Spontaneous miniature outward currents in mechanically dissociated rat Meynert neurons

Abstract

1. Spontaneous miniature outward currents (SMOCs) were observed in mechanically dissociated rat Meynert neurons using nystatin perforated patch recordings under voltage-clamp conditions.

2. SMOCs were blocked by apamin, a selective blocker of small conductance Ca²⁺-activated K⁺ (SK) channels, but not by blockers for other types of Ca²⁺-activated K⁺ channel.

3. Ryanodine (10-100 μm) reduced both the amplitude and frequency of SMOCs. Caffeine (1 mm) increased the SMOC frequency. Blockers of the sarco/endoplasmic reticulum Ca²⁺-ATPase completely abolished SMOCs, indicating a requirement for functioning sarco/endoplasmic reticulum (SR/ER) Ca²⁺ stores.

4. Both Cd²⁺-containing and Ca²⁺-free solutions partially inhibited SMOC frequency, a result which suggests that Ca²⁺ influx contributes to, but is not essential for, SMOC generation.

5. Thus, SMOCs are SK currents linked to ryanodine- and caffeine-sensitive SR/ER Ca²⁺ stores, and are only indirectly influenced by extracellular Ca²⁺ influx. The development of this new, minimally invasive mechanical dissociation method has revealed that SMOCs are common in native CNS neurons.

In neurons, as well as in other cells, calcium ions (Ca²⁺) regulate a wide range of processes including excitability, neurotransmitter release, synaptic plasticity, gene expression and even cell death (Berridge, 1997, 1998). Such Ca²⁺ signalling ranges from discrete, very brief changes in highly localized intracellular Ca²⁺ concentration ([Ca²⁺]_i) within a cell to more global, long lasting elevations of [Ca²⁺]_i, which may oscillate as repetitive Ca²⁺ waves (Kostyuk & Verkhratsky, 1994; Bootman & Berridge, 1995; Berridge, 1997). Focal increases in [Ca²⁺]_i observed in several cell types are characterized by a rapid rising phase followed by a slower recovery (Berridge, 1997).

Spontaneous miniature outward currents (SMOCs) (Mathers & Barker, 1984; Satin & Adams, 1987; Merriam et al. 1999) and spontaneous transient outward currents (STOCs) (Benham & Bolton, 1986; Saunders & Farley, 1992; Greenwood et al. 1995; ZhuGe et al. 1999) occur in peripheral neurons and smooth muscle cells. SMOCs/STOCs arise from Ca²⁺-activated K⁺ channel activity, which is triggered by brief, localized increases in [Ca²⁺]_i. Such Ca²⁺‘sparks’ reflect the periodic release of Ca²⁺ from the openings of ryanodine/caffeine-sensitive Ca²⁺ channels (ryanodine receptors, RyRs) on the sarco/endoplasmic reticulum (SR/ER) Ca²⁺ stores (Cheng et al. 1993; Nelson et al. 1995; Merriam et al. 1999). Ca²⁺ sparks may either contribute to global Ca²⁺ elevation during excitation or muscle contraction, or reduce global Ca²⁺ levels by triggering Ca²⁺-activated K⁺ channels and hyperpolarization (Perez et al. 1999). In arterial smooth muscle, STOC-induced hyperpolarization causes relaxation of the muscle and vasodilatation (Nelson et al. 1995). Ca²⁺ sparks occur in nerve growth factor (NGF)-differentiated PC12 cells and cultured hippocampal neurons (Koizumi et al. 1999), where they may play an important role in neuronal signalling and regulation. All of the component mechanisms thought to generate SMOCs are known to exist in neurons, including Ca²⁺-activated K⁺ channels, RyRs and SR/ER stores, whereas SMOCs or STOCs have not been observed in CNS neurons.

Spontaneous transient K⁺ currents have on rare occasions been observed in acutely, enzymatically dissociated CNS neurons in our institute. Following our development of a neuron dissociation method that avoids both enzymes and trituration (Rhee et al. 1999; Koyama et al. 1999), SMOCs were clearly evident in recordings from neurons acutely dispersed from the Meynert nucleus. In addition, SMOCs were only evident in nystatin perforated patch recordings and not during conventional open patch whole-cell recordings. Here, we used perforated patch recordings from mechanically dissociated Meynert neurons and investigated the intracellular signalling underlying these novel currents in mammalian CNS neurons.

METHODS

Preparation

Our institutional Ethics Review Committee for Animal Experimentation approved all the following experimental protocols in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences of the Physiological Society of Japan. Thirteen- to sixteen-day-old Wistar rats were decapitated under pentobarbital anaesthesia (50 mg kg⁻¹i.p.). The brain was quickly removed from the skull and was sliced in the coronal plane at a thickness of 400 μm with a microslicer (DTK-1000; Dosaka, Kyoto, Japan). The brain slices were incubated in a medium of the following composition saturated with 95 % O₂ and 5 % CO₂ at room temperature (21-24 °C) for at least 1 h (mm): 124 NaCl, 5 KCl, 1.2 KH₂PO₄, 24 NaHCO₃, 2.4 CaCl₂, 1.3 MgSO₄ and 10 glucose.

Following incubation, slices were transferred into a 35 mm culture dish (Primaria; Falcon, Lincoln Park, NJ, USA). The region of the nucleus basalis of Meynert was identified under a binocular microscope (SMZ-1; Nikon, Tokyo, Japan). The fine tip of a fire-polished glass pipette was placed lightly onto the surface of the Meynert nucleus and was horizontally vibrated at 10-20 Hz. The slice was removed from the dish after dissociation. Note that the method requires no enzyme treatment or trituration. Electrophysiological recordings were initiated after neurons adhered to the bottom of the dish, which was usually within 20 min following dissociation.

Electrical measurements

Electrical measurements were performed using the nystatin perforated patch recording method (Akaike & Harata, 1994). The electrodes were made from borosilicate capillary glass (1.5 mm o.d., 0.9 mm i.d., G-1.5, Narishige, Tokyo, Japan) using a vertical pipette puller (PB-7, Narishige) and filled with the following solution containing nystatin (mm): 20 N-methyl-d-glucamine-methanesulfonate (NMG-methanesulfonate), 80 potassium methanesulfonate, 70 KCl, 5 MgCl₂ and 10 Hepes. The pH of the internal solution was adjusted to 7.2 with Tris-OH. Nystatin was initially dissolved in acidified methanol at 10 mg ml⁻¹, and the stock solution was diluted with the internal solution just before use to a final concentration of 200 mg ml⁻¹. The resistance between the recording electrode filled with this pipette solution and the reference electrode was 5-7 MΩ. Neurons were visualized using phase contrast optics on an inverted microscope (IMT-2; Olympus, Tokyo, Japan). Current and voltage were measured under voltage clamp with a patch clamp amplifier (CEZ-2400; Nihon Kohden, Tokyo, Japan), monitored on both a storage oscilloscope (5100A; Iwatsu Electric, Tokyo, Japan) and a pen recorder (Recti-Horiz-8K; Nippondenki San-ei, Tokyo, Japan), and stored on magnetic tape (RD-120TE, TEAC, Tokyo, Japan). Stored currents were filtered at 1 kHz (E-3201A Decade Filter, NF Electronic Instruments, Tokyo, Japan) and digitized at 4 kHz using Digidata 1200 with pCLAMP software (version 6.0, Axon Instruments, Foster City, CA, USA). All experiments were performed at room temperature.

Data analysis

SMOCs were collected from digitized records in pre-set epochs before, during and after each experimental condition. Each epoch included at least 100 events except for the conditions in which event frequency was strongly inhibited. Inclusion criteria generally required a minimum event amplitude of 3 pA. In some cases, however, this threshold value was raised to 5 pA (see individual figure legends). SMOCs were automatically detected using MiniAnalysis software (Synaptosoft, Leonia, NJ, USA) and then all identified events were visually verified before the data were subjected to further analysis. This procedure minimized inclusion of occasional artificial events. Event amplitude was calculated by subtracting baseline current from the peak current value. Baseline current for each event was obtained by averaging the current level in a segment of 2.5 ms just preceding the event. When two or more events overlapped, the baseline current of the latter events was calculated by extrapolating the decay phase of the preceding event to baseline. Note that in this detection system, baseline current was calculated for every detected event. This procedure minimized the influence of changes in baseline current on event amplitude. Event frequency, mean amplitude, and key kinetic parameters such as rise time and 50 % decay time of individual SMOCs were also analysed. Experimental values were normalized to control values obtained in the same cell under the same conditions and are presented as means ± standard error of the mean (s.e.m.).

For analysis of concentration-inhibition curves, data were fitted to the following equation using a least squares method: I= 1 − (Cn_H/(Cn_H+ IC₅₀n_H)), where I is the current amplitude normalized to the control response without an antagonist, C is the concentration of antagonist, IC₅₀ is the concentration for half-maximal inhibition and n_H is the Hill coefficient.

External solutions

The ionic composition of the standard external solution was (mm): 150 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose and 10 Hepes. The ionic composition of the Ca²⁺-free external solution was (mm): 146 NaCl, 5 KCl, 5 MgCl₂, 2 EGTA, 10 glucose and 10 Hepes. Nominally Ca²⁺-free external solution did not include EGTA. The pH of these external solutions was adjusted to 7.4 with Tris-OH. Tetrodotoxin (TTX, 300 nm), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 3 μm) and bicuculline (3 μm) were routinely added to the external solutions throughout the experiments to block spontaneous synaptic currents observed in the mechanically dissociated neurons.

Drugs

The drugs used in the present study include BAPTA-AM and EGTA-AM purchased from Calbiochem (La Jolla, CA, USA), caffeine and CdCl₂ from Katayama Chemical (Osaka, Japan), charybdotoxin, iberiotoxin and apamin from Peptide Institute (Osaka, Japan), CNQX, cyclopiazonic acid (CPA), nystatin, ryanodine, thapsigargin, and TTX from Sigma (St Louis, MO, USA), bicuculline methochloride from Tocris Cookson (Bristol, UK), and tetraethylammonium chloride (TEA-Cl) from Tokyo Kasei (Tokyo, Japan). Drugs that are insoluble in water were first dissolved in DMSO and were then diluted in the external solution. The final concentration of DMSO was always less than 0.1 %, a solvent concentration that had no effect on membrane potential or electrical activities. Drug solutions were applied using a rapid application system termed the ‘Y-tube method’ (Murase et al. 1990). By this technique the external solution surrounding a neuron can be exchanged within 20 ms.

RESULTS

SMOCs in rat Meynert neurons

More than half of the dissociated Meynert neurons (56.6 %, n = 565) exhibited SMOCs at a holding potential (V_H) of −50 mV, which is near the resting potential of these neurons. Note that TTX and neurotransmitter antagonists for glutamate and GABA were present throughout. The current amplitudes of the SMOCs at this V_H varied within and across neurons but could be as large as 40 pA. SMOC rise times and decay times were much longer than is characteristic for postsynaptic currents generally observed in these mechanically dissociated neurons (Rhee et al. 1999). Thus, SMOCs were clearly evident even in drug-free conditions. Changing V_H reversed the direction of SMOCs to inward at membrane potentials more hyperpolarized than −85 mV (Fig. 1A). The reversal potential estimated from the I-V relationship averaged −82.6 ± 0.58 mV (n = 5) in the normal external solution. This value corresponds to the Nernst K⁺ equilibrium potential (E_K) of −85.7 mV for the experimental solutions and suggests that SMOCs are K⁺ currents.

Figure 1. Basic properties of SMOCs in dissociated rat Meynert neurons.

Aa, SMOCs at various holding potentials. b, the I-V relationship of the SMOC. Mean amplitude values were normalized to the value at a V_H of −40 mV (*). E_K was calculated using the Nernst equation and equalled −85.7 mV. Each point and vertical bar represents the mean ±s.e.m. from 4 neurons. Continuous line is a least squares linear regression fit to the data. Ba, effect of 1 μm charybdotoxin (ChTx), 1 μm iberiotoxin (IbTx) and 100 nm apamin at a V_H of −50 mV. b, apamin inhibited SMOCs in a concentration-dependent manner at a V_H of −50 mV. All responses were normalized to the mean amplitude of individual controls. Each point and vertical bar represents mean ±s.e.m. from 5 neurons. Continuous line is a least squares curve fit (described in Methods).

SMOCs in peripheral neurons were reported to be mediated by large conductance Ca²⁺-activated K⁺ (BK) channels (Mathers & Barker, 1984; Satin & Adams, 1987; Merriam et al. 1999). To determine the type of K⁺ channel producing SMOCs in rat Meynert neurons, we applied several Ca²⁺-activated K⁺ channel blockers. Neither charybdotoxin (1 μm, n = 7, Fig. 1Ba) nor iberiotoxin (1 μm, n = 5, Fig. 1Ba) applied for over 15 min altered the frequency or amplitude of SMOCs. TEA (1 μm) applied for over 15 min did not alter SMOC frequency or amplitude (n = 4, data not shown). In contrast, apamin depressed the SMOC mean amplitude in a concentration-dependent manner with a half-maximal inhibitory concentration (IC₅₀) of 2.8 nm(n = 5, Fig. 1B). These findings indicate that the SMOCs in Meynert neurons are mediated by small conductance Ca²⁺-activated K⁺ (SK) channels.

Involvement of Ca²⁺ stores in SMOCs

SMOCs/STOCs in peripheral neurons and smooth muscle cells are activated by Ca²⁺ sparks resulting from spontaneous Ca²⁺ release from internal Ca²⁺ stores through RyRs (Satin & Adams, 1987; Fletcher & Chiappinelli, 1992; Nelson et al. 1995; Kang et al. 1995; Merriam et al. 1999). Caffeine (1 mm) substantially increased SMOC frequency without altering SMOC amplitude (Fig. 2A and B). Caffeine also reversibly induced a steady outward current (Fig. 2A). Conversely, ryanodine, a blocker of RyRs (McPherson & Campbell, 1993), reduced both the amplitude and the frequency of SMOCs in rat Meynert neurons (Fig. 2C). The highest concentration of ryanodine (100 μm) completely and reversibly inhibited SMOCs. Thus SMOC activity is strongly influenced by RyRs.

Figure 2. RyRs are involved in SMOC generation.

A, at a V_H of −50 mV, caffeine (1 mm) enhanced SMOC frequency while not affecting mean amplitude. B, summary of the effect of caffeine from 5 neurons. Data were normalized to the individual control values. The boxes and the vertical bars represent means ±s.e.m. C, at a V_H of −50 mV, 1 μm ryanodine did not affect the SMOCs, but a continuous application of 10 μm ryanodine inhibited the SMOCs, and 100 μm ryanodine further suppressed the SMOCs.

Since caffeine and ryanodine modulated SMOC activity, the intracellular Ca²⁺ stores of the SR/ER may be involved in SMOC regulation and/or genesis. In vascular smooth muscle cells, both ryanodine and thapsigargin almost completely inhibited the STOCs (Nelson et al. 1995), suggesting the importance of the SR/ER in generating STOCs. Ca²⁺-free external solution should eventually deplete intracellular Ca²⁺ stores, and application of caffeine should accelerate this depletion. Exposure of Meynert neurons to Ca²⁺-free external solution rapidly reduced the frequency of SMOCs (Fig. 3A). A high-dose pulse of caffeine (10 mm) elicited a large transient outward current (Fig. 3A) and when the caffeine pulse was repeated this outward current was progressively attenuated. Following the first caffeine pulse in Ca²⁺-free solution SMOC activity disappeared (Fig. 3A). Re-introduction of normal external solution rapidly restored SMOC activity. These findings support the hypothesis that Ca²⁺ released from the SR/ER through RyRs plays an important role in generating SMOCs.

Figure 3. Depletion of Ca²⁺ stores inhibits SMOCs.

V_H was −50 mV. A, repetitive application of a high concentration of caffeine (10 mm) in nominally Ca²⁺-free external solution. B, thapsigargin (a) and cyclopiazonic acid (CPA, b) eliminated SMOCs within 2 min after application. The effect of CPA was reversible.

The sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA pump) is responsible for Ca²⁺ uptake from the cytoplasm and transfer into the SR/ER Ca²⁺ store (Tsien & Tsien, 1990; MacLennan et al. 1997). Thapsigargin (Thastrup et al. 1990; Pozzan et al. 1994) and cyclopiazonic acid (CPA) (Seidler et al. 1989; Garaschuk et al. 1997) act as irreversible and reversible SERCA pump blockers, respectively. In rat Meynert neurons, thapsigargin (100 nm) inhibited and finally irreversibly eliminated the SMOC activity (Fig. 3Ba). CPA (10 μm) rapidly eliminated SMOC activity, but SMOCs completely recovered within 7 min of reperfusion with normal solution (Fig. 3Bb). Together, these data indicate that SR/ER, RyR and SERCA function are essential to SMOC activity in rat Meynert neurons.

Effect of Ca²⁺ influx on SMOCs

Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCCs) followed by Ca²⁺-induced Ca²⁺ release (CICR) from RyRs is the primary step for triggering SMOCs in cardiac parasympathetic neurons (Merriam et al. 1999). In Meynert neurons, application of 100 μm Cd²⁺ to block VDCCs did not alter the SMOC amplitude (Fig. 4A and Ba), but reduced the frequency at depolarized membrane potentials (-20 to −50 mV; Fig. 4Ab and Bb). Thus, Ca²⁺ influx through VDCCs facilitates SMOC activity.

Figure 4. Role of Ca²⁺ influx through VDCCs.

A, SMOCs in the presence (b) or absence (a) of 100 μm Cd²⁺ at various V_H values. B, the relationship of relative current amplitude (a) and frequency (b) of SMOCs to V_H. All the amplitudes and frequencies were normalized to the control value of the respective cells observed at a V_H of −40 mV. Each point and vertical bar represents the mean ±s.e.m. from 4 neurons.

Ca²⁺-free external solution should eliminate Ca²⁺ influx. The baseline current shifted inward slightly in nominally Ca²⁺-free solution (Fig. 3A), and more clearly in Ca²⁺-free solution containing EGTA (Fig. 5A). Neither Ca²⁺-free external solution eliminated SMOCs (Fig. 3A and Fig. 5A), suggesting that the SMOCs do not directly depend on Ca²⁺ influx from the external solution. Moreover, the SMOC frequency was nearly doubled by caffeine in Ca²⁺-free external solution while the amplitude remained steady (Fig. 5). This finding was quite similar to the effect of caffeine in normal Ca²⁺ external solutions (Fig. 2A and B). Such results suggest that Ca²⁺ sparks which release Ca²⁺ from the SR/ER through RyRs may be the main trigger for SMOC generation.

Figure 5. Role of Ca²⁺ influx.

A, representative recorded trace during Ca²⁺-free external solution and caffeine application. The SMOCs were recorded from a neuron at a V_H of −50 mV. B, summary of the effects on SMOCs of Ca²⁺-free external solution and caffeine. Each value was normalized to the control value. The boxes and the vertical bars represent means ±s.e.m. from 4 neurons.

Augmentation of SMOC amplitude by Ca²⁺-free external solution

Removing external Ca²⁺ significantly increased the SMOC amplitude (Fig. 5). Since individual distributions of SMOC amplitude often showed two or more amplitude groups (Fig. 6A), we examined amplitude histograms of SMOCs in Ca²⁺-free external solution. In some neurons, Ca²⁺-free external solution created an apparently new SMOC group with larger peak amplitudes (Fig. 6A, left panel). In other neurons, amplitude histograms of SMOCs simply shifted rightwards in Ca²⁺-free external solution (Fig. 6A, right panel). In no case did the amplitude histogram shift leftwards.

Figure 6. Manipulation of cytosolic Ca²⁺ concentration.

Amplitude threshold for detection was set to 5 pA for all data described in the figure. V_H was −50 mV. A, 2 representative amplitude histograms showing the enhanced SMOCs in Ca²⁺-free external solution. B, representative scatter plots during EGTA-AM (10 μm) or BAPTA-AM (1 μm) application. C, time course of normalized frequency during application of EGTA-AM or BAPTA-AM. Each value was normalized to the frequency just before the application of drugs. Each point and vertical bar represents mean ±s.e.m. from 3 neurons (EGTA-AM) or 4 neurons (BAPTA-AM).

In order to test whether the amplitude augmentation was related to decreased mean cytosolic Ca²⁺ concentration ([Ca²⁺]_i) resulting from reduced influx, we applied membrane-permeant Ca²⁺ chelators. Although EGTA-AM and BAPTA-AM have a similar affinity for Ca²⁺, BAPTA has a much faster association rate than EGTA (Naraghi, 1997). Introduction of EGTA-AM (10 μm) only gradually inhibited the SMOC frequency and failed to eliminate SMOCs within a period of 40 min. BAPTA-AM (1 μm) rapidly decreased and then eliminated SMOCs within 30 min of application (Fig. 6C). Neither Ca²⁺ chelator increased the amplitude of the SMOCs. These findings suggest that the increased SMOC amplitude arising from removal of external Ca²⁺ is unrelated to the reduced [Ca²⁺]_i.

DISCUSSION

In this report, we investigated SMOCs in neurons that were mechanically dissociated from rat nucleus basalis of Meynert without using enzymes. This is the first report demonstrating SMOCs in mammalian central neurons and describes the role of Ca²⁺ in their regulation.

Mechanism for generating SMOCs

The proposed mechanism by which SMOCs in CNS neurons are generated and/or modulated is shown in Fig. 7. K⁺ currents through SK channels are activated by localized elevation of [Ca²⁺]_i. The main trigger for these Ca²⁺ transients is probably the spontaneous Ca²⁺ spark-like release through RyRs on SR/ER stores. Ca²⁺ influx through VDCCs is only indirectly related to SK channels. Considering the reported link between L-type VDCCs and SK channels in hippocampal neurons (Marrion & Tavalin, 1998) and the proposed functional triad of RyRs, VDCCs and Ca²⁺-activated K⁺ channels in bullfrog sympathetic neurons (Akita & Kuba, 2000), these three components may also form a functional unit in Meynert neurons.

Figure 7.

Proposed mechanism for the generation of SMOCs in rat Meynert neurons.

CNS SMOCs are K⁺ currents through SK channels

Unlike peripheral neurons (Mathers & Barker, 1984; Satin & Adams, 1987; Fletcher & Chiappinelli, 1992; Merriam et al. 1999) or various myocytes (Benham & Bolton, 1986; Nelson et al. 1995; Bychkov et al. 1997), SMOCs in rat Meynert neurons were completely blocked by apamin. Since charybdotoxin, iberiotoxin and TEA failed to inhibit SMOCs, Meynert neuron SMOCs may depend entirely on SK channels. This result agrees with the report that spontaneous hyperpolarization observed in rat dopaminergic neurons was specifically blocked by apamin (Seutin et al. 2000).

Contribution of ryanodine/caffeine-sensitive Ca²⁺ release from SR/ER

In rat Meynert neurons, 1 mm caffeine increased the SMOC frequency. Conversely, ryanodine applied in concentrations higher than 10 μm reversibly blocked SMOCs. These data are consistent with an important role for RyRs in generating SMOCs in these neurons. Experimental manoeuvres (CPA, thapsigargin or high concentrations of caffeine in Ca²⁺-free external solution) that depleted the SR/ER Ca²⁺ content inhibited SMOCs. Such results also suggest that Ca²⁺ release from intracellular stores significantly contributes to SMOC activation.

Chelation of intracellular free Ca²⁺ by addition of BAPTA-AM reduced both the amplitude and the frequency of SMOCs before eliminating the events after 20-30 min (Fig. 6B). EGTA-AM was ineffective. The difference of dynamic Ca²⁺ buffering between BAPTA and EGTA shows that the temporal and spatial domain of the Ca²⁺ transient is quite localized so that only BAPTA could effectively buffer the Ca²⁺ spark-like activity (Neher, 1998; Merriam et al. 1999). Electron micrographs in Helix neurons indicate that the gap between the plasma and SR/ER membrane is as small as 20 nm (Akaike et al. 1983).

Ca²⁺ influx plays a limited role in generating SMOCs

Cd²⁺, a blocker of VDCCs, reduced the SMOC frequency at holding potentials more depolarized than −50 mV. However, the majority of SMOCs were unaltered by VDCC blockade. In the presence of Cd²⁺, the remaining events still increased in frequency during depolarization. In contrast, Ca²⁺-free external solution had the additional effect of reducing SMOC frequency by 90 %.

Cd²⁺ at 100 μm completely blocked VDCCs in frog sympathetic neurons at a V_H of −50 mV (Thevenod & Jones, 1992). In Meynert neurons, 100 μm Cd²⁺ eliminated the I_Ca during current injection (data not shown). However, brief openings of incompletely or intermittently blocked VDCCs might be sufficient to modulate SMOC activity. Such remaining VDCC activity might account for the difference between Cd²⁺-containing and Ca²⁺-free external solutions.

Cd²⁺ application did not eliminate SMOCs. Ca²⁺-free external solution did not eliminate SMOCs until repeated exposure to caffeine apparently depleted Ca²⁺ stores. Thapsigargin- or CPA-treated neurons, which could no longer generate SMOCs, probably have functioning VDCCs but depleted Ca²⁺ stores. This observation suggests that Ca²⁺ stores are essential and VDCCs alone are not sufficient to support SMOC activity. Thus, Ca²⁺ entry through VDCCs may indirectly modulate SMOCs. However, the possibility that Ca²⁺ entry through VDCCs might trigger SMOCs indirectly by Ca²⁺-induced Ca²⁺ release (CICR) cannot be excluded since depletion of Ca²⁺ stores also blocks CICR. SMOCs may be a mixture of SK channel currents generated by either of two processes sharing the same RyR: spontaneous or CICR-induced Ca²⁺ sparks.

Augmentation of SMOC amplitude by Ca²⁺-free external solution

If [Ca²⁺]_i modulates SMOCs, the fact that SMOC amplitude significantly increased in Ca²⁺-free external solution is quite confusing. The baseline current was shifted inward during application of Ca²⁺-free external solution containing EGTA. This baseline shift was less obvious in nominally Ca²⁺-free external solution. There were no baseline shifts following application of EGTA-AM or BAPTA-AM. It should also be noted that in nominally Ca²⁺-free solution, the SMOC amplitude was not increased as much as that in the EGTA-containing solution.

Changes in baseline current did not affect the event detection or amplitude measurement. No amplitude histograms showed a leftward shift during application of Ca²⁺-free external solution. Thus, this observation was probably an actual increase in the SMOC amplitude rather than an artifact.

Our next question was whether this augmentation was due to an enhanced Ca²⁺ spark. If so, it may be hypothesized that lowered [Ca²⁺]_i increased the Ca²⁺ driving force from the SR/ER to the cytoplasm. The inward shift of baseline current during perfusion of Ca²⁺-free external solution containing EGTA can also be explained by a reduction of baseline Ca²⁺-activated K⁺ current. An alternative explanation regarding lowered [Ca²⁺]_i is that SK channels may have different sensitivities to Ca²⁺ sparks depending on the baseline [Ca²⁺]_i. However, the SMOC amplitude was never increased by applying EGTA-AM or BAPTA-AM (Fig. 6B). If lowered baseline [Ca²⁺]_i was responsible for this observation, the SMOC amplitude should have been augmented before the SMOCs were eliminated by BAPTA-AM application. This result suggests that the amplitude augmentation by Ca²⁺-free, EGTA-containing external solution is unlikely to be related to a lowered [Ca²⁺]_i.

The remaining possible explanations for this amplitude augmentation and baseline shift include the changed surface potential by Ca²⁺-free external solution containing EGTA. Complete depletion of Ca²⁺ from the membrane surface will mimic membrane depolarization and thus enhance the K⁺ driving force (Hille, 1992). An intramembrane voltage sensor would also see a change in field potential, which is equivalent to a membrane depolarization. This affects Na⁺ channels, K⁺ channels and Ca²⁺ channels and may account for the shift of the baseline current observed during application of Ca²⁺-free external solution containing EGTA. However, in this study we could not determine the ion(s) that caused the shift in the baseline current. Thus our explanation remains inconclusive. The side effect of EGTA itself should also be taken into consideration.

Experimental uniqueness of mechanically dissociated neurons

SMOCs/STOCs have almost exclusively been reported in cultured or isolated cells. The existence of SMOCs in more intact systems is unclear. It should be noted that open patch conventional recordings failed to reveal SMOCs in similarly dissociated rat Meynert neurons (results not shown). Thus, SMOCs appear to be sensitive to intracellular conditions that are disrupted by the conventional whole-cell methods used most commonly.

We originally developed this technique to preserve functioning native synaptic terminals on isolated neurons (Rhee et al. 1999; Koyama et al. 1999). This method has two advantages that may contribute to the successful observation of SMOCs. First, degradation of important functional proteins is avoided by eliminating proteolytic enzymes that are often used to dissociate cells. Second, the lack of trituration common in dissociation methods using enzymes reduces cellular trauma and preserves significant portions of proximal neuron processes. In these minimally disturbed isolated neurons that may represent important mechanisms in vivo, we have shown the common existence of SMOCs whose function, however, remains poorly understood.