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¹ Queensland Brain Institute, School of Biomedical Sciences, University of Queensland, Australia and Division of Neuroscience, John Curtin School for Medical Research, Australian National University, Canberra, Australia

	Abstract

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The long-term changes that underlie learning and memory are activated by rises in intracellular Ca²⁺ that activate a number of signalling pathways and trigger changes in gene transcription. Ca²⁺ rises due to influx via L-type voltage-dependent Ca²⁺ channels (L-VDCCs) and release from intracellular Ca²⁺ stores have been consistently implicated in the biochemical cascades that underlie the final changes in memory formation. Here, we show that pyramidal neurones in the basolateral amygdala express an L-VDCC that is active at resting membrane potentials. Subthreshold depolarization of neurones either by current injection or summating synaptic potentials led to a sustained rise in cytosolic Ca²⁺ that was blocked by the dihydropyridine nicardipine. Activation of metabotropic receptors released Ca²⁺ from intracellular Ca²⁺ stores. At hyperpolarized potentials, metabotropic-evoked store release ran down with repeated stimulation. Depolarization of cells to –50 mV, or maintaining them at the resting membrane potential, restored release from intracellular Ca²⁺ stores, an effect that was blocked by nicardipine. These results show that Ca²⁺ influx via a low-voltage-activated L-type Ca²⁺ current refills inositol 1,4,5-trisphosphate (IP₃)-sensitive intracellular Ca²⁺ stores, and maintains Ca²⁺ release and wave generation by metabotropic receptor activation.

(Received 3 October 2004; accepted after revision 11 November 2004; first published online 18 November 2004)
Corresponding author P. Sah: Queensland Brain Institute, School of Biomedical Sciences, St Lucia, Queensland 4072, Australia. Email: pankaj.sah{at}uq.edu.au

	Introduction

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Calcium is a ubiquitous second messenger that initiates many physiological processes from gene transcription to synaptic plasticity (Berridge et al. 2000). In most cells cytosolic Ca²⁺ is maintained at low levels (50–100 nM) and can rise either due to influx from the extracellular space or by release from intracellular stores (Berridge et al. 2000). Neurones have abundant intracellular Ca²⁺ stores (Blaustein & Golovina, 2001) within the smooth endoplasmic reticulum (Tsien & Tsien, 1990; Meldolesi, 2001). These stores can release Ca²⁺ in response to stimulation of metabotropic receptors (Finch et al. 1991), and can generate propagating Ca²⁺ waves (Finch et al. 1991; Nakamura et al. 1999; Power & Sah, 2002) that may invade the nucleus (Power & Sah, 2002) and have been suggested to be involved in the initiation of gene transcription (West et al. 2001). Once discharged, intracellular stores are replenished by the action of sarcoplasmic-endoplasmic reticulum ATPases (SERCA) that transport Ca²⁺ from the cytosol into the endoplasmic reticulum (Tsien & Tsien, 1990; Meldolesi, 2001). The source of this Ca²⁺ in nonexcitable cells is by Ca²⁺ influx via a plasmalemmal Ca²⁺ channel activated by store emptying, a mechanism called capacitative Ca²⁺ entry (Putney & McKay, 1999; Parekh, 2003). However, capacitative Ca²⁺ entry has not been demonstrated in central neurones, and the mechanisms that underlie refilling of intracellular stores in these cells are not well understood.

Neurones express a large diversity of voltage-gated Ca²⁺ channels that have been divided into low-voltage-activated (LVA) and high-voltage-activated (HVA) channels (Ertel et al. 2000). LVA (T-type) channels mediate rapidly inactivating currents that provide a transient Ca²⁺ influx in response to subthreshold depolarizations (Magee & Johnston, 1995; Perez-Reyes, 2003) whereas HVA (L, P/Q, N and R types) channels (Ertel et al. 2000) generate sustained currents that activate at suprathreshold membrane potentials (Magee & Johnston, 1995). These Ca²⁺ channels can also be distinguished pharmacologically, with LVA channels being selectively sensitive to low concentrations of nickel. Among the HVA channels, L-type channels are sensitive to dihydropyridines, N-type channels are blocked by -conotoxin, P/Q-type channels by agatoxin, while the R-type channels are insensitive to these agents. Functionally, LVA channels have been proposed to be involved in the generation of burst firing (Huguenard, 1996). Of the HVA channels, L-type voltage-dependent Ca²⁺ channels (L-VDCCs) have been implicated in the regulation of synaptic plasticity and gene transcription (Finkbeiner & Greenberg, 1998; Dolmetsch et al. 2001; West et al. 2001). A low-voltage-activated, voltage-dependent Ca²⁺ current that activates with pharmacological properties similar to that of L-type Ca²⁺ channels has also been described in hippocampal neurones (Avery & Johnston, 1996; Magee et al. 1996). These currents have now been described in a number of cell types (Lipscombe, 2002); however, their functional role in central neurones is not known.

Long-term synaptic plasticity within the basolateral amygdala (BLA) has been proposed as the cellular mechanism that underlies the storage of fear-related memory (Blair et al. 2001; Davis & Whalen, 2001; Sah et al. 2003). Infusion of L-VDCC antagonists into the BLA impairs acquisition of the fear-conditioned response in rats (Bauer et al. 2002; Shinnick-Gallagher et al. 2003) (but see Cain et al. 2002). Long-term potentiation induced by pairing trains of action potentials with synaptic stimulation is blocked by the dihydropyridine nicardipine (Weisskopf et al. 1999; Bauer et al. 2001, 2002). It has therefore been proposed that activation of high-voltage-activated L-VDCCs is in part necessary for the induction of synaptic plasticity in the amygdala (Blair et al. 2001). Here, we show that projection neurones in the BLA express a low-voltage-activated L-VDCC. Activation of this current at subthreshold membrane potentials refills inositol 1,4,5 trisphosphate (IP₃)-sensitive intracellular Ca²⁺ stores.

	Methods

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Brain slices were prepared from rats using standard techniques (Mahanty & Sah, 1999). Rats (21–28 days old) were anaesthetized with halothane and decapitated. All procedures were in accordance with the guidelines of the Institutional Animal Ethics Committee. The brain was removed and coronal slices (400 µm) were prepared using a vibratome. Slices were incubated at 33°C for 30 min, and then maintained at room temperature in an artificial cerebrospinal fluid (ACSF) solution containing (mM): 119 NaCl, 2.5 KCl, 1.3 MgCl₂, 2.5 CaCl₂, 1.0 Na₂PO₄, 26.2 NaHCO₃, 11 glucose, which was equilibrated with 95% CO₂, 5% O₂. Whole-cell recordings were made from the soma of basolateral amygdalar neurones from slices perfused with ACSF heated to 33°C using infrared differential interference videomicroscopy.

In Ca²⁺-imaging experiments, patch pipettes (2–5 M) were filled with an internal solution containing (mM): 135 KMeSO₄, 8 NaCl, 10 Hepes, 2 Mg₂ATP, 0.3 Na₃GTP (pH 7.3 with KOH, osmolarity 280–290 mosmol l^–1) and the Ca²⁺ indicator Oregon Green BAPTA-1 (Molecular Probes; 50 µM for whole-field and 100 µM for confocal experiments). Ca²⁺ currents were measured using an with an internal solution containing (mM): 135 Cs-MeSO₄, 8 NaCl, 10 Hepes, 2 Mg₂ATP, 0.3 Na₃GTP (pH 7.3 with KOH, osmolarity 280–290 mosmol l^–1) in the presence of 5–10 mM CsCl, 5–10 mM 4-aminopyridine (4AP), 5–10 mM TEACl, 500 nM TTX, 10 µM CNQX, 30 µMD-aminophosphonovalerate (APV), 10 µM bicuculline. The addition of TEA, CsCl and 4AP was accompanied by an iso-osmotic reduction of NaCl. Ca²⁺ current experiments were performed at near room temperature (23°C). Capacitive transients and leak currents were subtracted using a series of 5 mV hyperpolarizing steps of equal duration. In some experiments phosphocreatine (7 mM) and spermine (0.1 mM) were also added to the internal solution. Junction potentials were not corrected for. Electrophysiological signals were recorded with either an Axopatch 1D amplifier or a Multiclamp 700 amplifier (Axon Instruments), digitized at 10 kHz with an ITC-16 board (Instrutech), and controlled using Axograph (Axon Instruments). Electrophysiological data were analysed using Axograph. Trains of action potentials were evoked by repetitive injection of a suprathreshold (1–2 nA, 1–2 ms) current injection at the indicated frequency.

Whole-field fluorescence measurements were made using a monochromator-based imaging system, Polychrome II (TILL Photonics). Neurones were visualized using a BX50 microscope (Olympus) equipped with a x60 water immersion objective (NA 0.9, Olympus) and illuminated with 488 nm light. IP₃ was uncaged using a pulsed xenon arc lamp (TILL Photonics), which illuminated the entire field of view and discharged 80 J in 2 ms. Light from both the monochromator and the flash were delivered to the BX50 microscope via a quartz light guide and a custom epiflourescence attachment provided by TILL photonics. Images were acquired with an in-line transfer, cooled CCD camera (TILL photonics) in which the scan lines were binned by two in both horizontal and vertical directions giving a spatial resolution of 0.33 µm pixel^–1. Frames were collected at 25–33 Hz with an exposure time of 10–20 ms per frame. Images were analysed off line using Vision (TILL Photonics). Small regions of interest were selected over the extranuclear soma and the proximal dendrite (20–50 µm from the soma), and the fluorescence over this region was averaged.

Single- and two-photon confocal fluorescence images were obtained using a Zeiss Axioskop 2FS (x63 objective) with a 510 laser scanning head, and equipped with an argon laser for single-photon illumination, and a Verdi solid-state pump laser and a Mira 900-F femptosecond Ti:S pulsed laser (Coherent Scientific) for two-photon excitation. The excitation wavelengths were 488 nm for single-photon excitation, and 800 nm for two-photon excitation. When acquiring single-photon confocal data, the detector pinhole aperture was set to give a vertical resolution of <1.5 µm. Small segments were selected over each subcellular region, and the fluorescence over this region was averaged. Single- and two-photon confocal data were pooled.

Kinetic sequences were constructed over time for each of the selected regions. Kinetic sequences were calculated as the relative change in fluorescence over baseline fluorescence (F/F). F/F_t= (F_t–F₀)/(F₀–B) where F_t is the fluorescence at time t, F₀ is the average baseline fluorescence prior to the stimulus, and B is the background fluorescence measured in an adjacent extracellular region. In some cases the Ca²⁺ concentration was estimated using methods published by Maravall et al. (2000). To estimate Ca²⁺ changes ([Ca²⁺]) and resting Ca²⁺ ([Ca²⁺]₀), we used the following formulae:

(1)

(2)

R_f and K_D are the dynamic range and dissociation constant of the indicator, respectively. We used published values of indicators dynamic range R_f= 7.1 and K_D= 206 nM for Oregon Green BAPTA-1. Estimates of F_max and F_max were obtained by measuring the amplitude of a saturating fluorescence plateau evoked by a high-frequency train of action potentials (500 ms, 100 Hz).

-Conotoxin GVIA was purchased from Alomone Labs (Jerusalem, Israel). Caged myo-inositol 1,4,5-trisphosphate (caged IP₃) was purchased from Molecular Probes. All other drugs were purchased from Sigma. Stock solutions of nicardipine were prepared in ethanol. Stock solutions of caffeine, ryanodine and BayK 8644 were prepared in DMSO. Caged IP₃ and ruthenium red were added to the internal solution. Muscarine (10 µM) was applied by picospritzer (15–20 p.s.i., 300–600 ms; Parker Hannifin Fairfield, NJ, USA) application through a patch pipette. All other drugs were bath applied. -Agatoxin IVA and -conotoxin GVIA were coapplied with 0.1 mg ml^–1 cytochrome C (Sigma).

Statistical analyses were made using StatView (SAS Institute). Statistical comparisons were made using ANOVA or as otherwise indicated. Data are presented as means ±S.E.M.

	Results

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Whole-cell patch-clamp recordings were made from pyramidal neurones in the BLA loaded with the Ca²⁺ indicator Oregon Green BAPTA-1. These neurones had a resting membrane potential of –66.4 ± 0.2 mV (n= 221). Depolarization of neurones from the resting membrane potentials led to a slow, sustained increase in fluorescence, indicating a rise in free intracellular calcium ([Ca²⁺]_i) in the soma and proximal dendrite (Fig. 1A). Further depolarization generated an action potential that, as in other neurones (Markram et al. 1995; Sah & Clements, 1999), was accompanied by a large brief rapid rise of Ca²⁺ in the soma and proximal dendrite (Fig. 1A). Ca²⁺ rises to subthreshold depolarizations were similar in projection neurones from both the lateral and basal divisions of the BLA. To examine the voltage dependence of the subthreshold rise in cytosolic Ca²⁺, neurones were voltage clamped and given depolarizing voltage steps. From a holding potential of –80 mV, depolarization of cells beyond –70 mV generated a slowly rising, sustained rise in cytosolic Ca²⁺ that returned to baseline levels on return of the membrane potential (Fig. 1B and D). The Ca²⁺ rise was larger and faster in the proximal dendrite. The steady-state change in fluorescence at –50 mV was 0.22 ± 0.03 F/F in the proximal dendrite (n= 49) and 0.13 ± 0.01 F/F in the soma (n= 67), with an onset time constant of 131 ± 13 ms in the dendrite and 433 ± 43 ms in the soma for projection neurones in the basal nucleus. We estimated the resting [Ca²⁺]_i in the proximal dendrite to be 79 ± 10 nM, and depolarization of cells from –80 to –50 mV raised cytosolic Ca²⁺ by 38 ± 7 nM(n= 5). For comparison, projection neurones in the lateral amygdala showed a steady-state rise of 0.16 F/F in the proximal dendrite (n= 2), and 0.12 ± 0.03 F/F in the soma (n= 4). In CA1 pyramidal neurones, where sustained low-voltage-activated Ca²⁺ currents have been previously described (Magee et al. 1996), a depolarizing step from –80 to –50 mV evoked a steady-state rise in fluorescence of 0.09 ± 0.03 F/F in the proximal dendrite, and 0.07 ± 0.02 F/F in the soma (n= 4). Assuming that resting cytosolic Ca²⁺ is similar in all three cell types, these data suggest that subthreshold rises in Ca²⁺ are larger in projection neurones of the BLA. The voltage-dependent rise in Ca²⁺ was persistent as shown by the decrease in cytosolic Ca²⁺ when neurones were hyperpolarized from a steady holding potential of –50 mV (Fig. 1D). To examine the spatial extent of the voltage-dependent Ca²⁺, rise we used confocal imaging to follow the dendrites distally (Fig. 2A). Subthreshold depolarization evoked a rise in cytosolic Ca²⁺ in all compartments of the neurone, including nucleus and dendritic spines (Fig. 2B), with the largest rise observed in the proximal dendrites. Due to the slow time course of the response, it was not possible to determine whether these low-voltage-activated Ca²⁺ channels are located on the spines themselves, or whether Ca²⁺ in the spines diffused from the dendrite into the spine.

View larger version (40K): [in this window] [in a new window]   Figure 1.  Cytosolic Ca2+ is voltage dependent at subthreshold membrane potentials A, voltage and Ca2+ response to a series of current injections. Left, a prolonged subthreshold current injection causes a slowly rising change in cytosolic Ca2+, whereas a hyperpolarizing step causes a reduction in cytosolic Ca2+. A suprathreshold current injection leads to the generation of a single action potential (AP). The arrow indicates the rapid and large Ca2+ rise associated with AP generation. The Ca2+ response to a brief (10 ms) suprathreshold current injection is shown on the right. B, fluorescence responses in the soma and proximal dendrite to a series of 5 mV voltage steps from a holding potential of –80 mV. Whole-cell currents are shown above command potentials. C, mean plateau fluorescence response (indicated by the square bracket in B) plotted against voltage step potential. D, hyperpolarizing voltage steps from a holding potential of –50 mV reduce [Ca2+]i in a voltage-dependent manner (left). When held at –70 mV further hyperpolarization (right) has no effect on [Ca2+]i.

View larger version (65K): [in this window] [in a new window]   Figure 2.  All proximal neuronal compartments show voltage-dependent rises at subthreshold membrane potentials A, projection neurones in the basolateral amygdala (BLA) have multiple spiny dendritic processes. The image shown is a projection constructed from a series of two-photon z-stacks of a pyramidal neurone filled with Oregon Green BAPTA-1. The inset shows the highlighted dendritic segment at higher magnification showing many dendritic spines. B, cytosolic Ca2+ rises in all proximal neuronal compartments. Fluorescence responses evoked by a depolarizing voltage step from –80 to –50 mV in various cellular regions. Data shown in B is from a different cell than that shown in A.

View larger version (28K): [in this window] [in a new window]   Figure 3.  L-type Ca2+ channels mediate subthreshold Ca2+ transients Rises in Ca2+, plotted as F/F, to a voltage step from –80 to –50 mV. A, a high concentration (5 mM) of NiSO4 fully blocks the Ca2+ rise on subthreshold depolarization, but low concentration, which selectively blocks T-type currents (50 µM, B) has no effect.C, the L-type Ca2+ channel blocker nicardipine (10 µM) blocks the Ca2+ rise, whereas BayK 8644 (2.5 µM) potentiates the Ca2+ rise (D). E, the mean ±S.E.M. effect of nicardipine (nic), BayK 8644 (BAYK), 50 µM NiSO4 (low Ni), and 5 mM NiSO4 (high Ni) on the fluorescence rise in the soma (filled bars) and proximal dendrite (open bars). ***P < 0.001.

To isolate low-voltage-activated Ca²⁺ currents, wholecell recordings were made using a caesium-methanesulphonate-based internal solution in the presence of TTX, CsCl, 4AP, TEA, CNQX, APV and bicuculline. When neurones were held at –80 mV, depolarizing voltage steps to –50 mV evoked a small sustained inward current (Fig. 4). In some instances we observed that a small transient component, reminiscent of T-type Ca²⁺ current, was also present. The sustained component was reduced by nicardipine (45 ± 17%; n= 6; P < 0.05) and augmented by BayK 8644 (113 ± 67%; n= 5; P < 0.001) (Fig. 4D). We next tested whether L-VDCCs are necessary for subthreshold Ca²⁺ entry in the soma and proximal dendrite or are simply favoured during prolonged depolarizations which would inactivate T-type channels. To promote Ca²⁺ entry via T-VDCCs, neurones were voltage clamped at –80 mV, and 20 ms depolarizing pulses to –50 mV were given at 20 Hz. The subthreshold pulse train evoked a Ca²⁺ rise that was smaller than that evoked by the sustained depolarization (47 ± 18 and 33 ± 18% of the sustained step in soma and dendrite; n= 4). These results suggest that in BLA neurones, like other neurones, T-VDCCs may be preferentially located on more distal dendrites (Perez-Reyes, 2003). Together, these results show that the rise in cytosolic Ca²⁺ during subthreshold depolarizations is mediated principally by activation of a non-inactivating, low-threshold, dihydropyridine-sensitive, voltage-dependent Ca²⁺ channel.

View larger version (27K): [in this window] [in a new window]   Figure 4.  Dihydropyridine-sensitive currents activate at subthreshold potentials in BLA neurones A, a 600 ms voltage step from –80 to –50 mV evokes an inward current with a transient and steady-state component. Application of nicardipine reduces the steady-state component. B, currents elicited by a voltage step from –80 to –50 mV before and after application of BayK 8644. Note the persistence of the steady-state component and its augmentation by BayK 8644. C, current–voltage (I–V) plot of the transient and steady-state current elicited by voltage steps from a holding potential of –80 mV (n= 12). D, summary data show that steady-state current is reduced by nicardipine (n= 6) and augmented by BayK 9644 (n= 5). *P < 0.05, ***P < 0.001. All inward currents were abolished by application of 5 mM NiSO4 (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 5.  Subthreshold synaptic activity modulates [Ca2+]i via activation of L-type Ca2+ channels A, Ca2+ rise and voltage response evoked by an EPSP train (50 Hz, 2 s delivered to the lateral amygdala) is shown before during and after sequential application of D-aminophosphonovalerate (APV; 30 µM) and nicardipine (10 µM). B, voltage clamping the neurone at the resting membrane potential to block the depolarization associated with the EPSP train blocks the Ca2+ rise. The response to an EPSP train, the response to an identical tetanus under voltage clamp, and the response to a command potential simulating the control EPSP are overlaid. C, stimulation of the lateral amygdala (50 Hz 1 s) in the presence of glutamatergic blockers APV (30 µM) and CNQX (10 µM) evokes a summating IPSP (bottom trace) and is associated with a reduction in cytosolic Ca2+. The change in resting Ca2+ is blocked by application of nicardipine (bold). D, Ca2+ responses associated with an IPSP train are shown at –52 and –70 mV in current clamp (hyperpolarized). A change in membrane potential is required for the change in Ca2+ levels as voltage clamping the cell to –50 mV abolishes the Ca2+ response. A command potential simulating the control IPSP in voltage clamp reproduces the reduction in Ca2+ (simulated, bold).

View larger version (22K): [in this window] [in a new window]   Figure 6.  Ca2+ influx via low-threshold L-type Ca2+ channels fills Ca2+ stores A, Ca2+ release from intracellular stores by focal application of muscarine (10 µM) under voltage clamp (–80 mV) at 3 min intervals is shown recorded from the soma and proximal dendrite. The bars below the Ca2+ traces indicate the timing of the muscarine application. The hatched bars indicate the timing of the 1 min voltage steps to –50 mV that were initiated 2 min prior to the next muscarine puff. Nicardipine was applied immediately following the second depolarization. The muscarine-evoked Ca2+ rise was reduced by repeated application of muscarine, and replenished by a brief subthreshold depolarization (pre depol. versus post depol.). In the presence of nicardipine, subthreshold depolarization failed to replenish the Ca2+ response to muscarine (n= 5). B, summary data for subthreshold depolarization mediated store loading before and during application of nicardipine. Somatic peak responses were normalized to the response evoked by the initial application of muscarine. *P < 0.05 (Wilcoxon signed rank test; n= 9). An example of action-potential-mediated store filling is shown in C. Somatic Ca2+ rises evoked by focal application of muscarine (10 µM) under voltage clamp at hyperpolarized potentials (–75 to –80 mV) at 3 min intervals are shown in the presence of nicardipine (indicated by filled bar). Action potentials, but not subthreshold depolarization, could refill intracellular Ca2+ in the presence of nicardipine. D, summary data for action potential mediated store loading before and during application of nicardipine. Somatic peak responses were normalized to the response evoked by the initial application of muscarine.

View larger version (23K): [in this window] [in a new window]   Figure 7.  Ca2+ influx via low-threshold L-type Ca2+ channels fills ryanodine-sensitive Ca2+ stores A, Ca2+ release from intracellular stores by focal application of caffeine (20 mM) under voltage clamp (–80 mV) at 3 min intervals is shown recorded from the soma and proximal dendrite. The arrows below the Ca2+ traces indicate the timing of the caffeine application. The hatched bar indicates the timing of the 1 min voltage steps to –50 mV that were initiated 2 min prior to the next muscarine puff. B, summary data for subthreshold-depolarization-mediated store loading before and during application. The caffeine-evoked Ca2+ rise was reduced by repeated application of caffeine and replenished by a brief subthreshold depolarization (pre depol. versus post depol.). Somatic peak responses were normalized to the response evoked by the initial application of caffeine. *P < 0.05 (Wilcoxon signed rank test; n= 6). C, somatic Ca2+ rises evoked by focal application of muscarine (10 µM) at 5 min intervals are shown before and after application of ryanodine (indicated by the horizontal bar). Neurones were held at –55 mV to avoid run down of the muscarinic Ca2+ response. D, somatic Ca2+ rises evoked by focal application of muscarine (10 µM) with ruthenium red in the pipette are shown before and after application of ryanodine. Muscarine was applied at 5 min intervals under voltage clamp (holding potential, Vh, –55 mV).

Ca²⁺ can also be released from intracellular Ca²⁺ stores via activation of ryanodine receptors, which are abundant in neurones (Sharp et al. 1993). Caffeine activates ryanodine receptors (Zucchi & Ronca-Testoni, 1997) and antagonizes the actions of IP₃ (Parker & Ivorra, 1991). Focal application of caffeine evoked a rise in free intracellular Ca²⁺ in the soma and proximal dendrites (Fig. 7A), indicating the presence of ryanodine-sensitive Ca²⁺ stores. At hyperpolarized potentials, the caffeine-evoked Ca²⁺ response ran down with repeated stimulation, and was augmented by subthreshold depolarization (Fig. 7A and B). Following a subthreshold depolarization, the caffeine-evoked F/F increased from 41 ± 9 to 84 ± 26% and from 51 ± 14 to 81 ± 42% of the initial Ca²⁺ response in soma (n= 6) and proximal dendrite (n= 4), respectively. We next tested whether ryanodine receptors (RyR) and IP₃ receptors share a common pool of Ca²⁺. At low concentrations, ryanodine depletes RyR-sensitive Ca²⁺ stores by locking the RyR in the open state (Rousseau et al. 1987). Application of ryanodine (10 µM) reduced the muscarine-evoked Ca²⁺ rise to 9.4% of control (n= 4; Fig. 7C). However, the muscarine-evoked Ca²⁺ rise did not require RyR activation, since robust muscarine-evoked Ca²⁺ rises were observed when the RyR antagonist ruthenium red was included in the patch pipette (Fig. 7D). Furthermore, ruthenium red also occluded the action of ryanodine (96.4% of control; n= 2; Fig. 7D). Thus, the blockade of the IP₃-mediated response by ryanodine was due to store depletion, and, as in hippocampal pyramidal neurones (Power & Sah, 2002), ryanodine and IP₃ receptors in BLA neurones share a common Ca²⁺ pool.

Synaptic activation of metabotropic receptors on cortical and hippocampal neurones, and the subsequent release Ca²⁺ from IP₃-sensitive intracellular Ca²⁺ stores, also lead to the generation of propagating Ca²⁺ waves (Nakamura et al. 1999; Power & Sah, 2002; Larkum et al. 2003). Similarly, tetanic subthreshold stimulation of inputs to BLA neurones also generated a focal rise in Ca²⁺ in the proximal dendrite (10–50 µm from the soma) that began 300–1000 ms after the first stimulus and propagated as a wave toward the soma (Figs 8 and 9B). This Ca²⁺ wave closely resembled those described in hippocampal and cortical pyramidal neurones (Nakamura et al. 1999; Power & Sah, 2002; Larkum et al. 2003). In hippocampal and cortical pyramidal neurones, multiple trains of action potentials have been reported to replenish the generation of Ca²⁺ waves (Jaffe & Brown, 1994). However, in vivo, projection neurones in the basolateral complex are largely silent (Paréet al. 1995), and recordings during some forms of fear conditioning have indicated that most projection neurones do not reach threshold during the induction of fear conditioning (Rosenkranz & Grace, 2002). We therefore asked whether Ca²⁺ stores could be replenished solely by subthreshold Ca²⁺ entry. When neurones were voltage clamped at –70 mV, synaptically evoked Ca²⁺ waves ran down with repeated stimulation, consistent with a reduction in store Ca²⁺ content (Fig. 9A). Brief (1 min) subthreshold depolarization of the neurone to –50 mV resulted in a transient restoration of the synaptically evoked Ca²⁺ rise (Fig. 9A and D). Following the depolarizing voltage step, F/F increased from 29 ± 12 to 569 ± 429% of the initial Ca²⁺ response in the soma (n= 10; Wilcoxon signed rank; P < 0.01), and from 33 ± 12 to 192 ± 57% of the initial Ca²⁺ response in the proximal dendrite (n= 10; Wilcoxon signed rank, P < 0.01). In some neurones, synaptic stimulation at resting membrane potentials was unable to generate Ca²⁺ waves, but a brief depolarization of the cell to –50 mV enabled the neurones to generate waves (Fig. 9C). This finding suggests that in acute brain slices, basal store content is low in some neurones.

View larger version (71K): [in this window] [in a new window]   Figure 8.  Synaptic stimulation can evoke wave-like rises in intracellular Ca2+ A, neurone filled with Oregon Green BAPTA-1. B, graphical representation of a Ca2+ wave evoked by stimulation of the lateral amygdala (100 Hz 1 s) in the presence of CNQX (20 µM) and APV (60 µM). The ‘line scan’ image was generated from a sequence of whole-field images where ordinate positions correspond to the F/F of pixels along the line overlaying the neurone shown in a. Time is shown along the abscissa. Note the rise in Ca2+ initiates in the proximal dendrite (30 µm from the soma) and propagates bidirectionally into the soma and more distally. C, Ca2+ transients, plotted as F/F, in the proximal dendrite and extranuclear soma as indicated by regions of interest (boxes) shown in A.

View larger version (22K): [in this window] [in a new window]   Figure 9.  Brief subthreshold depolarizations augment synaptically evoked Ca2+ waves A, Ca2+ rises generated by stimulation of the lateral amygdala (100 Hz 1 s) in the presence of APV (30 µM) and CNQX (10 µM). Ca2+ waves evoked at 3 min intervals under voltage clamp (–70 mV) run down. The horizontal bar below the Ca2+ trace indicates the timing of the tetanus. The hatched area indicates a voltage step to –50 mV (1 min) initiated 2 min prior to the next tetanus. B, the synaptically evoked Ca2+ rises, measured in the soma and dendrite, are overlaid showing that the Ca2+ rise is initiated in the dendrite and propagates to the soma. C, in some neurones tetanic stimulation failed to evoke Ca2+ waves, and depolarization of the cell to –50 mV revealed a Ca2+ wave indicating that in some cells intracellular stores are not full. D, summary data showing the amplitude of the synaptically evoked Ca2+ rise pre and post a 1 min subthreshold depolarization. Values for individual neurones are shown as circles. Horizontal bar indicates mean amplitudes (n= 13). Note that the response is greater following subthreshold depolarization in nearly every neurone.

View larger version (26K): [in this window] [in a new window]   Figure 10.  Subthreshold depolarization loads inositol 1,4,5- trisphoshate (IP3)-sensitive Ca2+ stores in BLA neurones A, the amplitude of the Ca2+ responses evoked photolytic uncaging of IP3 plotted versus the UV flash number for a typical neurone. UV flashes were given at 2 min intervals. After 5 or 6 baseline responses (Vh–80 mV), neurones were depolarized to –50 mV for 1 min (indicated by hatched region) and returned to –80 mV for 1 min. B, uncaging of IP3 results in a large rise in intracellular Ca2+. IP3-evoked Ca2+ transients before (control) and after (Post depol) a 1 min depolarization to –50 mV are shown overlaid with the subsequent IP3-evoked Ca2+ transient (next). C, summary data show the depolarization-induced enhancement of IP3-evoked Ca2+ rise relative to the response immediately preceding the depolarization (n= 6). **P < 0.01.

View larger version (18K): [in this window] [in a new window]   Figure 11.  L-type voltage-dependent Ca2+ channels refill Ca2+ stores at resting potentials A, somatic Ca2+ release from intracellular stores by focal application of muscarine under voltage clamp (–80 mV). To deplete Ca2+ stores, muscarine was applied at 30 s intervals. To promote store depletion the muscarine concentration was raised to 20 or 50 µM. Holding the neurone at –70 mV for 10 min failed to revive the muscarine-evoked Ca2+ response. Applying a 2 min depolarization near threshold rescued the muscarine-evoked response. Following a second depletion phase, the neurone was held at –55 mV for 10 min. Subsequent application of muscarine evoked a robust Ca2+ rise indicating successful store filling. B, time course of store refilling. The median response is plotted against filling duration at –55, –60, –65 and –75 mV. Numbers in parentheses indicate the number of neurones at 5 and 10 min, respectively. C, summary data showing the recovery of the muscarine-evoked Ca2+ rise after 10 min at a holding potential of –55 mV in the presence and absence of nicardipine. *P < 0.05. Neurones in which the Ca2+ rise was not revived by a 2 min depolarization and paired with 1 Hz action potentials were excluded from the data set.

	Discussion

Top Abstract Introduction Methods Results Discussion References

Subthreshold depolarization of BLA pyramidal neurones, either by current injection or summating synaptic potentials, caused a sustained Ca²⁺ influx. This rise in Ca²⁺ was not due to influx via NMDA receptors or to activation of low-voltage-activated, T-type Ca²⁺ channels (Magee et al. 1995), since it was insensitive to APV and low concentrations of nickel. However this Ca²⁺ rise was blocked by the dihydropyridine nicardipine and potentiated by BayK 8644 showing that it is due activation of an L-type voltage-dependent Ca²⁺ current. Under voltage clamp we have identified a small persistent inward current with similar dihydropyridine sensitivity. While, L-type Ca²⁺ channels generally activate at suprathreshold membrane potentials, a low-threshold L-type Ca²⁺ current has previously been described in hippocampal pyramidal neurones (Avery & Johnston, 1996; Magee et al. 1996). Currents with these properties have recently been shown be due to the Cav1.3 gene product (1D) (Koschak et al. 2001; Xu & Lipscombe, 2001). In agreement with our data, 1D transcripts are also expressed in the BLA (Shinnick-Gallagher et al. 2003). These currents are involved in transmitter release in hair cells and in setting the threshold for action potential generation in the sinoatrial note (Platzer et al. 2000). However, the physiological role of this current in central neurones is not known.

Our results show that Ca²⁺ influx via low-threshold Ca²⁺ channels is sequestered within the ER, and is necessary to maintain the filling status of IP₃-sensitive Ca²⁺ stores. Filling of internal Ca²⁺ stores in neurones has been previously demonstrated during suprathreshold depolarizations that open high-threshold, voltage-dependent Ca²⁺ channels, and lead to large rises in cytosolic Ca²⁺ (Pozzo-Miller et al. 1996, 1997). In cultured hippocampal neurones, L-type VDCCs have been shown to contribute to the filling status of IP₃-sensitive Ca²⁺ stores (Rae et al. 2000). However, in those studies Ca²⁺ channels were activated by either increasing extracellular K⁺ or suprathreshold depolarization for several minutes. In BLA neurones, repetitive release of IP₃-sensitive Ca²⁺ stores at negative membrane potentials led to a clear decline in store content. A subthreshold depolarization caused only a modest rise in cytosolic free-Ca²⁺, but significantly replenished IP₃-sensitive Ca²⁺ stores. The rise in cytosolic Ca²⁺ during subthreshold depolarization was largely, but not fully blocked by nicardipine, consistent with the incomplete block of 1D channels by dihydropyridines (Xu & Lipscombe, 2001). However, the refilling of internal stores was abolished by nicardipine, showing that Ca²⁺ entering the cytosol via low-threshold L-type Ca²⁺ channels is in part sequestered within the ER.

Action potentials also activate high-threshold, voltage-dependent Ca²⁺ channels leading to large, brief rises in cytosolic Ca²⁺ (Markram et al. 1995). This Ca²⁺ is in part buffered by uptake into the ER. In BLA neurones, while trains of action potentials could reload intracellular stores, it was less robust than subthreshold-depolarization-evoked store loading. It is notable that while action potential trains are capable of filling IP₃-sensitive Ca²⁺ stores, projection neurones in the basolateral complex are largely silent in vivo (Paréet al. 1995). This suggests that, in BLA projection neurones, store content is largely controlled by subthreshold activation of dihydropyridine-sensitive Ca²⁺ channels.

The amygdala has been consistently implicated in fear conditioning, and long-term synaptic plasticity within the basolateral amygdala has been proposed as the cellular mechanism that underlies the storage of fear-related memory (LeDoux, 2000; Davis & Whalen, 2001). It is becoming increasing apparent that synaptic plasticity requires activation of intracellular Ca²⁺ stores (Rose & Konnerth, 2001; Barbara, 2002; Fitzjohn & Collingridge, 2002). These rises in Ca²⁺ have been suggested to have a role in the mechanisms that underlie synaptic plasticity and the initiation of gene transcription (West et al. 2001). In the BLA, dihydropyridine-sensitive, voltage-dependent Ca²⁺ channels and metabotropic receptor activation are necessary for fear conditioning and some forms of long-term potentiation (Weisskopf et al. 1999; Bauer et al. 2002; Fendt & Schmid, 2002; Rodrigues et al. 2002). Intracellular recordings during some forms of fear conditioning have shown that most projection neurones do not reach threshold during the induction of fear conditioning (Rosenkranz & Grace, 2002). Furthermore, action potentials which are evoked would be riding upon depolarizations. Our results show that brief subthreshold depolarizations, similar to what has been reported during the conditioned stimulus (Rosenkranz & Grace, 2002), can refill IP₃-sensitive Ca²⁺ stores. These channels are therefore well suited to provide a cellular memory trace of prior subthreshold synaptic activity. Thus, neurones that are sufficiently depolarized by the conditioned stimulus would be more apt to show an augmented Ca²⁺ response to the subsequent unconditioned stimulus due to release from intracellular stores. This may be the basis of the dyhydropyridine sensitivity of fear conditioning and LTP in the lateral amygdala.

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