Experience-dependent plasticity and modulation of growth regulatory molecules at central synapses

Abstract

Structural remodeling or repair of neural circuits depends on the balance between intrinsic neuronal properties and regulatory cues present in the surrounding microenvironment. These processes are also influenced by experience, but it is still unclear how external stimuli modulate growth-regulatory mechanisms in the central nervous system. We asked whether environmental stimulation promotes neuronal plasticity by modifying the expression of growth-inhibitory molecules, specifically those of the extracellular matrix. We examined the effects of an enriched environment on neuritic remodeling and modulation of perineuronal nets in the deep cerebellar nuclei of adult mice. Perineuronal nets are meshworks of extracellular matrix that enwrap the neuronal perikaryon and restrict plasticity in the adult CNS. We found that exposure to an enriched environment induces significant morphological changes of Purkinje and precerebellar axon terminals in the cerebellar nuclei, accompanied by a conspicuous reduction of perineuronal nets. In the animals reared in an enriched environment, cerebellar nuclear neurons show decreased expression of mRNAs coding for key matrix components (as shown by real time PCR experiments), and enhanced activity of matrix degrading enzymes (matrix metalloproteinases 2 and 9), which was assessed by in situ zymography. Accordingly, we found that in mutant mice lacking a crucial perineuronal net component, cartilage link protein 1, perineuronal nets around cerebellar neurons are disrupted and plasticity of Purkinje cell terminal is enhanced. Moreover, all the effects of environmental stimulation are amplified if the afferent Purkinje axons are endowed with enhanced intrinsic growth capabilities, induced by overexpression of GAP-43. Our observations show that the maintenance and growth-inhibitory function of perineuronal nets are regulated by a dynamic interplay between pre- and postsynaptic neurons. External stimuli act on this interaction and shift the balance between synthesis and removal of matrix components in order to facilitate neuritic growth by locally dampening the activity of inhibitory cues.

Figure 1. Size changes of PC axon terminals following exposure to EE.

(A-H) DCN neurons, visualized by SMI32 antibodies (blue), are contacted by PC axon terminals, stained by anti-calbindin antibodies (green). In both wild-type (A,B,E,F) and transgenic mice (C,D,G,H), PC terminals are enlarged after EE (B,F,D,H). (I) Size of PC axon boutons in the different experimental conditions (One Way Anova; N = 4 wild-type and transgenic ST; N = 10 wild-type EE and 9 transgenic EE mice). (J) Relative size increase of PC terminals induced by EE in wild-type and transgenic mice (Student's t-test; N = 10 wild-type, N = 9 transgenic mice). (K) Number of PC terminals/mm of post-synaptic membrane of DCN neurons (One Way Anova; N = 5 mice/experimental condition). *P<0.05; **P<0.01. Scale bars: 10 µm (A–D), 2 µm (E–H). WT: wild-type; TG: transgenic; ST: standard condition; EE: enriched condition; CaBP: calbindin; SMI32: neurofilament-H non-phosphorylated.

Figure 2. Changes of glutamatergic terminal density after EE.

(A–D₁) The micrographs show the distribution pattern of excitatory mossy fiber and olivocerebellar axon terminals, stained by anti-v-glut2 antibodies (red), in the DCN. DCN neurons are visualized by SMI32 antibodies (green). (E) Quantification of the density of glutamatergic terminals in the DCN in the different experimental conditions (One Way Anova; N = 4 ST and EE wild-type mice, N = 7 ST and EE transgenic mice). **P<0.01. Scale bar: 20 µm. WT: wild-type; TG: transgenic; ST: standard condition; EE: enriched condition; SMI32: neurofilament-H non-phosphorylated; v-glut2: second vesicular glutamate transporter.

Figure 3. PNNs decrease after EE. (A–D₁) PNNs, stained by WFA (red), enwrap DCN projection neurons, labeled by SMI32 antibodies (blue).

(E) Percentage of SMI32-positive neurons bearing a WFA-positive net in the different experimental conditions (One Way Anova; N = 5 mice/experimental condition). (F) Frequency distribution of PNNs subdivided in three categories according to their WFA staining intensity (see methods; χ²-test: 343.04 with 6 DF). (g–j₁) HABP staining of PNNs (red) around SMI32-positive neurons (blue). (K) Frequency distribution of HABP-positive nets subdivided in three categories according to their staining intensity (χ²-test: 38.77 with 6 DF). **P<0.01. Scale bar: 20 µm. WT: wild-type; TG: transgenic; ST: standard condition; EE: enriched condition; SMI32: neurofilament-H non-phosphorylated; WFA: Wisteria floribunda agglutinin; HABP: hyaluronan binding protein.

Figure 4. Morphology of PNN and PC terminals in cartilage LP1 knockout mice.

(A–B₁) show the appearance of WFA-positive nets (red), surrounding SMI32-positive DCN neurons (green) in wild-type (A,A₁) and cartilage LP1 knockout mice (B,B₁). (C) Frequency distribution of PNNs subdivided in three categories according to their WFA staining intensity (χ²-test: 52.26 with 2 DF). (D–E₂) Morphology of PC terminals (green) impinging upon DCN neurons (red) in wild-type (D-D₂) and cartilage LP1 knockout mice (E–E₂). (F) Size of PC axon terminals in the two strains (Student's t-test; N = 3 wild-type, N = 4 knockout mice). **P<0.01. Scale bars: 20 µm in A–B₁, 10 µm in D–E₂. WT: wild-type; Crtl1 KO: cartilage LP1 knockout; WFA: Wisteria floribunda agglutinin; SMI32: neurofilament-H non-phosphorylated; CaBP: calbindin.

Figure 5. Expression of mRNA for PNN components in the DCN following EE.

(A–I) Real-Time PCR experiments on DCN extracts show the level of expression of mRNA for cartilage LP1 (A–C: Student's t-test, N = 6–10/experimental condition), aggrecan (D–F: Student's t-test, N = 9/experimental condition), and HAS2 (G–I: Student's t-test, N = 6/experimental condition) in wild-type (A,D,G) or L7/GAP-43 mice (B,E,H) reared in ST conditions or EE. (C,F,I) compare basal level of the three molecules in the two mouse strains in ST conditions. *P<0.05; **P<0.01. Crtl1: cartilage LP1; HAS2: hyaluronan synthase 2.

Figure 6. Immunolabeling for MMP9 and MMP2 in the DCN.

(A,B,D,E) Immunohistochemistry for MMP9 (A,B) and for MMP2 (D,E) in wild-type (A,D) and transgenic (B,E) animals in ST conditions. (C,F) Quantification of the staining intensity for MMP9 (C; One Way Anova; N = 3 mice/experimental condition) and MMP2 (F; One Way Anova; N = 3 mice/experimental condition) in the four experimental conditions. *P<0.05; **P<0.01. Scale bar: 20 µm. WT: wild-type; TG: transgenic; MMP9: matrix metalloproteinase 9; MMP2: matrix metalloproteinase 2.

Figure 7. Relationship between MMP activity and PNN intensity in the DCN.

(A–D₂) show the ISZ staining (green) of DCN neurons and WFA labeling of PNNs (red) in the different experimental conditions. (E–H) Representative examples of the relationship between ISZ and WFA staining intensity in individual neurons (each plot reports data from a single animal for each group). (I) Regression lines calculated for each experimental group (N: wild-type ST = 106, wild-type EE = 167, transgenic ST = 148, transgenic EE = 176): the different regression lines have been plotted with the same intercept value (100), in order to compare the obtained slopes (One Way Anova on angular coefficients). Regression parameters are reported below the graph. Scale bars: 20 µm. WT: wild-type; TG: transgenic; ST: standard condition; EE: enriched condition; ISZ: In situ zymography; WFA: Wisteria floribunda agglutinin.

Figure 8. Morphology of PNNs in partially denervated DCN.

(A₁–D₂) DCN neurons in intact and denervated wild-type and L7/GAP-43 mice (14 days after propidium iodide injection): SMI32-positive DCN neurons are visualized in blue, calbindin-immunolabeled PC terminals are green (A₁–D₁), and WFA stained PNNs are red (A₂–D₂). (E) Frequency distribution of PNNs subdivided in three categories according to their WFA staining intensity (χ²-test: 188.1 with 6 DF). Scale bar: 20 µm. WT: wild-type; TG: transgenic; ST: standard condition; EE: enriched condition; CaBP: calbindin; SMI32: neurofilament-H non-phosphorylated; WFA: Wisteria floribunda agglutinin.