Notes

Information is stored in neural circuits through long-lasting changes in synaptic strengths [1,2]. Most studies of information storage have focused on mechanisms such as long-term potentiation and depression (LTP and LTD), in which synaptic strengths change in a synapse-specific manner [3,4]. In contrast, little attention has been paid to mechanisms that regulate the total synaptic strength of a neuron.

Here we describe a new form of synaptic plasticity that increases or decreases the strength of all of a neuron’s synaptic inputs as a function of activity. Chronic blockade of cortical culture activity increased the amplitude of miniature excitatory postsynaptic currents (mEPSCs) without changing their kinetics. Conversely, blocking GABA (gamma-aminobutyric acid)-mediated inhibition initially raised firing rates, but over a 48-hour period mEPSC amplitudes decreased, and firing rates returned to close to control values. These changes were at least partly due to postsynaptic alterations in the response to glutamate and apparently affected each synapse in proportion to its initial strength. Such ‘synaptic scaling’ may help to ensure that firing rates do not become saturated during developmental changes in the number and strength of synaptic inputs, as well as stabilizing synaptic strengths during Hebbian modification and facilitating competition between synapses.

We tested the idea that postnatal rat visual cortical pyramidal neurons in primary cell culture scale the strength of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-mediated synaptic currents up or down as a function of activity. To sample inputs from a large number of synapses, we used whole-cell voltage-clamp recordings to measure mEPSCs. In control cultures, there was a broad distribution of mEPSC amplitudes [10,11]. The effects of decreasing or increasing firing rates for 48 h on mEPSC amplitude are shown. On average, mEPSC amplitudes from cultures grown in tetrodotoxin (TTX), which completely abolished firing, increased to 192 ± 16% of control values, and there was a similar effect on area. Blockade of GABAA-mediated inhibition with bicuculline, which raised firing rates to 265 ± 10% of control values, significantly decreased mEPSC amplitude to 70 ± 4% of control values and also decreased mEPSC area. These data demonstrate that the quantal amplitude of AMPA synapses can be increased or decreased by changes in activity. Activity blockade did not significantly affect the amplitude of AMPA-mediated mEPSCs recorded from bipolar interneurons, indicating that excitatory inputs from pyramidal neurons are regulated differently depending on the nature of the target neuron.

Some forms of synaptic plasticity require calcium influx through NMDA (N-methyl-D-aspartate) receptors. To examine the effects of NMDA receptor blockade on mEPSC amplitude, cultures were grown in the NMDA antagonist AP5 (D(−)-amino-7-phosphonovalenic acid) for 48 h. AP5 did not influence firing rates (95 ± 24% of control values) and had no effect on mEPSC amplitude or area. In contrast, blocking AMPA receptors with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), which like TTX completely abolished firing, significantly increased mEPSC amplitude to 195 ± 24% of control values. These observations indicate that, in contrast to many forms of LTP and LTD, the change in synaptic strength produced by activity blockade is not simply the result of a change in NMDA receptor signaling.

Cumulative amplitude histograms showed that the entire distribution of mEPSC amplitudes shifted towards larger values for TTX-treated and CNQX-treated cultures, and towards smaller values for bicuculline-treated cultures. We examined mEPSC kinetics by determining the average mEPSC waveforms under the different conditions. Scaling and overlaying these averaged waveforms revealed no differences in the rise and decay kinetics, and individual measurements of rise times also showed no significant differences between conditions. In contrast to LTP and other rapid potentiation protocols, there were no significant differences in mEPSC frequency between conditions. The lack of effect on frequency and kinetics suggests that, in accord with other studies, altering activity for two days does not significantly influence the placement or number of excitatory synapses onto pyramidal neurons.

The time course of mEPSC regulation was determined by blocking activity for 15, 26, or 48 (64) hours. There was a progressive increase in mEPSC amplitude with treatment time, indicating that this process is both slow and cumulative.

Does the activity-dependent regulation of mEPSC amplitude produce a change in spike-mediated EPSCs? TTX treatment increased EPSC amplitude between pairs of monosynaptically connected pyramidal neurons from 89 ± 65 pA to 219 ± 52 pA. There was no effect on EPSC reversal potential (1.6 ± 1.0 and 2.2 ± 1.1 mV for control and TTX-treated pyramidal neurons, respectively).

The regulation of mEPSC amplitudes described above could act to stabilize firing rates, because raising firing rates decreases the strength of excitatory synaptic inputs, and vice versa. To test whether firing rates are regulated homeostatically in response to changes in activity, cultures were grown under control conditions or in bicuculline for 48 hours, and whole-cell current-clamp recordings were used to measure firing rates. Acute bicuculline treatment raised firing rates significantly, but after 48 hours in bicuculline, firing rates recorded in bicuculline declined significantly and were close to zero when bicuculline was removed. These data indicate that increased firing produces a regulatory response that returns firing rates to control levels. Previous studies have shown that chronic TTX treatment dramatically increases firing rates when the TTX is removed. The bidirectional regulation of quantal amplitude is likely to contribute significantly to the homeostatic regulation of firing rates.

Changes in mEPSC amplitude could be occurring through a postsynaptic change in glutamate responsiveness or a presynaptic change in the glutamate content of synaptic vesicles. To investigate this, pulses of glutamate were applied to pyramidal neurons in the presence of TTX and AP5, and the amplitudes of the resulting glutamate currents were measured. Chronic TTX treatment increased the current amplitude, suggesting that activity regulates mEPSC amplitude through a postsynaptic change in receptor number or function, although there may be additional presynaptic changes.

What is the relationship between the mEPSC amplitude distributions produced by different activity levels? The average quantal amplitude could be modified by changing synaptic strengths by a constant value (an additive function), by adding a random amount (a random additive function), or by scaling synaptic strengths by the same multiplicative factor (a multiplicative function). To distinguish between these possibilities, control amplitudes were plotted against TTX amplitudes or bicuculline amplitudes, and the resulting relationship fit using each of the above models. The data were well fit by linear functions with slopes of 2.73 (TTX) and 0.66 (bicuculline), but not by additive or random additive functions. When scaled multiplicatively by these slope values, the cumulative amplitude distributions for TTX and bicuculline were almost perfectly superimposable on the control distribution, suggesting that activity is globally scaling quantal amplitudes in a multiplicative manner.

Taken together, our data place several constraints on the biophysical mechanism of synaptic scaling. If this scaling occurs through a change in the function of existing AMPA receptors through phosphorylation or some other mechanism, this process must modify single-channel conductance without influencing mEPSC kinetics. Alternatively, if this scaling occurs through changes in the synthesis and insertion of glutamate receptors, or through conversion of existing receptors between inactive and active states, then receptors must be inserted or converted in proportion to the existing number of functional receptors.

Multiplicative scaling of synaptic strengths preserves relative differences between inputs, such as those produced by Hebbian modifications, while allowing a neuron to adjust the total amount of synaptic excitation it receives. This process may help developing neurons remain responsive to inputs both early in development when the number of synaptic inputs is small and later in development as the number rises. This may be important for allowing developing neurons to participate in the correlation-based mechanisms that contribute to circuit formation. Consistent with this, there is evidence that mEPSC amplitudes in hippocampal neurons decrease as the number of synaptic inputs increases. In addition, by contributing to a stabilization in firing rates, synaptic scaling may help to counteract the destabilizing effects of Hebbian synaptic modifications. This instability arises because synaptic potentiation increases postsynaptic firing rates, thus increasing the correlation with presynaptic activity and producing a positive feedback loop that eventually saturates even weakly correlated inputs. Finally, this process predicts that strengthening of some inputs will lead to a scaling down of all synaptic strengths. Synaptic weakening has been suggested to be the prelude to synapse elimination. By reducing weak inputs in response to the strengthening of others, synaptic scaling may contribute to the processes of synaptic competition and elimination.