Contribution of the hyperpolarization-activated current to the resting membrane potential of rat nodose sensory neurons

Abstract

1. The voltage- and time-dependent characteristics of the hyperpolarization-activated current (I_H) and its contribution to the resting membrane potential of neonatal rat nodose sensory neurons were investigated using the whole-cell tight seal method of voltage and current clamp recording.

2. I_H was found in all neonatal nodose neurons in vitro, contrary to previous reports where its presence was particular for A-type neurons. We used the presence of both tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents to distinguish C- from A-type neurons (TTX-S only). We obtained further support for the presence of I_H in C-type neurons with experiments in which I_H was demonstrated in a subset of neurons sensitive to capsaicin.

3. In both groups I_H activated at potentials negative to −50 mV, developed slowly with time and was inhibited by 1–5 mm extracellular caesium. At −120 mV, I_H activated with a fast time constant of 73 ± 3 ms in A-type neurons and 163 ± 37 ms in C-type neurons (P < 0.05). A second, slower time constant of 682 ± 83 ms was observed in A-type neurons and 957 ± 122 ms in C-type neurons.

4. A- and C-type neurons differed in the amplitude of I_H. The mean magnitude of I_H at −110 mV was −2338 ± 258 pA in A-type neurons but only -241 ± 40 pA (P < 0.001) in C-type neurons. This disparity persisted when currents were normalized for capacitance. The reversal potentials for I_H were −39 ± 4 mV for A-type neurons and −37 ± 5 mV for C-type neurons (P > 0.05).

5. During current clamp recording I_H caused time-dependent rectification in response to hyperpolarizing current injections from the resting membrane potential. CsCl abolished the rectification and hyperpolarized the resting potential of A-type neurons from −55 ± 3 mV to −61 ± 4 mV and C-type neurons from −62 ± 2 mV to −71 ± 3 mV. Taken together, the results in these studies indicate that I_H contributes to the resting membrane potential in all nodose neurons.

A population of neurons whose cell bodies are contained within the nodose sensory ganglia serves as a primary afferent limb of the baroreceptor reflex. These neurons monitor arterial pressure by increasing their discharge when arterial pressure rises. Approximately 15 % of the baroreceptor neurons have myelinated axons, A-type neurons, while the remainder have unmyelinated axons, C-type neurons (Andresen et al. 1978). The two groups have quite different discharge properties in response to changes in arterial pressure (Brown et al. 1978). These discharge properties are the result of activation, inactivation, and/or deactivation of a diverse population of ion channels. Thus far, the kinetic properties of voltage-dependent calcium (Mendelowitz & Kunze, 1992; Bacal & Kunze, 1994), sodium (Baccaglini & Cooper, 1982; Ikeda et al. 1986; Ikeda & Schofield, 1987; Schild & Kunze, 1997) and outward potassium currents (Cooper & Shrier, 1989; McFarlane & Cooper, 1991) have been described and implemented in a model system that characterizes the contributions of individual ionic currents to firing patterns in A- and C-type neurons (Schild et al. 1994). In the absence of available experimental data, the model presented by Schild et al. (1994) rests on the assumption that the underlying current setting the resting membrane potential for nodose neurons is primarily due to a background leak current, permeable to both sodium and calcium cations.

In this study we sought the identity of the current(s) that set the resting membrane potential for nodose neurons. One of the current(s) that may contribute to the resting membrane potential is the hyperpolarization-activated current. The reason for this hypothesis is the following. When the neurons are given a hyperpolarizing current injection, there is an instantaneous hyperpolarization followed by a slow depolarization, described as time-dependent rectification (Stansfeld & Wallis, 1985; Puizillout & Gambarelli, 1989; Undem & Weinreich, 1993). This time-dependent rectification may be an indication of the hyperpolarization-activated current (I_f, I_h, I_q), similar to that established in sinoatrial node cells (DiFrancesco, 1985) and other central neurons (Spain et al. 1987; Bobker & Williams, 1989; McCormick & Pape, 1990a, b; Banks et al. 1993; Travagli & Gillis, 1994) and peripheral neurons (Ingram & Williams, 1994; Pearce & Duchen, 1994; Scroggs et al. 1994). This current is known to be active near -60 mV (Pape, 1996), which is also within the region of the resting membrane potential of nodose neurons. However, previous investigations have suggested that time-dependent rectification is prevalent only in A-type nodose neurons while C-type neurons show little to no time-dependent rectification (Stansfeld & Wallis, 1985; Puizillout & Gambarelli, 1989; Undem & Weinreich, 1993). This suggests that the resting membrane potential for C-type nodose neurons may be set by other ionic currents, such as a cation leak current used in a model of Schild et al. (1994).

Even though I_H has been extensively characterized in cardiac cells and in a variety of neurons, there are differences in the kinetics, activation threshold and reversal potential of I_H among various cell types (Pape, 1996). Therefore in the present study, we provide a detailed characterization of I_H for both A- and C-type nodose neurons. In addition, we demonstrate that the hyperpolarization-activated cation current contributes an inward current at the resting membrane potential of neonatal rat nodose sensory neurons in both A- and C-type neurons, as identified by their selective pharmacological sensitivity to tetrodotoxin or capsaicin during voltage clamp measurements. The difference in properties of I_H between A- and C-type neurons may explain the lack of time-dependent rectification in some nodose neurons, as previously suggested.

METHODS

Isolation of nodose neurons

Nodose ganglia were excised from neonatal (1–3 days old) Sprague- Dawley rats and the neurons were isolated and cultured, as previously described (Bacal & Kunze, 1994). Briefly, neonatal rats were asphyxiated by CO₂ inhalation and the nodose ganglia were surgically removed. The dissected ganglia were placed in nodose complete medium (NCM) consisting of Dulbecco's modified Eagle's medium/F-12 (Gibco BRL), 5 % fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT, USA), 0.1 % serum extender (Collaborative Research, Bedford, MA, USA) and 1 % penicillin/ streptomycin (Gibco BRL). The medium was replaced with Earle's balanced saline solution (EBSS; Gibco BRL) containing 5 mg ml⁻¹ trypsin, 12 mM cysteine, 0.5 mM EDTA and 1.5 mM CaCl₂, incubated for 30 min at 37°C. Following incubation, the medium was replaced with 3 ml NCM supplemented with 1.5 mg ml⁻¹ albumin. The tissue was triturated with a fire-polished Pasteur pipette to disperse the cells and subsequently placed into 35 mm Petri dishes containing poly-D-lysine-treated glass coverslips. The cells were kept in a humidified incubator (5 % CO₂-95 % air, at 37°C) and fed every other day with NCM. Some cultures were treated with NCM supplemented with 8 ng ml⁻¹ nerve growth factor (NGF; Collaborative Research). No statistical difference was observed for the expression of I_H among the cells with or without NGF. Therefore the data from the two groups of nodose neurons were pooled.

Electrophysiology

Electrophysiological experiments were performed on isolated neurons 1–2 days post-culture. By day 3, nodose neurons have developed processes; therefore space clamp was not possible. Isolated nodose sensory neurons were voltage and/or current clamped using the whole-cell tight seal patch technique. Polished electrodes were made from Corning no. 8161 or 7052 glass electrodes using a Narishige electrode puller. The electrodes, when filled with potassium aspartate solution, generally had a resistance of 1–3 MΩ for 8161 glass and 3–5 MΩ for 7052 glass. The reference electrode was a silver-silver chloride plug linked to the bath by a 150 mM KCl bridge. An Axopatch 1C patch clamp amplifier (Axon Instruments) was used with the 80 dB decade⁻¹ low-pass filter set at a -3 dB frequency of 10 kHz. pCLAMP software was used to apply voltage and current clamp protocols in order to measure the whole-cell currents and whole-cell membrane potentials, respectively. The series resistance was generally between 3 and 12 MΩ. The capacitance and series resistance were approximately 40–60 % compensated where stated and no leak subtraction was performed. For data presented, the voltage error did not exceed ±3 mV. Junction potentials were corrected as described by Nehr (1992). A multibarrel gravity-fed perfusion system was used to exchange extracellular solutions at a rate of approximately 1 ml min⁻¹. All recordings were done at room temperature.

Data analysis

Activation current traces were fitted with a biexponential function of the form:

using the Chebyshev technique within pCLAMP v. 6 software, where A₀ is the offset constant, A₁ and A₂ are the amplitudes, and τ₁ and τ₂ are the respective time constants. The instantaneous tail currents at -80 mV were used to construct the steady-state activation curve for I_H (see Figs 1C and 7C). The instantaneous tail currents were first normalized for each cell by using the formula:

where I_-50 is the minimal current for I_H and I_-140 is the maximal current for I_H. After normalizing the tail currents for each cell, the data points for each cell were fitted with a Boltzmann function:

where V_½ is the half-activation potential of I_H and k_S is the slope factor.

Figure 1. Whole-cell voltage clamped recordings of the hyperpolarization-activated current (I_H) in isolated neonatal rat nodose sensory neurons.

I_H was measured in solution 1 (refer to Methods) in the absence (A) or presence (B) of 1 mM CsCl. The intracellular pipette contained high potassium aspartate solution (see Methods). The neuron was held at -40 mV and pulsed to different test potentials between -40 and -140 mV for 1 s duration in 10 mV step increments (inset in A). After each test potential, the neuron was stepped to -80 mV for 750 ms before returning to the holding potential. This voltage clamp protocol was used for subsequent studies regarding resting membrane potential and ionic contributions to I_H, unless otherwise stated. C, representative current traces illustrating the caesium-sensitive current, attained by subtracting the current traces in the presence of CsCl (B) from the current traces in the absence of CsCl (A) at each test potential. D, mean ±s.e.m. (n= 16) current-voltage relationship measured at 1 s after start of test pulse in control (□) and in CsCl (▪) and resultant current (control - CsCl traces) at the beginning (^) and end of the pulse (•).

Figure 7. The Boltzmann fit of I_H.

A, voltage dependence of activation determined at -80 mV step following return from different prepulse potentials (see Fig. 1). B, the mean normalized current from the instantaneous current traces at -80 mV (A, ^) was fitted with a Boltzmann function for A- (^) and C-type (•) neurons.

The reversal potential for each extracellular ionic condition during voltage clamp measurements was determined by calculating the membrane potential at the zero current. This was accomplished by a linear fit between two current measurements on each side of the zero current. Data are presented as means ±s.e.m. Statistical significance was determined at P < 0.05 using Student's t test.

Solutions

For voltage clamp studies, the following bath solutions were used. Solution 1 (mM): 137 NaCl, 5.4 KCl, 1 MgCl₂, 0.02 CaCl₂, 10 glucose and 10 Hepes, pH 7.4 with NaOH. Solution 2 was the same as solution 1 with equimolar substitution of NaCl by NMDG-Cl; and solution 3 was the same as solution 2 but NMDG-Cl was replaced by NMDG-MeSO₄, CaCl₂ by calcium gluconate and MgCl₂ by magnesium gluconate. The pipette contained (mM): 145 potassium aspartate, 5 NaCl, 1.95 CaCl₂, 2.2 EGTA, 2 MgCl₂, 10 glucose and 5 Hepes, pH 7.3 with KOH.

For current clamp studies, the bath solution was the same as solution 1 except for the addition of 2 mM CaCl₂; the pipette solution was the same as above. All chemicals, including tetrodotoxin and capsaicin, were purchased from Sigma.

RESULTS

Current-voltage characteristics of I_H under voltage clamp

When a nodose neuron was voltage clamped at -40 mV, there was a small outward potassium current due to the activation of both the delayed rectifier potassium channels and the transient outward potassium channels, as previously reported in these neurons (Cooper & Shrier, 1989). As the neuron was hyperpolarized in 10 mV step increments, this outward potassium current deactivated within milliseconds and, subsequently, a slow non-inactivating inward current appeared (Fig. 1A). This hyperpolarization-activated inward current (I_H) increased as the membrane potential was further hyperpolarized. The inward current approached a near steady level within 1 s. The voltage was then stepped to -80 mV from each test potential, resulting in the I_H tail currents. The inward current at the hyperpolarized potentials was similar to the hyperpolarization-activated current found in other cells (DiFrancesco, 1985; Pape, 1996). In those reports, I_H was sensitive to extracellular CsCl. Superfusion of nodose neurons with 1 or 5 mM CsCl added to the extracellular bath solution did not affect the outward potassium current at -40 mV (Fig. 1B). On the other hand, I_H was completely inhibited in the presence of caesium. The inward I_H tail currents, produced by the step to -80 mV, were also inhibited. To isolate the hyperpolarization-activated inward current from the outward potassium currents, the current traces in the presence of caesium were subtracted from the current traces in the absence of caesium (Fig. 1C). The caesium-sensitive current, representing I_H, had an onset component that gradually increased towards a near steady value within 1 s. The I-V relation of the caesium-sensitive current at the near steady level showed that I_H was activated at membrane potentials more negative than -50 mV. In a group of 16 neurons, I_H reached a mean current amplitude of -542 ± 124 pA at -140 mV (Fig. 1D, n= 16).

In our study all nodose sensory neurons recorded under voltage clamp conditions expressed functional I_H. This is in contrast with previous voltage recordings in nodose neurons where investigators found time-dependent rectification, an index of I_H, only in A-type neurons and a few or no C-type neurons. In previous studies, the A- and C-type nodose neurons were distinguished according to the conduction velocity of their axons. We used the presence of both tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) voltage-dependent sodium current to distinguish C-type neurons from A-type neurons (Stansfeld & Wallis, 1985; Schild & Kunze, 1997). The same voltage clamp protocol as described earlier was used to demonstrate I_H (Fig. 2A). Cells were then held at -80 mV and stepped to depolarized potentials in 10 mV increments. There was a rapid activation and inactivation of the fast transient sodium currents at -40 mV and at more depolarized membrane potentials (Fig. 2B). Following the inactivation of the sodium currents, outward potassium currents appeared. It was not possible to obtain quantitative values for the sodium currents under these solution conditions due to poor voltage control. We were, however, able to demonstrate the elimination of the inward sodium current in 13 of 61 nodose neurons when 1 μm tetrodotoxin was perfused in the extracellular bath. The fast transient sodium current was completely inhibited while the outward potassium current was unaffected (Fig. 2C). These 13 TTX-S neurons were considered A-type neurons and had a large I_H. On the other hand, the remainder of the population of nodose neurons (48/61) also displayed I_H (Fig. 2D) but the sodium currents were only partially inhibited by 1 μm tetrodotoxin (Fig. 2F). The remaining inward current that activated near -20 mV was the TTX-R sodium current (Ikeda & Schofield, 1987; Schild & Kunze, 1997). The inward current was not a calcium or sodium current conducting through voltage-gated calcium channels, because the bath calcium concentration of 20 μm was sufficient to block sodium conductance through voltage-dependent calcium channels. Therefore, these neurons were classified as C-type neurons, since they expressed both the TTX-S and TTX-R sodium channels. No neurons were found to have only the TTX-R sodium current without the TTX-S sodium current.

Figure 2. I_H is present in both A- and C-type neurons.

Representative whole-cell recording of two neurons, one with a prominent I_H (A, 13 out of 61) and a second with a smaller I_H (D, 48 out of 61). Voltage protocol shown in inset in A. B and C, the same neuron as illustrated in A, or E and F, the same neuron as in D, but with test potentials from -80 to +40 mV for 75 ms in 10 mV step interval from a holding potential of -80 mV (inset in C). The recordings were performed in solution 1 in the absence (A, B, D and E) or presence (C and F) of 1 μm tetrodotoxin. Since the sodium current was too fast and large, adequate voltage clamp was compromised.

Another pharmacological marker used to identify C-type neurons was capsaicin, a substance known to depolarize nociceptive neurons (Wood et al. 1988). Again using the I_H voltage clamp protocol, we examined five nodose neurons that functionally expressed a very small amount of I_H at the hyperpolarized potentials (Fig. 3A). When these neurons were held at -80 mV and step depolarized, they displayed the fast transient sodium currents and the kinetically slower outward potassium currents (Fig. 3B). When 10 μm capsaicin was perfused with the bath solution, a large inward current was induced at the holding potential (-80 mV). The capsaicin-induced current reversed near 0 mV, as expected for activation of a non-selective cation channel (Fig. 3C). The effect of capsaicin was reversible upon washout (Fig. 3D). All five capsaicin-sensitive neurons had both the TTX-S and TTX-R sodium currents.

Figure 3. I_H was found in capsaicin-sensitive nodose neurons.

A, representative whole-cell recording of a neuron with a small magnitude I_H at voltages between -40 and -130 mV (inset in A). B, C and D, the same neuron as in A but with test potentials from -80 to +50 mV for 250 ms in 10 mV step interval from a holding potential of -80 mV (inset in C). The recordings were performed in solution 1 in the absence (A, B and D) or presence (C) of 10 μm capsaicin. Notice the change in current and time scale with I_H (A) or control and capsaicin treatment (B, C and D).

In comparing the current-voltage relationships, I_H in A-type nodose neurons was significantly larger than that in C-type neurons. At -110 mV, I_H in A-type nodose neurons had a mean amplitude of -2338 ± 258 pA while I_H in C-type neurons had a mean amplitude of only -241 ± 40 pA (Fig. 4A, P < 0.001). The difference was not explained by cell size. Using capacitance measurement as an index for cell size, there was no correlation between the amplitude of I_H and cell size (Fig. 4B). However, there was a tendency for A-type neurons to be larger than C-type neurons, where the mean capacitance was 53 ± 4 pF (n= 13) and 40 ± 2 pF (n= 48; P < 0.05), respectively. This was consistent with reports for dorsal root ganglia (Pearce & Duchen, 1994; Scroggs et al. 1994) but deviated from studies in nodose neurons (Carobi, 1996).

Figure 4. Magnitude of I_H in tetrodotoxin-sensitive and -resistant neurons.

A, mean magnitude of I_H at -110 mV for neurons with only tetrodotoxin-sensitive sodium currents (TTX-S) or for neurons with both tetrodotoxin-sensitive and -resistant sodium currents (TTX-R). *P < 0.001; n is given in parentheses. B, amplitude of I_H at -110 mV was plotted with respect to the capacitance of the neuron. Neurons with only tetrodotoxin-sensitive sodium currents (•, n= 13), with tetrodotoxin-sensitive and -resistant sodium currents (^, n= 43; ▵, n= 5) or with capsaicin-sensitive channels (▵, n= 5) are illustrated.

The activation kinetics of I_H were calculated by fitting the activation current traces at each hyperpolarized membrane potential to a biexponential function (Fig. 5A). In A-type neurons at -70 mV, the fast time constant for I_H was 337 ± 38 ms with an amplitude of 151 ± 41 pA and the second time constant was 2184 ± 777 ms with an amplitude of 136 ± 47 pA (Fig. 5B and C, n= 7; notice the time held at each test potential was extended to 3.5 s). The amplitude of both components increased as the membrane potential was further hyperpolarized, with the fast τ component contributing to a larger extent than the slow τ component at -120 mV. In addition, both the slow and fast time constants decreased by more than 67 % as the membrane potential was hyperpolarized to -120 mV. Since I_H was still increasing slightly even at 3.5 s, the slow time constant may be an underestimation, but protocols for longer durations compromised the whole-cell patch. Also, further hyperpolarization to -130 or -140 mV for greater than 2.5 s resulted in a loss of the tight seal.

Figure 5. I_H is voltage and time dependent.

A, biexponential fits (continuous lines) of I_H current traces of an A-type neuron at various test potentials for 3.5 s in a bath containing 2 mM CaCl₂ to block the outward potassium tail-currents (see Fig. 10). B, amplitude 1 (□) and amplitude 2 (^) were plotted at each test potential (n= 7); C, the rate constants, τ₁ (▪) and τ₂ (•), were plotted at each test potential (n= 7) demonstrating the voltage- and time-dependent activation of I_H; inset, the fast τ₁ (▪) was plotted with respect to membrane potential (V_m) on an expanded time scale.

The small amplitude of I_H in C-type neurons made it difficult to obtain fits to the data over the voltage range near threshold. Therefore, the activation kinetics for a group of C-type neurons were compared with A-type at -120 mV where the amplitude of the current was greatest. The activation kinetics of C-type neurons were slower than those for A-type neurons (Fig. 6). I_H in A-type neurons had a fast τ component of 73 ± 3 ms with an amplitude of 1482 ± 237 pA (n= 6), while I_H in C-type neurons had a fast time constant of 163 ± 37 ms and an amplitude of 38 ± 12 at -120 mV (n= 4; P < 0.05). The slow τ component for A-type neurons was 682 ± 83 ms with an amplitude of 393 ± 47 pA and that for C-type neurons, 957 ± 122 ms with an amplitude of 68 ± 14 pA (P < 0.05).

Figure 6. Activation kinetic of I_H was different in tetrodotoxin-sensitive and -resistant neurons.

A, fast time constant of activation of I_H (□) and its amplitude (▪) at -120 mV for neurons with only tetrodotoxin-sensitive sodium currents (TTX-S; n= 10) or neurons with both tetrodotoxin-sensitive and -resistant sodium currents (TTX-R; n= 10). B, second or slow time constant (□) and its amplitude (▪) of I_H at -120 mV for neurons with only TTX-S sodium currents (n= 10) or neurons with both TTX-S and TTX-R sodium currents (n= 10). *P < 0.05; **P < 0.001 for comparison of TTX-S and TTX-R.

The voltage dependence of activation was not different between A- and C-type neurons. This was determined by fitting the amplitude of normalized tail currents at -80 mV (Fig. 7A) to a Boltzmann function. The Boltzmann fit illustrated that I_H was active at membrane potentials more negative than -50 mV and approached saturation by -140 mV. For A-type neurons the half-maximal voltage of I_H (V_½) was -88 ± 3 mV with a slope factor (k_S) of 10.7 ± 0.5 mV (n= 8); and for C-type neurons the values were V_½= -87.4 ± 2.4 mV and k_S= 11.8 ± 0.9 mV (n= 8).

Since I_H was an inward current at -60 mV, this suggested that the reversal potential for I_H was more depolarized than the reversal potential for potassium alone (E_K= -83 mV). The reversal potential of I_H was determined by activating I_H at -120 mV and stepping thereafter in depolarizing increments of 10 mV towards -70 mV (Fig. 8A). At membrane potentials positive to -70 mV, the tetrodotoxin-sensitive and tetrodotoxin-resistant sodium currents were activated. This resulted in a loss of adequate voltage control and therefore made the calculation of the reversal potential for I_H difficult at those potentials. Hence, only data points between -120 and -70 mV were used for calculating the reversal potentials for I_H expressed in C-type neurons. The reversal potential for I_H in A-type neurons were calculated from -120 to -50 mV in the presence of 1 μm tetrodotoxin to inhibit the sodium current. Furthermore, the same protocol was repeated for each neuron in the presence of extracellular caesium in order to eliminate the contributions of other currents (Fig. 8B and C). The instantaneous currents of the caesium-sensitive traces (Fig. 8D) were best fitted with a linear regression. The extrapolation of the linear regression to 0 pA gave a projected mean reversal potential of I_H of -39 ± 3 mV in A-type neurons (n= 3) and -37 ± 5 mV in C-type neurons (n= 4; P > 0.05).

Figure 8. The reversal potential of I_H.

The reversal potential was determined by clamping the neuron to -120 mV for 1 s and depolarizing in 10 mV increments to -70 mV (inset in B). The voltage protocol was performed on neurons perfused with solution 1 in the absence (A) or presence (B) of 5 mM CsCl. C, the isolation of I_H was obtained from the difference between current traces in B and A.D, mean (n= 13) instantaneous currents of I_H (C, •) for A- (^; n= 3) or C-type (•; n= 4) neurons was plotted with respect to membrane potential and a linear regression was performed.

Extracellular ions contributing to I_H and the resting membrane potential

I_H has well documented permeability characteristics. The following sets of experiments were performed in a mixed group of neurons, both A- and C-type, using these characteristics to obtain support for a role for I_H in determining the resting membrane potential. Additionally, we explored possible contributions of other channels to the resting membrane potential.

When the nodose neurons were perfused with a bath containing the impermeant cation N-methyl-D-glucamine (Fig. 9B) instead of Na⁺ (Fig. 9A), no significant effect was seen on the peak tail currents of the outward potassium currents. I_H, however, was markedly reduced in the absence of Na⁺. In a bath with K⁺ and Na⁺, I_H was activated at -60 mV and more hyperpolarized, but in a bath with K⁺ and NMDG⁺, I_H was not evident until -80 mV. In this group of cells, I_H had a mean current amplitude of -1130 ± 299 pA at -140 mV in the presence of K⁺ and Na⁺, but decreased to -568 ± 152 pA when Na⁺ was replaced with NMDG⁺ (n= 15). The zero current potential of the nodose neurons in a bath containing both K⁺ and Na⁺ was -52 ± 1 mV (Fig. 9A) and shifted to a more hyperpolarized value, -57 ± 1 mV, when the sodium in the bath solution was replaced by the impermeant cation NMDG⁺ (Fig. 9C; n= 15; P < 0.005). This suggested that sodium ions permeated the I_H channel, and also, that I_H was active at the resting membrane potential of nodose sensory neurons. In addition, the application of TTX did not affect the conductance and amplitude of I_H or alter the zero current potential (data not shown). When the extracellular saline contained sodium ions but no potassium ions (solution 1 without potassium chloride), I_H was negligible (data not shown). This result was in agreement with a previous study in cardiac cells where the investigators reported that I_H required the presence of extracellular potassium (Maruoka et al. 1994).

Figure 9. Decreasing Na⁺ affected both the zero current potential and I_H.

Whole-cell voltage clamped measurements of I_H in the presence (A; solution 1) and absence (B; solution 2) of Na⁺ from -40 to -140 mV. C, mean (n= 15) currents measured at the steady-state component in the presence (•) and absence (^) of Na⁺. The arrow in B indicates the inward tail currents of the outward potassium currents.

Nodose neurons have a resting membrane potential close to the activation range of low threshold calcium channels. Therefore, we wanted to know if extracellular calcium ions contributed to the resting membrane potential and/or affected the conductance of I_H. When the nodose neuron was perfused with an extracellular potassium solution (solution 2, Methods) containing only 20 μm CaCl₂, a deactivation of the inward potassium tail currents (arrow in Fig. 10A) was followed by the activation of I_H in response to hyperpolarizing voltage steps. When the same neuron was perfused with an extracellular potassium solution containing 2 mM CaCl₂ (Fig. 10B), the inward potassium tail currents were inhibited, but the kinetics and amplitude of I_H were not affected. Therefore, unlike sodium and potassium cations, calcium ions were not conducted through I_H channels. However, the increase in Ca²⁺ caused a depolarizing shift in the zero current potential from -55 ± 2 to -48 ± 3 mV, when calculated in the presence of 20 μm and 2 mM CaCl₂, respectively (Fig. 10C; n= 6; P < 0.005). This suggested that another inward current, such as the previously identified low threshold calcium current (Mendelowitz & Kunze, 1992), also contributes to the resting membrane potential of nodose neurons. Similar results were obtained when calcium was added to a bath containing both potassium and sodium ions, such that the zero current potential changed from -54 ± 3 mV to -48 ± 3 mV in the presence of 20 μm and 2 mM CaCl₂, respectively (n= 6; P < 0.05).

Figure 10. Increasing Ca²⁺ affected the zero current potential but not I_H.

Whole-cell voltage clamped recordings of nodose sensory neurons in the presence of 20 μm (A, solution 2) and 2 mM (B, solution 2) Ca²⁺ in the extracellular potassium solution. The arrow (A) showed the inward potassium tail currents. C, mean (n= 6) currents measured at the steady-state component in the presence of 20 μm (^) and 2 mM (•) Ca²⁺.

In other central and peripheral neurons, the combination of I_H with other inward currents, such as the inward rectifier potassium current (I_K(IR)) and/or a leak current, contributed to setting the resting membrane potential. The perfusion of BaCl₂, a divalent salt that blocks I_K(IR), inhibited the inward tail currents but had no effect on the steady-state current in nodose neurons. For example, the mean steady-state current was -831 ± 141 and -795 ± 149 pA in the absence and presence, respectively, of 100 μm BaCl₂ at -140 mV (n= 8). This suggested that nodose neurons did not express I_K(IR); however, the presence of I_H may have masked the I_K(IR), if the I_K(IR) was substantially smaller than I_H. To diminish I_H without possibly affecting other potassium currents, we replaced extracellular chloride with gluconate in a bath without Na⁺ (Fig. 11). Previous investigators have shown that the replacement of extracellular chloride ions (Cl⁻) with large anions abolished I_H in cardiac cells (Frace et al. 1992) and thalamic relay neurons (McCormick & Pape, 1990a). For nodose neurons, the replacement of Cl⁻ with gluconate attenuated I_H at all membrane potentials (Fig. 11B). The tail currents of outward potassium currents (arrow in Fig. 11B) were not affected by the removal of Cl⁻ and furthermore, no I_K(IR) was evident. The decrease in the amplitude of I_H at the hyperpolarized membrane potentials also suggested that chloride ions were not permeable through I_H channels, similar to a previous report in cardiac cells (Frace et al. 1992). The exchange of extracellular chloride with gluconate shifted the zero current potential from -59 ± 2 to -64 ± 3 mV (Fig. 11C; n= 6; P < 0.005), and interestingly, produced an increase in the outward potassium current. The latter may account for the shift in membrane potential toward E_K. Even in the presence of both K⁺ and Na⁺, I_H was attenuated when Cl⁻ was substituted with gluconate and similar results were obtained when Cl⁻ was replaced with the large anion aspartate (data not shown).

Figure 11. The replacement of Cl⁻ with gluconate affected both the zero current potential and I_H.

A, representative current traces of whole-cell recordings of a nodose neuron in a potassium solution containing either Cl⁻ (solution 2; A) or gluconate (solution 3; B). The arrow (B) showed the inward potassium tail currents. C, mean (n= 6) currents measured at the steady-state component in the presence (^) and absence (•) of Cl⁻.

I_H under current clamp measurements

From the voltage clamp experiments, I_H appeared to be active at the resting membrane potential (V_rest). If this is the case, the inhibition of I_H with caesium should cause a hyperpolarization of the nodose neurons while at rest. This was demonstrated using the whole-cell current clamp technique to measure the membrane potential in the absence or presence of caesium chloride. In physiological saline, the V_rest was -55 ± 3 mV for A-type neurons (n= 4) and -62 ± 2 mV for C-type neurons (n= 12). When relatively small hyperpolarizing current injections were given, the membrane potential was hyperpolarized to a near steady-state level (Fig. 12A). However, when greater hyperpolarizing currents were applied to the neurons, there was an initial hyperpolarization followed by a slow depolarization, described as time-dependent rectification. The slow kinetics of the depolarizing component were in agreement with the slow activation of I_H. Upon perfusion with 5 mM CsCl (Fig. 12B), there was a significant hyperpolarization of the resting membrane potential to a mean of -61 ± 4 mV for A-type neurons (n= 4; with and without CsCl, P < 0.001) and -71 ± 3 for C-type neurons (n= 12; with and without CsCl, P < 0.001), suggesting the inhibition of an inward current at the V_rest. In addition, the time-dependent rectification during hyperpolarizing current injections was abolished in the presence of extracellular caesium. The input resistance, calculated from the linear regression of the I-V relation between 0 and -100 pA at the quasi-steady-state (Fig. 12C), was increased from 1.0 ± 0.3 GΩ (absence of CsCl) to 2.4 ± 0.2 GΩ (presence of CsCl) (n= 7; P < 0.001). The inhibition of an inward current was consistent with the inhibition of I_H by CsCl in the voltage clamp studies.

Figure 12. Time-dependent rectification was caused by the activation of I_H and was blocked by CsCl during current clamp recordings.

Representative whole-cell current clamped recordings of nodose sensory neurons in the absence (A, solution 1 with 2 mM CaCl₂) or presence (B) of 5 mM CsCl during hyperpolarizing current injection from 0 to -40 pA in 10 pA step interval (inset in A). C, current-voltage relation of the same neuron at steady state in the absence (^) and presence (•) of 5 mM CsCl. Linear regression was performed to calculate the input resistance for the neuron. Similar results were obtained for 7 neurons.

DISCUSSION

I_H contributes to the resting membrane potential of nodose sensory neurons

The resting membrane potential is determined by a steady-state relationship between the inward current(s) and outward current(s). In this study, we show that the hyperpolarization-activated inward current participates in determining the resting membrane potential of nodose sensory neurons. The evidence to support the hypothesis that I_H is active at the resting membrane potential is as follows. First, I_H has an activation threshold near -60 mV, a potential where nodose neurons are at rest, as shown in this study and in those of other investigators (Stansfeld & Wallis, 1985; Marsh et al. 1987; Puizillout & Gambarelli, 1989; Undem & Weinreich, 1993). Since I_H does not inactivate, this implies that I_H is tonically active at the resting membrane potential. Second, the replacement of extracellular Na⁺ or Cl⁻, which both reduce the contribution of I_H to the total current, results in a shift of the zero current potential to a more hyperpolarized value. Finally, the strongest evidence showing the contribution of I_H at the resting membrane potential is the hyperpolarization of the membrane potential and the decrease in membrane conductance upon perfusion with caesium, an inhibitor of I_H.

At the concentrations used in this study CsCl (1–5 mM) blocks only I_H at the resting membrane potential and consequently enables the outward current(s), presumably potassium current(s), to hyperpolarize the neurons. The neurons hyperpolarize to about -70 mV instead of E_K (-83 mV), suggesting an additional inward current is present. This caesium-insensitive inward current was considered a small leak current introduced by an incomplete seal between the glass electrode and the cell membrane.

Outward current balancing inward current at the resting membrane potential

The Boltzmann fit of I_H for both A- and C-type neurons indicates only about 2 % of this inward current is active at -60 mV. This suggests that the magnitude of the outward current opposing I_H is probably very small at the resting membrane potential. Furthermore, this outward current must be a current that does not completely inactivate at prolonged steady-state conditions. An outward current that fits such criteria is the inward rectifier potassium current. Inwardly rectifying potassium currents have been found in central and other peripheral neurons, where they are important in setting the resting membrane potential (for example Scroggs et al. 1994). Inward tail currents were present upon hyperpolarization from -40 mV to more negative values. However, they were eliminated when a holding potential of -50 mV was used instead of -40 mV. This indicated that these inward currents result from potassium current that is active at -40 mV but not at -50 mV and that they are not the result of the inwardly rectifying potassium current. Furthermore, when we exposed nodose neurons to BaCl₂, we observed no effect on the steady-state currents of nodose neurons, also suggesting the lack of an inward rectifier.

If the inward rectifier is smaller than I_H though, the current may be masked by I_H at the hyperpolarized potentials (-80 to -140 mV). However, when we substantially reduced I_H by eliminating Na⁺ and replacing extracellular Cl⁻ with gluconate, an inward rectifier potassium current was still not evident. These experiments suggest these neurons do not have an inward rectifier potassium current.

Other potassium channel inhibitors, such as α-dendrotoxin and charybdotoxin, were not effective at the resting membrane potential although they have been shown to block potassium channels in these neurons. Therefore, the outward current that balances I_H at the resting membrane potential still needs to be determined for nodose neurons.

Expression of I_H in nodose sensory neurons

There are several ways to explain the differences between our studies and those from other groups where I_H was not observed in C-type neurons. Marsh et al. (1987) identified C-type sensory neurons using capsaicin. In those studies, no time-dependent rectification was present during hyperpolarizing current injection. Scroggs et al. (1994) and Pearce & Duchen (1994) suggested that an inward rectifier potassium current and not I_H is present in capsaicin-sensitive neurons of the dorsal root ganglion. The most likely explanation for results of these studies resides in the small magnitude of I_H in C-type neurons. The presence of seal leak in either current clamp or voltage clamp recordings can easily obscure the small I_H. The activation of I_H at near threshold potentials is slow and might not be apparent in that voltage range. At more negative potentials the appearance of inward rectification would be dependent on the relative contributions of I_H to the total current at those potentials.

Ingram & Williams (1996) found the expression of I_H only in medium-size to large nodose neurons isolated from adult guinea-pig and concluded that the small neurons did not have I_H. However, they used caesium in the pipette to block outward potassium currents. Under these conditions the outward potassium currents become inward currents due to the change in the reversal potential for potassium ions, making it very difficult to isolate I_H from potassium currents. In addition, I_H is known to run down during whole-cell recordings (DiFrancesco, 1985; Ingram & Williams, 1996). The run-down of I_H has the most impact on C-type neurons, since I_H is substantially less in C-type neurons. In our experiments, the beginning of run-down was evident within 10–15 min after acquiring the whole-cell recording as monitored by changes in threshold for activation and in the amplitude of the current-voltage relationship. Therefore, no data were used when these changes were apparent. Finally, the age of the animal, culture conditions and/or the preparation of the neurons may be an explanation for the differences. There have been reports of developmental changes of I_H in some species. Developmental changes are unlikely since in a preliminary subset of experiments in adult rat nodose neurons, all neurons, including A- and C-type neurons, expressed I_H (data not shown). Additional support for the presence of I_H in C-type neurons is provided in a recent study of unmyelinated vagal and sural nerve fibres. A caesium-sensitive and time-dependent inward rectification was demonstrated in these axons (Grafe et al. 1997).

Functional importance of I_H

The resting membrane potential of nodose neurons lies between -50 and -65 mV, a range where a significant percentage of voltage-dependent sodium, calcium and potassium channels are inactivated. Hyperpolarization of the resting membrane potential may remove the inactivation of these voltage-dependent channels and consequently increase the number of channels available for activation during a depolarizing stimulus. On the other hand, a slight depolarization may increase the number of inactivated voltage-dependent channels, thereby reducing the ability of the neuron to discharge during an excitatory stimulus. In addition, I_H is probably important during the after-hyperpolarization phase of the action potential, where I_H opposes the ability of outward potassium currents to drive the membrane potential past the resting membrane potential. Weinreich & Wonderlin (1987) have shown that the inhibition of the slow after-hyperpolarization in rabbit nodose neurons consequently causes an increase in discharge during depolarizing current injections. Furthermore, the increase in excitability of the neuron is not due to a decrease in the conductance of the Ca²⁺-activated outward potassium channel, a current that participates in the slow after-hyperpolarization. Instead, the alteration in discharge is linked to a cAMP-dependent mechanism, identified by forskolin stimulation of adenylyl cyclase or by prostaglandin D₂ or E₂. Ingram & Williams (1996) have demonstrated that the amplitude of I_H in adult nodose neurons is increased with prostaglandin E₂ and cAMP analogues. This may explain the inhibition of the slow after-hyperpolarization in rabbit nodose neurons.

The major difference in the I_H current between A- and C-type neurons is the magnitude of the current. The projected reversal potentials and the voltage dependence of activation are similar in A- and C-type cells and fall within the range previously reported in other neurons (Pape, 1996). An additional difference is that C-type neurons appear to activate more slowly than A-type. The activation process is voltage-dependent, increasing with hyperpolarization. We find that in both A- and C-type neurons I_H activates with two time constants. Recently two groups have reported cloning from brain several members of what now appears to be a family of I_H channels (Ludwig et al. 1998; Santoro et al. 1998). The heterologous expression of the cloned channels showed differences in voltage-dependent characteristics such as threshold and rate of activation. Such differences within the resting potential range can have substantial effects on neuronal function. Whether they exist in the native cell and whether A- and C-type neurons express distinct members of the I_H family remain to be determined.

In conclusion, I_H contributes towards setting the resting membrane potential of nodose sensory neurons at a critical range where a portion of voltage-dependent sodium, potassium, and calcium channels are inactivated. The modulation of the current may alter the excitability of the neuron by either increasing or decreasing the relative amount of inactivated channels. In addition, the input resistance at the resting membrane potential is very high (≥1 GΩ); therefore any abrupt increase of this cation inward current may induce enough current to elicit an action potential. The characterization of the time- and voltage-dependent properties of I_H provided in the present study may now be used to refine the nodose neuronal model and to investigate the proposals for the functional role described above. The hyperpolarization-activated current has been integrated in a thalamocortical neuronal model (Bal & McCormick, 1996) where it is important for the oscillatory behaviour of the neuron. However, the current may have a different role when expressed in a non-oscillatory cell such as the nodose neuron.