The A-current modulates learning via NMDA receptors containing the NR2B subunit

Abstract

Synaptic plasticity involves short- and long-term events, although the molecular mechanisms that underlie these processes are not fully understood. The transient A-type K(+) current (I(A)) controls the excitability of the dendrites from CA1 pyramidal neurons by regulating the back-propagation of action potentials and shaping synaptic input. Here, we have studied how decreases in I(A) affect cognitive processes and synaptic plasticity. Using wild-type mice treated with 4-AP, an I(A) inhibitor, and mice lacking the DREAM protein, a transcriptional repressor and modulator of the I(A), we demonstrate that impairment of I(A) decreases the stimulation threshold for learning and the induction of early-LTP. Hippocampal electrical recordings in both models revealed alterations in basal electrical oscillatory properties toward low-theta frequencies. In addition, we demonstrated that the facilitated learning induced by decreased I(A) requires the activation of NMDA receptors containing the NR2B subunit. Together, these findings point to a balance between the I(A) and the activity of NR2B-containing NMDA receptors in the regulation of learning.

Figure 1. Kv4.2 expression and I_A activity are reduced in the hippocampus of dream^−/− mice.

Basal expression of kv4.2 mRNA (A) and Kv4.2 protein (B) in the hippocampus of wt (white bars) and dream^−/− (gray bars) mice. Gapdh mRNA and actin protein served as internal controls. (C) The two-step voltage clamp protocol used to activate whole-cell voltage-dependent K⁺ currents, and representative examples of the outward currents in CA1 pyramidal neurons from wt and dream^−/− mice. The first depolarizing step activated a mixed current (I_Total) with a transient A-type component (I_A; asterisks) and sustained non-inactivating K⁺ current (I_KDR). I_A was inactivated during the second depolarizing pulse (arrowheads) leaving only the sustained I_KDR. (D) Traces on the left show the superimposition of the sequential outward current responses (depolarizing pulse segments) illustrated in C. Traces on the right show I_A current traces calculated by subtracting I_KDR from I_Total for the same neurons. (E) Summary data comparing the peak current density (amplitude normalized to cell capacitance) of I_A and I_KDR in wt and mutant neurons (n = 30 and 23 neurons from 6 mice of each genotype, respectively). * p<0.05, ** p≤0.01.

Figure 2. The reduction of I_A facilitates learning in the object recognition test.

(A, B) The object recognition memory test was performed using a 5 min training session in wt (white bars) and dream^−/− (gray bars) mice. Discrimination indices during short-term memory (STM, A) and long-term memory (LTM, B) sessions (1 and 24 h after training, respectively) are shown. Administration of anisomycin (aniso, black pointed bars) before the training sessions blocked the facilitation of LTM in dream^−/− mice. (C, D) Administration of 4-AP before the training sessions facilitated short-term memory (STM) in wt mice. Discrimination indices during STM (C), and LTM (D) sessions are shown. n = at least 8 per group. ** p<0.01.

Figure 3. Kv4.2 channel blockade induces alterations in basal oscillatory hippocampal activity.

(A) Power spectra of hippocampal local field activity recorded from the CA1 pyramidal layer of wt, dream^−/− and 4-AP-treated wt mice during exploratory behavior. Two seconds of the basal electrocorticogram recordings are also shown. (B) Relative spectrum quantification (mean ± SEM) in the low theta and theta ranges for wt, dream ^−/− and 4-AP-treated mice. n = 6 per group. * p≤0.05, ** p≤0.01.

Figure 4. Reduced I_A decreases the stimulus threshold for short-term synaptic plasticity.

(A) A single high-frequency stimulation (HFS, five trains, 200 Hz, 100 ms, at a rate of 1/s) evoked LTP at the CA3-CA1 synapse, lasting for up to 2 h in dream^−/− mice (gray symbols). Anisomycin administration (white symbols) before HFS reduced the extension of LTP. A summary of the percent change in the fEPSP slope (mean ± SEM) and representative recordings at different times after a single HFS train in wt and dream^−/− mice, in the presence or absence of anisomycin, is shown. (B) Effect of 4-AP administration on high-frequency stimulation (HFS) in wt mice. 4-AP administration evoked lasting short-term potentiation in wt mice. A summary of the changes in the fEPSP slope (mean ± SEM) and representative recordings at different times after a single HFS pulse in vehicle and 4-AP-treated mice is shown. n = 6 per group. *p≤0.05, ** p≤0.01.

Figure 5. The facilitation of learning induced by decreased I_A is mediated by NR2B-containing NMDA receptors.

The object recognition memory test was performed with a 5 min training session in dream^−/− mice pretreated with Ro26-6981 or the vehicle alone (left graph, white and black bars, respectively), and in mice pretreated with 4-AP-treated in the presence or absence of Ro25-6981 (right graph). The discrimination indices during short-term memory (STM) are shown. n = at least 8 per group. * p≤0.05, *** p≤0.001.