Perineuronal nets are under the control of type-5 metabotropic glutamate receptors in the developing somatosensory cortex

Abstract

mGlu5 metabotropic glutamate receptors are highly functional in the early postnatal life, and regulate developmental plasticity of parvalbumin-positive (PV⁺) interneurons in the cerebral cortex. PV⁺ cells are enwrapped by perineuronal nets (PNNs) at the closure of critical windows of cortical plasticity. Changes in PNNs have been associated with neurodevelopmental disorders. We found that the number of Wisteria Fluoribunda Agglutinin (WFA)⁺ PNNs and the density of WFA⁺/PV⁺ cells were largely increased in the somatosensory cortex of mGlu5^-/- mice at PND16. An increased WFA⁺ PNN density was also observed after pharmacological blockade of mGlu5 receptors in the first two postnatal weeks. The number of WFA⁺ PNNs in mGlu5^-/- mice was close to a plateau at PND16, whereas continued to increase in wild-type mice, and there was no difference between the two genotypes at PND21 and PND60. mGlu5^-/- mice at PND16 showed increases in the transcripts of genes involved in PNN formation and a reduced expression and activity of type-9 matrix metalloproteinase in the somatosensory cortex suggesting that mGlu5 receptors control both PNN formation and degradation. Finally, unilateral whisker stimulation from PND9 to PND16 enhanced WFA⁺ PNN density in the contralateral somatosensory cortex only in mGlu5^+/+ mice, whereas whisker trimming from PND9 to PND16 reduced WFA⁺ PNN density exclusively in mGlu5^-/- mice, suggesting that mGlu5 receptors shape the PNN response to sensory experience. These findings disclose a novel undescribed mechanism of PNN regulation, and lay the groundwork for the study of mGlu5 receptors and PNNs in neurodevelopmental disorders.

Fig. 1. Up-regulation of PNNs in the somatosensory cortex of mGlu5^−/− mice at PND16.

a Thunder high resolution image showing WFA⁺ PNNs (green) and PV⁺ neurons (red) (counterstained with DAPI). b–c Stereological counting of WFA⁺ PNNs in the somatosensory cortex (b, c). Scale bar = 100 μm (b). In c *p < 0.01 vs. mGlu5^+/+ (Student’s t test; t5 = −5.475). d, e Fluorescent WFA staining and assessment of WFA⁺ PNN density; d scale bar = 50 μm. e *statistically significant vs. the corresponding mGlu5^+/+ sections (Student’s t test); t6 = −4.92, p < 0.01 (AP + 0.98 mm); t6 = −6.055, p < 0.01 (AP + 0.02 mm); t6 = −3.75, p = 0.01 (AP – 1.06 mm); t6 = −4.906, p = 0.003 (average counts). f–h WFA (green) and PV (red) staining and assessment of WFA⁺/PV⁺ and PV⁺ cell density. f Thunder high resolution tailing images counterstained with DAPI. Scale bar = 25 μm. g *Statistically significant vs. the corresponding mGlu5^+/+ sections (Student’s t test); t5 = −3.192, p = 0.024 (AP + 0.98 mm); t5 = −3.928, p = 0.011 (AP + 0.02 mm); t5 = −3.57, p = 0.016 (AP – 1.06 mm); t5 = −4.891, p = 0.005 (average counts). i Immunoblot analysis of PV. Immunostaining in d, e and in f–h was performed in independent groups of mice.

Fig. 2. No difference in WFA⁺ PNNs between mGlu5^−/− and wild-type mice in the somatosensory cortex at PND21 and PND60.

a, b WFA⁺ PNN density at PND21. c, d Stereological WFA⁺ PNN counting in the somatosensory cortex at PND60. e–i WFA⁺ PNN, WFA⁺/PV⁺ and PV⁺ cell density at PND60. Green = WFA; red = PV. Scale bars = 50 μm (e and upper panels in g); = 25 μm (g lower panels); = 100 μm (a, c). Immunostaining in e, f, in g, h, and in i was performed in three independent experiments.

Fig. 3. Pharmacological blockade of mGlu5 receptors increases WFA⁺ PNN density in the somatosensory cortex at PND16 but not at PND21.

Mice were treated i.p. with either saline or MTEP (3 mg/kg) daily from PND7 to PND14 and killed at PND16. a WFA (green) and PV (red) staining in vehicle and MTEP treated mice. Scale bar = 100 μm (upper panels) and 50 μm (lower panels). b, c *Statistically significant vs. the corresponding mGlu5^+/+ sections (Student’s t test); in b t5 = −4.248, p = 0.008 (AP + 0.98 mm); t5 = −12.576, p = <0.01 (AP + 0.02 mm); t5 = −2.473, p = 0.056 (AP – 1.06 mm); t5 = −5.867, p = 0.002 (average counts). In c t5 = −5.556, p = 0.003 (AP + 0.98 mm); t5 = −3.101, p = 0.027 (AP + 0.02 mm); t5 = −2.181, p = 0.081 (AP – 1.06 mm), t5 = −6.982, p < 0.001 (average counts). e–h WFA/PV immunostaining in mice treated daily with MTEP or vehicle from PND16 to PND21. e Scale bar = 100 μm. Immunostaining in c, d and g, h was performed in sections adjacent to the levels indicated in b and f, respectively.

Fig. 4. Expression of genes and proteins involved in the formation or degradation of PNNs in the somatosensory cortex of mGlu5^+/+ and mGlu5^−/− mice at PND16 and PND60.

a mRNA levels (copy numbers normalized by GAPDH) *Statistically significant vs. mGlu5^+/+ (Student’s t test); Nptx2, t8 = −2.15; p = 0/03; NPAS4, t8 = −4.87; p = 0.0012; Egr1, t9 = −3.63; p = 0.005; Acan, t9 = −2.73; p = 0.02. b, c MMP-9 mRNA and protein levels. *Statistically significant vs. mGlu5^+/+ (Student’s t test); b t8 = −2.08; p = 0.03; c t6 = 2.905; p = 0.027. d Gelatin zymography of MMP-9 activity. e mRNA levels of Nptx2, NPAS4, Egr-1, Acan, and MMP-9 in the somatosensory cortex of the two genotypes at PND60. *Statistically significant vs. mGlu5^+/+ (Student’s t test); t8 = 3,162; p < 0.05; f Immunoblot of MMP-9 in the somatosensory cortex at PND60.

Fig. 5. mGlu5 receptors shape the PNN response to sensory experience in the somatosensory cortex at PND16.

a–d WFA⁺, WFA⁺/PV⁺, and PV⁺ cell density in the ipsilateral (Ipsi) or contralateral (Contra) somatosensory cortex of mGlu5^+/+ and mGlu5^−/− mice after unilateral whisker stimulation from PND9 to PND16. a green = WFA and red = PV; scale bar = 100 μm. b, c *statistically significant vs the respective contralateral side. Immunostaining in c and d was performed in sections adjacent to levels indicated in b. e–h WFA⁺, WFA⁺/PV⁺ and PV⁺ cell density in the ipsilateral (Ipsi) or contralateral (Contra) somatosensory cortex of mGlu5^+/+ and mGlu5^−/− mice after unilateral whisker trimming from PND9 to PND16. e green = WFA and red = PV; scale bar = 100 μm. f, g statistically significant vs the respective side in mGlu5^−/− mice (*) or vs the ipsilateral side of mGlu5^−/− mice (#). Immunostaining in g and h was performed in sections adjacent to levels indicated in f. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post hoc test. All F values are in the Supplementary Materials.