Characterization of last‐order premotor interneurons by transneuronal tracing with rabies virus in the neonatal mouse spinal cord

Abstract

We characterized the interneurons involved in the control of ankle extensor (triceps surae [TS] muscles) motoneurons (MNs) in the lumbar enlargement of mouse neonates by retrograde transneuronal tracing using rabies virus (RV). Examination of the kinetics of retrograde transneuronal transfer at sequential intervals post inoculation enabled us to determine the time window during which only the first-order interneurons, i.e., interneurons likely monosynaptically connected to MNs (last-order interneurons [loINs]) were RV-infected. The infection of the network resulted exclusively from a retrograde transport of RV along the motor pathway. About 80% of the loINs were observed ipsilaterally to the injection. They were distributed all along the lumbar enlargement, but the majority was observed in L4 and L5 segments where TS MNs were localized. Most loINs were distributed in laminae V-VII, whereas the most superficial laminae were devoid of RV infection. Contralaterally, commissural loINs were found essentially in lamina VIII of all lumbar segments. Groups of loINs were characterized by their chemical phenotypes using dual immunolabeling. Glycinergic neurons connected to TS MNs represented 50% of loINs ipsilaterally and 10% contralaterally. As expected, the ipsilateral glycinergic loINs included Renshaw cells, the most ventral neurons expressing calbindin. We also demonstrated a direct connection between a group of cholinergic interneurons observed ipsilaterally in L3 and the rostral part of L4, and TS MNs. To conclude, transneuronal tracing with RV, combined with an immunohistochemical detection of neuronal determinants, allows a very specific mapping of motor networks involved in the control of single muscles.

Identification of a cellular node for motor control pathways

“…This approach is based on co-infecting motorneurons with genetically modified rabies virus (RabΔG) and adeno-associated virus (AAV) encoding glycoprotein (AAV:G) 11,12 . Experiments were performed on mice between postnatal days 0–15 (P0–P15) because this time window provides the most efficient trans-synaptic labeling, with a minimum of neuronal toxicity, and because the distribution of premotor neurons is similar between pups and adults 13,14 .…”

Organization of Cerebral Projections to Identified Cerebellar Zones in the Posterior Cerebellum of the Rat

“…The injectate contained a mixture of 1 part 1% CTb (low salt; List Biological Laboratories, 1% w/v in 0.2 M phosphate buffer, pH 7.4) and 4 parts RV Prevosto et al, 2009). For these experiments the "French" subtype of Challenge Virus Standard (CVS) strain was used (for a clarification on the different CVS strains, see Ugolini, 2010), which has been used in previous RV tracing experiments (Ugolini, 1995;Ugolini et al, 2006;Ruigrok et al, 2008;Salin et al, 2008Salin et al, , 2009Coulon et al, 2011). RV consisted of a cell culture supernatant in minimal essential medium titrating 4 ϫ 10 7 plaque-forming units/ml.…”

“…See also Apps and Hawkes (2009 monoclonal antibody diluted 1:5000 in PBS that contained 2% normal horse serum and 0.5% Triton X-100 (PBSϩ). This antibody (31G10) was isolated in the laboratory Virologie Moléculaire et Structurale (formerly Génétique des Virus), Gif-sur-Yvette, France, during the fusion experiment described by Raux et al (1997) and has been used in many tracing studies using rabies virus (Viemari et al, 2004;Ruigrok et al, 2008;Coulon et al, 2011). Subsequently, sections were rinsed before they were incubated in a rabbit anti-mouse horseradish peroxidase (P260 Dako, 1:200 in PBSϩ, 90 min at RT) After rinsing, the vials were incubated for 20 -25 min in a 3,3Ј-diaminobenzidine-tetrahydrochloride solution (DABϩ: 0.025% DAB and 0.005% H 2 O 2 in 0.05 M PB, at RT) for visualization.…”

“…Previous reports have indicated that infection of first-order neurons requires more time than the sequentially occurring labeling of second-and thirdorder labeling (Ugolini, 2010). Moreover, species type, type and titer of the rabies virus used, and characteristics of the pathway investigated may all induce variations in the speed of uptake, transport, replication and transneuronal infection characteristics (Ugolini, 1995(Ugolini, , 2010Ruigrok et al, 2008;Coulon et al, 2011). In the present study, we want to describe the disynaptic cerebrocerebellar connection, dominated by the cerebro-ponto-cerebellar pathway, which required that the virus should not exceed secondorder infection stages.…”

“…In the present study, we want to describe the disynaptic cerebrocerebellar connection, dominated by the cerebro-ponto-cerebellar pathway, which required that the virus should not exceed secondorder infection stages. Based on earlier experience Salin et al, 2008;Coulon et al, 2011), three survival times, 30, 50, and 70 h, were selected for initial examination. Fig.…”