European Journal of Neuroscience

European Journal of Neuroscience, Vol. 39, pp. 61–71, 2014

doi:10.1111/ejn.12399

MOLECULAR AND SYNAPTIC MECHANISMS

Adaptive and non-adaptive changes in activity-deprived presynaptic terminals €zel Horellou,1,2,3 Olivier Pascual,4,5,6 Antoine Triller1,2,3 and Serge Marty1,2,3 Su

rieure, 46 rue d’Ulm, 75005 Paris, France Institute of Biology of the Ecole Normale Supe INSERM U1024, Paris, France 3 CNRS UMR8197, Paris, France 4 INSERM U1028, Lyon Neuroscience Research Center, Neuro-oncology & Neuro-inflammation team, 69000 Lyon, France 5 CNRS 5292, Lyon Neuroscience Research Center, Neuro-oncology & Neuro-inflammation team, Lyon, France 6 University Lyon1, Lyon, France 1 2

Keywords: GluA2, mouse, RIM1/2, synaptic vesicles, tetrodotoxin

Abstract How the number of docked vesicles is regulated is still unclear. Following chronic activity blockade the number of docked vesicles increases, providing a model through which to address this issue. We tested the hypotheses that the number of docked vesicles is regulated with the size of the terminal, and by the level of Rab3-interacting molecule 1/2 (RIM1/2). We immobilized mouse hippocampal slice cultures by high-pressure freezing after 3 days of tetrodotoxin treatment and analysed them by electron microscopy. The number of docked vesicles, the size of the active zones and the amount of GluA2 were increased after activity blockade. However, there was no modification of either the total number of synaptic vesicles or the area of presynaptic profiles. Surprisingly, immunocytochemistry showed no change in the mean level of RIM1/2 per terminal but its distribution was modified. Additionally, there was no modification of the mean frequency or amplitude of miniature excitatory postsynaptic currents, but the distribution of amplitudes was modified. These results indicate a specific homeostatic regulation of the synaptic junction. The number of docked vesicles does not seem to be regulated by the amount of RIM1/2. The modification of the distribution, but not the amount, of RIM1/2 may explain the contradiction between the morphological and electrophysiological findings.

Introduction In presynaptic terminals, some of the synaptic vesicles (SVs) are docked to an area of the plasma membrane called the active zone (AZ), located in front of the postsynaptic density (PSD). The number of docked vesicles correlates with the size of the AZ (Branco et al., 2010; Holderith et al., 2012). At least some of these docked vesicles are thought to be the SVs that are ready to release their neurotransmitter content (Schikorski & Stevens, 2001). Importantly, the probability of neurotransmitter release (Pr) has been well correlated with the size of the AZ and with the number of docked SVs, both in dissociated cultures and in acute slices (Murthy et al., 2001; Branco et al., 2010; Holderith et al., 2012). The Pr is an essential determinant of synaptic efficiency, and an increase in Pr may underlie the long-term potentiation of synaptic transmission (Emptage et al., 2003; Enoki et al., 2009). However, how the number of docked SVs is regulated remains to be analysed. Homeostatic changes take place at synapses in response to chronic modifications in neuronal activity (Turrigiano et al., 1998). The Pr is increased following chronic blockade of neuronal activity in dissociated cultures, and this homeostatic change of synaptic

Correspondence: Serge Marty, as above. E-mail: [email protected] Received 13 June 2013, accepted 26 September 2013

efficiency is associated with an increased number of docked vesicles (Murthy et al., 2001). Therefore, this model could allow the study of the regulation of docked SVs numbers. It was initially proposed that increased docking of SVs following chronic activity blockade was part of a general synaptic scaling. Indeed, it was accompanied by an increase in the area of the AZ, in the size of the presynaptic terminal and in the total number of SVs (Murthy et al., 2001). However, a more recent study proposed that Pr and the number of docked SVs are correlated but neither are related to the total number of SVs (Branco et al., 2010). The molecular modifications taking place in activity-deprived presynaptic terminals were recently studied in dissociated cultures (Lazarevic et al., 2011). Contrary to several other AZ proteins that were down-regulated, Rab3-interacting molecule 1/2 (RIM1/2) were found to be up-regulated by activity blockade in a subpopulation of terminals. They are good candidates for regulating the number of docked SVs because the amount of RIM1/2 correlates with the size of the AZ, and the number of docked SVs is reduced in RIM1/2 double-knockout (Schoch et al., 2002; Kaeser et al., 2011; Holderith et al., 2012). In this study, we aimed to test the synaptic scaling hypothesis and to analyse the potential involvement of RIM1/2 in regulating the number of docked SVs. On hippocampal slice cultures taken from postnatal day 7 mice, we blocked neuronal activity for 3 days starting at 11 days in culture. The slices were then immobilized by

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd

62 S. Horellou et al. high-pressure freezing (HPF) to analyse presynaptic terminals by electron microscopy (EM) without aldehyde-associated artifacts (Siksou et al., 2013). We observed that some parameters of presynaptic terminals present homeostatic modifications, with an increased size of the AZ and an increased number of docked vesicles. In contrast, other parameters, such as the total number of SVs and the area of presynaptic profiles, were not modified by activity blockade. Strikingly, the mean RIM1/2 immunoreactivity (IR) per terminal was not modified, but the distribution of RIM1/2 IR was altered.

Materials and methods

NaHCO3, 26 mM; glucose, 10 mM) containing 1 lM TTX and 100 lM of the GABAA receptor antagonist picrotoxin (1128; Tocris Bioscience). Neurons were recorded in voltage-clamp (
70 mV) mode in the whole-cell configuration using a Multiclamp 700B amplifier (Axon, Sunnyvale, CA, USA). Recordings were filtered at 2–5 kHz and acquired at 5–10 kHz. Currents were recorded in voltage-clamp. Throughout the experiment the access resistance was periodically tested; if it was > 20 MΩ or changed by > 20% the cell was discarded. Data were acquired using the Clampex 10 software and analysed using the Clampfit 10 program (Axon). HPF, cryosubstitution and embedding

Animals All experimental procedures followed the guidelines of the European Communities Council Directive (86/609/EEC) regarding the care and use of animals for experimental procedures, the Services Veterinaires de Paris (permission # 75-882), and were approved by the Ethics Committee # 5 of Comite National de Reflexion Ethique sur l’Experimentation Animale. Hippocampal slice cultures Hippocampal slice cultures from postnatal day 7 C57/Bl6 mice (Janvier, France) were prepared according to the protocol developed by Stoppini et al. (1991). After anesthesia by isoflurane inhalation (Virbac, France), the pups were decapitated and their brains rapidly transferred to 0.12 M phosphate-buffered saline (PBS; 0.9% NaCl, pH 7.4) containing 0.5% D-glucose. The hippocampi were dissected and 400lm slices perpendicular to the axis of the hippocampus were sectioned using a McIllwain tissue chopper (Mickle Laboratory, Surrey, UK). Slices were placed on Millicell-CM inserts (Millipore, Billerica, MA, USA; PICM03050) and maintained over slice culture medium for 2 weeks. The slice culture medium was made of 10.63 g/L of Eagle’s minimum essential medium. (Cellgro 50019PC, Mediatech Inc., Manassas, VA, USA) reconstituted in ultrapure water (milliQ water, Millipore), with heat-inactivated horse serum, 20%; L-glutamine (Invitrogen 21103049; Life Technologies, Saint Aubin, France), 1 mM; CaCl2, 1 mM; MgSO4, 2 mM; insulin (Sigma I5500), 1 mg/L; ascorbic acid (Sigma A4034), 61 lM; D-glucose, 11 mM; NaHCO3, 5 mM; and Hepes (Sigma H4034), 30 mM (Poncer et al., 2002). The pH was adjusted to 7.27. The medium was exchanged every 2–3 days. Pharmacological treatments After 11 days in culture, slice cultures were treated with a mixture of glutamate receptor (GluR) antagonists or with the sodium channel blocker tetrodotoxin (TTX) for 3 days. The NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 0106; Tocris Bioscience, Bristol, UK) was used at 50 lM. The AMPA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7sulfonamide (NBQX) disodium salt (1044; Tocris Bioscience) was used at 50 lM. TTX (1069; Tocris Bioscience) was used at 1 lM. Electrophysiological recordings After 14 days in culture, slices were detached from the MillicellCM membrane with a razor blade (Siksou et al., 2009) and were transferred to an electrophysiology setup. To record miniature excitatory postsynaptic currents (mEPSCs), the slices were perfused with oxygenated artificial cerebrospinal fluid (NaCl, 124 mM; KCl, 3.1 mM; NaH2PO4, 1.25 mM; CaCl2, 2 mM; MgCl2, 1 mM;

After 14 days in culture, slices were immersed in slice medium and detached from the Millicell-CM membrane with a razor blade (Siksou et al., 2009). Whole slices were immediately transferred to a high-pressure chamber and frozen in an HPF machine (HPM 100; Leica, Vienna, Austria). After freezing, the samples were rapidly transferred to liquid nitrogen for storage. Cryosubstitution and embedding were performed in a Leica EM AFS2 apparatus. Two different protocols were used for morphological or immunocytochemical studies, as described previously (Siksou et al., 2007). Briefly, for morphological analysis cryosubstitution was performed in acetone containing 0.1% tannic acid at
90 °C for 4 days, followed by acetone containing 2% osmium during the last 7 h. The slices were warmed (5 °C/h) to
20 °C and incubated for an additional 16 h, before being warmed (10 °C/h) to 4 °C. At 4 °C, the slices were washed in acetone and then warmed to room temperature (20 °C). They were then embedded in araldite. For immunocytochemistry, cryosubstitution was performed in methanol containing 1.5% uranyl acetate at
90 °C for 4 days. The slices were warmed (4 °C/h) to
45 °C and embedded in Lowicryl (HM20 kit, CAT 15924, Polysciences, Warrington, PA, USA). Aldehyde fixation and embedding After 14 days in culture, the Millipore inserts were immersed in PBS containing 2% paraformaldehyde and 2% glutaraldehyde for 1 h at 4 °C and rinsed in PBS. The slices were then transferred into a solution containing 2% osmium for 2 h at 4 °C. After washes, they were stained ‘en bloc’ with a solution containing 2% uranyl acetate for 1 h at 4 °C. After washes, samples were dehydrated in graded ethanol followed by acetone, and incubated in 50% acetone– 50% araldite for 1 h, followed by 10% acetone–90% araldite for 2 h. They were then incubated twice in araldite for 2 h before being embedded. Sectioning Transverse sections were cut in the middle of the CA1 area, allowing a good control of the depth of sampling relative to the surface of the slice. Sections were cut using a Leica EM UC6. For light microscopy, 0.5-lm-thick semithin sections were collected on glass slides. For the ultrastructural analysis on single sections, 40- or 70nm-thick ultrathin sections were collected on 400-mesh copper grids. For the morphological analysis on serial sections, pyramids were prepared using a Diatome Trim 45 knife to collect a 200 9 50 lm first section. Fifty-nanometre serial sections (21–49 sections) were collected on formvar-coated nickel slot grids. For immunocytochemistry 70-nm ultrathin sections were collected on formvar-coated 400mesh nickel grids.

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 61–71

Changes in activity-deprived presynaptic terminals 63 Immunocytochemistry For immunocytochemistry, the sections were incubated for 30 min in blocking solution (905.002; Aurion, Wageningen, The Netherlands). After washes in incubation buffer of 0.2% BSA-c (900.099; Aurion) in PBS, the sections were incubated overnight in incubation buffer containing antibodies against RIM1/2 (1 : 1000; 140-203; Synaptic Systems, Goettingen, Germany) or GluA2 (1 : 500; 182103; Synaptic Systems). After washes in incubation buffer, the sections were incubated for 2 h in gold-conjugated secondary antirabbit antibodies, mouse IgG (1 : 50; BritishBiocell International, Cardiff, UK). The sections were washed, fixed in 1% glutaraldehyde (TAAB, Poole, UK) in PBS for 5 min, and washed before being air-dried. Counterstaining and examination of the sections Semithin sections were stained with 0.5% Toluidine Blue and observed with a Leica DMRD upright microscope. Pictures were collected using a Princeton Instruments CCD camera (RTECCD1300Y/ES; Princeton Instruments, NJ, USA). All ultrathin sections were stained by incubation with 5% uranyl acetate in 70% methanol for 5 min and then with lead citrate (0.08 M lead nitrate, 0.12 M sodium citrate in CO2-free dH2O) for 5 min. The ultrathin sections were observed with a Philips TECNAI 12 (FEI, Eindhoven, the Netherlands) and pictures were collected with a 1300 9 1030 Dualvision 300W CCD camera (Gatan, Evry, France). Sampling of the synapses The experiments were focused on spine synapses. Spine synapses were discriminated from shaft synapses on the basis of the small size of the postsynaptic compartment and its lack of mitochondria and microtubules (Peters et al., 1991). The ultrathin sections used for electron microscopy did not include synapses in their entire depth. Thus, synapses analysed in this study are referred to as synaptic profiles. After HPF, segregation patterns of nuclei that are probably due to the nucleation of small ice crystals (Hohenberg et al., 1996) start at ~ 5 lm from the border of the slices and increase towards the centre. Thus, analyses in these sections were performed within 5 lm of the border of the slices (Siksou et al., 2009). Quantifications Quantifications were performed blindly. To quantify the effect of activity blockade on the number of docked vesicles and on the length of AZ, counts were performed on 40-nm-thick sections from seven control slices and from five TTX-treated slices, taken from two independent experiments. A total of 239 control synapses and 163 TTX-treated synapses were analysed. The length of AZs was measured using ImageJ software (Rasband, 1997). The mean numbers were calculated for each slice. The statistical significance of differences in number of docked SVs per synaptic profile or length of AZ between control and TTX-treated slices was calculated using a two-tailed unpaired t-test on the means. The area of AZs was measured on 50-nm-thick serial sections using the Reconstruct software (Fiala, 2005). Measurements were performed in synapses from two control slices, two TTX-treated slices and three slices treated with GluR antagonists. A total of 145 AZs were measured in control slices, 121 in TTX-treated slices and 159 in slices treated with GluR antagonists.

Quantification of the effect of activity blockade on GluA2 immunostaining was performed on sections from three control slices and from three TTX-treated slices, taken from two independent experiments. The number of gold immunoparticles was counted in a total of 124 presynaptic profiles in control slices and 138 presynaptic profiles in TTX-treated slices. The mean numbers were calculated for each slice. The statistical significance of differences between control and TTX-treated slices was calculated using a two-tailed unpaired t-test on the means. Quantification of the effects of activity blockade on the area of presynaptic profiles and on the total number of SV profiles per presynaptic terminal was performed on 40-nm-thick sections from four control slices and from three TTX-treated slices, taken from two independent experiments. The total number of SV profiles was counted in 116 control synapses and 107 TTX-treated synapses. The area of presynaptic terminals was measured using ImageJ software in 126 control synapses and 106 TTX-treated synapses. The mean numbers were calculated for each slice. The statistical significance of differences between control and TTX-treated slices was calculated using a two-tailed unpaired t-test on the means. Neuronal nuclei were counted in transverse semithin sections through the middle of the CA1 area, in the pyramidal cell layer. Counts were performed in sections from six control and six adjacent TTX-treated slices, taken from two independent experiments. For each slice counts were performed on five semithin sections. The mean numbers were calculated for each slice. The statistical significance of difference in neuron number between control and TTXtreated slices was calculated using a two-tailed paired t-test on the means. The height of the slices was measured at the level of the stratum radiatum, in transverse semithin sections through the middle of the CA1 area as above, using ImageJ software. Counts were performed in sections from four control and four adjacent TTX-treated slices. The mean numbers were calculated for each slice. The statistical significance of difference in height of stratum radiatum between control and TTX-treated slices was calculated using a two-tailed paired t-test on the means. The density of synaptic profiles was counted in the stratum radiatum at 50–100 lm from the pyramidal cell bodies, in 70-nm-thick ultrathin sections through the middle of the CA1 area as above. Counts were performed in sections from three control and three adjacent TTX-treated slices. Photomicrographs of 7.74 lm2 were taken at 20 500 9 magnification every two fields in one square of the 400-mesh grid. Synaptic profiles crossing two sides of a picture were excluded, while those intersecting the two other sides were included. Synaptic profiles were counted in a total of 536 photomicrographs (287 from control and 249 from TTX-treated slices). The mean numbers were calculated for each slice. The statistical significance of difference in the density of synaptic profiles between control and TTX-treated slices was calculated using a two-tailed paired t-test on the means. Quantification of the effect of activity blockade on RIM immunostaining was performed on sections from five control slices and from five TTX-treated slices, taken from two independent experiments. The number of gold immunoparticles was counted in a total of 166 presynaptic profiles in control slices and 185 presynaptic profiles in TTX-treated slices. The mean numbers were calculated for each slice. The statistical significance of differences between control and TTX-treated slices was calculated using a two-tailed unpaired t-test on the means. The distribution of presynaptic profiles according to their bead content was compared between the control and TTX-treated slices using the chi-square and Fisher’s exact test. The distance

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 61–71

64 S. Horellou et al. A

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Fig. 1. Effects of activity blockade on SV docking and AZ length. (A) Example of a synapse made by a presynaptic terminal on a dendritic spine. (B and C) Examples of presynaptic terminals from (B) control and (C) TTX-treated slices. The docked vesicles are the SVs in contact with the presynaptic plasma membrane, indicated by black arrows. The AZ is the portion of the presynaptic plasma membrane facing the PSD, delineated by the white arrowheads. (D–F) Quantification of (D) the number of docked vesicles, (E) the distribution of the number of docked vesicles per presynaptic profile and (F) the length of the AZ in control slices and in slices treated with TTX. Note the significant increase in the length of the AZ and in the number of docked vesicles after TTX treatment. *P < 0.05, **P < 0.01. Scale bars, 200 nm (A), 100 nm (in B for B and C).

of gold particles from the plasma membrane at the AZ was measured using ImageJ software for 131 beads in control synapses and 162 beads in TTX-treated synapses. For miniature quantification, a total 10 neurons were recorded in TTX conditions vs. 11 neurons in control condition from four independent organotypic slice cultures. Miniature synaptic currents were detected using the Clampfit (Axon) template procedure. Amplitude

and frequency were averaged over a period of 1–5 min. Events smaller than 5 pA were rejected and not taken into account for the analysis. The statistical significance of differences in the mean frequency or amplitude of mEPSCs between control and TTX-treated slices was calculated using an unpaired t-test. The distribution of mEPSCs according to their amplitude was compared between the control and TTX-treated slices using the Pearson’s chi-square test.

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 61–71

Changes in activity-deprived presynaptic terminals 65 A

B

Fig. 2. Effects of activity blockade on the area of AZs. (A) Example of serial sections through a presynaptic terminal from a control slice. The AZ is the portion of the presynaptic plasma membrane facing the PSD, delineated by the white brackets. (B) Distribution of area of AZs in control slices and in slices treated with TTX or with antagonists of GluRs. Scale bar, 200 nm.

Results Activity blockade increased synaptic vesicle docking and active zone size It has been shown previously that activity blockade increases the number of docked vesicles and the size of AZs in dissociated neuronal cultures (Murthy et al., 2001). We first aimed to determine whether the same regulations occur in cultured slices from postnatal day 7 mice after 2 weeks of maturation in vitro (Buchs et al., 1993; Muller et al., 1993). To analyse the morphology of presynaptic terminals without aldehyde-associated artifacts we immobilized hippocampal slices using HPF. As described previously, the slices are well frozen in their superficial 5 lm after HPF (Siksou et al., 2009). Therefore, morphological analysis of presynaptic terminals was performed in this area (Fig. 1A). Ultrathin sections of 40 nm allowed quantification of the number of docked vesicles per synaptic profile. Vesicles apposed to the plasma membrane were considered as docked (Fig. 1B and C). The mean number of docked vesicles was increased after TTX treatment (1 lM; Fig. 1D). It was 2.05  0.33 (mean  SD) in control slices and 2.71  0.34 in TTX-treated slices (unpaired Student’s t-test on the effect of TTX, t10 = 3.38, P = 0.007). Histogram analysis showed a shift toward higher number of docked SVs in TTX-treated slices (Fig. 1E).

The length of the AZ was measured as the length of the presynaptic membrane facing the PSD (Fig. 1B and C). It was also increased after TTX treatment for 3 days (Fig. 1F). The mean AZ length was 164  14 nm (mean  SD) in control slices and 192  22 nm in TTX-treated slices (unpaired Student’s t-test on the effect of TTX, t10 = 2.67, P = 0.023). The area of AZs was measured on serial sections using the Reconstruct software (Fiala, 2005) (Fig. 2A). In TTX-treated slices, there was a shift in the distribution of AZ areas toward higher values when compared to control slices (Fig. 2B). To determine whether this change was due to activity blockade or to TTX itself, we blocked synaptic transmission with a mixture of NMDA- and AMPA-type GluR antagonists (D-AP5 50 lM + NBQX 50 lM). Under these conditions, too, we observed a reduction in the proportion of small AZs and an increase in the proportion of large AZs (Fig. 2B). Thus, activity blockade increased the size of AZs and the number of docked vesicles in slice cultures after in vitro maturation. Activity blockade increased GluA2 immunostaining The increased area of AZs after TTX treatment reflects an increased area of PSDs. As the size of the PSD has been correlated with the amount of GluR IR (Takumi et al., 1999), we aimed to analyse the

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 61–71

66 S. Horellou et al. A

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Fig. 4. Effects of activity blockade on the area of presynaptic profiles and the total number of SVs in these profiles. (A and B) Examples of presynaptic terminals from (A) control and (B) TTX-treated slices. (C and D) Quantification of (C) the area of presynaptic profiles and of (D) the total number of SVs per presynaptic profile, in control slices and in slices treated with TTX. No significant effects were observed. Scale bar, 100 nm (in A for A and B).

(Fig. 3A). We found an increase in the mean number of gold particles per synaptic profile after TTX treatment (Fig. 3B). It was 0.65  0.13 (mean  SD) in synaptic profiles from control slices and 1.08  0.19 in TTX-treated slices (unpaired Student’s t-test on the effect of TTX, t4 = 3.32, P = 0.029). Accordingly, the distribution of the number of particles per synaptic profile was shifted toward higher values (Fig. 3C). Notably, the number of synaptic profiles with no IR was reduced.

C

Activity blockade did not modify the size of presynaptic terminals or the total number of synaptic vesicles

Fig. 3. Effect of activity blockade on GluA2 immunostaining. (A) Example of GluA2 immunostaining in a control slice. Note the three immunogold particles (arrows). (B and C) Quantification of (B) the mean number of beads per synaptic profile and of (C) the distribution of synapses according to their number of beads, in control slices and in slices treated with TTX. Note the reduced number of presynaptic profiles with 0 beads after TTX treatment (C). *P < 0.05. Scale bar, 100 nm.

effect of TTX treatment on synaptic GluR content. We analysed GluA2 IR because the level of this subunit may correlate more linearly with PSD size than GluA1 (Shinohara et al., 2008). This analysis was performed in the superficial 5 lm of frozen slices to avoid ice crystal artifacts. After GluA2 immunocytochemistry the immunogold particles were concentrated near the postsynaptic membrane

We then aimed to determine whether the increased number of docked vesicles following activity blockade resulted from a general increase in size and vesicular content of presynaptic terminals (synaptic scaling) (Pierce & Lewin, 1994; Murthy et al., 2001). The area of presynaptic terminals and the total number of SVs were analysed in 40nm-thick ultrathin sections after HPF immobilization (Fig. 4A and B). Similarly to the study of docked vesicles, this analysis was performed in the superficial 5 lm of the slices to avoid ice crystal artifacts. We found no increase in the area of presynaptic profiles after TTX treatment (Fig. 4C). It was 0.222  0.018 lm2 (mean  SD) in control slices and 0.188  0.018 lm2 in TTX-treated slices (unpaired Student’s t-test on the effect of TTX, t5 = 2.50, P = 0.055). We also found no effect of activity blockade on the total number of SVs per synaptic profile. It was 28  3 (mean  SD) in control slices and 30  4 in TTX-treated slices (unpaired Student’s t-test on the effect of TTX, t5 = 0.89, P = 0.413). Thus, the increased number of docked vesicles occurred without an increase in the total number of SVs or in presynaptic bouton size.

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 61–71

Changes in activity-deprived presynaptic terminals 67 B

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Fig. 5. Effects of activity blockade on the number of neuronal nuclei profiles and on synaptic density. (A) Transverse semithin section of a control slice in the middle of the CA1 area. Note the numerous neuronal nuclei (some indicated by asterisks) and the thick apical dendrites (some indicated by arrows). (B) Transverse ultrathin section of a control slice in the middle of the CA1 area. Note the three presynaptic terminals facing a postsynaptic density (asterisks). (C–E) Quantification of (C) the density of neuronal nuclei profiles, of (D) the height of the stratum radiatum and of (E) the density of synaptic profiles in control slices and in slices treated with TTX. Note the significant increase in the density of synaptic profiles after TTX treatment. **P < 0.01. Scale bars, 10 lm (A), 200 nm (B).

Activity blockade increased synaptic density with no effect on neuronal survival The increased number of docked SVs after TTX treatment could be due to a reduction in the number of synapses established by the neurons, allowing an increased accumulation of AZ material at the remaining synapses. To quantify the number of neurons and synapses we also needed to access the inside of the slices. It is not possible to analyse these parameters in the depth of frozen slices because of ice crystal artifacts. Therefore, they were measured in aldehyde-fixed slices. The numbers of neuronal nuclei profiles were counted in transverse semithin sections of the pyramidal cell layer in the middle of the CA1 area (Fig. 5A). The mean number of neuronal nuclei profiles was not modified after TTX treatment (Fig. 5C). The mean number of neuronal nuclei profiles in the pyramidal cell layer was 91  13 (mean  SD) in control slices and 89  19 in TTX-treated slices (paired Student’s t-test on the effect of TTX, t5 = 0.26, P = 0.807). On the same sections the height of the slices was measured at the level of the stratum radiatum. It was not modified after TTX treatment (Fig. 5D). The mean height of the slices was 185  9 lm (mean  SD) in control slices and 187  6 lm in TTX-treated slices (paired Student’s t-test on the effect of TTX, t3 = 0.25, P = 0.822). The density of synaptic profiles was counted in ultrathin sections through the stratum radiatum, at 50–100 lm from the pyramidal cell bodies (Fig. 5B). The mean density of synaptic profiles was increased after TTX treatment (Fig. 5E). The mean density

of synaptic profiles per 7.74 lm2 was 1.7  0.4 (mean  SD) in control slices and 2  0.3 in TTX-treated slices (paired Student’s t-test on the effect of TTX, t2 = 20.22, P = 0.002). Thus, the increased number of docked vesicles per synaptic profile could not be simply explained by a reduced number of synaptic profiles.

Activity blockade modified the distribution but not the amount of RIM1/2 immunostaining in presynaptic terminals In dissociated neuronal cultures, RIM1/2 were the only proteins of the AZ upregulated at presynaptic terminals following activity blockade (Lazarevic et al., 2011). As they are good candidates for controlling the number of docked vesicles, we aimed to analyse whether the same regulation occurred in cultured slices. This analysis was performed in the superficial 5 lm of frozen slices similarly to the study of docked vesicles. After RIM1/2 immunocytochemistry, the immunogold particles were concentrated close to the AZ (Fig. 6A and B). Surprisingly, we found no increase in the number of gold particles per presynaptic profile after TTX treatment (Fig. 6C). We counted 2.1  0.4 beads per presynaptic profile (mean  SD) in control slices and 2.1  0.5 in TTX-treated slices (unpaired Student’s t-test on the effect of TTX, t8 = 0.14, P = 0.894). There was also no modification in the distribution of beads relative to the AZ (Fig. 6B). Thus, after TTX treatment the amount of RIM IR per presynaptic profile remained constant, in contrast to the increase in the number of docked vesicles. Given the increased length of AZs on synaptic profiles (Fig. 1F), this indicates

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 61–71

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Fig. 6. Effect of activity blockade on RIM1/2 immunostaining. (A) Example of RIM1/2 immunostaining in a control slice. Note the two immunogold particles (arrows). (B–D) Quantification of (B) the distance of beads from the plasma membrane at the AZ, of (C) the mean number of beads per presynaptic profile, and of (D) the distribution of synapses according to their number of beads, in control slices and in slices treated with TTX. Note in D the reduced numbers of presynaptic profiles with low (0–1) or high (> 7) numbers of beads after TTX treatment. Scale bar, 100 nm.

that the density of RIM1/2 beads per lm of active zone length decreased. However, the total amount of synaptic RIM1/2 increased as the density of synaptic profile increased. Even though the mean number of immunogold particles per presynaptic profile was not changed, the distribution of synapses according to their content in immunogold particles was modified (Fig. 6D). After TTX treatment there were fewer presynaptic profiles with 0–1 beads and also fewer profiles with > 7 beads. The frequency distribution was differed significantly between control and TTX-treated slices (Pearson’s chi-square test, X62 ¼ 12:95, P = 0.04; Fisher’s exact test, P = 0.03). Thus, TTX treatment did not modify the mean number of beads per presynaptic profile but it reduced the proportion of profiles with either low or high immunogold particle content. Activity blockade did not modify the mean frequency or amplitude of miniature excitatory postsynaptic currents but did change the distribution of amplitudes After TTX treatment we observed an increased number of docked vesicles per presynaptic profile, as well as an increased density of synaptic profiles (Figs 1D and 5E). These observations led us to expect a homeostatic regulation of presynaptic efficacy following TTX treatment, with an increased frequency of mEPSCs. Surprisingly, we found no effect of TTX treatment on the frequency of mEPSCs (Fig. 7A and B). We also observed after TTX treatment an increased GluA2 IR per synaptic profile (Fig. 3B). Thus, we expected a homeostatic increase in the mean amplitude of mEPSCs. Unexpectedly, we did not find such an increase (Fig. 7A and C). Thus, there was a discrepancy

between the morphological analysis of SV docking and GluA2 expression on one hand, and the electrophysiological analysis of mEPSCs on the other hand. However, TTX treatment modified the distribution of mEPSC amplitudes (Fig. 7D). After TTX treatment there was an increase in mEPSCs with either low (<
10 pA) or high (>
40 pA) amplitude. The frequency distribution was significantly different between control and TTX treated slices (Pearson’s chi-square test, X92 ¼ 79, P = 2.551 9 10
13). Thus, TTX treatment did not modify the mean amplitude of mEPSCs but did increase the proportion of mEPSCs with either low or high amplitude.

Discussion We observed several modifications of synapses after activity blockade in slice cultures. Notably, the number of docked vesicles and the size of the AZs were increased, in agreement with previous findings in dissociated cell cultures (Murthy et al., 2001). Accordingly, immunocytochemistry indicated that the amount of GluA2 in the postsynaptic membrane was also increased. However, we observed no modifications of either the total number of SVs per synaptic profile or the area of presynaptic profiles, indicating a specific regulation at the level of the synaptic junction. Surprisingly, immunocytochemistry showed no change in the mean level of RIM1/2 IR per synaptic profile after activity blockade. Thus, the amount of RIM1/2 does not seem to control the number of docked vesicles. Despite the fact that presynaptic terminals contained more docked vesicles, and also that there was a higher density of synaptic profiles, we observed no modification of the frequency of mEPSCs.

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 61–71

Changes in activity-deprived presynaptic terminals 69 A

B

C

D

Initial studies showed a coordinated modification of several morphological parameters of presynaptic terminals following activity blockade (Murthy et al., 2001). In addition to the number of docked vesicles and the size of the AZ, the size of presynaptic terminals and the total number of SVs in these terminals were increased. These observations supported the synaptic scaling hypothesis, an ultrastructural size principle postulating that modifications of presynaptic efficiency involve a growth or involution of all these parameters (Pierce & Lewin, 1994). However, a more recent study found that the probability of neurotransmitter release is correlated with the number of docked vesicles but not with the total number of SVs in boutons (Branco et al., 2010). In agreement with these last findings, we found that the size of presynaptic profiles and the total number of SVs in these profiles did not change following activity blockade (Fig. 4). Thus, the increased number of docked vesicles following activity blockade is probably due to a local regulation at the AZ rather than to a general presynaptic scaling. We found that the increased number of docked vesicles and the increased size of the AZ were accompanied by an increased GluA2 IR on the postsynaptic side (Fig. 3B). This is not surprising, given the correlation between the size of the AZ and the size of the PSD (Lisman & Harris, 1993) and between the size of the PSD and the amount of GluR (Takumi et al., 1999). It is also in agreement with previous studies showing the insertion of GluRs during homeostatic plasticity in dissociated cultures (Wierenga et al., 2005; Gainey et al., 2009). Our findings indicate that activity blockade induced a coordinated pre- and postsynaptic growth of the synaptic junction. The number of docked vesicles and RIM1/2 levels are differentially regulated after activity blockade

Fig. 7. Effects of activity blockade on mEPSCs. (A) Examples of mEPSCs recorded from a control and from a TTX-treated slice. (B–D) Quantification of (B) the mean  SEM frequency of mEPSCs, of (C) the mean  SEM amplitude of mEPSCs and of (D) the distribution mEPSC amplitudes, in control and TTX-treated slices. Note in D the increased number of mEPSCs with low (<
10 pA) and high (>
40 pA; inset) amplitude after TTX treatment.

Additionally, even though the amount of GluA2 increased at PSDs, no change in the mean mEPSCs amplitude was detected. We propose an explanation for the apparent contradiction between the morphological and electrophysiological findings, based on a modification of the distribution of RIM1/2 in presynaptic terminals. Specific homeostatic regulation of the synaptic junction after activity blockade Previous studies in dissociated neuronal cultures showed an increased size of the AZ and an increased number of docked vesicles following activity blockade (Murthy et al., 2001). Recent studies indicate that long-term dissociated neuronal cultures may not reach the maturation attained by slice cultures (Rose et al., 2013). For instance, the resting pool of SVs seems to disappear with maturation in slice but not in cell cultures. Our findings indicate that the homeostatic regulation of docked vesicles and AZ size is maintained after synapse maturation in slice culture (Fig. 1D and F).

The molecular mechanism regulating the number of docked vesicles remains poorly understood. It has been shown that among several AZ proteins, RIM1/2 were the only ones to be upregulated following activity blockade in dissociated neuronal cultures (Lazarevic et al., 2011). RIM1/2 are potential candidates for controlling the number of docked vesicles because the number of such vesicles is reduced in RIM1/2-knockout neurons, with no effect on AZs (Kaeser et al., 2011). Furthermore, the amount of RIM1/2 correlates with the size of the AZ (Holderith et al., 2012). Thus, a simple scenario emerged in which an increased size of the AZ would recruit more RIM1/2, which would in turn recruit more vesicles. As a first step to test this hypothesis, we analysed RIM1/2 IR following activity blockade in slice culture. To our surprise we found that, unlike the number of docked vesicles, the amount of RIM1/2 IR or its distance to the AZ were not modified in activity-deprived terminals (Figs 1D and 6B and C). It suggests that, under our conditions, the number of docked vesicles is not regulated by the amount of RIM1/2. We observed an increased number of docked vesicles per terminal and also an increased density of synaptic profiles (Figs 1D and 5E). The increased size of the AZ and PSD could increase the density of synaptic profiles by inducing a bias in the counting (Guillery & Herrup, 1997). Nevertheless, there is a contradiction between these morphological observations and the absence of effect on mEPSC frequency (Fig. 7B). Similarly, a recent study showed no effect of activity blockade on mEPSCs frequency in slice cultures (Arendt et al., 2013). Based on GluA1 immunostaining in dissociated cultures, the authors proposed that activity deprivation induces the growth and/or maintenance of synapses that are devoid of AMPA receptors, the so-called ‘silent synapses’. However, using GluA2 immunocytochemistry we found no evidence for the appearance of silent synapses after activity blockade. Rather, the proportion of

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 61–71

70 S. Horellou et al. unlabelled synaptic profiles was reduced (Fig. 3C). One potential reason for the discrepancy between the two studies is that GluA1 is not linearly correlated with the size of the PSD, accumulating more at larger PSDs (Shinohara et al., 2008). Another potential reason is that they analysed GluA1 immunostaining in dissociated cultures, which present more immature characteristics than slice cultures (Rose et al., 2013). Synapses without AMPA receptors are a characteristic of the immature hippocampus, and their number is strongly reduced during postnatal maturation (Petralia et al., 1999). Rather than the appearance of silent synapses, we propose another explanation for the absence of effect of activity blockade on mEPSC frequency in slice cultures. In control conditions Pr correlates with both the number of docked vesicles and the level of RIM1/2 IR (Murthy et al., 2001; Lazarevic et al., 2011; Holderith et al., 2012). After activity blockade we observed a differential regulation of the number of docked vesicles, which increased, and of the amount of RIM1/2 IR, which was not modified. The absence of effect of activity blockade on the frequency of mEPSCs suggests that RIM1/2, rather than the number of docked vesicles, sets Pr. This hypothesis is supported by the fact that RIM1/2 is a crucial determinant of Pr through the recruitment voltage-gated Ca2+ channels to the AZ (Schoch et al., 2002, 2006; Kaeser et al., 2011) and through the activation of Munc13, which increases the propensity of SVs to fuse with the plasma membrane (Deng et al., 2011). Activity blockade regulates the distribution of RIM1/2 IR differently from that of AZ size or GluA2 IR

synapses with higher levels of GluRs are sampled more efficiently during electrophysiological recordings. RIM1 and 2 are important determinants of release probability (Deng et al., 2011; Kaeser et al., 2011). TTX treatment homogenized the amount of RIM1/2 at synapses, with fewer synapses containing either low or high levels of RIM1/2. This indicated that synapses with low amounts of GluRs could be sampled more efficiently. It would counteract the homeostatic increase in amplitude that is observed at all synapses. The change in the distribution of mEPSC amplitudes following TTX treatment supports this hypothesis. There is an increased proportion of mEPSCs with high amplitude (> 40 pA; Fig. 7D), in agreement with the up-regulation of GluA2 at synapses (Fig. 3B). However, mEPSCs with low amplitude (<
10 pA) are also more numerous, explaining the absence of effect on the mean amplitude. The increased sampling of low-amplitude events, despite the reduced proportion of synapses with low amounts of GluA2, suggests that Pr is increased at small synapses containing few GluRs. This is in agreement with the homogenization of RIM1/2 IR at synapses.

Acknowledgements We thank Mohamed Doulazmi for his help with statistical analysis. We are also grateful for financial support as grants from Agence Nationale de la Recherche ANR 11BSV401902, and from idex Paris Science et Lettres/ CNRS to S.M.

Abbreviations

In control conditions, the amount of RIM1/2 IR is correlated with the size of the AZ (Holderith et al., 2012). We found that activity blockade modified the distribution of RIM1/2 IR levels to the benefit of median values: there were fewer synapses with low or high IR (Fig. 6D). By contrast, the size of the AZ was shifted toward higher values (Fig. 2B). It indicates that, following activity blockade, there is no longer coordination between the amount of RIM1/2 IR and the size of AZs. How neuronal activity normally adjusts RIM1/2 level to the size of the AZ remains to be studied. A recent study indicates that presynaptic terminals contain two pools of RIM1, a small immobile pool and a larger mobile one (Spangler et al., 2013). Thus, RIM1/2 could rapidly redistribute between synapses. It would be interesting to analyse the involvement of membrane depolarization, calcium influx or NMDAR activation in regulating RIM1/2 IR levels at the AZ. Activity blockade also regulated differentially the distribution of RIM1/2- and GluA2 IR at synapses. In control conditions, Pr and levels of RIM1/2 and GluR IR are correlated to the size of the AZ or PSD (Takumi et al., 1999; Holderith et al., 2012). However, activity blockade homogenized the levels of RIM1/2 IR at median values (Fig. 6D) while it shifted GluA2 IR levels toward higher values (Fig. 3C). It has been previously demonstrated that neuronal activity coordinates Pr and postsynaptic GluA2 content at synaptic junctions (Tokuoka & Goda, 2008). Based on the fact that RIM1/2 level is a crucial determinant of Pr (Deng et al., 2011; Kaeser et al., 2011), this coordination could be due to the activity-dependent regulation of RIM1/2 distribution according to the size of synaptic junctions. The absence of effect of TTX treatment on the mean amplitude of mEPSCs (Fig. 7C) was in contrast with the strong up-regulation of synaptic GluA2 IR (Fig. 3B). A decorrelation of Pr and GluR level following TTX treatment could resolve this contradiction. In control conditions Pr and GluR IR are correlated with the size of the AZ and PSD (Takumi et al., 1999; Holderith et al., 2012), so that

AZ, active zone; EM, electron microscopy; GluR, glutamate receptor; HPF, high-pressure freezing; IR, immunoreactivity; mEPSC, miniature excitatory postsynaptic current; PBS, phosphate-buffered saline; Pr, probability of neurotransmitter release; PSD, postsynaptic density; RIM1/2, Rab3-interacting molecule; SV, synaptic vesicle; TTX, tetrodotoxin.

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© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 61–71