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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ABSTRACT |
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Saitow, Fumihito,
Shin'Ichiro Satake,
Junko Yamada, and
Shiro Konishi.
-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
-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
2-adrenoceptor antagonist, ICI118,551, but not
the
1-adrenoceptor antagonist, CGP20712A,
blocked the NE-induced IPSC facilitation, suggesting that the
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
-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
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
-adrenergic receptors on cerebellar interneurons
(Kondo and Marty 1998
; Llano and Gerscenfeld
1993
; Mitoma and Konishi 1996
, 1999
), but the
precise subtype(s) of
-receptors has not yet been determined.
-Adrenoceptors are seven-transmembrane-spanning G-protein-coupled
receptors and classified into three subtypes,
1,
2, and
3. The mammalian CNS contains mainly
1- and
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 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
-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
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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METHODS |
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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 M 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 M
when filled with
ACSF. Patch electrodes used for the whole cell voltage-clamp recordings
from BCs had resistances of 5-7 M
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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RESULTS |
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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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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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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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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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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.
-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
-adrenoceptor antagonist, propranolol, blocked the NE-induced
enhancement (Mitoma and Konishi 1999
). Using selective
-receptor antagonists (Dooley et al. 1986
;
O'Donnell and Wanstall 1980
), we attempted to further characterize the
-adrenoceptor subtype(s) responsible for the noradrenergic facilitation of GABAergic transmission. ICI118,551, a
2-antagonist, markedly inhibited the action of
the
-agonist ISP on the GABAA IPSCs
(P < 0.05, n = 8, Fig.
5A), whereas CGP20712A, a
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
2-adrenergic receptor mediates the enhancement of GABAergic transmission between cerebellar interneuron BCs and PCs.
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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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Effects of NE on BC spike firings and spike-triggered IPSCs in PCs
We then sought to determine the effects of the -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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We also analyzed the amplitude distributions of the BC spike-triggered
IPSCs before and after -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
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,
2-adrenoceptors mediate either facilitation or
depression of spike-triggered IPSCs at individual BC-PC
GABAA synapses. The proportion of IPSC
enhancement following
-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
-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 -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
-adrenoceptor-mediated cyclic AMP-dependent mechanism (Mitoma and Konishi 1996
, 1999
).
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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
-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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DISCUSSION |
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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
2-adrenoceptors on the
presynaptic BCs in a manner dependent on an increase in the
intracellular cyclic AMP level. Furthermore our data suggest that
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
-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
-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
-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 -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
-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
-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.
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FOOTNOTES |
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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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