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. 2019 Sep 26;9(1):13939.
doi: 10.1038/s41598-019-50404-9.

Elimination of the four extracellular matrix molecules tenascin-C, tenascin-R, brevican and neurocan alters the ratio of excitatory and inhibitory synapses

Christine Gottschling  1 , David Wegrzyn  1 , Bernd Denecke  2 , Andreas Faissner  3
Affiliations

Elimination of the four extracellular matrix molecules tenascin-C, tenascin-R, brevican and neurocan alters the ratio of excitatory and inhibitory synapses

Christine Gottschling et al. Sci Rep. .

Abstract

The synaptic transmission in the mammalian brain is not limited to the interplay between the pre- and the postsynapse of neurons, but involves also astrocytes as well as extracellular matrix (ECM) molecules. Glycoproteins, proteoglycans and hyaluronic acid of the ECM pervade the pericellular environment and condense to special superstructures termed perineuronal nets (PNN) that surround a subpopulation of CNS neurons. The present study focuses on the analysis of PNNs in a quadruple knockout mouse deficient for the ECM molecules tenascin-C (TnC), tenascin-R (TnR), neurocan and brevican. Here, we analysed the proportion of excitatory and inhibitory synapses and performed electrophysiological recordings of the spontaneous neuronal network activity of hippocampal neurons in vitro. While we found an increase in the number of excitatory synaptic molecules in the quadruple knockout cultures, the number of inhibitory synaptic molecules was significantly reduced. This observation was complemented with an enhancement of the neuronal network activity level. The in vivo analysis of PNNs in the hippocampus of the quadruple knockout mouse revealed a reduction of PNN size and complexity in the CA2 region. In addition, a microarray analysis of the postnatal day (P) 21 hippocampus was performed unravelling an altered gene expression in the quadruple knockout hippocampus.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Expression of the synaptic puncta vGlut1 and PSD95 in hippocampal neurons after 14 and 21 DIV. (a,a’,a”,a”’,b,b’,b”,b”’,c,c’,c”,c”’,d,d’,d”,d”’) Representative images of the immunocytochemical stainings of the presynaptic protein vGlut1 (puncta appearing in magenta) and the postsynaptic protein PSD95 (green puncta) after 14 DIV and 21 DIV for the four conditions (Nwt/wt/Awt/wt, Nwt/wt/Ako/ko, Nko/ko/Awt/wt, Nko/ko/Ako/ko) are shown. The white puncta reflect the colocalization of both proteins and indicate structural synapses. The PNNs were visualized using aggrecan (blue), which is visible in (a,a”,b,b”,c,c”,d,d”) for PNN-bearing neurons. In (a’,a”’,b’,b”’,c’,c”’,d’,d”’), neurons devoid of PNNs are exemplified. Next to each of the micrographs is a higher magnification of one exemplary neurite (white box in each image) showing the individual synaptic puncta in more detail. Scale bar: 50 µm in D”’. (e,f,g,h) Analysis of the relative increase or decrease of the number of synaptic puncta in percent after 14 (e,f) and 21 DIV (g,h). PNN-wearing knockout neurons after 14 DIV (e) combined with wildtype astrocytes (Nko/ko/Awt/wt) and knockout astrocytes (Nko/ko/Ako/ko) showed a significant rise of excitatory synapses. The same applies for PNN-free neurons after 14 DIV (f). PNN-wearing knockout neurons (g) co-cultivated with knockout astrocytes (Nko/ko/Ako/ko) showed a significant enhancement of structural synapses in culture compared to the control (Nwt/wt/Awt/wt) after 21 DIV. No significant differences were revealed after 21 DIV for the number of synaptic puncta of PNN-negative knockout neurons (h) compared to the control condition. Statistics: Five independent experiments (biological replicates N = 5) were performed choosing randomly 20 neurons with (n = 20) and 20 neurons without PNNs (n = 20) per each condition. In sum, 800 neurons were analysed. Data are expressed as mean ± SEM (ANOVA and Scheffé post hoc test for PSD-95 data sets and Kruskal-Wallis test for vGlut1 and Colocalization data sets, p ≤ 0.05).
Figure 2
Figure 2
Expression of the synaptic puncta vGAT and gephyrin in hippocampal neurons after 14 and 21 DIV. (a,a’,a”,a”’,b,b’,b”,b”’,c,c’,c”,c”’,d,d’,d”,d”’) Representative images show the immunocytochemical stainings of hippocampal neurons after 14 and 21 DIV with the presynaptic marker vGAT (magenta puncta) and the postsynaptic marker gephyrin (green puncta) to detect inhibitory synapses in all four conditions (Nwt/wt/Awt/wt, Nwt/wt/Ako/ko, Nko/ko/Awt/wt, Nko/ko/Ako/ko). Colocalization of these proteins leads to the appearance of white puncta, which we define as structural inhibitory synapses in the neuronal cultures. To distinguish between neurons with (a,a”,b,b”,c,c”,d,d”) and without PNNs (a’,a”’,b’,b”’,c’,c”’,d’,d”’), aggrecan (blue) was used to visualize the PNNs. The higher magnification next to each image (white box in each image) displays one representative neurite to allow for a more detailed view on the distribution of the synaptic puncta. Scale bar: 50 µm in d”’. (e,f,g,h) The total number of the vGAT, gephyrin and colocalized puncta was quantified and the percentage of the increase/decrease of the number of synaptic puncta was thus determined after 14 (e,f) and 21 DIV (g,h). Irrespective of whether the neurons were covered by PNN (e) or not (f), the knockout neurons showed a significantly reduced number of synaptic puncta. PNN-wearing knockout neurons co-cultivated with knockout astrocytes for 21 DIV (Nko/ko/Ako/ko) exhibited a significant lower number of gephyrin puncta in comparison to the control (Nwt/wt/Awt/wt) (g), whereas the number of structural synapses was not significantly altered. Knockout neurons combined with knockout astrocytes and without PNNs showed no significant change in the number of their synaptic puncta (h). Statistics: Five independent experiments (N = 5) were performed choosing randomly 20 neurons with (n = 20) and 20 neurons without PNNs (n = 20) per each condition. In sum, 800 neurons were analysed. Data are expressed as mean ± SEM (Kruskal-Wallis test, p ≤ 0.05).
Figure 3
Figure 3
Cultivation of hippocampal neurons on MEAs. (a,b) Illustrates a standard MEA (electrode grid 8 × 8) for measuring the network activity of cells, e.g. of hippocampal neurons. In the centre of the MEA a glass ring (6 mm high) is located containing the electrode field on which the neurons were cultivated in 1 ml medium. Each MEA is equipped with 60 electrodes, including one internal reference electrode (red star in A). Each electrode is made of titanium nitride and has a diameter of 30 µm. The spacing between each electrode is 200 µm. (c,d,e) The exemplary microscopic images illustrate the growth of the hippocampal neurons on the MEA that elaborate a complex network after 14 DIV, as shown at higher magnification. (e) Neurons form numerous synaptic connections with each other. Scale bar (c,d): 200 µm. Scale bar (e): 100 µm. (f) Representative image of the activity of 60 electrodes shown by the spike sorter. Each small square displays the activity of one individual electrode. The occurrence of bursts can be detected through the emergence of simultaneous activity at the majority of electrodes. The red star marks the reference electrode. (g) Higher magnification of the neuronal activity of a singular electrode. Action potentials occur in the form of spikes and were registered when their amplitude exceeded the triggering threshold by 4.5-fold of the standard deviation.
Figure 4
Figure 4
Spontaneous neuronal network activity after 14 and 21 DIV measured via MEA. Using MEA analysis, the spontaneous activity of neuronal networks derived from the hippocampus of either wildtype or quadruple knockout mice was examined. Different parameters were detected and quantified for the four conditions (Nwt/wt/Awt/wt, Nwt/wt/Ako/ko, Nko/ko/Awt/wt, Nko/ko/Ako/ko) including the number of spikes (a), the number of bursts (b), the spike frequency (c), the spike frequency in bursts (d), the percentage of spikes in bursts (e) and the mean burst duration (f). Most analysed parameters were enhanced in the neuronal networks of knockout neurons cultured with knockout astrocytes (Nko/ko/Ako/ko) compared to the control (Nwt/wt/Awt/wt). For example the number of spikes increased significantly to almost twice the control level after 14 DIV as well as 21 DIV and the number of bursts in culture was significantly enhanced too. This also resulted in a higher spike frequency as well as in a higher percentage of spikes in bursts in the knockout neurons grown with knockout astrocytes. The only parameter that remained unchanged between these two conditions was the mean burst duration. Statistics: Five independent experiments (N = 5) were performed with all in all 60 electrodes (n = 60) on each of one or two MEAs per condition. Data are expressed as mean ± SEM (Kruskal-Wallis test, p ≤ 0.05).
Figure 5
Figure 5
Expression of PNNs in the developing murine hippocampus in vivo. (a–j) Immunohistochemical detection of PNNs in the hippocampus of postnatal wildtype and knockout mice (P15, P20, P25, P30, P35) using the lectin WFA as marker for PNNs (magenta). Beyond a general expression of PNNs next to the CA1 region and single WFA-positive cells in the hippocampus as well as the cortex, a distinct PNN area was detected in the CA2 region of the hippocampus. This PNN area is noticeably smaller in the hippocampus of the quadruple knockout mice, which is also clearly visible in the higher magnification of this region within each image. The antibody NF200 (green) was utilized for the detection of neurons. The nuclei were marked by bisbenzimide (blue, Hoechst). Scale bar: 500 µm in a and 250 µm in inset of a. (k–n) Close-up images of the immunohistochemical WFA and aggrecan staining in the wildtype CA2 region of P20 mice. Images were taken using a confocal laser scanning microscope with a 400× (k,m) and 630x magnification (l,n). The both PNN markers aggrecan and WFA clearly show a strong fluorescence signal around the soma of CA2 neurons. Scale bar: 100 µm in k,m and 50 µm in l,n.
Figure 6
Figure 6
PNN area size in different developmental stages of the murine hippocampus. (a,b) Representative images illustrate the immunohistochemical detection of PNNs in the hippocampus of a P30 wildtype mouse. WFA (magenta) was used as a marker to detect the PNNs surrounding individual neurons. The area size in the CA2 region of the hippocampus was measured as indicated by the line surrounding the PNN-positive territory (b, white line). Neurons were visualized using an antibody to NF200 (green) and Bisbenzimide (Hoechst) was used to detect the nuclei. Scale bar in A: 500 µm. (c,c’, d,d’) Representative images of the WFA-staining converted into white for a better visualization of the PNN fluorescence intensity of the wildtype (c,c’) and knockout (d,d’) hippocampus at P25. The higher magnification of the WFA-positive area in the CA2 region (white box) indicates a higher intensity of the PNN fluorescence in the wildtype compared to the knockout hippocampus. Scale bar: 500 µm in C, D and 250 µm in C’, D’. (e) Quantitative in vivo analysis of the PNN area size in the hippocampal CA2 region of postnatal mice (P15, P20, P25, P30 and P35). Except at P35, all other analysed postnatal stage of the hippocampus showed a significantly decreased area size of the PNNs in the CA2 region of knockout mice. At P35, the PNN areas size was not significantly different between knockout and wildtype. (f) Quantitative analysis of the PNN fluorescence intensity (CTCF) in the CA2 region of the hippocampus of mice at P15, P20, P25, P30 and P35. The fluorescence intensity was significantly reduced within the hippocampus of knockout compared to wildtype mice. The only exception was at P35, when no significant alteration of the PNN fluorescence intensity was detectable. Statistics: Three independent experiments (biological replicates N = 3) were performed and the PNN area size of four hippocampi (n = 4) was examined. Data are expressed as mean ± SEM (F-test and unpaired student’s t-test, p ≤ 0.05).
Figure 7
Figure 7
Hierarchical clustering of genes in the P21 hippocampus of quadruple knockout and wildtype mice. (a) The microarray analysis revealed that 438 genes are significantly altered in the quadruple knockout mouse. These are depicted in form of a volcano plot, which includes upregulated genes (red dots), downregulated genes (blue dots) and unaffected genes (grey). (b) The hierarchical clustering of genes in the P21 hippocampus uncovered a differential gene expression pattern in the quadruple knockout compared to the wildtype mouse using three dependent hippocampus samples of siblings (n = 3). The cluster heat map comprises significantly up- and downregulated genes of interest concerning the annotations “ECM” as well as “neurons/synapses” including Gpc3, Gabrq, Gad2, Wnt7, Syt9, Sod3, Dnm3, Cript, Sema4c, Calr, Cplx3, Col4a3, Col27a1, Adamts13, Grin2d, Col1a2, Spon2 and Rapsn. The colour shift of light blue to light red indicates the expression change of these genes, that is genes that appear in blue were down- whereas genes illustrated in red were upregulated in the hippocampus of the quadruple knockout mouse. (c) Linear fold change of exemplary ECM molecules (Spon2, Col1a2, Col4a3, Col27a1, Gpc3 and Calr) and (d) neuron related molecules (Adamts13, Cript, Sod3, Wnt7a) in the P21 quadruple knockout hippocampus. (e) Linear fold change of representative genes belonging to the field of neurons and their synapses (Rapsn, Cplx3, Grin2d, Dnm3, Sema4c, Syt9, Gad2, Cntnap4, Gabrq, SLC17A7, SLC32A1 and GPHN) in the quadruple knockout hippocampus.
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