INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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 native I_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 and I_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 and I_KIR are developmentally regulated, similar to that shown previously for I_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.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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).

Solutions

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 and I_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(-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 block I_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 M (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 M). 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 formula
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 = 10⁷, 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 formula
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 Q value. 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 at F_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 of P < 0.05 unless otherwise stated. In some cases, an ANOVA was performed for group comparisons.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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).

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 rectifying I_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 of I_h (Guido et al. 1998; Washio et al. 1999). In voltage-clamp recording in normal ACSF (Fig. 1B), 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. 1B, inset). The peak amplitude of the steady-state inward relaxation was age-dependent and showed robust inward rectification with larger voltage steps.

View larger version (26K): [in this window] [in a new window]   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 2A 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. 2B 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. 2B).

View larger version (25K): [in this window] [in a new window]   Fig. 2. Mes V neurons exhibit fast (IKIR) and slow (Ih) inward rectifiers. A and C: 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 IKIR below -100 mV, indicated by the inward current deviation from leak current, but produced minimal effects on the slow inward rectifier, Ih. Note the lack of effect of ZD 7288 on the IKIR. · · · , in B 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 and D) further confirming the presence of I_h. Although ZD 7288 increased input resistance somewhat, it did not block I_KIR (Fig. 2D, left).

Identification and postnatal development of fast and slow inward rectifying currents

FAST INWARD RECTIFYING CURRENT (I_KIR). To characterize I_KIR and I_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⁺-sensitive I_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⁺-sensitive I_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.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Ba2+-sensitive IKIR increases during early development. A: Ba2+-sensitive IKIR 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 IKIR 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).

SLOW INWARD RECTIFYING CURRENT (I_H). To characterize more clearly the electrophysiological properties of the slowly activating inward current, I_h, we applied hyperpolarizing voltage command steps in the modified ACSF used to isolated I_KIR. To block other K⁺ currents including I_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. 2B). Figure 4A shows the mean I_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⁺-sensitive I_KIR, I_h was also voltage dependent. Additionally, current density and conductance were developmentally regulated (Fig. 4B; Table 2).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Ih density increases postnatally. A: voltage-dependent activation of Ih density (inset, measured as a difference current) for 2 age groups in modified ACSF. B: summary of Ih 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).

VOLTAGE-DEPENDENT ACTIVATION OF I_H. 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 formula
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).

View larger version (22K): [in this window] [in a new window]   Fig. 5. Activation curve for Ih. A: activation curve for Ih 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 Rnorm = {1 + exp[(Vm  V1/2)/k]}1 (Vm, applied step command potential; V1/2, 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.

I_H KINETICS. The activation and deactivation kinetics of I_h were estimated using a long step (2-5s) protocol to allow I_h to approximate steady state. As shown in Fig. 6A, in most instances, the time course of activation and deactivation were well fitted with double exponential function of the form
where I_h(t) is the current at time t, C is a constant value, and A₁, and A₂ are amplitude coefficients for each time constant. Figure 6B indicates that for both age groups, the fast and slow time constants for activation (_fast and _slow) were voltage dependent as shown by others (Dickson et al. 2000; Kjaerulff and Kiehn 2001). Both time constants significantly decreased with age (_fast, P2-4, 277.1 ± 8.5 ms vs. P10-12, 209.5 ± 11.0 ms at -105 mV; P < 0.0001; _slow, 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).

View larger version (24K): [in this window] [in a new window]   Fig. 6. Ih activation and deactivation kinetics. Aa: Ih was evoked by a long voltage step (5 s) protocol (bottom). Ih activation time course was well fitted by a double-exponential function during the steps between -100 and -120 mV (see RESULTS). Ab: Ih 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 for Ih activation as a function of voltage. C: composite plot of fast and slow deactivation time constants for Ih (each data point represents 4 neurons).

Functional roles for I_h in control of cellular excitability

CONTRIBUTION OF I_H TO RESTING MEMBRANE POTENTIAL. The activation data presented in Fig. 5 suggest that I_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 and 3). However, those data on I_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.

LOW-FREQUENCY MEMBRANE RESONANCE. 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 exhibit I_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.

View larger version (35K): [in this window] [in a new window]   Fig. 7. Ih 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 below Fres and eliminated the resonant peak [holding potential (Vhold) -70 mV]. Ca: subthreshold membrane potential response to ZAP input current in the presence of 4-AP (30 µM) (see RESULTS) (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).

I_H MEDIATES LOW-FREQUENCY RESONANCE. To test the hypothesis that I_h mediates the low-frequency resonance, the effects of the potent I_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. 7B), 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).

CONTRIBUTION OF I_H TO ACTION POTENTIAL AHP. Previous studies in other neurons revealed a critical role for I_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 for I_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.

View larger version (19K): [in this window] [in a new window]   Fig. 8. Ih 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 Ih prolonged the duration of late AHP for both age groups.

View larger version (31K): [in this window] [in a new window]   Fig. 9. Ih 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 F with that in B and C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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 and I_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 for I_KIR in control of membrane excitability and resting potential due to its very negative activation threshold in Mes V neurons, we will argue that I_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_h suggests 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 both I_h and I_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_h activated at a more depolarized membrane potential compared with I_KIR, similar to that reported for amygdala neurons (Womble and Moises 1993). However, due to rapid time course and small amplitude of the repolarizing I_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 traverse I_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 in V_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_h in Mes V neurons was described well by two different kinetic components that had time constants ranging from hundreds of milliseconds to seconds (Fig. 6B), 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. 6C). These data suggest that multiple I_h channels with different kinetic and gating properties are expressed in Mes V neurons.

Roles for I_h in control of neuronal excitability

CONTRIBUTION OF I_H TO RESTING MEMBRANE POTENTIAL. In normal ACSF, the V_1/2 for I_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 of I_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 of I_h on membrane excitability. In theory, suppression of a tonically active resting I_h would slightly hyperpolarize neurons but lead to an increase in R_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 decrease in excitability that occurs as a result of loss of inward I_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 of I_h channels will be determined by the relative activation of those channels at rest and during spike discharge.

I_H PARTICIPATES IN CONTROL OF SPIKE FREQUENCY. 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 the F-I relationship downward throughout the current range tested, suggesting a significant participation of I_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 of I_h (Fig. 8B). 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 for I_h. Because I_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 and F).

Functional implications for I_h 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 of I_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_KIR contributed 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 of I_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. The I_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, Q values), 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 in I_h channels as a result of environmental stimuli, such as injury, could contribute to oral-motor dysfunction.