beta -Adrenergic Receptor-Mediated Presynaptic Facilitation of Inhibitory GABAergic Transmission at Cerebellar Interneuron-Purkinje Cell Synapses

Fumihito Saitow, Shin'Ichiro Satake, Junko Yamada, 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


    ABSTRACT
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ABSTRACT
INTRODUCTION
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Saitow, Fumihito, Shin'Ichiro Satake, Junko Yamada, and Shiro Konishi. beta -Adrenergic Receptor-Mediated Presynaptic Facilitation of Inhibitory GABAergic Transmission at Cerebellar Interneuron-Purkinje Cell Synapses. J. Neurophysiol. 84: 2016-2025, 2000. Norepinephrine (NE) has been shown to elicit long-term facilitation of GABAergic transmission to rat cerebellar Purkinje cells (PCs) through beta -adrenergic receptor activation. To further examine the locus and adrenoceptor subtypes involved in the NE-induced facilitation of GABAergic transmission, we recorded inhibitory postsynaptic currents (IPSCs) evoked by focal stimulation with paired-pulse (PP) stimuli from PCs in rat cerebellar slices by whole cell recordings and analyzed the PP ratio of the IPSC amplitude. NE increased the IPSC amplitude with a decease in the variance of the PP ratio, which was mimicked by presynaptic manipulation of the transmission caused by increasing the extracellular Ca2+ concentration, confirming that the presynaptic adrenergic receptors are responsible for the facilitation. Pharmacological tests showed that the beta 2-adrenoceptor antagonist, ICI118,551, but not the beta 1-adrenoceptor antagonist, CGP20712A, blocked the NE-induced IPSC facilitation, suggesting that the beta 2-adrenoceptors on cerebellar interneurons, basket cells (BCs), mediate the noradrenergic facilitation of GABAergic transmission. Double recordings were performed from BCs and PCs to further characterize the regulation of the GABAergic synapses. First, on-cell recordings from BCs showed that the beta -agonist isoproterenol (ISP) increased the frequencies of the spontaneous spikes in BCs and the spike-triggered IPSCs in PCs recorded with the whole cell mode. The amplitude of the spike-triggered IPSCs decreased or increased depending on the individual GABAergic synapses examined. Forskolin invariably increased both the amplitude and the frequency of the spike-triggered IPSCs. Double whole cell recordings from BC-PC pairs showed that ISP mainly caused an increase in the amplitude of the IPSCs evoked in the PCs by an action current in the BCs produced in response to voltage steps from -60 to -10 mV. Our data suggest that the noradrenergic facilitation of GABAergic transmission in the rat cerebellar cortex is mediated, at least in part, by depolarization and action potential discharges in the BCs through activation of the beta 2-adrenoceptors in BCs coupled to intracellular cyclic AMP formation.


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INTRODUCTION
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Patterns created by neuronal firings are essential for signal processing in neural circuitries. Neurotransmitters, such as amino acids, monoamines and neuropeptides, through activation of diverse receptor subtypes, can modulate the strength of synaptic transmission in the nervous system and thereby influence the firing patterns generated in individual neuronal pathways. Recently it has been shown that GABAergic synapses between rat cerebellar interneurons and Purkinje cells (PCs) are positively regulated by the monoamines norepinephrine (NE) and serotonin (5-HT) (Llano and Gerschenfeld 1993; Mitoma et al. 1994) and downregulated by the activation of glutamate receptors (Konishi et al. 1996; Satake et al. 2000). The activity of the PCs, the sole output neuron from the cerebellar cortex, is therefore reciprocally regulated by monoamine and glutamate receptor systems. NE and 5-HT liberated by cerebellar afferent inputs have been shown to exert long-lasting facilitation of the GABAergic transmission at basket cell (BC)-PC inhibitory synapses, leading to tonic inhibition of the output from the cerebellar cortex (Mitoma and Konishi 1996, 1999). In contrast, the excitatory amino acid released from the climbing fiber, another afferent input, appeared to cause not only typical direct excitation of the PCs but also inhibition of the GABAergic inputs converging on the same PCs (Satake et al. 2000). Patterns of cerebellar output signals from the PC thus appear to be profoundly influenced by the neurotransmitters liberated by the two afferent inputs derived from the brain stem to the cerebellar cortex.

Monoaminergic neurons in the brain stem project to various regions in the mammalian CNS that include the cerebellar cortex (Olson and Fuxe 1971; Pickel et al. 1972). Among the monoamines, NE was first shown to inhibit the activity of PCs (Siggins et al. 1971) and was subsequently reported to act directly on the PCs to elicit a slow postsynaptic hyperpolarizing response, thereby mediating the effect of electrical stimulation in the locus coeruleus producing a long-lasting inhibition of PC activity. However, it has recently been demonstrated that NE and 5-HT act on GABAergic interneurons to cause presynaptic facilitation of the inhibitory transmission to the PCs (Mitoma and Konishi 1999; Saitow et al. 1998), although direct postsynaptic action of NE has also been reported (Cheun and Yeh 1996). NE enhanced the GABAergic transmission through the activation of beta -adrenergic receptors on cerebellar interneurons (Kondo and Marty 1998; Llano and Gerscenfeld 1993; Mitoma and Konishi 1996, 1999), but the precise subtype(s) of beta -receptors has not yet been determined. beta -Adrenoceptors are seven-transmembrane-spanning G-protein-coupled receptors and classified into three subtypes, beta 1, beta 2, and beta 3. The mammalian CNS contains mainly beta 1- and beta 2-adrenoceptors (Gibbs and Summers 2000; Nicholas et al. 1996) to which selective receptor antagonists are now available (Dooley et al. 1986; O'Donnell and Wanstall 1980). Therefore for better understanding of the molecular basis underlying the monoaminergic modulation of GABAergic synapses, it is crucial to determine the locus of NE-induced facilitation and the adrenoceptor subtypes involved.

This is the first of a series of papers that deal with the site of action, receptor subtypes, and ionic mechanisms underlying the noradrenergic facilitation of inhibitory GABAergic synapses between cerebellar interneurons and PCs. The paper describes our attempt to further examine whether pre- or postsynaptic mechanisms mediate the NE-induced enhancement of GABAergic transmission. For this purpose, we employed paired-pulse (PP) stimulation to evoke inhibitory postsynaptic currents (IPSCs) that were recorded from the PCs in rat cerebellar slices by the whole cell voltage-clamp technique and analyzed the PP ratio of the evoked IPSCs. Our data showed that NE decreased the variance of the PP ratio of the IPSC amplitude and that this effect is mimicked by an increase in extracellular Ca2+ concentration, a manipulation of increasing the synaptic strength by a presynaptic mechanism. We also carried out pharmacological experiments using adrenergic receptor antagonists, which provided evidence of beta 2-adrenoceptors being responsible for the enhancement of the IPSCs by NE. Then we performed two modes of simultaneous pair recordings from BCs and PCs to examine the actions of NE on presynaptic GABAergic interneurons. On-cell recordings from BCs with the cell-attached mode demonstrated that NE causes a robust increase in the frequency of spike discharges of the BCs, resulting in a concurrent increase in the frequency of IPSCs in the PCs. The amplitude of BC-spike-driven IPSCs recorded from the PCs was either decreased or increased by NE in the individual BC-PC pairs when the spike activity of BCs was recorded with the cell-attached mode. However, isoproterenol (ISP), a beta -adrenergic agonist, increased the IPSC amplitude in the majority of BC-PC pairs when both cells were voltage-clamped with the whole cell recording mode. The adenylyl cyclase activator forskolin mimicked the actions of NE and ISP in increasing the frequency of BC spiking and the spike-driven IPSCs in the PCs. Taken together, our data suggest that NE activates beta 2-adrenergic receptors on the BCs and increases cyclic AMP formation, thereby causing BC depolarization and consequent increase in the frequencies of the BC spike discharges and spike-driven IPSCs in PCs. Some of the present results have appeared in a preliminary form (Saitow et al. 1998).


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Preparation

Experiments were performed using thin slices of the cerebellar cortex prepared from 13- to 21-day-old Wistar rats. Animals of either sex were deeply anesthetized with halothane, and their brains were rapidly removed. Parasagittal slices with the thickness of 250 µm were cut using a vibratome (VT1000S, Leica, Nussloch, Germany) at ~4°C in Na+-deficient saline that contained (in mM) 299.2 sucrose, 3.4 KCl, 0.3 CaCl2, 3.0 MgCl2, 10 HEPES, 0.6 NaH2PO4, and 10 glucose. This solution appeared to decrease tissue damage occurring due to the excessive excitation during slicing. The slices were kept for 1 h in a humidified and oxygenated chamber with an interface of artificial cerebrospinal fluid (ACSF) that contained (in mM) 138.6 NaCl, 3.4 KCl, 2.5 CaCl2, 1.0 MgCl2, 21.0 NaHCO3, 0.6 NaH2PO4, and 10.0 glucose. The pH of the ACSF was maintained at 7.4 by bubbling with 95% O2-5% CO2 gas. In most experiments, the slices were superfused with ACSF to which 5 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) had been added to eliminate glutamatergic excitatory synaptic responses.

Patch-clamp recording

Individual slices were transferred to a recording chamber attached to the stage of a microscope (BX50WI, Olympus, Tokyo, Japan) and continuously perfused with the oxygenated ACSF at a flow rate of 1.5 ml/min and temperature of 25-27°C. Patch electrodes were pulled from thin-walled glass tubing (GD-1.5, Narishige, Tokyo, Japan) with a pipette puller (PP-83, Narishige). Patch electrodes used for whole cell voltage-clamp recordings from PCs had resistances of 3-6 MOmega when filled with an internal solution containing (in mM) 150.0 Cs methanesulphonate, 5.0 KCl, 0.1 K-EGTA, 5.0 Na-HEPES, 3.0 Mg-ATP, and 0.4 Na-GTP (pH 7.4). PCs were visually identified under Nomarski optics with a water-immersion objective (×63 N. A., 0.90, Olympus, Japan). Extracellular spike activity in BCs was observed by patch-electrode recordings with the tight-seal cell-attached configuration. BC spike-triggered IPSCs in PCs were recorded by the whole cell patch-clamp technique. Glass electrodes used for the cell-attached recordings had resistances of 7-9 MOmega when filled with ACSF. Patch electrodes used for the whole cell voltage-clamp recordings from BCs had resistances of 5-7 MOmega when filled with an internal solution containing (in mM) 150.0 potassium methanesulphonate, 5.0 KCl, 0.1 K-EGTA, 5.0 Na-HEPES, 3.0 Mg-ATP, and 0.4 Na-GTP (pH 7.4). Recordings of BC spike activity were performed in the lowest third of the molecular layer in the cerebellar cortex where BCs have shown to be located (Palay and Chan-Palay 1974). Membrane currents and extracellular spike activity were recorded with a patch-clamp amplifier (EPC-7, HEKA, Lambrecht, Germany) and a voltage-clamp amplifier (Axoclamp2A, Axon Instruments, Foster City, CA), respectively. Signals were digitized by the pClamp6 program through an A/D converter, Digidata 1200 (Axon Instruments). Data were acquired on the computer disk for off-line analysis. No corrections for liquid junction potential and series resistance were employed. The leak current were continuously monitored, and data were not included if this parameter changed 200 pA. Signals obtained from double recordings in BC-PC pairs were continuously stored during the experiments on a videotape recorder after digitizing using a PCM data recorder (NF Electronic Instruments, Japan). All signals were filtered at 2 kHz and sampled at 5 kHz. The membrane currents of the PCs were held at -50 mV, and IPSCs were evoked by stimulation (10-30 V and 60-100 µs) via the glass microelectrodes (tip diameter, 1-2 µm) filled with ACSF and placed within the molecular layer. The amplitude ratio of the second to the first IPSCs evoked by PP stimulation with an inter-stimulus interval of 50 ms was defined as the PP ratio. The ratio was calculated by measuring the first peak amplitude of averaged IPSCs if they exhibited multiple peaks (see e.g., Fig. 4B). Presynaptic whole cell recordings were made from BCs that were held at -60 mV and stimulated with short voltage pulses (2-5 ms) to -10 mV at a constant frequency of 0.1 Hz to evoke an unclamped sodium action potential (Pouzat and Hestrin 1997).

Drugs

All drugs used for the testing of their effects on synaptic responses were applied by superfusion. The chemicals were obtained from the following sources: (+)-bicuculline, isoproterenol, forskolin, 1,9-dideoxyforskolin, norepinephrine, and D-(-)-2-amino-5-phosphonovaleric acid (D-AP5) from Sigma (St. Louis, MO); CGP20712A and ICI118,551 from Research Biochemicals International (Natick, MA); CNQX from Tocris Cookson (Bristol, UK); and tetrodotoxin from Sankyo (Tokyo, Japan). CNQX and forskolin dissolved in dimethyl sulfoxide at 100 mM were stored at -20°C and diluted before the experiments.

Statistics

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


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NE-induced changes in the paired-pulse ratio of IPSCs and its variance

Stimulation via glass microelectrodes placed in the inner zone of the molecular layer produced outward synaptic currents in PCs held at the membrane potential of -50 mV. These synaptic responses were almost completely abolished by application of the GABAA receptor antagonist bicuculline (5 µM), suggesting that they are produced by activation of GABAA receptors; they are therefore referred to as GABAA IPSCs. In the control medium, the GABAA IPSC exhibited the rise time (10-90% of the amplitude) of 2.6 ± 0.1 ms and the decay time constant of 25.1 ± 2.0 ms (n = 62). Application of NE at a concentration of 10 µM increased the amplitude of the GABAA IPSCs in a majority of PCs tested (see Fig. 1): the extent of enhancement by NE was 185 ± 17% (n = 22) of the control IPSCs recorded before NE application, while there was no discernible effect in the remaining cells (n = 6, see Fig. 1E). However, NE caused no significant change in kinetics of the IPSCs: the rise time of GABAA IPSCs before and after NE application were 2.6 ± 0.1 and 2.5 ± 0.1 ms, respectively (n = 14, P > 0.7). The enhancement of IPSCs by NE was not affected by treatment with 5 µM CNQX and 50 µM D-AP5 as reported previously (Mitoma and Konishi 1999), indicating that ionotoropic glutamate receptor-mediated synaptic mechanism is unlikely to be involved in the NE-induced facilitation of GABAA IPSCs. Mean PP ratio of IPSCs determined in the control medium was 0.99 ± 0.07 (n =48), which was decreased to 0.93 ± 0.07 during the NE-induced enhancement of GABAergic transmission in the control medium containing the extracellular Ca2+ concentration ([Ca2+]o) of 2.5 mM, although the decrease was not statistically significant (P > 0.07, n = 11, see following text).



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Fig. 1. Effects of norepinephrine (NE) on the amplitude and paired-pulse (PP) ratio of inhibitory postsynaptic currents (IPSCs) evoked by PP stimulation and recorded from Purkinje cells (PCs). A and C: time courses of changes in the peak amplitudes of the 1st (open circles) and 2nd IPSCs (filled circles; A) and the PP ratio (C). NE (10 µM) was applied during the period indicated in the graph. The PP ratio represents the amplitude ratio of the 2nd to the 1st IPSCs recorded from a PC. The horizontal straight lines indicate the averaged PP ratio before, during, and after NE application. The averaged PP ratio and its fluctuation decreased following NE-induced increase in the IPSC amplitude. B: superimposed traces of consecutive IPSCs recorded before (a) and during NE application (b). Traces in a and b were obtained at the time points indicated in A. D: effects of NE on the PP ratio variance of IPSCs. The variance determined during NE application was expressed as the ratio to the averaged variance determined during the control period before NE application. Each column represents the mean ± SE obtained from 11 independent experiments. E: distribution of the effects of 10 µM NE on IPSCs in individual PCs tested. The potentiation ratio was determined as a ratio of mean amplitude of IPSCs recorded in each PC for 3 min during NE application to that of IPSCs before application (n = 28).

The PP ratio of synaptic responses changes in association with presynaptic manipulations of the synaptic strength, whereas it remains unaltered following postsynaptic manipulation of synaptic transmission (Manabe et al. 1993). However, the PP ratio of GABAA IPSCs was not dramatically altered by NE in the 2.5-mM [Ca2+]o medium as described in the preceding text, showing that the PP ratio may not serve as a reliable index to determine the locus of NE action under these conditions. Nevertheless it appeared that fluctuations of the IPSC amplitude became smaller following NE application as compared with those of the control IPSC amplitude in 2.5 mM [Ca2+]o (Fig. 1A), which suggested that the decrease in the IPSC fluctuations results in a reduction of the coefficient of variation (CV). The observation prompted us to compare a parameter, variance of the PP ratio of IPSCs evoked by PP stimulation, before and following NE application. Consequently, we found that NE significantly decreased the variance of the PP ratio (Fig. 1D, P < 0.01, n = 11), which is consistent with the observation that fluctuations of the GABAA IPSC amplitude became less pronounced during NE-induced enhancement than in the control medium.

Presynaptic manipulation mimics the effects of NE on the PP ratio fluctuation

To assess the validity of this parameter, we then examined how the PP ratio of IPSCs fluctuates in response to presynaptic and postsynaptic manipulations of the strength of GABAergic inhibitory transmission. When inhibitory transmission was presynaptically attenuated by reducing [Ca2+]o from 2.5 to 1.0 mM, the mean PP ratio increased from 0.75 ± 0.03 at 2.5 mM [Ca2+]o to 0.96 ± 0.05 at 1.0 mM [Ca2+]o (Fig. 2, A and B). The variance of the PP ratio also increased as GABAergic transmission was suppressed following reduction of the [Ca2+]o from 2.5 to 1.0 mM (Fig. 2, B and D; P < 0.01, n = 13).



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Fig. 2. Effects of changes in the [Ca2+]o and the membrane potential on the amplitude and PP ratio of IPSCs. A and C: time courses of changes in the peak amplitudes of the 1st (open circle ) and 2nd IPSCs (, A) and the PP ratio (C) determined in a PC. The [Ca2+]o was changed between 2.5 and 1.0 mM, and the membrane potential (Vm) was shifted from -50 to -10 mV as indicated. B: superimposed traces of consecutive IPSCs recorded during the periods a-c indicated in A. a, 2.5 mM [Ca2+]o at -50 mV; b, 1.0 mM [Ca2+]o at -10 mV; and c, 2.5 mM [Ca2+]o at -10 mV. D: effects of change in the [Ca2+]o on the PP ratio variance of IPSCs. The PP ratio variance determined in a 2.5-mM [Ca2+]o medium was expressed as the ratio to the averaged variance in a 1.0-mM [Ca2+]o medium. Each column represents the mean ± SE obtained from 13 independent experiments.

In the excitatory glutamatergic synapses in the rat hippocampus, Schultz (1997) reported that there was a significant inverse correlation between the PP ratio and the magnitude of long-term potentiation. We also tested the relationship between the extent of enhancement of GABAA IPSCs and the change of the PP ratio caused by NE. Figure 3 compares the effects of NE on the IPSC amplitude, PP ratio and its variance in 1.5- and 2.5-mM [Ca2+]o medium. In association with the enhancement of GABAA IPSCs by 10 µM NE, the mean PP ratio decreased from 1.36 to 1.10 (Fig. 3C), and the variance of the PP ratio decreased from 0.31 to 0.14 in the 1.5-mM [Ca2+]o medium, indicating that the NE-induced change in the PP ratio was larger in the 1.5-mM than in the 2.5-mM [Ca2+]o medium. The NE-induced facilitation of GABAergic transmission was also associated with a significant decrease in the PP ratio in the 1.5-mM [Ca2+]o medium (Fig. 3D, P < 0.003, n = 5).



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Fig. 3. Comparison of the effects of NE application and increase in the [Ca2+]o on the amplitude and PP ratio of IPSCs. A and C: time courses of changes in the peak amplitudes of the 1st (open circle ) and 2nd IPSCs () (A) and the PP ratio (C) determined in a PC. The effect of NE (10 µM) was first determined in a 1.5-mM [Ca2+]o medium, and then the [Ca2+]o was switched from 1.5 to 2.5 mM during the periods indicated. B: superimposed (top) and averaged traces (bottom) of consecutive IPSCs recorded during the periods a-c indicated in A. a, control IPSCs at 1.5-mM [Ca2+]o; b, responses recorded during NE application at 1.5 mM [Ca2+]o; and c, responses recorded at 2.5 mM [Ca2+]o. D: effects of NE in a 1.5-mM [Ca2+]o medium on the PP ratio of IPSCs. Data were obtained from 5 independent experiments in a control artificial cerebrospinal fluid (ACSF) containing 1.5 mM [Ca2+]o and during 10 µM NE application in the control ACSF of 1.5 mM [Ca2+]o.

Postsynaptic manipulation does not alter the PP ratio fluctuation

We next examined how PP ratio fluctuations are affected when the GABAergic transmission is modulated by postsynaptic mechanisms. Application of the GABAA receptor antagonist bicuculline (2 µM) suppressed GABAA IPSCs and reduced their amplitude to ~30%. In contrast, neither the average PP ratio of the GABAA IPSCs nor the variance of PP ratio was affected by bicuculline (Fig. 4, B and D). The result suggests that postsynaptic manipulation of GABAergic transmission causes no significant alterations in the PP ratio or its fluctuations (P > 0.4, n = 13). Although the presence of presynaptic GABAA receptors was reported in cerebellar GABAergic synapses (Glitsch and Marty 1999; Pouzat and Marty 1999), it seems that they play a little part in the modulation of GABAA IPSCs evoked by single-shock stimulation used under the present experimental conditions.



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Fig. 4. Effects of the GABAA antagonist bicuculline on the amplitude and PP ratio of IPSCs. A and C: time courses of changes in the peak amplitudes of the 1st (open circle ) and 2nd IPSCs () (A) and the PP ratio (C) determined in a PC. Bicuculline (2 µM) was applied during the period indicated. B: superimposed traces of consecutive IPSCs recorded before (a) and during bicuculline application (b). Records in a and b were obtained at the time points indicated in A. D: effects of bicuculline on the PP ratio variance of IPSCs. Each column represents the mean of the PP ratio variance ± SE (n = 10), determined as in Fig. 1. n.s., statistically no significant differences between the values determined in the control and 2 µM bicuculline-containing ACSF (P > 0.4, n = 13).

It might be possible that the increase in PP ratio fluctuation observed in a low-Ca2+ medium is due to the increase in the driving force of the GABA receptor channels because it would increase when the amplitude of the IPSCs decreased under a low [Ca2+]o. This possibility was tested in the experiment shown in Fig. 2, in which the [Ca2+]o was kept at 1.0 mM, and the recorded PCs were depolarized from -50 to -10 mV to increase the driving force of the GABAA receptors. As expected, the amplitude of the IPSCs increased at the depolarized membrane potential, whereas the mean PP ratio and its variance were not changed significantly by the membrane depolarization (P > 0.3, n = 5). In contrast, the increase in the [Ca2+]o from 1.0 to 2.5 mM at the depolarized membrane potential of -10 mV reduced the PP ratio fluctuation as well as its variance, while the treatment increased the amplitude of the IPSCs (Fig. 2, A and C), which suggests that presynaptic manipulation of GABA release but not a shifting of the driving force of postsynaptic GABAA receptor channels contribute to the PP ratio fluctuation of the evoked GABAA IPSCs. Furthermore there was no significant correlation between the PP ratio variance and the IPSC amplitude (r2 = 0.095, n = 25, data not shown), which excludes the possibility that the IPSC amplitude per se affects the PP ratio fluctuation.

beta -Adrenoceptor subtype involved in the NE-induced enhancement

Previously it was shown that ISP mimicked the effect of NE of enhancing the GABAA IPSCs and that a nonselective beta -adrenoceptor antagonist, propranolol, blocked the NE-induced enhancement (Mitoma and Konishi 1999). Using selective beta -receptor antagonists (Dooley et al. 1986; O'Donnell and Wanstall 1980), we attempted to further characterize the beta -adrenoceptor subtype(s) responsible for the noradrenergic facilitation of GABAergic transmission. ICI118,551, a beta 2-antagonist, markedly inhibited the action of the beta -agonist ISP on the GABAA IPSCs (P < 0.05, n = 8, Fig. 5A), whereas CGP20712A, a beta 1-antagonist, was without significant effect on the ISP-induced enhancement of the IPSCs (P > 0.2, n = 7, Fig. 5B). Thus it is likely that the beta 2-adrenergic receptor mediates the enhancement of GABAergic transmission between cerebellar interneuron BCs and PCs.



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Fig. 5. Effects of beta -adrenoceptor antagonists on isoproterenol (ISP)-induced facilitation of IPSCs. ISP (20 µM) was superfused during the periods indicated by the horizontal bars. beta 1 antagonist, CGP20712A (10 µM), in A and beta 2 antagonist, ICI118,551 (10 µM), in B were applied during the periods indicated by the open bars. open circle  and , 2 sets of experiments in which the effects of ISP on the relative peak amplitudes of IPSCs were examined in the control ACSF and in the presence of each of the beta -antagonists. Each point represents the mean ± SE obtained from independent experiments (control, n = 6; ICI118,551, n = 8; and CGP20712A, n = 7).

Simultaneous recordings of synaptic activity from BCs and PCs

To analyze the mechanism underlying the NE-induced enhancement of GABAergic transmission, we preformed two types of simultaneous recordings from BCs and PCs. First, we recorded spontaneous spike activity from BCs by on-cell recordings with the cell-attached mode and the IPSCs triggered in PCs by the BC spikes using the whole cell mode. BCs were identified based on anatomical and electrophysiological criteria: they were located in the inner zone of the molecular layer, and the average diameter of their cell bodies was 12.4 ± 0.5 µm (n = 28). All of the recorded BCs exhibited spontaneous spike discharges with frequencies ranging from 0.3 to 20 Hz, which is consistent with previous findings (Llano and Marty 1995; Pouzat and Hestrin 1997). We obtained 70 pairs of BC-PC double recordings and analyzed 41 pairs that showed a high success rate (>90%) of the synaptic interaction. A typical example is illustrated in Fig. 6A, where each of the spikes recorded extracellularly in a presynaptic BC elicited an IPSC in a postsynaptic PC: 1,116 spikes were observed during 15 min of recording, and every spike produced an IPSC in the PC (success rate, 1.0 in 2.5-mM [Ca2+]o medium). On average, the frequencies of the spike firings in the BC and the spontaneous IPSCs in the PC were 3.9 ± 0.7 Hz (n = 24) and 30.8 ± 3.7 Hz (n = 16), respectively. This indicates that multiple BC synaptic inputs converge on a single PC with the ratio of BCs synaptically connected to a PC being ~8:1 as estimated from the ratio of event frequencies. The spike-driven IPSCs were sensitive to the blocking action of bicuculline (data not shown) and completely abolished by application of TTX (1 µM). Therefore it appears that BCs are capable of generating TTX-sensitive action potentials that contribute to powerful GABAA-receptor-mediated synaptic connections between the BCs and PCs, in contrast to the relatively low success rate of transmission at cerebellar interneuron-interneuron connections as previously reported (Kondo and Marty 1998).



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Fig. 6. Double recordings from synaptically connected basket cell (BC)-PC pairs. A: spike activities in a BC (b) and IPSCs in a PC (a) were recorded simultaneously using the cell-attached and whole cell modes, respectively, as illustrated in the inset. Arrowheads indicate BC spike-driven IPSCs recorded in a PC. B: effects of the beta -agonist ISP on the frequencies of the spike discharges in a BC and spontaneous IPSCs in a PC. ISP (10 µM) was superfused during the period indicated by the horizontal bar. Each column and filled circle represent the frequency of presynaptic BC spikes and PC IPSCs, respectively. a and b, Continuous recordings of spontaneous IPSCs in a PC obtained for the periods indicated by arrows in the graph before (a) and during ISP application (b).

Effects of NE on BC spike firings and spike-triggered IPSCs in PCs

We then sought to determine the effects of the beta -adrenoceptor agonist ISP on the presynaptic BC spikes and the IPSCs in the postsynaptic PCs. Application of ISP (10 µM) markedly increased the frequencies of both BC spikes and spontaneous IPSCs in PCs as illustrated in an example of BC-PC pair recording (Fig. 6B): the frequencies of BC spike and PC-IPSC were increased by 3.8- and 1.6-fold, respectively. Interestingly, ISP produced two different effects on the IPSC amplitude (Fig. 7, A and B). In four of seven BC-PC pairs tested, ISP application decreased the amplitude of BC-spike-driven IPSCs to 56 ± 19% (ranged from 50 to 85%), while in the remaining pairs, it was increased to 160 ± 21% (ranged from 110 to 215%). NE also produced effects similar to ISP on BC spike-driven IPSCs: although NE invariably increased the frequencies of BC spike and PC-IPSCs in all the pairs tested, it reduced and increased the amplitude of spike-driven IPSCs in 7 and 5 of 12 pair recordings, respectively (data not shown).



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Fig. 7. Effects of the beta -agonist ISP on the amplitude of BC spike-driven IPSCs recorded in PCs. A and B: ISP-induced facilitation (A) and depression (B) of spike-triggered IPSCs. Thirty successive sweeps triggered by BC spikes were superimposed before (a) and during 10 µM ISP application (b). IPSCs were displayed by aligning with half-amplitude of the BC spikes. The histograms under the records illustrate amplitude distributions of spike-triggered IPSCs, where n and CV (coefficient of variation) represent total number of IPSCs triggered by the BC spikes in a PC and the CV of IPSC amplitude. Responses in c indicate the average responses in a (control, thin trace) and b (in the presence of ISP, thick trace).

We also analyzed the amplitude distributions of the BC spike-triggered IPSCs before and after beta -agonist stimulation. In the BC-PC pair where ISP elicited facilitation of GABAergic transmission, the CV of the IPSC amplitudes decreased (Fig. 7A), whereas it increased in association with the ISP-induced suppression of spike-triggered IPSCs (Fig. 7B). The changes in the CV of IPSCs are in accord with the observations that the fluctuation of IPSC amplitudes is inversely correlated with the synaptic strength (Figs. 1 and 2). It is therefore likely that the activation of beta 2-adrenergic receptors by ISP and NE results in mechanistically distinct effects on BC-PC GABAergic synapses: first, it consistently increases spontaneously occurring IPSCs, and second, beta 2-adrenoceptors mediate either facilitation or depression of spike-triggered IPSCs at individual BC-PC GABAA synapses. The proportion of IPSC enhancement following beta -agonist stimulation was less in BC spike-triggered IPSCs than in stimulation-evoked IPSCs. This might be due to the fact that a single PC receives multiple GABAergic synaptic contacts from multiple BCs and that beta -agonists tend to enhance more readily the stimulation-evoked IPSCs resulting from activation of multiple synaptic inputs than the single BC input-driven IPSC.

Forskolin mimics the ISP-induced effects on BCs and PCs

Since beta -adrenoceptors have been shown in many cell types to activate an intracellular transduction mechanism involving cyclic AMP as a second messenger, we tested the effects of forskolin, a diterpene activator of adenylyl cyclase (AC) (Zhang et al. 1997). As illustrated in Fig. 8A, forskolin (20 µM) caused a marked increase in the frequency of spike discharges recorded from BCs (open column) and consequently increased the frequency of spontaneous IPSCs recorded in the BCs (filled circles), indicating that AC activation enhances the action potential generation in the BCs. Furthermore forskolin was capable of increasing the amplitude of BC-spike-triggered IPSCs (Fig. 8B) to 110-160% of the control IPSCs, and enhancement of GABAergic transmission was observed in all of the BC-PC pairs tested (n = 5). An inactive analogue of forskolin, 1,9-dideoxyforskolin (20 µM) (Seamon and Daly 1986), produced slight decrease or no discernible change in the frequency of spike discharges (n = 3). As we observed previously, 1,9-dideoxyforskolin did not affect the amplitudes of stimulation-evoked IPSCs and spontaneous IPSCs (Mitoma and Konishi 1999). The observations are consistent with the results from previous studies showing that GABAA IPSCs in PCs evoked by electrical stimulation was increased by NE through a beta -adrenoceptor-mediated cyclic AMP-dependent mechanism (Mitoma and Konishi 1996, 1999).



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Fig. 8. Forskolin-induced facilitation of spike discharges in BCs and spike-driven IPSCs in PCs. A: effects of the adenylyl cyclase activator forskolin on the frequencies of the spike discharges recorded in a BC and spontaneous IPSCs simultaneously recorded in a PC. Forskolin (20 µM) was superfused during the period indicated by the horizontal bar. Each column and filled circle represent the frequency of presynaptic BC spikes and PC IPSCs, respectively. B: 30 successive sweeps triggered by the BC spike activity and recorded from a PC. IPSCs were displayed as in Fig. 7. The traces were obtained for the periods indicated by the arrows in A before (a) and during forskolin application (b). Traces in c indicate the average responses in a (control, thin trace) and b (in the presence of forskolin, thick trace). The CV of IPSC amplitudes decreased from 8.1 during the control period to 4.7 after the forskolin treatment.

Effects of ISP on IPSCs with voltage-clamped BCs

As described in the preceding text, NE and ISP produced two different effects on the amplitude of spike-triggered IPSCs and electrical-stimulation-evoked IPSCs (Figs. 1 and 7). Similar discrepant effects of NE on GABAergic transmission have also been reported at cerebellar stellate cell-stellate cell synapses (Kondo and Marty 1998), although the underlying mechanism has yet to be determined. To address this issue, we exploited double whole cell recordings from BC-PC pairs, where we examined the effects of NE on IPSCs produced by a Na+-dependent action current. Stimulation of the BC with a fast Na+-dependent inward current evoked by a short pulse of depolarization to -10 mV elicited an IPSC in the PC if they were synaptically connected (248 ± 121 pA in control medium, n = 15). The amplitude of IPSC gradually increased for ~5 min after initiation of double whole cell recordings and exhibited a time-dependent decrease in some BC-PC pairs to 84 ± 22% of peak amplitude (n = 7, see Fig. 9D) during the period of 20-min recording. Figure 9 shows a typical example of the facilitatory action of ISP on the BC action-current-triggered IPSCs recorded from the PC. In 12 of 15 BC-PC pairs tested, ISP increased the amplitude of the triggered IPSCs to 137 ± 28%, while it caused no changes or a slight decrease in the IPSC amplitude to 91 ± 22% in the remaining pairs. Facilitation of the triggered IPSCs following ISP application was statistically significant (P < 0.02, n = 15). Fluctuations (CV) of IPSC amplitudes sharply decreased during the ISP-induced facilitation of spike-triggered IPSCs (Fig. 9, A and B), which is consistent with the change observed with the forskolin-induced facilitation (Fig. 8B). The observation is in clear contrast to the effects of NE and ISP on the amplitude of the BC-spike-triggered IPSCs; namely, the proportion of BC-PC pairs that exhibited facilitation of the IPSC amplitude in response to application of catecholamines was profoundly increased (from ~40 to 80%) when the mode of recordings from the BC was changed from the cell-attached to the whole cell voltage-clamp mode. This could be explained by the possibility that activation of beta -adrenergic receptors by NE and ISP results in depolarization of voltage-unclamped BCs under on-cell recording, which, in turn, causes a conduction block of the action potentials entering the BC nerve terminals with multiple ramifications, thereby decreasing the strength of GABAergic transmission onto PCs.



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Fig. 9. Effects of the beta -agonist ISP on PC IPSCs triggered by Na+-dependent action currents in a BC. Double whole cell voltage-clamp recordings were obtained from a synaptically connected BC-PC pair. A and B: PC IPSCs were evoked by BC action currents in response to a voltage step from -50 to -10 mV in the control (A) and 10 µM ISP-containing ACSF (B). The CV of IPSC amplitudes decreased from 16.9 during the control period to 6.4 after the ISP application. C: the averaged traces of IPSCs in A (thin trace) and B (thick trace) recorded from a PC were superimposed (top), and presynaptic whole cell currents in a BC produced in response to the depolarizing voltage pulse were averaged (bottom). D: time courses of changes in the peak amplitudes of IPSCs triggered by the BC spikes in a PC. ISP (10 µM) was applied during the period indicated by a horizontal bar.


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Previously, we reported that GABAA receptor-mediated inhibitory transmission at BC-PC synapses is profoundly enhanced by endogenous monoamines released by electrical stimulation from afferent input terminals in the rat cerebellar cortex (Mitoma and Konishi 1996, 1999). In this study, we further examined the receptor subtype involved in the facilitation of GABAergic transmission induced by exogenous catecholamines and the mechanisms underlying the noradrenergic facilitation. Our findings provide evidence that the NE-induced facilitation of GABAA IPSCs at the BC-PC synapse is mediated by the activation of beta 2-adrenoceptors on the presynaptic BCs in a manner dependent on an increase in the intracellular cyclic AMP level. Furthermore our data suggest that beta 2-adrenoceptor activation by NE and ISP results in depolarization of the presynaptic BCs, which leads to the increase in its action potential firings and consequently, increase in the frequency of IPSCs in the postsynaptic PCs.

Variance of the PP ratio

It is crucial to determine the locus of monoaminergic facilitation for further analysis of the underlying mechanism. Thus we explored the validity of using variance of the PP ratio as an index to examine the site of action of adrenergic agonists on the cerebellar GABAergic synapses. Although the PP ratio has been considered to serve as a good indicator (Manabe et al. 1993), we found that fluctuation of the PP ratio is a more sensitive indicator than the PP ratio itself to detect presynaptic changes in the strength of the GABAergic transmission to PCs. The PP ratio fluctuated more in low-[Ca2+]o than in high-[Ca2+]o medium. In contrast, postsynaptic changes in the synaptic strength induced by GABAA receptor antagonists and shifting of the driving force for the receptor channels resulted in no significant change in the PP ratio variance. The basis for the variance of the PP ratio has been derived from previous studies on the quantal nature of neurotransmission at various synapses, where the usefulness of statistical parameters, such as the coefficient of variation (CV) (Isaacson and Walmsley 1995; Martin 1966) and (CV)-2 associated with amplitude fluctuation of the synaptic responses (Barnes-Davies and Forsythe 1995; Lupica et al. 1992; Malinow and Tsien 1990; Manabe et al. 1993) has been vigorously tested. Thus it seemed reasonable to expect that the variance of the PP ratio changes depending on the variance of the amplitude distribution caused by presynaptic manipulations of the strength of transmission and serves as an index for determining the locus of synaptic modulation. In fact, the assumption was consistent with our observations that the CV of IPSC amplitude distributions changed as synaptic strength of GABAergic transmission was modified by catecholamines and forskolin (see Figs. 7 and 8). Another advantage of examining the PP ratio variance is that this parameter changes rapidly during the course of manipulation of the synaptic strength and therefore does not require collection of a large number of sample responses at the steady state level (see Fig. 4 for example). However, elaborate statistics must be utilized to obtain more detailed information about parameters such as the quantal size, q, the number of release sites, n and the probability of release, p (Silver et al. 1998).

The observation that facilitation of the GABAA IPSCs by NE was associated with a significant decrease in the PP ratio variance but not in the PP ratio itself in 2.5 mM [Ca2+]o suggests a presynaptic locus of NE-induced facilitation of GABAA IPSCs through the increase in probability of GABA release from the cerebellar interneuron BCs. This is consistent with the results of previous studies showing that NE and ISP enhanced GABAergic transmission without altering the sensitivity of GABAA receptors in PCs and that they increased the frequency of miniature IPSCs without changes in their mean amplitude (Mitoma and Konishi 1996, 1999).

Double recordings from BC-PC pairs

Because our analysis of the PP ratio fluctuation pointed to presynaptic BCs as the target of NE released by afferent inputs to the cerebellar cortex, the next question we asked was how NE and beta -agonists affect the electrical activity of BCs. Thus we performed double recordings from BC-PC pairs with two modes, which revealed three main effects of beta -adrenoceptor activation on the BCs. 1) The BCs exhibited TTX-sensitive spontaneous firings when recorded with the cell-attached mode, and application of NE or ISP invariably increased the frequency of the spike discharges of the BCs. 2) The BC-spike triggered GABAA IPSCs in the PC with a high success rate, and the amplitude of the BC-spike-driven IPSCs was either increased or decreased by NE and ISP application. 3) When the BC was voltage-clamped at the holding potential of -60 mV using the whole cell recording mode and was stimulated by an action current evoked by a short depolarizing pulse, synchronous IPSCs could be recorded from the PC. ISP increased the amplitude of the IPSCs triggered by the BC action currents in most of the BC-PC pairs tested (12 of 15). This is in contrast to the observation that the amplitude of the BC-spike-triggered IPSCs was increased or decreased when the BC activity was recorded with the cell-attached and voltage unclamped mode. From these findings, it is suggested that the BC fires TTX-sensitive repetitive action potentials, resulting in a tonic inhibitory influence on the PCs, and that NE-mediated activation of beta -adrenergic receptors in the BCs enhances the spike firing through a depolarizing action of NE on the BCs. This possibility has been tested in the subsequent report (Saitow and Konishi 2000).

Another conspicuous feature of the beta -adrenoceptor-mediated actions on the BC is the dual effect of NE on BC spike-driven IPSCs, namely, enhancement and suppression of GABAergic transmission at BC-PC synapses. The inhibitory effect of NE might be explained by the blockade of spontaneous action potentials due to NE-induced depolarization of the BC. The effect of NE on BC spike-triggered IPSCs was in contrast to that of the AC activator forskolin: the latter compound mimicked only one aspect of NE actions, namely, enhancement of GABAergic transmission (see the following paper). The difference in effects between NE and forskolin might be explained by the following possibilities. First, the discrepancy would be due to differential cellular localization of beta -adrenoceptors and adenylyl cyclase in the BC or different levels of cyclic AMP produced by the two compounds, leading to diverse (or contrasting) effects. Increase in the intracellular cyclic AMP levels has been shown to elicit distinct physiological responses. For example, increased intracellular cyclic AMP formation was reported to influence the neuronal excitability at cerebellar synapses (Chen and Regehr 1997). Modulation of membrane excitability by cyclic AMP has been reported to involve cyclic nucleotide-mediated modifications of several ion channels, such as closure of Ca2+-activated potassium channels responsible for a slow afterhyperpolarization (Knöpfel et al. 1990; Madison and Nicoll 1982) and activation of hyperpolarization-activated cationic channels that regulate the membrane potential and the action potential firing (Banks et al. 1993; Ingram and Williams 1996; McCormick and Pape 1990; Wang et al. 1997). Second, it might be conceivable that beta -adrenoceptor activation by catecholamines recruits a cellular mechanism distinct from the forskolin-induced cyclic AMP-dependent pathway, although there has been no evidence for this notion.

An alternative possibility might be that NE-induced depolarization of BCs causes liberation of GABA into BC-PC synapses and leads to activation of GABAB autoreceptors, thereby causing inhibition of GABAA IPSCs. NE has been reported to inhibit inhibitory transmission to GABAergic interneurons in the sensorimotor cortex (Bennett et al. 1997, 1998). This action was attributed to activation of presynaptic GABAB autoreceptors following NE-induced GABA release because a GABAB receptor antagonist blocked the NE-induced depression of IPSCs without altering the NE-induced increase in the frequency of spontaneous IPSCs (Deisz and Prince 1989; Harrison et al. 1990). However, it appears unlikely that the NE-induced inhibition of BC spike-driven IPSCs involves GABAB autoreceptors because NE decreased the mean amplitude of BC spike-driven IPSCs to 90-75% of control even after pretreatment with the GABAB antagonist, CGP55845A (n = 3) (unpublished observations). The most likely explanation is that the increase in BC spikes causes an enormous increase in GABA release that would reduce releasable pools of GABA in BC terminals. Further experiments are needed to verify these possibilities.

Under physiological conditions, the overall effect of the activation of noradrenergic afferent inputs would be to exert a tonic inhibition of the PC through repetitive release of GABA caused by action potential discharges in the BC as well as facilitation of GABAergic transmission in some BC-PC pairs. It appears that the noradrenergic afferent input to the cerebellar cortex reinforces GABAergic inhibitory influence from the BCs to the PCs and profoundly affects the pattern of output signals generated in the PCs, the exclusive efferent neuronal system from the cerebellar cortex. Thus monoaminergic modulation of GABAergic transmission at BC-PC synapses appears to play a critical role in motor coordination associated with the cerebellar system.


    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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