Electrophysiological effects of SKF83959 on hippocampal CA1 pyramidal neurons: potential mechanisms for the drug's neuroprotective effects

Abstract

Although the potent anti-parkinsonian action of the atypical D₁-like receptor agonist SKF83959 has been attributed to the selective activation of phosphoinositol(PI)-linked D₁ receptor, whereas the mechanism underlying its potent neuroprotective effect is not fully understood. In the present study, the actions of SKF83959 on neuronal membrane potential and neuronal excitability were investigated in CA1 pyramidal neurons of rat hippocampal slices. SKF83959 (10-100 µM) caused a concentration-dependent depolarization, associated with a reduction of input resistance in CA1 pyramidal neurons. The depolarization was blocked neither by antagonists for D₁, D₂, 5-HT(2A/2C) receptors and α₁-adrenoceptor, nor by intracellular dialysis of GDP-β-S. However, the specific HCN channel blocker ZD7288 (10 µM) antagonized both the depolarization and reduction of input resistance caused by SKF83959. In voltage-clamp experiments, SKF83959 (10-100 µM) caused a concentration-dependent increase of Ih current in CA1 pyramidal neurons, which was independent of D₁ receptor activation. Moreover, SKF83959 (50 µM) caused a 6 mV positive shift in the activation curve of Ih and significantly accelerated the activation of Ih current. In addition, SKF83959 also reduced the neuronal excitability of CA1 pyramidal neurons, which was manifested by the decrease in the number and amplitude of action potentials evoked by depolarizing currents, and by the increase of firing threshold and rhoebase current. The above results suggest that SKF83959 increased Ih current through a D₁ receptor-independent mechanism, which led to the depolarization of hippocampal CA1 pyramidal neurons. These findings provide a novel mechanism for the drug's neuroprotective effects, which may contributes to its therapeutic benefits in Parkinson's disease.

Figure 1. SKF83959 induced depolarizing response in CA1 pyramidal neuron in hippocampal slices.

A. Resting membrane potential recorded in a representative neuron. The upper trace shows the membrane potential of the neuron. The resting potential was −60 mV, whereas the input resistance change was monitored in the upper trace through injecting hyperpolarizing current pulses (400 ms, −50 pA, lower trace) every 10 sec (the downward deflections). The black bar denotes the perfusion with SKF83959 (50 µM). To exclude the change of input resistance caused indirectly by depolarizing response, the membrane potential during SKF83959 application was manually clamped to the pre-drug level. Bicuculline (5 µM) was added in ASCF to suppress the spontaneous IPSPs. B. Bar graphs showing the maximal depolarization caused by SKF83959 (50 µM) in the presence of TTX (0.5 µM, n = 5), SCH (D1 receptor antagonist SCH23390, 10 µM, n = 5), Rac (D2 receptor antagonist raclopride, 10 µM, n = 5), Mes (5-HT2A/2C receptor antagonist mesulergine, 10 µM, n = 5), Pra (Alpha1-adrenoceptor antagonist prazosin, 10 µM, n = 5), or following intracellular dialysis of GDP-β-S (0.5 mM, n = 6). C. Bar graphs showing the input resistance in the control (Ctrl) and during perfusion with SKF83959 (SKF, 50 µM, n = 10, *P<0.05).

Figure 2. Effects of SKF83959 on subthreshold response of CA1 pyramidal neurons.

A. Superimposed responses to prolonged hyperpolarizing current pulses (400 ms, −50 pA) recorded from a representative neuron before (black) and during (gray) the perfusion with SKF83959 (50 µM), showing the enhanced voltage sag (arrow). The resting potential of the neuron was −60 mV, and the membrane potential was manually clamped to compensate the SKF83959-induced depolarizing response. B. Bar graphs showing the voltage sag ratios in the presence and absence of SKF83959 (50 µM). The voltage sag ratio was quantified as the peak voltage deflection divided by the steady-state voltage deflection. C. The results were obtained from another neuron in the presence of ZD7288 (10 µM). The resting potential of the neuron was −66 mV. Note that ZD7288 completely abolished the voltage sag. D. Bar graphs showing the maximal depolarizing response caused by SKF83959 (50 µM) in the presence (n = 6) and absence (n = 8) of ZD7288 (10 µM). E. Bar graphs showing the input resistance of CA1 pyramidal neurons measured in the presence of ZD7288 (10 µM) or in the presence of ZD7288 (10 µM) and SKF83959 (50 µM). n = 8 for each group. ** P<0.01. Ctrl, Control; SKF, SKF83959; ZD, ZD7288.

Figure 3. SKF83959 increased Ih current in hippocampal CA1 pyramidal neurons.

A. Current family of Ih recorded from a representative neuron in the presence and absence of SKF83959 (50 µM). The neuron was hold at −20 mV and current traces were elicited with a series of 1.5-s hyperpolarizing voltage steps from −20 mV to −120 mV with increment of 10 mV followed by a voltage step to −80 mV to measure the tail currents. B. Averaged current/voltage (I/V) relationship of Ih plotted in the presence and absence of SKF83959 (50 µM). C. Plot of the amplitude of Ih against time in a representative neuron. The black bar denotes the perfusion with SKF83959 (50 µM). The neuron was hold at −45 mV, and Ih was elicited with 1.5-s hyperpolarizing voltage steps to −105 mV every 30 sec. The inset shows the superimposed current traces taken at the time indicated by the two arrows. Scale bars: 0.5 s, 250 pA. D. Bar graphs showing the maximal steady-state amplitude of Ih in the presence of different concentrations of SKF83959. *P<0.05, **P<0.01, ***P<0.001 vs. Control.

Figure 4. Effects of SKF83959 on the kinetic properties of Ih current.

A. the activation curves of Ih plotted in control and in the presence of SKF83959 (50 µM). The neurons were held at −20 mV. Ih currents were elicited with 1.5-sec hyperpolarizing steps to various potentials followed by a voltage step to −80 mV to measure the tail currents. Normalized amplitude of the tail current was plotted as the function of the test potentials and fitted with the Boltzmann equation: I/Imax = 1/[1+exp(V-V1/2)/s], where I/Imax is the normalized amplitude of the tail current, V is the test potential, V1/2 is the half-activation potential, and s is the slope factor. B. Plot of the activation time constant (τf) of Ih against the test potentials. The trace of Ih current was fitted with bi-exponential functions. n = 6 for each symbol. *P<0.05, **P<0.01 vs. Control.

Figure 5. Increase of Ih by SKF83959 was independent of activation of D1-like receptors.

The neurons were held at −45 mV, and Ih current was elicited with 1.5-s hyperpolarizing voltage steps to −105 mV every 30 sec. In each panel, the amplitude of Ih was plotted against time. The black bars denotes the perfusion with SKF83959 (50 µM), whereas the gray bar denotes the application of various agents: A. perfusion with D1-like receptor antagonist SCH23390 (10 µM), n = 11; B. Intracellular dialysis of GDP-beta-S (0.5 mM), n = 9; C. intracellular dialysis of GppNHp (0.5 mM), n = 6; D. intracellular dialysis of high concentrations of cAMP (100 µM), n = 6; E. intracellular dialysis of Rp-cAPM (100 µM), n = 6.

Figure 6. SKF83959 suppressed the somatic excitability of CA1 pyramidal neurons in hippocampal slices.

A. Train of action potentials of a representative neuron in response to a prolonged depolarization current pulse (150 pA, 300 ms) prior to (a) and after (b) perfusion with SKF83959 (50 µM). The resting potential was −69 mV in (a). The membrane potential was compensated by injecting steady hyperpolarizing current in (b). The two traces were superimposed at the bottom (c). B. Plot of the number of action potentials against the current intensities in another neuron. C. Plot of the latency of the first spike against the current intensities in the same neuron shown in B. The latency was defined as the time between the onset of depolarizing current pulse and the time of threshold of the first spike. D. Bar graph showing the rheobase currents measured prior to and after perfusion with SKF83959 (50 µM). *P<0.05 vs. Control.

Figure 7. Effect of SKF83959 on action potentials of CA1 pyramidal neurons in hippocampal slices.

A. Superimposed action potentials elicited in a representative neuron prior to and after perfusion with SKF83959 (50 µM). For comparison, the membrane potential was compensated. B. Bar graph showing the amplitude of action potentials prior to and after perfusion with SKF83959 (50 µM). The amplitude was defined as the voltage difference between the threshold and peak of the action potential. C. Bar graph showing the half-width of action potentials prior to and after perfusion with SKF83959. The half-width was measured as the width of half-maximal spike amplitude. D. Bar graph showing the threshold of action potentials prior to and after perfusion with SKF83959 (50 µM). The threshold was defined as the first point on the upstroke of action potential with a rising rate exceeded 50 mV/ms. In B, C, D, n = 8, **P<0.01 vs. Control.