Quantitative estimate of synaptic inputs to striatal neurons during up and down states in vitro

Abstract

Up states are prolonged membrane potential depolarizations critical for synaptic integration and action potential generation in cortical and striatal neurons. They commonly result from numerous concurrent synaptic inputs, whereas neurons reside in a down state when synaptic inputs are few. By quantifying the composition, frequency, and amplitude of synaptic inputs for both states, we provide important constraints for state transitions in striatal network dynamics. Up and down states occur naturally in cortex-striatum-substantia nigra cocultures, which were used as an in vitro model in the present study. Spontaneous synaptic inputs during down states were extracted automatically in spiny projection neurons and fast spiking interneurons of the striatum using a newly developed computer algorithm. Consistent with a heterogeneous population of synaptic inputs, PSPs and PSCs showed no correlation in amplitude and rise time and occurred at relatively low frequencies of 10-40 Hz during the down state. The number of synaptic inputs during up states, estimated from the up-state charge and the unitary charge of down-state PSCs, was 217 +/- 44. Given the average up-state duration of 284 +/- 34 msec, synaptic input frequency was approximately 800 Hz during up-states for both neuronal types. Many down-state events reversed at the chloride reversal potential and were blocked by GABA(A) antagonists. The high correlation between up- and down-state reversal potential suggests that despite these drastic changes in synaptic input frequency, the ratio of inhibitory to excitatory currents is similar during both states.

Figure 1.

Automatic detection of spontaneous synaptic events. A, Schematic illustration of detection algorithm and critical parameters. The difference between the minimum and maximum of membrane potential is computed for a time period, τ₁ (equal to twice the minimum event distance, τ_min). An event is detected if the difference is greater than the amplitude threshold, Fσ, where σ is the SD of the noise and F is a multiplication factor. The peak value is computed as the local maximum within the expected event duration τ₂. The search for a subsequent synaptic event begins once the membrane potential decays from the peak value by ΔV_m = 4σ. B, Number of events detected as a function of minimum event distance, τ_min (n = 4 spiny projection neurons). Note that the number of events varies very little when τ_min is reduced from 10 msec to 5 msec or increased to 15 msec. C, Scatter plot reveals no correlation between mean event amplitude and mean event frequency. This suggests that differences in event frequency between neurons are not caused by differences in event amplitude. D, Similarity of noise from perceptually synaptic input-free data segments (▪, SD, 3.4 pA) and noise from data segments in which ionotropic glutamate and GABA receptors are blocked (□, bath application of 10 μm DNQX, 50 μm APV, and local application of 10 mm picrotoxin; SD, 2.9 pA). For the population, noise SD is 2 ± 0.18 pA. E, Effect of threshold on amplitude and charge of unitary synaptic events confirms that F = 5 is the “best” threshold for PSC detection. An increase in F to 6 significantly decreases the charge and amplitude of the mean unitary PSC by >20%, whereas a decrease in F to 4 nonsignificantly increases the charge and amplitude of the mean unitary PSC by only ∼10%.

Figure 2.

Differences in electrophysiological characteristics of spiny projection neurons (SP) and fast spiking interneurons (FS) recorded with whole-cell patch pipettes in current clamp. A, Responses of an SP and an FS to somatic current-pulse injections. B, Steady-state current-voltage plot for SP and FS. C, Latency to first action potential as a function of current injection for each neuron. Note the increased latency for SP (gray lines) compared with FS (black lines) independent of current strength. D, Scatter plot of maximum observed latency to first action potential versus action potential (AP) width. E, Scatter plot of down-state membrane potential versus time to maximal spike afterhyperpolarization (AHP). Note that spiny projection neurons and fast spiking interneurons form separate clusters in both of these two-dimensional parameter spaces.

Figure 3.

Spontaneous synaptic events in striatal neurons during the down state (current clamp) in spiny projection neurons (SP; A) and fast spiking interneurons (FS; B). Traces represent contiguous segments. Activity is characterized by the presence of spontaneous events that resemble synaptic inputs at down-state potential. ▴, Identified events; ▵, putative events missed by the algorithm or misclassified. Down-state potential is -93 mV (SP) and -57 mV (FS), respectively. Note the occurrence of multiple PSPs with an IEI too short for the membrane potential to return to resting potential in the FS. C, Rise time versus amplitude is uncorrelated for both spiny projection neurons and fast spiking interneurons, suggesting a heterogeneous population of synapses. D, Summary of PSP slope characteristics for all neurons. Low R² values indicate the absence of correlation between PSP amplitude and PSP rise time. Similarly, relatively high coefficient of variation (CV) values demonstrate large variations in PSP slope for all neurons.

Figure 4.

Density function of spontaneous postsynaptic events for the population of spiny projection neurons (SP) and fast spiking interneurons (FS) in current clamp. Amplitude (A), rise time (B), and population IEI (C) distributions do not differ between both classes.

Figure 5.

Spontaneous synaptic currents in striatal neurons during the down state in voltage clamp in a spiny projection neuron (SP; A) and a fast spiking interneuron (FS; B). Traces represent contiguous segments. Activity is characterized by the presence of spontaneous events that resemble synaptic currents. ▾, Identified events; ▿, putative events missed by the algorithm or misclassified. Holding potential is -78 mV (SP) and -66 mV (FS), respectively. C, Rise time versus amplitude is uncorrelated for both SP and FS, suggesting a heterogeneous population of synapses. D, Mean amplitude is highly correlated with mean slope (Pearson's r = 0.95), indicating a small variation in mean rise time among neurons.

Figure 6.

Density function and cumulative distribution of PSCs for the population of spiny projection neurons (SP) and fast spiking interneurons (FS). Amplitude distribution (A), rise time distribution (B), and IEI distribution (C) do not differ between neuronal types.

Figure 7.

Striatal neurons receive ∼200 synaptic inputs during up states. A, B, Example up states for spiny projection neurons (SP) and fast spiking interneurons (FS). Top trace, Current clamp. Bottom trace, Voltage clamp. V_H = -77 mV for SP and -66 mV for FS. C, D, Summary statistics for total number of synaptic inputs during up states (C) and average synaptic input frequency (D) for both neuronal types. E, Up-state duration is correlated with the number of synaptic inputs (r = 0.79) for the population of neurons (excluding one outlier with a charge of -0.67), suggesting that input frequency during up states is stationary.

Figure 8.

Up and down states receive a significant number of GABAergic inputs. Spontaneous activity in a spiny projection neuron (SP; left) and fast spiking interneuron (FS; right) during down state (A, B) and upstate (C, D) at different holding potentials. E, In the presence of the glutamate receptor antagonists CNQX (20 μm) and APV (50 μm), all spontaneous PSCs were reversed and blocked by local application of picrotoxin. Bars indicate clamp voltage and application of picrotoxin. Insets show traces on an expanded time scale.

Figure 9.

The correlation in population reversal potential between up and down states suggests that the fraction of GABAergic inputs is stable in both states for each neuron. A, Up-state charge for spiny projection neurons (SP) and fast spiking interneurons (FS) reverses between -60 and 0 mV. Numbers indicate the number of neurons tested for each holding potential. B, Reversal potential for up-state charge correlates highly with reversal potential of charge for unitary down-state PSCs (R² = 0.90).