4-Chloro-m-cresol, an activator of ryanodine receptors, inhibits voltage-gated K⁺ channels at the rat calyx of Held

Preparation and solutions

Transverse brainstem slices (175–250 µm thick) were prepared from Wistar rats (8–10 days old) killed by decapitation under halothane anesthesia. The slicing solution was the same as described previously (Forsythe & Barnes-Davies, 1993), except that equiosmolar concentrations of sucrose were substituted for NaCl and 6 mm MgCl₂ was the only divalent ion in the solution. The artificial cerebrospinal fluid (aCSF) contained (in mm): 125 NaCl, 2.5 KCl, 10 glucose, 26 NaHCO₃, 1.25 NaH₂PO₄, 2 CaCl₂, 1 MgCl₂, 3 myo-inositol, 0.5 ascorbic acid and 2 Na-pyruvate (pH 7.4, gassed with 95% O₂/5% CO₂). Soon after slicing, slices were incubated at 37 °C for 60 min in aCSF and thereafter stored at room temperature (24–28°C). For recording TIFCs evoked with a bipolar electrode, bicuculline methiodide (10 µm), strychnine hydrochloride (0.5 µm) and kynurenic acid (2 mm) were added to block inhibitory synaptic inputs and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor saturation (Kaneko & Takahashi, 2004). Tetrodotoxin (1 µm) was added for recording presynaptic K⁺ currents (I_pK). For recording presynaptic Ca²⁺ currents (I_pCa) and TIFCs simultaneously, tetrodotoxin (1 µm), tetraethylammonium (TEA)-Cl (10 mm), equimolar replacement of NaCl, d-(–)-2-amino-5-phosphonopentanoic acid (50 µm) and kynurenic acid (1 or 2 mm) were added. 4-CmC was dissolved in dimethylsulphoxide to a final concentration of less than 0.1% and the same concentration of dimethylsulphoxide was included in control solutions. The postsynaptic patch pipette solution contained (in mm): 110 CsF, 30 CsCl, 10 HEPES, 5 EGTA, 1 MgCl₂ and 5 QX-314 (pH 7.3, adjusted with CsOH). For recording action potentials (APs), the presynaptic patch pipette solution contained (in mm): 87.5 K-gluconate, 10 K-glutamate, 32.5 KCl, 10 HEPES, 0.5 EGTA, 12 Na₂-phosphocreatine, 1 MgCl₂, 2 Mg-ATP and 0.5 Na-GTP (pH 7.3, adjusted with KOH). For recording I_pK, the pipette solution contained (in mm): 97.5 K-gluconate, 32.5 KCl, 10 HEPES, 5 EGTA, 12 Na₂-phosphocreatine, 1 MgCl₂, 2 Mg-ATP and 0.5 Na-GTP (pH 7.3, adjusted with KOH). For recording I_pCa, the pipette solution contained (in mm): 110 Cs-gluconate, 10 Cs-glutamate, 30 CsCl, 10 HEPES, 0.5 EGTA, 12 Na₂-phosphocreatine, 1 MgCl₂, 3 Mg-ATP and 0.5 Na-GTP (pH 7.3, adjusted with CsOH) or 100 CsCl, 10 Cs-glutamate, 40 HEPES, 10 TEA-Cl, 0.5 EGTA, 12 Na₂-phosphocreatine, 1 MgCl₂, 2 Mg-ATP and 0.5 Na-GTP (pH 7.3, adjusted with CsOH).

Tetrodotoxin and ryanodine were obtained from Wako Pure Chemical (Osaka, Japan). Kynurenic acid and d-(–)-2-amino-5-phosphonopentanoic acid were obtained from Tocris Cookson (Bristol, UK). 4-CmC was obtained from Calbiochem (Darmstadt, Germany).

Recording

Whole-cell patch-clamp recordings were made from medial nucleus of the trapezoid body principal neurones, presynaptic calyces or simultaneously from both structures. When slices were ready for use, they were transferred to a recording chamber and continuously perfused with aCSF at a speed of 1.3–1.6 mL/min. Principal neurones and calyces were visually identified using a 60× water immersion lens (Olympus, Tokyo, Japan) attached to an upright microscope (Axioskop; Zeiss, Goettingen, Germany). Voltage-clamp and current-clamp recordings were performed using a patch-clamp amplifier (Axopatch 200B or Multiclamp 700A; Axon Instruments, Union City, CA, USA). Pipettes for pre- and postsynaptic recordings had resistances of 3–8 and 2–5 MΩ, respectively. Pre- and postsynaptic series resistances were 5–18 and 3–15 MΩ, respectively. The postsynaptic series resistance was not compensated for but data were discarded when the series resistance changed by >30%. The presynaptic series resistance was routinely compensated by 70–80%. Leak currents were subtracted for presynaptic currents by the scaled pulse (P/8) protocol. The liquid junction potential between the pipette solution and aCSF was not corrected. For recording single postsynaptic currents, TIFCs were evoked every 20 s by extracellular stimulation of presynaptic axons using a bipolar tungsten or platinum electrode. Only those TIFCs showing all-or-none responses to changes in stimulus intensity were selected. For recording I_pK or I_pCa, calyces were voltage-clamped at a holding potential of −70 or −80 mV and depolarizing voltage sttif were applied every 15–20 s. The presynaptic APs shown in Fig. 2A were elicited by a brief (1 ms) depolarizing pulse and those shown in Fig. 7A were evoked by afferent fibre stimulation. All experiments were performed at room temperature.

Data analysis

Records were low-pass filtered at 5 kHz and digitized at 20–100 kHz by a Digidata 1320A analogue/digital converter with pCLAMP9 software (Axon Instruments). Data were analysed using Clampfit (Axon Instruments). All values are given as means ± SEM. Statistical comparisons were made using Student's paired t-test, unless otherwise noted. A value of P < 0.05 was taken as the level of significance.

Immunocytochemistry

Immunocytochemical procedures were as previously described (Ishikawa et al., 2003). Briefly, Wistar rats (8 or 14 days old) were transcardially perfused with a fixative (4% paraformaldehyde and 0.1% picric acid in 0.1 m Na-phosphate buffer, pH 7.4). After fixation, rats were decapitated and tissue blocks were postfixed and cryoprotected. Transverse slices (30 µm thick) were cut using a cryostat (CM3050; Leica, Nussloch, Germany). The sections were then processed for blocking the tissues with phosphate-buffered saline containing 5% skimmed milk and 0.3% Triton X-100, followed by application of primary antibodies (Chemicon, Temecula, CA, USA). Presynaptic RyRs were detected with rabbit anti-type 1, type 2 or type 3 RyR antibody (diluted 1 : 500) and anti-synaptophysin antibody (diluted 1 : 200). Goat secondary antibodies (Invitrogen, Eugene, OR, USA) (diluted 1 : 200) conjugated with alexa fluor 488 and 568 were used to visualize RyR and synaptophysin signals, respectively. As neither the epitope peptides of RyRs nor their sequences were available, we treated slices only with anti-synaptophysin primary antibody (with no RyR antibody) and subsequently with the mixture of secondary antibodies as a control for background signals.

Stained sections were viewed using a 100 × oil-immersion objective (numerical aperture 1.35) attached to a confocal laser-scanning microscope (FLUOVIEW FV300; Olympus). Emission wavelengths were 510–550 nm for RyRs and > 610 nm for synaptophysin. For the RyR signal acquisition, the argon laser power (488 nm, 10 mW) was reduced to 0.18% (for type 1 RyR), 1.2% (for type 2 RyR and negative control) or 0.42% (for type 3 RyR) using an acousto-optic tunable filter module and neutral density filter. For imaging synaptophysin immunoreactivity, the krypton laser power (568 nm, 30 mW) was reduced to 2.4%. Single slice images (512 × 512 pixel, 95 nm/pixel) were averaged after passing a Kalman filter. The photomultiplier tube voltage was 800 V throughout.

Presynaptic facilitatory effect of 4-chloro-m-cresol

In patch-clamp whole-cell recordings from medial nucleus of the trapezoid body principal neurones, TIFCs were evoked by stimulating input nerve fibers. Bath application of 4-CmC (10–1000 µm) dramatically enhanced the peak amplitude of TIFCs, in a concentration-dependent manner (Fig. 1). In these experiments, the low-affinity glutamate receptor antagonist kynurenic acid was included in the aCSF to minimize saturation and desensitization of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors by transmitter glutamate (Kaneko & Takahashi, 2004). The facilitatory effect of 4-CmC reached a maximum at around 500 µm and the half-maximal effective concentration was 116 µm (Fig. 1B). 4-CmC (500 µm) concomitantly decreased the paired-pulse ratio (the second amplitude relative to the first) of TIFCs (50 ms interstimulus interval) from 0.75 ± 0.07 to 0.34 ± 0.05 (n = 6; P < 0.01; Fig. 1C), suggesting that its site of action is presynaptic. In addition to its facilitatory effect, 4-CmC prolonged the synaptic delay (i.e. the time from the stimulus artifact to the TIFC onset; Fig. 1C, see also Fig. 7).

Effects of 4-chloro-m-cresol on presynaptic action potentials and K⁺ currents

To identify the presynaptic target of 4-CmC, we first recorded action potentials (APs) elicited by a brief (1 ms) depolarizing pulse in the calyceal nerve terminal. Bath application of 4-CmC (500 µm) markedly slowed the repolarizing phase of APs, with their half-widths (durations at 50% of peak amplitude) undergoing threefold prolongation from 0.43 ± 0.05 to 1.3 ± 0.2 ms (n = 5; P < 0.01; Fig. 2). However, 4-CmC had no effect on the peak amplitude of APs (from 125 ± 2.4 to 127 ± 2.5 mV; P = 0.21) or the resting membrane potential (from −76.0 ± 1.2 to −74.8 ± 1.3 mV; P = 0.073). It also had no significant effect on the AP rise time kinetics, with the maximum rate of rise remaining similar before (0.488 ± 0.024 V/ms) and after (0.440 ± 0.017 V/ms) 4-CmC applications (P = 0.098).

The dramatic prolongation of the AP duration by 4-CmC suggests that it might affect I_pK. After blocking Na⁺ currents using tetrodotoxin (1 µm), we recorded I_pK evoked by depolarizing square pulses (Ishikawa et al., 2003). 4-CmC (500 µm) attenuated I_pK by 51–69%, between −40 and +20 mV, with the magnitude of inhibition being 52.6 ± 3.6% at +20 mV (n = 5; measured at 10 ms from the current onset; Fig. 3). Together these results suggest that 4-CmC prolongs presynaptic AP duration by inhibiting I_pK, thereby facilitating nerve-evoked transmitter release.

Effects of 4-chloro-m-cresol on presynaptic Ca²⁺ currents and excitatory postsynaptic currents

In addition to the inhibitory effect on I_pK, 4-CmC might increase I_pCa or stimulate exocytotic mechanisms downstream of Ca²⁺ entry. To examine these possibilities, we performed paired whole-cell recordings from a presynaptic terminal and its postsynaptic target cell, and evoked TIFCs by I_pCa, after blocking Na⁺ and K⁺ conductance (Takahashi et al., 1996). As illustrated in Fig. 4, 4-CmC (500 µm) had little effect on the amplitude of I_pCa (−6.6 ± 2.0%, n = 5) or TIFCs (−10 ± 10%). These results suggest that the presynaptic facilitatory effect of 4-CmC on TIFCs is predominantly mediated by its inhibitory effect on voltage-gated K⁺ channels.

Effect of ryanodine on the presynaptic facilitatory effect of 4-chloro-m-cresol

Type 2 RyR is expressed in chick cerebellar parallel fibre and mossy fibre terminals (Ouyang et al., 1997), and type 3 RyR is expressed at the frog motor nerve terminals (Kubota et al., 2005). However, it is unknown whether RyR is expressed at the calyx of Held presynaptic terminal. We investigated this issue using type-specific antibodies. The calyceal terminal region can be identified with the synaptophysin immunofluorescence. In both postnatal day (P)8 and P14 rats, calyceal terminals showed clear signals to type 3 RyR but no immunoreactivity was observed for type 1 or type 2 RyR (Fig. 5). No age difference was observed for the intensity of type 3 RyR immunofluorescence signals between P8 and P14.

Given that type 3 RyR is expressed at the calyceal terminal, ryanodine might attenuate the presynaptic facilitatory effect of 4-CmC. However, ryanodine (20 µm) by itself had no effect on the amplitude of TIFCs (−1.2 ± 1.8%, n = 3; data not shown) and preincubation of slices with ryanodine (20 µm, > 20 min) did not affect the facilitatory effect of 4-CmC on TIFCs (n = 6, P = 0.56; Fig. 6). These results suggest that RyR-mediated Ca²⁺ release is not involved in the nerve-evoked transmitter release and facilitatory effect of 4-CmC on the release at this synapse.

Mechanism underlying prolonged synaptic delay by 4-chloro-m-cresol

As shown in Fig. 1C, 4-CmC prolongs synaptic delay. As 4-CmC blocks human muscle type Na⁺ channels at the half-maximal inhibitory concentration of 400 µm (Haeseler et al., 1999), the prolongation of synaptic delay by 4-CmC might result from slowing of axonal conduction velocity. To test this possibility, we made presynaptic recordings and evoked APs by presynaptic fibre stimulation. Bath-applied 4-CmC (1 mm) broadened presynaptic APs (see also Fig. 2A) without altering their onsets (Fig. 7A), indicating that 4-CmC has no effect on the axonal conduction velocity.

Expanding the duration of the presynaptic AP-like command pulse can delay the onset of TIFCs (Yang & Wang, 2006). To examine whether the AP broadening can explain the prolongation of synaptic delay, we made simultaneous pre- and postsynaptic voltage-clamp recordings and evoked I_pCa by two kinds of AP waveforms, one recorded before (control) and the other recorded after 4-CmC application (Fig. 7B). The 4-CmC AP waveform command pulse produced biphasic I_pCa, with a small step followed by a larger inward current. Its peak was significantly delayed from that of I_pCa evoked by control command pulse. The initial small step must be caused by a reduced driving force for Ca²⁺ currents at the peak of the command pulse (Katz & Miledi, 1971). The peak amplitude of I_pCa was similar between them. In parallel with the delayed peak of I_pCa, the onset of TIFCs evoked by the 4-CmC command pulse was delayed from the control. The mean magnitude of prolongation of synaptic delay in these experiments was similar to, although less variable than, those observed for TIFCs evoked by extracellular stimulation (P = 0.55, Mann–Whitney test; Fig. 7C). The TIFC amplitude evoked by the 4-CmC command pulse was larger than that evoked by control pulse (potentiation by 382 ± 92%, n = 4). This magnitude is similar to the maximal potentiation of TIFCs induced by bath-applied 4-CmC (334%, Fig. 1B). Thus, the 4-CmC-induced facilitation of transmitter release can be fully explained by the broadening of presynaptic AP duration.