Perisynaptic chondroitin sulfate proteoglycans restrict structural plasticity in an integrin-dependent manner

Abstract

During early postnatal development of the CNS, neuronal networks are configured through the formation, elimination, and remodeling of dendritic spines, the sites of most excitatory synaptic connections. The closure of this critical period for plasticity correlates with the maturation of the extracellular matrix (ECM) and results in reduced dendritic spine dynamics. Chondroitin sulfate proteoglycans (CSPGs) are thought to be the active components of the mature ECM that inhibit functional plasticity in the adult CNS. These molecules are diffusely expressed in the extracellular space or aggregated as perineuronal nets around specific classes of neurons. We used organotypic hippocampal slices prepared from 6-d-old Thy1-YFP mice and maintained in culture for 4 weeks to allow ECM maturation. We performed live imaging of CA1 pyramidal cells to assess the effect of chondroitinase ABC (ChABC)-mediated digestion of CSPGs on dendritic spine dynamics. We found that CSPG digestion enhanced the motility of dendritic spines and induced the appearance of spine head protrusions in a glutamate receptor-independent manner. These changes were paralleled by the activation of β1-integrins and phosphorylation of focal adhesion kinase at synaptic sites, and were prevented by preincubation with a β1-integrin blocking antibody. Interestingly, microinjection of ChABC close to dendritic segments was sufficient to induce spine remodeling, demonstrating that CSPGs located around dendritic spines modulate their dynamics independently of perineuronal nets. This restrictive action of perisynaptic CSPGs in mature neural tissue may account for the therapeutic effects of ChABC in promoting functional recovery in impaired neural circuits.

Figure 1.

Thy1-YFP expression in hippocampal organotypic slice cultures after 4 weeks in vitro. A, Overview of the CA1 field. A subpopulation of pyramidal neurons is labeled by YFP in their entirety, including somata, axons, dendrites, and dendritic spines. So, Stratum oriens; Sp, stratum pyramidale; Sr, stratum radiatum; Slm, stratum lacunosum moleculare. B, Deconvolved image of a dendritic segment in stratum radiatum (maximal intensity projection of a stack of 25 images, 0.2 μm z-step size). In 4-week-old slices, dendritic spines showed mature morphologies (i.e., stubby, thin, and mushroom).

Figure 2.

Distribution and digestion of CSPGs in long-term hippocampal organotypic slices. A, Hippocampal organotypic slices (after 28 DIV) were stained with WFA (red) to reveal CSPGs diffusely distributed around YFP-positive CA1 pyramidal dendrites in stratum radiatum (Sr), or aggregated in PNNs around parvalbumin-positive interneurons (blue) in stratum oriens (So) and pyramidale (Sp). B, Aggrecan, brevican, versican, and phosphacan-positive PNNs (red) were detected in 4-week-old slices in CA1 stratum oriens (So) and pyramidale (Sp). DAPI was used to visualize the strata. C, In slice cultures, the density of WFA-positive PNNs increased over time and became comparable with that observed in vivo in 8-week-old mice. Optical density analysis of immunostaining for CSPGs in the stratum radiatum of hippocampal slice cultures (28 DIV, white bars, 4 slices/protein) compared with fixed tissues from 5-week-old mice (gray bars; n = 3 mice). As in vivo, aggrecan and versican are the most abundant CSPGs in the stratum radiatum. D, Western blot of slice lysates shows digested chondroitin 4-sulfate glycosaminoglycan chains on three core proteins (bands A–C) after ChABC treatment, reflecting efficient CSPG digestion. Band D: GAPDH; 1, control; 2, penicillinase (control enzyme) long-term treatment; 3, penicillinase short-term treatment; 4, ChABC long-term treatment; 5, ChABC short-term treatment. Error bars indicate SEM.

Figure 3.

CSPG digestion-induced enhancement of spine dynamics is dependent on β1-integrins. A, Representative confocal images of living YFP-positive CA1 pyramidal apical dendrites in the different treatment conditions. The white arrows indicate SHPs. B, ChABC-mediated enhancement of spine motility was blocked by pretreatment with a β1-integrin blocking antibody. C, Fluctuations in length over time for representative spines. The average spine length was not affected by ChABC treatment. D, CSPG digestion resulted in a doubling of the percentage of motile spines. Spines were considered motile when the respective motility index was higher than the average motility index of the control group. E, ChABC induced the formation of SHPs. Blocking of glutamatergic receptors failed to inhibit formation of SHPs, while pretreatment with a β1-integrin blocking antibody prevented their appearance. LtT, Long-term treatment; ACSF, artificial CSF microinjection; β1Ab, β1-integrin blocking antibody microinjection; β1Ab+ChABC, β1-integrin blocking antibody microinjection followed by ChABC treatment. ***p < 0.001, Student's t test. Error bars indicate SEM.

Figure 4.

CSPG digestion promotes activation of β1-integrins and phosphorylation of FAK. A, A higher density of active β1-integrins and phosphorylated FAK-positive puncta was detected within the CA1 stratum radiatum after ChABC treatment compared with control slices (active β1-int: control, 2.35 ± 0.27 puncta, 5 slices; ChABC, 4.81 ± 0.7 puncta, 5 slices; phospho-FAK: control, 5.5 ± 0.76 puncta, 10 slices; ChABC, 9.9 ± 0.93 puncta, 10 slices). ChABC treatment did not change the level of expression of native FAK and β1-integrin. Active β1-integrin puncta were significantly larger in treated slices compared with control slices (control, 0.28 ± 0.01 μm², 5 slices; ChABC, 0.33 ± 0.01 μm², 5 slices). B, Active β1-integrin-positive puncta were often associated with phosphorylated FAK-positive puncta in CA1 stratum radiatum after ChABC treatment. DAPI staining was used to visualize the CA1 strata. The white arrows indicate active β1-integrin-positive puncta (green) colocalizing with phosphorylated FAK-positive puncta (red). C, In ChABC-treated slices, phosphorylated FAK-positive puncta (red) were detected in association with YFP-positive dendritic spines (green) and the synaptic marker PSD-95 (blue). On average, 60.1 ± 2.09% of phosphorylated FAK puncta colocalized with PSD-95 puncta after CSPGs digestion compared with 41.28 ± 2.26% in control slices. D, High-magnification images show phosphorylated FAK-positive puncta (red) and PSD-95-positive puncta (blue) in the stratum radiatum of the CA1 field in control and ChABC-treated slices. **p < 0.01, ***p < 0.001, Student's t test. Error bars indicate SEM.

Figure 5.

Local CSPG digestion around selected dendritic segments is sufficient to enhance spine dynamics. A, ChABC was microinjected in the stratum radiatum (Sr). A glass micropipette was positioned in the stratum radiatum of the CA1 field at 50 μm from the imaged dendrite (green). HCS CellMask Deep Red stain (blue) was included in the pipette to monitor the ejection. B, Example of imaged dendrites located 50 μm far from the ejection site but not included in the digestion area and used as control (white arrows indicate the dendrite). Effective CSPG digestion was confirmed by staining with the antibody 2B6 (green) and WFA (red). C, This approach resulted in the successful digestion of CSPG around segments of apical dendrites as revealed by positive staining for proteoglycan 4-sulfate disaccharides in the stratum radiatum (2B6; green staining highlighted by white arrows) and loss of WFA binding sites inside the digestion area, while preserving the integrity of PNNs (WFA, red) in stratum oriens (So) and pyramidale (Sp). D, SHPs appeared on both thin and mushroom spine subtypes shortly after local CSPG digestion and persisted for the entire recording period (i.e., 2 h). E, F, Local ChABC injection enhanced spine motility (E) and induced SHPs (F) only on dendritic segments included in the digestion area. *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. Error bars indicate SEM.