Acid-sensing ion channels in neurones of the rat suprachiasmatic nucleus

Abstract

We used reduced slice reparations to study ASIC-like currents in the rat central clock suprachiasmatic nucleus (SCN). In reduced SCN preparations, a drop of extracellular pH evoked a desensitizing inward current to excite SCN neurones to fire at higher rates. Under voltage-clamped conditions, all SCN neurones responded to a 5 s pH step to 6.4 with an inward current that decayed with an average time constant of 1.2 s to 10% of the peak at the end of step. The current was blocked by amiloride with an IC₅₀ of 14 μm and was carried mainly by Na⁺, suggesting an origin of ASIC-like channels. The SCN neurones were sensitive to neutral pH, with 94% of cells responding to pH 7.0 with an inward current. The study of sensitivity to pH between 7.0 and 4.4 revealed a two-component dose-dependent H⁺ activation in most SCN neurones, with the first component (85% in amplitude) having a pH₅₀ of 6.6, and the second (15%) a pH₅₀ of 5. The ASIC-like currents were potentiated by lactate and low Ca²⁺, but were inhibited by Zn²⁺. RT-PCR analysis demonstrated the presence of mRNA for ASIC1a, 2a, 2b, and 3 in SCN. Compared to other central neurones, the unique presence of ASIC3 along with ASIC1a in SCN neurones may contribute to the high pH sensitivity and unusual inhibition by Zn²⁺. The high pH sensitivity suggests that the SCN neurones are susceptive to extracellular acidification of physiological origins and that the ASIC current might play a role in regulating SCN excitability.

The acid-sensing ion channels (ASICs) are proton-gated Na⁺ channels, acting as key receptors for extracellular H⁺ in both central and peripheral neurones (Krishtal, 2003; Wemmie et al. 2006). ASICs belong to the voltage-insensitive, amiloride-sensitive ENaC/DEG (epithelial Na⁺ channel/degenerin) family, with four genes encoding six transcripts, ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4 (Kellenberger & Schild, 2002 and references therein). ASIC2b or ASIC4 cannot form functional homomeric channels responding to H⁺ (Lingueglia et al. 1997; Akopian et al. 2000; Gründer et al. 2000), but can modulate other ASIC subunits (Lingueglia et al. 1997; Deveal et al. 2004; Donier et al. 2008). ASIC1a, ASIC2a and ASIC2b are abundant in brain and spinal cord neurones, and ASIC3 is additionally found in the peripheral neurones (Wemmie et al. 2006; Diochot et al. 2007). ASIC1a is particularly important in brain areas in which the subunit composition of ASICs has been determined (Baron et al. 2002; Wemmie et al. 2003; Askwith et al. 2004), whereas ASIC3 appears to confer the high pH sensitivity in peripheral neurones (Price et al. 2001; Benson et al. 2002; Xie et al. 2002).

The hypothalamic suprachiasmatic nucleus (SCN) is the central clock that coordinates the peripheral oscillators to control circadian rhythms in mammals (Reppert & Weaver, 2002; Yoo et al. 2004; Guilding & Piggins, 2007). These clock neurones vary their firing activity across the time of day, exhibiting a circadian rhythm in spontaneous firing rate (Inouye & Kawamura, 1979; Green & Gillette, 1982; Groos & Hendriks, 1982; Shibata et al. 1982). As ∼50% of the total signalling energy is spent on fuelling the sodium pump to restore the ionic gradients for spike generation (Atwell & Laughlin, 2001), a daily rhythm in firing rate suggests a rhythm in Na⁺/K⁺-ATPase pump activity to meet the changing demands. Indeed SCN neurones exhibit in-phase oscillation of Na⁺/K⁺-ATPase pump activity and firing activity (Wang & Huang, 2004) as well as daily metabolic rhythms in glucose uptake (Schwartz & Gainer, 1997) and ATP contents (Yamazaki et al. 1994). ATP hydrolysis during energy metabolism produces H⁺ (Alberti & Cuthbert, 1982) and causes extracellular acidification (Dmitriev & Mangel, 2004 and references herein) that may have effects on the external H⁺ targets including the ASIC channels.

This study aimed to determine the effects of extracellular acid shifts on the SCN neurones as well as the expression of ASICs. For investigating the acid responses, we modified our reduced slice preparations (Wang & Huang, 2006) to allow for rapid change of solutions. The cell-attached and whole-cell current-clamped recording techniques were used to determine the acid effects on firing rate and membrane potentials and the voltage-clamped recording technique was used to characterise the acid-evoked currents.

Methods

Reduced SCN preparations

All experiments were carried out according to the guidelines of the Institutional Animal Care and Use Committee of Chang Gung University School of Medicine. Sprague–Dawley rats (17–26 days old) were kept in a temperature-controlled room under a 12: 12 light: dark cycle (light on 07.00–19.00 h). Lights-on was designated Zeitgeber time (ZT) 0. For daytime and night-time recordings, the animal was killed at ZT 2 and ZT 10, respectively. A total of 50 rats were used in this study. An animal was carefully restrained by hand to reduce stress and killed by decapitation using a small rodent guillotine without anaesthesia, and the brain was put in an ice-cold artificial cerebrospinal fluid (ACSF) prebubbled with 95% O₂–5% CO₂. The ACSF contained (in mm): 125 NaCl, 3.5 KCl, 2 CaCl₂, 1.5 MgCl₂, 26 NaHCO₃, 1.2 NaH₂PO₄, 10 glucose. A coronal slice (200–300 μm) containing the SCN and the optic chiasm was cut with a Vibroslice (Campden Instruments, Lafayette, IN, USA), and was then incubated at room temperature (22–25°C) in the incubation solution, which contained (in mm): 140 NaCl, 3.5 KCl, 2 CaCl₂, 1.5 MgCl₂, 10 glucose, 10 Hepes, pH 7.4, bubbled with 100% O₂. To obtain reduced preparations for rapid change of solutions, we first excised a small piece of tissue from the SCN using a fine needle (Cat no. 26002-10, Fine Science Tools, Foster City, CA, USA), and then further trimmed down to smaller pieces containing tens to hundreds of cells with a short strip of razor blade. The reduced preparation was then transferred to a recording chamber for recording. The SCN neurones of the reduced preparation could be identified visually with an inverted microscope (Olympus IX70, Japan).

Electrical recordings

The reduced SCN preparation was perfused with bath solution containing (in mm): 140 NaCl, 3.5 KCl, 2 CaCl₂, 1.5 MgCl₂, 10 glucose, 10 Hepes, pH adjusted to 7.4 with NaOH. To prepare the extracellular solutions with pH below 6.0, the Mes (4-morpholineethanesulfonic acid) buffer was used in place of Hepes, and no difference was observed in the acid-evoked currents at pH 5.0 between the two pH buffers (not shown). Two different modes of solution change were used. The results shown in Fig. 1 were obtained with a home-made seven-barrel pipette, which completed the solution change in ∼1 s as judged from the measurement of junction potential. The rest were obtained with a fast-step SF-77B perfusion system with a three-barrel pipette (Warner Instruments, Hamden, CT, USA). The solution change could be completed within 70 ms judging from the inward current activated by 1 mm glutamate.

Figure 1. Acid effects on SCN neurones.

A and B, firing responses to the application of pH 6.4 solution of two representative cells recorded in the cell-attached (A) and the current-clamped (B) mode. Note the similar firing pattern, due to depolarisation block of spike generation, in both recording modes. C, desensitizing current response to the application of pH 6.4 solution of a representative cell voltage-clamped at a holding potential of −52 mV. The dashed line is the zero voltage level.

All recordings were made at room temperature (22–25°C). For the cell-attached recording, the patch electrode was filled with the bath solution or with the patch solution as described below. Membrane potentials and currents were recorded with the patch-clamp technique in the whole-cell current-clamped and voltage-clamped modes, respectively. The signal was low-pass filtered at 1–5 kHz and digitised on-line at 2–10 kHz via a 12-bit A/D digitizing board (DT2821F-DI, Data Translation, Marlboro, MA, USA) with a custom-made program written in the C Language. The patch solution contained (in mm): 20 NaCl, 1 CaCl₂, 2 MgCl₂, 110 potassium gluconate, 11 EGTA, 10 Hepes, 3 Na-ATP, 0.3 Na-GTP, pH adjusted to 7.3 with KOH. The measured liquid junction potential was −12 mV (Neher, 1992) and was corrected for in the presentation of data. Pipette resistance was 4∼5 MΩ. For measuring the reversal potential of voltage-dependent Na⁺ current and acid-evoked current, the internal solution of 20 Na⁺/110 K⁺ was replaced with 65 Na⁺/65 Cs⁺, and pH was adjusted with NaOH (Fig. 4). Data were analysed and plotted with custom-made programs written in Visual Basic 6.0 and the commercial software GraphPad PRISM (GraphPad Software, San Diego, CA, USA). Data are given as means ±s.e.m.

Figure 4. The ASIC-like current is carried mainly by Na⁺.

A, both the ASIC-like current (upper left panel) and the voltage-activated Na⁺ current (upper right panel) reversed the direction of current flow at approximately the same potential (lower panel) in a representative cell. The ASIC-like current was activated by a 5 s pH step to 6.4 at holding potentials of −72, −52, −32, −12, −2, +8, +18, +28 and +48 mV (from bottom to top, respectively). The voltage-dependent Na⁺ current was activated by stepping to more depolarised potentials between −72 and +38 mV at 10 mV increments. Both currents had zero amplitude near +10 mV as indicated by the arrows. Note that the amplitude of ASIC-like current was magnified 10 times for better comparison. B, the averaged current–voltage relation indicated a reversal potential of ∼+13 mV, close to the predicted reversal potential of +19 mV for a Na⁺-selective channel. Each data point represents the mean ±s.e.m. of 7 cells.

Data analysis

The decay time constant was obtained by fitting an exponential equation to the decay phase of the acid-evoked currents. For the biphasic decay phases of the current (Fig. 2A, right panel), the transient component was fitted with

Figure 2. Acid-evoked currents in SCN neurones.

A, examples of acid-evoked currents from two representative SCN neurones in response to a 5 s pH step from 7.4 to 6.4. B and C, histograms showing the distribution of decay time constants (B) and I_5s/I_peak (the current amplitude at the end of 5 s pH step/peak amplitude) ratios (C). The biphasic neurones are included in the plots.

I(t) =I_oexp(−t/τ) +I_sustained, where I(t) is the current amplitude at time t, I_o is the initial current amplitude measured at ∼80–90% of the peak amplitude, τ is the decay time constant, and I_sustained is the sustained current at the end of 5 s pH step. The rest (Fig. 2A, left panel) were fitted with I(t) =I_o exp(−t/τ), assuming that the acid-evoked current will eventually decay to zero level in time points after the 5 s pH change.

The theoretical curve for the dose-dependent amiloride block was calculated with the equation

Percentage blockade =[Amiloride]/([Amiloride]+ IC₅₀) (Fig. 3B), where IC₅₀ is the half-block concentration. The curve for the one-component dose-dependent H⁺ activation is calculated with the equation

Figure 3. Amiloride blockade of the acid-evoked current.

A, the acid-evoked current was reversibly blocked by 100 μm amiloride in a representative neurone. B, dose-dependent block by amiloride. The theoretical curve is calculated with the equation assuming a one-to-one binding of amiloride to and blockade of the channel, yielding an IC₅₀ value of 14 μm. Each data point represents the mean ±s.e.m. of at least 6 cells. C, amiloride reversibly blocked the acid response. Dashed lines are the zero voltage levels

Percentage activation =[H⁺]ⁿ/([H⁺]ⁿ+ EC₅₀) (Fig. 5Aa), where n is the Hill coefficient and EC₅₀ is the H⁺ concentration for half-maximal activation. The value of pH₅₀ is determined as −log EC₅₀. The curve for the two-component dose-dependent H⁺ activation is calculated with the equation

Figure 5. pH dependency of the ASIC-like current.

A, two representative SCN neurones showing two different pH-dependent activations of ASIC-like currents. The current amplitude in one cell (Aa, left panel) had saturated between pH 5.9 and 4.4 and could be accounted for by a one-component pH-dependent activation. The theoretical curve for the dose–response relation is calculated with a pH₅₀ of 6.67, assuming a three-to-one binding of H⁺ to and activation of the channel (Aa, right panel). The current amplitude in the second cell (Ab, left panel) levelled off between pH 5.9 and 5.4 but then doubled its amplitude at pH 4.4, and could only be described as a two-component pH-dependent activation. The theoretical curve for the dose–response relation is calculated with pH₅₀ of 6.6 and 5, with the amplitudes being 36% and 64%, respectively, for the first and second components (see Methods). A Hill coefficient of 3 was assigned for both components. All data points were obtained by normalizing to the peak current amplitude at pH 4.4. B, dose–response relation of the ASIC-like current amplitudes pooled from all 29 cells. The theoretical curve for the dose–response relation is calculated with pH₅₀ of 6.6 and 5, with the amplitudes being 85% and 15%, respectively, for the first and second components. Each data point represents the mean ±s.e.m. of 12 cells (Aa), 4 cells (Ab), and 25–29 cells (B). C, a representative cell showing the current response to pH 5.4, 4.4, 3.9, and 3.4, with current amplitude decreasing with lower pH (left panel). Right panel: superimposition of current traces after proper scaling to reveal a decrease in the decay time constant with lower pH. The scaling factor were 117%, 152%, and 217% for the current trace at pH 4.4, 3.9, and 3.4, respectively.

Percentage activation =A₁[H⁺]^n₁/([H⁺]^n₁+ EC1₅₀) +A₂[H⁺]^n₂/([H⁺]^n₂+ EC2₅₀) (Fig. 5Ab and B), where A₁ is the amplitude for the first component and A₂ the amplitude for the second component. The values of pH₅₀ are then taken as −log (EC1₅₀) and −log (EC2₅₀).

RT-PCR analysis of ASICs expression

Total RNA of SCN was extracted using the Absolutely RNA Nanoprep kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's guide; total RNA of brain and DRG was extracted using the NeucleoSpin RNA II kit (Macherey-Nagel, Duren, Germany). The quality and quantity of RNA were determined by measuring the A₂₆₀: A₂₈₀ ratio. RNA samples were treated with DNaseI for 13–15 min at 25°C to eliminate genomic DNA contamination. The resulting RNA was reverse-transcribed (RT) to cDNA using ReverTra Ace (Toyobo, Osaka, Japan) with random primers in a total volume of 20 μl. One-tenth of the RT products were used as templates (2 μl) to perform the PCR. RT reaction with omission of reverse transcriptase was used as templates for negative control PCR. Primers used for RT-PCR were as follows: ASIC1a forward 5′-CTACTTCTGCTATCACCACGTCAC-3′ and reverse 5′-GGATCTCATACCTGTTGTTGAGC-3′, ASIC1b forward 5′-GAAGAGGAAGAGAAGGAAATGGAG-3′ and reverse 5′-GTGTGGGTAGCTGAGGTAATAAGC-3′, ASIC2a forward 5′-CAACCTTCCAGTATCCAGATCTTC-3′ and reverse 5′-ACCACTTCATCCACCTTGGTAAC-3′, ASIC2b forward 5′-CGGGCCACTGGCTAGGGCTGCTG-C-3′ and reverse 5′-GTACTTGCAGGAGAGCAGCATA-3′, ASIC3 forward 5′-CTGCTTACCATCCTTGAGATCC-3′ and reverse 5′-GAGAGTGTCTTGGTGACAGCA-3′, and ASIC4 forward 5′-ACTCAGCGATGCTGATATCTTCC-3′ and reverse 5′-GGTAGGTATTCCTCCTGCTGGTGGATG TC-3′. The thermal cycling condition of RT-PCR was 94°C for 3 min, followed by 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and then 72°C for 7 min. PCR amplified products were electrophoresed in 1.5% agarose gels, stained with ethidium bromide, and photographed.

Results

Acid effects on excitability of SCN neurones

To determine the acid effects on SCN neurones, the control solution of pH 7.4 was replaced with a solution of pH 6.4 (Fig. 1). Figure 1A shows the response of a representative SCN neurone recorded in the cell-attached mode. A drop in the external pH to 6.4 evoked a burst of action potential-like currents from the otherwise silent cell. The current amplitudes gradually decreased and eventually vanished altogether, suggesting a depolarisation block of spike generation. The acid-induced depolarisation block was confirmed with a cell recorded in the current-clamped mode (Fig. 1B). However, the acid-induced depolarisation was transient even in constant pH 6.4. Such a desensitizing response to pH drop was indeed observed in a representative voltage-clamped SCN neurone held at a potential of −52 mV (after correction for a −12 mV junction potential) (Fig. 1C). The two silent cells (Fig. 1A and B) were chosen to highlight the depolarisation block and the decaying nature of the acid response. Other spontaneous-firing neurones were similarly excited by pH 6.4 to increase their firing rate, with different degree of depolarisation block (see Fig. 3C).

Acid-evoked currents in SCN neurones

To study the acid-evoked current, we used a fast perfusion system to examine the effects of a step-like drop in the external pH on the SCN neurones under voltage-clamp conditions. Two actions were taken to avoid the possible problem of slow perfusion associated with this preparation. First, we selected only the cells on the edge of the tissue. Second, we determined the speed of perfusion by measuring the current response to 1 mm glutamate and compared the glutamate response with that of pH 6.4. For a total of eight cells, the t_1/2 (half-time to peak) values for 1 mm glutamate and pH 6.4 responses averaged 25 ± 2 ms (n= 8) and 331 ± 60 ms (n= 8), respectively. The rapid activation of glutamate current indicated a rapid change of solution in the reduced preparations. To characterise the acid-evoked currents, each application of low pH solution was separated by at least 70 s to minimise accumulated inactivation with repeated stimulation. However, decline of the acid-evoked current did occur during the course of experiment. In all SCN neurones (n= 104) voltage-clamped at a holding potential of −52 mV, a 5 s pH step to 6.4 evoked an inward current that invariably decayed to a lower level (Fig. 2). However, the decay time constant and the ratio of I_5s/I_peak (the current amplitude at the end of 5 s pH step/peak amplitude) varied among cells. Figure 2A shows two examples of the acid-evoked currents recorded from representative cells. In the majority of cells (90%; 94/104), the currents activated and then inactivated with a seemingly monophasic time course (left panel), with the decay time constant averaging 1.26 ± 0.06 s (n= 94) and the I_5s/I_peak ratio averaging 10 ± 1% (n= 94). A subset of cells (10%; 10/104) clearly had a biphasic decay time course, characterised by both transient and sustained components (right panel). The transient component desensitised with a decay time constant of 0.67 ± 0.07 s (n= 10; P < 0.05, Student's t test) to an I_5s/I_peak ratio of 14 ± 3% (n= 10). Pooled, the I_5s/I_peak ratio amounted to 10 ± 1% (n= 104) of the peak amplitude (87 ± 9 pA; n= 104). Figure 2B and C show histograms of the distribution of decay time constants and I_5s/I_peak ratios, respectively.

Amiloride blockade of the acid-evoked currents

Figure 3 shows the effect of the diuretic amiloride, a non-specific blocker of the ENaC/DEG family (Kellenberger & Schild, 2002), on the acid-evoked current in SCN neurones. A 5 s pH step to 6.4 in the absence of drug always preceded and followed the application of amiloride to determine its block of the acid-evoked current at pH 6.4 to prevent the declining current (and/or accumulated inactivation) from interfering with interpretation. The current in drugs was normalised to the average of the preceding and following control pH applications when necessary. Figure 3A shows an example of 100 μm amiloride on the acid-evoked current in a representative cell. For a total of 10 neurones, 100 μm amiloride reversibly blocked the current by 81 ± 2% (n= 10). Figure 3B shows the dose-dependent blockade by amiloride of the acid-evoked currents. The theoretical curve, assuming a one-to-one binding of amiloride to and blockade of the channel, yielded an IC₅₀ value of 14 μm. Figure 3C shows the acid response of a spontaneous-firing neurone and its blockade by amiloride. As indicated, a 3 s step-like drop to pH 6.4 increased spontaneous firing and induced depolarisation block (left panel and right panel), and the application of 100 μm amiloride markedly reduced the acid response (middle panel). The remaining depolarisation and increase in spontaneous firing in 100 μm amiloride was due to an incomplete block of the acid-evoked current at this concentration. On the other hand, 0.5 μm capsaicin, a specific TRPV1 (transient receptor potential channel vanilloid subfamily, member 1) activator (Caterina et al. 1997; Tominaga et al. 1998), had no effect on the membrane current in all six cells responsive to pH 6.4 (not shown), suggesting a lack of involvement of TRPV1 in the acid-evoked current. Together the results suggest an ASIC-like nature of the acid-evoked currents in SCN neurones.

The ASIC-like current is carried mainly by Na⁺

To determine the Na⁺ dependency of the ASIC-like current (Kellenberger & Schild, 2002), internal solution of 20 Na⁺/110 K⁺ was replaced with 65 Na⁺/65 Cs⁺. The reversal potential for the ASIC-like current was determined by stepping the pH from 7.4 to 6.4 for 5 s at various holding potentials between −72 mV and +48 mV (Fig. 4). The result of such a series of ASIC-like currents from a representative neurone is shown in Fig. 4A (upper left panel). For comparison, the voltage-dependent TTX-sensitive Na⁺ current was also activated in the same cell by stepping from a holding potential of −82 mV to more depolarised potentials between −72 and +38 mV at 10 mV increments (upper right panel). The current–voltage relations indicated that both the ASIC-like current (amplified 10 times for better comparison) and the voltage-dependent Na⁺ current reversed at ∼+10 mV (lower panel). Another cell had both currents reversed at ∼+12 mV. Figure 4B shows the averaged current-voltage relation of the ASIC-like current obtained from a total of seven cells, with the reversal potential at ∼+13 mV, close to the predicted value of +19 mV for a Na⁺-selective channel. The slope conductance between −72 mV and +8 mV averaged 3.06 ± 0.79 nS (n= 7). Additionally, the reversal potential of the ASIC-like current with an internal solution of 20 Na⁺/110 K⁺ was ∼+40 mV (n= 3; not shown), also close to the predicted value of +49 mV for a Na⁺-selective channel. The results indicated that the ASIC-like current in SCN neurones is carried mainly by Na⁺.

pH dependency of the ASIC-like current

The pH-dependent activation of the ASIC-like current was determined by lowering the external pH from 7.4 to 7.0, 6.7, 6.4, 5.9, 5.4 and 4.4 (Fig. 5). Almost all SCN neurones (94%, 31 out of 33 cells) responded to pH 7.0 with an inward current averaging 5.0 ± 0.8 pA (n= 33). Consistently, the smallest acid shift that excited SCN neurones to fire at a higher rate ranged between pH 7.2 and pH 7.1 as recorded in the cell-attached condition (n= 16; Wang & Huang, unpublished results). Nonetheless, not all SCN neurones (n= 29) showed the same pH dependency. In 38% of cells (11/29), lower external pH appeared to increase the amplitude of ASIC-like currents which saturated between pH 5.9 and pH 4.4 (Fig. 5Aa, left panel). The dose–response relation thus obtained could be accounted for with a Hill equation assuming a pH₅₀ of 6.67 and a Hill coefficient of 3 (Fig. 5Aa, right panel). In the remaining cells (62%, 18/29), the ASIC-like currents appeared to level off between pH 5.9 and pH 5.4 and then increased further at pH 4.4. The extent of increase in current amplitude between pH 5.4 and 4.4 varied among these 18 cells, with four of them doubling the amplitude (Fig. 5Ab, left panel) and the remaining 14 cells showing less than 25% increase. Since currents activated at pH lower than 4.4 always had a smaller amplitude than at pH 4.4 (Fig. 5C), we thus constructed the dose–response relation by normalizing the peak current amplitudes relative to those at pH 4.4. The theoretical curve for the dose-response relation of these four cells was calculated with a Hill equation assuming two populations of channels, the first component having an amplitude of 36% and a pH₅₀ of 6.6 and the second component having an amplitude of 64% and a pH₅₀ of 5, with a Hill coefficient of 3 for both components (Fig. 5Ab, right panel). Since no data point was determined at between pH 5.4 and 4.4, a Hill coefficient of 3 was assigned according to the recent finding of trimeric assembly of ASIC1a channels (Jasti et al. 2007). Figure 5B shows the pH dependency of the ASIC-like currents pooled from all 29 cells, with the first component having an amplitude of 85% and a pH₅₀ of 6.6 and the second component having an amplitude of 15% and a pH₅₀ of 5.0, with a Hill coefficient of 3 for both components.

Note that the proportion of the second component could be an underestimate for two reasons. First, the current tends to become smaller during the course of experiments, although care was taken by reversing the order of solution application for the pH dependency experiments. Second, the pH-dependent desensitisation may also compromise the current amplitude measured at pH 4.4 as describe below. Figure 5C shows the response of a representative cell to the application of pH 5.4, 4.4, 3.9 and 3.4 solutions, with the current amplitude decreasing with lower pH (left panel). Superimposition of the current traces after proper scaling revealed that the rate of desensitisation became faster with lower pH (right panel). For this particular cell, the decay time constant decreased from 711 ms to 451 ms, 319 ms and 150 ms at pH 5.4, 4.4, 3.9 and 3.4, respectively. The pH-dependent desensitisation may in part account for the smaller amplitude at pH lower than 4.4. A similar decrease in the amplitude and decay time constant was also observed in three other cells, with an additional cell having smaller amplitude but similar rate of desensitisation with lower pH. Note that cells deteriorated and even died during the application of solutions more acidic than pH 3.9.

Modulation of the ASIC-like current

The ASIC channels are subject to modulation by a variety of substances from the extracellular domain, with differing sensitivity to modulation depending on the combination of ASIC subunits (Wemmie et al. 2006). A pH₅₀ of 6.6 is similar to that for ASIC1a and ASIC3 (Babini et al. 2002; Immke & McCleskey, 2003), suggesting an involvement in the ASIC-like currents in SCN neurones. Since ASIC1a or ASIC3 is enhanced by lactate, an effect attributed to its chelation of external Ca²⁺ (Immke & McCleskey, 2001), we tested the effects of lactate and low Ca²⁺ on the ASIC-like current in SCN neurones (Fig. 6A and B). As indicated in Fig. 6A, 30 mm lactate enhanced the ASIC-like current activated by a pH step to 6.4 in a representative cell (left panel). On average, 30 mm lactate increased the current to 144 ± 7% (n= 6; P < 0.005, paired t test) (right panel). Figure 6B shows the effect of 0.5 mm Ca²⁺ on the ASIC-like current in a different SCN neurone (left panel). On average, lowering external Ca²⁺ from 2 mm to 0.5 mm enhanced the ASIC current to 129 ± 8% (n= 6; P < 0.005, paired t test) (right panel).

Figure 6. Modulation of the ASIC-like current by lactate (A), low Ca²⁺ (B), and Zn²⁺ (C).

A, 30 mm lactate potentiated the current response to pH 6.4 in a representative cell (left panel). Right panel: summary of experiments showing the potentiation by 30 mm lactate of the current response to pH 6.4 (n= 6). B, 0.5 mm Ca²⁺ potentiated the current response to pH 6.4 in a representative cell (left panel). Right panel: summary of experiments showing the potentiation by 0.5 mm Ca²⁺ of the current response to pH 6.4 (n= 6). C, 300 μm Zn²⁺ inhibited the current response to pH 5.4 in a representative cell (left panel). Right panel: summary of experiments showing the inhibition by 300 μm Zn²⁺ of current responses to pH 6.4 (n= 5), 5.4 (n= 14), and 4.4 (n= 2).

On the other hand, the second minor component of a pH₅₀ of 5 suggests an involvement of ASIC2a (Hesselager et al. 2004). Since Zn²⁺ has been shown to potentiate the homomeric and heteromeric ASIC2a-containing channels (ASIC2a, ASIC1a+2a, and ASIC2a+3) but not the homomeric ASIC1a or ASIC3 (Baron et al. 2001), which is activated by lower pH, we have thus examined the effects of Zn²⁺ on the ASIC-like currents activated by pH 6.4, 5.4, and 4.4. For a total of 21 cells, however, the ASIC-like currents were all reduced by 300 μm Zn²⁺. Figure 6C shows a representative current response to pH 5.4 and its inhibition by 300 μm Zn²⁺ (left panel). On average, 300 μm Zn²⁺ reduced the current to 77 ± 5% (n= 5; P < 0.001, ANOVA, followed by Bonferroni's test for selected pairs comparison), 71 ± 2% (n= 14; P < 0.001, ANOVA, followed by Bonferroni's test for selected pairs comparison), and 65% (n= 2), respectively, in response to pH drop to 6.4, 5.4 and 4.4 (right panel).

Expression of ASIC subunits in SCN

We then used RT-PCR to determine the expression of ASIC transcripts in SCN (Fig. 7). Positive control reactions were performed using cDNA of rat brain (ASIC1a, 2a, 2b, and 4) and DRG (ASIC1b and 3) to determine the primer efficiency and anneal temperature. These primers were used to examine the gene transcription of ASIC subunits in SCN. The RT-PCR of SCN showed positive signals with primers of ASIC1a, 2a, 2b and 3. In addition, ASIC3 transcripts showed a splice variant form (containing 332 bp of intron sequence) in SCN as compared with the RT– (with omission of reverse transcriptase) control. Single-cell RT-PCR analysis indicates that at least some SCN neurones expressed ASIC2a (data not shown).

Figure 7. RT-PCR analysis of ASIC-encoding mRNA in SCN.

RT-PCR was used to detect the expression of six ASIC subunits (1a, 1b, 2a, 2b, 3, and 4) in SCN. Positive controls were performed using cDNA from rat brain or DRG. The expected PCR product sizes for each ASIC subunit were 173, 234, 203, 212, 209, and 326 bp, respectively. Of note, a splice variant of ASIC3 transcript was found with expected size 541 bp. Negative controls were performed using RT products with omission of reverse transcriptase to examine the contamination of genomic DNA.

Discussion

We used reduced SCN preparations to characterise the acid-evoked currents. Our results show that the central clock SCN neurones are sensitive to extracellular acid shifts. The high acid sensitivity is reflected by the pH dependency of the major component of ASIC currents with a pH₅₀ of 6.6. An additional component of ASIC currents appear at pH lower than 5.4. Although the ASIC currents are potentiated by lactate and low Ca²⁺, they are inhibited by Zn²⁺. RT-PCR analysis demonstrates the expression of ASIC1a, 2a, 2b and 3 in SCN neurones. To our knowledge, this is the first demonstration of ASIC3 in a specific brain area (ASIC3 mRNA was previously reported in the mouse brain; Drew et al. 2004).

In reduced SCN preparations, a drop of extracellular pH evokes a desensitizing inward current to excite the SCN neurones to fire at higher rates. The acid-evoked inward current in response to a 5 s pH step to 6.4 desensitises with a decay time constant of ∼1.2 s to ∼10% of the peak amplitude at the end of 5 s step. Most cells (90%) have monophasic inactivation time courses, and the rest (10%) have biphasic inactivation kinetics with both transient and sustained components. It is not known at present how the different acid responses correlate with the physiology of SCN neurones. Characterisation of the acid-evoked currents suggests that they are most likely to be mediated via the ASIC-like channels. Firstly, the current is blocked by amiloride, with an IC₅₀ of 14 μm. Secondly, capsaicin does not generate any current in neurones responsive to pH 6.4, suggesting a lack of TRPV1 involvement. Furthermore, the acid-evoked current is carried mainly by Na⁺.

Investigation of pH dependency revealed two components of ASIC-like currents in most SCN neurones. The first, major component (85%) has a pH₅₀ of 6.6, whereas the second, smaller component (15%) has a pH₅₀ of 5. The dose–response relation was constructed by taking the current at pH 4.4 as the maximum, an assumption supported by the observation that all currents activated at pH lower than 4.4 become smaller and desensitise at a faster rate. A decrease in the current amplitude with lower pH has been reported for the ASIC-like current in the human SJ-RH30 skeletal muscle cell line (Gitterman et al. 2005). There are also similar observations of faster desensitisation with lower pH (Zhang & Canessa, 2002; Hesselager et al. 2004; Gitterman et al. 2005). The pH-dependent desensitisation may depend on the combination of ASIC subunits as indicated in the heterologous expression system (Hesselager et al. 2004). Furthermore, the ASIC-like current may enter a long-lived inactivated state, the fraction of channels entering which state increasing with lower pH, as in the homomeric ASIC1a-mediated current (Chen & Gründer, 2007).

SCN neurones are sensitive to neutral pH, with 94% SCN neurones responding to pH 7.0 with an inward current, and the smallest acid shift that excites SCN neurones to fire at a higher rate being between pH 7.2 and 7.1. Although we do not know the exact combination of ASIC subunits for the ASIC currents, the SCN neurones express both ASIC1a and ASIC3, with each being potentially able to form homomeric channels to generate currents with a pH₅₀ of 6.6 (Babini et al. 2002; Immke & McCleskey, 2003). Consistent with this idea, both lactate and low Ca²⁺ potentiate the ASIC-like currents in SCN neurones. Lactate has been shown to enhance ASIC1a or ASIC3 by chelating extracellular divalent ions (Immke & McCleskey, 2001), and external Ca²⁺ proposed to block the activation of ASIC channels (Immke & McCleskey, 2003). Nevertheless, recent studies suggest that Ca²⁺ also blocks the channel pores (Paukert et al. 2004; Zhang et al. 2006). The ASIC3-like current in the cardiac sympathetic afferents has a pH₅₀ of 6.6 (Sutherland et al. 2001) and is potentiated by lactate and low Ca²⁺ (Immke & McCleskey, 2001). It is possible that ASIC3 contributes to the high pH sensitivity of SCN channels. In line with this idea, a decrease in pH sensitivity has been demonstrated for the ASIC-like current in ASIC3 null sensory neurones (Price et al. 2001; Benson et al. 2002; Xie et al. 2002).

For comparison, the ASIC currents in most central neurones have a pH₅₀ lower than 6.5 (with few exceptions, see Varming, 1999; Escoubas et al. 2000), and appear to be a mixture of ASIC1a and ASIC2a at least in the hippocampal neurones (Baron et al. 2002; Wemmie et al. 2003; Askwith et al. 2004). This agrees with the high expression levels of ASIC1a, ASIC2a, and ASIC2b in brain areas including the hippocampus (see Deveal et al. 2004 and references therein). Zn²⁺ has been shown in the heterologous expression system to potentiate the homomeric and heteromeric ASIC2a-containing channels (ASIC2a, ASIC1a+2a, and ASIC2a+3) and to inhibit homomeric ASIC1a, with no effect on homomeric ASIC3 (Baron et al. 2001). A mixture of ASIC1a and ASIC2a confers the potentiation by Zn²⁺ of ASIC currents in other central neurones: ASIC currents potentiated by Zn²⁺ in 30% cortical neurones (Chu et al. 2006) and in 80% hippocampal neurones (Baron et al. 2002).

The possible participation of ASIC2a in SCN channels is suggested by the second, small component of the ASIC currents that does not reach saturation at pH 5.4. Interestingly, unlike the potentiation by Zn²⁺ in other central neurones, the ASIC currents in all 21 SCN neurones are inhibited by 300 μm Zn²⁺, a response reminiscent of homomeric ASIC1a than ASIC3 channels (Baron et al. 2001). Nevertheless, in view of the expression of ASIC1a, 2a, 2b and 3, it is highly unlikely that the ASIC currents in SCN neurones are all (21/21) mediated by the homomeric ASIC1a channels. It seems more likely that the presence of ASIC3 and ASIC1a contributes to the inhibition by Zn²⁺. It would be interesting to see if heteromeric ASIC1a+3 or ASIC1a+2a+3 channels were inhibited by Zn²⁺ as in the SCN currents. It would also be interesting to know if this unusual response to Zn²⁺ is of physiological relevance, since the presence of releasable Zn²⁺ has been demonstrated in SCN (Huang et al. 1993). Further work is needed to better determine the exact combination of ASICs in mediating the ASIC current in SCN neurones.

The ASIC channels may play (patho)-physiological roles in sensory transduction in the peripheral nervous system and neurotransmission in the central nervous system (Krishtal, 2003; Wemmie et al. 2006). Furthermore, ASICs may interact or co-assemble with other channel types to alter cell function (Su et al. 2006; Meltzer et al. 2007; Petroff et al. 2008). While the function of ASIC channels in SCN neurones is not known, the high pH sensitivity suggests that they are susceptive to extracellular acidification of physiological origin. Specifically, 94% of SCN neurones respond to pH 7.0 with an inward current, and mild extracellular acid shifts to pH between 7.2 and 7.1 are able to excite SCN neurones to increase spontaneous firing. In the central nervous system, a drop of 0.2 pH unit could be reached by activity-evoked extracellular acidification (Chesler & Kaila, 1992; Chesler, 2003). An even greater acidification of ∼0.5 pH unit has been shown in the rabbit retina in the physiological setting (Dmitriev & Mangel, 2001). Importantly, the extracellular acidification is controlled by the circadian clock in the retina (Dmitriev & Mangel, 2001) and the production of H⁺ is associated with metabolism (Dmitriev & Mangel, 2004). As major energy expenditure is used to fuel the sodium pump (Atwell & Laughlin, 2001), the activity of which oscillates between day and night in SCN (Wang & Huang, 2004), one might predict a similar oscillation of extracellular acid shifts in SCN as in retina. Our preliminary results with pH-sensitive microelectrodes indicated a difference of 0.2 pH unit between the SCN in a hypothalamic slice and suprefusate (Su & Huang, unpublished results). Such a mild acidification could potentially alter SCN excitability via the activation of ASIC channels. On-going experiments are being carried out to better examine this issue.

Glossary

Abbreviations

ASIC

acid-sensing ion channel

SCN

suprachiasmatic nucleus

TRPV1

transient receptor potential channel vanilloid subfamily, member 1

Author contributions

Study conception and design: C.C.C., R.C.H.; acquisition of data: C.H.C., Y.T.H.; analysis and interpretation of data: all authors; drafting of the manuscript: C.C.C., R.C.H.; critical revision for important intellectual content: all authors; final approval of the version to be published: all authors. All experiments were done in Chang Gung University.