Cholinergic interneurons control the excitatory input to the striatum

Abstract

How the extent and time course of presynaptic inhibition depend on the action potentials of the neuron controlling the terminals is unknown. We investigated this issue in the striatum using paired recordings from cholinergic interneurons and projection neurons. Glutamatergic EPSCs were evoked in projection neurons and cholinergic interneurons by stimulation of afferent fibers in the cortex and the striatum, respectively. A single spike in a cholinergic interneuron caused significant depression of the evoked glutamatergic EPSC in 34% of projection neurons located within 100 microm and 41% of cholinergic interneurons located within 200 microm. The time course of these effects was similar in the two cases, with EPSC inhibition peaking 20-30 ms after the spike and disappearing after 40-80 ms. Maximal depression of EPSC amplitude was up to 27% in projection neurons and to 19% in cholinergic interneurons. These effects were reversibly blocked by muscarinic receptor antagonists (atropine or methoctramine), which also significantly increased baseline EPSC (evoked without a preceding spike in the cholinergic interneuron), suggesting that some tonic cholinergic presynaptic inhibition was present. This was confirmed by the fact that lowering extracellular potassium, which silenced spontaneously active cholinergic interneurons, also increased baseline EPSC amplitude, and these effects were occluded by previous application of muscarinic receptor antagonists. Collectively, these results show that a single spike in a cholinergic interneuron exerts a fast and powerful inhibitory control over the glutamatergic input to striatal neurons.

Figure 1.

Positioning of recording and stimulating electrodes in oblique brain slices and typical electrophysiological features of an LAI and an MSN. A, To investigate the effects of LAI spikes on glutamatergic input to MSN, the bipolar stimulating electrode (represented as 2 black circles) was placed in the medial agranular cortex, close to the white matter (wm), in oblique slices cut as described by Kawaguchi et al. (1989). Whole-cell recordings were obtained from an LAI (dark gray) and an MSN (light gray) in the dorsorostral portion of the striatum. Cell sizes are exaggerated for clarity. B, To investigate the effects of LAI spikes on glutamatergic input to another LAI, the bipolar stimulating electrode was placed in the striatum. Whole-cell recordings were obtained from two LAIs (dark gray). C, Membrane potential changes induced in an LAI by current injections (+30 and −180 pA); typical features of responses include a depolarizing sag that develops during the hyperpolarizing step, a rebound depolarization after the end of this step, and a long-lasting afterhyperpolarization after a train of spikes. No current was injected before and after the steps. Dashed gray line indicates resting membrane potential. D, Responses of an MSN to current injections (±100 pA); the typical waveforms observed are characterized by a much larger voltage deflection in response to the depolarizing step, a slow depolarizing ramp that develops during the depolarizing step, and the delayed appearance of an action potential. No current was injected before and after the steps. Dashed gray line indicates resting membrane potential.

Figure 2.

A single spike in an LAI inhibits evoked EPSC in a neighboring MSN. A, Simultaneous current-clamp recordings illustrate the lack of detectable effects of spikes in an LAI on the membrane potential of a neighboring MSN. The MSN was kept at its resting membrane potential (−79 mV, no current injected), whereas the LAI was slightly hyperpolarized (−60 mV) with a steady current injection and then depolarized for 500 ms to elicit action potentials. B, Average results obtained in 13 LAI–MSN pairs for the effects of a single LAI spike on MSN EPSC. These pairs were selected because (1) significant effects were observed for some intervals and (2) at least three different intervals were tested. Plotted is the EPSC amplitude (in percentage of baseline value, evoked with no spike in the LAI) as a function of the interval between the spike and the EPSC. Note that the positive axis runs leftwards from the origin in this and in the next figure. A positive value of the interval indicates that the LAI spike preceded the EPSC. C, Overall average effects of individual LAI spikes on MSN EPSC amplitude as a function of the interval between these events, for all of the pairs in which significant effects were observed for at least one interval. D, A representative example of the paired recording experiments performed to investigate the effects of individual LAI spikes on MSN EPSCs. Top traces are individual examples of current-clamp recordings from an LAI, illustrating the temporal position of the spike (if present) with respect to the evoked EPSC. Bottom traces are superimposed voltage-clamp recordings from the MSN (V_h of −80 mV), showing EPSCs evoked at different intervals after the LAI spike. Gray bars represent the interval between the LAI spike peak and the MSN EPSC peak (∼10, 20, 30, 40, and 60 ms). Electrical stimulation artifacts were removed in this and subsequent figures.

Figure 3.

Effects of multiple spikes and neostigmine on EPSC inhibition. A, In this LAI–MSN pair, a single spike in the LAI significantly reduced the MSN EPSC amplitude for intervals of ∼25 ms (left and middle traces; 10 MSN responses are superimposed for each experiment illustrated in this figure; LAI spikes are truncated). Longer depolarizing current steps elicited 10 spikes and were timed so that the last spike occurred 20–25 ms before the peak of the evoked EPSC (right traces). The MSN EPSC amplitude after 10 LAI spikes was significantly larger than the one after a single spike. B, In a different LAI–MSN pair, a single spike in the LAI significantly reduced MSN EPSC amplitude (interval of 20 ms). In the presence of neostigmine, the MSN EPSC amplitude in the absence of LAI spikes was significantly decreased, and the inhibitory effects of an LAI spike were strongly reduced (from 27 ± 4 to 15 ± 3%). These effects were partially reversed on neostigmine washout.

Figure 4.

A single spike in an LAI inhibits evoked EPSC in a neighboring LAI. A, An example of the lack of detectable direct interactions between two neighboring LAIs. The two LAIs were simultaneous recorded in current clamp and slightly hyperpolarized (at approximately −60 mV) to prevent spontaneous firing; one of them was then depolarized for 500 ms to elicit action potentials. B, Average effects of a single spike in an LAI on the evoked EPSC amplitude in a neighboring LAI. Data are from nine LAI–LAI pairs in which (1) significant effects were observed for some intervals and (2) at least three different intervals were tested. The EPSC amplitude (in percentage of control) is plotted as a function of the interval between the spike peak and the EPSC peak. C, Average effects of LAI spikes on neighboring LAI EPSC amplitude as a function of the interval, for all of the pairs in which significant effects were observed for at least one interval. D, A representative example of the experiments performed to investigate these effects. Top traces are individual current-clamp recordings from an LAI, in which spikes (when present) were elicited with brief current injections at different intervals before an EPSC was evoked in a neighboring LAI. Bottom traces are superimposed recordings from the second LAI (voltage clamp; V_h = −60 mV), showing the EPSCs evoked at different intervals after the spike in the other LAI. Gray bars represent the intervals between the LAI spike peak and the other LAI EPSC peak (∼10, 20, 30, and 40 ms).

Figure 5.

Effects of an LAI single spike on the EPSP evoked in the same cell. A, Average effects of a single spike in an LAI on the amplitude of the evoked EPSPs recorded in the same neuron, as a function of the interval between the two events. Data are from 14 experiments in which at least three different intervals were tested. B, Overall average effects for all of the cells in which significant effects were observed for at least one interval. C, A representative example of these experiments, in which an EPSP was evoked at variable times after an action potential was elicited in the same cell by a short current injection. Spikes are truncated. Gray bars represent the interval between the peak of the spike and that of the EPSP (∼10, 20, and 40 ms).

Figure 6.

Spike-induced inhibition of evoked EPSCs in MSNs is mediated by muscarinic receptors. A, Traces from a representative experiment in which significant MSN EPSC suppression was induced by an LAI spike in control solution, for an interval of ∼30 ms (gray bars). In the presence of atropine, the baseline MSN EPSC amplitude was significantly increased, and a spike in the LAI no longer affected such EPSC. These effects were reversed on atropine washout. B, Average effects of application of atropine or methoctramine in 24 LAI–MSN pairs, in which significant spike-induced suppression of MSN EPSC was observed in control solution. The spike–EPSC interval was constant for each pair (range of 20–30 ms).

Figure 7.

Spike-induced inhibition of evoked EPSCs in LAI is mediated by muscarinic receptors. A, A representative experiment in which significant EPSC suppression was induced in an LAI by a spike generated by a neighboring LAI in control solution (interval of ∼20 ms; gray bars). In the presence of methoctramine, the LAI EPSC amplitude was significantly increased, and a spike in the other LAI no longer affected this EPSC. These effects were reversed on methoctramine washout. B, Average effects of application of atropine or methoctramine in 13 LAI–LAI pairs, in which significant spike-induced suppression of EPSC was observed in control solution. The spike–EPSC interval (range of 20–30 ms) was constant for each pair.

Figure 8.

Afterspike reduction of evoked EPSPs in LAIs is partly mediated by muscarinic receptors. A, A representative experiment showing the effects of methoctramine on afterspike reduction of evoked EPSPs in an LAI. In control solution, an EPSP was evoked after a spike was triggered in the same cell; spike–EPSP interval was ∼40 ms (gray bars; the spikes are truncated in these traces). In the presence of methoctramine, the EPSP after a spike is still significantly smaller than the one evoked without a preceding spike, but the spike-induced inhibition is significantly reduced. These effects are reversed on washout. B, Average effects on afterspike reduction of EPSP in 23 LAIs in which either atropine or methoctramine were applied.

Figure 9.

Effects of low potassium on evoked EPSCs and their spike-induced modulation. A, In this representative experiment, paired recordings were obtained from an LAI and an MSN. Only the MSN synaptic currents are shown (10 superimposed traces are presented for each condition), without a spike in the LAI (no gray bar) or with one (gray bars represent time between peak of spike and peak of EPSC). In control solution, a spike in the LAI significantly decreased the amplitude of the evoked EPSC in the MSN (at an interval of ∼20 ms). In the presence of low potassium solution, the EPSC amplitude in the absence of an LAI spike was significantly increased, and the inhibitory effects of a spike in the neighboring LAI were still present. These effects were reversed on washout. B, Average effects of low potassium solution on MSN EPSC amplitude in the absence, or presence, of a spike in a neighboring LAI. C, Traces form a paired recording experiment from two LAIs. In control solution, a spike in one LAI (recorded in current-clamp mode) significantly decreased the amplitude of the evoked EPSC in the other LAI (recorded in voltage clamp). The spike–EPSC interval was ∼30 ms. Superimposed traces from the LAI recorded in voltage clamp are shown, in the absence (no gray bar) or presence of a spike in the other LAI (in this case, gray bars represent time between peak of spike and peak of EPSC). In the presence of low potassium solution, the EPSC amplitude in the absence of a spike in the other LAI was significantly increased, and the inhibitory effects of a spike in the neighboring LAI were still present. These effects were reversed on washout. D, Average effects of low potassium solution on LAI EPSC amplitude in the absence, or presence, of a spike in a neighboring LA. E, In an MSN, evoked EPSCs were significantly increased in amplitude by atropine application. However, subsequent application of low potassium solution (still in the presence of atropine) failed to affect EPSC amplitude.