The relationship between duration of initial alcohol exposure and persistence of molecular tolerance is markedly nonlinear

Abstract

The neuronal calcium- and voltage-activated BK potassium channel is modulated by ethanol, and plays a role in behavioral tolerance in vertebrates and invertebrates. We examine the influence of temporal parameters of alcohol exposure on the characteristics of BK molecular tolerance in the ventral striatum, an important component of brain reward circuitry. BK channels in striatal neurons of C57BL/6J mice exhibited molecular tolerance whose duration was a function of exposure time. After 6 h exposure to 20 mm (0.09 mg%) ethanol, alcohol sensitivity was suppressed beyond 24 h after withdrawal, while after a 1 or 3 h exposure, sensitivity had significantly recovered after 4 h. This temporally controlled transition to persistent molecular tolerance parallels changes in BK channel isoform profile. After withdrawal from 6 h, but not 3 h alcohol exposure, mRNA levels of the alcohol-insensitive STREX (stress axis-regulated exon) splice variant were increased. Moreover, the biophysical properties of BK channels during withdrawal from 6 h exposure were altered, and match the properties of STREX channels exogenously expressed in HEK 293 cells. Our results suggest a temporally triggered shift in BK isoform identity. Once activated, the transition does not require the continued presence of alcohol. We next determined whether the results obtained using cultured striatal neurons could be observed in acutely dissociated striatal neurons, after alcohol administration in the living mouse. The results were in remarkable agreement with the striatal culture data, showing persistent molecular tolerance after injections producing 6 h of intoxication, but not after injections producing only 3 h of intoxication.

Figure 1.

Conductance, voltage dependence, and pharmacology of BK channels in cultured striatal neurons. A, Digitally captured image of rat P8 striatal neurons 1 week in culture. B, Time spent in the open state (inside-out configuration; 10 μm free Ca²⁺) increases as the membrane is depolarized. C and O represent the closed and open states, respectively. C, Plot of BK channel current amplitude as a function of membrane potential. A linear fit of this relationship (r = 0.99) gave a BK channel unitary conductance of 230 pS. D, Pharmacological dissection of the macroscopic potassium current in striatal neurons. Macroscopic currents were evoked by stepping from a holding potential of −60 mV to +80 mV. The largest trace is the total current, recorded in normal Locke's solution containing 2.2 mm calcium. Addition of the I_A channel inhibitor 4-AP (1 mm) removes the fast inactivating I_A. Perfusion with 100 nm Ibtx specifically blocks BK channels leaving a 4-AP- and Ibtx-resistant current. Finally, addition of 100 mm TEA-Cl, a blocker of voltage-dependent potassium channels, leaves a small residual current (smallest trace). In all subsequent figures, I_A is subtracted from the macroscopic current by inclusion of 1 mm 4-AP.

Figure 2.

Persistence of acute tolerance is a function of ethanol exposure time. A–C, BK channel activity (NP_o) in cell-attached patches before and after challenge with 50 mm EtOH in neurons that were preexposed to 20 mm EtOH for 3 h and with ethanol withdrawn for 4 h (A), preexposed to 20 mm EtOH for 6 h and with ethanol withdrawn for 4 h (B), or preexposed to 20 mm EtOH for 6 h and with ethanol withdrawn for 24 h (C). All traces were recorded at a holding potential of −60 mV. C and O represent the closed and open state, respectively. D, Plot of aggregate single-channel recording data. Note: In D, a value of 1 on the ordinate represents maximal tolerance, while higher numbers represent recovery from tolerance (i.e., return of potentiation).

Figure 3.

Six, but not 3 h of 20 mm EtOH induces an upregulation of STREX mRNA during withdrawal. mRNA levels in cultured striatal neurons were measured by real-time PCR with primers to α-STREX and to the β1 subunit. Immediately after 6 h of exposure to 20 mm EtOH, STREX mRNA increased approximately threefold compared with control. Bar graphs represent the mean ± SEM for six independent experiments performed in triplicate. Statistical significance was determined using one-way ANOVA; *p < 0.05.

Figure 4.

Kinetic properties of BK channels differ from control during withdrawal from a 6 h, but not a 3 h exposure to 20 mm EtOH. A–C, A series of six consecutive BK traces evoked by depolarizing channels that were naive (A), preexposed to 20 mm EtOH for 3 h and with ethanol withdrawn for 24 h (B), or preexposed to 20 mm EtOH for 6 h and with ethanol withdrawn for 24 h (C). Channel activity was recorded in an outside-out patch with 10 μm free Ca²⁺ in the recording electrode. Cumulative current was compiled from 100 single-channel traces. Current activation was best fit with a single exponential. The use of different voltage steps used in A and B versus C reflects the need to maintain constant P_o values in light of the shift in the P_o–V curve after transition to PMT. Compiled currents shown in D–F were obtained at the same potentials as the traces shown in A–C.

Figure 5.

There is a leftward shift in G–V relationship present after withdrawal from a 6 h but not 3 h exposure to 20 mm EtOH. Plots of G–V relationship for macroscopic currents obtained with 1 μm free Ca²⁺ (left graph), or 10 μm free Ca²⁺ (middle graph). Graph on right was obtained with 10 μm free Ca²⁺ in the recording pipette and 100 nm Ibtx in the bath. BK currents were evoked from a holding potential of −70 mV with +10 mV (left and middle graph) or +20 mV (right graph) incremental voltage steps. Macroscopic currents were measured at steady-state condition, 450–490 ms after the beginning of the voltage step. Bath contained 1 mm 4-AP to block I_A current.

Figure 6.

Mean open times (MOT) and closed times (MCT) are altered during withdrawal from a 6 h but not a 3 h exposure to 20 mm EtOH. A–C, Representative traces from single BK channels recorded in cell-attached patch-clamp mode from naive channels (A), and neurons preexposed to 20 mm EtOH for 3 or 6 h, and with ethanol withdrawn for 24 h (B, C). Channel activity was evoked by stepping from a holding potential of −60 mV to a potential of +70 mV for the traces recorded in A and B or to +50 mV for the traces recorded in C. The dwell time distributions of single-channel patches are shown in the histograms below the current traces. Open and closed times were computed with half-amplitude threshold analysis. Curves were fitted using a maximum-likelihood minimization routine. The open times distribution could be well fitted with a single-component function while the closed time distribution was well fitted with a double exponential.

Figure 7.

Biophysical properties of BK α splice variants exogenously expressed in HEK293 cells. A–C, Macroscopic BK currents: Insertless (A), ALCOREX (B), or STREX (C), evoked from a holding potential of −60 mV. D, G–V relationship of Insertless (circles), ALCOREX (triangles), and STREX (squares). E, F, Bar graphs of time constants of activation (E) and mean open time (F), for BK α isoforms in HEK cells and for BK in striatal neurons. All measurements in HEK cells were performed similarly to those previously described for neurons.

Figure 8.

Effect of 8-bromo-cAMP, a PKA activator, on striatal currents after various exposures to 20 mm EtOH. A–E, Macroscopic currents from neurons that are naive (A), EtOH exposed for 3 h (B), EtOH exposed for 3 h and withdrawn for 24 h (C), EtOH exposed for 6 h (D), and ethanol exposed for 6 h and withdrawn for 24 h (E). Traces are before (black) and after (gray) perfusion with 250 μm 8-bromo-cAMP. All macroscopic currents were recorded in the presence of 1 mm 4-AP, stepped from a holding potential of −60 mV to +80 mV, with 1 μm free Ca²⁺ in the recording pipette. F, Tabulation of the effects of 8-bromo-cAMP.

Figure 9.

In vivo EtOH injections mimic in vitro results. A, Blood alcohol concentrations in C57BL/6J mice: naive (no injection: n = 4), 1 h after the third injection of EtOH (n = 4), 3 h after the third injection (n = 3), and 1 h after the sixth EtOH injection (n = 4). One-way ANOVA, Tukey post hoc, ***p = 0.001. Hourly EtOH intraperitoneal injections were administered. The first injection (1.8 g/kg, i.p.) was followed by booster injections (1.2 g/kg, i.p.) every hour for two or five subsequent injections (see Results for further explanation). B, Effect of 50 mm EtOH challenge on BK channel activity in dissociated striatal neurons obtained from mice 24 h after in vivo alcohol exposure for either 3 or 6 h. There is significant molecular tolerance remaining after the 6 h exposure, but not after the 3 h exposure. Calculation of NP_o was performed with Igor Pro 6.1 Wave Metrics software. Statistical differences were determined using Student's t test: **p < 0.002.