G protein-activated inwardly rectifying potassium channels mediate depotentiation of long-term potentiation

Abstract

Excitatory synapses in the brain undergo activity-dependent changes in the strength of synaptic transmission. Such synaptic plasticity as exemplified by long-term potentiation (LTP) is considered a cellular correlate of learning and memory. The presence of G protein-activated inwardly rectifying K⁺ (GIRK) channels near excitatory synapses on dendritic spines suggests their possible involvement in synaptic plasticity. However, whether activity-dependent regulation of GIRK channels affects excitatory synaptic plasticity is unknown. In a companion article we have reported activity-dependent regulation of GIRK channel density in cultured hippocampal neurons that requires activity of NMDA receptors (NMDAR) and protein phosphatase-1 (PP1) and takes place within 15 min. In this study, we performed whole-cell recordings of cultured hippocampal neurons and found that NMDAR activation increases basal GIRK current and GIRK channel activation mediated by adenosine A₁ receptors, but not GABA_B receptors. Given the similar involvement of NMDARs, adenosine A₁ receptors, and PP1 in depotentiation of LTP caused by low-frequency stimulation that immediately follows LTP-inducing high-frequency stimulation, we wondered whether NMDAR-induced increase in GIRK channel surface density and current may contribute to the molecular mechanisms underlying this specific depotentiation. Remarkably, GIRK2 null mutation or GIRK channel blockade abolishes depotentiation of LTP, demonstrating that GIRK channels are critical for depotentiation, one form of excitatory synaptic plasticity.

Synaptic plasticity, the ability of neurons to modify the efficacy of synaptic transmission, is thought to provide the cellular basis for the profound influence of experience over information processing and storage in the brain (1, 2). For example, long-term potentiation (LTP), a long-lasting increase in excitatory synaptic strength after heightened synaptic activity (3), is believed to be the cellular correlate of learning and memory. Since the discovery of LTP in the hippocampus, a region known to be essential for learning and memory, LTP has been extensively characterized for the molecular mechanisms of its induction and expression that involves Ca²⁺ influx through NMDA receptors (NMDARs), leading to a high level of intracellular Ca²⁺ concentration, and activation of calcium/calmodulin-dependent kinase II (CaMKII) (3).

Whether the excitatory synaptic activities generated by high-frequency stimulation (HFS) result in LTP depends on the pattern of synaptic inputs impinging on the postsynaptic CA1 neurons shortly afterward; LTP of field excitatory postsynaptic potential (fEPSP) of CA1 neurons fails to develop if the HFS of the Schaffer collateral nerve fibers is followed within minutes by low-frequency stimulation (LFS) (4, 5). This form of synaptic plasticity, called depotentiation, may be a mechanism to abort the LTP according to events that take place immediately after the signals for LTP induction. Together with long-term depression, depotentiation may contribute to plasticity mechanisms that prevent the saturation of synaptic potentiation and increase the flexibility and storage capacity of neuronal circuits. Like LTP, depotentiation is input-specific and depends on NMDAR activation (6). Extensive studies of depotentiation have revealed an essential role for A₁ receptors activated by ambient adenosine and a requirement for the activity of protein phosphatase-1 (PP1) but not protein phosphatase-2B (PP2B) (4, 6). Because A₁R is coupled to Gi/o that can inhibit adenylate cyclase to reduce protein kinase A (PKA) activity (7), it has been suggested that extracellular adenosine mediates depotentiation by activating A₁ receptor-dependent inhibition of PKA, thereby inducing dephosphorylation of AMPA receptor subunit GluR1 (6). However, this hypothesis has not been tested experimentally, and it remains an open question whether the A₁ receptor- and PP1-dependent depotentiation involves any ion channels other than glutamate receptors.

G protein-activated inwardly rectifying K⁺ (GIRK) channels regulate neuronal excitability by mediating inhibitory effects of G protein-coupled receptors (GPCRs) for neurotransmitters and neuromodulators, including GABA_B receptors and adenosine A₁ receptors (8). In the study reported in a companion article (9), we have demonstrated that synaptic NMDAR activation increases surface expression of GIRK channels by stimulating PP1- but not PP2B-dependent dephosphorylation of GIRK2 subunit, thereby enhancing their delivery from recycling endosomes to the plasma membrane. In this study, we show that the same protocol for inducing synaptic NMDAR activation in cultured hippocampal neurons also elevated basal GIRK current and GIRK channel activation induced by adenosine A₁ receptors but not GABA_B receptors, suggesting that NMDAR-induced GIRK surface expression may lead to increased GIRK channel activity. Because activity-dependent GIRK surface expression in cultured hippocampal neurons involves NMDARs, adenosine A₁ receptors, and PP1, the same molecular players implicated in depotentiation in hippocampal slices (4, 6), we hypothesized that GIRK channels may mediate depotentiation by serving as a downstream target of A₁ receptors and PP1. Indeed, GIRK2 knockout mouse hippocampal slices or wild-type hippocampal slices treated with the GIRK channel blocker tertiapin (TP) failed to display depotentiation of LTP of fEPSP, revealing a critical role of GIRK channels in excitatory synaptic plasticity.

Results

NMDAR Activation Increases Basal GIRK Current in Cultured Hippocampal Neurons.

In the companion article (9), we demonstrate that NMDAR activation in cultured hippocampal neurons increases surface expression of GIRK channels by stimulating PP1-dependent dephosphorylation of GIRK2 Ser-9, thereby promoting Rme1-dependent transport of GIRK2-containing channels from recycling endosomes to the plasma membrane. To determine whether NMDAR-induced GIRK surface expression leads to increased GIRK channel activity, we measured basal GIRK current by performing electrophysiological recording of cultured hippocampal neurons (10). After exposure to 2-amino-5-phosphonovaleric acid (APV) for 3–4 days [days in vitro (DIV) 11–14], cultured hippocampal neurons displayed little action potential firing (Fig. 1A) and very small TP-sensitive basal GIRK current (Fig. 1B) in the control artificial cerebrospinal fluid (ACSF) bathing solution containing APV. Replacing this bath solution with the fresh bathing solution without APV so as to activate synaptic NMDARs by synaptically released glutamate (9) immediately caused multiple action potentials, followed with high-frequency burst discharges (Fig. 1A) and increased basal GIRK current by ≈4-fold in 10 min, from 0.14 ± 0.06 pA/pF to 0.50 ± 0.11 pA/pF (P < 0.05; Fig. 1 B–D). In contrast, the basal GIRK current (0.20 ± 0.1 pA/pF) did not significantly change when we replaced the APV-containing bath solution with fresh bath solution still containing APV (0.23 ± 0.1 pA/pF, APV control). Thus, the NMDAR-induced GIRK channel surface expression was accompanied with an increase in basal GIRK current.

Fig. 1.

NMDAR activation increases basal GIRK current in cultured hippocampal neurons. (A) An example of action potential firing in a hippocampal neuron recorded before and after removal of APV. (B) An example of TP-sensitive basal GIRK current before and after removal of APV (10 min). Representative traces of whole-cell current (Middle) with (red) or without (black) 120 nM TP, were obtained when the membrane potential was stepped to −120 mV for 100 ms from the holding potential of −70 mV (Top). (Bottom) TP-sensitive current obtained by digital subtraction. (C) Representative basal GIRK current amplitude as a function of time. Whereas the current remained constant in size after control solution change (control; ○) to fresh bath solution containing APV, the basal GIRK current increased in amplitude after removal of APV to activate synaptic NMDAR (●). (D) Quantification of basal GIRK current density (pA/pF) recorded from the same neurons before and after removal of APV (n = 5; *, P < 0.05).

NMDAR Activation Increases GIRK Current Stimulated by Adenosine A₁ Receptors but Not GABA_B Receptors in Cultured Hippocampal Neurons.

Because GIRK channels mediate postsynaptic potassium current upon stimulation of GABA_B receptors and adenosine A₁ receptors in hippocampal neurons (11), we next examined whether NMDAR-induced GIRK surface expression is accompanied with any increase in GIRK channel activation by GPCRs. In dissociated hippocampal culture each pyramidal neuron tested under APV control condition yielded an outward GIRK current of a different amplitude in response to the adenosine A₁ receptor agonist 2-chloro-N6-cyclopentyl adenosine (CPA) at 5 μM (2–80 pA) or adenosine at 100 μM (4–20 pA), or the GABA_B receptor agonist baclofen at 50 μM (15–70 pA) while the neuron was held at −50 mV. Most of the adenosine and CPA responses were smaller than the baclofen responses, consistent with previous observations (Fig. 2 A and B) (11, 12).

Fig. 2.

NMDAR activation increases GIRK current stimulated by adenosine A₁ receptors but not GABA_B receptors in cultured hippocampal neurons. (A) Sample traces of outward GIRK currents recorded at −50 mV from the same neuron in dissociated hippocampal culture before and 15 min after removal of APV. The GIRK current was induced by adenosine A₁ receptor agonist, CPA (5 μM), adenosine (100 μM), and GABA_B receptor agonist, baclofen (50 μM). (B) The outward GIRK currents (pA) induced by CPA, adenosine, and baclofen, with lines connecting the measurements of the currents recorded from the same neuron before and after removal of APV for 15 min. (C) Normalized outward GIRK current induced by CPA (n = 11), adenosine (n = 5), and baclofen (n = 11) before and after removal of APV. The agonist-induced current after removal of APV was normalized to that before removal of APV for each neuron. *, P < 0.05; ***, P < 0.001.

We then removed APV while recording from the same neuron and examined the agonist-induced GIRK current after 15 min of synaptic NMDAR activation (Fig. 2 A and B). Whereas individual neurons displayed large variability in the absolute current amplitudes (Fig. 2B), normalizing the agonist-induced current after removal of APV by the agonist-induced current before removal of APV for each neuron revealed that synaptic NMDAR activation significantly increased GIRK current evoked by CPA (153% ± 10% of control response before NMDAR activation, paired t test P < 0.005) and adenosine (176% ± 24%, paired t test P < 0.05) but not baclofen (89% ± 15%, paired t test P > 0.05) (Fig. 2C). This specific effect on A₁ receptor-induced GIRK current may indicate that synaptic NMDAR activation in cultured hippocampal neurons selectively increased GIRK channels that are coupled to A₁ receptors but not GABA_B receptors, because GIRK channel activation requires its physical association with multiple Gβγ subunits that diffuse in a membrane delimited fashion from nearby receptors in the same microdomain or macromolecular complex as the GIRK channel (13). It is also possible that NMDAR activation affects not only GIRK channel surface expression but also the surface expression of A₁ receptors or the coupling between the GIRK channels and the adenosine A₁ receptors.

GIRK Channels Are Required for Depotentiation of LTP.

Depotentiation of LTP of fEPSP in rat hippocampus has been shown to require activity of NMDARs, adenosine A₁ receptors, and PP1 (4, 6) (Fig. 3). Because we found that synaptic NMDAR activation not only increases GIRK surface expression in a PP1-dependent pathway (see ref. 9) but also enhances A₁ receptor-induced GIRK current (Fig. 2), we wondered whether NMDAR-dependent regulation of GIRK channels may contribute to depotentiation by enhancing GIRK channel activation by A₁ receptors. To test whether depotentiation requires GIRK channel function, we stimulated Schaffer collaterals and performed extracellular recording of fEPSP in CA1 stratum radiatum of hippocampal slices from the wild-type and GIRK2 knockout mice (14). After establishing a stable baseline of fEPSP, we applied HFS (100 Hz for 1 s, twice) and found that the HFS induced LTP in hippocampal slices from not only wild-type (154.5% ± 10.1% of baseline fEPSP slope, 30 min after the onset of HFS, P < 0.005; Figs. 4 A and C and 5C) but also GIRK2 null mice (138.7% ± 5.1%, P < 0.005; Figs. 4 B and C and 5C). When LFS (2 Hz for 10 min) was delivered within 1–2 min of HFS, it caused depotentiation and thus prevented LTP expression in wild-type hippocampal mouse slice (103.0% ± 6.0% of baseline fEPSP slope, 30 min after the onset of LFS) (Figs. 4 D and F and 5C), as reported in rat hippocampal slices (4, 6). This depotentiation was blocked by treating wild-type slices with A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (DP, 100 nM) during LFS (134.8% ± 12.7%, n = 4, P < 0.05; Fig. 5 A and C), confirming the role of A₁R in depotentiation (6). In contrast, LFS caused no depotentiation in GIRK2 null hippocampal slice (137.9% ± 7.1%, P < 0.005; Figs. 4 E and F and 5C), or wild-type hippocampal slices treated with the GIRK channel blocker TP (50 nM, 144.8% ± 21.7%, P < 0.05; Fig. 5 B and C), revealing a requirement of GIRK channels in depotentiation of LTP.

Fig. 3.

Involvement of NMDAR, PP1, and adenosine A₁ receptor for both GIRK channel regulation in cultured hippocampal neurons and depotentitation of LTP in hippocampal slices. (A) In cultured hippocampal neurons, NMDAR activation increases GIRK surface expression by stimulating PP1-dependent dephosphorylation of the GIRK channel GIRK2 subunit, thereby increasing insertion of GIRK channels from recycling endosomes to plasma membrane (see ref. 9). NMDAR activation also increases GIRK current stimulated by adenosine A₁ receptors but not GABA_B receptors (Fig. 2). (B) LFS delivered within minutes of HFS diminishes the propensity for LTP of fEPSP induced by HFS, leading to depotentiation. Such LFS-induced depotentiation requires the activity of NMDARs, A₁ receptors, and PP1 (4, 6).

Fig. 4.

GIRK2 null mutant mice failed to display depotentiation of LTP of fEPSP. (A and B) Example of LTP of fEPSP at the Schaffer collateral–CA1 synapses induced by HFS (100 Hz for 1 s, twice) in a hippocampal slice from the wild-type mice (+/+, black open circle, A) or GIRK2 knockout mice (−/−, red open circle, B). Sample field potential responses taken at the time points indicated as 1 and 2 are shown above. (C) Summary of LTP induced by HFS in hippocampal slices from wild-type mice (+/+, black open circle, n = 5 from 4 mice) and GIRK2 knockout mice (−/−, red open circle, n = 4 from 3 mice). (D) Example of depotentiation induced by LFS (2 Hz for 10 min applied within 1–2 min of HFS) in a hippocampal slice from the wild-type mice (+/+, black open circle). (E) No depotentiation was induced by LFS in a hippocampal slice from GIRK2 knockout mice (−/−, red open circle). The field potential recordings at the time points indicated as 1 and 2 are shown above in D and E. (F) Summary of LFS-induced depotentiation in the wild-type mice (+/+, black open circle, n = 11 from 8 mice) and the absence of LFS-induced depotentiation in GIRK2 knockout mice (−/−, red open circle, n = 6 from 3 mice). LTP persisted in hippocampal slices from GIRK2 null mutant mice but not wild-type mice even after HFS was followed by LFS. (Scale bars: 0.5 mV, 20 ms.)

Fig. 5.

GIRK channel activity is required for depotentiation of LTP. (A) LFS failed to induce depotentiation in a wild-type mouse hippocampal slice exposed to the A₁ receptor antagonist DPCPX (DP, 100 nM). (B) LFS also failed to induce depotentiation in a wild-type mouse hippocampal slice treated with the GIRK channel blocker TP (50 nM). (C) Quantification of the extent of fEPSP potentiation after HFS that induced LTP in both wild-type and GIRK2 null mutant hippocampal slices (LTP) or HFS followed by LFS that resulted in depotentiation in wild-type hippocampal slices (Depotentiation). The fEPSP slope after stimulation is normalized with the fEPSP slope before stimulation to yield the %fEPSP slope. Comparison of hippocampal slices from wild-type (+/+, grey), GIRK2 knockout mutant (−/−, red), and the wild-type hippocampal slices treated with the GIRK channel blocker TP (+/+, 50 nM, n = 7 from 4 mice) or the A₁ receptor antagonist DPCPX (+/+, 100 nM, n = 4 from 3 mice) reveals that depotentiation requires both A₁ receptor and GIRK channel function. ***, P < 0.005; *, P < 0.05.

Discussion

Implications for Neuronal Excitability.

In this study we demonstrate that the same paradigm for synaptic NMDAR activation that increases GIRK channel density within 15 min in a PP1- but not PP2B-dependent manner, as reported in ref. 9, also increased the basal GIRK current within 15 min of APV withdrawal. Interestingly, synaptic NMDAR activation also increased GIRK channel activation by adenosine A₁ receptors, but not GIRK channel activation by GABA_B receptor. We cannot exclude the possibility that the lack of effect of NMDAR on GIRK channel activation by GABA_B receptors could be caused by an insufficient amount of Gβγ available for full activation of GIRK channels, which requires the interaction of one GIRK channel with multiple Gβγ subunits (13), given that activation of GABA_B receptors produces a large and saturating current in hippocampal neurons (12). Regardless, the NMDAR-induced increase in GIRK current evoked by maximal activation of A₁ receptors using 2 different agonists may have arisen from an increase of surface GIRK channels that are specifically coupled to A₁ receptors and/or an increase of A₁ receptors. Because A₁ receptors and GABA_B receptors do not exhibit strictly additive effects in inducing GIRK currents, it has been proposed that they share a common population of GIRK channels (12). However, considering the technical limitations of somatic voltage clamping (15) of acutely dissociated neurons, which have lost their distal dendrites and spines (12) that not only contain GIRK channels in close proximity to GABA_B receptors (16, 17) but also A₁ receptors (18, 19), the possibility remains that A₁ receptors and GABA_B receptors may form distinct complexes with GIRK channels that are differentially regulated by neuronal activity in the hippocampus.

The NMDAR-induced increase of GIRK channel activation by adenosine, a major neuromodulator of neuronal excitability (7), could strongly influence neuronal activity in dendrites and spines. Because GIRK1 and GIRK2 are found in close proximity to GABA_B receptors in dendritic spines, but not in soma and dendritic shafts (16, 17), GIRK channels on dendritic shafts are likely coupled to other GPCRs such as A₁ receptors (11). Considering the dendritic and postsynaptic distribution of A₁ receptors (18, 19) and GIRK channels (16, 17), the basal activity of GIRK channels on dendrites (20), and the tonic GIRK channel activation caused by A₁ receptor stimulation by ambient adenosine in hippocampal neurons (21), NMDAR-induced increase of GIRK channel activation by A₁ receptors may represent a homeostatic response to dampen membrane excitability in dendrites and spines and shunt synaptic integration via adenosine-induced GIRK channel activity (21–23) without altering signaling via GABAergic interneurons.

Because neuronal depression mediated by A₁ receptors contributes to neuroprotection (24), the NMDAR-mediated enhancement of GIRK channel activation by A₁ receptors could be important for a number of pathophysiological situations in which GIRK channels play a role, such as seizure susceptibility and pain responsiveness (25). Finally, dynamic control of neuronal excitability by NMDAR-induced GIRK surface expression may also serve as an adaptive mechanism to regulate behavior given that GIRK channels are implicated in cocaine and ethanol seeking behavior, depression, and anxiety (25), whereas A₁ receptors are involved in analgesia, anxiety, and sleep–wake activity (26).

Implications for Neuronal Plasticity.

NMDAR-induced GIRK surface expression in spines (see ref. 9) suggests possible roles of GIRK channels in synaptic plasticity, a cellular correlate of learning and memory (1, 2). In this study, we demonstrate that GIRK channels are required for one specific form of synaptic plasticity, LFS-induced depotentiation of LTP of fEPSP in hippocampal CA1 neurons, a process that resembles activity-dependent regulation of GIRK surface expression (see ref. 9) and GIRK channel activity (Figs. 1 and 2) in the involvement of NMDARs, adenosine A₁ receptors, and PP1 (4, 6). Thus, depotentiation likely involves GIRK channel activation by postsynaptic A₁ receptors (11) in addition to modulation of AMPA receptor phosphorylation (6, 27). Given that both GIRK channels (16, 28) and A₁ receptors (18, 19) reside on the dendritic spines and shafts, and that GIRK channel activation by adenosine attenuates the EPSP by ≈30% (21), LFS-induced NMDAR activation could lead to depotentiation by enhancing GIRK channel activation by A₁ receptors at or near excitatory synapses, thereby shunting the synaptic inputs (21, 22) and/or back-propagating dendritic action potentials so as to dampen or reverse processes for the induction or expression of LTP (29, 30).

Studies of molecular mechanisms of excitatory synaptic plasticity such as depotentiation have focused primarily on the regulation of glutamate receptor phosphorylation and trafficking (27). Upon LTP induction, GluR1-containing AMPA receptors are inserted into synaptic membranes, and phosphorylation of GluR1 subunit is necessary for maintaining LTP by stabilizing the inserted receptors at the synapses (27). Because LTP induced by HFS (100–200 Hz), such as the one used in our studies, requires intracellular Ca²⁺ elevation from both NMDARs and voltage-gated calcium channels (VGCCs) (31, 32), A₁R-dependent depotentiation of LTP induced by LFS within a brief period after HFS for LTP induction (4, 33, 34) might reduce or prevent subsequent VGCC and NMDAR activation, because of enhanced GIRK channel activity upon stimulation of A₁ receptors by ambient adenosine. The resulting decrease in intracellular Ca²⁺ concentration may hamper CaMKII activation, whereas the LFS-induced activation of PP1 would cause dephosphorylation of not only GIRK2 but also GluR1 subunits (27), ultimately causing depotentiation.

In this study we have uncovered a critical requirement of GIRK channels in depotentiation of LTP of fEPSP, a form of synaptic plasticity that depends on PP1 stimulation by NMDAR and ambient adenosine activation of A₁ receptors. Previous studies have found that the slow inhibition mediated by GABA_B receptors and GIRK channels in hippocampal CA1 neurons is potentiated in a NMDAR- and CaMKII-dependent process (35). The crucial roles of GIRK channels in both excitatory and inhibitory synaptic plasticity using different signaling pathways may be more readily fulfilled with the neurons' capacity to dynamically regulate GIRK channels according to the pattern of their synaptic activities. It will be important to understand how modulation of GIRK channels and other ion channels besides glutamate receptors contribute to synaptic plasticity (36), to fully elucidate the cellular mechanisms that allow experience to influence the processing and storage of information in the brain.

Methods

Electrophysiological Recordings of GIRK Channels in Cultured Hippocampal Neurons.

Primary cultures of hippocampal neurons from 18-day embryonic rats were prepared as described (9), and 10⁵ cells were plated onto each 12-mm coverslip (Warner Instrument) pretreated with nitric acid and precoated with poly-l-lysine (0.1 mg/mL; Sigma–Aldrich). Neurons were maintained in Neurobasal media containing B27 extract, 2 mM glutamine, 100 units/mL penicillin, and 100 units/mL streptomycin (Invitrogen). Whole-cell patch-clamp recordings of basal and agonist-induced GIRK currents were performed in cultured hippocampal neurons (DIV 11–14) that had been treated with 200 μM DL-APV in neurobasal medium for 3–4 days. The resting potential was between −45 and −55 mV at room temperature. Recording of basal and agonist-induced GIRK current was obtained with an Axopatch 200B amplifier in conjunction with pClamp 8.0 software (Axon Instruments) as described (10). In brief, a pyramidal neuron was voltage clamped at −70 mV in an APV-containing external ACSF bath solution [(pH 7.4, osmolarity 300; 10 mM Hepes-free acid, 145 mM NaCl, 2.5 mM KCl, 10 mM glucose, 1 mM MgCl₂, 2 mM CaCl₂, 0.1 mM picrotoxin, 0.005 mM strychnine, 0.2 mM DL-APV, 0.005 mM 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX)], and then shifted to −120 mV for 1 s in the presence or absence of 120 nM TP, to obtain TP-sensitive basal GIRK current. To record the basal GIRK current in the same neuron after synaptic NMDAR activation, another voltage step was applied after bath solution was changed to an “APV removal” ACSF bath solution lacking APV (0.5 mL/min) (pH 7.4, osmolarity 300; 10 mM Hepes-free acid, 145 mM NaCl, 2.5 mM KCl, 10 mM glucose, 2 mM CaCl₂, 0.1 mM picrotoxin, 0.005 mM strychnine, 0.1 mM glycine, 0.005 mM NBQX) (9) for 10 min during which the current clamp (i = 0) configuration was switched on (without voltage clamp) for 5 min to allow depolarization of the tested neuron to be monitored. To record agonist-induced GIRK current, CPA (5 μM; Tocris), adenosine (100 μM; Sigma), or baclofen (50 μM; Tocris) was first perfused to the neuron in the APV-containing bath solution at holding potential −50 mV. After washing out the agonists with the APV-containing bath solution, the same procedure was repeated after incubating the same neuron in the APV removal bath solution for 15 min.

Electrophysiological Recordings of fEPSP.

The use and care of animals in this study follows the guideline of the Institutional Animal Care and Use Committee at the University of California, San Francisco. The GIRK2^−/− mice from GIRK2^+/− parents (C57BL/6) were analyzed. Control animals were GIRK2^+/+ littermates of GIRK2^−/− mice and C57BL/6 mice. The genotype was determined by PCR amplification of genomic DNA isolated from mouse tails. Recordings of fEPSP of hippocampal slices from wild-type and GIRK2 knockout mice were performed as described (4, 6). A₁ receptors or GIRK channels were blocked by DPCPX (100 nM) or TP-Q (50 nM). Signals filtered at 5 kHz using the amplifier circuitry were sampled at 10 kHz and analyzed with Clampex 9.2 (Axon Instrument). fEPSP slopes were measured during the initial 1–2 ms of the fEPSP, and the average of 3 subsequent responses was calculated. The % field potential slope was obtained by averaging the fEPSP slopes from 30 to 50 min after HFS or LFS stimulation.

Statistical Analysis.

All data are reported as mean ± SEM. Sample size n refers to the number of neurons analyzed in electrophysiological recordings from cultured hippocampal neurons or the number of hippocampal slices used in extracellular recording of fEPSP. ANOVA and post-ANOVA Tukey's multiple comparison tests were performed to determine the statistical significance for any difference between the groups of 3 or more, whereas Student's t test or paired t test was performed for groups of 2 by using Prism4 (Graphpad).