Multiple cerebellar zones are involved in the control of individual muscles: a retrograde transneuronal tracing study with rabies virus in the rat

Abstract

To identify cerebellar regions that are involved in the control of limb muscles, rabies virus was injected into the tibialis anterior (TA), the gastrocnemius (GC) or, for comparison, into the flexor digitorum (FD) muscles of the rat. Progression of retrograde transneuronal infection at supraspinal levels was assessed after variable time spans and was divided into three groups. Initially, infected neurons were observed in the reticular formation, lateral vestibular nucleus, red nucleus and motor cortex (group 1). Group 2 was characterized by labelling within the cerebellar nuclei as well as of two vermal strips of Purkinje cells (PCs). Double-labelling with zebrin enabled identification of these strips as the lateral part of the A1- and B-zone. For TA both zones were ipsilateral, whereas for GC the A1 strip predominated contralaterally. Group 3 infections showed additional labelling of multiple, in part bilateral, identifiable strips of PCs in vermis, paravermis and hemisphere. FD injections resulted in less robust labelling of vermal strips and more pronounced labelling within paravermal and hemispheral zonal regions. Only sporadic labelling in corresponding regions of the inferior olive and no labelling of cortical interneurons or granule cells was observed. Prolonged infection was seen to result in degeneration of PCs and possibly of motoneurons. We conclude that vermal, paravermal as well as hemispheral zones of the cerebellar cortex converge upon motoneurons that innervate a particular muscle. In addition, individual zones may control motorpools of different muscles and thus contribute to muscle synergies.

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

“…RV has proved to be a powerful retrograde transneuronal tracer for characterizing synaptically connected networks in adult rodents (Kučera et al, 1985;Ugolini, 1995;Tang et al, 1999;Graf et al, 2002;Morcuende et al, 2002;Bras et al, 2008;Ruigrok et al, 2008) and primates (Grantyn et al, 2002;Moschovakis et al, 2004;Strick, 2006, 2009;Ugolini et al, 2006) after peripheral inoculations. The effectiveness and reliability of the RV tool are largely because RV is transported only retrogradely in the central nervous system (CNS) (reviewed in Kelly and Strick, 2000;Ugolini, 2010) and does not infect the sympathetic neurons after a peripheral inoculation (Kučera et al, 1985;Tang et al, 1999;Ruigrok et al, 2008), in contrast to both herpes simplex type 1 virus (Ugolini, 1992) and PRV (Rotto-Percelay et al, 1992;Kerman et al, 2003). In the present study, a conventional laboratory strain of RV (Challenge Virus Standard [CVS]) was used in neonate mice to map the lumbar premotor network connected to motoneurons (MNs) innervating ankle extensor muscles (triceps surae [TS]).…”

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.…”

“…These modules are based on the matching organizations of projections from the olivocerebellar climbing fibers to longitudinal strips or zones of the Purkinje cells, the projections from these Purkinje cells to the cerebellar nuclei and their subsequent and selective projection patterns Teune et al, 2000;Sugihara and Shinoda, 2004;Voogd and Ruigrok, 2004;Pijpers et al, 2005;Apps and Hawkes, 2009;Ruigrok, 2011). The resulting parasagittally organized circuits are referred to as modules, some of which have been subdivided into micromodules, and are seen as the functional units of the cerebellum (Apps and Garwicz, 2005;Ruigrok et al, 2008;Ruigrok, 2011). Recent studies show that the distribution of pontocerebellar connections, at least partly, may be closely related to that of climbing fibers (Odeh et al, 2005;.…”