Anatomical development of the cerebellothalamic tract in embryonic mice

Abstract

Cerebellar projections to the thalamus are a pivotal connection in cerebello-cerebral interactions. Apart from its role in coordinating sensorimotor integration in the adult brain, the cerebello-thalamic projection has also been implemented in developmental disorders, such as autism spectrum disorders. Although the development of the cerebellum, thalamus and cerebral cortex have been studied in many species, a detailed description of the ontogeny of the mammalian cerebello-thalamic tract (CbT) is currently missing. Here we investigated the development of the CbT at embryonic stages using transgenic Ntsr1-Cre/Ai14 mice and in utero electroporation (IUE) of wild type mice. Wide-field, confocal and 3D light-sheet imaging of immunohistochemical stainings showed that CbT fibers arrive in the prethalamus between E14.5 and E15.5, but only invade the thalamus after E16.5. We quantified the spread of CbT fibers throughout the various thalamic nuclei and found that at E17.5 and E18.5 the ventrolateral, ventromedial and parafascicular nuclei, but also the mediodorsal and posterior complex become increasingly innervated. Several CbT fiber varicosities colocalize with vGluT2, indicating that already from E18.5 the CbT synapse in various thalamic nuclei. Our results contribute to the construction of a frame of reference on the anatomical development of the CbT, which will help to guide future experiments investigating neurodevelopmental disorders.Significance statementUsing various microscopic approaches, we investigated the anatomical development of the fiber tract between the cerebellum and thalamus, one of the major mammalian brain connections. Our results show that in mice cerebellar axons wait outside of the thalamus from embryonic day (E)15.5 until E17.5 before invading the thalamic complex. Cerebellar axons establish vGluT2-positive synapses at E18.5 throughout various thalamic nuclei, each of which subsequently develops its connections with dedicated cerebral cortical regions. Our data thereby advocate the cerebellar influence on the maturation of the thalamus and connected cerebral cortex. This knowledge can help to guide future experiments into neurodevelopmental disorders affecting cerebello-thalamo-cortical networks.

Diverse inhibitory projections from the cerebellar interposed nucleus

“…All specimens exhibited dense terminal fields in the ventromedial (VM) and anterior ventrolateral (VAL) nuclei of the thalamus ( Teune et al, 2000 ; Aumann et al, 1994 ; Houck and Person, 2015 ; Kalil, 1981 ; Low et al, 2018 ; Stanton, 1980 ). Additionally, we observed terminals in intralaminar thalamic structures including centromedial (CM), paracentral (PCN), mediodorsal (MD), parafascicular (PF), ventral posterior (VP), and posterior (PO) nuclei ( Teune et al, 2000 ; Chen et al, 2014 ; Dumas et al, 2019 ). Int-Ntsr1 neurons formed nucleocortical mossy fibers in multiple lobules across the cortex ( Figure 3G–I ; Gao et al, 2016 ; Houck and Person, 2015 ; Tolbert et al, 1978 ; Low et al, 2018 ; Sathyamurthy et al, 2020 ).…”

Temporal dynamics of the cerebello‐cortical convergence in ventro‐lateral motor thalamus

“…2007) as well as glutamatergic, thalamus‐projecting CN neurons (Houck & Person, 2015; Dumas et al . 2019). The colony was originally purchased from the MMRRC repository and maintained by backcrossing with C57Bl/6 OlaHsd mice.…”

“…All animal protocols were approved by the Dutch national experimental animal committee (DEC). For all experiments Tg (Ntsr1-cre)GN220Gsat (Ntsr1-Cre) transgenic mice were used, which in combination with Cre-dependent AAV constructs allows expression in L6 neurons (Gong et al 2007) as well as glutamatergic, thalamus-projecting CN neurons (Houck & Person, 2015;Dumas et al 2019). The colony was originally purchased from the MMRRC repository and maintained by backcrossing with C57Bl/6 OlaHsd mice.…”

Widespread inhibitory projections from the cerebellar interposed nucleus

“…All specimens exhibited dense terminal fields in the ventromedial (VM) and anterior ventrolateral (VAL) nuclei of the thalamus (Teune et al 2000; Aumann et al, 1994; Houck & Person, 2015; Kalil, 1981; Low et al, 2018; Stanton, 1980). Additionally, we observed terminals in intralaminar thalamic structures including: centromedial (CM), paracentral (PCN), mediodorsal (MD), parafascicular (PF), ventral posterior (VP), and posterior (PO) nuclei (Teune et al, 2000; Chen et al, 2014; Dumas et al, 2019). Int-Ntsr1 neurons formed nucleocortical mossy fibers in multiple lobules across the cortex (Fig.…”

“…Finally, we compared these populations to putative excitatory neuron cell sizes. First, we validated Ntsr1-Cre (gn220) as a cerebellar nuclear cre driver line labeling non-GABAergic neurons (Dumas et al, 2019). Examination of publicly available dual fluorescent in situ hybridization expression showed non-overlap of Ntsr1-Cre expression with Gad1 ( Figure S1A).…”