Development of Inward Rectification and Control of Membrane Excitability in Mesencephalic V Neurons

Susumu Tanaka, Nanping Wu, Chie-Fang Hsaio, Jack Turman Jr., Scott H. Chandler


The present study was performed to assess the postnatal development and functional roles of inward rectifying currents in rat mesencephalic trigeminal (Mes V) neurons, which are involved in the genesis and control of oral-motor activities. Whole cell voltage-clamp recordings obtained from Mes V neurons in brain stem slices identified fast (I KIR) and slow (I h) inward rectifying currents, which were specifically blocked by BaCl2 (300–500 μM) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD 7288, 10 μM), respectively. The whole cell current density for these channels increased between postnatal days 2 to 12 (P2-P12), and the time courses forI h activation and deactivation were each well described by two time constants. Application of ZD 7288 produced membrane hyperpolarization in the majority of cells and prolonged afterhyperpolarization repolarization. Additionally, in the presence of ZD 7288, spike frequency was decreased and adaptation was more pronounced. Interestingly, these neurons exhibited a voltage-dependent membrane resonance (<10 Hz) that was prominent around resting potential and more negative to rest and was blocked by ZD 7288. These results suggest that I hcontributes to stabilizing resting membrane potential and controlling cell excitability. The presence of I himparts the neuron with the unique property of low-frequency membrane resonance; the ability to discriminate between synaptic inputs based on frequency content.


Mesencephalic trigeminal (Mes V) neurons are important neurons involved in the generation and control of oral-motor activities. They innervate peripheral jaw-closer muscle spindles and periodontal mechanoreceptors (Corbin and Harrison 1940; Jerge 1963) and centrally project to trigeminal jaw-closer motoneurons and brain stem trigeminal interneurons involved in oral-motor activity (Dessem and Taylor 1989; Luo et al. 1995). Due to their unique location within the brain stem, in addition to their classical proprioceptive sensory function, they have the potential to function as interneurons, as well (Kolta et al. 1990, 1995;Manni et al. 1982). Recently, in vitro studies demonstrated that in response to maintained membrane depolarization these neurons produce high-frequency spike discharge, sometimes exceeding 150 Hz (Pedroarena et al. 1999; Wu et al. 2001), similar to that observed during jaw movements (Hidaka et al. 1999), and rhythmical burst discharge starting around postnatal day 6 (P6) (Wu et al. 2001).

Inward rectification in response to membrane hyperpolarization is a common membrane property of many central and peripheral neurons, including Mes V neurons (Del Negro and Chandler 1997;Khakh and Henderson 1998). Typically, a nonselective cationic current, I h, and a fast K+ selective current,I KIR, are the basis for this property (Hogg et al. 2001; Kjaerulff and Kiehn 2001; Travagli and Gillis 1994; Womble and Moises 1993). Electrophysiological characteristics of these currents vary considerably in kinetics and pharmacological sensitivity (Hogg et al. 2001; Kjaerulff and Kiehn 2001; Travagli and Gillis 1994; Womble and Moises 1993) and have been implicated in control of resting potential, spike discharge, and pacemaker activity, among others (Bal and McCormick 1997; Corotto and Michel 1998; Gasparini and DiFrancesco 1997;Ghamari-Langroudi and Bourque 2000; McCormick and Pape 1990; Thoby-Brisson et al. 2000). Additionally, activation of I h is the basis for the observed subthreshold membrane resonance in neocortical (Hutcheon et al. 1996) neurons, as well. The genes encoding I h channels, called HCN1–4, have recently been cloned in mammals (Ludwig et al. 1998; Santoro et al. 1998), and when expressed in heterologous systems, they produce channels with distinct kinetic properties (reviewed in Santoro and Tibbs 1999). The expression of different combinations of these channels could possibly explain the diverse properties of nativeI h channels in many different types of neurons (Santoro et al. 2000).

Following each action potential, Mes V neurons exhibit a very large, peak early afterhyperpolarization (AHP), traversing membrane potentials greater than –70 mV (Wu et al. 2001). Therefore depending on the properties of I h andI KIR, these currents could participate in control of Mes V neuronal excitability, thus influencing the spike frequency and burst duration during burst discharge. Therefore to test the hypothesis that these currents participate in control of membrane excitability and spike discharge, we examined in detail the properties of these currents during early postnatal development in rat Mes V neurons. We show that I h andI KIR are developmentally regulated, similar to that shown previously forI h in hypoglossal motoneurons (Bayliss et al. 1994), and due to their voltage-and kinetic properties, I h participates in setting membrane potential and maintaining high-frequency spike discharge during excitation.


Tissue preparation

Whole cell recordings from Mes V neurons within brain stem slices were performed using Sprague-Dawley rats (P2–12). The preparation of the slices was similar to the method described before (Del Negro and Chandler 1997, 1998; Wu et al. 2001). Briefly, animals were anesthetized by halothane inhalation and decapitated, and the brain stem was removed in oxygenated ice-cold cutting solution (composition in the next section). Coronal slices (300 μm) were cut with a microslicer (DSK Microslicer), incubated (37°C, 40 min), and placed into a room temperature (22–24°) oxygenated incubation solution (see following text).


Cutting solution was composed of (in mM) 126 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 1 CaCl2, 5 MgCl2, and 4 lactic acid (Schurr et al. 1988). Normal ACSF contained (in mM) 124 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 2 CaCl2, and 2 MgCl2. The incubation solution was identical to normal ACSF with the exception that an additional 4 mM lactic acid added. In voltage-clamp experiments, to isolate inward rectifying currents (I h andI KIR) from other voltage- and time-dependent currents, the recording solution was replaced by a specific solution (modified ACSF) containing (mM) 114 NaCl, 3 KCl, 26 NaHCO3, 10 glucose, 2 CaCl2 or BaCl2, 2 MgCl2, 10 TEA-Cl, 0.1 CdCl2, 2 4-aminopyridine (4-AP), and 0.0005 TTX. NaH2PO4 was omitted to avoid precipitation in this solution. All solutions were equilibrated with 95% O2-5% CO2 and adjusted pH to 7.3. Patch pipettes were filled with (in mM) 115 K-gluconate, 25 KCl, 9 NaCl, 10 HEPES, 0.2 ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 MgCl2, 3 K2-ATP, and 1 Na-GTP, pH ≅ 7.25 (22–24°C). Osmolarity was adjusted to 280–290 mosM.

Drugs were bath-applied at the concentrations indicated: 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD 7288) (10 μM; Tocris Cookson), 4-AP (30–40 μM; Sigma). Ba2+ (300–500 μM) was added directly to the bath in some experiments to blockI KIR.

Patch-clamp recording technique

Mesencephalic trigeminal sensory nuclei were located bilaterally and dorsally in the coronal slice as an ellipsoid region under low magnification (×5) as previously described (Del Negro and Chandler 1997; Wu et al. 2001). Mes V neurons were easily distinguished from surrounding neurons on the basis of their location, pseudounipolar soma, and size (Del Negro and Chandler 1997; Wu et al. 2001) under visual control using infrared differential interference contrast video microscopy (Stuart et al. 1993). Patch electrodes were fabricated from thick-walled borosilicate grass (OD: 1.5 mm; ID: 0.86 mm) to a tip resistance of 2–4 MΩ (Sutter Instruments P-97, Novato, CA). An Axopatch-1D patch-clamp amplifier and pCLAMP acquisition software (Axon instruments, Foster City, CA) were utilized for voltage- and current-clamp recordings, and all signals were grounded by a 3 M KCl-agar bridge electrode (Ag/AgCl wire). Space-clamp artifacts were minimized by the spherical size and lack of dendritic processes in these neurons.

For time-domain analyses, we used a low-pass Bessel filter with a corner frequency of 3 dB at 2 kHz (voltage clamp) or 5 kHz (current clamp). Membrane input resistance (R inp) was calculated in voltage-clamp by applying 5-mV step pulse (10 ms) from a holding potential of –70 mV. Cell capacitance (C m) was determined from the integral of the high-resolution capacitive current in response to 15-ms hyperpolarizing step voltage command. Series resistance (R s) was evaluated by analyzing the decay time constant of the capacitance transients (uncompensated R s <15 MΩ).R s was compensated by 80% and monitored during the course of experiments. Liquid junctional potential (≅ −5 mV), resulting from the K-gluconate containing electrode, was measured and corrected off-line (Neher 1992).

Data analysis

Whole cell voltage and current signals were digitized and recorded with pCLAMP acquisition software (v8.1, Axon Instruments) for subsequent analysis. Inward rectifying currents were induced from a series of hyperpolarizing 5- to 10-mV voltage step commands from a holding potential of –55 to –140 mV. We used a short-duration (300 ms) V-clamp protocol (except for time constant analysis when a step duration of 2–5 s was used) even though the currents did not fully reach a steady-state phase because the cell was unstable during strong hyperpolarization negative to –120 mV. The interval between command pulses was 15 s.

Frequency-domain analysis (Puil et al. 1986, 1988) was performed by injecting a computer generated ZAP input current of changing frequencies between 0 and 250 Hz into neurons around or more negative to resting potential and recording the resulting voltage responses. To carefully analyze the subthreshold membrane properties in the absence of spikes, the amplitude of the ZAP input function was adjusted to keep the peak-to-peak voltage responses smaller than 10 mV. ZAP input current was generated with the formulal(t)=asin(bt3),0tT Here, a and b are adjustable parameters controlling the amplitude and bandwidth of the input current, respectively. T was a finite duration. In our case,a = 5, b = 10−7,t = 8 s, T = 10 s. We used a low-pass filter of 0.5 kHz to reduce the noise of the input current. The results with and without the low-pass filter were identical. The current and voltage records were digitized at frequencies of 10 kHz. Impedance (Z) was calculated from the ratio of the fast Fourier transforms (FFT) of the voltage response and the input current using the formulaZ=FFT(V)/FFT(I) The magnitude of the impedance was plotted against frequency to give a frequency-response curve (FRC). Once the FRC was obtained the resonant behavior, if present, was quantified by measuring the resonant frequency (F res) and the Qvalue. The F res was defined as the frequency at the peak of the hump in the FRC. The Q value was calculated by measuring the impedance atF res and dividing that by the magnitude of the impedance at the lowest frequency measured (Koch 1984; Hutcheon et al. 1996;Wu et al. 2001).

Data analyses were assessed with a combination of software [StatView (SAS Institute, Cary, NC), and Microsoft Excel]. Results were reported as means ± SE and comparison of means was assessed with the Student's t-test set at a level of significance ofP < 0.05 unless otherwise stated. In some cases, an ANOVA was performed for group comparisons.


Passive membrane properties of developing Mes V neurons

For the present study whole cell recordings were performed from P2 to P12 rat Mes V neurons through out the rostral-caudal extent of the nucleus to avoid sampling bias. Generally, after establishing whole cell configuration, cells were held at a potential of –70 mV in normal ACSF, and various membrane properties were measured. Cells that showed an initial resting potential equal to, or more negative, than –55 mV and an evoked action potential amplitude greater than or equal to, 80 mV were accepted for further study. Resting membrane potential (E rest) did not vary significantly with age. However, input resistance (R inp) significantly decreased and this was accompanied by an increase in cell size as measured by an increase in cell capacitance (C m) among P2–4, P6–8, and P10–12 age groups (P < 0.0001; Table 1), as described, previously (Wu et al. 2001).

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Table 1.

Summary of passive membrane properties in Mes V neurons

Characterization of inward rectification in Mes V neurons

Hyperpolarizing current pulses (0.2–0.5 nA) consistently produced voltage- and time-dependent depolarizing sag during current-clamp recording in normal ACSF, indicative of an underlying inward rectifyingI h described previously in Mes V neurons (Khakh and Henderson 1998) (Fig.1A). The time course of relaxation from the initial peak amplitude to a steady-state value was faster in older compared with younger animals, indicating developmental regulation ofI h (Guido et al. 1998;Washio et al. 1999). In voltage-clamp recording in normal ACSF (Fig. 1 B), a family of hyperpolarizing voltage steps ranging from –140 to –60 mV in 10-mV increments, from a holding potential of –55 mV, induced an instantaneous current jump that was followed by a slowly developing steady-state current for both age groups (Fig. 1 B, inset). The peak amplitude of the steady-state inward relaxation was age-dependent and showed robust inward rectification with larger voltage steps.

Fig. 1.

Mesencephalic V (Mes V) neurons exhibit inward rectification. Current- (A) and voltage-clamp (B) recordings from postnatal P2 and P11 neurons in normal artificial cerebrospinal fluid (ACSF). A: voltage- and time-dependent depolarizing sag in response to hyperpolarizing current pulses (450 ms). The sag occurred more rapidly and was more prominent in P11 neurons (←).B: current responses evoked by hyperpolarizing voltage steps between –140 and –60 mV from a holding potential of –55 mV. Instantaneous currents are indicated (*) following capacitive transients (inset, expanded time scale). In this and subsequent figures, slowly developing steady-state inward currents were measured at the end of the pulse (**). Tail currents were observed at −75 mV after termination of pulse.

Pharmacological identification of fast and slow inward rectifying currents

In most neurons examined (33/40), a fast inward rectifying current was evident and was blocked by low doses of Ba2+, suggesting the presence of I KIR. Figure 2 A illustrates a typical example of superimposed current records in normal ACSF evoked in response to 5-mV hyperpolarizing voltage step increments from a holding potential of –55 to –140 mV before, and after, Ba2+ application (300–500 μM), while Fig.2 B shows the associated instantaneous (measured from *) and steady-state I-V (measured from **) relationships. Typically, the instantaneous current responses deviated from linearity ( · · · indicate extrapolated leakage current) at voltages more negative than –100 mV. As shown, Ba2+ blocked this deviation. In contrast, the steady-state I-V relationship showed inward rectification starting around –90 mV. Although, Ba2+ increased input resistance, it produced no effect on the steady-state inward rectification that represents I h (Fig.2 B).

Fig. 2.

Mes V neurons exhibit fast (I KIR) and slow (I h) inward rectifiers. A andC: membrane current responses in response to hyperpolarizing step voltage commands between –60 and –140 mV from a holding potential of –55 mV before (A) and after Ba2+ application (500 μM) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (C, ZD 7288, 10 μM), respectively. B and D: instantaneous (*) and steady-state (**) current-voltage relationships obtained from data shown in A and C. External Ba2+ reduced the instantaneous leak current (extrapolated · · · ) and I KIR below –100 mV, indicated by the inward current deviation from leak current, but produced minimal effects on the slow inward rectifier,I h. Note the lack of effect of ZD 7288 on the I KIR. · · · , inB and D, are extrapolated leak currents obtained from linear region of I-V relationship around –60 mV. Data obtained in normal ACSF from 2 different P11 neurons.

In contrast, the bradycardic agent, ZD 7288 (10 μM), which is highly specific antagonist of I h in other systems (Harris and Constanti 1995), prominently reduced the steady-state inward rectification (Fig. 2, C andD) further confirming the presence ofI h. Although ZD 7288 increased input resistance somewhat, it did not blockI KIR (Fig. 2 D, left).

Identification and postnatal development of fast and slow inward rectifying currents


To characterize I KIR andI h in detail, in the following experiments, we applied a modified ACSF to the bath to eliminate potential contamination of the inward rectifying currents by other currents (see methods). To isolate Ba2+-sensitiveI KIR, using the protocol shown in Fig.2, we obtained leak-subtracted difference currents by the following method. First we leak-subtracted the total membrane current in control conditions, then we repeated this in Ba2+(300–500 μM) conditions, and obtained the difference current. The reversal potential (E KIR) for Ba2+-sensitiveI KIR was determined from the intersection of instantaneous I-V curves obtained before, and after, addition of Ba2+. The measured reversal potentials in 3 mM external K+ were similar between two extreme age groups (P2–3, −95.1 ± 1.6 mV,n = 7; P10–12, −96.9 ± 1.2 mV,n = 8) and close to predicted potassium equilibrium potential (i.e., –91.9 mV). In 5 and 10 mM external K+ (n = 3), we found the measured reversal potential to shift in the depolarizing direction (5 mM, −77.1; 10 mM, −62.7 mV) and was within 3 mV of the equilibrium potential predicted from the Nernst relationship for K+, suggesting that the fast inward rectifying current is a K+ current resembling KIR.

For both age groups, Ba2+-sensitiveI KIR was voltage dependent and close to fully activated around –140 mV (Fig.3 A). The magnitude of the current (P2–3, 23.5 ± 2.6 pA, n = 7 vs. P10–12, 106.0 ± 11.0 pA at –140 mV, n = 9;P < 0.0001), and current density (current divided byC m) increased with age (Fig.3 B; Table 2). In addition,G KIR was quantified with the equationGKIR=I/(VEKIR) where V is applied step command potential, andE KIR is the approximated reversal potential. As shown in Fig. 3 B, G KIR(measured at –140 mV) also increased, substantially, with age.

Fig. 3.

Ba2+-sensitive I KIR increases during early development. A: Ba2+-sensitiveI KIR density plotted against command potentials for 2 age groups. Difference current density-voltage relationship revealed that activation started around the calculated potassium equilibrium potential (i.e., –91.9 mV) for both age groups (P2–3 and P10–12). B: summary of Ba2+-sensitive I KIR current density and conductance changes as a function of age. Each column represents mean ± SE and the number of neurons examined (in parentheses) for each age group. Measurements taken at –140mV. *, statistical difference compared with P2–3 neurons (P < 0.001, ANOVA).

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Table 2.

Electrophysiological properties of Ih and IKIRin Mes V neurons


To characterize more clearly the electrophysiological properties of the slowly activating inward current, I h, we applied hyperpolarizing voltage command steps in themodified ACSF used to isolatedI KIR. To block other K+ currents includingI KIR, Ca2+ was replaced by Ba2+, a nonspecific blocker of K+ conductances, in the extracellular solution. This produced a nearly linear instantaneous I-V relationship over the voltage range examined (similar to Fig. 2 B). Figure4 A shows the meanI h current density-voltage relationship, obtained by subtraction of instantaneous current from the steady-state current (inset) for the P2–3 and P10–12 age groups. Similar to the Ba2+-sensitiveI KIR,I h was also voltage dependent. Additionally, current density and conductance were developmentally regulated (Fig. 4 B; Table 2).

Fig. 4.

I h density increases postnatally.A: voltage-dependent activation ofI h density (inset, measured as a difference current) for 2 age groups in modified ACSF.B: summary of I h current density and conductance changes as a function of age. Each column represents mean ± SE and the number of cells examined (in parentheses) for each age group. Measurements taken at –140 mV. *, statistical difference compared with P2–3 neurons (P < 0.001, ANOVA).

The reversal potential (E h) forI h was determined by the method employed by others (Banks et al. 1993; Bayliss et al. 1994; Dickson et al. 2000). Briefly,E h was obtained from the extrapolated intersection of two instantaneous I-V curves induced by voltage steps from a holding potential whereI h is not activated (−45 mV) and from a potential where I h is activated (−95 mV; data are not shown). There were small differences in meanE h values between P2–3 and P10–12 age groups that were statistically significant (Table 2), but more depolarized to previous measurements in Mes V neurons recorded in normal extracellular bathing solutions (-57 mV) (Khakh and Henderson 1998).


Voltage-dependent activation of I h was further examined by measuring the tail currents evoked after stepping back to –75 mV in response to a series of preceding hyperpolarizing voltage steps as described previously (Hsiao et al. 1997). Tail current amplitudes were normalized using the following formulaRnorm=(RRmin)/(RmaxRmin) where R max is the maximal tail current and R min is the minimal tail current (see Fig. 5, inset). Normalized current amplitudes were plotted as a function of the prepulse potential. As shown in Fig. 5,I h activation curves obtained from young (P2–3) and older (P10–12) age groups were well-fitted with a single Boltzmann function. For both age groups the onset of activation and the voltage where maximal activation occurred were similar (Fig.5). However, the half-maximal activation voltage (V 1/2) was shifted to the right and the slope factor (k) was decreased for the older age group (Table 2).

Fig. 5.

Activation curve for I h. A: activation curve for I h conductance based on tail current analysis for 2 age groups. Normalized tail currents measured at –75 mV after termination of a family of hyperpolarizing voltage steps (see inset and results) and plotted against prepulse potential. Mean activation curves for P2–3 (○, n = 8) and P10–12 (●,n = 12) age groups were fitted with single Boltzmann functions of the form R norm = {1 + exp[(V mV 1/2)/k]}−1(V m, applied step command potential;V 1/2, half-maximal activation voltage;k, slope factor). Note that the half-maximal activation was shifted to a more depolarized potential in P10–12 compared with P2–3 age group.


The activation and deactivation kinetics ofI h were estimated using a long step (2–5s) protocol to allow I h to approximate steady state. As shown in Fig.6 A, in most instances, the time course of activation and deactivation were well fitted with double exponential function of the formIh(t)=A1e(t/τfast)+A2e(t/τslow)+C where I h(t) is the current at time t, C is a constant value, andA 1, andA 2 are amplitude coefficients for each time constant. Figure 6 B indicates that for both age groups, the fast and slow time constants for activation (τfast and τslow) were voltage dependent as shown by others (Dickson et al. 2000; Kjaerulff and Kiehn 2001). Both time constants significantly decreased with age (τfast, P2–4, 277.1 ± 8.5 ms vs. P10–12, 209.5 ± 11.0 ms at –105 mV; P < 0.0001; τslow, P2–4 1986.0 ± 60.3 ms vs. P10–12, 1729.2 ± 45.9 ms at –105 mV; P < 0.0001, factorial ANOVA) as shown previously (Hogg et al. 2001; Washio et al. 1999).

Fig. 6.

I h activation and deactivation kinetics.Aa: I h was evoked by a long voltage step (5 s) protocol (bottom).I h activation time course was well fitted by a double-exponential function during the steps between –100 and –120 mV (see results). Ab:I h deactivation time course induced by depolarizing voltage steps from a holding potential of –110 mV was well fitted by a double-exponential function (see results).B: composite plot of fast and slow time constants forI h activation as a function of voltage.C: composite plot of fast and slow deactivation time constants for I h (each data point represents ≥4 neurons).

The time course of deactivation also showed fast and slow components that were both voltage dependent and increased somewhat with age (τfast, P2–4, 128.7 ± 9.7 ms vs. P10–12, 156.8 ± 3.0 ms at –70 mV; P < 0.0001; τslow, P2–4, 615.1 ± 38.6 ms vs. P10–12, 686.6 ± 31.5 ms at –70 mV; P < 0.01, factorial ANOVA; Fig. 6 C). Our results are similar to those shown previously in adult Mes V neurons (Khakh and Henderson 1998).

Functional roles for Ih in control of cellular excitability


The activation data presented in Fig. 5 suggest thatI h does not contribute to control of resting potential because, for most Mes V neurons, the mean resting potential is around –65 mV (Tables 1 and3). However, those data onI h were obtained in a modified ACSF solution to specifically isolate I h. Interestingly, other studies using normal ACSF showed a more positive onset for activation of I h that would suggest its participation in control of resting potential (Gasparini and DiFrancesco 1997). Therefore to determine if I h contributes to resting potential, we conducted the following experiments in normal ACSF.

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Table 3.

Effect of ZD 7288 on resting membrane potential

In a subset of P10–12 neurons (n = 6), we re-examined the activation curve for ZD 7288-sensitive currents obtained by subtraction of the currents prior to, and after, addition of ZD 7288 (10 μM) in normal ACSF using the methods presented in Fig. 5. We found that compared with V 1/2 obtained in modified ACSF (V 1/2 = −105.0 mV), in normal ACSF the value was shifted in the depolarizing direction (V 1/2 = −96.9 mV), similar to that previously reported (Khakh and Henderson 1998). Furthermore, the onset of activation was approximately –60 mV instead of –80 mV. This would suggest that I hcan participate in control of resting potential. To substantiate this, we examined the effects of the I hblocker, ZD 7288 on resting potential in normal ACSF.

As shown in Table 3, application of ZD 7288 (10 μM, 10–15 min) produced a hyperpolarizing shift >1 mV in the resting membrane potential in more than half of the cells for both age groups (mean change: P2–4, −5.1mV; P10–12, −5.4 mV). In spite of the slightly depolarized mean membrane potential in the older age group, a greater percentage of cells showed a hyperpolarizing shift in response to the channel blocker for that age group. The greater percent change in the older animals is consistent with our observed developmental increase inI h current density.

In some neurons, we artificially hyperpolarized the membrane potential from rest to approximately –72 mV; a region that should activateI h in normal ACSF. As expected, in all neurons (n = 9), application of ZD 7288 produced a membrane hyperpolarization of >10 mV, irrespective of age group tested. These effects of ZD 7288 suggest a significant contribution ofI h to resting membrane potential in Mes V neurons.


Membrane resonance is a property that indicates the ability of a neuron to discriminate between inputs on the basis of their frequency content (Hutcheon and Yarom 2000). Frequency domain analysis showed, previously, that Mes V neurons exhibit a voltage-dependent high-frequency resonance (∼50–100 Hz) at potentials just subthreshold to action potential generation (between –50 and –45 mV) (Wu et al. 2001). However, in other types of neurons, the presence of a low-frequency resonance (<10 Hz) at voltages negative to resting potential was observed and is mediated by activation of I h (Hutcheon et al. 1996). Because Mes V neurons exhibitI h around resting potential, the possibility exists that this current, in combination with the passive membrane properties, produces the necessary conditions for low frequency resonance in these neurons. This would endow these neurons with the ability to discriminate inputs on the basis of frequency content; enhancing appropriate synaptic potentials while suppressing spurious activity. To test this hypothesis, frequency domain analysis was performed.

Figure 7 A shows the impedance-frequency relationship in response to a ZAP current input as a function of membrane voltage. As shown, a resonant peak (F res) in the FRC is observed ∼5 Hz and is largest between –65 and –75 mV. TheF res was similar for both older and younger age groups (Table 4). Although the peak magnitude of the impedance is less for the older animals, theQ value was actually greater, and this is summarized in Table 4. This suggests that older animals can selective amplify incoming oscillatory signals occurring around Fres more effectively compared with younger animals.

Fig. 7.

I h is responsible for low-frequency membrane resonance. A: 3-dimensional representation of subthreshold membrane resonance at holding potentials between –65 and –80 mV in a P10 neuron. Frequency-response curve was produced in normal ACSF using ZAP input currents (see methods). Peak magnitude of impedance was voltage dependent with the highest value occurring around –70 mV. B: application of ZD 7288 (10 μM, 10 min) increased impedance magnitude of FRC at frequencies belowF res and eliminated the resonant peak [holding potential (V hold) –70 mV].Ca: subthreshold membrane potential response to ZAP input current in the presence of 4-AP (30 μM) (seeresults) (inset, FRC). Cb: action potentials (truncated, ■) produced at the peak of the membrane response after a small increase in ZAP input current (holding of –70 mV). D: Ba2+ (500 μM) increased the overall impedance magnitude of the FRC without affecting resonance (holding potential –80 mV).

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Table 4.

Resonant properties of Mes V neurons for different age groups


To test the hypothesis that I hmediates the low-frequency resonance, the effects of the potentI h blocker ZD 7288 (10 μM) was examined on the impedance-frequency relationship. Typically, ZD 7288 abolished the resonant peak and increased the impedance magnitude in both young (P2–3, n = 3, data not shown) and older age groups (P10–12, n = 7) predominately at frequencies less than the resonant frequency (Fig. 7 B), thus transforming the shape of the FRC into one resembling a low-pass filter. This suggests that I h is, indeed, the resonant current (Hutcheon and Yarom 2000;Hutcheon et al. 1996).

To determine if the low-frequency resonance is capable of generating spikes within the resonant frequency range, in the presence of 30 μM 4-AP to eliminate activation of I 4-AP, a noninactivating K+ current (Wu et al. 2001), the amplitude of the ZAP current was increased slightly until spikes were produced (Fig. 7 Cb, •). In all cases (n = 3), spikes occurred only within the frequency range of maximal impedance (Fig. 7 C and inset).

In contrast to ZD 7288, application of Ba2+(300–500 μM) did not abolish the resonant peak but did prominently increase the overall impedance at frequencies on both sides of the resonant peak (Fig. 7 D). As expected, Ba2+ also increased the input resistance of all neurons most likely from suppression ofI K-leak (control, 104.5 ± 6.7 vs. Ba2+, 140.1 ± 13.5 MΩ;P < 0.01).


Previous studies in other neurons revealed a critical role forI h in generating the action potential AHP and regulating repetitive discharge characteristics (Corotto and Michel 1998; Ghamari-Langroudi and Bourque 2000; McCormick and Pape 1990). We showed previously that the AHP following an action potential is generated by a variety of K+ conductances, including a 4-AP-sensitive K+ conductance and a Ca2+-activated K+conductance (Del Negro and Chandler 1997; Wu et al. 2001). Furthermore, Mes V neurons exhibit strong adaptation in response to a maintained current pulse (Del Negro and Chandler 1997). An interesting difference between Mes V neurons and trigeminal motoneurons (TMNs) during repetitive discharge is the larger amplitude AHP following each action potential within a spike train for Mes V neurons (Chandler et al. 1994;Del Negro and Chandler 1997; Wu et al. 2001). In these neurons, the peak amplitude attained for the early AHP traverses the activation range forI h. Therefore the possibility arises that I h actually contributes to control of spike frequency by virtue of its slow kinetics and voltage dependence. To test this hypothesis, we examined the effects of block of I h on individual action potential AHPs and repetitive discharge characteristics in both P2–4 and P10–12 age groups.

To evaluate the role of I h in generating the AHP following an action potential in Mes V neurons, we recorded single action potentials (AP) using short-duration depolarizing step pulses (3 ms, 0.2–0.5 nA) from a resting potential (E rest) in young (P2–4) and older (P10–12) neurons. Figure 8 Ashow a typical example from each age group. During action potential repolarization in P2–4 neurons, an early, short-duration afterhyperpolarization (e-AHP) was followed by a later, long duration AHP (l-AHP). These afterpotentials were separated by an afterdepolarization (ADP; Fig. 8 A, box), similar to that shown in trigeminal motoneurons (Chandler et al. 1994). Although an early and late AHP were also identified in P10–12 neurons, the transition of the two AHPs was not clearly defined. Furthermore, the peak amplitude of e-AHP (measured fromE rest) was smaller and the time for recovery from the peak of e-AHP toE rest, representing the duration of AHP, was longer in P2–4 compared with P10–12 age groups, indicating a developmental regulation of ionic conductances underlying action potential AHP.

Fig. 8.

I h accelerates afterhyperpolarization (AHP) repolarization. A: single action potential was evoked by short-duration (3 ms) depolarizing current pulse in P2 and P12 neuron. Neurons from both age groups showed an early, short-duration AHP (e-AHP) and a subsequent later, longer-duration AHP (l-AHP).Inset: example of waveforms from both age groups and arrows indicate measurements taken. Membrane potential was adjusted to be approximately the same for both neurons. Note that the early and late components were clearly separated by an afterdepolarization (ADP, *) for the P2 neuron, while the transition between the 2 AHPs was not clearly defined for the P12 neuron. B: superimposed mean AHP waveforms (n = 5) following single action potentials before, and after, application of ZD 7288 (10 μM) for a P3 and P11 neuron. Suppression of I h prolonged the duration of late AHP for both age groups.

External application of ZD 7288 (10 μM) prolonged the duration of the AHP and partially suppressed the peak amplitude of the e-AHP for both P3 and P11 neurons (Fig. 8 B). The prolonged duration mainly resulted from a change in the time course of the late AHP. Table5 summarizes the effects of ZD 7288 on AHP characteristics for the two age groups.

View this table:
Table 5.

Effect of Ih blocker on action potential properties

These data suggest that block of I hshould affect repetitive discharge characteristics during a spike train. This is demonstrated in Fig. 9. After application of ZD 7288 the duration of a spike train and instantaneous frequency of spikes within the spike train in response to constant current pulse were decreased and the slope of the frequency- time relationship increased (Fig. 9, A and B). After ZD 7288, the slope of the mean frequency-current relationship was reduced, as well (Fig. 9 C). This effect was not a result of a decrease in R inp because this actually increased after ZD 7288 application (control, 98 MΩ; ZD 7288, 174 MΩ, n = 14, see Fig. 2 D).

Fig. 9.

I h participates in control of repetitive discharge. A: effects of ZD 7288 on sag response and spike discharge in response to current pulse (1 s). Note that ZD 7288 suppressed sag response and enhanced adaptation. Holding potential was –55 mV (P11 neuron). B: instantaneous frequency-time relationship prior to, and after, ZD 7288. Note that the time axis is truncated to 600 ms compared with the 1-s duration pulse for clarity.C: mean frequency (last 5 interspike intervals, indicated by bar in A)—current relationship prior to, and after, ZD 7288. Mean frequency was increased with the current intensities and decreased by ZD 7288 throughout the applied current range. D: spike discharge in response to current pulse application in the presence of 4-AP (40 μM) prior to, and after, ZD 7288. E and F: frequency-time and current relationships for data shown in D. Compare the effects of ZD 7288 on data taken from E and Fwith that in B and C.

To obtain further evidence that I hactivation, as a result of action potential repolarization to very negative values, participates in control of spike frequency and adaptation, we blocked the early AHP with 4-AP (Fig. 9 D), previously shown to eliminate the large amplitude early AHP in Mes V neurons (Wu et al. 2001) and then applied ZD 7288 (Fig.9 D, right). During 4-AP conditions,I h was minimally activated and the spike frequency-time and current relationships were hardly altered after ZD 7288 application (compare Fig. 9, B andC to E and F). These data further support a role for I h in control of repetitive spike discharge characteristics.


The present study demonstrated in Mes V neurons from postnatal rats P2–P12 the presence of two developmentally-regulated different types of hyperpolarization activated inward rectifying currents, identified as I h andI KIR based on their unique electrophysiological and pharmacological properties, similar to that shown previously in both central and peripheral neurons (Hogg et al. 2001; Kjaerulff and Kiehn 2001;Travagli and Gillis 1994; Womble and Moises 1993). Although it is difficult to attribute a precise role forI KIR in control of membrane excitability and resting potential due to its very negative activation threshold in Mes V neurons, we will argue thatI h has significant effects on membrane excitability through its contributions to resting potential, individual spike, and spike train characteristics. Furthermore, the presence of low-frequency resonance mediated by I hsuggests that Mes V neurons can discriminate between, and selectively amplify, low-frequency inputs originating from peripheral (spindle, periodontal receptor) and central synaptic sources. The salient features of our arguments are put forth in the following text.

The majority of Mes V neurons exhibited bothI h andI KIR during the developmental period tested (P2–12). Although both currents were present in Mes V neurons, in the older age group (P10–12) I hactivated at a more depolarized membrane potential compared withI KIR, similar to that reported for amygdala neurons (Womble and Moises 1993). However, due to rapid time course and small amplitude of the repolarizingI KIR tail currents, we were not able to characterize their voltage dependence reliably. It is unlikely that this channel would contribute to spike discharge or resting potential since the maximum peak AHP and resting potential do not traverseI KIR activation threshold.

I h channels in Mes V neurons show clear developmental increases in current density, as well as maximal conductance (Fig. 4) as previously reported in other cell types (Bayliss et al. 1994; Richter et al. 1997; Washio et al. 1999). Interestingly, the half-maximal activation was significantly shifted to more depolarized values during postnatal development (Fig. 5) in contrast to some previous studies (Bayliss et al. 1994; Cerbai et al. 1999; Washio et al. 1999). The shift inV 1/2 suggests that during development, Mes V neurons become more efficient in stabilizing membrane potential in response to phasic inhibitory inputs, as well as more effective in controlling membrane excitability during spike discharge (discussed in the following text).

The time course for activation of I hin Mes V neurons was described well by two different kinetic components that had time constants ranging from hundreds of milliseconds to seconds (Fig. 6 B), similar to that previously reported in Mes V neurons (Khakh and Henderson 1998). Interestingly, the activation time constants associated previously with HCN2 and HCN4 isoform expression in other CNS neurons (Santoro et al. 2000) were in the same range as that found for Mes V neurons. In contrast, deactivation exhibited two time constants that showed a more restricted range of values (Fig. 6 C). These data suggest that multiple I h channels with different kinetic and gating properties are expressed in Mes V neurons.

Roles for Ih in control of neuronal excitability


In normal ACSF, the V 1/2 forI h was shifted closer to resting potential. In response to ZD 7288, more than half of the neurons hyperpolarized ∼5 mV (Table 3), suggesting a tonic activation ofI h at rest (Dickson et al. 2000; Gasparini and DiFrancesco 1997;Ghamari-Langroudi and Bourque 2000; Lupica et al. 2001). This is in contrast to a previous study on Mes V neurons where suppression of I h did not alter resting potential (Khakh and Henderson 1998). It is difficult to predict the effects of suppression of a resting level ofI h on membrane excitability. In theory, suppression of a tonically active restingI h would slightly hyperpolarize neurons but lead to an increase inR inp as a result of closure of active channels, thus making the neuron more responsive to small synaptic inputs at more negative potentials (Gasparini and DiFrancesco 1997) and enhancing the frequency-current relationship (Maccaferri and McBain 1996). However, in Mes V neurons this would be counterbalanced to some extent by the decreasein excitability that occurs as a result of loss of inwardI h activated by individual spike AHPs during a spike train (Fig. 9, discussed in the following text). In physiological conditions, the net effect of modulation ofI h channels will be determined by the relative activation of those channels at rest and during spike discharge.


In the majority of cells, ZD 7288 reduced steady-state discharge frequency, produced rapid adaptation and early termination of repetitive discharge (Fig. 9). Furthermore, ZD 7288 shifted theF-I relationship downward throughout the current range tested, suggesting a significant participation ofI h in control of spike frequency (Gasparini and DiFrancesco 1997;Ghamari-Langroudi and Bourque 2000; Hogg et al. 2001). These effects most likely resulted from prolongation of the late AHP repolarization phase as a result of block ofI h (Fig. 8 B). In contrast to trigeminal motoneurons (Chandler et al. 1994;Hsaio et al. 2002), in Mes V neurons the peak amplitude of the AHP following each action potential during a spike train is large and traverses the activation range forI h. BecauseI h reversal potential is positive to resting potential, and I h deactivation is on the order of hundreds of milliseconds (Fig. 6), activation of inward I h would effectively hasten the repolarization of the late AHP toward resting potential, thereby shortening the interspike interval and contribute to the high rates of discharge observed in these neurons. In support of this, in the presence of 4-AP to block the rapid, large amplitude AHP and a resting 4-AP-sensitive K+ conductance (Del Negro and Chandler 1997), as expected, the frequency-current relationship and frequency adaptation were not effected by ZD 7288 (Fig. 9, B and C vs. E andF).

Further support for activation of I hduring a spike train comes from the observation that ZD 7288 enhanced individual spike duration and slowed the rate of action potential repolarization (Table 5). The deactivation time constant forI h is orders of magnitude slower than that for spike repolarization (Fig. 6). Therefore in the absence of ZD 7288, tonic activation of I h would produce outward current during the rising and falling phase of the spike at potentials depolarized toI h reversal potential, thus leading to more rapid action potential repolarization. BecauseI h current density was greater in Mes V neurons from older animals, this could, partly, account for the shorter action potential duration observed in those animals. Although difficult to assess, the prolongation in AHP duration after ZD 7288 could have resulted, partly, from enhancement of the Ca2+-dependent K+ current present in these neurons (Del Negro and Chandler 1997) as a result of the increased Ca2+ influx during the action potential.

Functional implications for Ih and membrane resonance during jaw movements

Previously, we showed that Mes V neurons exhibit high-frequency (>50 Hz), voltage-dependent membrane resonance at depolarized membrane potentials (Wu et al. 2001) as a result of a complex interaction between a 4-AP-sensitive K+ current and the passive membrane properties. Furthermore, we demonstrated that the resonance was amplified by a voltage-dependent activation of a persistent Na+ current that is responsible for producing subthreshold oscillations and high-frequency discharge at depolarized membrane potentials. In the present study, we found that at resting potential and more negative to resting potential, low-frequency resonance (∼4–5 Hz) is present. Application of ZD 7288 revealed that this resonance resulted from activation ofI h; in the presence of ZD 7288 the resonance observed in the frequency-response curve was abolished and the curve was transformed into one characteristic of a low-pass filter. It is unlikely that activation of I KIRcontributed to production of this resonance because its block with Ba2+ enhanced the resonance throughout most of the frequency range examined; suggesting its role as a high-pass filter. Similar observations on the presence of resonance were reported in neocortical neurons (Hutcheon et al. 1996).

The presence of low-frequency membrane resonance should have important effects on the integrative behavior of Mes V neurons during oral-motor activity. Resonance endows a neuron with the ability to discriminate between input signals based on their frequency content (Hutcheon and Yarom 2000). Mes V neurons are unique in that their cell bodies are located within the CNS, thus potentially endowing them with the capability of functioning as interneurons, as well as in the more traditional role as sensory proprioceptive neurons. Mes V neurons exhibit rhythmical burst discharges of <5 Hz during fictive mastication (Kolta et al. 1990). If this is associated with low-frequency synaptic drive potentials from a central pattern generator, similar to that observed in trigeminal motoneurons (Chandler and Goldberg 1982), then the presence ofI h-mediated resonance would amplify the membrane drive potentials and facilitate spike bursting. This would be particularly effective if the drive potentials consisted of a hyperpolarizing component. TheI h-mediated resonance would hasten the time course of the inhibition and facilitate post inhibitory rebound spike discharge. In contrast, asynchronous high-frequency subthreshold synaptic activity would be functionally suppressed, preventing spurious excitation of Mes V neurons.

Finally, the observation that the resonance is enhanced in the older compared with younger population examined (Table 4, Qvalues), suggests a maturation in the ability to fine tune and discriminate between jaw movement related inputs during development. Given that I h channels are targets for modulation by various neuromessengers (Pape 1996), the presence of I h and membrane resonance engenders Mes V neurons with great flexibility in control of membrane excitability during normal reflex and centrally produced jaw movements. It is interesting to speculate that abnormal function inI h channels as a result of environmental stimuli, such as injury, could contribute to oral-motor dysfunction.


Special thanks goes to M. Castillo for technical assistance.

This work was supported by National Institute of Dental and Craniofacial Research Grant DE-06193. S. Tanaka was supported by the Japan Society for the Promotion of Science.


  • Address for reprint requests: S. H. Chandler, Dept. of Physiological Science, UCLA 2851 Slichter Hall Los Angeles, CA 90095-1568 (E-mail: schandler{at}


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