate the head muscles, demonstrating that this ion channel is essential for mammalian proprioceptive mechanotransduction.

Proprioceptive feedback mainly derives from groups Ia and II muscle spindle (MS) afferents and group Ib Golgi tendon organ (GTO) afferents, but the molecular correlates of these three afferent subtypes remain unknown. We performed single cell RNA sequencing of genetically identified adult proprioceptors and uncovered five molecularly distinct neuronal clusters. Validation of cluster-specific transcripts in dorsal root ganglia and skeletal muscle demonstrates that two of these clusters correspond to group Ia MS afferents and group Ib GTO afferent proprioceptors, respectively, and suggest that the remaining clusters could represent group II MS afferents. Lineage analysis between proprioceptor transcriptomes at different developmental stages provides evidence that proprioceptor subtype identities emerge late in development. Together, our data provide comprehensive molecular signatures for groups Ia and II MS afferents and group Ib GTO afferents, enabling genetic interrogation of the role of individual proprioceptor subtypes in regulating motor output.

We thank Ardem Patapoutian and Paul Heppenstall for providing the Piezo2 fl/fl and Advillin-Cre mice respectively, and Salvador Sala for helping with current kinetic analysis. Mireille Tora and Eva Quintero are acknowledged for excellent technical assistance and Carlos Ramos and Sergio Javaloy for illustrations. We also thanks all members of the Sensory Transduction and Nociception Group for useful comments and suggestions. The authors gratefully acknowledge the financial support received by the Spanish Government projects: SAF2016-77233R (A.Gomis and F.V.

Anatomical and physiological analyses have long revealed differences between proprioceptive groups Ia, II, and Ib sensory neurons, yet the molecular correlates of these three muscle afferent subtypes remain unknown. We performed single cell RNA sequencing of genetically identified adult proprioceptors and, using unbiased bioinformatics approaches, detected five molecularly distinct neuronal clusters. Validation of cluster-specific transcripts in dorsal root ganglia (DRG) and skeletal muscle provides evidence these clusters correspond to functionally distinct muscle spindle (MS) or Golgi tendon organ (GTO) afferent proprioceptors. Remarkably, while we uncovered just one type of GTO afferents, four of the five clusters represent MS afferents, thus demonstrating a previously unappreciated diversity among these muscle proprioceptors. In vitro electrophysiological recordings reveal just two broadly distinct proprioceptor types, and suggest that the refinement of functional subtype diversity may occur along multiple axes of maturation. Lineage analysis between proprioceptor transcriptomes at different developmental stages show little or no correlation for transcripts that define adult MS or GTO afferents, supporting the idea that proprioceptor subtype identity emerges late in development. Together, our data provide the first comprehensive molecular signature for groups Ia and II MS afferents and group Ib GTO afferents, and offer new strategies for genetic interrogation of the role of these individual proprioceptor subtypes in regulating voluntary motor behavior.

total dimensions of 600 Â 600 Â 100 mm. Pillars of different height with cavities on top, which act as attachment sites for the somata of the neurons, are connected to each other through channels to guide the neurites from one pillar to the next in a precise predefined way. The inner diameter of the channels, 3.6 mm, is tailored to fit the axon-diameter of 2-3 mm. To specifically enhance cell attachment, poly-D-lysin is printed directly into the cavities of the pillars. The substrate is cultured with granule cells from murine cerebella. Cell activity after at least 5 days in vitro is shown through basic patch-clamp measurements. The work presented here is a prove of principle for an approach to build complex, tailor-made 3D neural circuits.

The formation of the visual system is a complex multistep process that includes proper assembly between retinal ganglion cell (RGC) axon terminals and their relay neurons in the different visual nuclei of the brain. RGC axons reach the main image-forming nuclei (IFN) —the superior colliculus and the lateral geniculate nucleus— at perinatal stages and extensively arborize to then refine throughout the first postnatal weeks. Spontaneous activity generated in the immature retina plays an essential role in the fine tune refinement of exhuberant axonal arborizations but the molecular mechanisms underlying this activity-dependent remodeling process remain poorly characterized. RGC axons, in addition to innervate IFN, target non-image forming nuclei (NIFN), but the impact of spontaneous retinal activity in the development of these accessory nuclei has not yet been described. Here, by genetically altering spontaneous activity in the RGCs of mice we demonstrate that correlated retinal activity also shapes the connectivity of the non-image forming circuit and identify the transcriptional programs modulating this process. Together, our data contribute to a better understanding of the molecular mechanisms ruling activity-dependent axon refining in the building of visual circuits.