Noradrenergic modulation of the hyperpolarization-activated cation current (Ih) in dopamine neurons of the ventral tegmental area

Abstract

Alterations in the state of excitability of midbrain dopamine (DA) neurons from the ventral tegmental area (VTA) may underlie changes in the synaptic plasticity of the mesocorticolimbic system. Here, we investigated norepinephrine's (NE) regulation of VTA DA cell excitability by modulation of the hyperpolarization-activated cation current, Ih, with whole cell recordings in rat brain slices. Current clamp recordings show that NE (40 microM) hyperpolarizes spontaneously firing VTA DA cells (11.23+/-4 mV; n=8). In a voltage clamp, NE (40 microM) induces an outward current (100+/-24 pA; n=8) at -60 mV that reverses at about the Nernst potential for potassium (-106 mV). In addition, NE (40 microM) increases the membrane cord conductance (179+/-42%; n=10) and reduces Ih amplitude (68+/-3% of control at -120 mV; n=10). The noradrenergic alpha-1 antagonist prazosin (40 microM; n=5) or the alpha-2 antagonist yohimbine (40 microM; n=5) did not block NE effects. All NE-evoked events were blocked by the D2 antagonists sulpiride (1 microM) and eticlopride (100 nM) and no significant reduction of Ih took place in the presence of the potassium channel blocker BaCl2 (300 microM). Therefore, it is concluded that NE inhibition of Ih was due to an increase in membrane conductance by a nonspecific activation of D2 receptors that induce an outward potassium current and is not a result of a second messenger system acting on h-channels. The results also suggest that Ih channels are mainly located at dendrites of VTA DA cells and, thus, their inhibition may facilitate the transition from single-spike firing to burst firing and vice versa.

Fig. 1. Effects of norepinephrine (NE; 40 µM) on membrane potential, holding current and membrane conductance of VTA dopamine (DA) cells

A. Bath application of NE hyperpolarizes spontaneously firing VTA DA cells. B. NE evokes an outward current on VTA DA cells when held at − 60 mV. C. Current vs. voltage curves showing the effect of NE on membrane cord conductance. Observe that NE increases the membrane conductance (increased slope). The curves also show what is evident from parts A and B. That is, NE evokes an outward current at − 60 mV (reduction in the current amplitude) and hyperpolarizes (change of intercepts on the potential axis) the probed cells. The membrane potential coordinate of the point of intersection of the two IV-curves represents the reversal potential of the conductance activated by NE (see part D). The curves were constructed by plotting the instantaneous membrane current against the command potentials. Points on each curve represent the average ± s.e.m. from a sample of six cells. The smooth lines show linear fits. D. Current vs. voltage curve obtained by subtracting the NE curve from the control curve for each of the six cells in part C. Points on the curve represent average ± s.e.m.. This curve represents the current vs. voltage relation of the conductance activated by NE. The reversal potential (intercept on the potential axis) for this conductance is − 96 ± 8 mV (n = 6), which is very close to the predicted Nernst potential for K⁺ (− 106 mV). The smooth line shows a linear fit.

Fig. 2. NE (40 µM) inhibits I_h

A. Voltage clamp recording showing the current response of a VTA DA cell when held at − 60 mV and commanded to − 120 mV for 1 second. Bath application of NE (2 min.) induced an outward current of 96 pA (change in holding current), a 1.7-fold increase in membrane conductance and a 30% reduction in I_h amplitude. The membrane conductance was calculated from the amplitude of the instantaneous current (between …. and ----). B. The time course of I_h inhibition is parallel to that of the NE-induced increase in membrane conductance (increase in instantaneous current (I_ins) amplitude). The data used to plot the graph is from a recording similar to the one shown in part A. C. NE significantly reduced I_h amplitude (2-way ANOVA, F= 249, df = 1, 108; P < 0.0001). Data points represent average ± s.e.m. of ten cells.

Fig. 3. Alpha adrenergic antagonists did not block the NE-induced outward current, increase in membrane conductance or I_h inhibition

Data points on each graph represent average ± s.e.m. of five cells. A. At 40 µM alpha-1 antagonist prazosin did not affect I_h amplitude. B. At 40 µM alpha-2 antagonist yohimbine did not affect I_h amplitude. C. In the presence of prazosin, NE evokes an outward current, increases the membrane conductance and inhibits I_h. D. In the presence of yohimbine, NE evokes an outward current, increases the membrane conductance and inhibits I_h. E. NE significantly reduced I_h amplitude in the presence of prazosin (2-way ANOVA, F=534.9, df = 1, 48;P < 0.0001). F NE significantly reduced I_h amplitude in the presence of yohimbine (2-way ANOVA, F=99.72, df = 1, 48;P < 0.0001). G. Maximal I_h inhibition (− 120 mV) in the presence of NE and prazosin or NE and yohimbine was not different from that of NE alone (1-way ANOVA, MS= 0.3481, df = 2, 19;P = 0.9956).

Fig. 4. In presence of D₂ antagonist eticlopride (100 nM) NE did not affect I_h

Data points in part A and C represent average ± s.e.m. of five and ten cells, respectively. A. At 100 nM eticlopride has no effect on I_h amplitude. B. Eticlopride blocks the NE-induced outward current, increase in membrane conductance and I_h inhibition. C. Summary of NE effects on I_h in the presence of eticlopride.

Fig. 5. NE (40 µM) inhibits I_h without affecting its voltage and time dependence

A. NE inhibits I_h without affecting its activation curve, indicating a voltage independent inhibition (2-way ANOVA, F= 0.2384, df = 1, 48; P = 0.6276). The level of I_h activation for a given command potential and condition were calculated as the ratio between the I_h tail (I_tail) current at the command step and the tail current at − 120 mV (I_{tail max}.). The half activation voltage and slope factor (k) were − 82 mV and k = 8 mV in control and in NE. The smooth curves show the Boltzmann fit. Data points represent average ± s.e.m. of five cells. B. The time course of I_h was fitted by single exponential function. The activation time constants (τ_h) obtained from the fit are plotted against command potentials. NE did not affect the time dependence of I_h, suggesting a voltage independent inhibition (2-way ANOVA, F=0.4951, df = 1, 32; P = 0.4868). The smooth curves show a single exponential fit. Data points represent average ± s.e.m. of five cells.

Fig. 6. In the presence of the K⁺ channel blocker BaCl₂ (300 µM), NE did not affect I_h

Data points in part A and C represent average ± s.e.m. of five cells. A. At 300 µM BaCl₂ causes a small diminution of I_h amplitude. B. BaCl₂ blocks the NE-induced outward current, increase in membrane conductance and I_h inhibition. C. Summary of NE effects on I_h in the presence of BaCl₂.