Dysregulated IP₃ Signaling in Cortical Neurons of Knock-In Mice Expressing an Alzheimer's-Linked Mutation in Presenilin1 Results in Exaggerated Ca²⁺ Signals and Altered Membrane Excitability

Abstract

Disruptions in intracellular Ca²⁺ signaling are proposed to underlie the pathophysiology of Alzheimer's disease (AD), and it has recently been shown that AD-linked mutations in the presenilin 1 gene (PS1) enhance inositol triphosphate (IP₃)-mediated Ca²⁺ liberation in nonexcitable cells. However, little is known of these actions in neurons, which are the principal locus of AD pathology. We therefore sought to determine how PS1 mutations affect Ca²⁺ signals and their subsequent downstream effector functions in cortical neurons. Using whole-cell patch-clamp recording, flash photolysis, and two-photon imaging in brain slices from 4-5-week-old mice, we show that IP₃-evoked Ca²⁺ responses are more than threefold greater in PS1_M146V knock-in mice relative to age-matched nontransgenic controls. Electrical excitability is thereby reduced via enhanced Ca²⁺ activation of K⁺ conductances. Action potential-evoked Ca²⁺ signals were unchanged, indicating that PS1_M146V mutations specifically disrupt intracellular Ca²⁺ liberation rather than reduce cytosolic Ca²⁺ buffering or clearance. Moreover, IP₃ receptor levels are not different in cortical homogenates, further suggesting that the exaggerated cytosolic Ca²⁺ signals may result from increased store filling and not from increased flux through additional IP₃-gated channels. Even in young animals, PS1 mutations have profound effects on neuronal Ca²⁺ and electrical signaling: cumulatively, these disruptions may contribute to the long-term pathophysiology of AD.

Introduction

Inositol triphosphate (IP₃) is a ubiquitous second messenger that functions by binding to receptors (IP₃Rs) on the endoplasmic reticulum (ER) membrane to cause liberation of sequestered Ca²⁺ (Berridge, 1998, 2002). The resultant cytosolic Ca²⁺ transients serve numerous signaling functions in neurons, including modulation of membrane excitability (Yamamoto et al., 2002; Stutzmann et al., 2003), synaptic plasticity (Fujii et al., 2000; Miyata et al., 2000; Nishiyama et al., 2000), and gene expression (Mellstrom and Naranjo, 2001). Moreover, disruptions in this pathway are implicated in Alzheimer's disease (AD) and other neurodegenerative disorders (Abe, 1997; Mattson et al., 2000; LaFerla, 2002). Therefore, factors that modulate or disrupt IP₃-mediated Ca²⁺ signaling are expected to exert powerful physiological and possibly pathological effects on the nervous system.

Among the proteins known to modulate IP₃-mediated Ca²⁺ liberation, recent attention has focused on presenilin 1 (PS1), an ER-localized transmembrane protein required for the proteolysis of amyloid precursor protein (APP) (Selkoe, 2001). Mutant forms of the presenilin 1 (PS1) gene, which account for the majority of early-onset AD cases (Campion et al., 1995; Selkoe, 2001), have been shown to enhance Ca²⁺ transients in various isolated cell systems that include transfected PC12 cells (Guo et al., 1996), fibroblasts from human familial AD patients (Ito et al., 1994; Hirashima et al., 1996), mutant PS1 knock-in (PS1_KI) mouse fibroblasts (Leissring et al., 2000), cultured hippocampal neurons (Guo et al., 1999b), and oocytes overexpressing mutant PS1 (Leissring et al., 1999). Moreover, PS1 mutations have been shown to alter neuronal functions, including increased susceptibility to toxicity (Guo et al., 1999b, Grilli et al., 2000) and facilitation of long-term potentiation in hippocampal slices (Parent et al., 1999; Barrow et al., 2000; Oddo et al., 2003).

Taken together, these data lend strong support to the proposal that disruptions of Ca²⁺ signaling contribute to the pathogenesis of AD (Khachaturian, 1994; Leissring et al., 1999; Mattson and Chan, 2001; LaFerla, 2002). However, because of technical limitations, previous work has primarily been restricted to cultured cell systems and nonexcitable cells. To validate the involvement of Ca²⁺ signaling in AD and elucidate the underlying mechanisms, it is crucial to extend these studies to cortical neurons that have developed normally in an intact brain. We addressed this issue using a combination of video-rate two-photon microscopy, flash photolysis of caged IP₃, and whole-cell electrophysiology (Nguyen et al., 2001; Stutzmann et al., 2003) to image Ca²⁺ signals and record corresponding changes in membrane excitability in layer V projection neurons in slices of mouse brain frontal cortex. Neurons from knock-in mice expressing a mutant form of PS1 (PS1_M146V) showed appreciably larger (more than threefold) Ca²⁺ signals and membrane potential responses to photoreleased IP₃ than did age-matched nontransgenic (non-tg) animals, and the proportion of neurons responding to IP₃ was substantially greater. These actions were restricted to IP₃-sensitive ER Ca²⁺ stores and did not involve changes in voltage-gated Ca²⁺ influx or cytosolic Ca²⁺ buffering, supporting a role for a specific IP₃-sensitive Ca²⁺ signaling disruption in the pathogenesis of AD.

Materials and Methods

Transgenic mice and slice preparation. The derivation and characterization of the PS1_M146V knock-in (PS1_KI) mice and the nontransgenic (non-tg) controls have been described previously (Guo et al., 1999a). The PS1_KI mice express the targeted allele at normal physiological levels in the absence of endogenous PS1 but do not show an overt neuropathology. Age-matched (4-5 week postnatal) non-tg control mice were of the same background strain (C57BL/6). Brain slices were prepared as described (Stutzmann et al., 2003), in adherence with protocols approved by the University of California Irvine Institutional Animal Care and Use Committee. Briefly, mice were deeply anesthetized with halothane and decapitated. The brains were quickly removed and placed in ice-cold artificial CSF (aCSF) containing (in mm): 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 10 d-glucose, 25 NaHCO₃, 2 CaCl₂, 1.2 MgSO₄, pH 7.3-7.4, bubbled with 95%O₂/5% CO₂. Slices (300 μm) were cut from coronal sections containing the prefrontal cortex using a Vibroslice (Camden Instruments). The aCSF was bubbled with 95% O₂/5% CO₂ and superfused at room temperature (22-24°C) at a flow rate of ∼3 ml/min.

Whole-cell recordings and solutions. Visualized whole-cell patch-clamp recordings were performed using an infrared-differential interference contrast (IR-DIC) setup. Patch pipettes (3-4 MΩ) were filled with intracellular solution containing (in mm): 135 K-methlysulfonate, 10 HEPES, 10 Na-phosphocreatine 10, 2 MgCl₂, 4 NaATP, and 0.4 NaGTP (pH adjusted to 7.3-7.4 with KOH), as well as 50 μm fura-2 (Molecular Probes, Eugene, OR) and 10 μm caged IP₃ (Molecular Probes). Signals were acquired at 1 kHz using an Axopatch 1C amplifier and analyzed using Clampex 8.1 and Clampfit 8.1 software (Axon Instruments, Union City, CA).

Ca²⁺ imaging and flash photolysis. Imaging was performed using a custom-made video-rate two-photon microscope, as described (Nguyen et al., 2001). In brief, excitation was provided by trains (80 MHz) of ultra-short (∼100 fsec) pulses at 780 nm from a Ti:sapphire laser (Tsunami; Spectra-Physics, Mountain View, CA). The laser beam was scanned at a frame-scan rate of 30 frames per second and focused onto the specimen through a 40× water-immersion objective (numerical aperture = 0.8). Emitted fluorescence light was detected by a wide-field photomultiplier to derive a video signal that was captured and analyzed by the MetaMorph package (Universal Imaging, West Chester, PA). For clarity, images and traces of fura-2 fluorescence are expressed as inverse pseudo-ratios: F₀/ΔF or F₀/F (F is the fluorescence at any given pixel and time, F₀ is the average resting fluorescence at that pixel before stimulation, and ΔF is the decrease of fluorescence on stimulation), so that increases in [Ca²⁺] correspond to increasing ratios. Ratio values in the present experiments appeared well below dye saturation levels. Most cells showed peak F₀/ΔF values <2.5, whereas maximum values >5.0 were seen in response to strong stimuli. Data are expressed as mean ± 1 SEM, and statistical comparisons were made using a two-tailed t test with 5% confidence interval for normally distributed data, or χ² tests for nonparametric data.

Photolysis of caged IP₃ was accomplished by flashes of UV light (340-400 nm) derived from a 100 W Hg arc lamp coupled to an electronically controlled shutter (Uniblitz). The irradiance at the specimen was ∼50 mW/mm^-2, focused as a uniform circle (radius ∼50 μm) centered on the imaging field. Stimulus strength was regulated by the flash duration. On the basis of previous calibration (Parker and Ivorra, 1992), a flash of 10 msec duration would photolyse ∼4% of the total caged IP₃, resulting in an intracellular concentration of free IP₃ of ∼0.4 μm.

Immunoblot analysis. Detailed Western blot methodology has been described previously (Oddo et al., 2003). Cortical layers from PSI_KI and non-tg control mice were homogenized in a solution of 50 mm Tris, pH 8.0, in purified water containing 0.7 mg/ml Pepstatin A supplemented with a complete Mini protease inhibitor tablet (Roche 1836153). The soluble fraction of the supernatant was saved, and the pellet was spun again in a solution of 10 mm Tris, pH 7.5, 150 mm NaCl, and 2% Triton plus 0.7 mg/ml Pepstatin A in H₂0 again at 100,000 × g for 1 hr at 4°C. Proteins in the supernatant and membrane fractions were resolved by SDS-PAGE (10% bis-Tris, 10% Tris-glycine; Invitrogen) under reducing conditions and transferred to nitrocellulose membrane. The membrane was incubated in a 5% solution of nonfat milk for 1 hr at 20°C. After overnight incubation with primary antibody, the blots were washed in Tween TBS and incubated at 20°C with secondary antibody. Protein extracts were monitored by quantitative immunoblotting. Four samples were analyzed for each group, and all samples were normalized to β-actin. Antibody sources and dilutions are as follows: anti-calsenilin (1:100) (Zymed Laboratories Inc.), anti-calbindin (1:5000) (Chemicon), anti-IP₃ against all receptor subtypes (1:200) (Sigma), and anti-SERCA 2b (1:45,000). Secondary antibodies were obtained from Sigma (anti-rabbit 1:20,000 and anti-mouse 1:50,000).

Results

Ca²⁺ signals were imaged from both non-tg and PS1_KI mice (n = 28 and 25 neurons, respectively). Neurons in the medial prefrontal cortex were studied because of their importance in cognitive functions (Goldman-Rakic, 1995; Rainer et al., 1998) and their vulnerability in AD-related cognitive decline and aging (Pearson et al., 1985; Grill and Riddle, 2002; Bussiere et al., 2003). Moreover, we had previously characterized the physiological dynamics of IP₃-Ca²⁺ signaling in these cells (Stutzmann et al., 2003). Records were obtained from cells in non-tg and PS1_KI mice that showed electrophysiological properties characteristic of regularly spiking pyramidal neurons (McCormick et al., 1985), with respective resting membrane potentials of -63.6 ± 1.08 and -65.6 ± 1.4 mV and input resistances of 216.7 ± 11.7 and 186 ± 12.1 MΩ (differences not statistically significant).

Enhanced IP₃-evoked Ca²⁺ signals in neurons expressing PS1 mutations

Neurons were loaded with fura-2 (50 μm) and caged IP₃ (10 μm) via the whole-cell pipette, and flashes of UV light of varying duration were applied to photorelease IP₃. This approach allowed us to directly control [IP₃] within a single cell, thereby circumventing ambiguities associated with the application of G_q-coupled agonists. The resulting Ca²⁺ transients were monitored from the soma (excluding nucleus) and proximal apical dendrite (Stutzmann et al., 2003). Because fura-2 shows a decrease in fluorescence with increasing [Ca²⁺] with two-photon excitation at 780 nm, images and measurements are presented as a pseudo-ratio (F₀/F and F₀/ΔF; see Materials and Methods) so that increasing cytosolic free [Ca²⁺] corresponds to an increasing ratio.

Ca²⁺ signals in representative neurons from age-matched (4-5 week) non-tg and PS1_KI mice are illustrated in Figure 1, demonstrating some of the key similarities and differences in Ca²⁺ signaling between these groups. Photorelease of IP₃ with all flash durations tested (7-100 msec, 50 mW/mm^-2) resulted in a markedly larger Ca²⁺ response in the PS1_KI neuron when compared with the non-tg neuron. However, spike-evoked Ca²⁺ transients during spike trains (100 pA depolarizing current for 500 msec; n = 5-6 spikes) were of similar amplitude, despite the difference in IP₃-evoked responses. Different than the somatic localization of IP₃-evoked Ca²⁺ transients, signals resulting from Ca²⁺ influx through voltage-gated plasma membrane channels were larger in the proximal dendrite than in the soma.

Figure 1.

Ca²⁺ signals evoked by flash photorelease of IP₃ and by trains of action potentials in strongly responding non-tg and PS1_KI cortical neurons. A, Two-photon image showing resting fluorescence of a non-tg neuron loaded with fura-2. The annular region (excluding nucleus) from which Ca²⁺ measurements were taken is outlined in blue. B, C, Images and corresponding Ca²⁺ transients evoked by photolysis flashes with respective durations of 20 and 100 msec. Images show fluorescence ratios (F/ΔF) and are pseudocolored so that increasing ratios (increasing free [Ca²⁺]) are depicted by increasingly “warm” colors. Each ratio image was formed from ∼10 averaged video frames during stimulation and ∼20 averaged control frames. Traces show measurements of fura-2 fluorescence ratios from the region marked in A. Photolysis flashes were delivered when marked by the arrows. D, Ca²⁺ transients in the same neuron resulting from a train of action potentials evoked by depolarizing current injection (100 nA, 500 msec). E-H, Corresponding images and traces in a PS1_KI neuron. I, Superimposed traces show fluorescence ratio signals in the soma of a non-tg cortical neuron, in response to photolysis flashes with durations of 7, 10, 15, 30, 50, and 100 msec. J, Similar traces from a PS1_KI neuron obtained using the same flash durations.

Figure 2A shows a scatter plot of amplitudes of IP₃-evoked Ca²⁺ responses from individual neurons (PS1_KI, n = 14; non-tg, n = 15) evoked by a range of flash durations. For each flash duration, the proportion of cells showing Ca²⁺ responses was greater in the PS1_KI mice, and the average responses were larger. Pooled data derived from all of these neurons are plotted in Figure 2, B and C. Both the non-tg and PS1_KI neurons displayed a characteristic nonlinear dependence of Ca²⁺ signal amplitude on [IP₃] (Stutzmann et al., 2003), with a similar threshold flash duration required to evoke detectable signals (Fig. 2B). In contrast to this marked enhancement of IP₃-evoked Ca²⁺ signals in the soma, Ca²⁺ signals elicited by action potential trains (500 msec, 0.1 nA) were only slightly enhanced in PS1_KI neurons (by 22 and 6% in the soma and dendrite, respectively; differences not statistically significant). Across all flash durations, the PS1_KI neurons on average gave larger responses than non-tg neurons at all suprathreshold flash durations, with the potentiation being greatest (372%) for strong (50 msec) flashes (Fig. 2C). The IP₃-evoked Ca²⁺ signals in the proximal dendrites were also enhanced in the PS1_KI cells with moderate to long flash durations, whereas the spike-evoked signals were unchanged (Fig. 2D).

Figure 2.

Enhancement of IP₃-evoked Ca²⁺ signals in PS1_KI neurons. Data in this figure were derived by pooling measurements from all neurons examined (i.e., including nonresponding and weakly responding cells). A, Distribution of IP₃-Ca²⁺ responses evoked by photolysis flashes with durations of 10, 20, 50, and 100 msec. Filled symbols (non-tg, gray; PS1_KI, black) are measurements from individual neurons; corresponding means and SE are shown as open symbols. B, Mean peak amplitude of fluorescence signals from the soma measured from non-tg (open circles; n = 15) and PS1_KI neurons (filled squares; n = 14) and plotted as a function of flash duration. Amplitudes of Ca²⁺ signals evoked by trains of action potentials are shown at the right. Error bars indicate 1 SEM. C, Bar graph showing average peak amplitudes of IP₃-evoked Ca²⁺ responses in PS1_KI neurons as a percentage of that in non-tg neurons at corresponding flash durations. Data for action potential-evoked signals are shown at the right. D, Mean peak amplitude of fluorescence signals from the proximal apical dendrite measured from non-tg (open circles; n = 11) and PS1_KI neurons (filled squares; n = 13).

PS1 mutation alters the distribution of IP₃-evoked Ca²⁺ responses

IP₃-evoked Ca²⁺ signals show considerable variability among different neurons (Fig. 2A) (Stutzmann et al., 2003). To further characterize the effects of the PS1 mutation on the distribution of cell responses, we categorized neurons as “nonresponders” (which fail to give Ca²⁺ signals even with strong photorelease of IP₃), “weak responders” (which respond only to strong photorelease-flash >30 msec), or strong responders (which demonstrate a clear IP₃-evoked Ca²⁺ response even at short flash durations). The proportions of non-, weak, and strong responders for the non-tg cells (n = 15) was 0.2, 0.33, and 0.47, respectively, whereas the corresponding proportion for PS1_KI cells (n = 14) was 0.07, 0.0, and 0.93 (differences significantly different; p < 0.025; χ² = 7.77). To determine whether this shift in proportion of responding neurons accounts entirely for the observed overall enhancement of Ca²⁺ signals in the PS1_KI mice, we replotted the dose-response relationships after excluding nonresponding neurons (Fig. 3A). Within this population, an appreciable potentiation of Ca²⁺ responses to strong photolysis flashes remained, although PS1_KI and non-tg groups showed nearly identical Ca²⁺ signals in response to action potential trains.

Figure 3.

Amplitudes and kinetics of IP₃-evoked Ca²⁺ signals in responsive PS1_KI and non-tg neurons. Data in this figure were derived after excluding neurons that failed to responded to IP₃. A, Dose-response relationships of IP₃-evoked Ca²⁺ signals in non-tg (gray circles; n = 4-12) and PS1_KI (black squares; n = 8-14) neurons. B, Mean rising slopes of IP₃- and spike-evoked Ca²⁺ responses for non-tg (open circles; n = 4-12) and PS1_KI (filled squares; n = 8-14) neurons. C, Mean decay time constants of IP₃- and action potential-evoked Ca²⁺ signals in non-tg (open circles; n = 4-12) and PS1_KI neurons (filled squares; n = 8-14) derived from single exponential fits to the decay of fluorescence signals. D, Representative Ca²⁺ traces showing response latencies from non-tg (left) and PS1_KI neurons (right) in response to photolysis flashes with durations of 20, 50, and 100 msec. Latencies were measured as the interval between the onset of the flash artifact and time at which a straight-line fit to the rising phase of Ca²⁺ signals (dotted lines in the PS1_KI traces) intercepted the baseline. E, Mean latencies in non-tg (gray) and PS1_KI (black) neurons after flash durations of 20, 50, and 100 msec (n = 5-12 cells per group). Bars marked by asterisk are significantly different (p < 0.05) between these two groups.

Ca²⁺ kinetics in PS1_KI neurons

We measured kinetic parameters including initial rate of rise, decay time constant, and latency of IP₃-evoked Ca²⁺ signals, pooling data from strong and weak responders in each group. The upstroke of IP₃-evoked responses was enhanced by almost 200% in PS1_KI neurons (Fig. 3B); however, no differences were apparent between non-tg and PS1_KI neurons in the rate of decay of IP₃-evoked Ca²⁺ signals, although the decay rate for action potential-evoked Ca²⁺ transients was slightly slower in the PS_KI neurons. In both non-tg and PS1_KI neurons the latency to onset of Ca²⁺ liberation after photorelease of IP₃ shortened progressively with increasing flash duration (Fig. 3D), but PS1_KI neurons on average showed shorter latencies (Fig. 3E).

IP₃-mediated hyperpolarization is enhanced in PS1_KI neurons

IP₃-evoked Ca²⁺ liberation leads to opening of Ca²⁺-dependent K⁺ channels, thereby resulting in membrane hyperpolarization and a decreased electrical excitability that strongly affects neuronal spiking patterns (Stutzmann et al., 2003). In both non-tg and PS1_KI neurons the membrane hyperpolarization increased progressively with increasing flash duration, but responses were significantly greater in PS1_KI neurons than in non-tg cells (Fig. 4A). This potentiation was most prominent at longer flash durations (Fig. 4B).

Figure 4.

IP₃-evoked hyperpolarizing membrane potential responses are enhanced in PS1_KI neurons. A, Representative traces of membrane potential in a non-tg neuron (left column) and a PS1_KI neuron (right) in response to photolysis flashes with durations as indicated in milliseconds. B, Bar graph showing average IP₃-evoked hyperpolarizations evoked by these photolysis flashes, pooled from all non-tg (gray bars; n = 15-17) and PS1_KI (black bars; n = 11-15) neurons.

Levels of IP₃R and Ca²⁺ signaling proteins

To further elucidate the mechanisms underlying the enhancement of IP₃-mediated Ca²⁺ signaling in PS1_M146V mice, we determined whether the levels of selected key proteins implicated in cellular Ca²⁺ signaling were altered. We monitored the steady-state levels of the following proteins obtained from cortical homogenates: IP₃ receptor, calsenilin, SERCA-2b, and calbindin-D. IP₃Rs directly mediate calcium release from the ER, SERCA2b and calsenilin interact with presenilin to modulate calcium signaling (Buxbaum et al., 1998; Leissring et al., 2000), and calbindin-D is a high-affinity calcium buffering protein found in interneurons and subpopulations of cortical pyramidal neurons (Fujimaru and Kosaka, 1996; Hof et al., 1999). Densitometric analysis of Western blots revealed that the levels of these candidate proteins were not significantly different (p > 0.05) in 4- to 5-week-old PS_KI and non-tg mice (data not shown).

Discussion

We used two-photon imaging and flash photolysis of caged IP₃ to demonstrate that IP₃-evoked Ca²⁺ transients in cortical neurons are substantially and specifically potentiated in transgenic mice expressing an AD-linked mutation of the presenilin gene. Our results offer mechanistic insights into the locus of PS1 modulation of Ca²⁺ signaling and reveal changes in downstream electrical excitability in a cell type and brain region sensitive to AD pathology.

The peak amplitude and rate of rise of IP₃-evoked Ca²⁺ signals were much greater, on average, in neurons from PS1_KI mice (>350%) than in age-matched non-tg mice. In contrast, Ca²⁺ signals evoked by trains of action potentials were not very different. Thus, the actions of the PS1 mutant appear specific to IP₃-mediated ER Ca²⁺ liberation rather than involving general changes in cytoplasmic Ca²⁺ regulation such as alterations in cytosolic Ca²⁺ buffering capacity or voltage-gated Ca²⁺ influx. Immunoblot analyses showed that IP₃R levels were not increased in the cortical homogenates of PS1_KI mice, suggesting that the enhanced Ca²⁺ liberation did not arise simply through an increase in numbers of release channels (Leissring et al., 1999). Moreover, we did not observe a leftward shift in dose-response curves for IP₃ action that might suggest an increase in sensitivity of IP₃R. Instead, our results are consistent with previous observations in nonexcitable and cultured cells (Leissring et al., 2000; Mattson et al., 2000; Herms et al., 2003) that point to an overfilling of ER Ca²⁺ stores. The underlying mechanisms remain unclear because we are presently unable to measure ER store levels directly, but they likely do not involve increases in the number or activity of the SERCA pumps that accumulate Ca²⁺ into the ER, because levels of SERCA 2b protein were unaltered by the PS1_M146V mutation in cortical homogenates, as were the rate constants of cytosolic Ca²⁺ clearance after both IP₃- and action potential-evoked signals. It remains possible, however, that a change in “set point” of the SERCA pumps could cause a continued uptake at basal cytosolic-free Ca²⁺ levels, resulting in enhanced store filling over time.

Another mechanism that may contribute to increased store filling is downregulation of the passive leak of Ca²⁺ from the ER. The ER Ca²⁺ store content is set by a balance between active uptake through SERCA pumps and passive leak into the cytosol. Experiments in nonexcitable cells using thapsigargin to assess the Ca²⁺ leak by measuring the rise in cytosolic [Ca²⁺] after block of SERCA pumps showed little change after PS1_M146V expression (Guo et al., 1999b; Leissring et al., 2000; Yoo et al., 2000; Camello et al., 2002). Neurons, however, use the ER leak more predominantly to maintain homeostasis than do nonexcitable cells (Simpson et al., 1995; Solovyova et al., 2002), so that even subtle changes may have profound effects on intraluminal Ca²⁺ levels. It is also possible that increased amounts of Ca²⁺ buffering proteins in the ER lumen could increase the total Ca²⁺ storage capacity for any given luminal-free Ca²⁺ concentration.

A further finding was the increase in the proportion of neurons responding to moderate levels of IP₃. The PS1 mutation results, therefore, not only in an exaggeration of IP₃-evoked Ca²⁺ signals in responding neurons but appears to shift weak and nonresponders to strong responders. The heterogeneity of IP₃-evoked Ca²⁺ responses likely reflects the recent Ca²⁺ filling or release history of the ER (Yoshizaki et al., 1995; Garaschuk et al., 1997), and we had demonstrated that nonresponding neurons could sometimes be rescued by allowing the ER to fill via Ca²⁺ entry during action potentials (Stutzmann et al., 2003). Thus, ER Ca²⁺ stores in most neurons from PS1_KI mice are likely overfilled, with possible subsequent consequences for neuronal signaling, metabolism, and neuropathology.

Finally, we examined the effects of the PS1 mutation on downstream Ca²⁺-dependent modulation of membrane excitability. In non-tg neurons, photoreleased IP₃ evokes a transient membrane hyperpolarization via activation of Ca²⁺-sensitive K⁺ conductances, although the spatial relationship between the intracellular Ca²⁺ stores and the K⁺ channels and the specific class(es) of K⁺ channel involved are not known (Sah, 1996; Stutzmann et al., 2003). The magnitude of the membrane hyperpolarization was significantly larger in neurons from PS1_KI mice, presumably because of the enhanced Ca²⁺ release from ER stores. This hyperpolarization continued to increase even with flash durations longer than required to evoke near-maximal Ca²⁺ signals, possibly as a result of the steeply nonlinear Ca²⁺ dependence of the underlying conductance change (Stutzmann et al., 2003). The enhanced IP₃-evoked hyperpolarization leads to an interesting reciprocal effect on neuronal excitability. PS1_M146V increases the “excitability” of the ER, yet this is transduced as an inhibition of plasma membrane electrical excitability that will lead to changes in spike patterning and reduced responsiveness to excitatory synaptic inputs.