Cocaine sensitization inhibits the hyperpolarization-activated cation current Ih and reduces cell size in dopamine neurons of the ventral tegmental area

Abstract

The progressive augmentation of motor activity that results from repeated cocaine administration is termed behavioral sensitization. This phenomenon is thought to be a critical component in compulsive drug taking and relapse. Still, the cellular mechanisms that underlie sensitization remain elusive. Cocaine abuse, nonetheless, is known to evoke neuroplastic adaptations in dopamine (DA) neurotransmission originating from the midbrain's ventral tegmental area (VTA). Here, we report that concomitant with the development of locomotor sensitization to cocaine the hyperpolarization-activated cation current (I(h)) amplitude is depressed by ∼40% in VTA DA cells. Such effect did not result from a negative shift in I(h) voltage dependence. Nonstationary fluctuation analysis indicates that this inhibition was caused by an ∼45% reduction in the number of h-channels with no change in their unitary properties. The cocaine-induced I(h) depression was accompanied by a reduction in cell capacitance of similar magnitude (∼33%), leaving h-current density unaltered. Two implications follow from these data. First, I(h) inhibition may contribute to cocaine addiction by increasing bursting probability in DA cells and this effect could be intensified by the decrease in cell capacitance. Second, the cocaine-induced diminution of DA cell capacitance may also lead to reward tolerance promoting drug-seeking behaviors.

Fig. 1.

Repeated exposure to cocaine leads to the development of locomotor sensitization. A: 7-day cocaine (15 mg/kg) ip injection protocol results in locomotor sensitization. Rats were first placed in the recording cages for 15 min to allow for habituation, and data (means ± SE) were recorded at 5-min intervals. Then they were immediately injected with saline solution (0.9%) or cocaine, and motor activity was captured at 10-min intervals for 60 min. The motor activity measured at each time interval represents the sum of horizontal + stereotype activities that each animal executed during interval completion. Asterisks are for the comparison between days 1 and 7 of cocaine animals: 1-way ANOVA: F(15,128) = 2.84, P < 0.001; post hoc comparison: Newman-Keuls multiple comparison test. B: summary bar graph showing that total activity on day 7 is significantly different from that of day 1 in cocaine-treated animals. Similarly, on day 1 total activity is statistically different between saline and cocaine animals. Here total activity represents the sum of the activity measured at each particular time interval shown in A. Data (means ± SE) were analyzed by unpaired t-tests. **P < 0.01, ***P < 0.001.

Fig. 2.

Cocaine sensitization leads to the reduction of the hyperpolarization-activated cation current (I_h) in ventral tegmental area (VTA) dopamine (DA) neurons. A₁: representative voltage-clamp traces showing the effects of cocaine sensitization onto I_h of VTA DA neurons. As shown, I_h amplitude is defined as the difference between the steady-state current (I_ss) and the instantaneous current (I_ins). A₂: summary bar graph of the example presented in A₁ showing that cocaine sensitization reduces I_h amplitude. B: I_h current amplitudes increase with membrane hyperpolarization; however, this behavior is clearly decreased after cocaine sensitization. C: analysis of I_h electric charge transfer when cells were voltage clamped at −130 mV (duration: 1 s) also reveals that cocaine sensitization significantly reduces I_h amplitude. D: I_ins was not altered in cocaine animals relative to saline animals. E: I_ss amplitude was decreased in cocaine animals, in contrast to saline animals. F: analysis of I_ins electric charge transfer when cells were voltage clamped at −130 mV (duration: 1 s) also shows that cocaine sensitization does not alter I_ins. G: summary bar graph showing that cocaine sensitization does not alter input resistance (R_N) at −60 mV. **P < 0.01, saline vs. cocaine.

Fig. 3.

Reversal potential of I_h in VTA DA neurons is not altered by repeated cocaine exposure. A_1,2,3: example illustrating the voltage clamp procedure to determine I_h reversal potential (E_h) through tail current analysis in VTA DA neuron of a cocaine-treated rat. To avoid contamination of I_h tail currents (I_tail) with other currents, these experiments were performed in the presence of TTX (1 μM), TEA (2 mM), 4-aminopyridine (2 mM), and MgCl₂ (20 mM) in artificial cerebrospinal fluid (ACSF). B: summary plot of the relationship between the I_h tail current amplitude and the command potential for saline animals. C: summary plot of the relationship between the I_h tail current amplitude and the command potential for cocaine animals. D: bar graph showing that E_h was not altered in cocaine animals compared with saline animals.

Fig. 4.

Cocaine sensitization decreases I_h conductance in VTA DA neurons without any effect on its voltage- and time dependence. A: cocaine sensitization results in a significant decrease of I_h maximal conductance (G_h,max; at −130 mV). B: I_h conductance (G_h) increases with membrane hyperpolarization; nonetheless, this behavior is visibly decreased after cocaine sensitization. C: activation curves of saline animals vs. cocaine animals showing that cocaine sensitization does not lead to shifts in I_h voltage dependence. D: as seen in C, the potential of half-activation (V_1/2) remains unaffected after cocaine sensitization. E: cocaine sensitization does not alter the steepness of I_h voltage dependence, i.e., slope factor. F: the activation rate (τ_activation) of I_h at −130 mV remains unaltered after cocaine sensitization. G: I_h activation rate decreases with membrane hyperpolarization; evidently, this behavior is not affected by cocaine sensitization. Current traces between −100 mV and −70 mV were not analyzed since time dependence at these test potentials was not exponential for all cells. **P < 0.01, saline vs. cocaine.

Fig. 5.

Repeated cocaine exposure decreases the number of I_h channels in VTA DA neurons with no change in single-channel properties. A and B: examples of nonstationary noise analysis of I_h current for saline and cocaine animals, respectively. Gray curves represent parabolic fits to the data points. C: noise analysis reveals that the number of I_h channels is significantly decreased after repeated cocaine injections. D–F: noise analysis shows that I_h unitary current (i_h), single-channel conductance (γ), and open probability (P_o) are not altered after repeated cocaine injections. **P < 0.01, saline vs. cocaine.

Fig. 6.

Repeated cocaine exposure reduces the membrane capacitance of VTA DA neurons; however, the I_h current density remains unaltered. A: examples illustrating cell membrane capacitance (C_m) analysis for saline and cocaine animals. The first 40 ms of the capacitive transients were fitted with a sum of 2 exponential functions (not shown). The time constant (τ_fast) of the fastest exponential term was used to estimate C_m according to the following expression: C_m = τ_fast[1/R_S + 1/R_N], where R_S is the series resistance and R_N is the input resistance. B: C_m is significantly decreased after repeated cocaine injections. C: I_h current density is not affected after repeated cocaine injections. **P < 0.01, saline vs. cocaine.