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GABAergic regulation of cerebellar NG2 cell development is altered in perinatal white matter injury

Abstract

Diffuse white matter injury (DWMI), a leading cause of neurodevelopmental disabilities in preterm infants, is characterized by reduced oligodendrocyte formation. NG2-expressing oligodendrocyte precursor cells (NG2 cells) are exposed to various extrinsic regulatory signals, including the neurotransmitter GABA. We investigated GABAergic signaling to cerebellar white matter NG2 cells in a mouse model of DWMI (chronic neonatal hypoxia). We found that hypoxia caused a loss of GABAA receptor–mediated synaptic input to NG2 cells, extensive proliferation of these cells and delayed oligodendrocyte maturation, leading to dysmyelination. Treatment of control mice with a GABAA receptor antagonist or deletion of the chloride-accumulating transporter NKCC1 mimicked the effects of hypoxia. Conversely, blockade of GABA catabolism or GABA uptake reduced NG2 cell numbers and increased the formation of mature oligodendrocytes both in control and hypoxic mice. Our results indicate that GABAergic signaling regulates NG2 cell differentiation and proliferation in vivo, and suggest that its perturbation is a key factor in DWMI.

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Figure 1: Changes in cerebellar myelination following neonatal hypoxia.
Figure 2: Altered development of NG2 cells following neonatal hypoxia.
Figure 3: Loss of cerebellar GABAergic neurons following neonatal hypoxia.
Figure 4: Reduced GABAergic synaptic activity in cerebellar white-matter NG2 cells and GABAergic interneurons following neonatal hypoxia.
Figure 5: The carbachol-induced increase in NG2 cell sIPSC frequency reflects an increased activity of local white-matter interneurons.
Figure 6: Modulation of cerebellar white-matter NG2 cell development by GABAergic drugs and NKCC1 deletion.
Figure 7: Effects of tiagabine and vigabatrin on NG2 cell proliferation and loss of mature oligodendrocytes following hypoxia.

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Acknowledgements

We thank D. Bergles (Johns Hopkins School of Medicine) for providing NG2DsRed and Pdgfra-creERT2 mice, M. Turmaine for assistance with electron microscopy, and L.-J. Chew for comments on the manuscript. This work was supported by the US National Institutes of Health (R01NS045702, P01NS062686 and P30HD040677 to V.G., K08NS073793 to J.S., and R01NS038118 to D.S.), the Wellcome Trust (086185/Z/08/Z to S.G.C.-C. and M.F.), the MRC (MR/J002976/1 to S.G.C.-C. and M.F., MR/J012998/1 to M.F. and S.G.C.-C.) and the DFG (HU 800/8-1 to C.A.H.). Part of the electron microscopy was performed at the Virginia Commonwealth University Department of Anatomy and Neurobiology Microscopy Facility, which is supported, in part, with funding from US National Institutes of Health and the National Institute of Neurological Disorders and Stroke Center core grant 5P30NS047463. M.Z. was in receipt of a Charlotte and Yule Bogue Research Fellowship.

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Authors and Affiliations

Authors

Contributions

M.Z., J.S., S.G.C.-C., M.F. and V.G. designed the experiments. M.Z. and P.L. performed the electrophysiology. M.Z. and J.S. carried out the immunocytochemistry and drug treatment studies. J.S. and V.G. generated the Pdgfra-creERT2; Nkcc1loxP/loxP; Rosa26-yfp mouse. M.Z., J.S. and J.L.D. performed electron microscopy. B.M. performed culture experiments. J.E. performed genotyping. L.H. and D.S. provided the Nkcc1−/− mouse brain tissue. C.A.H. provided the Nkcc1loxP/loxP mice. M.Z., J.S. and M.F. analyzed the experimental data. M.Z. and M.F. prepared the figures and wrote the manuscript, with contributions from S.G.C.-C., J.S. and V.G.

Corresponding authors

Correspondence to Stuart G Cull-Candy, Mark Farrant or Vittorio Gallo.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Transient changes in cerebellar area following neonatal hypoxia.

Representative midline sagittal sections (50 μm) of cerebellum from normoxic (left) and hypoxic (right) mice at P7, 11, 15 and 30. Sections were stained with cresyl violet and photographed using a dissecting microscope. Red arrowheads point to the intercrural fissure (ict) that normally separates lobules VI and VII. Plots to the right illustrate the area of the vermis determined by freehand selection in ImageJ. Columns indicate mean values from three normoxic and three hypoxic mice at each age. Error bars indicate SEM. Fractional areas following hypoxia were 0.93 at P7, 0.69 at P11, 0.98 at P15 and 0.96 at P30. ** P < 0.01 (Welch two-sample unpaired t test). Assuming uniform tissue loss in all dimensions, these measures would correspond to fractional cerebellar volumes at P7, 11, 15 and 30 following hypoxia of 0.85, 0.57, 0.97 and 0.94. Although these data suggest reduced cerebellar volume at P7 and 11, correction of the cell counts presented in Fig. 2, Fig. 3, Fig. 7 and Supplementary Fig. 6 for these calculated volume changes did not alter the qualitative interpretation; the outcome of all statistical tests remained significant. The original, uncorrected, counts are presented in the figures.

Supplementary Figure 2 Neonatal hypoxia results in delayed maturation of Purkinje cells and loss of MBP without apoptosis or axonal injury.

(a) Representative confocal images of sagittal sections from normoxic and hypoxic mice (P7-P30) showing calbindin immunofluorescence (green). ML, molecular layer; PCL, Purkinje cell layer; IGL, internal granule cell layer. Scale bars 50 μm. Quantification of Purkinje cell numbers and Purkinje cell layer thickness are presented in Fig. 1d and e. (b) Representative confocal images showing NF200 (green) and MBP (red) immunofluorescence (P7-P30). Images below represent NF200 and MBP overlay merged with DAPI. Abbreviations as in (a), plus WM, white matter. Scale bars 50 μm. Quantification of NF200 and MBP immunofluoresecence is presented in Fig. 1g and h. (c) Immunolabeling of cerebellar sagittal sections from normoxic and hypoxic mice (P11) with calbindin (green) and caspase-3 (red). Images indicate no increase in the expression of caspase-3 in the Purkinje cell layer. Scale bar 50 μm. (d) Representative confocal images of cerebellar white matter sections from normoxic and hypoxic GAD65-GFP mice (P11) labeled with caspase-3. No increase in apoptosis was detected following hypoxia. Apoptotic caspase-3+ cells (white arrowheads) are present in the external granule cell layer and in the white matter in both normoxia and hypoxia tissue. Scale bar 25 μm. (e) Confocal images of cerebellar sections from normoxic and hypoxic mice (P11) labeled with the axonal injury marker β-amyolid precursor protein (β-APP) and calbindin. Scale bar 50 μm. A small number of β-APP+ cells were seen in the internal granule cell layer of both normoxic and hypoxic mice (white arrowheads). In comparing sections from 5 normoxic and 7 hypoxic mice, no increase in β-APP expression was detected.

Supplementary Figure 3 Effects of neonatal hypoxia on MBP and axonal myelination at P30.

(a) Representative western blot analysis of MBP and NF200 from P11 and P30 cerebellar lysates. Actin was used as a loading control. (b) Pooled data from 3 independent experiments (each from a single mouse) showed a decrease at P11 in the MBP to NF200 ratio following hypoxia (t3.24 = 3.96, P = 0.03; unpaired Welch two-sample t test). At P30 the MBP to NF200 ratio was not significantly different following hypoxia (t1.23 = 3.65, P = 0.29). (c) Electron microscopy images from P30 cerebellar white matter. (d) Left, scatter plot of g ratios of individual axons versus axon diameter; pooled data from 3 normoxic and 3 hypoxic mice (blue and red, respectively). Fitted lines are linear regressions and shaded areas indicate 95% confidence bands. Right, plot of normalized cumulative probability of g ratio for the 3 normoxic mice (blue dashed lines) and the 3 hypoxic mice (red dashed lines). Solid lines are the averaged probability plots and the shaded areas denote the s.e.m. Hypoxia increased the g ratio from 0.80 ± 0.007 to 0.85 ± 0.005 (t5.1 = 3.77 P = 0.0084).

Supplementary Figure 4 Western blot analysis shows loss of CNPase and MBP following neonatal hypoxia.

(a) Western blot analysis of myelin markers CNPase and MBP in whole cerebellum from normoxic and hypoxic animals (P7-P30). Full membranes were cut to probe for both CNPase (210 kDa-40 kDa) and MBP (40 kDa-12 kDa) (dashed line). The same blots were then re-probed for the loading control actin (210 kDa-40 kDa). (b) Pooled data from 3 independent experiments (3 animals) at each age showed significant decreases at all ages in the density of CNPase and MBP (relative to actin). For CNPase at P7 t2.38 = 6.29, P = 0.016; at P11 t2.14 = 9.03, P = 0.0097; at P15 t3.07 = 6.06, P = 0.0084; at P30 t3.18 = 4.66, P = 0.016. For MBP at P7 t2.10 = 5.56, P = 0.028; at P11 t2.68 = 22.31, P = 0.0004; at P15 t3.48 = 14.46, P = 0.0003; at P30 t3.80 = 8.81, P = 0.0011. Graphs show mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001.

Supplementary Figure 5 Reduced frequency of Purkinje cell simple spike firing following neonatal hypoxia.

(a) Selected records illustrating representative Purkinje cell firing patterns. Each record is a 20 s segment from a longer cell-attached recording in voltage-clamp (at least 2 mins). The traces are from a Purkinje cell in a slice from a normoxic mouse (P11) and from two cells in slices from hypoxic mice (both P10). To the right are the corresponding inter-spike interval (ISI) histograms, together with the mean interspike interval (ISI), coefficient of variation of ISI (CV) and the coefficient of variation of adjacent intervals (CV2) and mean firing rate for the cells illustrated. As expected for Purkinje cells from young mice (P10-11), all cells in slices from normoxic mice exhibited a purely ‘tonic’ firing pattern. By contrast, ~30% of cells in slices from hypoxic mice displayed a ‘tonic-silent’ firing pattern, with periods of tonic firing interspersed by silent periods of ~1-10 s. None of the cells displayed a ‘trimodal’ (inactive/tonic/burst) pattern of firing. (b) Box-and-whisker plots showing the pooled data for mean firing rate and variability (n = 40 and 35 cells from 3 normoxic and 3 hypoxic mice, respectively). Box-and-whisker plots indicate the median value (black line), the mean (black cross), the 25-75th percentiles (box) and the 10-90th percentiles (whiskers). *** P < 0.001, Wilcoxon Mann-Whitney Rank Sum test. Frequency was reduced from 9.8 ± 0.7 Hz to 4.4 ± 0.6 Hz, Z = 5.50, P = 3.67e–9. ISI CV was increased from 0.12 ± 0.01 ms to 0.50 ± 0.08 ms, Z = −4.67, P = 1.03e–6. ISI CV2 was increased from 0.12 ± 0.01 ms to 0.29 ± 0.04 ms Z = −3.70, P = 0.00021. (c) Scatter plot (log axes) of firing rate against ISI CV, illustrating the varied effect of hypoxia; a subset cells exhibited properties similar to those recorded from normoxic mice while a majority of cells (23/35) had lower firing rates and increased CV.

Supplementary Figure 6 Neonatal hypoxia reduces interneuron proliferation in the molecular layer, internal granule cell layer and white matter.

(a) Timeline showing the experimental design for BrdU injections. (b) Representative confocal images of sagittal cerebellar sections from normoxic and hypoxic GAD65-GFP mice at P11. The sections (obtained 72 hrs following BrdU pulse) contain BrdU+ cells (red) in the molecular layer (ML) and internal granule cell layer (IGL). Overlay images of GAD65 (green) and BrdU+ (red) reveal proliferating interneurons (yellow) (white arrowheads). Scale bar 50 μm. (c) Same as b, but for cerebellar white matter. (d) Pooled data. Following hypoxic treatment the number of proliferating interneurons was reduced in the molecular layer (t5.96 = 5.01 P = 0.0019) and internal granule cell layer (t6.14 = 7.77, P = 0.00030). (e) Pooled data. Following hypoxic treatment the number of proliferating interneurons was reduced in the white matter (t2.61 = 5.3 P = 0.045). All tests Welch two-sample unpaired t tests, n = 6 normoxic- and 6 hypoxic mice. Graphs show mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001.

Supplementary Figure 7 Effects of TTX and hyperosmotic sucrose on IPSC frequency in NG2-cells.

(a) Raster plot illustrating mIPSCs recorded from an NG2-cell in a P7 NG2DsRed mouse and the increase in their frequency in response to application of hyperosmotic sucrose (500 mM; gray bar). Records i-iii in the lower panel illustrate representative mIPSCs recorded at +30 mV from the time periods indicated by the red bars. Similar results were obtained in four cells from three mice; sucrose application increased the mIPSC frequency from 0.059 ± 0.025 Hz to 0.56 ± 0.012 Hz (t3 = 3.97, P = 0.028, Welch two-sample paired t test). (b, c) Pooled data showing the effect of TTX on 100 μM carbachol-induced increases in IPSC frequency. TTX both prevented (b) and reversed (c) the action of carbachol. Symbols in gray denote data that has either been described in the main text or, in the case of c, is a subset of data already shown (Fig. 5a).

Supplementary Figure 8 Altered NG2-cell development in vivo and in vitro following GABAergic drugs.

(a) Representative confocal images of sagittal sections from NG2DsRed mice at P11 following treatment (P5–11) with daily injections of the GABAA receptor antagonist bicuculline (1 mg/kg i.p.), the GABA-T inhibitor vigabatrin (100 mg/kg i.p.) or the GAT-1 inhibitor tiagabine (50 mg/kg i.p.) (see Methods). NG2-cells (red) were co-labeled with Olig2 (an oligodendrocyte lineage marker; white), Ki67 (a marker of proliferation; white), CC1 (a marker of mature oligodendrocytes; green) and DAPI (blue). White boxes illustrate the approximate regions in which cells were counted (see Methods and Fig. 6a). Scale bars 100 μm. (b) Representative confocal images of purified mouse NG2-cell cultures prepared from P1–P3 CD1 pups (see Methods). Cultures were treated with saline (left) or 100 μM muscimol (right). Cells were labeled with NG2 (red) and BrdU (white). Scale bars 50 μm. Bar graph (right) shows pooled data from 5 cultures, each prepared from 5 mice. Selected culture wells were treated either with saline (control; n = 5), 100 μM muscimol (n = 5) or 50 μM bicuculline (n = 3). One-way ANOVA (Welch heteroscedastic F test) showed a significant difference among the groups (F2, 5.37 = 10.34, P = 0.014), and subsequent pairwise comparisons showed an effect of muscimol on the percentage of NG2-cells that were BrdU+ (t7.54 = 3.68, P = 0.014, n = 5 cultures from 5 mice each) but no effect following bicuculline pretreatment (t4.74 = −0.78, P = 0.47, n = 5 and 3). *P < 0.05 (Welch two-sample t test with Holm's sequential Bonferroni correction for multiple comparisons).

Supplementary Figure 9 Altered NG2-cell development in vivo in NKCC1–/– mice.

Representative confocal images of sagittal sections from NKCC1–/– mice and control littermates (NKCC1+/+) at P7, 11 and 15. Sections were co-labeled with NG2 and Olig2 or Ki67. In each case, the right hand columns show sections labeled with CC1. White boxes illustrate the approximate regions in which cells were counted (see Methods and Fig. 6b). Scale bars 100 μm.

Supplementary Figure 10 Treatment with vigabatrin and tiagabine following neonatal hypoxia reverses NG2-cell proliferation and loss of mature oligodendrocytes.

(a-c) Representative confocal images of sagittal sections from NG2DsRed mice at P15 following hypoxia (P3–11) and subsequent treatment (P11–15) with vehicle (a), the GABA-T inhibitor vigabatrin (100 mg/kg; b) or the GAT-1 inhibitor tiagabine (50 mg/kg; c). NG2- cells (red) were co-labeled with Olig2 (an oligodendrocyte lineage marker; white), Ki67 (a marker of proliferation; white), CC1 (a marker of mature oligodendrocytes; green) and DAPI (blue). White boxes illustrate the approximate regions in which cells were counted (see Methods and Fig. 7). Scale bars 100 μm.

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Zonouzi, M., Scafidi, J., Li, P. et al. GABAergic regulation of cerebellar NG2 cell development is altered in perinatal white matter injury. Nat Neurosci 18, 674–682 (2015). https://doi.org/10.1038/nn.3990

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