1Department of Rehabilitation Medicine, Huashan Hospital, Fudan University, Shanghai, China. 2Stem Cell and Regenerative Medicine Laboratory, Ningbo Second Hospital, Zhejiang, China. 3University of Chinese Academy of Sciences, Beijing, China. 4Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China. 5Department of Rehabilitation Medicine, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. 6Department of Rehabilitation, Clinical Medical College, Yangzhou University, Jiangsu, China
The level of cerebellar activity in stroke patients has been shown to correlate with the extent of functional recovery. We reasoned that the cerebellum may be an important player in post-stroke rehabilitation. Because the neurons in the deep cerebellar nuclei (DCN) represent virtually all of the output from the cerebellum, in this study, using environmental enrichment (EE) to promote rehabilitation, we investigated the influence of the optogenetic neuronal modulation of DCN on EE-induced rehabilitation. We found that neuronal inhibition of the DCN almost completely blocked motor recovery in EE treated mice, but the stroke mice with neuronal activation of the DCN achieved a similar recovery level as those in the EE treated group. No difference was observed in anxiety-like behavior. Moreover, Htr2a in the DCN, the gene encoding 5-HT2A receptor, was shown to be a hub gene in the protein-protein interaction network identified using RNA-seq. This indicated that 5-HT2A receptor-mediated signaling may be responsible for DCN-dependent functional improvement in EE. We further verified this using the 5-HT2A receptor antagonist, MDL100907, to inhibit the function of 5-HT2A receptor in the DCN. This treatment resulted in impaired recovery in EE treated mice, who performed at a level as poor as the stroke-only group. Thus, this work contributes to an understanding of the importance of the DCN activation in EE-induced post-stroke rehabilitation. Attempts to clarify the mechanism of 5-HT2A receptor-mediated signaling in the DCN may also lead to the creation of a pharmacological mimetic of the benefits of EE-induced rehabilitation.
Qun Zhang,Jun-fa Wu,Qi-li Shi, et al. The Neuronal Activation of Deep Cerebellar Nuclei Is Essential for Environmental Enrichment-Induced Post-Stroke Motor Recovery[J]. Aging and disease,
2019, 10(3): 530-543.
Table 1 Primer sequences used for the validation of cerebellum-associated gene expression amongst upregulated genes.
Figure 1. Effects of EE on motor recovery after PT
(A) Time course of behavioral tests. Motor assessments included the rotarod, cylinder and rung walking tests. BL: baseline data for rotarod. (B) Time course of the latency to fall off the rotarod. (C) The percentage of laterality in the cylinder test. (D-E) The percentage of limb slips in the rung walking test, for both forelimb and hindlimb. Data are expressed as the mean±SEM. *PT vs. PT+EE groups. *p≤0.05; **p≤0.01; ***p≤0.001.
Figure 2. Effects of EE on anxiety-like behavior after PT
(A-C) The percentage of time mice spent in the open arm (A), closed arm (B) and center zone (C) of the elevated plus maze in each group. Data are expressed as the mean±SEM; *p≤0.05; **p≤0.01.
Figure 3. Effects of optogenetic DCN stimulation on motor recovery
(A) Time course of behavioral tests after optogenetic stimulation of the DCN. Motor assessments included the rotarod, cylinder and rung walking tests. BL: baseline data for rotarod. FI: fiber implantation. (B) Schematic depiction of the optogenetic DCN stimulation used to modulate motor function. (C) Representative image of viral injection location targeted to the DCN. Positive expression of ChR2-GFP was observed in the DCN (100X magnification, scale bar =1mm). The morphology of the neurons (green) was shown in the inserted image (400X magnification, scale bar = 60μm). (D) Time course of the latency to fall off the rotarod in each group. (E) Histogram of the falling off latency in each group on the 14th and 21th day after stroke. (F) The percentage of laterality in the cylinder test in each group. (G) Histogram of the laterality percentage in each group on the 14th and 21th day after stroke. (H and J) The percent of forelimb slips (H) and hindlimb slips (J) in the rung walking test in each group. (I, K) Histogram of the limb slips percentage in each group on the 14th and 21th day after stroke. Data are expressed as the mean±SEM. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001.
Figure 4. Effects of optogenetic DCN stimulation on anxiety-like behavior after PT
The percentage of time spent in the open arm (A), closed arm (B) and center zone (C) of the elevated plus maze. Data are expressed as the mean±SEM; *p≤0.05; **p≤0.01.
Figure 5. Hierarchical clustering of transcripts in the DCN of PT and PT+EE groups
(A) Heat map of differentially expressed genes in the PT vs. the PT+EE group. Each group included three independent biological replicates. Red: high, Green: low, log scale from -2 to +1. (B) Volcano plots of significant differential expression of mRNAs in the PT versus the PT+EE group. The x-axis represents log2-fold change, and the y-axis represents -log10 (p-value). Blue dots: genes that were lower in the PT+EE group compared to the PT group. Red dots: genes that were higher in the PT+EE group compared to the PT group. Grey dots: genes that were unchanged in the PT+EE and PT groups. (C) Representative distribution of genes in the high expression subset and low expression subset in the PT+EE vs. PT group. Blue column: genes that were lower in the PT+EE group compared to the PT group. Red column: genes that were higher in the PT+EE group compared to the PT group. (D) PPI (protein-protein interaction) network of the upregulated genes. The red nodes are known cerebellar target genes. (E) RT-PCR validation of the expression of cerebellar-associated genes, from the list of upregulated genes. Data are expressed as the mean fold-change±SEM from three separate experiments, conducted in triplicate. **p≤0.01; ***p≤0.001.
Figure 6. Effects of the 5-HT2A receptor antagonist MDL100907 on motor recovery after PT
(A) Relative expression of Htr2a mRNA in each group. (B) Time course of behavioral tests after MDL100907 administration to the DCN. Motor assessments included the rotarod, cylinder and rung walking tests. BL: baseline data for rotarod. PI: osmotic pump implantation. (C) Schematic depiction of the mini osmotic pump implantation filled with MDL100907. (D) The latency to fall off the rotarod in each group. (E) Histogram of the falling off latency in each group on the 14th and 21th day after stroke. (F) The percentage of laterality in cylinder test. (G) Histogram of the laterality percentage in each group on the 14th and 21th day after stroke. (H, J) The percentage of the forelimb (H) and hindlimb (J) slips in the rung walking test. (I, K) Histogram of the error slips percentage in each group on the 14th and 21th day after stroke. *p≤0.05; **p≤0.01, ****p≤0.0001.
Figure 7. Effect of the 5-HT2A receptor antagonist MDL100907 on anxiety-like behavior after PT
The percentage of time mice spent in the open arm (A), closed arm (B) and center zone (C) of the elevated plus maze. Data are expressed as the mean±SEM; *p≤0.05; **p≤0.01; ***p≤0.001.
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