Segmental organization of vestibulospinal inputs to spinal interneurons mediating crossed activation of thoracolumbar motoneurons in the neonatal mouse.

Vestibulospinal pathways activate contralateral motoneurons (MNs) in the thoracolumbar spinal cord of the neonatal mouse exclusively via axons descending ipsilaterally from the vestibular nuclei via the lateral vestibulospinal tract (LVST; Kasumacic et al., 2010). Here we investigate how transmission from the LVST to contralateral MNs is mediated by descending commissural interneurons (dCINs) in different spinal segments. We test the polysynaptic nature of this crossed projection by assessing LVST-mediated ventral root (VR) response latencies, manipulating synaptic responses pharmacologically, and tracing the pathway transynaptically from hindlimb extensor muscles using rabies virus (RV). Longer response latencies in contralateral than ipsilateral VRs, near-complete abolition of LVST-mediated calcium responses in contralateral MNs by mephenesin, and the absence of transsynaptic RV labeling of contralateral LVST neurons within a monosynaptic time window all indicate an overwhelmingly polysynaptic pathway from the LVST to contralateral MNs. Optical recording of synaptically mediated calcium responses identifies LVST-responsive ipsilateral dCINs that exhibit segmental differences in proportion and dorsoventral distribution. In contrast to thoracic and lower lumbar segments, in which most dCINs are LVST responsive, upper lumbar segments stand out because they contain a much smaller and more ventrally restricted subpopulation of LVST-responsive dCINs. A large proportion of these upper lumbar LVST-responsive dCINs project to contralateral L5, which contains many of the hindlimb extensor MNs activated by the LVST. A selective channeling of LVST inputs through segmentally and dorsoventrally restricted subsets of dCINs provides a mechanism for targeting vestibulospinal signals differentially to contralateral trunk and hindlimb MNs in the mammalian spinal cord.

Vestibular-mediated synaptic inputs and pathways to sympathetic preganglionic neurons in the neonatal mouse.

To assess when vestibulosympathetic projections become functional postnatally, and to establish a preparation in which vestibulosympathetic circuitry can be characterized more precisely, we used an optical approach to record VIIIth nerve-evoked synaptic inputs to thoracic sympathetic preganglionic neurons (SPNs) in newborn mice. Stimulation of the VIIIth nerve was performed in an isolated brainstem-spinal cord preparation after retrogradely labelling with the fluorescent calcium indicator Calcium Green 1-conjugated dextran amine, the SPNs and the somatic motoneurons (MNs) in the thoracic (T) segments T2, 4, 6, 8, 10 and 12. Synaptically mediated calcium responses could be visualized and recorded in individual SPNs and MNs, and analysed with respect to latency, temporal pattern, magnitude and synaptic pharmacology. VIIIth nerve stimulation evoked responses in all SPNs and MNs investigated. The SPN responses had onset latencies from 90 to 200 ms, compared with much shorter latencies in MNs, and were completely abolished by mephenesin, a drug that preferentially reduces polysynaptic over monosynaptic transmission. Bicuculline and picrotoxin, but not strychnine, increased the magnitudes of the SPN responses without changing the onset latencies, suggesting a convergence of concomitant excitatory and inhibitory synaptic inputs. Lesions strategically placed to test the involvement of direct vestibulospinal pathways versus indirect pathways within the brainstem showed that vestibulosympathetic inputs in the neonate are mediated predominantly, if not exclusively, by the latter. Thus, already at birth, synaptic connections in the vestibulosympathetic reflex are functional and require the involvement of the ventrolateral medulla as in adult mammals.

Segmental patterns of vestibular-mediated synaptic inputs to axial and limb motoneurons in the neonatal mouse assessed by optical recording.

Proper control of movement and posture occurs partly via descending projections from the vestibular nuclei to spinal motor circuits. Days before birth in rodents, vestibulospinal neurons develop axonal projections that extend to the spinal cord. How functional these projections are just after birth is unknown. Our goal was to assess the overall functional organization of vestibulospinal inputs to spinal motoneurons in a brainstem-spinal cord preparation of the neonatal mouse (postnatal day (P) 0-5). Using calcium imaging, we recorded responses evoked by electrical stimulation of the VIIIth nerve, in many motoneurons simultaneously throughout the spinal cord (C2, C6, T7, L2 and L5 segments), in the medial and lateral motor columns. Selective lesions in the brainstem and/or spinal cord distinguished which tracts contributed to the responses: those in the cervical cord originated primarily from the medial vestibulospinal tracts but with a substantial contribution from the lateral vestibulospinal tract; those in the thoracolumbar cord originated exclusively from the lateral vestibulospinal tract. In the thoracolumbar but not the cervical cord, excitatory commissural connections mediated vestibular responses in contralateral motoneurons. Pharmacological blockade of GABA(A) receptors showed that responses involved a convergence of excitatory and inhibitory inputs which in combination produced temporal response patterns specific for different segmental levels. Our results show that by birth vestibulospinal projections in rodents have already established functional synapses and are organized to differentially regulate activity in neck and limb motoneurons in a tract- and segment-specific pattern similar to that in adult mammals. Thus, this particular set of descending projections develops several key features of connectivity appropriately at prenatal stages. We also present novel information about vestibulospinal inputs to axial motoneurons in mammals, providing a more comprehensive platform for future studies into the overall organization of vestibulospinal inputs and their role in regulating postural stability.

Xenotransplantation of human stem cells into the chicken embryo.

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

Chimeric animal models in human stem cell biology.

The clinical use of stem cells for regenerative medicine is critically dependent on preclinical studies in animal models. In this review we examine some of the key issues and challenges in the use of animal models to study human stem cell biology-experimental standardization, body size, immunological barriers, cell survival factors, fusion of host and donor cells, and in vivo imaging and tracking. We focus particular attention on the various imaging modalities that can be used to track cells in living animals, comparing their strengths and weaknesses and describing technical developments that are likely to lead to new opportunities for the dynamic assessment of stem cell behavior in vivo. We then provide an overview of some of the most commonly used animal models, their advantages and disadvantages, and examples of their use for xenotypic transplantation of human stem cells, with separate reviews of models involving rodents, ungulates, nonhuman primates, and the chicken embryo. As the use of human somatic, embryonic, and induced pluripotent stem cells increases, so too will the range of applications for these animal models. It is likely that increasingly sophisticated uses of human/animal chimeric models will be developed through advances in genetic manipulation, cell delivery, and in vivo imaging.