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First published online February 18, 2009
doi: 10.1242/10.1242/jcs.037382

Journal of Cell Science 122, 735-744 (2009)
Published by The Company of Biologists 2009
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The function of glutamatergic synapses is not perturbed by severe knockdown of 4.1N and 4.1G expression

Christian Wozny1,*,{ddagger},§, Jörg Breustedt1,*, Friederike Wolk2,*, Frédérique Varoqueaux2, Susann Boretius3, Aleksandar R. Zivkovic1, Antje Neeb4, Jens Frahm3, Dietmar Schmitz1, Nils Brose2 and Aleksandra Ivanovic2,{ddagger}

1 Neuroscience Research Center, Charité–Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany
2 Max-Planck-Institut für Experimentelle Medizin, Abteilung Molekulare Neurobiologie, DFG Center for Molecular Physiology of the Brain, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany
3 Biomedizinische NMR Forschungs GmbH, Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, D-37077 Göttingen, Germany
4 Institut für Toxikologie und Genetik, Forschungszentrum Karlsruhe, Hermann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany


Figure 1
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  		Fig. 1. Targeting of genes encoding 4.1G and 4.1N. (A,D) Schematic representations of the mouse 4.1G and 4.1N genomic loci, targeting vectors, targeted alleles, and excision of the first coding exon by Cre recombinase. LoxP sites are indicated by grey triangles, the neomycin selection cassette is flanked by frt sites (black arrowheads) for possible excision by flip recombinase. Ex, exon. (A) Targeting strategy for gene encoding 4.1G. The first coding exon has been excised. The positions of AccI restriction sites and the 5' Southern probe (Probe 1) are shown. (B) Southern blot analysis of 4.1G. Probe P1 was used for hybridisation. The signal for the 4.1G WT allele resulted in a 6598 bp band and the recombinant allele resulted in a 4569 bp band. (C) Genotyping of 4.1G WT and mutant mice by PCR. In 4.1G WT animals primers p1 and p2 generated a 144 bp band. In recombinant alleles and after Cre recombination a 408 bp fragment is synthesized with primers p3 and p4. (D) Targeting strategy for gene encoding 4.1N. The first coding exon has been excised. The positions of ScaI restriction sites and the 5' Southern probe (Probe 2) are marked. (E) Southern blot analysis with Probe 2 after ScaI digestion of stem cell DNA. The introduction of the neomycin selection cassette resulted in an additional ScaI restriction site and a shift of the 8904 bp WT band to a 4471 bp band. (F,G) Genotyping of 4.1N mutants. Genotyping of mouse tail DNA with primers p5 and p6 resulted in a 243 bp WT band and no band was synthesized after knockout of 4.1N (F). In genomic PCRs with primers p7 and p8, a 1239 bp band for the recombinant allele (flox/flox–/–) and a 708 bp band after Cre recombination (Cre–/–) were detected (G).

 

Figure 2
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  		Fig. 2. Anatomical differences in young 4.1G/N double-mutant mice are compensated during development. (A) Anatomical 3D MRI at age 3 weeks. T1-weighted (upper panel) and T2-weighted (lower panel) horizontal sections from 3D MRI data sets of WT and 4.1G/N double-mutant (DKO) mice reveal no obvious differences in overall brain morphology (n=4, for WT and DKO). (B) 3D MRI of adult mice. T1-weighted (upper panel) and T2-weighted (lower panel) horizontal sections from 3D MRI data sets of WT and 4.1G/N double-mutant mice reveal no obvious differences in overall brain morphology (n=3, for WT and DKO). (C) Quantitative in vivo MRI volumes based on the images shown in A, error bars indicate s.d. The whole-brain volume was significantly reduced in the 4.1G/N double mutants at the age of 3 weeks (**P=0.0074, determined by Student's t-test) but not in adult mutants. (D) The cerebellar volume was significantly reduced in the 4.1G/N double mutants at the age of 3 weeks (*P=0.039, determined by Student's t-test, error bars indicate s.d.) but not in adult mutants. (E) The ventricle volume was not altered in 3-week-old 4.1G/N double-mutant or in adult mice.

 

Figure 3
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  		Fig. 3. Synaptic morphology in the hippocampus of 4.1G/N double-mutant mice. (A) Properly aligned pre- and postsynaptic specializations in stratum radiatum of hippocampal area CA1. The left panel shows representative micrographs of the CA1 area of control (WT, n=3) and 4.1G/N double-mutant (DKO, n=3) sections after double labeling for glutamatergic excitatory postsynapses (stained for ProSAP1, red) and presynapses (stained for VGLUT1/2, green). Scale bars: 8 µm. (B) Quantification of isolated and colocalized ProSAP1 and VGLUT1/2 puncta in the CA1 region of control (WT, white, n=3) and 4.1G/N double-mutant mice (DKO, grey, n=3). The total numbers of synapses were not significantly different in the two experimental groups. Error bars indicate s.d. (C,D) Ultrastructural analysis of CA1 neurons in 4.1G/N double-mutant mice and WT controls at the age of 3 weeks. Mature synaptic specializations were observed in both groups; quantification was performed in the stratum radiatum. The number of synapses per 100 µm2 and the length and the width of PSDs were similar in both experimental groups. Error bars indicate s.d. The numbers within the bars in D indicate the number of synapses. Scale bar: 500 nm.

 

Figure 4
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  		Fig. 4. Expression pattern of 4.1G and 4.1N in WT and 4.1G/N double-mutant brains. (A) Western blot analysis using a 4.1G-specific antibody detecting an epitope located within the first coding exon. 4.1G is expressed in all examined regions of the central nervous system (upper panel). OB, olfactory bulb; CX, cortex; CPu, striatum; Hi, hippocampus; Thal, thalamus; Hthal, hypothalamus; Co, colliculus; Cb, cerebellum; BS, brain stem; SpC, spinal cord. No signal was detected in 4.1G/N double-mutant mice (lower panel). (B) Western blot analysis using the 4.1N monoclonal antibody. 4.1N is expressed in all examined brain regions with minor expression in BS and SpC. The short variant of the two 4.1N isoforms was still present at low levels in the 4.1G/N double-mutant animals.

 

Figure 5
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  		Fig. 5. Residual expression of a shorter 4.1N form in the 4.1G/N double-mutant hippocampus. (A-C) Hippocampal preparations from 3-week-old WT (left panel) or 3-week-old 4.1G/N double mutant (right panel) were loaded and stained on western blots with the following antibodies. (A) Anti-4.1N exon 2, which is directed against the first 15 amino acids of the N-terminus; (B) Anti-4.1N monoclonal antibody, which recognizes a C-terminal region of the spectrin-actin-binding domain; (C) Anti-4.1N CTD, which is detecting the very C-terminus of 4.1N. (D) Densitometric quantification of data pooled from B and C. The error bars indicate s.e.m. **P<0.01; ***P<0.001; Student's t-test. (E) Schematic representation of the murine full-length 4.1N protein. The positions of the epitopes of the antibodies used in A-C are indicated. U1, unique region 1; FERM, Four-point-one, ezrin, radixin, moesin homology domain; FA, FERM adjacent region; U2, unique region 2; SABD, spectrin-actin-binding domain; U3, unique region 3; CTD, C-terminal domain. Unique regions are shown in yellow, conserved areas are in blue. (F) Exon usage map of the mouse 4.1N gene in brain. Numbering is derived from mouse Epb41l1 as described (Ramez et al., 2003Go). The colour code is modelled on the domain structure shown in E. Dotted boxes denote nontranscribed exons. The positions of the specific 5'RACE primers and the specific 3'RACE primers are indicated by arrows. (G) Schematic representation of the obtained RACE transcripts from WT and 4.1G/N double mutants.

 

Figure 6
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  		Fig. 6. GluR levels are altered in 4.1G/N double-mutant mice. (A) Reduced GluR1 levels in whole brain synaptosomal preparations. Protein levels in synaptosomal fractions of 3-week-old mice expressed as percentage of WT levels. Error bars indicate s.d. (WT, n=6; DKO, n=6; **P<0.01). (B) Reduced GluR1 and GluR2/3 levels in PSD preparations from young adult hippocampi. Protein levels are expressed as percentage (± s.d.) of WT levels (WT, n=7; DKO, n=7; *P<0.05).

 

Figure 7
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  		Fig. 7. Synaptic responses in area CA1 in WT and 4.1G/N double-mutant mice. (A) Input-output curves for basal synaptic transmission in area CA1 of the hippocampus. Sample traces are shown for the input (fiber volley) and the output (fEPSP). No significant difference was found between WT and 4.1G/N double mutants in the fEPSP slopes at various afferent volley amplitudes (WT, n=10; DKO, n=13). (B) Ratio of AMPA and NMDA currents is not altered in 4.1G/N double mutants (AMPA/NMDA ratio: WT, n=13; DKO, n=11). Sample traces from CA1 pyramidal cells are shown. (C) Paired-pulse facilitation (PPF) is unchanged in 4.1G/N double-mutant mice. PPF in field recordings is not significantly changed in double-mutant mice at intervals varying from 50 mseconds to 500 mseconds (WT, n=7; DKO, n=10). (D) Whole-cell recordings from CA1 pyramidal cells do not reveal differences in PPF between WT and 4.1G/N double mutants at an interstimulus interval of 50 mseconds (WT, n=15; DKO, n=15). Stimulus artefacts were removed.

 

Figure 8
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  		Fig. 8. mEPSCs recorded from CA1 pyramidal neurons in WT and 4.1G/N double-mutant animals. (A,B) Miniature EPSCs (mEPSCs) were recorded in the presence of the sodium channel blocker tetrodotoxin (TTX, 1 µM) and the AMPAR-desensitization inhibitor cyclothiazide (CTZ, 100 µM). mEPSCs showed no change in frequency (A) or amplitude (B). (C) Recordings of WT and 4.1G/N double mutants cells at –60 mV (for details see text). A cumulative frequency distribution of mEPSCs amplitudes is shown in (A). n.s., not significant.

 

Figure 9
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  		Fig. 9. Extrasynaptic AMPA currents are not reduced in 4.1G/N double-mutant mice. (A,B) Whole-cell currents evoked by bath application of 100 nM AMPA. A single example is shown in A and a summary plot in B. AMPA was applied for 5 minutes in the presence of 100 µM cyclothiazide and 1 µM TTX. (C) Maximal AMPA whole-cell current is not altered in 4.1G/N double mutants. The numbers in parentheses indicate the number of mice studied. n.s., not significant.

 

Figure 10
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  		Fig. 10. LTP is not impaired in 4.1G/N double-mutant animals. (A) Depicted are representative results from WT (open circles) and 4.1G/N double-mutant (filled circles) animals. LTP was induced by tetanic stimulation, i.e. 100 pulses at 100 Hz, four 4 times, 20 seconds apart. Traces on top of the graph are averages of 7-10 consecutive responses each, taken from control and at the end of the recording period. (B) Summary plot of 10 experiments in WT and 11 experiments in 4.1G/N double-mutant mice. No differences in synaptic plasticity were detected between both groups.

 

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© The Company of Biologists Ltd 2009