Investigation of the role of intracellular Ca²⁺ stores in generation of the muscarinic agonist-induced slow afterdepolarization (sADP) in guinea-pig olfactory cortical neurones in vitro

Abstract

1. Intracellular recordings were made from guinea-pig olfactory cortical brain slice neurones to assess the possible role of intracellular Ca²⁺ stores in the generation of the slow post-stimulus afterdepolarization (sADP) and its underlying tail current (I_ADP), induced by muscarinic receptor activation.

2. Caffeine or theophylline (0.5–3 mM) reduced the amplitude of the I_ADP (measured under ‘hybrid' voltage clamp) induced in the presence of the muscarinic agonist oxotremorine-M (OXO-M, 10 μM) by up to 96%, without affecting membrane properties or muscarinic depolarization of these neurones.

3. The L-type Ca²⁺ channel blocker nifedipine (1, 10 μM) also inhibited I_ADP (by up to 46%), while ryanodine (10 μM) (a blocker of Ca²⁺ release from internal stores) produced a small (∼10%) reduction in I_ADP amplitude; however, neither 10 μM dantrolene (another internal Ca²⁺ release blocker) nor the intracellular Ca²⁺ store re-uptake inhibitors thapsigargin (3 μM) or cyclopiazonic acid (CPA, 15 μM) affected I_ADP amplitude.

4. IBMX (100 μM), a phosphodiesterase inhibitor, also had no effect on I_ADP. Furthermore, inhibition of I_ADP by caffeine was not reversed by co-application of 100 μM adenosine.

5. Caffeine (3 mM) or nifedipine (10 μM) reduced the duration of presumed Ca²⁺ spikes revealed by intracellular Cs⁺ loading. When applied in combination, nifedipine and caffeine effects were occlusive, rather than additive, suggesting a common site of action on L-type calcium channels.

6. We conclude that Ca²⁺-induced Ca²⁺ release (CICR) from internal stores does not contribute significantly to muscarinic I_ADP generation in olfactory cortical neurones. However caffeine and theophylline, which enhance CICR in other systems, blocked I_ADP induction. We suggest that this action might involve a combination of L-type voltage-gated Ca²⁺ channel blockade, and a direct inhibitory action on the putative I_ADP K⁺ conductance.

Introduction

A wide variety of mammalian central neurones have been shown to exhibit afterdepolarizing responses following stimuli ranging from a single spike (Higashi et al., 1993; Haj-Dahmane & Andrade, 1997) to multiple spike discharges (Constanti et al., 1993; Libri et al., 1996). Such afterpotentials are important in governing neuronal repetitive firing, and can either occur intrinsically (Costa et al., 1991; Li & Hatton, 1997), or appear following activation of various receptor systems e.g. muscarinic receptors (Constanti et al., 1993; Haj-Dahmane & Andrade, 1998), metabotropic glutamate receptors (Greene et al., 1994; Libri et al., 1997), 5-hydroxytryptamine (5-HT) receptors (Araneda & Andrade, 1991) or even GABA_A-operated channels (Cerne & Spain, 1997). The dependence of some of these spike afterdepolarizations on cellular Ca²⁺ entry has been confirmed by studies on cortical neurones (Schwindt et al., 1988; Andrade, 1991), as well as other systems such as dorsal root ganglia (White et al., 1989) and supraoptic nucleus neurones (Li et al., 1995). Furthermore, it has been reported that L-type voltage-gated Ca²⁺ channels and release of Ca²⁺ from intracellular stores (Ca²⁺⁻-induced Ca²⁺ release; CICR) play an important role in generation of intrinsic depolarizing afterpotentials in the latter neurone type (Li & Hatton, 1997).

In vitro exposure of adult olfactory cortical neurones to muscarinic (or metabotropic glutamate) agonists has been shown to induce postsynaptic excitatory effects such as a slow depolarization, repetitive spike discharge, and appearance of a slow post-stimulus afterdepolarizing potential (sADP), which was strongly reliant on Ca²⁺ influx, and believed to involve a Ca²⁺-dependent de- and subsequent re-activation of a novel potassium conductance (Constanti & Bagetta, 1991; Constanti et al., 1993; Libri et al., 1994; 1996). This phenomenon was thought to contribute to the characteristically prolonged muscarinic (or metabotropic) excitation of olfactory and perhaps other mammalian cortical neurones, and may therefore be implicated in the formation of memory traces, or the induction and maintenance of epileptiform discharges under certain conditions (Constanti et al., 1993; Libri et al., 1997). However, the mode of Ca²⁺ entry or the possible involvement of releasable intracellular Ca²⁺ stores in generation of the olfactory cortical sADP have not been investigated.

Methylxanthines such as caffeine and theophylline have a wide range of pharmacological actions on central neurones (e.g. direct interaction with voltage-gated Ca²⁺ channels, inhibition of phosphodiesterases and direct antagonism of adenosine receptors; for review see Fredholm et al., 1999). However, one of the most well known actions of these compounds is the mobilization of Ca²⁺ from neuronal intracellular stores through interaction with intracellular ryanodine receptors (for reviews see Nehlig et al., 1992; Sawynok & Yaksh, 1993; Kuba, 1994). In view of the findings of Li & Hatton (1997) showing that caffeine-induced Ca²⁺ release from internal stores could enhance the amplitude of Ca²⁺-dependent depolarizing afterpotentials recorded in supraoptic nucleus neurones, and also taking into account previous reports of the role of neuronal Ca²⁺ entry and intracellular Ca²⁺ release in the generation of the slow afterhyperpolarization (sAHP) in guinea-pig vagal neurones (Sah & McLachlan, 1991), rabbit otic ganglion cells (Yoshizaki et al., 1995) or hippocampal pyramidal cells (Tanabe et al., 1998), we were interested in examining the possible role of Ca²⁺ release from caffeine-sensitive intracellular stores in olfactory cortical sADP generation. The effects of caffeine, and some other agents which modulate calcium entry/release from intracellular stores, upon muscarinic induction of the sADP and its underlying inward tail current (I_ADP, measured under ‘hybrid' voltage clamp) were therefore investigated under the present conditions. A preliminary report of these findings has recently been presented to the British Pharmacological Society (Postlethwaite et al., 1999).

Methods

Preparation and storage of brain slices

Slices of guinea-pig olfactory cortex were prepared and stored as described previously by Constanti et al. (1993). Briefly, adult albino guinea-pigs (250–400 g, of either sex) were anaesthetized using halothane, decapitated, and the brain rapidly removed. A rectangular block of tissue was then prepared incorporating the lateral olfactory tract, the olfactory cortex and surrounding regions, which was stuck onto the cutting stage of a Campden vibroslice/M tissue cutter with cyanoacrylate glue. Transverse slices of ∼450 μm thickness were then cut in ice cold (∼4°C Krebs solution), transferred to a storage chamber and incubated in oxygenated Krebs at 32°C for 30 min–1 h before transferring to the recording chamber maintained at 30°C. The composition of the Kreb's solution was (mM): NaCl 118; KCl 3; CaCl₂ 1.5; NaHCO₃ 25; MgCl₂.6H₂O 1 and D-glucose 11 (bubbled with 95% O₂ : 5% CO₂, pH 7.4).

Recording and stimulation

Recordings were taken from neurones in cell layers II–III of the periamygdaloid area of the slices with glass microelectrodes filled with 4 M potassium acetate (tip resistance 50–80 MΩ), using conventional intracellular current-clamp/voltage-clamp techniques. An Axoclamp 2A sample-and-hold preamplifier was used with switching frequency 2–3 kHz, and 30% duty cycle. Membrane resistance and firing properties were measured by passing positive or negative current pulses of increasing intensities (∼0.5–3 nA, 160 ms duration) and the resulting electrotonic potential recorded. During applications of muscarinic or metabotropic receptor agonists, the membrane potential was routinely depolarized to −70 mV (close to firing threshold) by applying steady depolarizing current; tests with other compounds, or experiments to test the effects of caffeine on the muscarinic-induced depolarization, were carried out at resting potential (∼−84 mV).

For evoked synaptic responses, a bipolar nichrome wire electrode (50 μM diameter, insulated except at the tip) was used to deliver orthodromic stimuli to association fibres in cortical layer III projecting to layer II–III neurones. Postsynaptic potentials (PSPs) were evoked using stimuli of increasing intensity (5–30 V) and constant duration (200 μs) delivered by a Digitimer isolated DS2 stimulator. Recordings of synaptic potentials were routinely taken at −84 mV membrane potential for comparison, and stimulus strength was adjusted so that the evoked excitatory PSP was just subthreshold for evoking an orthodromic action potential. All recordings of membrane and synaptic responses were taken before and during application of drugs so that each neurone served as its own control.

Membrane current and voltage signals were sampled and then fed to a computer (Viglen 4DX2-66, Viglen Ltd., U.K.) via a Digidata 1200 analogue-to-digital interface (Axon instruments, Foster City, CA, U.S.A.) using pCLAMP 6.03 software (Axon instruments) for recording and subsequent off-line analysis. Data were also recorded on a Gould RS3200 ink-jet chart recorder. Data are presented as mean±s.e.mean, and where appropriate, differences between groups are expressed as percentage change in comparison to control. Statistical significance between data groups was assessed by paired t-tests performed on the original recorded data, prior to any transformation or calculation of percentages.

Drugs

Drugs used in this study included caffeine, theophylline and nifedipine (all from Sigma Ltd, U.K.), 3-isobutyl-1-methylxanthine (IBMX), ryanodine, dantrolene, thapsigargin, cyclopiazonic acid (CPA) (all kindly donated by Sigma Ltd, U.K.), oxotremorine-M iodide (OXO-M) (Semat Ltd, U.K.), 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (Tocris Cookson Ltd, Bristol, U.K.) and ω-conotoxin GVIA (ω-CTx GVIA) (Alomone Laboratories, Jerusalem, Israel). Except where specified, all drugs were prepared in Krebs solution from stock solutions predissolved in water. Caffeine and theophylline were routinely dissolved directly into Krebs solution on the day of the experiment, whereas IBMX, thapsigargin, dantrolene and nifedipine were pre-dissolved in dimethylsulphoxide (DMSO); 1S,3R-ACPD was pre-dissolved in 1 : 1 equivalent of 100 mM sodium hydroxide (NaOH) solution. ω-CTx GVIA was dissolved in distilled water at 100 μM; small aliquots were then prepared and stored at −20°C until required. The aliquots were diluted to a final concentration of 200 nM in bathing medium immediately before use. To minimize nonspecific binding of toxin to plastic tubes, all toxin solutions contained 0.1 mg/ml cytochrome C and 1 mg/ml lysozyme (both from Sigma Ltd., U.K.). (Magistretti et al., 1999). Nifedipine, dantrolene and cytochrome C were weighed and dissolved in semi-darkness, due to their light sensitive nature, and subsequently stored in containers wrapped in silver foil to exclude light. Precautions were also taken to minimise their exposure to light during applications of these agents to cells. Final bath concentrations of DMSO (up to 0.5%), NaOH (up to 0.1%), cytochrome C (0.1 mg/ml) or lysozyme (1 mg/ml) had no deleterious effects on neuronal membrane properties or muscarinic/metabotropic responsiveness.

Results

Intrinsic neuronal membrane properties

Stable intracellular recordings of 1–5 h duration were obtained from a total of 63 guinea-pig olfactory cortical neurones. In control solution, neurones electrophysiologically identified as ‘deep' (layer II–III) cells (Libri et al., 1994) had a mean resting membrane potential of −84.3±0.3 mV, spike amplitude (measured from baseline) of 114±0.8 mV, and a mean resting input resistance (calculated from <20 mV hyperpolarizing electrotonic potentials) of 40.0±1.5 MΩ; their electrophysiological properties and firing behaviour were typical for pyramidal cells in rodent cerebral cortex (Connors & Gutnick, 1990). Neurones electrophysiologically identified as superficial pyramidal cells (n=7) were previously reported to be unresponsive to muscarinic or metabotropic agonists (Libri et al., 1994), so were regularly discarded.

Effects of caffeine on muscarinic or metabotropic glutamate receptor responses

A prolonged application (2–5 min) of the muscarinic receptor agonist OXO-M (10 μM) or the metabotropic glutamate agonist 1S,3R-ACPD (10 μM) to the olfactory neurones resulted in a slow membrane depolarization (mean=8.3 ±0.4 mV in OXO-M, n=36; or 5.0±1.5 mV in 1S,3R-ACPD, n=3) with superimposed repetitive spike discharge (∼10 Hz), coupled with an increase in membrane input resistance (8.5±3.6% in OXO-M or 22.4±7.6% in 1S,3R-ACPD, measured from −70 mV membrane potential). Exposure of these responding neurones to 3 mM caffeine (15 min) did not notably alter the slow depolarization response profile and subsequent repetitive firing induced at −70 mV membrane potential by either OXO-M (n=3) or 1S,3R-ACPD (n=3; not shown). Caffeine (3 mM) also had no significant effect on the muscarinic depolarization amplitude measured from a more negative membrane potential (−84 mV), where no induced firing occurred (mean amplitude=13±4 mV in OXO-M, and 12.5±0.5 in OXO-M plus 3 mM caffeine; P>0.05; n=3; Figure 1A). 3 mM caffeine, applied alone in control solution (n=5), also had no measurable effect on the intrinsic membrane properties of recorded cells.

Figure 1.

Effects of 3 mM caffeine on the muscarinic depolarization, sADP and I_ADP observed in olfactory cortical neurones. (A) Shows the effect of applying 10 μM oxotremorine-M (OXO-M, 6 min; indicated by solid bar below trace) to a neurone at the resting membrane potential (−84 mV) in control solution, and in the presence of 3 mM caffeine. Note that caffeine had no effect on the amplitude of the slow muscarinic depolarization of the cell (continuous chart recording; chart speed was slowed during drug washout). (B) (Different neurone maintained at −70 mV membrane potential). A prolonged depolarizing stimulus (1.6 s, +2 nA) induces a post-stimulus sAHP in control solution (left panel). In the presence of 10 μM OXO-M, the same current injection induces a post-stimulus sADP with superimposed repetitive firing (middle panel). In 3 mM caffeine (15 min) the sADP and associated repetitive firing induced by OXO-M were dramatically reduced (right panel); (in both cases, a small direct negative current was applied at the open arrow leading to a gradual hyperpolarization of the cell to the original ‘holding' potential. (C) Corresponding slow tail currents measured under ‘hybrid' voltage-clamp at −70 mV. Left panel shows the outward tail current underlying the sAHP recorded in control solution. Middle panel shows the slow inward tail current underlying the sADP (I_ADP) recorded in OXO-M. Further addition of caffeine produced a clear reduction in the I_ADP amplitude (right panel).

Effects of caffeine on sADP and I_ADP

As previously reported (Constanti et al., 1993; Libri et al., 1997), application of 10 μM OXO-M or 10 μM 1S,3R-ACPD induced the appearance of a sADP in response to a large (1.6 s) depolarizing stimulus, which replaced the sAHP evoked in control solution (Figure 1B). The mean sADP amplitude (measured at −70 mV) was 8.7±0.4 mV (range 4–12 mV) in OXO-M (n=38) and 7.0±1.5 mV (range 4–9 mV) in the presence of 1S,3R-ACPD (n=3). The underlying inward tail current (I_ADP) revealed under ‘hybrid' voltage clamp (Pennefather et al., 1985), at −70 mV holding potential, had a mean amplitude of 0.31±0.02 nA (range 0.10–0.67 nA) and mean duration 37.9±1.6 s (range 20–67 s) in OXO-M (n=42) (Figure 1C) and 0.28±0.01 nA amplitude, 27.3±3.2 s duration in 1S,3R-ACPD (n=3, not shown). Induction of the sADP (and I_ADP) were readily reversible upon washout of either OXO-M or 1S,3R-ACPD from the bathing solution.

Despite its lack of effect on muscarinic agonist responses, application of caffeine consistently resulted in a dramatic reduction in the amplitude of the OXO-M-induced sADP and its underlying tail current I_ADP (Figure 1B,C). This reduction was concentration-dependent, being reduced by 31.6±1.4% and 30.2±3.0% in 0.5 mM (n=3), 26.7±14.5% and 51.5±15.5% in 1 mM (n=3), 60.8±5.8% and 74.5±9.1% in 2 mM (n=3) and 64.1±15.0% and 74.2±8.1% in 3 mM caffeine respectively (n=8) (all reductions significantly different from control, by paired t-tests performed on original untransformed data; P<0.05) (Figure 2A). Caffeine (3 mM) also significantly reduced the sADP and I_ADP induced by 10 μM 1S,3R-ACPD (77.8±13.8% and 73.3±15.0% respectively; n=3, P<0.05, by t-tests: not shown). All effects were readily reversible after 15–20 min of caffeine washout.

Figure 2.

The inhibition of the I_ADP by caffeine or theophylline is concentration-dependent. (A) shows the percentage reduction in I_ADP amplitude in the presence of 0.5 mM, 1 mM, 2 mM (n=3 at each concentration) or 3 mM (n=8) caffeine respectively. (B) shows the similar reduction of I_ADP amplitude in 0.5 mM, 1 mM, 2 mM or 3 mM theophylline (n=3 at each concentration). Data are presented as mean percentage reduction of the peak I_ADP amplitude (±s.e.mean) induced by caffeine or theophylline in the presence of 10 μM OXO-M. All measurements were performed at a holding membrane potential of −70 mV, following a ‘hybrid' voltage clamp protocol; a set current stimulus of +2 nA, 1.6 s was used throughout.

Theophylline, another methylxanthine which can release Ca²⁺ from intracellular stores (Smith et al., 1983), also significantly inhibited I_ADP in a concentration-dependent reversible manner, with a similar potency to caffeine; mean reduction was 41.1±8.4% in 0.5 mM, 68.9±9.3% in 1 mM, 85.5±0.4% in 2 mM and 96.2±3.9% in 3 mM theophylline (P<0.05, n=3; t-tests; Figure 2B).

Effects of caffeine on evoked synaptic potentials

Synaptic potentials could be elicited using an external stimulating electrode placed in layer III of the cortical slice. Focal stimulation (10–30 V; 0.2 ms) of the local association fibres resulted in the appearance of the characteristic excitatory/inhibitory postsynaptic potential (e.p.s.p/i.p.s.p) complex as described previously (Libri et al., 1996; 1997). No repetitive spike discharges were superimposed on the e.p.s.p, even following stimuli of maximal intensity (30 V) or depolarizing the membrane potential up to −60 mV (n=3); the threshold for appearance of an action potential was around −65 mV. The mean amplitude of evoked e.p.s.ps in control solution at −84 mV membrane potential was 14.6±0.7 mV (n=28). In the presence of 10 μM OXO-M, the mean e.p.s.p amplitude was reduced by 58±3% (n=28), and in 10 μM 1S,3R-ACPD it was reduced by 80±10% (n=3) (both reductions significant; P<0.05, t-tests; c.f. Libri et al., 1997). By contrast, in 3 mM caffeine, there was a clear and consistent augmentation of evoked synaptic transmission, the peak e.p.s.p amplitude being enhanced by 93±33% (n=5, P<0.05, t-test) (most probably due to inhibition of endogenous adenosine activity on presynaptic adenosine receptors; Motley & Collins, 1983; Haas & Greene, 1988) (Figure 3).

Figure 3.

Postsynaptic potentials (p.s.ps) recorded intracellularly at −84 mV membrane potential in response to orthodromic electrical stimuli (25 V, 0.2 ms) delivered locally to association fibre terminals; the stimulus strength was just subthreshold for evoking action potentials in control solution. Superimposed traces show p.s.ps evoked during the control period (C), after a 2 min bath-application of 10 μM oxotremorine-M (OXO-M) or after 15 min in 3 mM caffeine. A 30 min washout period was allowed between each drug application. Note the reduction in p.s.p amplitude induced by OXO-M, and the marked enhancement of synaptic transmission in the presence of caffeine (p.s.p now exceeding spike threshold).

Blockade of Ca²⁺ entry suppresses I_ADP

Blockade of L-type voltage-gated Ca²⁺ channels has been shown to inhibit the sAHP in hippocampal CA3 cells (Tanabe et al., 1998); the possible contribution of these channels towards sADP generation in olfactory neurones was therefore investigated by application of the selective L-type calcium channel blocker nifedipine (1 and 10 μM; Bean, 1989; Kostyuk, 1999). The amplitude of the OXO-M-induced I_ADP was reduced in a concentration-dependent fashion by application of nifedipine at 1 μM or 10 μM concentration. In 1 μM nifedipine, the mean I_ADP amplitude was reduced from 0.10±0.1 nA to 0.08±0.01 nA, a reduction of 28.5±1.5% (n=3) while at 10 μM nifedipine, the mean I_ADP amplitude was reduced from 0.39±0.09 nA to 0.21±0.05 nA, a decrease of 41.7±4.8% (n=4) (both reductions significantly different from control; P<0.05; Figure 4); the effects of nifedipine were reversed after 40 min of washout.

Figure 4.

Inhibitory effects of the L-type Ca²⁺ channel blocker nifedipine on the sADP and I_ADP measured in a single olfactory cortical neurone. (A) shows the control sADP and I_ADP recorded in 10 μM OXO-M at −70 mV membrane holding potential respectively. (B) shows the sADP and I_ADP recorded in the same neurone in the presence of 10 μM OXO-M and 10 μM nifedipine; note the reduction in sADP and I_ADP amplitude. Stimulus parameters were +2 nA, 1.6 s throughout.

By contrast, the peptide toxin ω-conotoxin GVIA (ω-CTx-GVIA) (200 nM, n=4), which is known to selectively block N-type Ca²⁺ channels in central neurones (Magistretti et al., 1999), produced only a small and variable reduction in the OXO-M-induced I_ADP (amplitude decreased from 0.16±0.04 nA to 0.13±0.05 nA, a mean reduction of 18.8%); this effect was not significantly different from control (P>0.05). Note that this concentration of ω-CTx-GVIA was previously found to reduce the amplitudes of Ca²⁺-dependent spike afterdepolarizations in supraoptic nucleus neurones (Li & Hatton, 1997).

Effects of ryanodine receptor blockers and Ca²⁺ store uptake inhibitors on I_ADP

The possible role of CICR in generating the olfactory cortical sADP and the underlying I_ADP was investigated using the membrane-permeable inhibitors of intracellular Ca²⁺ release, ryanodine and dantrolene (Henzi & MacDermott, 1992). Further to this, the effects of intracellular Ca²⁺ store depletion were also tested by using the membrane permeable Ca²⁺ store uptake inhibitors thapsigargin and cyclopiazonic acid (CPA; Markram et al., 1995). Bath-application of ryanodine (10 μM, 15 min) reduced the I_ADP amplitude from 0.38±0.06 nA to 0.35±0.06 nA, a small but significant amount (mean reduction=9.9±4.2%, P<0.05, n=7, t-test; Figure 5A), while application of dantrolene (10 μM, n=3) for up to 40 min failed to reduce the sADP or I_ADP amplitudes (Table 1; Figure 5B).

Figure 5.

Effects of intracellular Ca²⁺ release modulators on I_ADP amplitude in the presence of 10 μM OXO-M. Left panels of each section show the control I_ADP evoked in OXO-M and right panels show I_ADP in the presence of drug. (A) Superfusion of 10 μM ryanodine caused a small but significant depression of the I_ADP (∼10%), whereas 10 μM dantrolene (B), 3 μM thapsigargin (C) or 15 μM cyclopiazonic acid (CPA) (D) had no obvious effect. In each experiment, I_ADP was recorded under ‘hybrid' voltage clamp at −70 mV holding potential, and was elicited using set stimulus parameters (+2 nA, 1.6 s). Scale bars refers to all recordings; (A–D were obtained from different neurones).

Table 1.

The effects of intracellular Ca²⁺ release modulators upon peak I_ADP amplitude recorded in olfactory cortical neurones

Thapsigargin and CPA have previously been shown to suppress neuronal Ca²⁺ uptake into intracellular stores, by inhibiting the Ca²⁺. ATPase in the endoplasmic reticulim, making less Ca²⁺ available for subsequent release (Markram et al., 1995). However, neither thapsigargin (3 μM, n=7) nor CPA (15 μM, n=3) reduced the amplitude of I_ADP, even after 30–40 min exposure to these agents (Table 1; Figure 5C,D).

Furthermore, in an attempt to determine whether the observed blocking actions of caffeine or ryanodine on the I_ADP could be mediated by Ca²⁺ release from intracellular stores, we examined the effects of these compounds in the presence of 3 μM thapsigargin; however, prior exposure (15 min) of neurones to this agent had no significant effect on the I_ADP-blocking actions of either 3 mM caffeine (n=3) or 10 μM ryanodine (n=3) (P>0.05 in both cases; t-tests).

Other possible actions of caffeine

Caffeine is known to have several different sites of action (Fredholm et al., 1999) apart from its classical interaction with intracellular Ca²⁺ store release channels (Sawynok & Yaksh, 1993; Kuba, 1994). Thus, caffeine also inhibits the enzyme phosphodiesterase, leading to an increase in intracellular cyclic AMP concentration (Sawynok & Yaksh, 1993), as well as being a direct antagonist at adenosine receptors (Daly et al., 1981; Greene et al., 1985). To test the former possibility, some experiments were carried out in the presence of the broad-spectrum phosphodiesterase inhibitor IBMX. Out of three neurones tested with 100 mM IBMX (in the continued presence of 10 μM OXO-M), none showed any reduction of the sADP or I_ADP amplitude compared to OXO-M alone, even after 30 min IBMX exposure; this suggested that phosphodiesterase inhibition could not account for the block of the I_ADP by caffeine.

In light of the previously reported inhibition of the hippocampal sAHP (and its underlying I_AHP) by caffeine (Haas & Greene, 1988), and its enhancement by applied adenosine (Haas & Greene, 1984), the possibility that caffeine might be affecting I_ADP by behaving as an antagonist of endogenously-released adenosine (possibly important for I_ADP generation) was also investigated (c.f. Motley & Collins, 1983). In these experiments, the I_ADP was initially recorded in the presence of 10 μM OXO-M alone, and then in the presence of 3 mM caffeine, which produced an almost complete blockade of the tail current (mean reduction=86.8±5.6%, n=3) and enhanced the evoked synaptic potentials (mean enhancement=144.2±66.7%, relative to amplitude in OXO-M; n=3) (Figure 6). Following this, 100 μM adenosine was added in an attempt to counteract any possible antagonism of endogenous adenosine receptors produced by caffeine. After 15 min exposure, the e.p.s.p amplitude was significantly reduced from 18.1±1.0 mV to 5.9±2.1 mV (mean reduction=73.1±14.7% relative to the enhanced level in caffeine/OXO-M; n=3; P<0.05, t-test), suggesting that adenosine was reversing the effect of caffeine presynaptically. However, there was no detectable difference between the I_ADP amplitude recorded in caffeine, or with added adenosine, suggesting that caffeine blockade of adenosine receptors was not responsible for the observed reduction in the I_ADP (Figure 6D).

Figure 6.

Attempts to reserve the effects of caffeine with applied adenosine. (A) shows the I_ADP tail current (top) and the evoked synaptic potential (bottom) recorded in control solution. (B) I_ADP and evoked synaptic potential recorded in the presence of 10 μM OXO-M alone. (C) Inhibition of the I_ADP and enhancement of the e.p.s.p. produced by adding 3 mM caffeine. (D) Application of 100 μM adenosine failed to reverse the inhibition of the I_ADP by caffeine, although the e.p.s.p. was reduced. Top scale bars refer to all current traces and bottom scale bars refer to all voltage traces. I_AHP and I_ADP were elicited under ‘hybrid' voltage clamp at −70 mV holding potential using set stimulus parameters (+2 nA, 1.6 s). Synaptic potentials were all recorded at −84 mV membrane potential, using identical stimulus parameters throughout (2.0 V, 0.8 ms).

A possible direct blockade of Ca²⁺ channels by caffeine

There are several reports showing a direct interaction of caffeine with calcium currents in cell membranes of muscle cells (Zholos et al., 1991; Varro et al., 1993; Yoshino et al., 1996), indicating an overall decrease in conductance through these channels. To examine whether caffeine was inhibiting I_ADP by blocking voltage-activated Ca²⁺ currents in the olfactory cortical neurones, cells were recorded with 2 M caesium chloride-filled electrodes. It has previously been shown that intracellular Cs⁺ loading blocks voltage-gated K⁺ channels, thereby delaying repolarization after an action potential, and revealing a characteristic plateau potential resulting from activation of voltage-gated Ca²⁺ channels (Galvan et al., 1985; Libri et al., 1996). Cells impaled with caesium chloride electrodes were routinely left for at least 15 min to allow the Cs⁺ to diffuse into the cell interior, after which the evoked action potential duration was lengthened accordingly (Figure 7A,B, left panels). The spike half-width, measured at half peak amplitude from baseline, was taken as a standard measure during the Cs⁺-loading experiments, and care was taken to ensure that all recordings were made at the same membrane potential. Superfusion of the neurones with 1 or 10 μM nifedipine significantly reduced the half-width of the first action potential in a concentration-dependent manner: at 1 μM, the mean half-width was reduced from 65.7±9.1 ms to 50.1±10.5 ms (mean reduction 27.3±7.1%; P<0.05, n=5; not shown), whereas at 10 μM, the half-width was reduced from 55.5±8.0 ms to 35.4±8.5 ms (mean reduction 42.8±8.8%, n=8; P<0.05, t-test), indicating that the plateau resulted, in part, from the activation of L-type voltage-gated Ca²⁺ channels (Figure 7A, middle panel). Likewise, superfusion of cells with 3 mM caffeine resulted in a significant reduction of the first spike half-width from 57.3±4.4 ms to 33.0±10.8 ms (45.0±15.0% reduction, n=3; P<0.05, t-test; Figure 7B, middle panel). No further reduction of the spike half-width was observed on applying 1 μM nifedipine in the presence of 3 mM caffeine (n=4; P>0.05; t-test; Figure 7A, right panel). Also, in the presence of 10 μM nifedipine, addition of 3 mM caffeine exerted no further significant reduction in spike half-width (n=3; P>0.05; t-test; Figure 7B, right panel). These data strongly suggest that caffeine was predominantly blocking voltage-gated (possibly L-type) Ca²⁺ conductances in the olfactory cortical neurones.

Figure 7.

Prolonged action potentials recorded with caesium chloride-filled microelectrodes, revealing calcium plateaus. (A) Depolarizing electrotonic potential evoked in control solution (left panel), with a clear calcium plateau present on the first and subsequent action potentials. In the presence of 10 μM nifedipine (15 min) a clear reduction is observed in the half-width of the initial spike (middle panel); however, no further change in first spike half-width occurred on co-applying nifedipine with 3 mM caffeine (right panel). (B) Recordings made from a different neurone show a similar reduction in initial spike half-width in 3  mM caffeine that was unaffected by further adding 1 μM nifedipine. Scale bars alongside refer to all traces. Current stimulus parameters were identical for each trace (+0.5 nA, 160 ms).

Discussion

The sADP and I_ADP in responding neurones

The sADP and its underlying tail current I_ADP could be readily revealed by application of a muscarinic or metabotropic (glutamate) agonist in neurones from layer II–III of the guinea-pig olfactory cortex as previously reported (Constanti & Bagetta, 1991; Constanti et al., 1993; Libri et al., 1997). The mechanism of induction of the sADP was originally proposed to involve a Ca²⁺-dependent inactivation of a novel ongoing K⁺ conductance (during the prolonged spike burst), with a subsequent slow reactivation of this current over several seconds, to give the sADP its characteristic long-lasting deflection (Constanti & Bagetta, 1991; Constanti et al., 1993). The principal aim of the present series of experiments was to establish whether CICR from intracellular Ca²⁺ stores contributes to this sADP induction process. To this end, a series of compounds were tested, which either augment or inhibit Ca²⁺ release from intracellular stores in other systems.

Caffeine inhibition of the sADP and I_ADP

Caffeine is now widely acknowledged as an agent which can enhance CICR from ryanodine-sensitive stores (for review see Fredholm et al., 1999), leading to profound effects on the activity of neurones in which this process is essential for normal function. Early studies on intracellular Ca²⁺ release used a relatively high dose of caffeine as standard (10 mM; Marrion & Adams, 1992; Nohmi et al., 1992); however, in several experiments where this concentration was applied to olfactory neurones, it led to rapid depolarization and cell death. For this reason, the concentrations of caffeine used in the present work were lowered, although at these doses, it became less likely that this agent was directly interfering with intracellular Ca²⁺ release.

3 mM caffeine was consistently found to inhibit I_ADP (and the concomitant sADP) induced by either OXO-M or 1S,3R-ACPD in a reversible manner; thus caffeine was not acting directly on either receptor system to cause the inhibition, an observation which was further confirmed by the apparent lack of inhibition of the muscarinic (or metabotropic) agonist-induced depolarizations by caffeine during these recordings. It may be noted however, that caffeine has been previously found to directly antagonize the effects of carbachol and the binding of ³H-N-methylscopolamine in rat pancreatic acini (Grosfils et al., 1996). Also, the inhibition of the OXO-M-induced I_ADP was concentration-dependent over the range of caffeine doses used (0.5–3 mM). The inhibition of I_ADP by caffeine was mimicked by theophylline, another member of the methylxanthine group; this inhibition was similar to that of caffeine, and was dose-dependent over a similar concentration range. Thus, the block of I_ADP by caffeine was not specific to this methylxanthine. Considering that both caffeine and theophylline can augment release of Ca²⁺ from intracellular stores (Endo, 1977; Smith et al., 1983; Kuba, 1994), and taking into account the proposed mechanism of induction of I_ADP (i.e. raised intracellular Ca²⁺ leading to a decrease in K⁺ conductance; Constanti et al., 1993), it seems very unlikely that these agents were further raising the intracellular Ca²⁺ level during a long depolarizing stimulus, since this would be expected to increase the I_ADP, amplitude, as seen in the case of the Ca²⁺-dependent depolarizing afterpotential recorded in supraoptic nucleus neurones (Li & Hatton, 1997). In addition, depletion of putative intracellular Ca²⁺ stores in the olfactory neurones by prior application of thapsigargin had no effect on the blocking action of caffeine on I_ADP, thereby suggesting that intracellular Ca²⁺ stores play little or no role in this inhibitory process. Caffeine and theophylline were therefore acting on other systems to cause I_ADP inhibition. Even if the olfactory cortical I_ADP had some other underlying ionic mechanism such as an increase in nonspecific cationic conductance, as proposed by Haj-Dahmane & Andrade (1998) in prefrontal cortex neurones, then one would still expect an increase in I_ADP amplitude in caffeine, since this cortical afterdepolarization was also dependent on intracellular Ca²⁺ concentration.

Intracellular Ca²⁺ stores – a role in I_ADP generation?

The importance of Ca²⁺ influx in the generation of the sADP and I_ADP allowed speculation about the possible functional role of CICR from intracellular stores in this process. Thus, application of the Ca²⁺ release channel blocker ryanodine caused a small (∼10%), yet significant, decrease in amplitude of I_ADP, implying that Ca²⁺ stores present within the olfactory neurones, were contributing towards generation of the slow afterpotential response. If so, then these putative stores would appear to have only a very limited role in this type of cellular activity, unlike the generation of depolarizing afterpotentials in the supraoptic nucleus (Li & Hatton, 1997) or the sAHP in hippocampal neurones (Torres et al., 1996; Tanabe et al., 1998). Furthermore, there was no significant reduction of I_ADP amplitude in the presence of the CICR blocker dantrolene, or the intracellular Ca²⁺ store depletors thapsigargin or CPA, again suggesting a minimal involvement of these stores in cortical I_ADP generation (c.f. supraoptic neurones or hippocampal neurones where these agents clearly reduced afterpotential amplitudes; Li & Hatton, 1997; Torres et al., 1996; Tanabe et al., 1998). The small blocking effect of ryanodine on I_ADP also persisted in the continued presence of thapsigargin, suggesting that intracellular Ca²⁺ release was not involved.

One possibility was that ryanodine reduced I_ADP by directly blocking Ca²⁺ currents in the plasma membrane, thereby preventing Ca²⁺ entry; however, there is currently no evidence that this alkaloid can directly block voltage-activated Ca²⁺ channels in neurones (e.g. Kawai & Watanabe, 1989). Vais et al. (1996) reported that ryanodine (and several other ryanoid compounds) could interfere with K⁺ fluxes in locust and mouse muscle membranes, suggesting that ryanodine could exert a weak block of the putative K⁺ conductance underlying the sADP; if this were the case, then one would expect to see a small depolarization during ryanodine superfusion. Since the observed reduction of I_ADP was very small, any effects of altering conductance through this type of channel would probably be masked by other background leak currents present, making such a depolarization difficult to detect.

The possible involvement of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores (Henzi & MacDermott, 1992) in I_ADP generation would have been difficult to assess, as there are currently no membrane permeant agents available which affect these sites. The use of heparin in recording pipettes, to inhibit IP₃-sensitive Ca²⁺ release (Abdul-Ghani et al., 1996; Li & Hatton, 1997) was considered impractical, since it was impossible to guarantee that recordings were always being made from ‘type 1' responding olfactory neurones (expressing I_ADP) at the outset (Libri et al., 1994).

Other pharmacological actions of caffeine

Caffeine is known to have a number of pharmacological actions in addition to augmenting intracellular Ca²⁺ release. Firstly, caffeine inhibits phosphodiesterase activity, thus leading to a buildup of intracellular cyclic adenosine monophosphate (cyclic AMP) within treated cells (for reviews see Nehlig et al., 1992; Sawynok & Yaksh, 1993). In order to examine whether this action of caffeine was responsible for I_ADP, inhibition, a more selective inhibitor of phosphodiesterase, IBMX, was added to the bathing solution in the presence of OXO-M. At this concentration, IBMX was expected to be a potent inhibitor of the phosphodiesterase (Beavo & Reifsnyder, 1990), while having little or no effect on intracellular calcium release per se, normally observed at millimolar concentrations (Usachev & Verkhratsky, 1995). However in our experiments, application of IBMX (in OXO-M) failed to reduce the amplitude of I_ADP, even after 20–30 min exposure; this would suggest that inhibition of phosphodiesterase (and increase of intracellular cyclic AMP) was not responsible for I_ADP inhibition.

It is interesting to note that IBMX can also act as an adenosine receptor antagonist at the concentration used in the present study (Schwabe et al., 1985; Prestwich et al., 1987). Considering that caffeine shares this action (Greene et al., 1985; Haas & Greene, 1988) it seems rather unlikely that pharmacological inhibition of endogenously-released adenosine (Motley & Collins, 1983) was involved in the effect. Furthermore, in experiments where a high concentration of adenosine was used in an attempt to overcome any possible antagonism of endogenous adenosine receptors imposed by caffeine, there was no reversal of the I_ADP depression, even though the e.p.s.p amplitude was clearly decreased (Figure 6D) (the latter was most likely due to activation of presynaptic (inhibitory) adenosine receptors; c.f. McCabe & Scholfield, 1985).

Caffeine affects calcium entry into neurones during generation of I_ADP

The original experiments of Constanti et al. (1993) clearly showed that neuronal Ca²⁺ entry was essential for the generation of I_ADP, insofar as removal of Ca²⁺ from the external bathing medium abolished the afterdepolarizing response. In the present study we found that 10 μM nifedipine (a potent and selective L-type calcium channel blocker; Bean, 1989; Kostyuk, 1999) inhibited the I_ADP by 42%, indicating a clear importance of these channels in I_ADP generation. However, the selective N-type calcium channel blocker ω-CTx GVIA (at 200 nM) only reduced I_ADP by an average of 20%, and in a highly variable fashion, suggesting that N-channels were less generally involved in I_ADP generation in these cells.

Since caffeine can directly interfere with inward calcium currents in muscle preparations (Zholos et al., 1991; Varro et al., 1993; Yoshino et al., 1996), it is conceivable that it was blocking Ca²⁺ influx through voltage-sensitive Ca²⁺ channels, and leading to an inhibition of the I_ADP. In recordings made in Cs⁺-loaded cells (to block outward repolarizing K⁺ conductances; Galvan et al., 1985; Libri et al., 1996), caffeine was able to significantly reduce the half-width of the initial spike in an evoked spike train, suggesting a blockade of voltage-gated Ca²⁺ currents. For comparison, 10 μM nifedipine produced a similar degree of spike shortening as was seen in the presence of caffeine (43 and 45% inhibition in nifedipine or caffeine respectively).

In additional experiments where 1 μM nifedipine was added to cells while in the presence of 3 mM caffeine, no additive blockade of the Ca²⁺ spike plateau was observed, suggesting that caffeine and nifedipine were acting at a common site, i.e. the L-type calcium channel. Moreover, applying 3 mM caffeine in the presence of 10 μM nifedipine exerted no further reduction in the Ca²⁺ spike half-width. It is thus likely that inhibition of I_ADP by caffeine may partly result from a direct block of an L-type Ca²⁺ conductance similar to that affected by nifedipine. However, it is worth noting that nifedipine inhibited I_ADP amplitude by ∼42% (similar to the 43% reduction in spike half-width), whereas caffeine was able to reduce I_ADP amplitude by ∼75% (c.f. spike half-width inhibition of 45%). A further blocking mechanism for caffeine was therefore suggested.

Block of I_ADP K⁺ conductance by caffeine?

One further action of caffeine which could explain the residual inhibition of I_ADP is a direct block of the putative I_ADP K⁺ conductance (Constanti et al., 1993). Caffeine is known to block certain types of K⁺ current, without interfering with intracellular signalling mechanisms (Greene et al., 1985; Yamamoto et al., 1995); e.g. the neuronal delayed rectifier (I_DR) and the transient outward current (I_A) in neurones (Reiser et al., 1996), or I_A in smooth muscle cells (Noack et al., 1990). It should be noted that these currents were claimed to be independent of intracellular Ca²⁺ concentration (which must always be a consideration when testing caffeine). Interestingly, theophylline has also been shown to block a Ca²⁺-independent K⁺ conductance in dissociated cortical neurones (Munakata & Akaike, 1993).

In conclusion, it is clear from the present experiments, that the olfactory cortical I_ADP tail current has a complicated mechanism of induction. It appears that Ca²⁺ entry during prolonged depolarizing stimuli takes place, at least in part, via L-type voltage-gated Ca²⁺ channels, and that agents capable of manipulating release of Ca²⁺ from intracellular stores have little or no effect on I_ADP generation. Similarly, inhibition of intracellular phosphodiesterase, or pharmacological manipulation of adenosine receptors in the slices does not affect I_ADP. It is therefore likely that the inhibition of I_ADP observed in caffeine was due in part, to a block of voltage-gated Ca²⁺ currents (reducing Ca²⁺ entry into the neurones), and also a direct block of the putative K⁺ conductance underlying the I_ADP. Further studies will be required to gain a full understanding of the mechanism(s) underlying this unusual afterpotential phenomenon.

Abbreviations

1S,3R-ACPD

1S,3R-aminocyclopentane-1,3-dicarboxylic acid

cyclic AMP

3′ : 5′-cyclic adenosine monophosphate

CICR

Ca²⁺-induced Ca²⁺ release

CPA

cyclopiazonic acid

DMSO

dimethylsulphoxide

E.P.S.P

excitatory post-synaptic potential

I_ADP

afterdepolarization current

I_AHP

afterhyperpolarization current

IBMX

3-isobutyl 1-methylxanthine

OXO-M

oxotremorine-M

PSP

postsynaptic potential

sADP

slow afterdepolarization

sAHP

slow afterhyperpolarization

ω-CTx GVIA

ω-conotoxin GVIA