Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
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Takahashi, Kanji A. and David J. Linden. Cannabinoid Receptor Modulation of Synapses Received by Cerebellar Purkinje Cells. J. Neurophysiol. 83: 1167-1180, 2000. The high density of cannabinoid receptors in the cerebellum and the degradation of motor coordination produced by cannabinoid intoxication suggest that synaptic transmission in the cerebellum may be strongly regulated by cannabinoid receptors. Therefore the effects of exogenous cannabinoids on synapses received by Purkinje cells were investigated in rat cerebellar slices. Parallel fiber-evoked (PF) excitatory postsynaptic currents (EPSCs) were strongly inhibited by bath application of the cannabinoid receptor agonist WIN 55212-2 (5 µM, 12% of baseline EPSC amplitude). This effect was completely blocked by the cannabinoid CB1 receptor antagonist SR 141716. It is unlikely that this was the result of alterations in axonal excitability because fiber volley velocity and kinetics were unchanged and a cannabinoid-induced decrease in fiber volley amplitude was very minor (93% of baseline). WIN 55212-2 had no effect on the amplitude or frequency of spontaneously occurring miniature EPSCs (mEPSCs), suggesting that the effect of CB1 receptor activation on PF EPSCs was presynaptically expressed, but giving no evidence for modulation of release processes after Ca2+ influx. EPSCs evoked by climbing fiber (CF) stimulation were less powerfully attenuated by WIN 55212-2 (5 µM, 74% of baseline). Large, action potential-dependent, spontaneously occurring inhibitory postsynaptic currents (sIPSCs) were either severely reduced in amplitude (<25% of baseline) or eliminated. Miniature IPSCs (mIPSCs) were reduced in frequency (52% of baseline) but not in amplitude, demonstrating suppression of presynaptic vesicle release processes after Ca2+ influx and suggesting an absence of postsynaptic modulation. The decrease in mIPSC frequency was not large enough to account for the decrease in sIPSC amplitude, suggesting that presynaptic voltage-gated channel modulation was also involved. Thus, while CB1 receptor activation reduced neurotransmitter release at all major classes of Purkinje cell synapses, this was not accomplished by a single molecular mechanism. At excitatory synapses, cannabinoid suppression of neurotransmitter release was mediated by modulation of voltage-gated channels in the presynaptic axon terminal. At inhibitory synapses, in addition to modulation of presynaptic voltage-gated channels, suppression of the downstream vesicle release machinery also played a large role.
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INTRODUCTION |
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Marijuana has been used by humans since 2700 BC or
earlier and is presently one of the most commonly used recreational
drugs in the world (reviewed in Adams and Martin 1996). It's principal active ingredient,
(
)-trans-
9-tetrahydrocannabinol
(Gaoni and Mechoulam 1964
), is one of a class of
cannabinoid drugs made up of numerous natural and synthetic analogues.
The effects of cannabinoid administration are varied and include
altered perception of sight and sound, euphoria, anxiolysis, and
loosening of associations. Cannabinoids also induce effects such as
degraded motor coordination and timing that suggest a possible
cerebellar influence.
Cannabinoids act as agonists at specific cannabinoid receptors, two of
which, CB1 (Matsuda et al. 1990) and CB2 (Munro
et al. 1993
), have been cloned. Although both CB1 and CB2 are
found in the periphery, only CB1 is found in the central nervous system (CNS) where its density is particularly high and its distribution widespread (Herkenham 1992
; Herkenham et al.
1991
; Katona et al. 1999
; Mailleux and
Vanderhaeghen 1992
; Matsuda et al. 1993
;
Pettit et al. 1998
; Tsou et al. 1998
).
The brain regions that have the highest densities of CB1 are the basal
ganglia, cerebellum, hippocampus, and parts of the olfactory cortex.
The moderate-to-high density of CB1 in the cerebellum, basal ganglia,
and motor cortex may largely explain the motor deficits associated with
cannabinoid intoxication (reviewed in Herkenham 1992
;
Loewe 1946
).
In the cerebellum, the vast majority of CB1 is found at the presynaptic
terminals received by Purkinje cells, the axons of which are the sole
output of the cerebellar cortex. GABAergic axon collaterals form a
pericellular basket around the Purkinje cell body and a tighter
structure, called the pinceau, around the axon initial segment
(Palay and Chan-Palay 1974). Basket cell axons and
terminals make up this structure, although a small proportion of
stellate cells also contribute (Paula-Barbosa et al.
1983
). CB1 receptor density is highest by far in the cerebellar
pinceau and is moderately high in the pericellular basket (Tsou
et al. 1998
). This is consistent with the reported lack of
cannabinoid receptor mRNA expression by Purkinje cells and the rather
high expression in cells of the molecular layer that appear to be
stellate and basket cells (Mailleux and Vanderhaeghen
1992
; Matsuda et al. 1993
). CB1 immunoreactivity
is moderately high in the molecular layer and low in the granule cell
layer. However, granule cell layer expression of cannabinoid receptor
mRNA is uniform and high. This suggests CB1 localization in parallel
fibers (PFs), which are the axons of granule cells in the molecular
layer. There is low expression of cannabinoid receptor mRNA in the
inferior olive (Herkenham 1992
; Herkenham et al.
1991
; Mailleux and Vanderhaeghen 1992
;
Matsuda et al. 1993
; Pettit et al. 1998
;
Tsou et al. 1998
).
CB1 activation has been widely shown to decrease adenylyl cyclase
activity and reduce cAMP accumulation via
Gi/Go activation in
heterologous expression systems and in neurons including both primary
cultures and acute preparations of cerebellar cells (Childers and Deadwyler 1996; Deadwyler et al. 1993
;
Howlett 1984
; Howlett and Fleming 1984
;
Pacheco et al. 1993
). CB1 receptors have also been found
to modulate voltage-gated ion channels. Using cultured hippocampal
neurons, heterologous expression systems, and neuroblastoma and
neuroblastoma/glioma cell lines, it has been demonstrated that CB1
activation can decrease N and P/Q type Ca2+
channel activity and increase KIR activity
through direct interaction with G protein
subunits. All of these
effects are pertussis toxin-sensitive and therefore mediated by
Gi/Go (Caulfield and Brown 1992
; Henry and Chavkin 1995
;
Mackie and Hille 1992
; Mackie et al.
1995
; Shen and Thayer 1998
; Twitchell et
al. 1997
). In primary hippocampal cultures, cannabinoid
application has been shown to relieve the resting inactivation of
KA channels, and this may be mediated through a
cAMP dependent pathway (Deadwyler et al. 1995
;
Hampson et al. 1995
; reviewed by Childers and
Deadwyler 1996
).
CB1 receptor activation has been found to suppress synaptic
transmission at a number of excitatory and inhibitory synapses. CB1
receptor activation acting presynaptically strongly suppressed EPSC
amplitude at synapses between hippocampal neurons in dispersed culture.
However, in these neurons, IPSC amplitude was unaffected (Shen
and Thayer 1998; Shen et al. 1996
). Similarly,
in acute slices of rat hippocampus, CB1 activation was found to
suppress synaptic input from CA3 to CA1 neurons by acting on
presynaptic Ca channels (Sullivan 1999
). This was
sufficient to prevent induction of long-term potentiation and long-term
depression by standard protocols (Misner and Sullivan
1999
). In addition, CB1 activation was shown to suppress
transmission between cerebellar PFs and Purkinje cells
(Lévénès et al. 1998
). In rat corpus
striatum, recurrent collaterals from the axons of medium spiny neurons
form autapses. IPSCs evoked by stimulating these collaterals were
strongly suppressed by CB1-mediated presynaptic inhibition
(Szabo et al. 1998
). CB1 activation was also reported to
suppress inhibitory transmission between striatal neurons and their
targets in substantia nigra pars reticulata and globus pallidus
(Chan et al. 1998
; Miller and Walker
1995
, 1996
). CB1 activation was also shown to
suppress IPSCs in the neurons of the rostral ventromedial medulla, a
location that participates in the processing of nociceptive information (Vaughn et al. 1999
). Finally, CB1 activation reduced
the release of [3H]-GABA from hippocampal
slices stimulated with an electrical field (Katona et al.
1999
).
Given the high-density of CB1 on the axons or axon terminals of Purkinje cell afferents and the behavioral deficits produced by cannabinoid intoxication that suggest cerebellar involvement, we examined CB1 receptor-mediated modulation of synapses received by Purkinje cells. This was explored electrophysiologically in both whole-cell patch clamp recordings of Purkinje cells and field potential recordings of PFs.
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METHODS |
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Slice preparation and whole-cell patch-clamp recording
Sagittal slices of cerebellar vermis (200 µm thick) or
horizontal cerebellar slices (400 µm thick) were prepared from male and female Sprague-Dawley rats (Harlan) using standard methods (Dittman and Regehr 1996; Konnerth et al.
1990
; Perkel et al. 1990
) modified as follows.
Normal external saline consisted of (in mM) 129 NaCl, 3.0 KCl, 2 CaCl2, 1.75 MgCl2, 1 NaH2PO4, 26.3 NaHCO3, 20 glucose, pH 7.40, 321 mmol/kg, and was
gassed with 95% O2/5%
CO2. Dissections and sectioning were performed in
ice cold external saline in which CaCl2 had been
replaced with MgCl2. Slices were placed submerged
on small slips of nylon mesh supported on a ledge in a custom-built
storage chamber containing normal external saline incubated at
~35°C and gassed with 95% O2/5%
CO2. After all slices were placed in the chamber,
solution was removed until an interface condition was achieved and the
top of the chamber was covered with parafilm. After incubating for
30-60 min at 35°C, the slices were left at room temperature for at
least an additional 30 min before use. Slice health was maximized by
storing slices at room temperature in an interface condition. At least
10 min before recording, slices were submerged and secured with a
silver wire-nylon thread harp in a recording chamber containing ~2.7 ml of saline .
Visualized whole-cell patch recordings were conducted at room
temperature using standard methods (Konnerth et al.
1990; Perkel et al. 1990
). External saline was
delivered to the chamber at 3 ml/min with a peristaltic pump. All
external saline used in the recording chamber contained either 0.02 or
0.1% DMSO and the concentration of DMSO was always kept constant
throughout each recording. When 0.1% DMSO was used,
ddH2O was also added to compensate for the
increase in osmolality. Purkinje cells were visualized using an
Axioskop FS upright microscope equipped with a water-immersion 40×
objective. Patch pipettes were pulled from borosilicate capillary tubing and heat polished. Membrane current was recorded using an
Axopatch 200A amplifier controlled with Axodata software and analyzed
off-line using Axograph software (Axon Instruments, Burlingame, CA).
PFs and climbing fibers (CFs) were stimulated with a monopolar stimulating electrode consisting of a patch pipette filled with filtered external saline. To avoid contamination, stimulating electrodes were changed after every experiment during which a drug was
added to the recording chamber.
Drugs
R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)-methyl]-pyrrolo[1, 2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone
mesylate [R(+)-WIN 55212-2] and
S()-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)-methanone-mesylate [S(
)-WIN 55212-3] were obtained from Research Biochemicals Inc. (Natick, MA). Stocks (10 mM) were made in DMSO and stored in 50-µl aliquots at
20°C. A fresh 10-mM stock of
N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide (SR 141716) in DMSO was made on each day of use. As cannabinoids tend
to stick to surfaces they contact, most tubing was replaced and all
other exposed parts were thoroughly cleansed with ethanol and water
after each cannabinoid recording. Bicuculline methiodide was obtained
from Research Biochemicals Inc. and was added directly to external
saline on the day of use. Aqueous aliquots of 1 mM TTX (Alexis
Biochemicals, San Diego, CA) were kept at
20°C.
PF-evoked EPSC recordings
Purkinje cells in sagittal sections of postnatal day (P)
15-19 rat cerebellar vermis were whole-cell patch clamped with 3-5.5 M pipettes in external saline containing 20 µM bicuculline
methiodide. Pipette saline consisted of (in mM) 8 KCl, 125 K-gluconate,
5 MgCl2, 10 HEPES, 5 NaOH, 4 Na2ATP, 0.4 Na3GTP, and sucrose to bring osmolality to 287-290
mmol/kg, pH
7.23. Liquid junction potential was
12 mV
(pipette negative) and was compensated on-line. Series resistance
(Rs) compensation, used not so much to cancel Rs as to keep it constant, was set to 2-3 M
, 50%
compensation, and 60 µs lag. Rs was typically 7-10 M
after compensation and was kept constant throughout each experiment
with gentle suction or adjustment of Rs compensation.
PF-EPSCs inevitably contained a voltage-activated component, and large
EPSCs evoked Na+ spikes that escaped voltage clamp.
Therefore baseline EPSC amplitude was kept relatively small (typically
300 to
500 pA). Vhold was set at
57 mV, which was
very close to Vrest, to reduce both inactivation and
sensitization of voltage-gated currents. With these adjustments, space
clamp was poor but adequate. In some pilot experiments, Cs-based
internal saline was used to improve space clamp, but slow perfusion of
dendrites produced a gradually increasing EPSC amplitude that often
took >30 min to stabilize. To stimulate PF-evoked EPSCs, the tip of
the stimulating electrode was placed in the inner two-thirds of the
molecular layer. To obtain stable, spike-free EPSCs, the following
protocol was empirically derived (Fig.
1A): every 10 s,
Vhold was stepped from
57 to
80 mV. PFs were stimulated 2000 ms after the onset of hyperpolarization. After another 350 ms,
Vhold was stepped from
80 to
85 mV for 150 ms to
measure Rs and Ri, and then returned to
57
mV. In analysis, EPSCs and
5 mV steps were analyzed separately, with
each event baselined to the 100 ms interval that preceded it. Data were
filtered at 5 kHz with the four-pole, low-pass Bessel filter integral
to the Axopatch 200A and acquired at 5 kHz. Data are presented as
mean ± SE.
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Experiments were conducted as follows (Fig. 1C): after
10 min of stable baseline recording of PF EPSCs, the external saline reservoir was replaced with either another bottle of control external saline or one containing the cannabinoid receptor agonist WIN 55212-2 for
15 min. Finally, the reservoir was switched to external saline
containing 1.0 µM TTX, which eliminated the EPSC and isolated the
stimulus artifact. Off-line, the stimulus artifact, averaged from
10
traces in TTX, was subtracted from all traces in the experiment to
improve accuracy. Within each experiment, the effect of switching to a
second external saline reservoir was calculated as 100%
X (the average of all data 10.3-14.3 min after the
second saline entered the recording chamber) divided by the average of all data 0.7-5.7 min before the second saline entered the recording chamber. Experiments in which Rs or membrane conductance
(Gm) changed by >12% between these periods were discarded.
Fiber volley recordings
These experiments were conducted in 400-µm-thick horizontal
sections of cerebellum from P15-16 rats with methods modified from
Dittman and Regehr (1996). Submerged recordings were made using saline
containing 200 µM CdCl2 to eliminate
Ca2+ currents and Ca2+
channel-dependent synaptic contributions. Because 200 µM
Cd2+ precipitates out of normal
NaHCO3/NaH2PO4-based
salines, an oxygenated HEPES-based saline was used consisting of (in
mM) 149 NaCl, 3.0 KCl, 2 CaCl2, 1.75 MgCl2, 20 glucose, 10 HEPES, 0.02 bicuculline methiodide, 1 ml/l DMSO, pH 7.40, 322 mmol/kg. All stimulating and
recording electrodes consisted of patch-type pipettes with a tip
diameter of 1-2 µm filled with filtered external saline. Stimulus
intensity was
15 to
100 µA (200 µs duration) and was kept well
below saturation. A stimulating electrode and two recording electrodes
were placed in the vermis in the inner half of the molecular layer,
typically 300-500 µm apart (Fig.
2A). Estimated fiber volley
velocity was between 15 and 22 cm/s, consistent with previous reports
in intact cerebellum (reviewed in Ito 1984
). Recordings
were made with an Axoclamp 2A amplifier in bridge mode. Signals were
low-pass filtered at 1 kHz before computer acquisition at 10 kHz. PFs
were stimulated every 10 s. After
30 min of baseline recording,
the external saline reservoir was switched to a control or
cannabinoid-containing solution for another 30 min, and then to 1 µM
TTX to isolate the stimulus artifact for subsequent digital subtraction. Within each experiment, the effect of switching to a
second external saline reservoir was calculated from the average of all
data 0.7-5.7 min before and 24.3-29.3 min after the second saline
entered the recording chamber. Recordings in which the baseline was not
stable were discarded.
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CF-evoked EPSC recordings
Methods were similar to those used in PF EPSC experiments. To
improve voltage clamp, the tip diameter of patch pipettes was increased
(Rpipette = 3-5 M). A monopolar stimulating
electrode was placed in the granule cell layer and its position and
intensity adjusted (
5 to
40 µA, 200 µs) to elicit a large
all-or-none CF EPSC uncontaminated by a PF EPSC component. In many
experiments, a second electrode was placed in the molecular layer to
elicit pure PF EPSCs. External saline contained 20 µM bicuculline and 0.1% DMSO. After positioning the stimulating electrodes,
Vhold was changed to
20 mV. This caused the
amplitude of both CF-evoked and PF-evoked EPSC amplitudes to decrease
severalfold, caused spiking to disappear, and caused
Gm to greatly increase. The cell was allowed to
accommodate to this new Vhold as long as
necessary (>10 min) before data was collected. Thereafter, CF EPSC
amplitude and Rs were stable, although
Gm had a tendency to drift a little over time.
Every 20 s, the following stimulation protocol was applied (see
Fig. 3A): after collecting a
100-ms baseline, a pair of stimuli 100 ms apart were delivered to the
CF. PFs were stimulated 1 s after the second CF stimulation. After
an additional 500 ms, a
5 mV, 150 ms voltage step was imposed on the
cell to measure Rs and Ri.
In some experiments, PF stimulation was omitted and the
5 mV voltage
step occurred 400 ms after the second CF stimulation. After recording a
stable baseline for
15 min, the external saline reservoir was
replaced with saline containing 5.0 µM WIN 55212-2 and recordings
were continued for
15 min before isolation of stimulus artifact with
1.0 µM TTX for subsequent digital subtraction. In control
experiments, each cerebellar slice was incubated in SR 141716 for
15
min before recording commenced. Within each experiment, the effect of
switching to external saline containing WIN 55212-2 was calculated from
the average of all data 0.7-5.7 min before and 10.3-14.3 min after
WIN 55212-2 entered the recording chamber. Experiments in which
Rs changed more than 10% or
Gm changed more than 20% were excluded.
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mEPSC experiments
mEPSCs were recorded in whole-cell patched Purkinje cells using
methods similar to those used in PF-evoked EPSC experiments. Vhold = 80 mV. To decrease Rs, increase
space clamp, and improve detection of small currents, we used P10-11
rats and patch pipettes with large tip diameters (1.8-2.5 M
) filled
with (in mM) 35.3 CsOH, 95 Cs2SO4, 4 MgSO4 · 7H2O, 10 EGTA, 4 CaCl2,
10 HEPES, 4 Na2ATP, 0.4 Na3GTP, 287-290
mmol/kg, pH
7.23. Liquid junction potential was measured to be
approximately
8 mV (pipette negative) and was compensated on-line.
Data were filtered extensively at 5 kHz with the four-pole Bessel
filter intrinsic to the Axopatch 200A, and externally at 200 Hz with an
additional eight-pole, low-pass Bessel filter. Recordings were
performed in external saline containing 20 µM bicuculline, 1.0 µM
TTX, and 1 ml/l DMSO. Every 5 s, 10 times per minute, 3200 ms of
data were acquired at 5 kHz. At 3050 ms, a
5 mV, 150 ms step was
applied from which Rs and Gm were derived.
After recording for
20 min in normal external saline, the reservoir
was switched to one containing 5.0 µM WIN 55212-2 for another 16 min.
Finally, 10 µM 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) was applied to confirm that detected events were mEPSCs and not noise or mIPSCs. To allow Cs+ in the Purkinje cell
time to equilibrate, an interval of
60 min was imposed between the
time the cell was whole-cell patch clamped and when WIN 55212-2 first
entered the recording chamber. Within each experiment, the effect of
switching to external saline containing WIN 55212-2 was calculated from
the average of all data 1-6 min before and 11-15 min after WIN
55212-2 entered the recording chamber. Data from each cell were
analyzed off-line using Axograph 3.5 software that used template-based
detection. Events smaller than
5 pA were discarded. A separate
template was created for each recording by averaging a large number of hand-selected unambiguous mEPSCs. Detection thresholds were
adjusted such that in the presence of 10 µM NBQX the total frequency
of detected events was <0.05 Hz. There was a wide range of basal mEPSC
frequencies among the cells, and recordings with very low frequencies
were not continued. Average Rs did not change by more than
4% in any of the accepted recordings . Each 3200-ms trace was visually
inspected and traces contaminated with transient noise were deleted. If
>3 in 10 traces were rejected in any minute-long bin, the entire
minute was deleted. Average mEPSC frequency and amplitude were
calculated at 1 min intervals.
sIPSC experiments
Methods were similar to those used in mEPSC experiments. P12-13
rats were used. Vhold = 70 mV. Pipette saline
consisted of (in mM) 35.9 CsOH, 121 CsCl, 4 MgCl2, 10 EGTA, 4 CaCl2, 10 HEPES, 4 Na2ATP, 0.4 Na3GTP, pH 7.22, 290 mmol/kg. Liquid junction
potential was measured to be approximately
4 mV with
kynurenate-containing external saline and was compensated on-line. To
allow CsCl to efficiently perfuse the cell, large-tipped patch pipettes
were used (1.6-2.5 M
). The four-pole, low-pass Bessel filter
intrinsic to the Axopatch 200A was set to 5 kHz, and in most cases
current was additionally filtered at 1 kHz with an external eight-pole, low-pass Bessel filter. At the end of each experiment, 20 µM
bicuculline was applied to confirm that detected events were IPSCs and
not noise or EPSCs. Detection thresholds were adjusted such that
detection frequency in bicuculline was <0.05 Hz. In mixed sIPSC/mIPSC
experiments (without TTX), in the absence of kynurenate, an amplitude
cutoff of
50 pA was used and smaller events, including almost all
EPSCs, were discarded. In the presence of 2 mM kynurenate, an amplitude cutoff of
20 pA was used. The few IPSCs that elicited action potentials were included in calculations of total frequency but omitted
from calculations of average amplitude. In mIPSC experiments, 1.0 µM
TTX and 2.0 mM kynurenate were added to all external salines. After
recording a baseline of
15 min, the external saline reservoir was
switched to one containing either more control saline, 5.0 µM WIN
55212-2, or 100 µM CdCl2 for at least 17 min,
and was then swiched to 20 µM bicuculline. Recordings in which
Rs was not constant or holding current changed
unusually were discarded. Because of the large size and high frequency
of IPSCs, Ri could not be determined accurately,
and all capacitative transients used to calculate Rs that were contaminated by IPSCs were
discarded. In no experiment did Rs change by
>7% after experimental manipulation.
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RESULTS |
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PF-evoked EPSCs
Whole-cell patch clamp recordings were made from Purkinje cells in
acute sagittal sections of cerebellar vermis from P15-19 rats. Regular
stimulation of PFs (Fig. 1A and
METHODS) produced EPSCs similar to those seen
previously (Konnerth et al. 1990; Llano et al.
1991
; Perkel et al. 1990
) and that were stable
for over an hour. In control experiments, switching the external saline reservoir to a second bottle of normal external saline had no effect on
PF-evoked EPSC (PF EPSC) amplitude (103 ± 3.7% of baseline, n = 5). Switching to external saline containing WIN
55212-2 resulted rapidly in a steep reduction in PF EPSC amplitude that
would typically continue for ~10 min before stabilizing. Subsequent
application of 1.0 µM TTX completely eliminated the PF EPSC. A
typical cannabinoid experiment is depicted in Fig. 1,
A-C. The effect of TTX could be washed out, but
in almost all cases the effect of WIN 55212-2 could not be washed out
(data not shown), probably because of its high lipophilicity.
Preincubation with 1.0 µM SR 141716, a CB1-selective antagonist,
completely blocked the effect of 5.0 µM WIN 55212-2 (Fig.
1D). This suggests that the effect of WIN 55212-2 is fully
CB1 receptor-mediated and not the result of a direct membrane effect,
a direct effect on Ca channels (Shen and Thayer 1998
),
or other non-CB1-receptor-mediated processes. WIN 55212-2 reduced PF
EPSC amplitude in a dose-dependent manner with the effect appearing to
approach saturation at high concentrations (5.0 µM: 12.3 ± 2.2% of baseline, n = 3; see Fig. 1E). The
effect of WIN 55212-2 at all concentrations differed significantly from the no-drug control by two-tailed, heteroscedastic t-test,
P < 10
4. In the presence of
1.0 µM SR 141716, 5.0 µM WIN 55212-2 had no effect (107 ± 2.60% of baseline, n = 4, P = 0.41).
Rs and Gm were stable and
always unaffected by WIN 55212-2 (Fig. 1F).
PF-fiber volleys
There are several mechanisms by which CB1 activation could produce
a decrease in PF EPSC amplitude. Two possibilities are CB1-mediated
reduction in PF excitability or action potential duration. These
possible mechanisms were assessed by examining the effect of WIN
55212-2 on fiber volleys recorded in stimulated bundles of PFs.
Slices containing long tracts of PFs were prepared by horizontal
sectioning of P15-16 cerebella. Two recording electrodes were placed
along the path of the stimulated axons so that fiber volley velocity
could be measured (Fig. 2A). Because perhaps 40% of the
PF in the rat is made up of synaptic varicosities (Ito 1984), it is theoretically possible that synaptic
Ca2+ channels might contribute to the fiber volley. To
isolate the fiber volley from any potential contribution from
Ca2+ channels or field excitatory postsynaptic potentials
(EPSPs), all experiments were performed in external saline containing
200 µM CdCl2, which was determined to be a saturating
concentration (data not shown). WIN 55212-2 had a variable effect on
fiber volley amplitude. In about half of the experiments, 5.0 µM WIN
55212-2 had no effect. In the remaining experiments, it caused a small decrease in amplitude (Fig. 2B). Fiber volley velocity
and kinetics were never affected, even in those recordings in which
there was a decrease in fiber volley amplitude. On average, the effect
of WIN 55212-2 on fiber volley amplitude was very small (5.0 µM: 93 ± 2% of baseline, n = 9; see Fig.
2C). But this was significantly different
(P = 0.017) from switching the external saline
reservoir to a second bottle of control saline (104 ± 3%,
n = 5). S(
)-WIN 55212-3, the inactive enantiomer
of R(+)-WIN 55212-2, had no effect (105 ± 6%,
n = 3), suggesting that the small effect of WIN
55212-2 was receptor mediated. However, this small and inconsistent
effect on fiber volley amplitude cannot account for the very large and extremely consistent PF EPSC results.
CF-evoked EPSCs.
CF stimulation elicited characteristically large, all-or-nothing
EPSCs that exhibited paired-pulse depression (Konnerth et al.
1990; Llano et al. 1991
; Perkel et al.
1990
) (Fig. 3, A and B). 5.0 µM WIN
55212-2 produced a moderate and somewhat variable decrease in CF-evoked
EPSC (CF EPSC) amplitude, which tended to recover slightly with
continued agonist application (Fig. 3C). In control
experiments, slices were incubated in 1.0 µM SR 141716 for
30 min
before switching to 5.0 µM WIN 55212-2 + 1.0 µM SR 141716. In the
presence of this CB1 antagonist, WIN 55212-2 had no effect on EPSCs. On
average, 5.0 µM WIN 55212-2 reduced the amplitude of the first CF
EPSC of each pair to 73.7 ± 5.4% of baseline (n = 7) compared with 99.8 ± 1.8% of baseline with SR 141716 pretreatment (n = 6; P = 0.0029). In
addition, 5.0 µM WIN 55212-2 increased EPSC2/EPSC1 to 109 ± 1.3% of baseline (changed paired-pulse depression from 78.6 ± 1.0% to 85.5 ± 1.7%) compared with 99.9 ± 0.8% of
baseline in controls (P = 0.0002; see Fig. 3D). When recorded concurrently as an internal control, as
before, PF EPSCs were severely reduced by WIN 55212-2 and this
reduction was completely blocked by SR 141716 (data not shown).
Rs was stable and unaffected by WIN 55212-2 in
both test recordings (99.3 ± 1.3% of baseline) and control
recordings with SR 141716 pretreatment (99.6 ± 0.8% of
baseline). Because Gm tended to drift
unpredictably in CF EPSC experiments at
20 mV (see
METHODS), inspection of individual records was
the most reliable way to look for an effect of WIN 55212-2 on
Gm. No effect was seen. On average,
Gm was 97.5 ± 2.6% of baseline in test
recordings and 92.1 ± 2.7% of baseline in control recordings.
mEPSCs
In a further effort to determine the mechanism by which CB1
receptor activation suppresses EPSCs, miniature EPSCs (mEPSCs) were
recorded. mEPSCs result from spontaneous fusion of
neurotransmitter-containing vesicles to the presynaptic terminal
membrane. mEPSCs recorded in Purkinje cells were found by Chen
and Regehr (1997) to be unaffected by externally applied
Cd2+, demonstrating that they are independent of
Ca2+ influx. A change in the
frequency of these spontaneous fusion events is taken to be evidence of
modulation of the synaptic vesicle release machinery downstream from
Ca2+ entry. A change in the amplitude of mEPSCs
is taken to be evidence of postsynaptic modulation. Most mEPSCs
probably reflect spontaneous vesicular release from PFs although some
may result from release from CFs (see DISCUSSION). In pilot
experiments (data not shown), asynchronous vesicular release from CFs
and PFs was induced by stimulating these afferents in external saline
in which CaCl2 was replaced with an equal amount
of SrCl2 (Goda and Stevens 1994
; Lévénès et al. 1998
). There were no
differences between CF-evoked and PF-evoked asynchronous EPSCs that
were sufficient to allow mEPSCs from these sources to be discriminated
from each other.
P10-11 rats were used. mEPSCs were very difficult to record in
Purkinje cells from rats older than P11. At all ages, on first breaking
into whole-cell mode, there was a great amount of noise of up to 15
pA, which probably reflected the activity of intrinsic conductances in
the Purkinje cell. This noise was impossible to distinguish from mEPSCs
with certainty. In very young Purkinje cells, this noise dissipated
rapidly, probably because of perfusion of the dendrites with
Cs+ from the pipette saline. In older cells, it
took much longer for this noise to dissipate. Additionally, as age
increased, the frequency of mEPSCs appeared to decrease, possibly
because of increased dendritic filtering. mEPSCs were detected in TTX
under low-noise recording conditions as very small currents that could be blocked by the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist, NBQX (Fig.
4A). The average baseline
mEPSC frequency and amplitude were 4.8 ± 1.7 Hz and
19.6 ± 1.3 pA (mean ± SE, n = 4), respectively. The
baseline distribution of mEPSCs in one Purkinje cell is plotted in Fig.
4B. A representative experiment is shown in Fig.
4C. After collecting a long, stable baseline, the external
saline reservoir was switched to 5.0 µM WIN 55212-2 for 16 min. There
was absolutely no effect on either mEPSC frequency or amplitude.
Subsequent application of 10 µM NBQX eliminated the mEPSCs,
confirming them to be true AMPA/kainate receptor-mediated events.
|
The combined experiments plotted in Fig. 4, D and
E, show that WIN 55212-2 had no effect. On average
(n = 4), mEPSC frequency and amplitude in 5.0 µM WIN
55212-2 were 103 ± 4.0% and 97.1 ± 3.9% of baseline,
respectively. To address the possibility that WIN-55212-2 might be
altering a subpopulation of mEPSCs, mEPSCs within each recording were
segregated into several bins by amplitude. Each bin was normalized to
its own baseline and then combined with the corresponding bins of the
other experiments. The binwidths used for this analysis were
5 to
10 pA,
10 to
20 pA,
20 to
30 pA,
30 to
50 pA, and
greater than
50 pA. In none of these amplitude ranges did 5.0 µM
WIN 55212-2 have any effect on mEPSC frequency. Recordings were stable;
on average, Rs was 102 ± 0.64% of baseline and
Ri was 112 ± 2.0% of baseline in WIN 55212-2.
It is possibile that no effect was seen on mEPSCs because P10-11 rats
were used whereas P15-19 rats were used in all other PF and CF
experiments. These ages span a period of rapid growth: cannabinoid
binding/mg of cerebellar homogenate (Bmax) was reported to
increase ~2.4-fold between P7 and P14 and another ~1.7-fold between
P14 and P21 (Belue et al. 1994). Therefore the effect of
5.0 µM WIN 55212-2 on EPSCs was reexamined in three Purkinje cells
from P10-11 rats using the same CsSO4-based internal
saline used for mEPSC recordings and the protocol shown in Fig.
2A at Vhold =
30 to
40 mV. The
effect in P10-11 rats was even larger than that seen in P15-19 rats,
perhaps because there was less glial membrane in these young slices to
impede WIN 55212-2 access to the relevant synapses. Results in WIN
55212-2 were as follows (% of baseline): PF EPSC, 10.0 ± 2.8; CF
EPSC1, 67.3 ± 7.1; CF EPSC2/CF EPSC1, 116 ± 8.3;
Rs 104 ± 0.5; Gm 100.7 ± 8.1. In
conclusion, no evidence was found that CB1 receptor activation
modulates the synaptic vesicle release machinery downstream of
Ca2+ entry at excitatory synapses impinging on Purkinje
cells. Furthermore, the absence of any effect on mEPSC amplitude
suggests that CB1 does not act postsynaptically at these synapses.
IPSCs
Purkinje cells receive powerful inhibitory input that comes mainly
from local GABAergic interneurons. There are about six basket cells and
17 stellate cells/Purkinje cell in cat (reviewed in Ito
1984). Another minor inhibitory input to Purkinje cell dendrites comes from Purkinje cell axon collaterals (reviewed in
Ito 1984
). Basket cells, stellate cells, and Purkinje
cells fire action potentials spontaneously (Llano and
Gershenfeld 1993
; Llinás and Sugimori
1980
; Pouzat and Hestrin 1997
). Basket and stellate cells express CB1 mRNA, but Purkinje cells do not
(Mailleux and Vanderhaeghen 1992
; Matsuda et al.
1993
). CB1 immunoreactivity colocalizes with the pericellular
basket and is especially intense on the pinceau (Tsou et al.
1998
). To determine the functional consequences of this
localization, the effect of CB1 activation on inhibitory currents was
examined. Purkinje cells were whole-cell patched and held at
70 mV
with a high [Cl
] pipette saline that shifted
the GABAA reversal potential to about 0 mV. Under
these conditions, GABAA activation produced inward currents. Spontaneous inhibitory currents were then recorded and
analyzed in much the same way as mEPSCs. Recordings of spontaneous IPSCs were similar to those reported previously (Farrant and
Cull-Candy 1991
; Konnerth et al. 1990
;
Vincent et al. 1992
). Spontaneous IPSCs were numerous
and much larger than mEPSCs. Average baseline IPSC frequency and
amplitude were 7.1 ± 2.3 Hz and
362 ± 31 pA (n = 3 cells). Run-down of IPSC amplitude over time was
prominent (see also Kano 1992
), especially after 35-50
min of recording, and appeared to correlate positively with the size of
the pipette tip. IPSCs fell into two classes, action
potential-dependent and mIPSCs (Fig.
5A). Very large TTX-sensitive,
action potential-dependent IPSCs reflect spontaneously arising action
potentials in basket and stellate cells. Most action
potential-dependent IPSCs were between
1 and
5 nA in amplitude and
were sometimes large enough to escape space clamp and evoke a
postsynaptic action potential. Smaller TTX-insensitive mIPSCs reflect
spontaneous vesicle fusion events at inhibitory terminals. mIPSCs were
generally less than
1 nA in amplitude. mIPSC frequency was much
greater than that of action potential-dependent IPSCs.
|
To get an overall view of the effect of CB1 activation on IPSCs, experiments were conducted as follows (Fig. 5, A-C). After recording a baseline of mixed large and small IPSCs, WIN 55212-2 was added to the recording chamber. In 5.0 µM WIN 55212-2, the large IPSCs disappeared and total spontaneous IPSC frequency decreased by more than half. This effect was completely reversed by subsequent application of 1.0 µM SR 141716 + 5.0 µM WIN 55212-2, indicating that it was CB1 receptor-mediated. To identify which IPSCs were dependent on spontaneous action potentials and which were mIPSCs, 1.0 µM TTX (or 1.0 µM TTX + 1.0 µM SR 141716 in repeated experiments) was then applied. In TTX, the large IPSCs again disappeared whereas small IPSCs remained. On the whole, TTX produced only a small decrease in the total frequency of IPSCs, indicating that mIPSCs far outnumbered action potential-dependent IPSCs. Because WIN 55212-2 produced a much larger decrease in total IPSC frequency than TTX, WIN 55212-2 must have substantially reduced mIPSC frequency. Bicuculline (20 µM) eliminated the IPSCs, proving them to be GABAA-mediated. Because spontaneously occurring IPSCs were studied, and because the cannabinoid-induced elimination of large TTX-sensitive IPSCs was observed on a background of unaffected mIPSC amplitude, it could not be determined whether action potential-dependent IPSCs were reduced in amplitude to within the range of mIPSCs, or whether they were eliminated altogether. Action potential-dependent IPSC amplitude must have been reduced by >75% because they were sometimes more than four-fold larger than the largest mIPSCs. This experiment was performed three times. In two recordings, 2 mM kynurenate was present throughout the experiment. All three results were consistent.
mIPSCs
In the previous experiment, the effects of WIN 55212-2 on a
mixture of action potential-dependent IPSCs and mIPSCs were studied. To study mIPSCs in isolation, we recorded from Purkinje cells in the
presence of 1.0 µM TTX and 2.0 mM kynurenate (Fig.
6A). Average baseline
frequency and amplitude were 3.1 ± 0.4 Hz and 128 ± 9 pA,
respectively. A typical baseline frequency versus amplitude
distribution is shown in Fig. 6B. The great majority of
mIPSCs were smaller than
200 pA, though some were larger than
700
pA. After recording a stable baseline, either more control saline, 5.0 µM WIN 55212-2, or 100 µM CdCl2 was
bath-applied (Fig. 6, C and D). In controls,
mIPSC frequency spontaneously increased late in two of four recordings,
driving the average change to 126 ± 15.6% of baseline
(n = 4). This increase was not accompanied by any
change in recording stability, and all events detected as mIPSCs were
eliminated by 20 µM bicuculline. In contrast, 5.0 µM WIN 55212-2 produced a large and rapid decrease in mIPSC frequency to 52.3 ± 8.6% of baseline (n = 4), which was significantly
different from control (P = 0.018). 100 µM
CdCl2 had no effect on IPSC frequency (101 ± 4.4%; n = 3; P = 0.26), suggesting
that WIN 55212-2-mediated suppression of mIPSC frequency is not
mediated by Ca2+ channel inhibition (Fig.
6C). Because mIPSC amplitude tended to run down over time,
it was important to be sure that any decrease in amplitude was
statistically significant. Neither WIN 55212-2 or
CdCl2 had a significant effect on IPSC amplitude
compared with control (Fig. 6D). Rs
remained constant (control, 101 ± 0.8%; WIN 55212-2, 101 ± 1.0%; CdCl2, 96.3 ± 1.1%).
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DISCUSSION |
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CB1 activation was found to presynaptically suppress all major
classes of synaptic input to cerebellar Purkinje cells. Cannabinoid suppression of PF-evoked EPSCs was large, consistent, and
dose-dependent. Several possible mechanisms were investigated.
Cannabinoid-induced decreases in fiber volley amplitude were too small
and inconsistent to account for the effect on PF EPSCs. In addition,
fiber volley velocity and kinetics were completely unaffected,
suggesting that voltage-gated Na+ channels and
delayed rectifier K+ channels were not involved.
If Na+ channels had been inhibited, a slowing of
action potential velocity and kinetics would have been expected to
result. A significant increase in delayed rectifier
K+ channel activity would also have been expected
to change fiber volley kinetics. It is more likely that the observed
cannabinoid-induced decrease in excitability is caused by a small
increase in resting KIR activity. If the increase
in KIR were small enough, it might decrease
axonal excitability without affecting kinetics and velocity, and the
decrease in fiber volley amplitude would reflect a proportional decrease in the number of activated PFs. CB1 activation has been shown
to increase KIR channel activity in other systems
(Garcia et al. 1998; Henry and Chavkin
1995
)
The fact that WIN 55212-2 had no effect on mEPSC amplitude suggests
that CB1 receptor activation suppresses EPSCs recorded in Purkinje
cells presynaptically. This conclusion is supported by the work of
others who found no CB1 mRNA in Purkinje cells (Mailleux and
Vanderhaeghen 1992; Matsuda et al. 1993
). It
should be noted that mEPSCs recorded in Purkinje cells could be the
result of spontaneous release at either granule cell axon or CF
terminals. There is no reliable way to discriminate between them.
However, because the ratio of granule cell synapses to CF synapses in
the Purkinje cell is on the order of 150:1 (reviewed in Strata
and Rossi 1998
), it is generally assumed that most mEPSCs
reflect granule cell axon terminal release. However, because CF
synapses tend to be closer to the Purkinje cell body than most PF
synapses, the amplitude of CF mEPSCs may tend to be larger than that of PFs, making them more likely to be detected by somatic whole-cell recording. In contrast, PF, single-vesicle release events occurring in
the tertiary branches of distal dendrites may often be attenuated by
dendritic filtering to the point of undetectability. Release probability after an action potential is unusually high in CFs (Silver et al. 1998
); however, it is not known whether
spontaneous release probability in the absence of action potentials is
larger at CF terminals than at PF terminals. Thus while most mEPSCs
recorded in Purkinje cells result from PF events, it is possible that a measurable fraction comes from CF terminals as well.
To summarize the CB1 effects on PF EPSCs, receptor activation
greatly suppressed PF EPSC amplitude while producing only a weak and
inconsistent decrease in PF excitability and no change in postsynaptic
sensitivity or membrane properties. We suggest, by elimination, that
CB1 receptor activation inhibits neurotransmitter release at PFs by
inhibiting Ca2+ channels and/or increasing the
activity of K+ channels at the axon terminal. CB1
activation was previously reported to decrease
Ca2+ channel activity and increase
K+ channel activity; however these effects were
evaluated in somata and not at axon terminals. K+
channel modulation is not well established to be a general mechanism of
modulation of release at vertebrate synapses (Miller
1998). Moreover, presynaptic modulation of PF-Purkinje cell
transmission via GABAB or adenosine A1 receptors
does not appear to involve presynaptic K+
channels (Dittman and Regehr 1996
; Wu and Saggau
1997
). Therefore inhibition of axonal Ca channels seems the
more likely mechanism of CB1-mediated suppression of PF EPSC amplitude.
These results differ in some ways from those of
Lévénès et al. (1998). 1 µM WIN
55212-2 reduced PF EPSC amplitude to 28.8% of baseline in the present
experiments, but only to 44.4% of baseline in their experiments. In
addition, although we report an average 7% decrease in fiber volley
amplitude, Lévénès et al. (1998)
saw
no change, although this difference is minor. More important, although
we observed no change in mEPSC frequency,
Lévénès et al. (1998)
reported a 23%
decrease. However, basal mEPSC frequency was extremely low in their
research (0.26 Hz at P15-21) compared with our work (~4.8 Hz at
P10-11) and the work of others also recording from Purkinje cells in
acute slices of rat cerebellum [3-5 Hz at P9-14 (Dittman and
Regehr 1996
), 5-8 Hz at P9-15 (Chen and Regehr
1997
), and ~2 Hz at P10 (Barbour 1993
)]. This
discrepancy, which is probably caused by their use of older Purkinje
cells, makes the mEPSC results of Lévénès et
al. (1998)
difficult to compare with the present findings.
CF EPSCs were suppressed by CB1 receptor activation, but to a lesser extent and with much more variability than at PF synapses. This is the first report of cannabinoid-induced suppression of this synapse. It is likely that inhibition of CF EPSCs results largely from modulation of axon terminal Ca2+ or K+ channels as indicated for PFs. Even though some component of the mEPSCs recorded in Purkinje cells is probably caused by spontaneous vesicle release from CF terminals, that fraction is likely to be small and indistinguishable from PF mEPSCs. Therefore the results of mEPSC analysis cannot be applied to CFs.
The partial recovery of CF EPSCs from cannabinoid-induced suppression
over time (Fig. 3C) may represent desensitization of CB1 in
high concentrations of agonist. High concentrations of cannabinoids
have occasionally been reported to be less effective than lower doses
in inhibiting evoked synaptic currents in striatum (Szabo et al.
1998) and adenylyl cyclase activity (Bidaut-Russell et
al. 1990
; Glass and Felder 1997
; Howlett
and Fleming 1984
). Desensitization of G-protein-coupled
receptors is a well-established phenomenon (reviewed in
Lefkowitz 1998
). However, because WIN 55212-2 was used
only at 5 µM in CF experiments, it remains to be confirmed that the
recovery seen in CF EPSC amplitude was truly concentration-dependent
and not time-dependent.
This is the first report of cannabinoid modulation of inhibitory
inputs to Purkinje cells. In 5.0 µM WIN 55212-2, spontaneously occurring, action potential-dependent IPSCs were strongly inhibited (>75% decrease in amplitude) and the frequency of mIPSCs was reduced by 48%. mIPSC amplitude was not significantly reduced, suggesting a
purely presynaptic mechanism of suppression. Suppression was reversed
with SR 141716, indicating a CB1-dependent mechanism. This is similar
to the effect of WIN 55212-2 (3 µM) on synapses received by neurons
of the rostral ventrobasal medulla, where a 44% decrease in mIPSC
frequency was observed with no significant alteration in mIPSC
amplitude (Vaughn et al. 1999).
The large inhibition of mIPSC frequency suggests an important role for
modulation of post-Ca2+ entry, vesicle release
processes at the axon terminals of basket and stellate cells. This
modulation may be mediated by lowering [cAMP]i
and reducing PKA activity. A number of studies have implicated increased synaptic terminal cAMP in increased neurotransmitter release
(Chavez-Noriega and Stevens 1994; Chavis et al.
1998
; Chen and Regehr
1997
). However, it seems unlikely that
the 48% decrease in mIPSC frequency is sufficient to explain the
>75% decrease in action potential-dependent IPSC amplitude.
Therefore CB1-activation probably also results in modulation of
voltage-gated channels in the axon terminals of inhibitory
interneurons. It is plausible that such channel modulation, if similar
in magnitude to that responsible for the large suppression of PF EPSC
amplitude, could add to the inhibition of vesicle release processes
after Ca2+ entry and severely reduce, if not
entirely prevent, action potential-dependent neurotransmitter release.
It is also possible that CB1 activation suppresses spontaneous action
potential generation in basket and stellate cells. This could be
mediated by somatic hyperpolarization.
CB1, then, joins several other G protein-coupled neurotransmitter
receptors known to presynaptically inhibit transmitter release at
PF-Purkinje cell synapses, including adenosine A1,
GABAB, and mGluR4 (Pekhletski et al.
1996). The most well-studied of these, by far, are the
adenosine A1 receptor and the GABAB receptor, which have been analyzed in detail using Ca2+
imaging of presynaptic terminals together with recording of EPSCs (Dittman and Regehr 1996
, 1997
). Whereas
both GABAB and adenosine A1 activation result in
suppression of N-type and P/Q-type Ca2+ channel
activity, only GABAB activation results in
decreased mEPSC frequency. mEPSC amplitude was unaffected by both
drugs, showing the locus of activity to be presynaptic.
Much less work has been done on presynaptic modulation of CF,
stellate cell, and basket cell inputs to Purkinje cells.
Takahashi et al. (1995) reported that adenosine and
baclofen were more effective in suppressing PF-evoked EPSCs than
CF-evoked EPSCs. CF-evoked EPSC amplitude has also been reported to
have been suppressed by the selective mGluR2/3 agonist DCG-IV and by
norepinephrine (Konishi et al. 1996
; Mitoma and
Konishi 1996
). Facilitation of inhibitory inputs to Purkinje
cells by serotonin and norepinephrine have been reported to occur via a
presynaptic mechanism involving increased
[cAMP]i and PKA activity (Konishi et al.
1996
; Llano and Gershenfeld 1993
; Mitoma
and Konishi 1996
; Mitoma et al. 1994
). However,
others have reported that serotonin and norepinephrine increase
Purkinje cell sensitivity to GABAA activation
postsynaptically (Cheun and Yeh 1992
; Kerr and
Bishop 1992
; Sessler et al. 1989
; Strahlendorf et al. 1991
). Presynaptic inhibition of
evoked IPSC amplitude and mIPSC frequency by the selective mGluR2/3
agonist DCG-IV has also been reported (reviewed in Konishi et
al. 1996
).
In summary, although CB1 activation reduces neurotransmitter release at all major classes of Purkinje cell synapses, this is not accomplished by a single molecular mechanism. Suppression of action potential-evoked neurotransmitter release at PF excitatory synapses received by the Purkinje cell is mediated largely by modulation of voltage-gated channels in the axon terminal. In contrast, at inhibitory synapses, modulation of vesicle release machinery after presynaptic Ca2+ influx also plays a significant role. All of these actions have the potential to contribute to the cerebellar behavioral symptoms of cannabinoid intoxication.
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ACKNOWLEDGMENTS |
---|
We thank K. Narasimhan, C. Aizenman, C. Hansel, E. Huang, S. Morris, P. Manis, K.-W. Yau, J. Baraban, and R. Huganir for helpful discussion and/or critical reading of the manuscript. We also thank D. Gurfel for skillful technical assistance. SR 141716 was a generous gift from Dr. P. Soubrie of the Sanofi Recherche, Montpellier, France, as was the R(+)-WIN 55212-2 used in some pilot experiments.
This work was supported by a contract from Sanofi Recherche and by grants from the National Institute of Mental Health (MH-01590) and the Develbiss Fund.
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FOOTNOTES |
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Address for reprint requests: D. J. Linden, Dept. of Neuroscience, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205.
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 29 July 1999; accepted in final form 14 October 1999.
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REFERENCES |
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