Disruption of cerebellar microzonal organization in GluD2 (GluRδ2) knockout mouse

Abstract

Cerebellar cortex has an elaborate rostrocaudal organization comprised of numerous microzones. Purkinje cells (PCs) in the same microzone show synchronous activity of complex spikes (CSs) evoked by excitatory inputs from climbing fibers (CFs) that arise from neurons in the inferior olive (IO). The synchronous CS activity is considered to depend on electrical coupling among IO neurons and anatomical organization of the olivo-cerebellar projection. To determine how the CF–PC wiring contributes to the formation of microzone, we examined the synchronous CS activities between neighboring PCs in the glutamate receptor δ2 knockout (GluD2 KO) mouse in which exuberant surplus CFs make ectopic innervations onto distal dendrites of PCs. We performed in vivo two-photon calcium imaging for PC populations to detect CF inputs. Neighboring PCs in GluD2 KO mice showed higher synchrony of calcium transients than those in wild-type (control) mice. Moreover, the synchrony in GluD2 KO mice hardly declined with mediolateral separation between PCs up to ~200 μm, which was in marked contrast to the falloff of the synchrony in control mice. The enhanced synchrony was only partially affected by the blockade of gap junctional coupling. On the other hand, transverse CF collaterals in GluD2 KO mice extended beyond the border of microzone and formed locally clustered ectopic synapses onto dendrites of neighboring PCs. Furthermore, PCs in GluD2 KO mice exhibited clustered firing (Cf), the characteristic CF response that was not found in PCs of wild-type mice. Importantly, Cf was often associated with localized calcium transients in distal dendrites of PCs, which are likely to contribute to the enhanced synchrony of calcium signals in GluD2 KO mice. Thus, our results indicate that CF signals in GluD2 KO mice propagate across multiple microzones, and that proper formation of longitudinal olivo-cerebellar projection is essential for the spatiotemporal organization of CS activity in the cerebellum.

A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo

“…In the cerebellar cortex, spontaneous calcium transients in Purkinje cell dendrites have been shown to be triggered by complex spikes evoked by climbing fiber inputs (Ozden et al ., 2009 ; Schultz et al ., 2009 ; Kitamura & Häusser, 2011 ). Cal-520 AM or OGB-1 AM was bolus loaded into the cerebellar molecular layer of the cerebellar cortex (Mukamel et al ., 2009 ; Ozden et al ., 2009 ; Schultz et al ., 2009 ; Hashizume et al ., 2013 ). Cal-520 or OGB-1 was penetrated into various cell types, including Purkinje cells, molecular layer interneurons and Bergmann glia.…”

“…Multi-cell bolus loading of calcium indicator dye was performed as described previously (Stosiek et al ., 2003 ; Sullivan et al ., 2005 ; Mukamel et al ., 2009 ; Ozden et al ., 2009 ; Schultz et al ., 2009 ; Hashizume et al ., 2013 ). Cal-520 acetoxymethyl ester (Cal-520 AM) (AAT Bioquest, Sunnyvale, CA, USA; excitation/emission wavelength, 492/514 nm; spectra available on manufacturer’s website, www.aatbio.com ; K d , 320 n m ; quantum yield, 0.75) or Oregon Green 488 BAPTA-1 acetoxymethyl ester (OGB-1 AM) (Invitrogen) was dissolved with 20% (for neocortical neurons) or 10% (for cerebellar Purkinje cells) w/v Pluronic F-127 (Invitrogen) in dimethyl sulfoxide.…”

‘Medusa head ataxia’: the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 2: Anti-PKC-gamma, anti-GluR-delta2, anti-Ca/ARHGAP26 and anti-VGCC

“…A recent study found that in GluRδ2 knockout mice, surplus CFs make ectopic innervations onto distal dendrites of PCs and a higher and more widespread synchrony of calcium transients in neighbouring PCs occurs. Moreover, transverse CF collaterals and CF signals transgress PC microzone borders, form ectopic synapses onto neighbouring PC dendrites and exhibit clustered firing not present in wild-type mouse [ 83 ]. Finally, mutations in the Grid2 gene have been found to be the cause of ataxia in ‘lurcher’ mice, which are characterised by apoptosis of PCs during postnatal development, and ‘hotfoot’ mice [ 84 , 85 ].…”

Maturation of Cerebellar Purkinje Cell Population Activity during Postnatal Refinement of Climbing Fiber Network

“…These aberrant CFs form ectopic synapses on distal dendrites of neighboring PCs (Miyazaki et al, 2010). A in vivo two-photon calcium imaging study revealed that spontaneous CF responses in populations of PC dendrites were abnormally synchronized in adult GluD2 KO mice (Hashizume et al, 2013), suggesting that the aberrant CF transverse collaterals contribute to the enhanced synchrony in mice lacking GluRD2. Given that functional aberrant CF transverse collaterals in GluD2 KO mice are formed above the P10-P14 period (Hashimoto et al, 2001), synchrony of CF responses should be normal before P10 and enhanced thereafter.…”