In vitro longitudinal lumbar spinal cord preparations to study sensory and recurrent motor microcircuits of juvenile mice

Abstract

In vitro spinal cord preparations have been extensively used to study microcircuits involved in the control of movement. By allowing precise control of experimental conditions coupled with state-of-the-art genetics, imaging and electrophysiological techniques, isolated spinal cords from mice have been an essential tool in detailing the identity, connectivity and function of spinal networks. The majority of the research has arisen from in vitro spinal cords of neonatal mice, which are still undergoing important postnatal maturation. Studies from adults have been attempted in transverse slices, however, these have been quite challenging due to the poor motoneuron accessibility and viability, as well as to the extensive damage to the motoneuron dendritic trees. In this work, we describe two types of coronal spinal cord preparations with either the ventral or the dorsal horn ablated, obtained from mice of different postnatal ages, spanning from pre-weaned to one month old. These semi-intact preparations allow recordings of sensory-afferent and motor-efferent responses from lumbar motoneurons using whole cell patch-clamp electrophysiology. We provide details of the slicing procedure and discuss the feasibility of whole-cell recordings. The in vitro dorsal and ventral horn-ablated spinal cord preparations described here are a useful tool to study spinal motor circuits in young mice that have reached the adult stages of locomotor development.

Pathophysiology of Dyt1 dystonia is mediated by spinal cord dysfunction

“…For whole cord recordings (P1 only), the cord was transected above the lumbosacral region, with lumbar dorsal or ventral roots slightly trimmed and kept intact. For single-cell recordings, the isolated lumbosacral cord was glued longitudinally to an agar block with the ventral or dorsal side up with intact roots, and an oblique cut above the L3 region was performed to allow visualization of the central canal 76 . The glued cord was then immersed in a vibratome chamber (Leica VT1200) with ice-cold aCSF (~2°C) comprising (in mM): 130 K-gluconate, 15 KCl, 0.05 EGTA, 20 HEPES, 25 D-glucose, 3 Na-kynurenic acid, 2 Na-pyruvate, 3 Myo-inositol, 1 Na-L-ascorbate, pH 7.4 with NaOH 77 .…”

“…For whole cord recordings (P1 only), the cord was transected above the lumbosacral region, with lumbar dorsal or ventral roots slightly trimmed and kept intact. For single-cell recordings, the isolated lumbosacral cord was glued longitudinally to an agar block with the ventral or dorsal side up with intact roots, and an oblique cut above the L3 region was performed to allow visualization of the central canal 76 Motoneurons distributed near the surface of the ventral and dorsal horn ablated preparations were targeted and visualized using an Eclipse E600FN Nikon microscope (Nikon) equipped with infrared differential interference contrast connected to a digital camera (Nikon DS-Qi1Mc).…”

Spinal microcircuits go through multiphasic homeostatic compensations in a mouse model of motoneuron degeneration

“…In oblique slices, motoneurons were clearly identifiable due to their large soma and anatomical clustering in the ventrolateral and dorsolateral regions (Figure 1A), whereas in longitudinal preparations they were distributed along the lateral rostro-caudal surface 33 . Functional identification was additionally performed, as motoneurons receive a characteristic monosynaptic excitation and disynaptic inhibition from motor efferents and sensory afferents following stimulation of ventral or dorsal roots respectively 22,33 . Putative Renshaw cells were identified by their location in the most ventral part of lamina VIII and by the expression of EGFP (Figure 3A-B).…”

“…The contribution of Renshaw cells to motoneuron conductance during high-frequency ventral root stimulation is maximum, thus the difference between the conductance during this period and motoneuron resting conductance was considered as measurement of the strength of recurrent inhibition. This method has been recently used, and was shown to be as efficient at estimating recurrent inhibition as the measurement of absolute synaptic currents in voltage clamp 33 . When measuring inhibition evoked by dorsal root stimulation, this method was not suitable, because it requires blockade of glutamate receptors in order to isolate the disynaptic inhibitory input from the monosynaptic excitation, which would have also blocked direct transmission to the Ia/b interneurons that are responsible for the disynaptic inhibition to motoneurons.…”

Pathophysiology of Dyt1- Tor1a dystonia in mice is mediated by spinal neural circuit dysfunction

“…This PDF file includes: Supplementary Materials and Methods Fig. S1 to S5 Table S1 References (66)(67)(68)(69)(70)(71)(72)(73)(74) Other Supplementary Material for this manuscript includes the following: Data file S1 Movies S1 to S7 MDAR Reproducibility Checklist View/request a protocol for this paper from Bio-protocol.…”