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Fig. 4. Reduction of endogenous gephyrin clusters after transfection of EGFP-gephyrin constructs. Frequency distribution of endogenous gephyrin clusters on dendrites of mock-transfected cells (A,D) and cells transfected with EGFP-geph-GC (B,E), EGFP-geph-E (C,F), EGFP-geph-GCEE (G) or EGFP-geph-L2B (H). Red, 6+6; green, 10+2 experiments. Dominant-negative effects are evidenced by a shift of the distribution towards lower cluster numbers per segment (see Table 1 for statistical analysis). Dendrites devoid of gephyrin clusters are highlighted in pale color. The strongest effect occurred after transfection of EGFP-geph-GC and EGFP-geph-E. In cells transfected with EGFP-geph-GCEE (G), dendrites devoid of endogenous gephyrin clusters were from cells with a strong transgene expression. The 10+2 experiments show that the reduction of gephyrin clusters also occurs after only 2 days of recombinant gephyrin expression, suggesting a rapid replacement of gephyrin in postsynaptic clusters. (I) Summary table indicating the proportion of dendrites with an almost complete loss of endogenous gephyrin clusters. Note that for EGFP-geph-E and EGFP-geph-GC, this fraction is approximately double after long-term expression (6+6) compared with short-term expression (10+2). Mock-transfected dendrites show no evidence from toxic effects of the transfection reagent after long-term treatment.

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