In vivo pharmacological manipulation of small conductance Ca(2+)-activated K(+) channels influences motor behavior, object memory and fear conditioning

Abstract

Small conductance Ca(2+)-activated K(+) channels (SK, K(Ca2.1), K(Ca2.2), K(Ca2.3)) are expressed at high levels in brain regions critical for learning and memory. The activation of dendritic SK channels limits the induction of synaptic plasticity that may underlie hippocampal and amygdala dependent memory. EBIO facilitates SK channel activation by increasing their sensitivity to calcium. The compound CyPPA selectively activates SK2 and SK3 channels in a similar manner. To date there has been no report of the effects of SK channel activators on memory. Therefore, the present study examined the effects of systemic EBIO on mice in a behavioral task battery. Significant effects of EBIO on memory and motor activity were validated and extended by examining the effects of systemic CyPPA. Systemic EBIO and CyPPA both produced a transient decline in locomotor behavior. Neither SK channel activator affected anxiety. EBIO (17.5 mg/kg) impaired the encoding, but not retrieval, of object memory in a spontaneous object recognition task. A similar impairment of object memory encoding was observed in CyPPA (15 mg/kg)-treated mice. These memory-impairing effects were not due to changes in motivation, attention or movement. Systemic EBIO did not affect contextual or cued fear memory after conditioning with a 3 tone (CS)-footshock (US) pairing protocol or a 1 CS-US pairing protocol. Interestingly, apamin (0.4 mg/kg) enhanced contextual fear memory in mice conditioned with a 1 CS-US pairing protocol. These results suggest that SK channel activation impairs the encoding of non-aversive memory but not memory for aversive events. These data support converging evidence that SK channels regulate cellular mechanisms of memory encoding.

Figure 1

Activation of SK channels with systemic EBIO depresses locomotor responding in C57BL/6NHsd mice. (A) Dose-dependent effect of EBIO on mean distance traveled in the 60 min period in the open field expressed as cm traveled per 5-min time bin. Post-hoc Tukey HSD tests revealed that mice treated with EBIO exhibited a significant decrease in distance traveled during the first 15 min of the session. Exploratory motor activity recovered to the level of the 1% DMSO vehicle group by 20 min in the 10 and 17.5 mg/kg EBIO-treated mice and by 30 min in the 25 mg/kg EBIO-treated mice. (B) Dose-dependent effect of EBIO on immobility over the 60 min period expressed as percent of time deemed immobile by the Ethovision software per 5-min time bin. Post-hoc Tukey HSD tests revealed that measures of immobility recovered to vehicle levels within 20 min for the 10 and 17.5 mg/kg dose groups and by 30 min for the 25 mg/kg dose group. Shaded region depicts the 20 min post-injection interval over which all doses of EBIO impaired exploratory motor behavior in mice. In light of this effect, a 20 min delay was imposed after EBIO administration and before behavioral testing for all subsequent experiments. Error bars are SEM. *, P < 0.05 vs. vehicle; #, P < 0.05 vs. 10 mg/kg EBIO; and $, P < 0.05 vs. 17.5 mg/kg EBIO. Vehicle, n = 8; 10 mg/kg EBIO, n = 8; 17.5 mg/kg EBIO, n = 8; 25 mg/kg EBIO, n = 7.

Figure 2

Activation of SK channels with systemic CyPPA depresses locomotor responding in C57BL/6NHsd mice. (A) Effect of CyPPA (15 mg/kg) on mean distance traveled during the 60 min period in the open field. Student’s t-test analysis determined locomotor depression of CyPPA to return to that of 5% Cremophor vehicle levels by 35 min. (B) Effect of CyPPA on immobility over the 60 min period. Students t-test determined that measures of mobility returned to vehicle levels by 35 min. Shaded region depicts the 30 min post-injection interval over which CyPPA impaired locomotor behavior in mice. In light of this effect, a 30-min delay was imposed after CyPPA administration and before behavioral testing for all subsequent experiments. Error bars are SEM. *, P < 0.05 vs. vehicle. Vehicle, n = 8; 15 mg/kg CyPPA, n = 8.

Figure 3

Activation of SK channels with systemic EBIO (17.5 mg/kg) or CyPPA (15 mg/kg) impaired object memory encoding (A) Mice treated with EBIO (n = 10) before the sample session exhibited significantly less novel object preference during the test session 24 h later compared to vehicle-treated mice (n = 12), as measured by the novel object preference ratio; (B) and the discrimination ratio. Both novel object exploration ratios were significantly different from chance for vehicle mice (both P < 0.001), but non-significantly different from chance for EBIO mice (both P > 0.1). (C) A naïve cohort of mice received a vehicle injection before the sample session and then an injection of EBIO (n = 7) or vehicle (n = 8) prior to the test session 24 h later. Vehicle- and EBIO-treated mice exhibited equivalent preference for the novel object as measured by the novel object preference ratio and (D) the discrimination ratio. Both vehicle and EBIO groups demonstrated novel object exploration ratios significantly above chance (all P < 0.01). (E) A final cohort of naïve mice received an injection of CyPPA (n = 8) or 5% Cremophor vehicle (n = 10) before the sample session. CyPPA-treated mice exhibited significantly less novel object preference during the test session as measured by novel object preference ratio; (F) and the discrimination ratio. Both novel object exploration ratios were significantly above chance for the vehicle (both P < 0.01) mice but not significantly different from chance for the CyPPA mice (both P > 0.1). Error bars are SEM. *, P < 0.05, vs. vehicle; n.s., ratio not significant from chance exploration (P > 0.05). Dashed line at 0.5 of A, C and E represents chance performance or a lack of discrimination between the novel and familiar object.

Figure 4

Activation of SK channels with EBIO (17.5 mg/kg), or blockade of SK channels with apamin (0.4 mg/kg) did not affect the encoding of contextual or cued fear memory during conditioning with a 3 CS-US pairing protocol. Mice that received EBIO (n = 23) or vehicle (n = 23) before conditioning exhibited equivalent freezing during (A) the context test 24 h later, and during (B) the tone test. Mice that received apamin (0.4 mg/kg, n = 10) or saline (n = 10) before conditioning with 3 CS-US pairs also exhibited equivalent freezing during (C) the context test 24 h later, and during (D) the tone test. Error bars are SEM.

Figure 5

Activation of SK channels with EBIO (17.5 mg/kg) did not affect the encoding of contextual or cued fear memory after conditioning with a 1 CS-US pairing protocol. However blockade of SK channels with apamin (0.4 mg/kg) did enhance the encoding of contextual fear memory after the 1 CS-US pairing protocol. Mice that received EBIO (n = 9) or vehicle (n = 8) before conditioning exhibited equivalent freezing during (A) the context test 24 h later, and during (B) the tone test. Mice that received apamin (0.4 mg/kg, n = 11) before conditioning exhibited significantly greater freezing during (C) the context test 24 h later compared to vehicle-treated mice (n = 9). There were no treatment effects on tone-elicited freezing during (D) the tone test. Error bars are SEM. *, P < 0.05 vs. vehicle.