Activation of early silent synapses by spontaneous synchronous network activity limits the range of neocortical connections

Abstract

During the early development of neocortical networks, many glutamatergic synapses lack AMPA receptors and are physiologically silent. We show in neocortical cultures that spontaneous synchronous network activity is able to convert silent synapses to active synapses by the incorporation of AMPA receptors into synaptic complexes throughout the network within a few minutes. To test the effect of synaptic activation on the connectivity of neuronal populations, we created separated neuronal networks that could innervate each other. We allowed outgrowing axons to invade the neighboring network either before or after the onset of synchronous network activity. In the first case, both subnetworks connected to each other and synchronized their activity, whereas in the second case, axonal connections failed to form and network activity did not synchronize between compartments. We conclude that early spontaneous synchronous network activity triggers a global AMPAfication of immature synapses, which in turn prevents later-arriving axons from forming afferent connections. This activity-dependent process may set the range of corticocortical connections during early network development before experience-dependent mechanisms begin elaborating the mature layout of the neocortical connections and modules.

Figure 7.

Development of connectivity between compartment cultures. A, Silicon template used for plating dissociated cells in defined spaced compartments. Each compartment had a size of 2 × 7 mm. The distance between compartments A and B was 1.5 mm, and the distance between A and C was 2.0 mm. After the cells had attached to the substrate, the silicon was removed from the culture dish. Axons could freely invade the cell-free regions, and reciprocal interconnections between neighboring compartments were established. B, Examples of neurons in compartment B that were stained by application of a dextran-coated stainless steal block onto compartment A. C, The ingrowing axons (left side) and the labeled cell bodies stained by dye application to compartment A are visible in a border field of compartment B (dashed line). Scale bars, 50 μm.

Figure 9.

Evaluation of compartment connectivity with paired patch-clamp recordings. A, B, Connectivity within 13- to 16-d-old cultures was assessed by recording simultaneously two neurons. Synchronous network activity lead to barrages of synaptic currents that were either synchronized between both neurons (A) or occurred out-of-phase and often with different frequency (B). C, In simultaneously recorded neurons located at two distant sites of the same compartment (i.e., within 1 network), the onset of synchronous network activity occurred in both cells within a 250 ms interval. Based on these recordings, the bin width for the following histograms were set to 250 ms (n = 5 cultures, 1 pair perculture; see indications of culture number in the top right side of each histogram in this figure). D, All A-B pairs (neurons in compartments separated by 1.5 mm) showed synchronous burst activity (most bursts in the two recorded cells fell within a 250 ms interval, as shown in A). E, A-B pairs (neurons in compartments separated by 1.5 mm) showed nosynchronous burst activity (delay > 1 s, as shown in B) if the initially interconnected networks were mechanically separated. F, G, From the 25 recorded A-C neuronal pairs (neurons in compartments separated by 2.0 mm), 20 were asynchronous (F; delay > 1 s), and five pairs (G) presented synchronous burst activity. H, After blockade of GABA_Aergic and glutamatergic receptor activity until 13 DIV, all A-C pairs presented synchronous burst activity at 13-16 DIV.

Figure 3.

Spontaneous synchronous [Ca²⁺]_i oscillations in cultured neocortical neurons. A, Fluorometric Ca²⁺ recordings of synchronous [Ca²⁺]_i oscillations of six neurons in a 12-d-old culture. B, Activity histogram of all neurons present in the same field as cells shown in A. During this recording, a maximum of 18 of 22 neurons showed a synchronous increase in their [Ca²⁺]_i. C, Development of network activity in a control culture, showing the percentage of synchronous and nonsynchronous neurons (nonsynchronous neurons showed changes in [Ca²⁺]_i during the recording period but did not participate in synchronous events). D, Development of network activity in cultures raised in the presence of the GABA_AR antagonist BMI (drug added at 5 DIV).

Figure 4.

Coexpression of NR1/GluR2/3 correlates with synchronized network activity between 6 and 15 DIV in three experimental conditions. A, In untreated control cultures, the percentage of simultaneously active neurons increased gradually between 6 and 15 DIV (black bars). Under BMI, the onset of synchronized activity was delayed until 15 DIV (light gray bars), whereas a 0 mm Mg²⁺ Ringer's solution increased the percentage of simultaneously active neurons between 6 and 12 DIV but not thereafter (medium gray bars) (^**p = 0.005; ^***p ≤ 0.001; χ² test; 5 analyzed fields from 2 cultures per time point; total number of cells examined in successive time points: control, n = 280, 114, 143, 135; BMI, n = 312, 218, 129, 136; 0 mm Mg²⁺, n = 141, 127, 176, 114). B, The percentage of colocalized NR1/GluR2/3 clusters increased in control cultures between 6 and 12 DIV (black bars). This increase was delayed by BMI treatment (light gray bars) and could be boosted by 0 mm Mg²⁺ stimulation (medium gray bars) (^**p = 0.028; ^***p ≤ 0.001; χ² test; 18 analyzed fields from 3 cultures per time point; total number of clusters examined in successive time points: control, n = 271, 318, 199, 216; BMI, n = 318, 449, 301, 259; 0 mm Mg²⁺, n = 338, 319, 204, 277). C, Comparison of NR1/synaptophysin and NR1/GluR2/3-containing clusters in 9 DIV cultures under control condition (Contr.), in cultures raised in the presence of BMI, and after 0 mm Mg²⁺ stimulation. Although there was no significant difference between the NR1/Glu2/3 and NR1/synaptophysin colocalization in control cultures and 0 mm Mg²⁺-stimulated cultures, there was a significant difference between NR1/synaptophysin- and NR1/GluR2/3-containing clusters in BMI-treated cultures (^***p ≤ 0.001; χ² test; 27 analyzed fields from 3 cultures, 9 DIV) (total number of clusters examined: NR1/synaptophysin, control, n = 724; BMI, n = 591; 0 Mg²⁺, n = 685; NR1/GluR2/3, control, n = 600; BMI, n = 638; 0 Mg²⁺, n = 655). n.s., Not significant.

Figure 1.

Colocalization of NR1/GluR2/3 (A-F) and NR1/synaptophysin (G-I) immunostaining in 9-d-old cultures. Each row of three images shows double labeling of a different field. Images of immunostained NR1 (A, D, G), GluR2/3 (B, E), and synaptophysin (H) clusters were combined (C, F, I) to show that some of the NR1-containing clusters are colocalized with GluR2/3-containing (C, F) or synaptophysin-containing (I) clusters (long arrows), whereas others are not (short arrows). Scale bar, 2 μm.

Figure 2.

Quantitative analysis of NR1/synaptophysin and NR1/GluR2/3 colocalization in 9-d-old cultures. A shows the relative number of NR1-immunoreactive clusters that contained either synaptophysin or GluR2/3. The percentage of NR1 cluster colocalized with GluR2/3 was statistically not different from the percentage of NR1 clusters colocalized with synaptophysin, suggesting that the majority of NR1/GluR2/3 clusters are synaptic (χ² test; 27 analyzed fields from 3 cultures). Total number of NR1 clusters examined for colocalization with GluR2/3, n = 600; for colocalization with synaptophysin, n = 724. B, Colocalization of NR1 clusters with clusters stained by antibodies against the GluR2/3 and the GluR1 subunit. Although NR1/GluR2/3 colocalization is comparable with NR1/GluR1 plus GluR2/3 colocalization, GluR1 colocalizes in a much lower fraction of NR1-containing clusters (χ² test; ^***p ≤ 0.001; 36 analyzed fields from 4 cultures; total number of NR1 clusters examined, n = 683 for NR1/GluR2/3, n = 616 for NR1/GluR1 plus GluR2/3, and n = 567 for NR1/GluR1).

Figure 5.

Activation of silent synapses by synchronous network activity. A, Cultures were raised in BMI from 5 to 9 DIV and transferred to 0 mm Mg²⁺ Ringer's solution before fixation. A significant change in the percentage of colocalized NR1/GluR2/3 clusters was evident after 15 min, and control levels were reached after 30 min of 0 mm Mg²⁺ stimulation. Total number of clusters examined: control (contr.), n = 287; BMI, n = 400; 0 Mg²⁺ at increasing delays, n = 582, 398, 436, 441, 453; the second graph in A shows the percentage of active neurons imaged in sister cultures in the presence of BMI (n = 340) or in a 0 mm Mg²⁺ Ringer's solution (n = 478). Although the 9-d-old cultures presented no synchronicity between the spontaneously active neurons under BMI, strong synchronized activity occurred under 0 mm Mg²⁺ stimulation. B, The fraction of NR1/GluR2/3 clusters was determined in 9 DIV cultures cultivated in the presence of the GABA_A receptor antagonist BMI (gray columns; n = 667) and compared with control cultures (black column; n = 556). Three hours before fixation, three sets of cultures were transferred to 0 mm Mg²⁺ Ringer's solution (third column; n = 519) or to 0 mm Mg²⁺ Ringer's solution containing either the NMDA receptor antagonist APV (fourth column; n = 561) or the AMPA receptor antagonist CNQX (fifth column; n = 669). Unstimulated cultures raised in the presence of BMI (no synchronous network activity) or stimulated cultures in the presence of APV (blocked NMDA receptors) showed a significantly lower fraction of NR1/GluR2/3 clusters compared with control cultures. Conversely, the fraction of NR1/GluR2/3 clusters in cultures raised in BMI and stimulated with 0 mm Mg²⁺ in either the absence or presence of CNQX before fixation did not differ significantly from control cultures. The stimulation in A and B was performed in BMI-treated cultures after removal of BMI (^***p ≤ 0.001; χ² test; 18 analyzed fields from 2 cultures per time point in all cases; for imaging, 5 fields from 1 culture were analyzed per time point). n.s., Not significant.

Figure 6.

Comparison between axonal outgrowth and synaptic activation time. A shows the maximal distance that individual axons had grown after a given time in culture. To determine the axonal growth rate, neurons were plated into a single compartment, and the straight distance from compartment border to the 50 longest axons was determined in seven cultures (n = 350; mean ± SEM). At approximately 8 DIV, the axons spanned a distance of 1.5 mm and needed additional 3 d to reach 2.0 mm distance from the border. B, The density of NR1/GluR2/3-containing clusters increased dramatically between 6 and 9 DIV (p ≤ 0.001), and no significant change occurred thereafter [Kruskal-Wallis one-way ANOVA on ranks, followed by a pairwise multiple comparison procedure (Tukey's test); 18 analyzed fields from 3 cultures per time point].

Figure 8.

Distribution of anatomical connections between different treated compartment cultures. The top inset (control) shows schematically the distribution of stained neurons under control condition. Dextran was applied to compartment A at 14-17 DIV. Histograms B and C show results from 10 completely reconstructed cultures. Each bin corresponds to the labeled neurons counted within 500-μm-wide stripes running parallel to the compartment long axis. Topographical order of histogram bins is indicated by the arrows below the x-axis. The origin of arrows indicates the border facing compartment A (see inset). In all B compartments (separated by a 1.5 mm gap from the A compartment), a high amount of neurons were consistently labeled (total 947). In C compartments (separated by a 2.0 mm gap from the A compartment), the number of labeled neurons was consistently very low (total 46). The bottom inset (BMI) shows schematically the distribution of stained neurons in cultures grown in the presence of the GABA_AR antagonist BMI from 5 to 15 DIV. In these cases, the number of labeled neurons was much higher in compartment C′ (total 439) compared with the control situation (C). (Number of neurons labeled in compartment B′ was 792.)