Excitability Increase Induced by beta -Adrenergic Receptor-Mediated Activation of Hyperpolarization-Activated Cation Channels in Rat Cerebellar Basket Cells

Fumihito Saitow and Shiro Konishi

Laboratory of Molecular Neurobiology, Mitsubishi Kasei Institute of Life Sciences and CREST, JST (Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation), Tokyo 194-8511, Japan


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INTRODUCTION
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Saitow, Fumihito and Shiro Konishi. Excitability Increase Induced by beta -Adrenergic Receptor-Mediated Activation of Hyperpolarization-Activated Cation Channels in Rat Cerebellar Basket Cells. J. Neurophysiol. 84: 2026-2034, 2000. In the preceding paper, we showed that norepinephrine (NE) enhances the spontaneous spike firings in cerebellar interneurons, basket cells (BCs), resulting in an increase in the frequency of BC-spike-triggered inhibitory postsynaptic currents (IPSCs) in Purkinje cells (PCs), and that the effects of NE on GABAergic BCs are mediated by beta 2-adrenergic receptors. This study aimed to further examine the ionic mechanism underlying the beta -adrenoceptor-mediated facilitation of GABAergic transmission at the BC-PC synapses. Using cerebellar slices obtained from 15- to 21-day-old rats and whole cell recordings, we investigated ionic currents in the BCs and the effects of the beta -agonist isoproterenol (ISP) as well as forskolin on the BC excitability. Hyperpolarizing voltage steps from a holding potential of -50 mV elicited a hyperpolarization-activated inward current, Ih, in the BC. This current exhibited voltage-dependent activation that was accelerated by strong hyperpolarization, displaying two time constants, 84 ± 6 and 310 ± 40 ms, at -100 mV, and was inhibited by 20 µM ZD7288. ISP and forskolin, both at 20 µM, enhanced Ih by shifting the activation curve by 5.9 and 9.3 mV toward positive voltages, respectively. Under the current-clamp mode, ISP produced a depolarization of 7 ± 3 mV in BCs and reduced their input resistance to 74 ± 6%. ISP and a cAMP analogue, Rp-cAMP-S, increased the frequency of spontaneous spikes recorded from BCs using the cell-attached mode. The Ih inhibitor ZD7288 decreased the BC spike frequency and abolished the ISP-induced increase in spike discharges. The results suggest that NE depolarizes the BCs through beta -adrenoceptor-mediated cAMP formation linking it to activation of Ih, which is, at least in part, involved in noradrenergic afferent-mediated facilitation of GABAergic synaptic activity at BC-PC connections in the rat cerebellum.


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INTRODUCTION
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An inwardly rectifying current through hyperpolarization-activated cation channels has been demonstrated in a variety of nerve cells (named Ih) as well as in cardiac pacemaker myocytes where this current was first reported and named If (DiFrancesco 1993). It has been proposed that Ih plays a role in the generation of spontaneous action potentials (DiFrancesco 1991; Ingram and Williams 1996; McCormick and Pape 1990) and that modulation of Ih activation results in profound influences on background cell firings (Banks et al. 1993; Jiang et al. 1993; Maccaferri and McBain 1996; McCormick and Wang 1991). Moreover, Ih is thought to provide a mechanism for limiting excessive hyperpolarization on a negative shift of membrane potential (Bayliss et al. 1994; Solomon and Nerbonne 1993) and to contribute to the resting membrane potential (Doan and Kunze 1999). Another noteworthy characteristic of this current is that its activation can be modulated by second messengers including intracellular cAMP formed through G-protein-coupled neurotransmitter receptor-mediated activation of adenylyl cyclase (AC). It has also been reported that Ih activation is responsible for serotonin (5-HT)-induced depolarization in spinal motoneurons (Larkman et al. 1995) and the control by monoamines of action potential firings in thalamic neurons (Pape and McCormick 1989).

In the preceding paper, we found that NE increased spike discharges in cerebellar basket cells (BCs); this resulted in an increase in the frequency of spike-triggered inhibitory postsynaptic currents (IPSCs) recorded in Purkinje cells (PCs). Previous reports as well as our own observations prompted us to explore whether modulation of Ih via NE-mediated beta -adrenoceptor activation is involved in the noradrenergic facilitation of GABAergic transmission at the BC-PC synapses. Previous studies have shown that the beta -adrenergic receptor agonist isoproterenol (ISP) increased the excitability of cerebellar GABAergic interneurons; this resulted in an increase in the frequency of inhibitory synaptic responses in postsynaptic target PCs (Kondo and Marty 1998; Llano and Gershenfeld 1993; Saitow et al. 1998). Similar facilitation of spontaneous inhibitory postsynaptic potentials by NE was reported in hippocampal CA1 pyramidal cells (Bergles et al. 1996). Activation of Ih has been implicated in the generation of spontaneous firings caused by NE in hippocampal interneurons (Maccaferri and McBain 1996). In the present study, we first characterized the properties of the hyperpolarization-activated current Ih in cerebellar interneuron BCs using thin slices obtained from the rat cerebellum and whole cell voltage-clamp recordings. Then we explored how the hyperpolarization-activated current Ih is modulated by beta -adrenoceptor activation in the BC. Our data showed that the beta -receptor agonist ISP accelerates persistent activation of Ih in the range of the resting membrane potential and thereby depolarizes the BC, resulting in increases in the frequencies of spontaneous spiking in BCs and spike-triggered IPSCs in PCs. A part of the results of this study has been reported as abstracts (Saitow and Konishi 1999; Saitow et al. 1998).


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METHODS
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Electrophysiology

The methods used are similar to those described in the preceding paper (Saitow et al. 2000). Using parasagittal slices cut from the cerebellum of 15- to 21-day-old rats, whole cell voltage-clamp recordings were obtained from PCs. In some experiments, cell-attached and current-clamp recordings were obtained from BCs. In all experiments with the exception of the spontaneous spike recordings, slices were superfused with artificial cerebrospinal fluid (ACSF) to which 1 µM tetrodotoxin (TTX) had been added to eliminate synaptic activity. The compositions of the ACSF and internal solutions filled in the patch electrodes and other conditions of the experiments are described in our preceding paper.

Data analysis

The activation kinetics of Ih were fitted with a function of the form
<IT>I</IT>(<IT>t</IT>) = <IT>A</IT><SUB>0</SUB> + <IT>A</IT><SUB>1</SUB> × exp(<IT>t</IT>&cjs0823;  &tgr;<SUB>fast</SUB>) + <IT>A</IT><SUB>2</SUB> × exp(<IT>t</IT>&cjs0823;  &tgr;<SUB>slow</SUB>) (1)
using a program based on the pClamp6 (Axon Instruments) software. To determine the Erev of Ih, we used an extrapolation procedure (Banks et al. 1993; Bayliss et al. 1994). The activation curves for Ih were fitted by Boltzmann functions of the form
<IT>I</IT><SUB>tail,norm</SUB> = 1 &cjs0823;   [1 + exp {(<IT>V</IT><SUB>m</SUB> − <IT>V</IT><SUB>0.5</SUB>) &cjs0823;   <IT>k</IT>}] (2)
where Vm is the membrane potential during the initial voltage step, V0.5 is the membrane potential at which Ih is half-activated, k is the slope factor, and Itail,norm is the normalized tail current amplitude according to the following relationship
<IT>I</IT><SUB>tail,norm</SUB> = (<IT>I</IT><SUB>tail</SUB> − <IT>I</IT><SUB>tail,min</SUB>) &cjs0823;   (<IT>I</IT><SUB>tail,max</SUB> − <IT>I</IT><SUB>tail,min</SUB>) (3)
where Itail,max is the tail current after the maximal inward current (following the step to -120 mV) and Itail,min is the tail current after the minimal inward current (following the step to -60 mV). Those parameters in Eq. 2 were calculated using the software KYPLOT provided by Dr. K. Yoshioka (http://www.qualest.co.jp/Download/KyPlot/kyplot_e.htm).

Drugs

The chemicals used were obtained from the following sources: isoproterenol (ISP), forskolin, norepinephrine, Rp-cAMP-S, and H-7 from Sigma; ZD7288 from Tocris Cookson; TTX from Sankyo. Forskolin dissolved in dimethyl sulfoxide at 100 mM was stored at -20°C and diluted before the experiments. TTX was prepared as a 10 mM stock solution and added to the ACSF at a final concentration of 1 µM.

Statistics

Numerical data are given as means ± SE, and n represents the number of independent experiments. The difference between the experimental groups were evaluated using Student's paired t-test.


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Properties of Ih in cerebellar GABAergic interneuron BCs

Recordings were obtained from 76 neurons that were identified as BCs. When recorded using the voltage- or current-clamp modes, all of these neurons exhibited a hyperpolarization-activated Ih or a sag during hyperpolarizing current injection (Fig. 1, A and B). Under voltage-clamp conditions, hyperpolarizing voltage commands to the BC produced a slow inward current in the potential range between -60 and -80 mV, and its amplitude and rate of activation increased with increasing the extent of hyperpolarization (Fig. 1B). The current reached a maximal amplitude at -120 to -140 mV without showing decay during voltage steps, which indicates that the activation of Ih in BCs is voltage dependent with very little, if any, inactivation. The activation kinetics of Ih were calculated by fitting each trace of the slow inward currents elicited by a series of hyperpolarizing voltage steps to either single- or multiexponential functions. The rate of activation was best fitted with a double-exponential function. As shown in Fig. 1B, both fast and slow time constants revealed a steep voltage dependence: tau fast = 84 ± 6 and 39 ± 3 ms at -100 and -140 mV, respectively, and tau slow = 310 ± 40 and 165 ± 15 ms at -100 and -140 mV, respectively (n = 8). The kinetics of Ih activation in the BC resembled that reported for other central neurons (Banks et al. 1993; Wang et al. 1997).



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Fig. 1. Properties of hyperpolarization-activated cation channel current Ih recorded from cerebellar basket cells (BCs). A: voltage sag (top) produced in response to hyperpolarizing current injections (inset). B: Ih induced by hyperpolarizing voltage commands from a holding potential of -50 to -80 mV with an increment of 10-mV steps. Double-exponential fits (red continuous lines) of Ih current traces induced by individual test hyperpolarization for 1.5 s (a). Voltage- and time-dependence of Ih activation (b). The rate constants, tau  fast (open circle) and tau  slow (filled circle), determined by fitting the current data with Eq. 1 (see METHODS) were plotted as the function of each test hyperpolarization command (n = 8). C: I-V relationships and reversal potential of Ih currents in a BC. Current traces recorded in response to a series of test voltage steps from 2 different holding potentials of -70 (a) and -100 mV (b). I-V relationships (c) for Ih determined from the data obtained at the holding potential of -50, -70 (a), and -100 mV (b). Instantaneous current amplitude measured immediately after the capacitive transient (a time point indicated by each symbol in a and b) following each voltage step was plotted against the membrane potential. Solid lines are the least-squares fits to the I-V data sets determined at different holding potentials of -50 (open circle), -70 (filled triangle), and -100 mV (filled circle). A vertical line on the membrane potential axis represents the point of intersection for the 3 regression lines, indicating the reversal potential Eh. D: inhibitory effect of ZD7288 on Ih currents recorded from a BC. Current responses (bottom) recorded during a voltage step (top) in the absence and the presence of ZD7288 (20 µM). The holding potential was -50 mV, and the voltage command to -100 mV was stepped back to -70 mV to observe tail currents (a). Time course of the blocking action of ZD7288 on Ih (b). Instantaneous current (open circle) due to the input resistance measured after the capacitive transient in the initiation of the voltage step, and fully activated Ih current (filled circle) measured at end of the voltage step, were plotted.

We next estimated the reversal potential of this current based on methods described previously (Banks et al. 1993; Bayliss et al. 1994). Assuming that instantaneous currents consisted of only Ih and leakage currents, an intersection of instantaneous I-V relationships determined at different holding potentials was taken as the reversal potential at which the driving force of Ih became zero. Figure 1C shows an example of such estimation. The instantaneous I-V relationship at each holding potential was fitted well with the linear regression, the slope of which represents the chord conductance underlying Ih. The extrapolated intersection of the three regression lines aided in the estimation of the Eh at -38 mV in this particular BC: the mean Eh was -41.4 ± 3.2 mV (n = 10), the value being positive relative to the resting potential of BCs (-53 ± 6 mV, n = 31).

We then examined the effects of ZD7288 on the hyperpolarization-activated current in the BCs. ZD7288 has been shown to cause bradycardia via its selective blocking action on If, an Ih equivalent current, in cardiac pacemaker cells (BoSmith et al. 1993). This compound has also been reported to block Ih of central neurons in the guinea pig substantia nigra, rat hippocampal CA1 region, and the cat ventrobasal thalamus (Gasparini and DiFancesco 1997; Harris and Constanti 1995; Williams et al. 1997). To test the effects of ZD7288, a constant voltage step was repetitively applied from a holding potential of -50 mV to -100 mV for 1 s and then stepped back to -70 mV for 500 ms. As shown in Fig. 1D, following the application of ZD7288, the Ih current induced by the hyperpolarizing voltage step decreased in amplitude in a time-dependent manner. The onset of the action of the compound was slow, taking approximately 3 min to begin to exert a discernible blockade, with a steady suppression of Ih occurring after 10 min. The effect of ZD7288 was long-lasting with no significant recovery even at 30 min.

Modulation of Ih in cerebellar BCs by isoproterenol

We found in the preceding study that beta 2-adrenergic receptor activation in the BCs elicits an increase in the frequency of spontaneous spike discharges (Saitow et al. 2000). Thus we attempted to further determine whether modulation of the hyperpolarization-activated current Ih is involved in the increase in BC excitability following beta -adrenoceptor activation. Application of the beta -agonist ISP (20 µM) caused a marked enhancement of Ih (Fig. 2A). The time constant of Ih became shorter during the ISP-induced enhancement.



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Fig. 2. Effects of the beta -adrenergic agonist isoproterenol (ISP) and the adenylyl cyclase (AC) activator forskolin on Ih activation. A: acceleration of Ih activation by ISP. Current traces were obtained during voltage steps from a holding potential of -50 mV to the range between -90 and -120 mV with an increment of 10-mV steps. Red current traces and arrows indicate the current traces recorded in the presence of 20 µM ISP. B: tail current analysis for determination of the Ih activation curve. Sample traces of current responses produced in response to the voltage-clamp protocol (inset). The membrane potential of a BC was held at -50 mV, stepped to a series of test potentials, and then poststepped to -70 mV. C and D: effects of ISP and forskolin on the Ih activation curve determined as in B. Both ISP and forskolin increased the Ih amplitude by shifting the activation curve by 6 and 9 mV toward positive voltages, respectively, without changing the slope factor: k = 9.2 and 9.3 before and after ISP application (n = 8), and k = 9.8 and 9.9 before and after forskolin application (n = 7), respectively. Open circles and black lines represent the normalized amplitudes of Ih determined by the tail currents in the control ACSF, and filled circles and red lines determined in the presence of 20 µM ISP (C) or 20 µM forskolin (D).

Furthermore ISP caused an inward shift of the holding current held at a membrane potential of -50 mV. Analysis of tail currents following hyperpolarizing voltage steps revealed that the increase in current magnitude by ISP was due to a rightward shift of Ih activation (Fig. 2, B and C). ISP shifted the half activation voltage V0.5 by +5.9 mV (Fig. 2C, n = 8). Since beta -adrenergic receptors are known to be coupled to AC, we examined the effect of the AC activator forskolin on the hyperpolarization-activated current in the BCs. Forskolin mimicked the action of the beta -adrenoceptor agonist ISP, and enhancement of Ih by the AC activator was more marked than that by ISP (Fig. 2D), shifting the half activation voltage V0.5 by +9.3 mV.

Effects of beta -adrenoceptor stimulation on the BC membrane potential

Consistent with the ISP-induced slow inward current under the voltage-clamp condition described in the preceding text, application of ISP produced depolarization in BCs and decreased their input resistance when recorded using the current-clamp mode (Fig. 3): the extent of ISP-induced depolarization was 7.0 ± 3.0 mV (n = 5). During the ISP-induced depolarization, the voltage sag in response to hyperpolarizing current injection decreased in the magnitude (Fig. 3B). These changes in BC membrane properties produced by ISP could be explained by the possibility that stimulation of beta -adrenergic receptors on the BCs by ISP enhances the persistent activation of Ih around the resting potential of the BCs. The decrease of the voltage sag might be due to the decrease of the input resistance following ISP application. Forskolin produced similar effects on the membrane potential of the BC (data not shown). Together, the results suggest that beta -adrenoceptor stimulation by ISP increases the intracellular cyclic AMP levels resulting in the acceleration of Ih activation and depolarization of the BC.



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Fig. 3. Effects of the beta -adrenoceptor agonist ISP on the membrane potential and the input resistance of the BC. A: depolarization and input resistance decrease following 20 µM ISP application. Membrane potential and responses to repetitive constant current pulses of -200 pA for 200 ms injected every 20 s recorded from a BC under the current-clamp mode. B: membrane voltage traces produced in response to the current injection before (a) and during ISP application (b). Both time points are indicated in A. The dashed line represents a level of membrane potential -60 mV. C and D: time courses of changes in the membrane potential (C) and the input resistance (D) following 20 µM ISP application. Each point represents the mean ± SE from independent experiments in different BCs (n = 5). ISP was applied by perfusion during the periods indicated by the horizontal lines.

Does ISP affect ionic currents other than Ih?

We then examined whether ionic mechanisms other than activation of Ih are involved in the depolarizing action of the beta -adrenoceptor agonist. To address this issue, we determined current responses induced by ISP and explored the effect of the Ih blocker ZD7288 on the ISP-induced current. Subtraction of I-V relationships induced by a voltage ramp between -140 and +40 mV before and after ISP application yielded the ISP-induced current and the reversal potential of the ISP response (Fig. 4, A and B). In the control medium, ISP produced an inward current and caused an increase in the input conductance. Application of ZD7288 markedly suppressed the I-V relationship induced by the voltage ramp (trace b in Fig. 4C), indicating that the compound inhibited most of the Ih in the BC (see also Fig. 1D). After treatment with ZD7288, ISP produced only a slight inward current (trace c in Fig. 4C) without changing the reversal potential (Fig. 4D). The ISP-induced current in the presence of ZD7288 might be attributed to residual Ih due to the incomplete blocking action of ZD7288. These observations suggest that ISP elicits depolarization of the BC through activation of ZD7288-sensitive hyperpolarization-activated cation channels with minimal contribution from other ionic mechanisms.



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Fig. 4. ISP-induced currents in the absence and the presence of the Ih inhibitor ZD7288. A: I-V relationships determined before (black trace) and after ISP application (red trace) by a constant voltage-ramp command (upper trace) between -140 and +40 mV at the rate of 0.01 Vs-1. B: an ISP-induced current obtained by subtracting the current b from a in A. C: effects of ZD7288 on the I-V relationships determined before and after ISP application. The voltage-ramp was applied successively (top), and the resulting currents were obtained before (a, black trace) and after the application of ZD7288 (b, red trace) and ZD7288 plus ISP (c, blue trace). D: a ZD7288-sensitive current (black trace) and ISP-induced current in the presence of ZD7288 (red trace) obtained by subtracting the current a from b, and the current c from b in C, respectively. Note that there was no discernible change in the reversal potential of the ZD7288-induced current and the ISP-induced current obtained in the presence of ZD7288.

Mechanism of beta -adrenoceptor-mediated excitability increase in the BC

It has been demonstrated that activation of Ih is caused by direct interaction between intracellular cAMP and hyperpolarization-activated cation channels (Bois et al. 1997; Ingram and Williams 1996; Ludwig et al. 1998). We therefore examined whether the beta -agonist ISP causes the increase in BC excitability via direct action of cAMP formed following beta -adrenoceptor stimulation or involvement of protein kinase A (PKA)-signaling cascades. Previous studies reported that noradrenergic facilitation of stimulation-evoked GABAA IPSCs was mediated by PKA-dependent pathways on the basis of observations that the beta -agonist-induced increase in the GABAA IPSC was abolished by a broad-spectrum protein kinase inhibitor, H-7 (Kondo and Marty 1997; Mitoma and Konishi 1999). We confirmed that treatment of cerebellar slices with H-7 (10 µM) for at least 30 min completely suppressed the ISP-induced facilitation of stimulation-evoked IPSCs (data not shown). We then examined the effect of the protein kinase inhibitor H-7 on the increase in BC excitability induced by the beta -agonist. H-7 did not cause any significant effect on the BC spike discharges: the frequency of spontaneous spiking was 3.7 ± 1.5 Hz in the control ACSF (n = 10) and 3.5 ± 0.9 Hz in the presence of H-7 (n = 23, P > 0.7). Therefore H-7 had no effect on the ability of BCs to discharge spontaneous spikes per se. ISP caused a significant increase in the frequency of spontaneous spikes after treatment with H-7 (Fig. 5B, open circles): in pooled data, the percent increase of spike frequency following H-7 plus ISP application was 148 ± 11% (P < 0.01, n = 10), and the degree of ISP-induced increase in BC firings was not significantly different from that observed in the absence of H-7 (filled circles; n = 5, P > 0.2). Furthermore ISP produced depolarization of BCs by 6.8 ± 2.0 mV (n = 4) in the presence of 10 µM H-7 (Fig. 5A): there was no discernible change in the extent of ISP-induced depolarization in the control ACSF (see Fig. 3) and the H-7-containing ACSF. It is therefore suggested that different mechanisms, i.e., H-7-sensitive and -insensitive processes, are involved in the ISP-induced increases in the evoked IPSCs and the spontaneous spike frequency.



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Fig. 5. ISP-induced depolarization and spike frequency increase of a BC in the presence of H-7, and effects of Ih modulators, ISP, Rp-cAMP-S, and ZD7288, on firing of spontaneous spikes recorded in BCs. A: effects of H-7 on ISP-induced increase in the frequency of spontaneous spikes in BCs. ISP (20 µM) was applied by superfusion during the period indicated by a horizontal bar in the absence of H-7 (filled circles, n = 5) and after treatment with 10 µM H-7 (open circles, n = 10). No significant difference was observed between the two groups (P > 0.2, n = 5). B: depolarization produced in a BC in response to application of 20 µM ISP in the presence of 10 µM H-7, a protein kinase inhibitor. Membrane potential and responses to repetitive constant current pulses of -200 pA for 200 ms injected every 20 s were recorded from a BC using current-clamp mode. C: effect of a cAMP analogue, Rp-cAMP-S, on the spontaneous spike frequency. D: inhibitory action of ZD7288 on the BC spontaneous spike firing and the ISP-induced BC excitability increase. Slices were superfused with 50 µM ZD7288 for 10 min, and then ZD7288 plus 20 µM ISP was applied for 5 min. Each point and vertical bar in C and D represent the mean of the spike frequency normalized to the value determined before the drug application and SE (n = 6 in C and D).

In the experiment illustrated in Fig. 5C, we further tested the effect of the cAMP analogue Rp-cAMP-S that has been shown to serve as a PKA inhibitor (Dostmann 1995). Rp-cAMP-S (100 µM), however, elicited an increase in the BC spike frequency with a relatively slow time course as compared with that of the ISP-induced effect: the percent increases of the spike frequency were 111 ± 3% after application of the cAMP analogue for 5 min and 148 ± 6.4% after application for 10 min (P < 0.01, n = 6) with a slow onset of its action, presumably due to the time required for access to intracellular active sites. The increase in BC spiking by Rp-cAMP-S is compatible with the action of this cAMP analog as a direct activator of Ih (Ludwig et al. 1998). Taken together, the observations suggest that the increase in the BC excitability following beta -adrenoceptor stimulation is mediated by the direct action of intracellular cAMP without dependence on PKA-mediated pathways.

Involvement of Ih activation in beta -adrenoceptor-mediated BC excitability increase

Finally, we investigated whether Ih activation is involved in beta -adrenoceptor-mediated modulation of the BC spontaneous spike activity. As shown in Fig. 5D, application of the Ih blocker ZD 7288 suppressed spontaneous spike discharges with a 2-3 min delay, similar to the effect observed on the hyperpolarization-activated current Ih (see Fig. 1D). The inhibitory action of the Ih inhibitor was associated with BC hyperpolarization of 12.5 ± 2.1 mV (n = 4), which was consistent with a slight outward current induced by the compound under voltage-clamped conditions. In three of six BCs on which the effect of ZD7288 was tested, a transient increase in the spike frequency was caused immediately after the application. However, after 10-min application, the Ih blocker invariably decreased the frequency of the spontaneous spikes to 35 ± 2% of the control (n = 6). Thereafter the inhibitory action of this compound further progressed to a level of almost complete suppression of BC spiking. The increase by ISP of BC spike discharges could no longer be observed in the presence of ZD7288 when its effect was tested during a period 10-20 min after the Ih blocker treatment (Fig. 5D). From these findings, it is suggested that persistent activation of Ih occurs in the BC in the range around the resting membrane potential, contributing to the generation of spontaneous spike activity, and that the ISP-induced increase in the frequency of BC spontaneous spike firing is mainly mediated by the enhancement of Ih activation.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The results of this study demonstrate that the inwardly rectifying current Ih through hyperpolarization-activated cation channels is, at least in part, responsible for the noradrenergic facilitation of GABAergic transmission at cerebellar BC-PC synapses. The BCs exhibited profound activity of hyperpolarization-activated cation channels that were activated in the range around the resting membrane potential. The beta -adrenergic receptor activation by NE appeared to result in depolarization of the BC by increasing intracellular cAMP levels, thereby enhancing persistent activation of Ih. It is therefore suggested that the acceleration of Ih underlies the increase in the frequencies of the BC spike discharges and subsequent BC spike-triggered IPSCs in PCs following activation of the noradrenergic afferent input to the rat cerebellar cortex.

Modulation of Ih in BCs by beta -adrenoceptor activation

Our data showed that hyperpolarizing voltage steps in BCs produce a slow inward current whose characteristics are similar to those of the hyperpolarization-activated cation channel current, Ih, described in previous studies on several neurons and nonneuronal cells (Banks et al. 1993; Bois et al. 1997; Doan and Kunze 1999). We identified the slow inward current in the BCs as Ih on the basis of its activation kinetics, reversal potential and pharmacological properties. The reversal potential of Ih estimated in the BCs was approximately -40 mV, which is in close agreement with values reported previously, suggesting that the current is due to mixed Na+-K+ permeability (Akasu and Shoji 1994; Bayliss et al. 1994; Maccaferri and McBain 1996; Yagi and Sumino 1998).

The observation that NE depolarized the BC in a manner sensitive to the Ih blocker ZD7288 suggests that NE accelerates activation of Ih at a level close to the resting potential of the BCs. Although the driving force of Ih in the BCs was relatively small, the NE-induced Ih activation had a profound effect on the spontaneous spike activity of the BC. Furthermore, the Ih inhibitor ZD7288 induced an outward current in voltage-clamped BCs and caused hyperpolarization of the BC under current-clamp conditions, thereby resulting in marked suppression of the spike discharges in the BCs (see Fig. 5C). The hyperpolarizing action of the Ih blocker ZD7288 has also been demonstrated in previous studies on other cells (Gasparini and DiFancesco 1997; Maccaferri and McBain 1996). It is therefore most likely that modulation by NE of Ih persistently activated at the resting potential produces depolarization and, in turn, causes profound enhancement of spontaneous firings in the BC.

beta -Adrenoceptor-stimulation accelerates activation of Ih currents via cAMP formation

Recent molecular cloning of hyperpolarization-activated cation channels has demonstrated that the cAMP-binding site located directly in a putative intracellular domain of the channel protein plays a critical role in the modulation of channel activity (Gauss et al. 1998; Ludwig et al. 1998; Santro et al. 1998). The cAMP-binding site of this channel appeared to be a target of beta -adrenergic receptors that are coupled to intracellular cAMP formation via AC activation. In support of this notion, the beta -adrenoceptor agonist ISP enhanced the activation of Ih in the cerebellar BCs, and the AC activator forskolin mimicked the action mediated by beta -adrenoceptor stimulation (Fig. 2), which suggests that activation of beta -adrenoceptor by NE accelerates the Ih activity through the formation of intracellular cAMP in the BCs. Similarly in the sinoatrial node of the heart, beta -adrenergic receptor-mediated AC activation was shown to accelerate pacemaker activity via modulation of the hyperpolarization-activated cation channel current If (DiFrancesco 1993). More recently, Beaumont and Zucker (2000) have reported that serotonin receptor activation leads to presynaptic cAMP formation and direct modulation of Ih channels in axons, thereby enhancing synaptic strength of the crayfish neuromuscular excitatory transmission. Thus it appears that regulation of AC activity through G-protein-coupled neurotransmitter receptors is critically involved in the control of not only cardiac pacemaker cells but also neurons including mammalian cerebellar BCs and invertebrate motor neurons. Because an increase in intracellular Ca2+ is also reported to cause persistent activation of Ih in thalamocortical neurons, resulting from a positive shift in the activation curve of this current (Lüthi and McCormick 1998), NE-induced depolarization as well as an increase in spike firings in the cerebellar BCs may also cause increases of intracellular Ca2+ concentrations through activation of low- and high-threshold Ca2+ channels and thereby induce further activation of Ih. Thus noradrenergic facilitation of Ih may serve as a positive feedback mechanism for the up-regulation of BC spike firing, leading to a powerful inhibitory influence on the PC that limits the output from the cerebellar cortex.

beta -Adrenoceptor-mediated excitability increase without involvement of PKA-dependent pathways

The finding that Rp-cAMP-S, known as a PKA inhibitor and a direct activator of Ih, mimicked the action of ISP of enhancing spike firing of the BCs (Fig. 5C) suggests that the beta -adrenoceptor-mediated increase in BC excitability is due to direct activation of the hyperpolarization-activated cation channels by cAMP without involvement of PKA-dependent pathways. This notion is further supported by the observation that ISP enhanced the spike activity of BCs in the presence of the nonselective protein kinase inhibitor, H-7 (Fig. 5B), which also excludes roles of protein kinase-dependent mechanisms in beta -agonist-induced facilitation of BC spiking. Previously, it was assumed that PKA-dependent phosphorylation is involved in the modulation of Ih in certain neurons (Chang and Cohen 1992; Tokimasa and Akasu 1990). However, it has recently been demonstrated that cAMP and its analogues including Rp-cAMP-S increase the activation of Ih current through their direct actions on hyperploarization-activated cation channels (Ingram and Williams 1996; Raes et al. 1997).

Previous studies have shown that PKA-dependent pathways are involved in beta -adrenergic receptor-mediated enhancement of stimulation-evoked GABAA IPSCs and of the frequency of miniature IPSCs recorded in cerebellar interneurons and PCs (Kondo and Marty 1997; Mitoma and Konishi 1999). The effect of beta -adrenoceptor activation on stimulation-evoked GABA release from BC nerve terminals is therefore distinguished from the beta -adrenoceptor-mediated increase in the BC excitability, suggesting that distinct mechanism(s) other than the acceleration of Ih current may underlie noradrenergic facilitation of GABAergic transmission at BC-PC synapses following neural stimulation. In fact, the Ih inhibitor ZD7288 completely blocked the beta -agonist ISP-induced increase in the BC spiking (Fig. 5D), whereas ISP increased the amplitude of stimulation-evoked IPSCs even in the presence of ZD7288 (unpublished observation). As the beta -adrenoceptor agonists NE and ISP did not influence the amplitude of miniature IPSCs as well as postsynaptic GABA receptor sensitivity in the PCs but increased the frequency of miniature IPSCs (Mitoma and Konishi 1999), it is unlikely that the enhancement of evoked IPSCs by beta -agonists is elicited by a postsynaptic mechanism. Thus one possibility might be that the increase in intracellular cAMP level caused by beta -adrenoceptor activation elicits dual actions on the presynaptic BCs: one the activation of hyperpolarization-activated cation channels and the other stimulation of neurotransmitter release machinery in nerve terminals, leading to enhanced GABA release. Further experiments are needed to precisely determine what signaling pathways mediate long-term facilitation of GABA release following activation of monoaminergic receptors on the cerebellar BC.


    FOOTNOTES

Address for reprint requests: S. Konishi, Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194-8511, Japan (E-mail: skonishi{at}libra.ls.m-kagaku.co.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 January 2000; accepted in final form 7 June 2000.


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ABSTRACT
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