 ± 2.2 and 15.6 ± 1.5 pA; P > 0.5 for both frequency and amplitude; n = 6). However, after pairing, U0126 induced a persistent and significant reduction in the frequency of sEPSCs. On average, 20 min after pairing the frequency of sEPSCs was 71% of controls (frequency varied from 0.21 ± 0.04 to 0.15 ± 0.04 Hz before and after pairing, respectively; P < 0.05), whereas the amplitude was not significantly modified (16.6 ± 2.2 and 14.9 ± 0.9 pA before and after pairing, respectively; P > 0.5) (Fig. 5 A). Similar effects were obtained with PD98059. Slices were preincubated for 10 min with 50 μM PD98059. Although PD98059 did not modify the frequency or amplitude of synaptic events (frequency values were 0.11 ± 0.02 and 0.09 ± 0.02 Hz and amplitude values were 14.9 ± 2.7 and 12.8 ± 1.8 pA before and after drug application, respectively; P > 0.5 for both frequency and amplitude), it prevented the potentiating effect of pairing and caused a significant reduction of the frequency of sEPSCs (mean frequency values were 0.15 ± 0.04 and 0.08 ± 0.02 Hz before and after pairing, respectively; 57% of controls; P < 0.05), but not in the amplitude (mean amplitude values were 15.2 ± 2.7 and 14.3 ± 3.1 pA before and after pairing, respectively; P > 0.5) of sEPSCs (n = 6) (Fig. 5 A).
To test whether ERK activation occurred into the postsynaptic cell, PD98059 was applied directly into the patch pipette (32). Additionally, in this condition, 50 μM PD98059 prevented the effects of ERK upon paired-induced potentiation of the frequency of sEPSCs (mean frequency values were 0.09 ± 0.03 and 0.07 ± 0.02 Hz before and after pairing, respectively; P > 0.1; mean amplitude values were 16.1 ± 1.3 and 13.8 ± 1.9 pA before and after pairing, respectively; P > 0.1; n = 5) (Fig. 5 A).
The involvement of ERK in GDP-induced changes in synaptic efficacy was further validated by immunocytochemical experiments, which demonstrated that the pairing procedure was able to induce ERK phosphorylation (n = 5), an effect that was prevented by 20 μM U0126 (n = 4) (Fig. 5 B). In the absence of pairing, no ERK phosphorylation was observed (Fig. 5 B). These data suggest that the persistent changes in frequency of sEPSCs after GDP pairing require the activation of the ERK pathway. ERK phosphorylation also was prevented by the TrkB receptor inhibitor k-252a in the patch pipette (n = 4) or in the bath (n = 3), suggesting that, in the present experiments, ERK phosphorylation was triggered by BDNF (SI Fig. 9).
The present data clearly show that, during the first week of postnatal life, correlated presynaptic (Schaffer collateral) and postsynaptic (GDPs) activity induced a persistent enhancement of synaptic efficacy at glutamatergic CA3–CA1 connections. This outcome required a transient rise of calcium in the postsynaptic cell and the involvement of BDNF and the ERK pathway.
The pairing procedure not only produced a strong potentiation of the sEPSCs evoked by the Schaffer collateral stimulation but also persistently enhanced the frequency of spontaneous glutamatergic events.
In CA3 pyramidal neurons, evidence has been provided that, during a restricted period of postnatal development, long-term changes in GABAergic synaptic transmission can be induced by repetitive depolarizing pulses (25, 33, 34). Although in these studies the conditioning protocol used for LTP induction could have been of physiological relevance, in the present case, the persistent enhancement of glutamatergic transmission at CA3–CA1 synapses depended on a calcium rise through a voltage-dependent calcium channel activated by the depolarizing action of GABA during GDPs. Interestingly, when the afferent stimulation was paired with theta bursts or plateau potentials in the postsynaptic cell to mimic GDPs, it failed to persistently enhance the frequency of sEPSCs. When paired with afferent stimulation, plateau potentials only briefly enhanced the frequency of sEPSCs, which declined to a control level a few minutes after pairing. In the adult hippocampus, repetitive voltage pulses to the postsynaptic cell have been shown to induce a short-lasting potentiation of excitatory synaptic transmission (35). However, unlike the present case, transient changes in synaptic strength observed in the adult primarily depended on postsynaptic mechanisms because membrane depolarization not only enhanced the frequency of miniature events but also the amplitude of responses to exogenously applied AMPA. Whatever the mechanisms, our results indicate that the way by which calcium enters into the cell is crucial for pairing-induced potentiation. We cannot exclude, however, that in the present study GDPs may concomitantly stimulate presynaptic terminals to further strengthen synaptic currents. This result may, at least in part, explain why depolarizing the postsynaptic cell with theta bursts or plateau potentials failed to persistently enhance synaptic strength.
In the present experiments, the involvement of BDNF in GDP-induced synaptic potentiation was demonstrated by the observation that scavengers of endogenous BDNF or blockers of protein kinase coupled to Trk receptors were able to prevent pairing-induced persistent enhancement of the frequency of sEPSCs. The present results do not indicate whether endogenous BDNF was released from the pre- or postsynaptic cell. However, evidence was recently provided that a moderate postsynaptic depolarization can elicit the release of BDNF from the postsynaptic cell in a calcium-dependent way (20). Once released, BDNF would affect TrkB receptors localized on both pre- and postsynaptic membranes. Although at the presynaptic level BDNF-induced activation of TrkB receptors would enhance transmitter release (20, 36, 37), at the postsynaptic level it would activate fast dendritic calcium transients (38) and different intracellular signaling pathways. One of the most common signaling pathways activated by BDNF is the MAPK/ERK cascade (28, 39). Activation of ERK would lead to transcriptional regulation and new protein synthesis (27) required for the enduring forms of synaptic plasticity (31). As in the late phase of LTP (31), in the present case the activation of ERK was targeted to the postsynaptic neuron as demonstrated by electrophysiological and immunocytochemical experiments. However, we cannot exclude a concomitant activation of ERK in the presynaptic neuron. The postsynaptic action of ERK may further support structural changes (triggered by BDNF activation of TrkB receptors), such as dendritic growth and increase in spine density (30, 40). The ERK pathway also can be activated by calcium entry by NMDA receptors (41). However, this event is unlikely because pairing-induced potentiation was independent of NMDA receptor activation. Therefore, BDNF can act presynaptically to alter the probability of glutamate release but also postsynaptically to produce the morphological modifications necessary for the formation of new synapses and the refinement of the adult neuronal hippocampal circuit.
In conclusion, our data demonstrate that network-driven GABAergic oscillations such as GDPs are essential for the functional maturation of glutamatergic synapses.
Experiments were performed on hippocampal slices from P2–P6 Wistar rats as previously described (13). Briefly, animals were decapitated after being anesthetized with an i.p. injection of 2 g/kg urethane. The brain was quickly removed from the skull and placed in ice-cold artificial cerebrospinal fluid containing 130 mM NaCl, 3.5 mM KCl, 1.2 mM NaH2PO4, 25 mM NaHCO3, 1.3 mM MgCl2, 2 mM CaCl2, and 25 mM glucose saturated with 95% O2 and 5% CO2 (pH 7.3–7.4). Transverse hippocampal slices (400 μm thick) were cut with a vibratome and stored at room temperature in a holding bath containing the same solution. After a recovery period of at least 1 h, an individual slice was transferred to the recording chamber, where it was continuously superfused with oxygenated artificial cerebrospinal fluid at a rate of 2 to 3 ml/min at 33–34°C.
Electrophysiological experiments were performed with CA1 pyramidal cells by using the whole-cell configuration of the patch-clamp technique in current- or voltage-clamp mode. Synaptic responses were evoked at 0.05 Hz by minimal stimulation of the Schaffer collateral. They were recorded in voltage-clamp conditions from a holding potential of −53 mV (E GABA). In most cases, paired stimuli were applied at 50-msec intervals. Patch electrodes were pulled from Borosilicate glass capillaries (Hingelberg, Malsfeld, Germany). They had a resistance of 4 to 6 MΩ when filled with an intracellular solution containing 135 mM K-gluconate, 20 mM KCl, 10 mM Hepes, 4 mM MgATP, 0.3 mM GTP, 0.5 mM EGTA, and 5 mM QX-314. In some experiments, recordings were performed with patch pipettes containing 20 mM BAPTA (Sigma–Aldrich, Milan, Italy). When 20 mM BAPTA was added to the pipette solution, K-gluconate was reduced from 135 to 115 mM. For plateau potential experiments, K-gluconate was substituted with CsMeSO4. In some experiments, cells were visualized with 100 μM Oregon green 488 BAPTA-1 (Molecular Probes, Eugene, OR).
Recordings were made with a patch-clamp amplifier (Axop