Voltage-gated transient outward currents in neurons with different firing patterns in rat superior colliculus

Abstract

1. We investigated the electrophysiological properties of transient outward currents (TOCs) in neurons with different firing patterns, regular-spiking, fast-spiking and late-spiking neurons, in the intermediate layer (SGI) of the superior colliculus using the whole-cell patch clamp technique in slice preparations obtained from young rats (post-natal days 17-22). 2. Analysis of inactivation kinetics and normalized amplitude revealed that TOCs in regular-and fast-spiking neurons had fast inactivation kinetics (decay time constants (mean +/- s.e.m.) of 13.8 +/- 1.5 and 11.4 +/- 1.2 ms, respectively) and low current densities (36.6 +/- 3.3 and 32.1 +/- 4. 9 pA pF-1, respectively). TOCs in late-spiking neurons, on the other hand, displayed a wide range of both inactivation kinetics (36.7 +/- 2.4 ms, with a range from 11.3 to 147.8 ms) and current density (54. 0 +/- 2.9 pA pF-1, with a range from 9.8 to 131.2 pA pF-1). 3. In regular-, fast- and late-spiking neurons having TOCs with slow time constants (> 50 ms, class II late-spiking neurons), the TOCs were sensitive to 4-aminopyridine (4-AP), with IC50 values of 2.9, 2.4 and 1.2 mM, respectively. In late-spiking neurons having TOCs with fast decay time constants (< 30 ms, class I late-spiking neurons), the TOCs were composed of at least two 4-AP-sensitive components (IC50 values of 0.2 microM and 3.6 mM). 4. Class I late-spiking neurons displayed non-inactivating outward currents which were highly sensitive to 4-AP. They changed their firing patterns to the regular-spiking mode, not only in response to low concentrations of 4-AP (< 50 microM), but also in response to dendrotoxin (200 nM), suggesting that non-inactivating outward currents contribute to the late-spiking property. However, the components of TOCs which were highly sensitive to 4-AP were also sensitive to dendrotoxin. These results suggest that both or either of the two currents contribute to the late-spiking property of class I late-spiking neurons. 5. Although class II late-spiking neurons also displayed non-inactivating outward currents, the late-spiking property was not abolished by low concentrations of 4-AP and dendrotoxin. They changed to a regular firing pattern in response to a high concentration of 4-AP (5 mM), suggesting that TOCs contribute to late-spiking property of class II late-spiking neurons. 6. The results suggest that TOCs with different properties contribute to the different firing patterns of SGI neurons.

Figure 1

Firing patterns of three neuron types in the SGI of the SC A-C, firing patterns of regular-, fast- and late-spiking neurons, respectively. Intensities of injected currents are indicated on the right. Note that a transient hyperpolarization occurred following the onset of the membrane depolarization in the late-spiking neuron (C, arrow). D, the amplitude of depolarization excluding the spike amplitude was plotted as a function of the ratio of the 1st ISI (a in insets) to the 2nd ISI (b in insets). Filled and open symbols denote neurons with the late-spiking property and the others, respectively (see insets). There was no significant difference in membrane potentials before injection of depolarized current pulses between neurons with late-spiking properties (−89.3 ± 0.6 mV, n = 42) and other neurons (−88.6 ± 0.7 mV, n = 45) (P > 0.1, t test). E, the normalized mean half-width was plotted as a function of the firing frequency. The mean membrane potential before injection of depolarized current pulses was −88.6 ± 0.7 mV (n = 45). In subsequent analysis, neurons located in the dotted area were regarded as regular-spiking neurons (see left inset) and those in the hatched area as fast-spiking neurons (see right inset).

Figure 6

Sensitivity to 4-AP of TOCs A1–D1, recordings of TOCs in the presence of different concentrations of 4-AP in regular-spiking, fast-spiking, class I late-spiking and class II late-spiking neurons, respectively. A2-D2, dose-response curves for 4-AP. The numbers in parentheses indicate the number of neurons tested. Note that the plots of the normalized peak amplitude of TOCs in class I late-spiking neurons against the concentration of 4-AP were fitted by the sum of two logistic functions (C 2).

Figure 2

TOCs isolated by the subtraction protocol A, whole-cell currents (upper traces) evoked by application of a series of depolarizing voltage steps with 10 mV intervals (lower traces) to a neuron following a 500 ms prepulse to −100 mV. B, sustained outward currents evoked by application of the same depolarizing voltage steps as in A following a prepulse to −30 mV. C, isolated TOCs obtained by subtraction of the traces shown in B from those shown in A. D, the decay phase of a current trace obtained by the subtraction protocol was fitted by the sum of two exponential functions. Double-exponential fits are superimposed on the decay phase of a TOC depicted by the dotted trace.

Figure 3

Inactivation kinetics and current density of TOCs in the three neuron types A-D, TOCs (dotted traces) and double-exponential fits superimposed on the decay phase in regular-spiking (A), fast-spiking (B) and late-spiking neurons (C and D). E, relationship between the current density and the decay time constant (τ₁). ○, regular-spiking neurons; •, fast-spiking neurons; , late-spiking neurons.

Figure 4

Recovery from inactivation of TOCs A, an example of whole-cell outward currents evoked by the double-pulse protocol. B, time course of recovery from inactivation. A double-exponential fit is superimposed on the plots. C-E, fast recovery time constants (τ_R1), slow recovery time constants (τ_R2) and the percentage of fast recovery components in each neuron type, respectively. Plots of the time constants in individual neurons (○), the mean time constants (▪) and s.e.m. (bars) are shown (C-E). Slow recovery time constants and the percentage of fast recovery components were not significantly different among the different neuron types (P > 0.1, ANOVA post hoc test).

Figure 5

Voltage dependence of activation, steady-state inactivation and decay time constant of TOCs A1-D1, plots of the mean normalized chord conductances as a function of the membrane potentials in regular-spiking, fast-spiking, class I late-spiking and class II late-spiking neurons, respectively. Boltzman fits are superimposed on the plots of activation (•) and inactivation (○) with bars indicating s.e.m. A voltage of half-maximal conductance and a slope factor are shown in the tables between the panels. A2–D2, plots of decay time constants as a function of test pulse potentials.

Figure 7

Effect of 4-AP on firing patterns of late-spiking neurons A1 and B 1, effect of 4-AP on late-spiking neurons with the 1st ISI shorter than 50 ms (40.2 ± 4.4 ms, n = 8) and longer than 150 ms (182.8 ± 26.0 ms, n = 4), respectively. A2 and B 2, plots of the ratio of the 1st ISI to the 2nd ISI (a/b) as a function of concentration of 4-AP. Individual symbols represent the plots obtained from individual neurons. Dashed lines indicate a ratio value of 1.0. C, the mean ratio of the 1st ISI to the 2nd ISI (a/b) against the concentration of 4-AP in late-spiking neurons with a 1st ISI < 50 ms (▪) and > 150 ms (□). D, effect of 10 μm 4-AP on late-spiking neurons with the 1st ISI < 50 ms. The late-spiking property was recovered 10 min after washing out 4-AP.

Figure 9

Effect of dendrotoxin on late-spiking neurons A, firing pattern of a late-spiking neuron with a short 1st ISI (31.1 ± 5.2 ms, n = 4) in control solution (1) and in the presence of 200 nm dendrotoxin (2). B, plots of the ratio of the 1st ISI to the 2nd ISI in the absence and presence of dendrotoxin. Individual symbols represent the plots obtained from individual neurons. The dashed line indicates the ratio value of 1.0. C, whole-cell outward currents in control solution (1) and in the presence of 200 nm dendrotoxin (2) in a class I late-spiking neuron. C 3 shows a dendrotoxin-sensitive current obtained by subtraction of C 2 from C 1. Note that both inactivating and non-inactivating currents are sensitive to dendrotoxin. D, firing pattern in a late-spiking neuron with a long ISI (212.9 ± 24.2 ms, n = 4) in control solution (1) and in the presence of 200 nm dendrotoxin (2). E, plots of the ratio of the 1st ISI to the 2nd ISI in the absence and presence of dendrotoxin.

Figure 8

Effect of 4-AP on non-inactivating outward currents Outward currents evoked by a test pulse to −10 mV following a prepulse to −30 mV. Non-inactivating outward currents are represented as outward currents evoked by a test pulse. A, non-inactivating outward currents in class I late-spiking neurons in the control solution (1), and in the presence of 30 μm (2) and 100 μm (3) 4-AP. A4 shows the three traces of A1–3 superimposed. Note that the non-inactivating outward current was decreased following application of 30 μm 4-AP, but no further decrease was seen with 100 μm 4-AP. B, non-inactivating outward currents in class II late-spiking neurons in control solution (1), and in the presence of 100 μm (2) and 3 mm (3) 4-AP. B 4 shows the three traces of B1–3 superimposed. Note that the non-inactivating outward current was decreased following application of 100 μm 4-AP, but no further decrease was seen with 3 mm 4-AP. An inactivating outward current, which was evoked by a prepulse, was mostly abolished by application of 3 mm 4-AP. The voltage protocol is shown at bottom left.