Marie-Claude Perreault

Evaluation of intracellular labeling with micron-sized particles of iron oxide (MPIOs) as a general tool for in vitro and in vivo tracking of human stem and progenitor cells.

Magnetic resonance imaging (MRI)-based tracking is increasingly attracting attention as a means of better understanding stem cell dynamics in vivo. Intracellular labeling with micrometer-sized particles of iron oxide (MPIOs) provides a practical MRI-based approach due to superior detectability relative to smaller iron oxide particles. However, insufficient information is available about the general utility across cell types and the effects on cell vitality of MPIO labeling of human stem cells. We labeled six human cell types from different sources: mesenchymal stem cells derived from bone marrow (MSCs), mesenchymal stem cells derived from adipose tissue (ASCs), presumptive adult neural stem cells (ad-NSCs), fetal neural progenitor cells (f-NPCs), a glioma cell line (U87), and glioblastoma tumor stem cells (GSCs), with two different sizes of MPIOs (0.9 and 2.84 μm). Labeling and uptake efficiencies were highly variable among cell types. Several parameters of general cell function were tested in vitro. Only minor differences were found between labeled and unlabeled cells with respect to proliferation rate, mitotic duration, random motility, and capacity for differentiation to specific phenotypes. In vivo behavior was tested in chicken embryos and severe combined immunodeficient (SCID) mice. Postmortem histology showed that labeled cells survived and could integrate into various tissues. MRI-based tracking over several weeks in the SCID mice showed that labeled GSCs and f-NPCs injected into the brain exhibited translocations similar to those seen for unlabeled cells and as expected from migratory behavior described in previous studies. The results support MPIO-based cell tracking as a generally useful tool for studies of human stem cell dynamics in vivo.

Organization of Functional Synaptic Connections between Medullary Reticulospinal Neurons and Lumbar Descending Commissural Interneurons in the Neonatal Mouse

The medullary reticular formation (MRF) of the neonatal mouse is organized so that the medial and lateral MRF activate hindlimb and trunk motoneurons (MNs) with differential predominance. The goal of the present study was to investigate whether this activation is polysynaptic and mediated by commissural interneurons with descending axons (dCINs) in the lumbar spinal cord. To this end, we tested the polysynapticity of inputs from the MRF to MNs and tested for the presence of selective inputs from medial and lateral MRF to 574 individual dCINs in the L2 segment of the neonatal mouse. Reticulospinal-mediated postsynaptic Ca2+ responses in MNs were reduced in the presence of mephenesin and after a midline lesion, suggesting the involvement of dCINs in mediating the responses. Consistent with this, stimulation of reticulospinal neurons in the medial or lateral MRF activated 51% and 57% of ipsilateral dCINs examined (255 and 352 dCINs, respectively) and 52% and 46% of contralateral dCINs examined (166 and 133 dCINs, respectively). The proportion of dCINs that responded specifically to stimulation of medial or lateral MRF was similar to the proportions of dCINs that responded to both MRF regions or to neither. The three responsive dCIN populations had largely overlapping spatial distributions. We demonstrate the existence of dCIN subpopulations sufficient to mediate responses in lumbar motoneurons from reticulospinal pathways originating from the medial and lateral MRF. Differential control of trunk and hindlimb muscles by the medullary reticulospinal system may therefore be mediated in part by identifiable dCIN populations.

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 GABAA 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.

Organization of common synaptic drive to motoneurones during fictive locomotion in the spinal cat

The basic locomotor rhythm in the cat is generated by a neuronal network in the spinal cord. The exact organization of this network and its drive to the spinal motoneurones is unknown. The purpose of the present study was to use time (cumulant density) and frequency domain (coherence) analysis to examine the organization of the last order drive to motoneurones during fictive locomotion (evoked by application of nialamide and dihydroxyphenylalanine (DOPA)) in the spinal cat. In all cats, narrow central synchronization peaks (half‐width < 3 ms) were observed in cumulants estimated between electroneurograms (ENGs) of close synergists, but not between nerves belonging to muscles acting on different joints or to antagonistic muscles. Coherence was not observed at frequencies above 100 Hz and was mainly observed between synergists. Intracellular recording was obtained from a population of 70 lumbar motoneurones. Significant short‐term synchronization was observed between the individual intracellular recordings and the ENGs recorded from nerves of the same pool and of close synergists. Recordings from 34 pairs of motoneurones (10 pairs belonged to the same motor pool, 11 pairs to close synergists and 13 pairs to antagonistic pools) failed to reveal any short‐lasting synchronization. These data demonstrate that short‐term synchronization during fictive locomotion is relatively weak and is restricted to close synergists. In addition, coherence analysis failed to identify any specific rhythmic component in the locomotor drive that could be associated with this synchronization. These results resemble findings obtained during human treadmill walking and imply that the spinal interneurones participating in the generation of the locomotor rhythm are themselves weakly synchronized. The restricted synchronization within the locomotor drive to motoneuronal pools may be a feature of the locomotor generating networks that is related to the ability of these networks to produce highly adaptive patterns of muscle activity during locomotion.

Differential origin of reticulospinal drive to motoneurons innervating trunk and hindlimb muscles in the mouse revealed by optical recording

To better understand how the brainstem reticular formation controls and coordinates trunk and hindlimb muscle activity, we used optical recording to characterize the functional connections between medullary reticulospinal neurons and lumbar motoneurons of the L2 segment in the neonatal mouse. In an isolated brainstem–spinal cord preparation, synaptically induced calcium transients were visualized in individual MNs of the ipsilateral and contralateral medial and lateral motor columns (MMC, LMC) following focal electrical stimulation of the medullary reticular formation (MRF). Stimulation of the MRF elicited differential responses in MMC and LMC, according to a specific spatial organization. Stimulation of the medial MRF elicited responses predominantly in the LMC whereas stimulation of the lateral MRF elicited responses predominantly in the MMC. This reciprocal response pattern was observed on both the ipsilateral and contralateral sides of the spinal cord. To ascertain whether the regions stimulated contained reticulospinal neurons, we retrogradely labelled MRF neurons with axons coursing in different spinal funiculi, and compared the distributions of the labelled neurons to the stimulation sites. We found a large number of retrogradely labelled neurons within regions of the gigantocellularis reticular nucleus (including its pars ventralis and alpha) where most stimulation sites were located. The existence of a mediolateral organization within the MRF, whereby distinct populations of reticulospinal neurons predominantly influence medial or lateral motoneurons, provides an anatomical substrate for the differential control of trunk and hindlimb muscles. Such an organization introduces flexibility in the initiation and coordination of activity in the two sets of muscles that would satisfy many of the functional requirements that arise during postural and non‐postural motor control in mammals.

Glutamatergic reticulospinal neurons in the mouse: developmental origins, axon projections, and functional connectivity

Subcortical descending glutamatergic neurons, such as reticulospinal (RS) neurons, play decisive roles in the initiation and control of many motor behaviors in mammals. However, little is known about the mechanisms used by RS neurons to control spinal motor networks because most of the neuronal elements involved have not been identified and characterized. In this review, we compare, in the embryonic mouse, the timing of developmental events that lead to the formation of synaptic connections between RS and spinal cord neurons. We then summarize our recent research in the postnatal mouse on the organization of synaptic connections between RS neurons and lumbar axial motoneurons (MNs), hindlimb MNs, and commissural interneurons. Finally, we give a brief account of some of the most recent studies on the intrinsic capabilities for plasticity of the mammalian RS system. The present review should give an updated insight into how functional specificity in RS motor networks emerges.

Contribution of morphology and membrane resistance to integration of fast synaptic signals in two thalamic cell types

Thalamocortical cells (TCs) and interneurons (INs) in the lateral geniculate nucleus process visual information from the retina. The TCs have many short dendrites, whereas the INs have fewer and longer dendrites. Because of these morphological differences, it has been suggested that transmission of synaptic signals from dendritic synapses to soma is more efficient in TCs than in INs. However, a higher membrane resistance (Rm) for the INs could, in theory, compensate for the attenuating effect of their long dendrites and allow distal synaptic inputs to significantly depolarize the soma. Compartmental models were made from biocytin filled TCs (n= 15) and INs (n= 3) and adjusted to fit the current‐ and voltage‐clamp recordings from the individual cells. The confidence limits for the passive electrical parameters were explored by simulating the influence of noise, morphometric errors and non‐uniform and active conductances. One of the useful findings was that Rm was accurately estimated despite realistic levels of active conductance. Simulations to explore the somatic influence of dendritic synapses showed that a small (0.5 nS) excitatory synapse placed at different dendritic positions gave similar somatic potentials in the individual TCs, within the TC population and also between TCs and INs. A linear increase in the conductance of the synapse gave increases in somatic potentials that were more sublinear in INs than TCs. However, when the total synaptic conductance was increased by simultaneously activating many small, spatially distributed synapses, the INs converted the synaptic signals to soma potentials almost as efficiently as the TCs. Thus, INs can transfer fast synaptic signals to soma as efficiently as TCs except when the focal conductance is large.

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.

Imaging synaptically mediated responses produced by brainstem inputs onto identified spinal neurons in the neonatal mouse

Descending inputs to spinal cord neurons in mammals have previously been characterized functionally using microelectrode recording of single neurons, a technique with high spatial and temporal resolution but low yield. Consequently our knowledge about the functional connections between the brain and the spinal cord has been accumulating at a very low pace. Here we describe a high throughput optical recording approach in an ex vivo brainstem-spinal cord preparation of the neonatal mouse that permits screening many spinal neurons simultaneously for synaptic inputs from descending axons. The fluorescent calcium indicator calcium green dextran amine was loaded retrogradely into specific spinal neuron populations, including motoneurons (MNs) of the medial and lateral motor columns and two populations of interneurons with descending axons (dINs) in the ventral funiculus. Focal electrical stimulation of brainstem neuron populations with descending axons generated synaptic responses revealed by transient increases in intracellular calcium concentration in all four populations of spinal neurons. The resultant fluorescence signals could be readily visualized in individual MNs directly through the ventral white matter. In the more deeply located dINs, responses could be readily visualized in individual neurons from the surface of an oblique cut through the spinal cord. The rapid optical investigation of functional connections between brainstem descending neurons and various populations of spinal neurons in the living mammalian preparation should help uncover some of the key features of supraspinal sensory and motor control and provide a valuable tool for examining the re-innervation of spinal neurons by descending axons after spinal cord regeneration.

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

•  When the body is tilted from horizontal to vertical, blood tends to accumulate in the legs, and blood pressure may fall in the upper body and head, a phenomenon known as orthostatic hypotension. •  The vestibulosympathetic reflexes triggered by stimulation of vestibular afferents help to counteract orthostatic hypotension, especially during its initial onset. •  We used an ex vivo preparation of the brainstem and spinal cord of the neonatal mouse, and high‐throughput optical recording to assess when vestibulosympathetic projections become functional postnatally. •  Vestibulosympathetic synaptic connections are present in the mouse, and are already functional at birth. The organization of the projections includes many of the key features seen in adult mammals. •  The demonstration of similarity between vestibulosympathetic pathways in mice and other mammals is exciting because mouse models provide the possibility to use powerful molecular genetic techniques to make discoveries that may be very relevant for human vestibulo‐autonomic dysfunction.