Cerebellar Purkinje Cell Activity Regulates White Matter Response and Locomotor Function after Neonatal Hypoxia

Abstract

Neonatal hypoxia (Hx) causes white matter (WM) injury, particularly in the cerebellum. We previously demonstrated that Hx-induced reduction of cerebellar Purkinje cell (PC) activity results in locomotor deficits. Yet, the mechanism of Hx-induced cerebellar WM injury and associated locomotor abnormalities remains undetermined. Here, we show that the cerebellar WM injury and linked locomotor deficits are driven by PC activity and are reversed when PC activity is restored. Using optogenetics and multielectrode array recordings, we manipulated PC activity and captured the resulting cellular responses in WM oligodendrocyte precursor cells and GABAergic interneurons. To emulate the effects of Hx, we used light-activated halorhodopsin targeted specifically to the PC layer of normal mice. Suppression of PC firing activity at P13 and P21 phenocopied the locomotor deficits observed in Hx. Moreover, histopathologic analysis of the developing cerebellar WM following PC inhibition (P21) revealed a corresponding reduction in oligodendrocyte maturation and myelination, akin to our findings in Hx mice. Conversely, PC stimulation restored PC activity, promoted oligodendrocyte maturation, and enhanced myelination, resulting in reversed Hx-induced locomotor deficits. Our findings highlight the crucial role of PC activity in cerebellar WM development and locomotor performance following neonatal injury.

Keywords: Purkinje cells; WM myelination; cerebellum; hypoxia; locomotor function; prematurity.

Figure 1.

Selective optical stimulation of cerebellar PCs results in the modulation of cellular activity in WM. A, B, Schematic representation of recording electrodes and optical fiber for optical stimulation in the mouse brain, made in Biorender. Before starting the in vivo extracellular electrophysiological recording in WM, selective stimulation of PCs was confirmed by placing an eight-electrode array in the PC layer. The representative raw traces depict the increase in PC spiking activity observed during the optical stimulation (t = 1 s; 25 Hz) with blue light (λ = 470 nm) and reduced spiking activity during stimulation (t = 2 s; continuous) with orange light (λ = 590 nm). C, The pAAV-Efla-DIO-hChR2(H134R)-mCherry-WPRE virus and (D) pAAV-double floxed-eNpHR-EYFP-WPRE-pA virus injected locally into the cerebellar PC layer of Pcp2-cre Nx mice. The mCherry (C, in red) tagged with ChR2 and halorhodopsin tagged to YFP (D, in green) are selectively expressed in PCs stained with DAPI (blue). Scale bar, 50 µm. E, F, PC stimulation alters WM cellular activity in normal development at P13 and P21, respectively. At P13, Nx mice (n = 10) displayed an average basal firing frequency of 16.24 Hz, which was increased to 21.36 Hz an indication of excited cells, whereas in inhibited cells basal firing frequency was reduced to 16.68 Hz from 20.97 Hz. Excited and inhibited cells represented 47.66 and 13.28% of the total WM cells captured during extracellular recordings. In 39.06% of cells, no significant changes in firing frequency were observed during PC stimulation (nonresponsive cells). During the development of Nx mice (P13–P21), the percentage of excitatory cells decreased to 34.78%, whereas the percentage of inhibitory cells increased to 16.3%. G, H, Overall responses of WM cells, after PC stimulation, in chronic neonatal Hx in both P13 and P21, respectively (n = 16). Excited cells were not identified at P13 or at P21. The percentage of inhibited cells increased from 18.78 to 37.84% from P13 to P21. The bar graphs represent the mean firing frequency with (±) SEM of overall WM cell responses before, during, and after selective optical stimulation with blue light (λ = 470 nm; 1 s; 25 Hz) on pAAV-Efla-DIO-hChR2(H134R)-mCherry-WPRE–expressing PCs of Nx and Hx Pcp2-cre mice. The pie charts show the percentage of WM cells that were excited, inhibited, or nonresponsive upon PC stimulation. I, J, Difference in WM inhibitory cell response between the Nx and Hx groups in both P13 and P21 mice, respectively, after PC stimulation. There are higher firing frequency rates of inhibitory WM cells in both P13 and P21 of Nx mice than in Hx mice before, during, and after stimulation of PCs. The average firing frequency of PC activity plots over time at two different developmental timepoints, P13 and P21, and the alteration of PC spontaneous electrical activity after optogenetic stimulation are shown in Extended Data Figure 1-1A–F. The graphical illustration in Extended Data Figure 1-2 shows the proposed mechanism by which increasing firing of PC leads to reduced inhibition of some WM cells, which results in the excitation of nearly 50% of cells in the WM of Nx mice.

Figure 2.

Immunohistochemical and electrophysiological phenotype of WM cells responding to PC stimulation. Cells were identified as GABAergic interneurons OPCs, based on the expression of cell-specific markers, as well as spike waveform and PCA analysis of firing activity. A, B, Immunostaining of cerebellar WM cells demonstrating selective coexpression of ChR2-mCherry (red) in NeuN-expressing cells (green) in GAD2-cre mice and Olig2-expressing cells (green) in PDGFRα-cre mice. C, D, Extracellular responses (raw traces) recorded from WM cells of Pcp2-cre mice are influenced by selective PC stimulation. Responses were shortened, based on differences in waveform shape, amplitude, and duration. The spike profiles were compared with either pAAV-Efla-DIO-hChR2(H134R)-mCherry-WPRE–expressing GABAergic interneurons in WM of GAD2-cre mice and pAAV-Efla-DIO-hChR2(H134R)-mCherry-WPRE–expressing OPCs in WM of PDGFRα-cre mice. Sorted spike waveforms from optically stimulated ChR2-mCherry–expressing GABAergic interneurons of GAD2-cre mice and OPCs of PDGFRα-cre mice (respectively) were compared with the responses recorded from Pcp2-cre mice in which cells were recorded upon PC stimulation. The representative plots of principal component 1 versus principal component 2 of the sorted waveforms from a single trace are shown for GABAergic interneurons from both GAD2-cre and Pcp2-cre mice and OPCs from PDGFRα-cre and Pcp2-cre mice, as well. The consistency of the waveform shape, quantified by direct comparisons of its principal components within pairs, confirmed that these were the same cellular types recorded under all conditions. Calibration, 500 mV × 0.1 ms. E, F, Measurements of the mean duration of waveforms with (±) SEM indicate no significant differences between GAD2-cre and Pcp2-cre mice and between PDGFRα-cre and Pcp2-cre mice. Calibration, 100 µm and 100 µs. Optimization of the viral infection rate was ensured through our previous protocol using an AAV infection rate as shown in Extended Data Figure 2-1A–E.

Figure 3.

Relative functional contribution of GABAergic interneurons and OPCs toward overall WM response to PC stimulation. A, The schematic represents the placement of the optical fiber (blue) for PC stimulation and the electrode array (red dots) on the mouse brain slice (not up to scale) for recording from WM at P13 and P21. B, C, Each multiunit extracellular recording from WM is sorted, based on the identification and validation methods for GABAergic interneurons and OPCs. The spikes are separated for both the Nx and Hx Pcp2-cre mice groups. The two representative profiles show the differences in spike shape and waveform duration of WM GABAergic interneurons (red) and OPCs (green). Bar graphs demonstrate that the duration of action potentials of GABAergic and OPCs of Nx mice were reduced in Hx conditions. D, E, Heat maps show the basal firing frequency distributions of all sorted waveforms, cell by cell over time (2,000 ms) for Nx and Hx mice, respectively. Strikingly the GABAergic interneuron average basal firing frequency was consistently between 20 and 60 Hz, whereas the OPC average basal firing frequency fell under 20 Hz. The pie charts indicate the percent of cells that display high firing frequency (light blue) and low firing frequency (dark blue). At P13, the functional contribution from GABAergic interneurons is 86.88 and 13.12% from OPCs for Nx mice. A switch is observed at P21, with 21.88% for GABAergic interneurons and 78.31% for OPCs. In Hx mice at P13, a decrease in the contribution of GABAergic neurons was observed (22.22%), with a concomitant increase in OPC contribution (77.78%). At P21, no GABAergic interneuron signature spikes were observed in WM recordings.

Figure 4.

Suppression of PC activity in normal Pcp2-cre mice reproduces the effects of Hx on WM cell response. A, The schematic indicates the placement of optic fiber (orange) in the mouse brain slice (not up to scale) to selectively inhibit PCs in pAAV-double floxed-eNpHR-EYFP-WPRE-pA mice by optical stimulation. The recording eight-electrode array placement in WM is shown by red dots. B, C, Representative spike profiles show two distinct waveform shapes, corresponding to GABAergic interneurons (red) and OPCs (green), with waveform templates with an average of 200 and 800 µs spike durations, respectively. The bar graph demonstrates lower waveform durations of GABAergic interneurons than OPCs under PC inhibition. D, The mean firing frequency graphs over time indicate inhibitory responses recorded in WM cells throughout the duration of inhibition of PC activity (0–10,000 ms) in both P13 and P21. Bar graphs of the firing frequency in both P13 and P21 before and during inhibition of PCs. E, The firing frequency distribution heat maps indicate that, at P13, both GABAergic interneurons (29.82%) and OPCs (70.18%) contributed toward WM cell response, but at P21, all the inhibitory responses were from OPCs alone. Scale bar, 100 µs.

Figure 5.

Suppression of PC activity mimics the effects of Hx on locomotor performance and a reduction in oligodendrocyte maturation and myelination. A, After the suppression of PC activity (green bar) and in Hx mice (pink bar), the average number of foot slips was significantly higher than in Nx mice (black bar; n = 10/group; 1-cm-wide beam). B, Similar results were obtained on a 2-cm-wide beam. C, D, Graphs represent the time to complete the task. E, On the rotarod test, locomotor performance was quantified as time latency to fall (seconds). In Nx mice with suppressed PC activity, time latency values are widely distributed over a range, with the lower average time comparable to normal Nx mice, whereas Hx-induced mice performed poorly with an average of only 100 s on the rotarod. F, Representative images of cerebellarWM from Nx, Hx, and Nx-muted mice, in which PC activity was suppressed. Tissue sections were immunostained at P25 with anti-NG2 for OPCs and anti-CC1 for mature OLs. DAPI was used for total cell counts and FluoroMyelin for myelin labeling. G, Quantification of the number of CC1+ and NG2+ cells in WM indicates that there was an approximately 50% reduction in CC1+ cell density in the Nx mice group in which PC activity was suppressed, as compared with Nx mice. This resembled the reduction in CC1+ cells observed in Hx mice. Conversely, a significant increase in NG2+ cell density was observed in Nx mice after PC activity reduction, similar to Hx mice. H, Changes in the ratio of CC1+ (gray) to NG2+ (pink) cell density indicate a reduction in the number of mature oligodendrocytes at P25 in the Nx mice with suppressed PC activity, as compared with Nx. I, The green mean fluorescent intensity plots show a 50% reduction in FluoroMyelin expression in WM after suppression of PC activity, similar to Hx mice. Scale bar, 100 µm. The effects of PC inhibition showed no difference in glial cell response and GABAergic cell numbers as indicated in Extended Data Figure 6-1. EM analysis, as shown in Extended Data Figure 5-1A,B, demonstrated a thicker myelination in Nx than in PC inhibited Nx-Muted mice, which further demonstrates that PC activity influences myelination.

Figure 6.

Selective chronic stimulation of PCs in Hx mice partially rescues locomotor malperformances and promotes OL maturation and myelination after Hx. A–D, Inclined beam test. Graphs represent the number of foot slips on a 1- and 2-cm-wide inclined beam and the time taken to complete the test. In both cases, a significant improvement was observed in the Hx-stimulated group, as compared with Hx mice. On a 2-cm-wide inclined beam, no significant difference was observed in the number of foot slips between Hx-stimulated and Nx mice. E, Rotarod test. Hx-stimulated mice displayed a significant improvement, as compared with Hx mice, although a complete rescue was not observed, when compared with Nx mice. F, Representative confocal images of cerebellar white matter brain slices from Nx, Hx, and Hx-stimulated mice. WM immunostaining with anti-NG2, anti-CC1, DAPI, and FluoroMyelin antibodies. G, Bar graphs represent the percentages of CC1+ and NG2+ cells in Nx, Hx, and Hx-stimulated mice. There is no significant difference in CC1+ mature OLs and NG2+ OPCs between Hx-stimulated and Nx mice. In contrast, the Hx mice displayed a reduction in CC1+ cells and an increase in NG2+ cells. H, The same trend was also observed in the ratio between CC1+ (gray) and NG2+ (pink) cells. I, The mean green fluorescent intensity plot from FluoroMyelin staining showed a significant increase in myelination in Hx-stimulated mice, as compared with Hx mice, with a very similar CTCF expression to Nx mice. Scale bar, 100 µm. PC stimulation in Hx mice showed a difference in glial cell response and GABAergic cell numbers as indicated in Extended Data Figure 6-1. EM analysis, as shown in Extended Data Figure 6-2A,B, demonstrated a thicker myelination in Hx-stimulated mice than in Hx mice, which further demonstrates that PC stimulation increases myelination.