Fig 2: Short-term application of active rMMP3 potentiates mIPSC amplitude, immobilizes synaptic GABA receptors and increases the size of synaptic gephyrin clusters. a On left: typical raw mIPSC recordings before and 30 min after rMMP3 or sham treatment. Dotted line represents the man amplitude of baseline mIPSC. On right: averaged mIPSC traces recorded before (grey) and 30 min after treatment (sham—black, rMMP3—green). b mIPSC amplitude recorded in the sham-treated group and in neurons that were incubated for 3 min with active rMMP3 (400 ng/ml). c Comparison of changes in mIPSC amplitude in the sham group and in neurons that were treated with rMMP3. d Changes in mIPSC amplitude measured before and 28–30 min after rMMP3 administration (green) or sham treatment (gray). e a1GABAA receptor trajectories before and after application of rMMP3. Extrasynaptic parts of trajectories are marked as black, synaptic parts are marked blue. Scale bar 500 nm. f Mean square displacement of synaptic a1GABAA receptors before (black) and 20 min after (green) rMMP3 incubation. g Interquartile range and median diffusion coefficient of synaptic a1GABAA receptors before and 20 min after short-term (2 min) incubation with rMMP3. N and n refer to the number of coverslips and analysed trajectories, respectively. h Cumulative probability distributions of the total residence time in the synapse by a1GABAA receptors before (black) and 20 min after (green) the application of rMMP3. The total imaging time (1 min) indicates the maximal possible time that the analysed receptors could be detected in the synapse. (Inset) Average total residence time before and after rMMP3 treatment. i Representative confocal maximum projection images obtained from neuronal cultures that were immunolabeled with antibodies against presynaptic vGAT (magenta) and postsynaptic gephyrin (green). The colocalization of gephyrin and vGAT (white) corresponds to the presence of gephyrin at GABAergic synapses. (Upper) Images of two typical portions of dendrites after sham treatment. (Lower) Images of two typical portions of dendrites that undergo 2 min 15 s incubation with rMMP3 (400 ng/ml). j The average area of gephyrin clusters at dendritic inhibitory synapses (normalized to sham). k Representative confocal maximum projection images obtained from neuronal cultures that were labeled with antibodies against presynaptic vGAT (magenta) and postsynaptic gephyrin (green). The upper, middle, and lower images show typical fragments of dendrites that were fixed 20 min after sham, NMDA, and NMDA + rMMP3 treatment, respectively. l Quantification of the average area of synaptic gephyrin clusters in neuronal cultures that were treated with sham solution, NMDA, and NMDA + rMMP3. Values are normalized to sham group. *p < 0.05, **p < 0.01, ***p < 0.001. ns nonsignificant. The data in c, j, l, were analyzed using t test vs. respective control. The data in d were analyzed using paired t tests. The data in f–h were analyzed using the Mann–Whitney U test. Numbers on the bars refer to the number of coverslips from at least three different batches of cultures

Fig 3: Inhibition of MMP3 activity blocks iLTP induction and gephyrin accumulation at GABAergic synapses in neuronal cultures. a, b Time course (a) and magnitude (b) of NMDA-induced iLTP recorded under control conditions and in the presence of the MMP3 inhibitor UK-356618 (t test) in cultured hippocampal neurons. The gray area marks the application of NMDA. c Changes in mIPSC amplitude measured before and 20–22 min after iLTP induction with NMDA in the control and UK-356618 groups (paired t test). d, f Representative confocal maximum projection images of neuronal cultures immunolabeled with antibodies against presynaptic vGAT and postsynaptic gephyrin. (Upper) Low-magnification image of the gephyrin signal in the whole neuron. Framed areas are magnified below. (Lower) Magnification of exemplary dendrites indicated above (marked with colored boxes). The colocalization of gephyrin (green) and vGAT (magenta) corresponds to the presence of gephyrin at GABAergic synapses (white). e, g Quantification of the area of synaptic gephyrin clusters in neurons that were treated with e sham solution, NMDA, UK-356618, and NMDA + UK-356618 (wild type) and g sham solution and NMDA (Mmp3-/-). Values are normalized to respective sham group, t test comparison vs. respective control. **p < 0.01, ***p < 0.001. ns nonsignificant. Numbers on the bars refer to the number of coverslips from at least three different batches of cultures

Fig 4: Relative loss of GABAergic synapses and Calbindin-immunoreactive cells. (a) Glutamatergic and GABAergic synapses were stained with Vglut1 (blue, white arrow) and Vgat (green, asterisk), respectively, for manual and automated quantification. Only synapses showing co-localization with Bsn (red) were counted. Confocal microscopy at 63×. (b) Processed immunofluorescence images for pre- and post-synapses of Fiji plug-in “Synapse Counter” and resulting co-localizations. Scale bars are 500 nm. (c) Relative quantification of GABAergic over glutamatergic synapses at 3 and 7 days after glutamate treatment as compared with time-matched sham-treated controls. (d,e) Relative amount of Calb1-positive neurons compared with Calb1-negative neurons in glutamate and sham-treated cultures after 7 days. Asterisks (*) indicate significance. ctrl—control, d—day, glu—glutamate.

Fig 5: Mover colocalization with presynaptic markers vesicular glutamate transporter 1 (vGluT1) and vesicular ?-aminobutyric acid (GABA) transporter (vGAT) in the mouse hippocampus. Immunofluorescence triple labeling of the mouse hippocampus. (A–C) Overview showing the heterogeneous mover expression pattern (A) and the corresponding vGluT1 (B) and vGAT staining (C). (D–I) Overlay of mover (magenta) and vGluT1 staining (green, upper panel) and mover (magenta) and vGAT (green, lower panel) in regions of the hippocampus where mover immunofluorescence was above average. (D) High colocalization of mover with vGluT1 in polymorph layer of DG, low colocalization of mover with vGAT. (E) High colocalization of mover with vGluT1 in stratum radiatum of CA3, low colocalization of mover with vGAT. (F) High colocalization of mover with vGluT1 in stratum lucidum of CA3, low colocalization of mover with vGAT. (G) High colocalization of mover with vGluT1 in stratum oriens of CA3, low colocalization of mover with vGAT. (H) High colocalization of mover with vGluT1 in stratum radiatum of CA1, low colocalization of mover with vGAT. (I) High colocalization of mover with vGluT1 in stratum oriens of CA1, low colocalization of mover with vGAT. (J) Visualization of the quantification of colocalization with the different markers using the Pearson’s correlation coefficient. Bars show average ± SEM; ***P < 0.001. PmL, polymorph layer of dentate gyrus; SR, stratum radiatum (of either CA3 or CA1); SLu, stratum lucidum of CA3; SO, stratum oriens (of either CA3 or CA1). Scale bar: (A–C) = 500 µm, (D–I) = 10 µm.