Hyperpolarization-Activated Inward Current in Neurons of the Rat's Dorsal Nucleus of the Lateral Lemniscus In Vitro

Xiao Wen Fu, Borys L. Brezden, and Shu Hui Wu

Laboratory of Sensory Neuroscience, Institute of Neuroscience, Carleton University, Ottawa, Ontario K1S 5B6, Canada

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Fu, Xiao Wen, Borys L. Brezden, and Shu Hui Wu. Hyperpolarization-activated inward current in neurons of the rat's dorsal nucleus of the lateral lemniscus in vitro. J. Neurophysiol. 78: 2235-2245, 1997. The hyperpolarization-activated current (Ih) underlying inward rectification in neurons of the rat's dorsal nucleus of the lateral lemniscus (DNLL) was investigated using whole cell patch-clamp techniques. Patch recordings were made from DNLL neurons of young rats (21-30 days old) in 400 µm tissue slices. Under current clamp, injection of negative current produced a graded hyperpolarization of the cell membrane, often with a gradual sag in the membrane potential toward the resting value. The rate and magnitude of the sag depended on the amount of hyperpolarizing current. Larger current resulted in a larger and faster decay of the voltage. Under voltage clamp, hyperpolarizing voltage steps elicited a slowly activating inward current that was presumably responsible for the sag observed in the voltage response to a steady hyperpolarizing current recorded under current clamp. Activation of the inward current (Ih) was voltage and time dependent. The current just was seen at a membrane potential of -70 mV and was activated fully at -140 mV. The voltage value of half-maximal activation of Ih was -78.0 ± 6.0 (SE) mV. The rate of Ih activation was best approximated by a single exponential function with a time constant that was voltage dependent, ranging from 276 ± 27 ms at -100 mV to 186 ± 11 ms at -140 mV. Reversal potential (Eh) of Ih current was more positive than the resting potential. Raising the extracellular potassium concentration shifted Eh to a more depolarized value, whereas lowering the extracellular sodium concentration shifted Eh in a more negative direction. Ih was sensitive to extracellular cesium but relatively insensitive to extracellular barium. The current amplitude near maximal-activation (about -140 mV) was reduced to 40% of control by 1 mM cesium but was reduced to only 71% of control by 2 mM barium. When the membrane potential was near the resting potential (about -60 mV), cesium had no effect on the membrane potential, current-evoked firing rate and input resistance but reduced the spontaneous firing. When the membrane potential was more negative than -70 mV, cesium hyperpolarized the cell, decreased current-evoked firing and increased the input resistance. Ih in DNLL neurons does not contribute to the normal resting potential but may enhance the extent of excitation, thereby making the DNLL a consistently powerful inhibitory source to upper levels of the auditory system.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The dorsal nucleus of the lateral lemniscus (DNLL) is a collection of neurons located within the auditory lemniscal pathway just ventral to the inferior colliculus. The DNLL is believed to play an important role in binaural processing and sound localization. DNLL neurons receive excitatory as well as inhibitory connections from multiple sources of the lower auditory brain stem bilaterally and the DNLL contralaterally. Neurons in the DNLL send inhibitory projections to both ipsilateral and contralateral inferior colliculus and to the contralateral DNLL (Glendenning et al. 1981; Hutson et al. 1991; Kudo 1981; Oliver and Shneiderman 1989; Shneiderman et al. 1988; Zook and Casseday 1987). The majority of neurons in the DNLL are sensitive to binaural stimulation (Aitkin et al. 1970; Brugge et al. 1970; Covey 1993; Markovitz and Pollak 1993). Lesion of the DNLL or suppression of activity of DNLL neurons greatly reduces the slopes of interaural intensity or time difference functions of neurons in the contralateral inferior colliculus and auditory cortex (Faingold et al. 1993; Glenn and Kelly 1992; Kelly and Li 1997; Kidd and Kelly 1996; Li and Kelly 1992), and elevates minimum audible angles for sound localization (Kelly et al. 1996). Because almost all DNLL neurons are GABAergic (Adams and Mugnaini 1984; Glendenning and Baker 1988; Moore and Moore 1987; Roberts and Ribak 1987; Thompson et al. 1985; Vater et al. 1992; Winter et al. 1995), the effects of suppression or lesion of the DNLL on binaural function of the inferior colliculus and auditory cortex and on sound localization probably are manifested through the inhibitory influence of the DNLL on responses in the upper levels of the auditory system (Kelly and Li 1997; Kidd and Kelly 1996; Li and Kelly 1992).

The membrane properties of DNLL neurons already have been examined in our previous studies (Wu and Kelly 1995a,b). DNLL neurons responded to depolarizing currents with a sustained train of action potentials and a long-duration afterhyperpolarization. Injection of negative current resulted in a graded hyperpolarization that exhibited a sag back toward the resting potential. Strong hyperpolarizing currents often were followed by a large rebound depolarization that could elicit a burst of action potentials.

The sag back toward resting potential after intracellular injection of hyperpolarizing current has been described in many central neurons as an inward rectification, i.e., an increased conductance on hyperpolarization (see review, Pape 1996). There are two general types of inward rectifying currents. One type is the classical inward rectifier discovered in muscle cells by Katz, which is a pure K+ conductance that is activated at values negative to the K+ equilibrium potential. It has faster kinetics and much greater sensitivity to barium than cesium. Another type, called Ih, has been identified in rod photoreceptors, sino-atrial node cells, Purkinje fibers of the mammalian heart, and many types of neurons. Ih current is a slowly developing inward current activated on hyperpolarization of the membrane beyond the resting potential. In many central neurons, the hyperpolarization sag, i.e., inward rectification by hyperpolarization from the resting membrane potential, has been postulated to be due to Ih. Ih current is a mixed potassium and sodium current and behaves in a way expected of a time- and voltage-dependent membrane conductance. The voltage dependence of the activation of Ih can be regulated by neurotransmitters and metabolic stimuli via the intracellular adenosine 3',5'-cyclic monophosphate (cAMP) pathway. A general pharmacological feature of Ih is its sensitivity to low concentration of extracellular cesium and its relative insensitivity to barium.

Our previous whole cell patch-clamp studies described several types of potassium currents, including A-current, Ca2+ dependent- and Ca2+ independent-sustained current, and detrodotoxin-sensitive current in DNLL neurons. The membrane excitability and spike repolarization of DNLL neurons are dependent on these currents (Fu et al. 1996a). In the present study, we used whole cell patch-clamp techniques to characterize the hyperpolarization-activated, mixed potassium and sodium Ih current that underlies inward rectification in DNLL neurons. Our purpose was to investigate the basic properties of this current and to assess its possible role in modulation of DNLL activity. Some of these data have been published in abstract form (Fu et al. 1996b).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

The rat's DNLL brain slice preparation has been described in detail in a previous report (Fu et al. 1996a). In the present study, transverse DNLL slices (400 µM) were cut from freshly dissected brains of 21- to 30-day-old Wistar rats with a Vibratome. Sectioning was performed with tissue immersed in 30°C oxygenated artificial cerebrospinal fluid (ACSF) that had the following composition (in mM): 129 NaCl, 3 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 20 NaHCO3, and 10 dextrose. The ACSF was infused continuously with 95%O2-5%CO2 to ensure adequate oxygenation of slices and to maintain a pH of 7.4.

For recordings, a slice was transferred to a circular recording chamber mounted on the stage of a Diastar microscope (Reichert) equipped with Zeiss water-immersion objectives. The chamber, which had a volume of ~200 µl, was continuously perfused with ACSF at a rate of 6-8 ml/min. In voltage-clamp experiments tetrodotoxin (0.3 µM) was added to block voltage-dependent sodium currents. Cesium- and barium-containing solutions were made by adding of 1 mM CsCl and 2 mM BaCl2, respectively, to the ACSF solution. The low-sodium (27.2 mM) solution was made by equimolar substitution of choline chloride for NaCl. The high-potassium solution (10 mM) was made by substitution of 7.5 mM KCl for NaCl (Kamondi and Reiner 1991). Experimental drugs and test solutions were added to the chamber by redirecting the flow of liquid through a system of tubing and stopcocks. All drugs were prepared with ACSF as the vehicle. Tetrodotoxin and 4-aminopyridine (4-AP) were obtained from Sigma.

Whole cell current- and voltage-clamp recordings from DNLL neurons were obtained with patch electrodes, which were made from 1.1-mm OD and 0.8-mm ID thin-walled glass tubing (Kimax-51, Kimble) with a vertical puller (Kopf Instruments). The recording electrodes had a tip diameter of ~1 µm and a resistance of 3-5 MOmega . The electrodes were filled with a solution containing the following (in mM): 100 K-gluconate, 30 KCl, 1 MgCl2, 5 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. The pH of the internal solution was adjusted to 7.25 with KOH.

A List EPC-7 (Medical Systems) or Axopatch 200A (Axon Instruments) amplifier was used for recording in the whole cell voltage- and current-clamp experiments. In current-clamp experiments, currents were injected to hold the cell membrane potential at an appropriate level, and additional current pulses were superimposed on the holding current. In voltage-clamp experiments, there was no compensation for series resistance because the currents rarely exceeded 300 pA, and the resulting voltage errors were <3 mV. The data were digitized on-line, stored, and analyzed on a PC computer with pClamp 6.0 (Axon Instruments). Averaged data are presented as means ± SE.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

The data represent recordings from 42 DNLL neurons. The mean resting potential was -65.0 ± 3.8 mV (n = 30). The mean input resistance was 408.0 ± 10.4 MOmega (n = 20).

With current clamp, injections of hyperpolarizing current into DNLL neurons led to an initial hyperpolarization followed by a slowly developing sag of the membrane potential, i.e., inward rectification. Figure 1A shows an example. Hyperpolarizing current pulses of sufficient magnitude resulted in a slowly developing depolarization sag of the electrotonic potential. The rate and magnitude of the sag depended on the amount of hyperpolarizing current injected, i.e., larger currents lead to larger and faster decays. Current-voltage (I-V) curves (Fig. 1B) showed an essentially linear current-voltage relationship for the initial hyperpolarization (Fig. 1A, bullet ), but a nonlinear current-voltage relationship for the final hyperpolarization (Fig. 1A, open circle ) due to a decrease in the input resistance of the membrane. Thirty-six of 42 DNLL neurons (86%) exhibited an observable sag after the injection of hyperpolarizing current. Thus the slowly developing inward rectification that constitutes the relaxation phase of hyperpolarization is a common electrical feature of the DNLL cell membrane. During voltage clamp in this cell (Fig. 1C), hyperpolarizing voltage steps of 10-mV increments from a holding potential of -50 mV elicited an instantaneous inward leak current. At membrane potential more negative than -70 mV, there was an additional, slowly activating inward current that did not inactivate during voltage steps of 900-ms duration (Fig. 1C). The instantaneous current jump (Iins; bullet ) measured immediately after the capacitive transient, the steady-state current (Iss; Delta ) measured at the end of the 900-ms voltage step, and the difference between Iins and Iss are plotted as a function of the membrane potential in Fig. 1D. The difference in the current measured at the beginning and the end of the voltage step represents the hyperpolarization-activated inward current, Ih (open circle ) (Fig. 1D, Ih = Iss - Iins). A marked inward current appeared at membrane potentials more negative than -70 mV. This slowly activating inward current presumably underlies the sag observed during hyperpolarization in current-clamp records. The voltage- and time-dependent properties and pharmacological characteristics of Ih in DNLL neurons will be shown below.


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FIG. 1. General characteristics of Ih. A: in current clamp, the voltage responses (top) to hyperpolarizing and depolarizing current pulses (bottom) were recorded in a dorsal nucleus of the lateral lumniscus (DNLL) neuron. Depolarizing sag reflects activation of Ih. Resting potential was -73 mV. B: peak (bullet ) and steady-state (open circle ) voltages were plotted as a function of injected currents. C: currents (top) were evoked from a holding potential of -50 to -140 mV by hyperpolarizing voltage steps (bottom). An inward current slowly developed during hyperpolarization. D: instantaneous current (Iins; bullet ) measured at the end of the capacitive transient and steady-state current (Iss; Delta ) measured at the end of voltage steps were plotted against the membrane potential measured at the same time. Difference between Iins and Iss, represents the time- and voltage-dependent inward current Ih (open circle ). E: currents (top) were evoked from a holding potential of -130 mV by voltage steps (bottom). F: I-V curves for instantaneous currents from C (bullet ) and E (black-triangle) are linear throughout the voltage range steps. Extrapolated intersection (up-arrow ) of instantaneous I-V curves gives an estimate of the reversal potential (Eh) of Ih.

Reversal potential of Ih

In many other types of neurons, Ih is carried jointly by sodium and potassium ions and has a reversal potential (Eh) more positive than the resting membrane potential under normal physiological conditions (Banks et al. 1993; Bayliss et al. 1994; Dekin 1993; Kamondi and Reiner 1991; Mayer and Westbrook 1983; McCormick and Pape 1990). We used the method described by Mayer and Westbrook (1983), Kamondi and Reiner (1991), and Bayliss et al. (1994) to estimate the reversal potential of Ih in a DNLL cell. The voltage-clamp experiment was conducted in the same DNLL neuron as shown in Fig. 1A. Iins was elicited by voltage steps from a holding potential of -50 mV at which Ih was not activated (Fig. 1C, bullet ), and then from a holding potential of -130 mV where Ih was strongly activated (Fig. 1E, black-triangle). Plots of Iins as a function of the membrane potential are presented in Fig. 1F. These points were fitted with linear regression. The slopes of the curves represent the membrane conductance at each holding potential. The increased conductance activated from a holding potential of -130 mV reflects activation of the conductance underlying Ih. The intersection of the two extrapolated instantaneous I-V curves gives an estimate of the reversal potential (Eh) of Ih, which was found to be -48 mV in this cell. This value of Eh is consistent with the hypothesis that Ih is a mixed cationic current.

Voltage dependence of Ih

We used the method described by Kamondi and Reiner (1991) to measure the voltage dependence of Ih in DNLL neurons. A two-step voltage-clamp protocol was used to examine the activation of Ih. The membrane potential was held at -50 mV and then stepped in -10-mV increments to potentials between -60 and -140 mV for 900 ms (Fig. 2A). This conditioning step led to a partial activation of Ih. After this prepulse, a test pulse was applied by stepping the membrane potential to -140 mV, where Ih appeared to be fully activated (I-140). The currents observed during the test pulse represents the proportion of Ih that was not fully activated during the conditioning pulse. The magnitude of Ih at a given holding membrane potential was calculated by subtracting the current (Iv) measured during the test pulse from the maximal value of Ih (I-140). The activation curve of Ih was obtained by plotting the normalized current (I/Imax) against the membrane potential (Fig. 2B). The data were fitted by a Boltzman function
<IT>I</IT>/<IT>I</IT><SUB>max</SUB>= 1/{1 + exp[<IT>k</IT>(<IT>V − V</IT><SUB>1/2</SUB>)]} (1)
where Imax is the maximum current recorded at -140 mV after the conditioning pulse, I is the amplitude of the current recorded at a membrane potential (V) after the prepulse,V1/2 is the membrane potential at which the current is half-maximally activated, and k is a slope factor. The experimental data were fitted to the form giving V1/2 = -90 mV for this neuron. An average of V1/2 was -78.0 ± 6.0 mV, and of k was 0.10 ± 0.06 (n = 6).


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FIG. 2. Voltage dependence of Ih. A: a 2-step protocol was used to determine the voltage-dependent activation of Ih. The 900-ms-duration prepulse to various holding potentials between -50 and -140 mV partially activated Ih. Current observed during the subsequent test pulse to -140 mV was used to calculate the amount of Ih at different membrane potentials. I-140 is the maximal current, indicated by the horizontal arrow, evoked by stepping the membrane potential to -140 mV, and IV is the current evoked during the -140 mV test pulse after a prepulse of membrane potential V, measured at the time indicated by the vertical arrow. Ih was calculated for each test pulse by subtracting IV from I-140. B: plot of relative Ih current (I/Imax) against the membrane potential from the cell shown in A. The data were fitted to Eq. 1 (see text), with a calculated V1/2 = -90 mV and K = 0.1 mV.

The voltage dependence of the time constant of Ih activation was examined with the voltage protocol shown in Fig. 3A. The currents were evoked by hyperpolarizing voltage steps from a holding potential of -50 mV to various levels between -60 and -140 mV. The data (full traces) were best fitted by a single exponential function (F test, P < 0.05) of the form
<IT>I</IT><SUB>t</SUB><IT>= I</IT><SUB>ss</SUB><IT>+ I</IT><SUB>h</SUB>exp(−<IT>t</IT>/τ) (2)
where It is the current at the time (t), Iss is the steady-state current at the end of the voltage step, Ih is the difference between Iss and Iins, and tau  is the time constant. In six DNLL neurons, the time constant for activation of Ih was 186 ± 11 ms at -140 mV, 216 ± 17 ms at -120 mV, and 276 ± 27 ms at -100 mV. Figure 3B shows the mean activation time constants plotted against the membrane potential for these six neurons. The activation kinetics of Ih in DNLL neurons showed voltage sensitivity in the membrane potential range from -100 to -140 mV, with the activation time constant decreasing as the membrane potential was further hyperpolarized.


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FIG. 3. Voltage sensitivity of activation kinetics of Ih. A: current evoked by voltage steps to various membrane potentials was fitted to Eq. 2 (see text). B: mean ± SE time constants at various voltage steps are plotted against the membrane potential (n = 6).

Effects of low sodium and high potassium on Ih

To investigate which cation species contributing to Ih in DNLL neurons, we examined the effects of varying extracellular concentration of sodium ([Na+]o) and potassium ([K+]o) on Ih. The results of a typical experiment with a DNLL neuron are illustrated in Fig. 4. After perfusion of a modified ACSF containing high potassium (10 mM), the instantaneous currents increased (Fig. 4B). In contrast, when [Na+]o was reduced to 27.2 mM, the instantaneous currents decreased (Fig. 4C). These changes were accompanied by a marked alteration in Eh measured with the same voltage protocol shown in Fig. 1, C and E. Eh was shifted from -48 to -15 mV when [K+]o was increased to 10 mM (Fig. 4D) and from -48 to -70 mV when [Na+]o was decreased to 27.2 mM (Fig. 4E). Similar shifts of Eh toward depolarization by high [K+]o and toward hyperpolarization by low [Na+]o were observed from five and three other cells, respectively. These results suggest that sodium and potassium are the charge carriers for Ih in DNLL neurons (see DISCUSSION).


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FIG. 4. Effects of low [Na+]o and high [K+]o on Eh. A: current traces evoked by voltage steps from holding potential of -50 mV in control artificial cerebrospinal fluid (ACSF). B and C: current traces evoked by voltage steps from holding potentials of -50 mV (top) and of -130 mV (bottom) in high-potassium (10 mM) solution (B) and in low-sodium (27.2 mM) solution (C). Symbols (bullet  and black-triangle) indicate the time point used for measurement of instantaneous current. D and E: instantaneous I-V curves obtained at holding potentials of -50 and -130 mV. Intersections (up-arrow ) of extrapolated best-fitted lines represent the estimated Eh. All data were obtained from the cell shown in Fig. 1.

Pharmacology of Ih

We examined the relative sensitivity of Ih of DNLL neurons to extracellular cesium (Cs+) and barium (Ba2+). The time- and voltage-dependent hyperpolarization-activated Ih current was blocked almost completely by 1 mM Cs+ in the DNLL neuron shown in Fig. 5A. Using the voltage protocol shown in Fig. 1C, plots of the amplitude of Ih (Ih = Iss - Iins) against the membrane potential in normal and in Cs+-containing ACSF in this cell are shown in Fig. 5B. Ih was diminished greatly by Cs+ at all membrane potentials more negative than -60 mV.


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FIG. 5. Effect of cesium on Ih. A: current evoked by hyperpolarizing voltage steps (bottom) from -50 to -140 mV in control ACSF (top) and after addition of 1 mM CsCl (middle). B: the amplitude of Ih is plotted against the membrane potential in control (bullet ) and in Cs+ containing ACSF (open circle ).

Similarly, barium also reduced Ih but had less effect than cesium. The protocol shown in Fig. 1C was used for measuring Ih in normal and in Ba2+-containing ACSF. The reduction of Ih by 2 mM barium is shown in Fig. 6B. Ba2+ reduced Ih at all membrane potentials more negative than -60 mV, but to a lesser degree than Cs+.


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FIG. 6. Effect of barium on Ih. A: current evoked by hyperpolarizing voltage steps (bottom) from -50 to -140 mV in control ACSF (top) and after an addition of 2 mM BaCl2 (bottom). B: amplitude of Ih is plotted against the membrane potential in control (bullet ) and in Ba2+-containing ACSF (open circle ).

The suppression of Ih by Cs+ and Ba2+ was more prominent when the cell membrane was more hyperpolarized than when the cell membrane was less hyperpolarized (Figs. 5B and 6B), indicating the effects of Cs+ and Ba2+ on Ih were voltage dependent. Both Cs+ and Ba2+ also reduced the whole cell resting conductance of DNLL neurons. Similar results were obtained with 2 mM 4-AP (not shown), which is a blocker for potassium channels. Thus Cs+ and Ba2+ also may block the leakage conductance of DNLL neurons nonspecifically.

The combined data for the effects of cesium and barium on Ih are shown in Fig. 7. The amplitude of Ih was measured at the level of maximal activation and was plotted as a percentage of the control amplitude. Ih was reduced to 40% (35 and 45%, n = 2) of the control in 1 mM cesium and to 71% (59-84%, n = 7) of the control in 2 mM barium. The reduction in Ih was significantly different for the cesium and barium groups (Mann-Whitney U = 0, P < 0.05). Thus the Ih observed in DNLL neurons was more sensitive to cesium than to barium.


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FIG. 7. Mean data for the effects of cesium and barium on Ih. Amplitude of Ih was measured at its maximal activation (-140 mV). Ih was decreased to 40% of the control in 1 mM cesium (n = 2) and to 71% of the control in 2 mM barium (n = 7) (see text for details).

Functional properties of Ih

To further investigate what role Ih may play in physiological functions of DNLL neurons, we examined the effects of cesium on spontaneous and current-evoked firing because cesium substantially suppresses or blocks Ih in many types of neurons (Banks et al. 1993; Bayliss et al. 1994; Dekin 1993; Kamondi and Reiner 1991; Mayer and Westbrook 1983; McCormick and Pape 1990; Mercuri et al. 1995; Strohmann et al. 1994), as well as in DNLL neurons (Fig. 5). Bath application of CsCl (2 mM) was used to determine the contribution of Ih to firing rate. Figure 8 shows that 2 mM cesium reduced the frequency of spontaneous activity when the cell membrane potential was held at -59 mV. The spontaneous firing rate of DNLL neurons was 5.6 ± 0.7 Hz in normal ACSF and was decreased to 1.3 ± 0.3 Hz (n = 3) with the brain slice of the DNLL in 2 mM CsCl, i.e., cesium perfusion reduced the spontaneous rate by 23% (t-test, P < 0.05). The alteration of firing rate of DNLL neurons by cesium at least in part may be attributed to blockade of Ih (see DISCUSSION).


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FIG. 8. Effect of cesium on spontaneous firing rate. Firing rate was 4 Hz in normal ACSF and was reduced to 2 Hz in 2 mM CsCl in this cell. Frequency of cell firing was determined by the number of the spikes during 2-4 s. Cell membrane potential was -59 mV.

The contribution of Ih to evoked discharge rate also was tested with CsCl in three DNLL neurons. Figure 9 shows an example of the experiments. The cell was held at four different membrane potentials (-60, -70, -80, and -90 mV) by injecting the appropriate amount of DC current. Two depolarizing current pulses (20 and 40 pA) of 200-ms duration were applied with the brain slice in normal and Cs+-containing ACSF (Fig. 9A). Two millimolar CsCl had no effect on the membrane potential, input resistance or firing rate of this cell at a holding membrane potential of -60 and -70 mV. As the holding potential was hyperpolarized to -80 or -90 mV, 2 mM cesium hyperpolarized the cell, increased the input resistance, and decreased the firing rate, i.e., the lower the holding membrane potential, the greater the effects of cesium. The number of spikes evoked by 40 pA in this DNLL neuron is plotted as a function of the holding potential under normal conditions and with 2 mM CsCl in Fig. 9B. Cesium reduced spike frequency to 50 and 20% of the control at holding potentials of -80 and -90 mV, respectively, but did not affect the firing rate at holding potentials of -60 and -70 mV.


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FIG. 9. Effect of cesium on firing evoked by depolarizing current. A: responses of a DNLL neuron to 2 depolarizing current pulses (20 and 40 pA in 200 ms) in normal and Cs+-containing ACSF. Note that CsCl had neither an effect on the firing rate nor an effect on the membrane potential at a holding potential of -60 and -70 mV. Cesium hyperpolarized the cell membrane and reduced the frequency of firing at holding potentials of -80 and -90 mV. B: number of spikes evoked by current injection of 40 pA is plotted as a function of the membrane potential. triangle , perfusion with normal ACSF; open circle , perfusion with Cs+-containing ACSF.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The major finding of the present study is the presence of a mixed cation current (Ih) that underlies the inward rectification caused by hyperpolarization in rat DNLL neurons. The data add to our previous results showing a variety of potassium channels in DNLL neurons and represent a further step in understanding the mechanisms that govern the membrane excitability of DNLL neurons.

In the present study, we have characterized the hyperpolarization-activated inward current (Ih) in DNLL neurons using whole cell patch-clamp recordings. Our data show a close temporal correspondence between the depolarizing sag of the hyperpolarizing electrotonic potential and the development of Ih, indicating that Ih underlies the inward rectification on hyperpolarization. Activation of Ih in DNLL neurons is voltage dependent. This voltage dependency is well described by a Boltzmann function and is similar to that of the corresponding membrane conductance in many other neurons (Banks et al. 1993; Bayliss et al. 1994; Dekin 1993; Kamondi and Reiner 1991; Maccaferri et al. 1993; Mayer and Westbrook 1983; McCormick and Pape 1990; Mercuri et al. 1995; Travagli and Gillis 1994; Womble and Moises 1993). Ih in DNLL neurons is activated at a voltage range negative to the resting potential, i.e., -70 to -140 mV. The threshold for activation of Ih in DNLL neurons is comparable with reported values for threshold of Ih activation in other types of neurons, including -69 mV for rat midbrain dopaminergic neurons (Mercuri et al. 1995), -70 mV for guinea pig bulbospinal neurons (Dekin 1993), and -80 mV for rat hypothalamus histaminergic neurons (Kamondi and Reiner 1991) and neurons of the medial nucleus of the trapezoid body (Forsythe and Barnes-Davies 1993). The value of the reversal potential of Ih current in DNLL neurons is also consistent with that in other types of neurons. Measurements of the reversal potential for Ih, estimated by extrapolation methods similar to our own, range from -17 mV in rat hippocampal neurons (Maccaferri et al. 1993) to -50 mV in cells from the sensorimotor cortex of the cat (Spain et al. 1987). The reversal potential of Ih in DNLL neurons was positive to the normal resting potential and was affected by extracellular potassium or sodium concentrations. These results indicate that Ih in DNLL neurons is a mixed potassium and sodium current. Although the possibility of contribution of the chloride current to Ih was not investigated in this study, it is not likely that the Ih has a significant chloride component (Pape 1996). The kinetics of Ih current in DNLL neurons was slow and voltage dependent and could be well described by single exponential processes. These kinetic properties are consistent with those of Ih observed in many other neurons (Bayliss et al. 1994; Kamondi and Reiner 1991; McCormick and Pape 1990; Mercuri et al. 1995; Travagli and Gillis 1994). Ih in DNLL neurons was suppressed greatly by extracellular cesium but was less sensitive to extracellular barium. Thus all the properties of Ih in DNLL neurons, including the voltage range of activation, the kinetics of activation, the reversal potential and its alteration with extracellular potassium and sodium concentrations, and the differential sensitivity to cesium and barium are comparable with those previously described for Ih in many other neurons (Bank et al. 1993; Bayliss et al. 1994; Dekin 1993; Kamondi and Reiner 1991; McCormick and Pape 1990; Strohmann et al. 1994). These observations suggest that the inward rectification in DNLL neurons results from the activation of Ih current rather than the classical inward rectifier.

In some types of neurons, Ih appears to assist in determining the resting potential. This is due to the fact that the resting potential of these neurons lies within the voltage range of Ih activation between -50 and -100 mV (Bayliss et al. 1994; Maccaferri et al. 1993; McCormick and Pape 1990; Strohmann et al. 1994; Travagli and Gillis 1994; Womble and Moises 1993). However, this is not the case for DNLL neurons. The normal resting potential of DNLL neurons is around -60 mV (Wu and Kelly 1995a), just outside the voltage range for activation of Ih. In addition, cesium had no effect on the resting potential or the firing of action potentials evoked by current injection when the cell membrane was held at -60 mV. But cesium did hyperpolarize the cell and decrease the current-evoked firing frequency only when the cell membrane potential was more negative than -70 mV (Fig. 9). These results suggest that Ih in DNLL neurons is not activated at the resting state. Because Ih in DNLL neurons was activated only at membrane potentials more negative than -70 mV, Ih may not assist in setting the normal resting potential for DNLL neurons. A similar conclusion was drawn from studies of Ih in rat hypothalamus histaminergic neurons and midbrain dopaminergic neurons, as the activation threshold of Ih in those neurons was also more negative than their resting potentials (Kamondi and Reiner 1991; Mercuri et al. 1995).

Ih has been shown to be a "pacemaker" current and to provide pacemaker depolarization during generation of rhythmic oscillatory activity in sino-atrial node cells, Purkinje fibers of heart, and thalamic neurons, suggesting that Ih plays a significant role in controlling autorhythmic activity (DiFrancesco 1981, 1986; McCormick and Pape 1990). DNLL neurons usually do not exhibit an autorhythmic oscillation, but some fire spontaneously both in vivo (Aitkin et al. 1970) and in vitro (Wu and Kelly 1995a,b). We hypothesize that the spontaneous activity of DNLL neurons could be regulated in part by Ih. The action potential of DNLL neurons usually is followed by an afterhyperpolarization (AHP) that exhibits either a single component or double components (an initial rapid hyperpolarization followed by a 2nd slower phase of hyperpolarization) (Wu and Kelly 1995a,b). The AHP of DNLL neurons could take the membrane potential to a value between -70 and -80 mV (Wu and Kelly 1995a,b) (Fig. 8), causing activation of Ih, acceleration of spike repolarization and an increase in firing. Pharmacological suppression of Ih by application of cesium reduced the spontaneous firing rate of DNLL neurons (Fig. 8) probably because of a prolonged AHP. Thus Ih in DNLL neurons increases the rate of spike repolarization. As a consequence, the level of DNLL spontaneous activity is enhanced. Travagli and Gillis (1994) have made a similar suggestion that the spontaneous activity is modulated in part by Ih current in neurons of the rat dorsal motor nucleus of the vagus (DMV neurons), as the spontaneous firing rate of DMV neurons expressing Ih was reduced with cesium. Although cesium also may block leak conductance or other types of voltage-dependent conductances, such as delayed rectifier and K+ inward rectifier channels (Pape 1996), our evidence of the voltage range of the AHP at which the Ih could be activated in DNLL neurons supports the idea that the Ih at least in part may regulate the spontaneous activity in DNLL neurons. A further study using various pharmacological blocking agents differentially affecting these conductances and Ih may disclose the exact fraction of contribution of Ih current to electrical activity of DNLL neurons.

Another possible role of Ih current in DNLL neurons might be to modulate synaptic excitation. DNLL neurons receive excitatory projections probably from neurons in the contralateral cochlear nucleus, the contralateral lateral superior olive, and the ipsilateral medial superior olive (Glendenning et al. 1981). Electrical stimulation of the lateral lemniscus, which is a major afferent pathway from the lower auditory brain stem, often elicits excitatory postsynaptic potentials (EPSPs) in DNLL neurons in a brain slice preparation (Wu and Kelly 1995a,b). Both short- and longer-latency EPSPs can be evoked in the same neuron. A single spike with relatively constant latency can be elicited from the short-latency EPSP, whereas a single or multiple spikes with highly variable latencies can be evoked from the longer-latency EPSP. Studies of the synaptic pharmacology of DNLL neurons suggest that the short-latency EPSP is mediated by glutamate via non-N-methyl-D-aspartate (NMDA) receptors, whereas the longer-latency EPSP is mediated through NMDA receptors (Fu et al. 1997; Wu and Kelly 1996). During synaptic excitation, especially the NMDA-receptor-mediated excitation with a long time course and multiple action potentials (Fu et al. 1997), Ih may increase the rate of spike repolarization and thus increase the firing rate. These longer excitatory events would be enhanced by Ih. Therefore, Ih in DNLL neurons would augment the influence of excitatory inputs. We speculate that an important role of Ih in DNLL neurons is to increase the efficacy of glutamatergic-mediated excitatory postsynaptic potentials.

Neurons in the DNLL are largely GABAergic and provide a major source of inhibitory projection to the auditory midbrain (Adams and Mugnaini 1984; Hutson et al. 1991; Oliver and Shneiderman 1989, 1991; Shneiderman et al. 1993). The inhibition from the DNLL exerts an important influence on binaural processing in upper levels of the central auditory system (Faingold et al. 1993; Glenn and Kelly 1992; Kelly and Li 1997; Kidd and Kelly 1996; Li and Kelly 1992) and plays an active role in sound localization (Ito et al. 1996; Kelly et al. 1996). By limiting prolonged hyperpolarization, activation of Ih in DNLL neurons would augment spontaneous and excitatory synaptic activity. As a result, a higher level of excitation in the DNLL would be translated into an enhanced inhibition of the postsynaptic neurons with which the DNLL neurons make contact. Thus Ih current may serve as a modulator to regulate excitation to an extent that makes the DNLL a consistently powerful inhibitory source to upper levels of the auditory system.

    ACKNOWLEDGEMENTS

  We thank Drs. B. Hu and J. B. Kelly for a critical reading of the manuscript.

  This research was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada and the Deafness Research Foundation.

    FOOTNOTES

  Address reprint requests to S. H. Wu.

  Received 18 November 1996; accepted in final form 1 July 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society