Microglia enable mature perineuronal nets disassembly upon anesthetic ketamine exposure or 60-Hz light entrainment in the healthy brain

Abstract

Perineuronal nets (PNNs), components of the extracellular matrix, preferentially coat parvalbumin-positive interneurons and constrain critical-period plasticity in the adult cerebral cortex. Current strategies to remove PNN are long-lasting, invasive, and trigger neuropsychiatric symptoms. Here, we apply repeated anesthetic ketamine as a method with minimal behavioral effect. We find that this paradigm strongly reduces PNN coating in the healthy adult brain and promotes juvenile-like plasticity. Microglia are critically involved in PNN loss because they engage with parvalbumin-positive neurons in their defined cortical layer. We identify external 60-Hz light-flickering entrainment to recapitulate microglia-mediated PNN removal. Importantly, 40-Hz frequency, which is known to remove amyloid plaques, does not induce PNN loss, suggesting microglia might functionally tune to distinct brain frequencies. Thus, our 60-Hz light-entrainment strategy provides an alternative form of PNN intervention in the healthy adult brain.

Keywords: 60-Hz; anesthesia; ketamine; light entrainment; microglia; ocular dominance plasticity; perineuronal net.

Figure 1.. PNN gradually declines with repeated ketamine and reinstates juvenile-like plasticity.

(A) Experimental timeline. (B) WFA-labeled female coronal brain sections after 6× saline, 1×, 2×, 3×, or 6× KXA. Scale bar: 200 μm, zoom-in (S1): 20 μm. (C) Percent mean change of PNN-coated cells ± SEM in S1. Linear regression model: P_♀ < .0001 with selected post-hoc comparisons. (D) WFA-labeled female brain sections after 3× saline or XA. Scale bar: 200 μm. (E) Percent mean change of PNN-coated cells ± SEM in S1. Two sample t-test: P_♀ = 0.1193. (F) Experimental timeline. (G) Mean contra-/ipsilateral eye ratios per animal ± SEM. Two-way repeated measure ANOVA P = 0.0341. (H-I) Mean monocular VEP magnitudes ± SEM with representative traces for (H) saline (contralateral, Friedman’s test, P = 0.1550, ipsilateral, one-way ANOVA, P = 0.9102), or (I) ketamine-treated mice (contralateral, Friedman’s, P = 0.0097 with selected post-hoc comparisons; ipsilateral, Friedman’s P = 0.7546). KXA, ketamine-xylazine-acepromazine. MD, monocular deprivation. S1(BF), somatosensory 1, barrel field. WFA, Wisteria floribunda agglutinin. Each dot represents one animal (3–5 animals per condition with exception of the control bar in Figure 1C, which shows the mean value of all controls. See also Figure S2B). SEM, standard error of the mean. *P < .05, **P < .01, ***P < .001, ^nsP > .05.

Figure 2.. Microglia interact with PNN-coated neurons upon repeated ketamine stimulation.

(A) Immunostaining for Iba1 (green) and parvalbumin (Pvalb, cyan) after 3× saline or KXA in primary somatosensory cortex S1. Zoom-in, single-plane orthogonal projection. Arrow, microglia in close distance of Pvalb⁺ neurons. Scale bar: 10 μm. (B) Minimal Pvalb⁺ neuron-microglia median distance in μm after treatment. Each condition 3 animals as indicated in legend, 5 microglia per animal. Linear regression model with animal ID as random effect: P_♀ < .0001, P_♂ < .0001. (C-D) Ex-vivo live imaging of brain slices of Cx3Cr1^{CreERT2 het}/Ai9 ^het mice dissected 2-hours after 2× KXA. Snapshots of microglia (green) and WFA (magenta) with orthogonal side projection. Scale bar: 10 μm. (D) Time course of mean WFA volume within microglia percentage ± SEM after treatment. Linear regression model: P < .0001 with selected post-hoc comparisons. (E-F) Mean percentage CD68 volume within microglia ± SEM after (E) saline or KXA or (F) 3× KXA recovery. See also Figures S5A and S5B. Linear regression model: (C) P < .0001, (D) P < .0001 with selected post-hoc comparisons. (G) Mean percentage of PNN volume within microglial CD68 ± SEM upon treatment. Linear regression model: P < .0001 with selected post-hoc comparisons. Each dot represents one animal (3–5 animals per condition with exception of the control bar in (E) and (G), which shows the mean value of all controls. See also Figures S5A and S6B, respectively). (H) Immunostaining for PNN (WFA, magenta), endosomal-lysosomal marker CD68 (blue), and microglia (Iba1, green) after 2× KXA. Below: single plane orthogonal projections. Scale bar: 5 μm. ***P < .001, **P < .01, ^nsP > .05.

Figure 3.. Microglia depletion or clopidogrel exposure prior ketamine prevents PNN loss.

(A-C) Microglia depletion with Csf1r-inhibitor PLX5622 and 3× saline or KXA ketamine treatment. (A) Experimental timeline. (B) Immunostaining for WFA. Scale bar: 200 μm. Next, zoom-in into S1. Scale bar: 20 μm. (C) Mean density of PNN-coated cells ± SEM. Grey and green dashed lines: reference for mean value of 3× saline or KXA, respectively, from Figures 1C and S1D. Two sample t-test: P= 0.3467. (D-G) Clopidogrel treatment, i.p. injected alone (3× clopidogrel) or 5 min before KXA application (3× (clopidogrel + KXA)). (D) Experimental timeline. (E) Immunostaining for WFA. Scale bar: 200 μm. (F) Mean density of PNN-coated cells ± SEM. Grey and green dashed lines: reference for mean value of 3× saline or 3× KXA, respectively, from Figures 1C and S1D. Two sample t-test: P=0.3808. Each dot represents an animal. (G) Higher magnification images for WFA (magenta or contrast-image) and Iba1 (green). Scale bar: 30 μm. ^nsP > .05.

Figure 4.. 60-Hz light flickering recapitulates microglia response observed under repeated ketamine exposure.

Light stimulation either with constant light (black) or light-flickering frequencies at 8 Hz (red), 40-Hz (blue), or 60-Hz (purple) in V1. (A) Experimental timeline. (B-C) Mean density of WFA-coated cells in percent ± SEM. (B) Linear regression model with selected post-hoc comparisons: P < .0001. (C) After microglia depletion. Two sample t-test: P=0.0926. Grey and purple dashed lines: reference for mean value of constant light or 60 Hz from Figure 4B, respectively. (D) Median minimal parvalbumin (Pvalb)⁺ neuron-microglia distance. Each condition 3 animals as indicated in legend, 5 microglia per animal. Linear regression model: P_♂ < .0001, P_♀ < .0001 with selected post-hoc comparisons. (E) Mean percent change of MMP-9 volume ± SEM within each cell type. Two sample t-test: Pvalb⁺-neurons, P < .0001. Microglia, P < .0001. (F) Mean percentage of CD68 volume ± SEM within microglia. Linear regression model: P < .0001 with selected post-hoc comparisons. (G) Mean percentage ± SEM of WFA volume within microglial CD68. Linear regression model: P < .0001 with selected post-hoc comparisons. (H-I) Immunostained PNN (WFA, magenta), microglia (Iba1, green), endosomal-lysosomal marker CD68 (blue, H) and Pvalb (cyan, I) after 60-Hz light flickering stimulation. Scale bar: 20 μm. Frame indicates zoom-in and single plane orthogonal projections. Scale bar: 7 μm (H) and 4 μm (I). ***P < .001, ^nsP > .05. With exception of (D), each dot represents an animal.