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Among the spinocerebellar projections vital for sensorimotor coordination of limbs and the trunk, the morphology of spinocerebellar axons originating from the lumbar cord has not been well characterized compared to those from thoracic and sacral cords. We reconstructed 26 single spinocerebellar axons labeled by biotinylated dextran injections into the gray matter of the lumbar spinal cord in mice. Axon terminals were mapped with the zebrin pattern of the cerebellar cortex. Reconstructed axons were primarily classified into ipsilaterally and contralaterally ascending axons, arising mainly from the dorsal and ventral horns, respectively. The majority of ipsilateral and contralateral axons took the dorsal‐medullary and ventral‐pontine pathways, respectively. The axons of both groups terminated mainly in the vermal and medial paravermal areas of lobules II–V and VIII–IXa, often bilaterally but predominantly ipsilateral to the axonal origin, with a weak preference to particular portions of zebrin stripes. The ipsilateral axons originating from the medial dorsal horn in the upper lumbar cord (n = 3) had abundant (43–147) mossy fiber terminals and no medullary collaterals. The ipsilateral axons originating from the lateral dorsal horn in the lower lumbar cord (n = 9) and the contralateral axons (n = 14) showed remarkable morphology variations. The number of their mossy fiber terminals varied from 2 to 172. Their collaterals, observed in 17 axons out of 23, terminated mainly in the medial cerebellar nucleus, nucleus X, and lateral reticular nucleus in various degrees. The results indicated that the lumbar spinocerebellar projection contains highly heterogeneous axonal populations regarding their pathway, branching, and termination patterns. [ABSTRACT FROM AUTHOR]

Reconstruction of the axonal projection patterns of single neurons has been an important tool for understanding both the diversity of cell types in the brain and the logic of information flow between brain regions. Innovative approaches now enable the complete reconstruction of axonal projection patterns of individual neurons with vastly increased throughput. Here, we review how advances in genetic, imaging, and computational techniques have been exploited for axonal reconstruction. We also discuss how new innovations could enable the integration of genetic and physiological information with axonal morphology for producing a census of cell types in the mammalian brain at scale. [ABSTRACT FROM AUTHOR]

The basilar pontine nucleus (PN) is the key relay point for the cerebrocerebellar link. However, the projection pattern of pontocerebellar mossy fiber axons, which is essential in determining the functional organization of the cerebellar cortex, has not been fully clarified. We reconstructed the entire trajectory of 25 single pontocerebellar mossy fiber axons labeled by localized injection of biotinylated dextran amine into various locations in the PN and mapped all their terminals in an unfolded scheme of the cerebellum in 10 mice. The majority of axons (20/25 axons) entered the cerebellum through the middle cerebellar peduncle contralateral to the origin, while others entered through the ipsilateral pathway. A small number of axons (1/25 axons) had collaterals terminating in the cerebellar nuclei. Axons projected mostly to a combination of lobules, often bilaterally, and terminated in multiple zebrin (aldolase C) stripes, more frequently in zebrin‐positive stripes (83.9%) than in zebrin‐negative stripes, with 66.5 mossy fiber terminals on the average. Axons originating from the rostral (plus medial and lateral), central and caudal PN mainly terminated in the paraflocculus, crus I and lobule VIb–c, in the simplex lobule, crus II and paramedian lobule, and in lobules II–VIa, VIII and copula pyramidis, respectively. The results suggest that the interlobular branching pattern of pontocerebellar axons determines the group of cerebellar lobules that are involved in a related functional localization of the cerebellum. In the hemisphere, crus I may be functionally distinct from neighboring lobules (simple lobule and crus II) in the mouse cerebellum based on the pontocerebellar axonal projection pattern. [ABSTRACT FROM AUTHOR]

Abstract: The spinocerebellar projection has an essential role in sensorimotor coordination of limbs and the trunk. Multiple groups of spinocerebellar projections have been identified in retrograde labeling studies. In this study, we aimed at characterizing projection patterns of these groups using a combination of anterograde labeling of the thoracic spinal cord and aldolase C immunostaining of longitudinal stripes of the cerebellar cortex in the mouse. We reconstructed 22 single spinocerebellar axons, wholly in the cerebellum and brain stem and partly, in the spinal cord. They were classified into three groups, (a) non‐crossed axons of Clarke's column neurons (NCC, 8 axons), (b) non‐crossed axons of marginal Clarke's column neurons (NMCC, 7 axons), and (c) crossed axons of neurons in the medial ventral horn (CMVH, 7 axons), based on previous retrograde labeling studies. While NCC axons projected mainly to multiple bilateral stripes in vermal lobules II–IV and VIII–IX, and the ipsilateral medial cerebellar nucleus, NMCC axons projected mainly to ipsilateral stripes in paravermal lobules II–V and copula pyramidis, and the anterior interposed nucleus. CMVH axons projected bilaterally to multiple stripes in lobules II–V with a small number of terminals but had abundant collaterals in the spinal cord and medullary reticular nuclei as well as in the vestibular and cerebellar nuclei. The results indicate that, while CMVH axons overlap with propriospinal and spinoreticular projections, NCC and NMCC axons are primarily spinocerebellar axons, which seem to be involved in relatively more proximal and distal sensorimotor controls, respectively. [ABSTRACT FROM AUTHOR]

The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain.

Advances in microscopy and sample preparation have led to the first ever mapping of individual neurons in the whole mouse brain.

Neural tissue engineering is an attractive therapeutic strategy following neurotrauma, with the potential to replace neural cells and circuitry in order to improve neurological function. We are building on our previously reportedmicro-tissue engineered neural networks (micro-TENNs),which are miniature constructs grown in vitro that consist of discrete neuronal populations spanned by long axonal tracts within a protective tubular micro-column. Micro-TENNs are the first transplantable neural networks thatmimic gray-white matter architecture, and thereforemay be useful for the targeted neurosurgical reconstruction of vulnerable neural populations and long-distance axonal pathways following trauma. In the current study, we advanced our micro-tissue engineering techniques be seeding microcolumns with engineered neuronal aggregates rather than traditional methodology of seeding with dissociated neurons lacking organization. Here, micro-columns with an agarose outer-shell (345-701μm) and collagen-laminin inner-core (180-350 μm) were seeded with aggregated or dissociated cerebral cortical neurons. Neuronal aggregates exhibited rapid neurite extension within the micro-columns, attaining outgrowth rates of > 500 μm/day versus < 100 μm/day from dissociated neurons. Aggregate micro-TENNs demonstrated improved neuronal architecture and more robust axonal tracts. Micro-TENNs built using aggregate or dissociated neurons were then stereotaxically microinjected to mimic cortical-thalamic pathways in rats. At 1 week and 1 month post-implant, neuronal network survival and host inflammatory/gliotic responses were assessed via immunohistochemistry and microscopy. Implanted micro-TENN neurons survived and maintained their axonal architecture. A modest astrogliotic response was observed at the micro-TENN-host tissue interface, and this response waned as the hydrogel encasement was gradually resorbed. Neural network survival, neurite integration, and host astrogliotic responses are currently being quantified based on the neuronal architecture andmicro-column dimensions. Micro-TENNs may represent a novel platform to treat a range of neurological disorders by simultaneously replacing neurons as well as their long-distance axonal connections. [ABSTRACT FROM AUTHOR]

Advances in microscopy and sample preparation have led to the first ever mapping of individual neurons in the whole mouse brain.

The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain.