Roles of GABA Signaling in Directing Basket Cell Axonal Projections Toward Purkinje Cells in the Cerebellum

doi:10.21203/rs.3.rs-95440/v1

Download PDF

Research

Roles of GABA Signaling in Directing Basket Cell Axonal Projections Toward Purkinje Cells in the Cerebellum

https://doi.org/10.21203/rs.3.rs-95440/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system, synthesized by two isoforms of glutamate decarboxylase (GAD): GAD65 and GAD67. GABA may act as a trophic factor during brain development, but its contribution to the development and maturation of cerebellar neural circuits is not known. To understand the roles of GABA in cerebellar development and associated functions in motor coordination and balance, we examined GAD65 conventional knock out (KO) mice and mice in which GAD67 was eliminated in parvalbumin-expressing neurons ( PV-Cre ; GAD67 flox/flox mice). We found aberrant subcellular localization of the Shaker-type K channel Kv1.1 in basket cell collaterals of PV-Cre ; GAD67 flox/flox mice and abnormal projections from basket cells to Purkinje cells in both mouse strains. Furthermore, PV-Cre ; GAD67 flox/flox mice exhibited abnormal motor coordination in the rotarod test. These results indicate that GABA signaling in the cerebellum during development is critical for establishing appropriate connections between basket cells and Purkinje cells and is associated with motor coordination in mice.

Cellular & Molecular Neuroscience

cerebellum

development

GABA

GAD67

GAD65

basket cells

Additionalfile1.JPG
Body weight changes and survival curves of PV-Cre; VGATflox/flox mice. (a) Average body weights of VGATflox/flox and PV-Cre; VGATflox/flox mice. Significant differences were observed between two genotypes [F(1,6) = 0.0106, P = 0.0106; two-way repeated measures ANOVA]. Results shown are the means ± SEMs (n = 4 mice per genotype). (b) Survival curves of VGATflox/flox (n = 4) and PV-Cre; VGATflox/flox (n = 4) mice (P = 0.0833, log-rank test). (c) Comparison of VGATflox/flox (left) and PV-Cre; VGATflox/flox (right) mice at postnatal day 12. PV-Cre; VGATflox/flox mice exhibited generalized convulsive seizures.
Additionalfile2.JPG
Confirmation of antibody specificity using knockout mice. (a) GAD67 immunohistochemistry of sagittal sections of wild-type and GAD67GFP/GFP mice at P0. GE, ganglionic eminence. Scale bar, 500 μm. (b) GAD65 immunohistochemistry in the cerebellar cortices of wild-type and GAD65–/– mice at 8 weeks old. Scale bar, 50 μm. ML, molecular layer; PL, Purkinje cell layer.
Additionalfile3.JPG
Normal perineuronal nets and no ectopic expression of tyrosine hydroxylase in PV-Cre; GAD67flox/flox mice. (a) Perineuronal nets in the deep cerebellar nuclei are unaltered in PV-Cre; GAD67flox/flox mice. The large neurons (identified by NeuroTrace; magenta) are enwrapped by perineuronal nets identified by an antibody against Wisteria floribunda agglutinin (WFA; green). Scale bar, 200 μm. (b) Immunohistochemistry of coronal cerebellar sections for zebrin II and tyrosine hydroxylase (TH). Purkinje cells in PV-Cre; GAD67flox/flox mice exhibit no ectopic expression of TH. Cellular expression pattern of zebrin II (green) is unaltered in PV Cre; GAD67flox/flox mice. Scale bar, 500 μm.
Additionalfile4.JPG
Normal VGluT1-positive parallel fiber synapses, VGluT2-positive climbing fiber synapses, and VGAT-positive inhibitory synapses in GAD65 KO mice. (a) Immunohistochemistry for GAD67 (cyan), GAD65 (yellow), and calbindin (CB; magenta) in the cerebellar cortices of wild-type (GAD65+/+) and GAD65 KO (GAD65–/–) mice. Scale bar, 50 μm. ML, molecular layer; PL, Purkinje cell layer. (b) Quantification of immunoreactive signals for GAD67 (left) and GAD65 (right) in the molecular layer of the cerebellum. (c) Quantification of immunoreactive signals for GAD67 (left), GAD65 (middle), and calbindin (right) in the PL of the cerebellum. (d) Immunohistochemistry for VGluT1 (cyan), VGluT2 (yellow), and calbindin (magenta) in the cerebellar cortices of wild-type (GAD65+/+) and GAD65 KO (GAD65–/–) mice. Scale bar, 20 μm. (e) Quantification of parallel fiber synapses and climbing fiber synapse density byVGluT1-positive staining intensity (left) and counts of VGluT2- positve puncta per 100 μm2 (right). (f) Immunohistochemistry for VGAT (green) and parvalbumin (PV; magenta) in the cerebellar cortices of wild-type (GAD65+/+) and GAD65 KO (GAD65–/–) mice. Scale bar, 20 μm. (g) Quantification of VGAT-positive puncta per 100 μm2 in the molecular layer of the cerebellum (left), and the numbers of PV-positive cells in the molecular layer (middle) and in the Purkinje cell layer (right). *P < 0.05, **P < 0.01.

Download PDF

Version 1

posted

You are reading this latest preprint version

Roles of GABA Signaling in Directing Basket Cell Axonal Projections Toward Purkinje Cells in the Cerebellum

Status:

Version 1

Abstract

Figures

Full Text

Supplementary Files

Status:

Version 1