Abstract
BackgroundThe neonatal mouse has become a model system for studying the locomotor function of the lumbar spinal cord. However, information about the synaptic connectivity within the governing neural network remains scarce. A neurotropic pseudorabies virus (PRV) Bartha has been used to map neuronal connectivity in other parts of the nervous system, due to its ability to travel trans-neuronally. Its use in spinal circuits regulating locomotion has been limited and no study has defined the time course of labelling for neurons known to project monosynaptically to motoneurons.Methodology/Principal FindingsHere we investigated the ability of PRV Bartha, expressing green and/or red fluorescence, to label spinal neurons projecting monosynaptically to motoneurons of two principal hindlimb muscles, the tibialis anterior (TA) and gastrocnemius (GC). As revealed by combined immunocytochemistry and confocal microscopy, 24–32 h after the viral muscle injection the label was restricted to the motoneuron pool while at 32–40 h the fluorescence was seen in interneurons throughout the medial and lateral ventral grey matter. Two classes of ipsilateral interneurons known to project monosynaptically to motoneurons (Renshaw cells and cells of origin of C-terminals) were consistently labeled at 40 h post-injection but also a group in the ventral grey matter contralaterally. Our results suggest that the labeling of last order interneurons occurred 8–12 h after motoneuron labeling and we presume this is the time taken by the virus to cross one synapse, to travel retrogradely and to replicate in the labeled cells.Conclusions/SignificanceThe study establishes the time window for virally - labelling monosynaptic projections to lumbar motoneurons following viral injection into hindlimb muscles. Moreover, it provides a good foundation for intracellular targeting of the labeled neurons in future physiological studies and better understanding the functional organization of the lumbar neural networks.
Highlights
The mouse lumbar spinal cord has become a model system in which to study locomotor function because the underlying neural networks can be activated chemically or electrically and studied in the absence of an instructive input drive
To distinguish between the labeling of motoneurons and other spinal neurons we compared the pattern of viral labeling with that of the motoneuron marker choline acetyltransferase (ChAT) or the retrograde marker Fluorogold
This approach allowed us to establish when motoneurons first became labeled with the virus and when the first non-cholinergic interneurons were labeled
Summary
The mouse lumbar spinal cord has become a model system in which to study locomotor function because the underlying neural networks can be activated chemically or electrically and studied in the absence of an instructive input drive. Methods for identifying the synaptic connections within the network remain elusive One approach to this problem is to use neurotropic viruses that can propagate trans-synaptically [7,8,9,10,11,12,13]. No study has systematically investigated the time course of the viral propagation into spinal motoneurons and/or interneurons and established its ability to label neurons - such as Renshaw cells - that are known to project monosynaptically to motoneurons. This is important because the time of viral transfer may vary in different groups of neurons. Its use in spinal circuits regulating locomotion has been limited and no study has defined the time course of labelling for neurons known to project monosynaptically to motoneurons
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