M-Current Inhibition in Hippocampal Excitatory Neurons Triggers Intrinsic and Synaptic Homeostatic Responses at Different Temporal Scales

Persistent alterations in neuronal activity elicit homeostatic plastic changes in synaptic transmission and/or intrinsic excitability. However, it is unknown whether these homeostatic processes operate in concert or at different temporal scales to maintain network activity around a set-point value. Here we show that chronic neuronal hyperactivity, induced by M-channel inhibition, triggered intrinsic and synaptic homeostatic plasticity at different timescales in cultured hippocampal pyramidal neurons from mice of either sex. Homeostatic changes of intrinsic excitability occurred at a fast timescale (1–4 h) and depended on ongoing spiking activity. This fast intrinsic adaptation included plastic changes in the threshold current and a distal relocation of FGF14, a protein physically bridging Nav1.6 and Kv7.2 channels along the axon initial segment. In contrast, synaptic adaptations occurred at a slower timescale (∼2 d) and involved decreases in miniature EPSC amplitude. To examine how these temporally distinct homeostatic responses influenced hippocampal network activity, we quantified the rate of spontaneous spiking measured by multielectrode arrays at extended timescales. M-Channel blockade triggered slow homeostatic renormalization of the mean firing rate (MFR), concomitantly accompanied by a slow synaptic adaptation. Thus, the fast intrinsic adaptation of excitatory neurons is not sufficient to account for the homeostatic normalization of the MFR. In striking contrast, homeostatic adaptations of intrinsic excitability and spontaneous MFR failed in hippocampal GABAergic inhibitory neurons, which remained hyperexcitable following chronic M-channel blockage. Our results indicate that a single perturbation such as M-channel inhibition triggers multiple homeostatic mechanisms that operate at different timescales to maintain network mean firing rate. SIGNIFICANCE STATEMENT: Persistent alterations in synaptic input elicit homeostatic plastic changes in neuronal activity. Here we show that chronic neuronal hyperexcitability, induced by M-type potassium channel inhibition, triggered intrinsic and synaptic homeostatic plasticity at different timescales in hippocampal excitatory neurons. The data indicate that the fast adaptation of intrinsic excitability depends on ongoing spiking activity but is not sufficient to provide homeostasis of the mean firing rate. Our results show that a single perturbation such as M-channel inhibition can trigger multiple homeostatic processes that operate at different timescales to maintain network mean firing rate

IGF-1 receptor regulates upward firing rate homeostasis via the mitochondrial calcium uniporter

Regulation of firing rate homeostasis constitutes a fundamental property of central neural circuits. While intracellular Ca2+ has long been hypothesized to be a feedback control signal, the molecular machinery enabling a network-wide homeostatic response remains largely unknown. We show that deletion of insulin-like growth factor-1 receptor (IGF-1R) limits firing rate homeostasis in response to inactivity, without altering the distribution of baseline firing rates. The deficient firing rate homeostatic response was due to disruption of both postsynaptic and intrinsic plasticity. At the cellular level, we detected a fraction of IGF-1Rs in mitochondria, colocalized with the mitochondrial calcium uniporter complex (MCUc). IGF-1R deletion suppressed transcription of the MCUc members and burst-evoked mitochondrial Ca2+ (mitoCa(2+)) by weakening mitochondria-to-cytosol Ca2+ coupling. Overexpression of either mitochondria-targeted IGF-1R or MCUc in IGF-1R-deficient neurons was sufficient to rescue the deficits in burst-to-mitoCa(2+) coupling and firing rate homeostasis. Our findings indicate that mitochondrial IGF-1R is a key regulator of the integrated homeostatic response by tuning the reliability of burst transfer by MCUc. Based on these results, we propose that MCUc acts as a homeostatic Ca2+ sensor. Faulty activation of MCUc may drive dysregulation of firing rate homeostasis in aging and in brain disorders associated with aberrant IGF-1R/MCUc signaling

Measuring the surface density of GIRK1/2 and Gβγ in <i>Xenopus</i> oocytes.

<p><b>(A)</b> Immunochemical estimation of the amount of GIRK1 in manually separated plasma membranes of <i>Xenopus</i> oocytes injected with 1 or 2 ng of GIRK RNA. Shown is a Western blot of 20 manually separated plasma membranes and 4 cytosols, and variable known amounts of the GST-fused distal C-terminus of GIRK1 (the antibody's epitope) used for calibration of the antibody-produced signal. There was a non-specific band at ~75 KDa in cytosols but not PM of uninjected oocytes (“uninj”). <b>(B)</b> Summary of quantitative analysis of GIRK1 in PM from Western blots of 7 separate experiments. The fully glycosylated band was observed in 4 out of 7 blots. Molar amounts of protein and PM densities from Western blots were calculated as detailed in Methods. The dark red bar is the GIRK1/2 surface density in the high-density group estimated from I_βγ (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004598#pcbi.1004598.t001" target="_blank">Table 1</a>), shown for comparison. <b>(C)</b> Examples of confocal images of oocytes expressing YFP-GIRK1/2 (5 ng RNA) and Gβγ-YFP (5 ng RNA). <b>(D)</b> Estimating YFP molecules density in PM using YFP-GIRK1/2 as molecular ruler. A representative experiment is shown. The left plot shows the measured intensities of YFP-GIRK1/2 and YFP-Gβ coexpressed with wt Gγ in a separate group of oocytes (5:1 ng RNA). The right plot shows the PM densities of YFP in the YFP-GIRK1/2 oocytes, calculated as follows: I_βγ was 14.5±2.1 μA (n = 6), corresponding to 11.4±1.6 channels/μm², or 22.8±3.3 YFP molecules/μm². The density of YFP in the YFP-Gβγ expressing oocytes was calculated based on relative intensities from the left plot. <b>(E)</b> Estimating the amount of endogenous Gβ and expressed YFP-Gβ or YFP-Gβ-XL (5 ng RNA) coexpressed with wt-Gγ, in manually separated plasma membranes. Protocol was similar to Fig 4A; wt purified recombinant Gβγ was used for calibration. In parallel to biochemical measurements, we also measured GIRK currents and YFP intensity in 5–15 oocytes expressing either YFP-GIRK1/2-Gβγ or YFP-Gβγ, as explained in D. <b>(F)</b> Summary of YFP-Gβγ surface density measurements in 4 experiments by the two methods, quantitative Westerns and confocal imaging with YFP-GIRK1/2 as the molecular ruler.</p

Estimated densities and calculated functional stoichiometries of the GIRK channel, Gβγ and Gα_i/o in oocytes, HEK293 cells and neurons.

<p>Comparison of cultured mouse hippocampal neurons, and in oocytes and HEK293 cells expressing GIRK1/2. <b>(A)</b> Cells were subdivided into four groups according to the indicated I_basal ranges, and channel densities were estimated assuming I_βγ = 2I_total and P_o,max = 0.105. Densities in Gα expression experiments in oocytes were estimated from I_total in control groups of oocytes expressing GIRK1/2 and m2R only. <b>(B, C)</b> Estimates of Gβγ and Gα available for GIRK activation in the 4 channel density groups. In oocytes and HEK293 cells I_evoked was elicited by ACh via m2R, in neurons—by baclofen acting on GABA_B receptors.</p

Schematic representation of the GPCR-G-protein-GIRK system.

<p>In resting state (no activated GPCR), the GIRK1/2 channel, a heterotetramer of 2 GIRK1 (grey) and 2 GIRK2 (green) subunits, is expected to interact with ~ 3–4 Gβγ subunits, two of which are bound to Gα^GDP subunits (GDP is shown by a yellow circle). For simplicity, the hypothetical Gβγ anchoring sites (which may be separate or partly overlapping with the Gβγ-activation sites) are not shown. The interaction of GIRK with Gβγ subunits is reversible. Gα^GDP can release the bound Gβγ in basal state, but since Gβγ-Gα^GDP interaction is of a high affinity, the probability of GIRK activation due to this process is relatively low. Thus, at any given time the channel is occupied by 2–3 Gβγ molecules (with an open probability of 6–26% of P_o,max as shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004598#pcbi.1004598.g002" target="_blank">Fig 2C</a>). GIRK overexpression leads to a decrease in GIRK:Gα ratio but does not change the GIRK:Gβγ ratio due to the additional recruitment of Gβγ by GIRK1/2, thus effectively increasing the proportion of channels occupied by > 3 Gβγ molecules, leading to an increase in “basal” open probability. The opposite process occurs upon overexpression of Gα, leading to a decrease in free Gβγ available for channel activation. On expression of Gβγ, its availability for channel activation increases, leading to higher fraction of 4 Gβγ-occupied channels with an open probability close to P_o,max. Activation of G-proteins by an agonist (grey pentagon) via a GPCR (magenta) leads to an exchange of GDP to GTP (red circle) on Gα molecules, and to the subsequent dissociation of the Gαβγ heterotrimer, liberating additional Gβγ for channel activation.</p

A Quantitative Model of the GIRK1/2 Channel Reveals That Its Basal and Evoked Activities Are Controlled by Unequal Stoichiometry of Gα and Gβγ

<div><p>G protein-gated K⁺ channels (GIRK; Kir3), activated by Gβγ subunits derived from G_i/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (I_evoked) and neurotransmitter-independent basal (I_basal) GIRK activities are physiologically important, but mechanisms of I_basal and its relation to I_evoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that I_basal and I_evoked are interrelated: the extent of activation by neurotransmitter (activation index, R_a) is inversely related to I_basal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/2 and Gβγ expressed in <i>Xenopus</i> oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates I_basal and I_evoked in <i>Xenopus</i> oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gβγ and fully accounts for the inverse I_basal-R_a correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gβγ dimers are available for each GIRK1/2 channel. In contrast, available Gα_i/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases. The persistent Gβγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gβγ, consistent with recruitment to the cell surface of Gβγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gβγ but only 2 Gα_i/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gβγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gβγ and Gα.</p></div

Single channel and whole-cell data reveal incomplete activation of GIRK1/2 by agonist compared to Gβγ.

<p><b>(A)</b> Activity of GIRK1/2 in a cell-attached patch of an oocyte expressing the channel, m2R and Gβγ, without an agonist in the pipette. Right panel shows a 2 minutes segment of record, with zoom (below) on a shorter segment. The amplitude distribution histogram of the same 2 min-segment is shown on the right. Red line shows a two-component Gaussian fit. I_single was determined as the difference between the fitted midpoints (μ) of the GIRK current peak on the right (μ2) and the left peak which corresponds to noise (μ1). <b>(B)</b> Activity of GIRK1/2 channels in a cell-attached patch of an oocyte expressing the channel and m2R and activated by 2 μM ACh present in the patch pipette. (Asterisks denote artifacts produced by capacity discharges of patch clamp headstage). The corresponding amplitude histogram of the 2 min-segment of the record is shown on the right. In A and B, GIRK1/2 was expressed at low densities (GIRK1, 10–50 pg RNA; GIRK2, 7–17 pg RNA) whereas RNAs of m2R (1–2 ng/oocyte) and Gβγ (5:1 ng/oocyte) were chosen to produce saturating concentrations of these proteins. Inward K⁺ currents are shown as upward deflections from zero level. In the traces shown, acquisition was at 20 KHz with 5 KHz analog filter. Very similar values of I_single were obtained with 2 KHz filtering (not shown). <b>(C)</b> Single channel currents (left plot) are identical with either ACh or Gβγ. <b>(D)</b> P_o is lower with ACh than with Gβγ (p = 0.029). Bars in C and D show mean±SEM, number of patches is shown above the bars. <b>(E)</b> Summary of whole-cell GIRK1/2 currents at three expression levels (densities). See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004598#pcbi.1004598.t001" target="_blank">Table 1</a> for details. <b>(F)</b> Left panel shows the I_total/I_βγ ratios at three channel densities, calculated from data of <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004598#pcbi.1004598.t001" target="_blank">Table 1</a>. The right panel shows the fractional open probabilities of channels occupied by 0–4 Gβγ, same as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004598#pcbi.1004598.g002" target="_blank">Fig 2C</a> but in a simple graphic form. The red dotted lines are drawn to allow direct comparison of the experimental data from the left panel with the estimates of fractional P_o from the right panel.</p

Basal and agonist-evoked GIRK currents in neurons and oocytes are inversely related.

<p>(<b>A</b>) A representative whole-recording of GIRK current in a neuron. Switching from low-K⁺ extracellular solution to a high-K⁺ solution led to the development of a large inward current probably carried by several ion channel types. Addition of baclofen elicited I_evoked. Arrows show the amplitudes of I_basal, I_evoked and I_total. Extent of activation, R_a, is defined as I_total/I_basal. (<b>B</b>) Inverse correlation between I_basal and R_a in oocytes and neurons. To allow direct comparison of I_basal in oocytes and neurons, currents in neurons were corrected for the 10 mV difference in holding potential, which was -70 mV in neurons and -80 mV in oocytes (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004598#sec019" target="_blank">Methods</a>). The correlation between R_a and I_basal was highly significant, p = 0.000000028 (neurons; n = 60; correlation coefficient = -0.633) and p = 0.0000002 (oocytes; n = 272; correlation coefficient = -0.728) by Spearman correlation test.</p

Dose-dependent activation of GIRK1/2 by coexpressed Gβγ: experiment and simulation.

<p>GIRK1/2 was expressed at 0.2 ng RNA. All data are mean ± SEM from one experiment. <b>(A)</b> Confocal images of Gβγ in giant excised plasma membranes stained with the anti-Gβ antibody. The intensity of all images was increased equally for a better viewing in this figure, but not in the process of image analysis. <b>(B)</b> Dose-dependence of Gβγ levels and I_βγ in oocytes injected with incrementing amounts of wt Gβγ RNA (0.05–30 ng per oocyte). Gβγ expression in the PM (grey bars) was measured from images shown in A, in 4–8 oocyte membranes, and I_βγ currents (red circles; right Y-axis) were measured in 12–16 oocytes. The dashed line shows the basal level of fluorescence, arising from the endogenous Gβγ. Note that, unlike in Western blots, in immunocytochemistry the antibody poorly recognized the endogenous Gβγ compared to the expressed bovine Gβγ. <b>(C)</b> Comparison of measured I_βγ and R_βγ (red circles) and simulated currents (curves). The relative Gβγ levels (from grey bars in B) have been converted into surface densities assuming that 5 ng Gβγ gives 30 molecules Gβγ/μm². The blue line presents the simulation using graded contribution model and amounts of Gα and Gβγ (prior to coexpression of Gβγ) calculated using the methods described above: channel density was calculated from I_βγ (13.75 channels/μm² with 5 ng Gβγ RNA in this experiment), and Gβγ and Gα were estimated from I_total and I_basal, giving 3.16 and 0.73 Gβγ:GIRK and Gα:GIRK ratios, respectively. For simulation with endogenous G proteins only and no Gβγ recruitment allowed (red, black and green lines), the channel density was the same and 1, 10 or 24 endogenous Gαβγ were assumed to be available for GIRK1/2.</p