BK β4 subunit influences tolerance: (A) αβ4 BK Single channel activity recorded at hyperpolarized potentials from HEK293 cells. (B) EtOH's effects on αβ4 BK channel activity averaged over several cells (n values indicated in graphs). (C) shows α BK single channel activity before (control) and during EtOH exposure. (D) Magnitude of EtOH's effects on α BK activity averaged over several cells (n values are indicated in graphs). Representative traces of BK activity before (control) and during EtOH exposure (EtOH). “C” and “O” refer to BK closed and open states, respectively. NPo indicates BK channel open probability under each experimental condition. In graphs (B and D) lightly shaded areas indicate where BK channel potentiation typically occurs, while the darker shaded areas show where tolerance is observed. EtOH effects are expressed as % of baseline.

EtOH-mediated decrease of MSN excitability exhibits tolerance in KO, but not WT mice. (A) Number of APs recorded from WT (filled columns) and KO (open columns) MSNs following a series of incremental (50 pA) current steps (50–300 pA). (B) Representative action potential trains evoked in a slice preparation (Slices) by a single 100 pA current step in WT (Left) and KO (Middle) mice before (control) and after 50 mM EtOH exposure (2 or 8 min). Two minutes after EtOH, the number of APs is smaller in both WT and KO mice. While KO mice MSN excitability partially recovers 8 min after EtOH exposure (Bottom Trace; Right), WT neuronal excitability remains depressed (Left, Bottom Trace). Results obtained in slices were reproduced on freshly isolated neurons from KO mice (Right; β4 KO/F.I. cells). (C) Averaged change in action potential number recorded in MSNs in slices and freshly isolated after 2 or 8 min EtOH exposure, presented as percent of control before EtOH exposure in MSNs from WT and β4 KO striatal slices; 5/7 neurons were ethanol sensitive and developed tolerance in β4 KO MSNs, whereas 7/9 MSNs from WT were ethanol sensitive and did not develop tolerance (*P < 0.05). The Inset shows the ratio of AP number at 2 and 8 min reported as fold recovery from ethanol; value below the broken bar indicates a further decrease of APs number at 8 min compared to 2 min EtOH (solid column; WT), while value above the line indicate a recovery (KO); F_1,10 = 27.6, P < 0.001. (D) 100 nM ChTx blocks EtOH effects on striatal MSNs AP patterns in slices (Left) and freshly isolated neurons (F.I cells, Right). (E) Number of APs measured every minute before (open circles) and during 50 mM EtOH exposure (solid circles) in presence of 100 nM ChTx. Data from slices and freshly isolated MSNs were combined.

EtOH consumption is higher in β4 KO. (A) EtOH consumption of WT (black squares) and β4 KO mice (white squares) using a restricted access single bottle self-administration assay. Measurements were taken every day for four days for 2 h after lights were turned off. (B) Average daily EtOH intake in male and female WT and KO mice. (C and D) Water, sucrose, and quinine intake of WT (black squares and circles) and KO (white squares and circles) mice, respectively (n = 5–8 per genotype). No significant difference was observed in either condition. One Way ANOVA, Tukey posthoc, * P < 0.05, ** P < 0.01, *** P < 0.001.

β4 subunit controls tolerance of BK single channel activity in freshly isolated striatal MSNs. (A) DNA agarose gel shows that only β4 expression is lacking in striatum isolated from β4 KO mice; whereas β1 mRNA is present in both WT and KO animals. “L” denotes the 100 bp marker on molecular weight ladder. Base pair number is indicated in the left hand margin. “B1” and “B4” refer to BK β1 and β4 subunits, respectively. “NORT” are negative controls with omitted reverse transcriptase and “X” indicates columns where no material was loaded. Single channel activity recorded from striatal MSNs acutely isolated from WT (B and C) or β4 KO (D and E) mice, respectively. (B and D) Representative traces of BK activity from WT and KO mice, before (control) and during EtOH exposure (EtOH). “C” and “O” refer to closed and open states. NPo indicates BK channel open probability. (C and E) graphs show magnitude of EtOH's effects averaged over several cells (n values are indicated in graphs). Lightly shaded areas indicate where BK channel potentiation typically occurs, while the darker shaded areas show where tolerance is observed. EtOH effects are expressed as a percentage of baseline.

Effects of EtOH on locomotor activity in male WT and β4 KO mice. (A) Ambulatory activity of WT mice 5, 10, and 15 min after a 2 g/kg i.p. EtOH injection on days 0, 1, and 4. Each data point represents 5 min summed activity. Asterisks above symbols indicate significant locomotor activity on both days 1 and 4 of EtOH injection at each time point (5, 10, and 15 min) compared to saline injection baseline (represented by the dashed line). Asterisk next to bracket indicates difference between days 1 and 4 at 15 min time point only. (B) Effects of the same EtOH dose at the same time points in KO mice. (C) Fifteen minute summed activity immediately after a daily ethanol injection. Note the rapid development of tolerance for KO mice upon repeated injections of 2 g/kg EtOH, compared to WT mice. (D) Blood alcohol concentration in WT and KO mice (n = 4) before (time 0) and at 30-min intervals after a single i.p. injection of 2 g/kg ethanol. One-way ANOVA, Tukey posthoc, * P < 0.05, *** P < 0.001.