A Ca(2+) threshold for induction of spike-timing-dependent depression in the mouse striatum

Abstract

The striatum is the principal input nucleus of the basal ganglia, receiving glutamatergic afferents from the cerebral cortex. There is much interest in mechanisms of synaptic plasticity in the corticostriatal synapses. We used two-photon microscopy and whole-cell recording to measure changes in intracellular calcium concentration ([Ca(2+)](i)) associated with spike-time-dependent plasticity in mouse striatum. Uncaging glutamate adjacent to a dendritic spine caused a postsynaptic potential at the soma and a rise in spine [Ca(2+)](i). Action potentials elicited at the soma raised both dendrite and spine [Ca(2+)](i). Pairing protocols in which glutamate uncaging preceded action potentials by 10 ms (pre-post protocol) produced supralinear increases in spine [Ca(2+)](i) compared with the sum of increases seen with uncaging and action potentials alone, or timing protocols in which the uncaging followed the action potentials (post-pre protocols). The supralinear component of the increases in [Ca(2+)](i) were eliminated by the voltage-sensitive calcium channel blocker nimodipine. In the adjacent parent dendrites, the increases in [Ca(2+)](i) were neither supralinear nor sensitive to the relative pre-post timing. In parallel experiments, we investigated the effects of these pairing protocols on spike-timing-dependent synaptic plasticity. Long-term depression (t-LTD) of corticostriatal inputs was induced by pre-post but not post-pre protocols. Intracellular calcium chelators and calcium antagonists blocked pre-post t-LTD, confirming that elevated calcium entering via voltage-sensitive calcium channels is necessary for t-LTD. These findings confirm a spine [Ca(2+)](i) threshold for induction of t-LTD in the corticostriatal pathway, mediated by the supralinear increase in [Ca(2+)](i) associated with pre-post induction protocols.

Figure 1.

Electrophysiological characterization of striatal spiny neurons. A, Photograph of the parahorizontal corticostriatal slice preparation, showing the position of the cortex, striatum, and corpus callosum (CC). The cells in the study were in the hatched region. B, Micrograph of a neuron injected with biocytin after recording in the whole-cell configuration, showing typical morphological features of spiny projection neurons (scale bar, 20 μm). Inset shows spiny distal dendrites (scale bar, 5 μm). C, Voltage responses to hyperpolarizing and depolarizing current pulses showing the characteristic responses of spiny projection neurons. D, Current (I) versus voltage (Vm) relation showing inward rectification typical of spiny projection neurons.

Figure 2.

Comparison of [Ca²⁺]_i transients induced in dendrites and dendritic spines by different protocols. A, Two-photon image of a spiny neuron, including the recording pipette attached at the soma. B, Higher-power view of the region indicated by the red box in A, showing dendritic spines (S) and adjacent dendrite (D). The dashed line indicates the direction of a line scan taken through the spine and dendrite. Scale bars: A, 20 μm; B, 1 μm. C, An example line scan image for the Ca²⁺-sensitive indicator Fluo-5F (green, top) and the Ca²⁺-insensitive indicator Alexa Fluor 488 (red, bottom) showing the response to uncaging of MNI-glutamate followed by three APs at Δt = 10 ms (pre-post protocol). D, Left to right, Somatic voltage recordings for a uEPSP and three APs at Δt = 10 ms (pre-post protocol), three APs and uEPSP at Δt = −30 ms (post-pre protocol), uEPSP alone, and three APs alone. E, Dendritic spine [Ca²⁺]_i measurements corresponding to conditions in D. F, Dendritic [Ca²⁺]_i measurements corresponding to conditions in D. Traces are averages of three successive responses. The equation f(t) = Ca_max*(exp[−t/tau1] − exp[−t/tau2])/(tau1 − tau2) was used for curve fittings, where Ca_max is the peak concentration, tau1 is the time constant for the rising phase, and tau2 is the time constant for the falling phase of the calcium transient.

Figure 3.

Spike-timing-dependent t-LTD induced by glutamate uncaging paired with postsynaptic APs. A, Representative example showing effects of pre-post pairing of uEPSP and postsynaptic firing of three APs (100 Hz, post) with Δt = 10 ms. Test EPSPs were evoked by electrical stimulation. Pairing involved uncaging of glutamate to produce a uEPSP <5.0 mV in amplitude. B, Group average (n = 6) in which each point is the average of three consecutive EPSPs (1 min).

Figure 4.

Spike-timing-dependent t-LTD in the striatum. A, Pre-post pairing induced t-LTD in the striatum. Top, Representative example of changes in EPSP amplitude induced by pairing electrically evoked EPSPs with postsynaptic firing of three APs (100 Hz, post) with Δt = 10 ms. Pairing was repeated 60 times at 0.1 Hz. Inset, Superimposed traces of an average of consecutive evoked EPSPs (average of 12 traces at indicated time points) before and after pairing protocols. Gray circles, EPSP amplitudes during pairing protocol. Membrane resistance and membrane potentials were monitored over the course of the experiment. Bottom, Group average (n = 13) in which each point is the average of three consecutive EPSPs (1 min). B, Post-pre pairing [electrically evoked EPSPs with postsynaptic firing of three APs (100 Hz, post) with Δt = −30 ms] induced no changes. Pairing was repeated 60 times at 0.1 Hz. Responses during pairing not shown due to preceding spikes. Same conventions as in A. Top, Representative example. Bottom, Group average (n = 10).

Figure 5.

Failure to induce t-LTD using unpaired stimuli or single action potentials. A, Unpaired presynaptic stimulation (EPSP alone, repeated at 0.1 Hz) does not induce t-LTD (n = 6). B, Unpaired postsynaptic stimulation (three action potentials at 100 Hz, repeated at 0.1 Hz) does not induce t-LTD (n = 9). C, Pre-post protocol fails to induce t-LTD if postsynaptic activity is a single action potential (n = 6). D, Post-pre protocol induces no change (n = 4). Insets, Superimposed traces of an average of consecutive EPSPs (12 traces) before (black) and 14 min after (red) pairing-protocols. Same conventions as in Figure 3.

Figure 6.

A spine [Ca²⁺]_i threshold for t-LTD. A, Plasticity (EPSP changes) produced by pairing an EPSP and a single AP at Δt = 10 ms (pre-post), a single AP and an EPSP at Δt = −10 ms (post-pre), and an EPSP (pre alone). B, Plasticity (EPSP changes) caused by pairing an EPSP and three APs at Δt = 10 ms (pre-post), three APs and an EPSP at Δt = −30 ms (post-pre), an EPSP (pre alone) and three APs (post alone). C, D, Changes in spine [Ca²⁺]_i evoked by the pairing protocols in A (n = 4) and B (n = 11). *p < 0.05, **p < 0.01.

Figure 7.

Postsynaptic [Ca²⁺]_i increase is necessary for pre-post t-LTD. A, Activation of the NMDA receptor is not necessary for the induction of the pre-post t-LTD. Bath application of the NMDA blocker d-APV (50 μm) did not block pre-post t-LTD (n = 12, filled circles). B, Intracellular Ca²⁺ chelators blocked pre-post t-LTD. Top, Representative example of showing that postsynaptic inclusion of a Ca²⁺ chelator, BAPTA (5 mm), significantly blocks pre-post t-LTD. Bottom, Group average of effects of postsynaptic Ca²⁺ chelators (average of cells loaded with BAPTA, n = 4 or EGTA, n = 2; filled circles). C, Nimodipine 10 μm significantly blocked pre-post t-LTD (n = 7; filled circles) but nimodipine 1 μm did not block pre-post t-LTD (n = 5; open circles). D, Effect of nimodipine on pre-post induced Ca²⁺ transients. Left, Upper trace, voltage trace from recorded cell; lower trace, comparison of Ca²⁺ transient under control conditions and in the presence of nimodipine 10 μm. Right, Group average data show significant effect of nimodipine 10 μm on pre-post induced Ca²⁺ transients. *p < 0.05, **p < 0.01, ***p < 0.001.