Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, California 92037
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ABSTRACT |
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Sullivan, Jane M..
Mechanisms of Cannabinoid-Receptor-Mediated Inhibition of
Synaptic Transmission in Cultured Hippocampal Pyramidal Neurons.
J. Neurophysiol. 82: 1286-1294, 1999.
Cannabinoids, such as marijuana, are known to impair learning and
memory perhaps through their actions in the hippocampus where
cannabinoid receptors are expressed at high density. Although cannabinoid receptor activation decreases glutamatergic synaptic transmission in cultured hippocampal neurons, the mechanisms of this
action are not known. Cannabinoid receptor activation also inhibits
calcium channels that support neurotransmitter release in these cells,
making modulation of these channels a candidate for
cannabinoid-receptor-mediated effects on synaptic transmission. Whole
cell patch-clamp recordings of glutamatergic neurons cultured from the
CA1 and CA3 regions of the hippocampus were used to identify the
mechanisms of the effects of cannabinoids on synaptic transmission. Cannabinoid receptor activation reduced excitatory postsynaptic current
(EPSC) size by ~50% but had no effect on the amplitude of
spontaneous miniature EPSCs (mEPSCs). This reduction in EPSC size was
accompanied by an increase in paired-pulse facilitation measured in low
(1 mM) extracellular calcium and by a decrease in paired-pulse
depression measured in normal (2.5 mM) extracellular calcium. Together,
these results strongly support the hypothesis that cannabinoid receptor
activation decreases EPSC size by reducing release of neurotransmitter
presynaptically while having no effect on postsynaptic sensitivity to
glutamate. Further experiments were done to identify the molecular
mechanisms underlying this cannabinoid-receptor-mediated decrease in
neurotransmitter release. Cannabinoid receptor activation had no effect
on the size of the presynaptic pool of readily releasable
neurotransmitter-filled vesicles, eliminating reduction in pool size as
a mechanism for cannabinoid-receptor-mediated effects. After blockade
of Q- and N-type calcium channels with -agatoxin TK and
-conotoxin GVIA; however, activation of cannabinoid receptors
reduced EPSC size by only 14%. These results indicate that cannabinoid
receptor activation reduces the probability that neurotransmitter will be released in response to an action potential via an inhibition of
presynaptic Q- and N-type calcium channels. This molecular mechanism
most likely contributes to the impairment of learning and memory
produced by cannabinoids and may participate in the analgesic,
antiemetic, and anticonvulsive effects of these drugs as well.
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INTRODUCTION |
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Cannabinoids, the active constituents of
marijuana, have a broad range of potential medical benefits, including
analgesic, antiemetic, and anticonvulsive effects (Hollister
1984; Howlett 1995
). However, their
well-established impairment of learning and memory (Howlett
1995
; Miller 1984
) reduces their usefulness as
therapeutic agents (Abood and Martin 1992
;
Hollister 1984
; Howlett 1995
). In the
hippocampus, where cannabinoid receptors are highly expressed
(Herkenham et al. 1990
, 1991
; Matsuda
1990
; Tsou et al. 1998
), cannabinoid receptor
activation blocks long-term potentiation of field potentials
(Collins et al. 1994
, 1995
; Devane et al.
1992
; Nowicky et al. 1987
; Stella et al.
1997
; Terranova et al. 1995
). Recent experiments
have shown that cannabinoid-receptor-mediated impairments of
hippocampal long-term potentiation (LTP) and long-term depression (LTD)
are due to a decrease in presynaptic neurotransmitter release
(Misner and Sullivan 1999
). Because hippocampal LTP and LTD are believed to underlie certain forms of learning and memory, identifying the molecular mechanism mediating the decrease in neurotransmitter release produced by cannabinoid receptor activation may provide insights into the mechanism underlying the learning and
memory impairments produced by marijuana. In the present paper, the
effects of cannabinoid receptor activation on synaptic transmission of
hippocampal neurons in culture were studied to elucidate the molecular
mechanisms of cannabinoid action.
Cannabinoid receptor activation inhibits glutamatergic synaptic
transmission in cultured hippocampal neurons through a presynaptic site
of action (Shen and Thayer 1999; Shen et al.
1996
), but the molecular mechanisms mediating these effects are
only partially understood. A clue to these mechanisms of action comes
from the observation that cannabinoid receptor activation also inhibits N- as well as P- and/or Q-type calcium channels in cultured hippocampal neurons (Twitchell et al. 1997
) and in other cellular
systems (Mackie and Hille 1992
; Mackie et al.
1995
; Pan et al. 1996
). Because these calcium
channels are known to support neurotransmitter release in hippocampal
neurons (Reid et al. 1998
; Reuter 1995
; Scholz and Miller 1995
; Takahashi and Momiyama
1993
; Wheeler et al. 1994
), inhibition of these
channels is a candidate mechanism by which cannabinoid receptor
activation reduces glutamatergic transmission. In addition, cannabinoid
receptor activation activates potassium channels (Deadwyler et
al. 1993
; Henry and Chavkin 1995
; Mackie
et al. 1995
), and modulation of these channels also may contribute to cannabinoid-receptor-mediated effects. Finally, cannabinoid receptor activation could decrease neurotransmitter release
by reducing the size of the pool of readily releasable neurotransmitter-filled vesicles.
Using whole cell patch-clamping of cultured rat hippocampal neurons, I have found that cannabinoid-receptor-mediated inhibition of hippocampal excitatory postsynaptic currents (EPSCs) is due to a decrease in the probability that neurotransmitter will be released in response to an action potential, with no accompanying change in the postsynaptic sensitivity to glutamate. This decrease in the probability of release is mediated primarily via an inhibition of both Q- and N-type calcium channels.
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METHODS |
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Culture preparation
Rat hippocampal neurons isolated from the CA1-CA3 regions were
cultured on microislands as described previously (Bekkers and Steven 1991; Furshpan et al. 1976
). Neurons were
plated onto a feeder layer of astrocytes that had been laid down 1-7
days earlier (see Levison and McCarthy 1991
) and grown
in high-glucose (20 mM) medium containing 10% horse serum. Neurons
were grown without mitotic inhibitors and used for recordings only
after a minimum of 21 days in culture because cannabinoid (CB1)
receptors are not predominantly localized along neuronal processes
until cells have been in culture for 3-4 wk (Twitchell et al.
1997
). To test for the involvement of inhibitory G proteins in
CB1-receptor-mediated effects, some cultures were treated for 16-24 h
with 250 ng/ml pertussis toxin [Research Biochemicals International
(RBI), Natwick, MA]; control sister cultures were treated with
heat-inactivated (15 min, 100°C) pertussis toxin. All drug effects
were tested on cells from at least two different cultures.
Electrophysiology
When a single neuron is grown on a small island of permissive substrate, it forms synapses on itself. Such connections are referred to as "autapses." All experiments were performed on isolated autaptic neurons.
Whole cell voltage-clamp recordings from autaptic neurons were carried
out using an Axopatch 1B amplifier (Axon Instruments, Burlingame, CA).
The extracellular solution contained (in mM) 119 NaCl, 5 KCl, 2.5 CaCl2 (except where noted), 1.5 MgCl2, 30 glucose, 20 HEPES, and 0.1 mM picrotoxin (to
block inhibitory GABAergic currents; RBI) plus 1 µM glycine. Bovine
serum albumin (1 mg/ml; Boehringer Mannheim, Indianapolis, IN, and
Sigma, St. Louis, MO) was added to the extracellular solution to reduce
nonspecific binding of WIN55,212-2, and toxins. A low concentration of
tetrodotoxin (TTX; 2 nM; RBI) was included to prevent spontaneous
firing of neurons and to ensure that the large EPSCs evoked in the
older autaptic neurons used for these experiments did not trigger
unwanted action potentials. For those experiments where the
extracellular calcium concentration was altered, the magnesium
concentration was adjusted to maintain a constant concentration of
divalent cations. WIN55,212-2 (RBI), WIN55,212-3 (RBI), SR141716A (a
generous gift from Dr. K. Mackie, University of Washington),
-conotoxin GVIA (Alomone Labs, Jerusalem, Israel), and
-agatoxin
TK (Alomone Labs) were all applied for 0.5-3 min using a puffer
pipette controlled by a picospritzer or, more rarely, by bath
application. WIN55,212-2, WIN55,212-3, and SR141716A solutions were
made up as 10 mM stock solutions in DMSO, stored at
20°C and used
at a final DMSO concentration of
0.01%. For experiments using the
puffer pipette, a vacuum pipette was used to clear the drug away
rapidly. All cells served as their own control for the effects of
WIN55,212-2 on EPSC size before and after SR141716A,
-agatoxin TK,
or
-agatoxin TK plus
-conotoxin GVIA application. Only cells that
showed strong recovery (typically >80%) within 5-10 min of the
termination of WIN55,212-2 application were used for data analysis.
Recording pipettes of 2-5 M were filled with (in mM) 121.5 K
gluconate, 17.5 KCl, 9 NaCl, 1 MgCl2, 10 HEPES, 0.2 EGTA, 2 MgATP, and 0.5 LiGTP. Most experiments were done using
perforated patches; for these experiments amphotericin B (solubilized,
Sigma) was included in the pipette solution. Access resistance was
monitored, and only cells with stable access resistance were included
in the data analysis. The membrane potential was held at
60 mV, and
EPSCs were evoked every 10 s by triggering an unclamped sodium spike with a 0.5-ms depolarizing step. Data were acquired at a rate of
2 kHz. The size of the recorded EPSCs was calculated by integrating the
evoked current to yield a charge value. Calculating the charge value in
this manner yields a direct measure of the amount of neurotransmitter
released while minimizing the effects of cable distortion on currents
generated far from the site of the recording electrode (the soma).
The amplitude and frequency of spontaneous mEPSCs were studied by recording continuously during 10-60 s in the presence of TTX (2 nM). Data for mEPSC analysis were acquired at a rate of 5 kHz. The peak amplitudes of the mEPSCs were measured off-line semiautomatically using an adjustable amplitude threshold. All deflections from baseline that were greater than threshold were detected. Selected events then were examined visually, and any spurious events were rejected manually, whereas any missed events were flagged for inclusion in the mean amplitude and frequency calculations. mEPSC frequencies were calculated by dividing the total number of mEPSC events by the total time sampled.
Effects of WIN55,212-2 on paired-pulse responses were studied by interleaving trials in which a single depolarizing pulse was applied with trials in which two depolarizing pulses were applied at a 45-ms interval. The responses to single pulses were used to generate a template that could be subtracted from the response to the subsequent two-pulse trial to yield an accurate measurement of the response to the second of the pair of pulses. The peak amplitude of the response to the second pulse was averaged over three or more trials and divided by the averaged peak amplitude of the response to the first pulse to give a paired-pulse ratio (either PPF or PPD, depending on the relative sizes of the 2 averaged responses) before and after WIN55,212-2 application.
Hypertonic solution application
The size of the readily releasable pool of vesicles was measured electrophysiologically by application of hypertonic solution (normal extracellular solution plus 500 mM sucrose) to an isolated autaptic neuron using a puffer pipette controlled by a picospritzer. A vacuum pipette was used to clear the hypertonic solution rapidly. The hypertonic solution was applied over the entire island on which the autaptic neuron was located to ensure that the same population of synapses was activated every time the solution was applied. This solution evoked a large initial transient current that declined to a low steady-state level over ~3 s; hypertonic solution was applied for 4-5 s to deplete the readily releasable pool fully. Data were acquired at a rate of 200 Hz. With the broad area of application of hypertonic solution and relatively slow data acquisition rate used for this study, individual mEPSCs were poorly resolved and were only seen superimposed on the larger current. The size of the readily releasable pool was calculated by integrating the current evoked by the hypertonic solution to yield a charge value. To estimate the readily releasable pool size more accurately, we corrected the integral of the current by subtracting away the amount of steady-state exocytosis that occurred during the hypertonic solution flow.
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RESULTS |
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Cannabinoid receptor activation decreases the size of EPSCs in cultured hippocampal neurons
To study the effects of cannabinoid receptor activation on
glutamatergic synaptic transmission, I applied the selective and potent
cannabinoid receptor agonist WIN55,212-2 (Compton et al. 1992; D'Ambra et al. 1992
) to autaptic
hippocampal CA1 and CA3 neurons grown in culture for a minimum of 21 days, when cannabinoid receptors have become predominantly localized to
neurites (Twitchell et al. 1997
). WIN55,212-2 (1 µM)
reduced EPSC size by 51.7 ± 3.2% (mean ± SE;
P < 0.001, paired t-test;
n = 34; Fig. 1).
Washout of the drug reversed these effects and allowed the EPSC to
recover to 96.1 ± 1.6% of its original size. Although every cell
tested responded to WIN55,212-2, the degree of inhibition ranged from 19 to 89%, and there was both cell-to-cell and batch-to-batch variability in the response of neurons to the drug. This variability may explain the discrepancy between the degree of inhibition reported here and the more complete inhibition reported previously (Shen and Thayer 1999
; Shen et al. 1996
).
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Two lines of evidence indicate that WIN55,212-2 inhibition of EPSCs
was mediated by CB1-type cannabinoid receptors (Matsuda et al.
1990). First, the effect of WIN55,212-2 was effectively blocked by the selective CB1 inhibitor, SR141716A
(Rinaldi-Carmona et al. 1994
): after application of 200 nM SR141716A, WIN55,212-2 produced only a 0.8 ± 2.8% reduction
of EPSC size (n = 4; Fig. 1). Second, 1 µM
WIN55,212-3, an inactive form of WIN55,212-2 (Pacheco et al.
1991
), had no significant effect on EPSC size, producing a
2.3 ± 3.5% reduction (n = 6; Fig. 1). This
latter control experiment was important because slightly higher (
3 µM) concentrations of active and inactive WIN55,212 can directly inhibit whole cell currents carried through Ca2+ channels
(Shen and Thayer 1998
). Thus at the 1 µM concentration of WIN55,212-2 that was used for the experiments reported here, inhibition of EPSC size was mediated exclusively via CB1 cannabinoid receptor activation.
The effects of WIN55,212-2 were blocked by treating cultures overnight with pertussis toxin (n = 5), whereas WIN55,212-2-mediated inhibition was unimpaired in cultures treated with heat-inactivated pertussis toxin (n = 4; data not shown). These results strongly suggest that cannabinoid-receptor-mediated inhibition of EPSC size is mediated via a pertussis toxin-sensitive inhibitory G protein.
Effect of cannabinoid receptor activation is not due to a change in postsynaptic responsiveness to glutamate
The WIN55,212-2-mediated decrease in EPSC size could be due to changes in the amount of neurotransmitter released presynaptically and/or to changes in the postsynaptic responsiveness to glutamate. The amplitude of spontaneous mEPSCs was used to monitor changes in postsynaptic responsiveness to glutamate after cannabinoid receptor activation. Because spontaneous mEPSCs are the postsynaptic response to single spontaneously released synaptic vesicles, any change in postsynaptic responsiveness to glutamate should be reflected as a change in the amplitude of mEPSCs.
There was no effect of 1 µM WIN55,212-2 on mEPSC amplitude measured
in the presence of TTX, added to inhibit spontaneous action potential
firing. Mean mEPSC amplitude was 25.6 ± 0.4 pA before drug
application and 25.8 ± 0.5 pA after WIN55,212-2 application (n = 6; Fig.
2A). The lack of effect of
WIN55,212-2 on mEPSC amplitude indicates that there was no
WIN55,212-2-mediated change in the sensitivity of postsynaptic
receptors to glutamate nor in the postsynaptic ionic driving force
(Thompson et al. 1993). These results are in agreement
with a previous report that application of WIN55,212-2 had no
significant effect on the response of cultured hippocampal neurons to
exogenous application of the glutamate receptor agonist kainate
(Shen et al. 1996
) and with a recent report that
application of WIN55,212-2 had no effect on the amplitude of mEPSCs
recorded in cerebellar slices (Levenes et al. 1998
).
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Interestingly, 1 µM WIN55,212-2 did produce a small but significant
(P < 0.05, paired t-test) decrease in the
frequency of mEPSCs, from 8.5 ± 2.0 mEPSCs per second before
application of WIN55,212-2 to 6.8 ± 1.6 per second after
application of drug (n = 6; Fig. 2B). This
decrease in mEPSC frequency also was observed after application of
WIN55,212-2 to cerebellar slices (Levenes et al. 1998)
and to hippocampal slices (Misner and Sullivan 1999
). All of these results, taken together, indicate that
cannabinoid-receptor-mediated reduction in EPSC size is not due to a
change in postsynaptic sensitivity to glutamate and therefore must be
due to a change in the amount of neurotransmitter released presynaptically.
Effect of cannabinoid receptor activation is due to a decrease in the probability of neurotransmitter release
Measuring the relative amplitude of the synaptic response to two
closely spaced action potentials provides a way to estimate the
synaptic probability of release. The size of the response to the second
action potential reflects a balance between two counteracting trends:
the effect of residual calcium entering the presynaptic axon terminal
during the first action potential and the effect of release triggered
by the first action potential (Debanne et al. 1996;
Mennerick and Zorumski 1995
; Zucker
1989
). The elevation of intracellular calcium concentration
will tend to enhance the probability of release, while the effect of
release in response to the first action potential will diminish the
probability of release (perhaps due to the depletion of synaptic
vesicles). If the probability of release of transmitter is high when
the first action potential arrives, then the depressing effect of release will tend to predominate and the second response will be
smaller than the first; this phenomenon is known as paired-pulse depression (PPD). If, in contrast, the probability of release is low
during the first stimulus, then the effects of elevated intracellular
calcium will tend to predominate and the second response will be larger
than the first; this phenomenon is known as paired-pulse facilitation (PPF).
Under normal recording conditions (2.5 mM Ca2+, 1.5 mM Mg2+), the response to the second of a pair of action potentials evoked at a 45-ms interval was only 68 ± 10% the size of the first response (n = 6; Fig. 3). This magnitude of PPD indicates a rather high probability of release in response to the first stimulus. After application of 1 µM WIN55,212-2 under these conditions, the size of EPSCs evoked by the first of the pair of stimuli was reduced by 55.5 ± 7.5% of the predrug control, but the response to the second stimulus was now 101 ± 10% of the size of the first response (P < 0.01, paired t-test). Thus application of WIN55,212-2 decreased PPD (increased the paired-pulse ratio), indicating that WIN55,212-2 decreased the probability of neurotransmitter release.
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When the extracellular calcium concentration was lowered from 2.5 to 1 mM (3 mM Mg2+), the response to the second
stimulus of the pair increased to 117 ± 12% the size of the
first response (n = 6; Fig. 3). This change from PPD to
PPF on lowering extracellular calcium indicates that the probability of
release in response to the first stimulus was reduced in the lower
calcium solution (Creager et al. 1980; Debanne et
al. 1996
; Manabe et al. 1993
; Mennerick
and Zorumski 1995
), allowing the effect of residual calcium to
predominate on the second stimulus. Under these lower calcium
conditions, application of WIN55,212-2 reduced the size of EPSCs
evoked by the first stimulus by 47.5 ± 6.0% but enhanced the
responses evoked by the second stimulus to 135 ± 13% the size of
the first (P < 0.01, paired t-test). Thus
application of WIN55,212-2 in low Ca2+ enhanced
PPF, consistent with its increase of the paired-pulse ratio in 2.5 mM
Ca2+. Taken together, these results indicate that
activation of cannabinoid receptors decreases the probability of
neurotransmitter release by a presynaptic action.
Effect of cannabinoid receptor activation is not due to a reduction in the size of the readily releasable pool of vesicles
One way that cannabinoid receptor activation could decrease the
probability of release is by reducing the number of synaptic vesicles
that are immediately available for release; this population of vesicles
is called the readily releasable pool. The resting probability of
release at a synapse is approximately linearly related to the size of
this pool (Dobrunz and Stevens; 1997; Murthy et
al. 1997
), so a decrease in pool size could account for the decrease in the probability of release observed after the application of WIN55,212-2.
To determine the effects of cannabinoid receptor activation on the size
of this pool, pool size was estimated by applying hypertonic solution
to these neurons and measuring the total charge transferred by synaptic
currents (see METHODS). Application of hypertonic solution
empties the readily releasable pool through a
Ca2+-independent release mechanism
(Rosenmund and Stevens 1996; Stevens and
Tsujimoto 1995
), so the size of the synaptic response to
application of hypertonic solution reflects the number of vesicles in
the pool. Provided there is no change in the postsynaptic sensitivity to glutamate, a change in the charge transferred by the synaptic response to hypertonic application indicates a change in the size of
the readily releasable vesicle pool. Application of 1 µM WIN55,212-2 had no effect on the size of the readily releasable pool, producing a
4.5 ± 4.8% increase in pool size (n = 6; Fig.
4). These results, considered in
conjunction with the lack of effect of WIN55,212-2 on mEPSC amplitude,
indicate that activation of cannabinoid receptors has no effect on the
size of the readily releasable vesicle pool and that some other
mechanism must underlie the WIN55,212-2-mediated decrease in the
probability of neurotransmitter release.
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Cannabinoid receptor activation decreases the probability of release by inhibiting presynaptic N- and Q-type calcium channels mediating neurotransmitter release
Because cannabinoid receptor activation inhibits N- and P- and/or
Q-type channels in hippocampal neurons (Twitchell et al. 1997) and because these channels have been shown to support
transmitter release in these cells (Reid et al. 1998
;
Reuter 1995
; Scholz and Miller 1995
;
Takahashi and Momiyama 1993
; Wheeler et al.
1994
), inhibition of one or more of these subtypes of calcium
channels is a good candidate mechanism for the WIN55,212-2-mediated
reduction of EPSC size. I therefore examined the effects of
WIN55,212-2 on EPSC size before and after selective blockade of each
of these channel subtypes. A decrease in the effects of WIN55,212-2
after selective channel blockade would indicate that this channel
subtype (or subtypes) plays a role in cannabinoid-receptor-mediated effects.
EFFECT OF P-TYPE CALCIUM CHANNEL BLOCKADE ON WIN55,212-2-MEDIATED
REDUCTION OF EPSCS.
To test the possible contribution of P-type calcium channel
inhibition to WIN55,212-2-mediated reduction of EPSCs, I investigated the effects of 25 nM -agatoxin TK, a concentration of toxin that should rapidly and potently block P-type Ca2+
channels (Randall and Tsien 1995
). Application of 25 nM
-agatoxin TK reduced EPSC size by only 7.7 ± 2.1%
(n = 6). The absence of an effect of
-agatoxin TK at
this concentration argues against P-type calcium channels contributing
to release in cultured hippocampal neurons, in agreement with previous
results (Reuter 1995
; Scholz and Miller
1995
; Wheeler et al. 1994
). These results
indicate that inhibition of P-type channels cannot be a mechanism for
the WIN55,2122
mediated reduction of EPSCs.
EFFECT OF Q-TYPE CALCIUM CHANNEL BLOCKADE ON WIN55,212-2-MEDIATED
REDUCTION OF EPSCS.
To test the possible contribution of Q-type calcium channel inhibition
to WIN55,212-2-mediated reduction of EPSCs, the concentration of
-agatoxin TK was increased to 0.5-1 µM. At concentrations >100
nM,
-agatoxin TK selectively blocks not only P-type but also Q-type
Ca2+ channels (Mintz et al. 1992
;
Wheeler et al. 1994
). Application of 0.5-1 µM
-agatoxin TK reduced EPSC size by 57.8 ± 5.7%
(n = 6), an effect comparable with that reported for
hippocampal neurons maintained in culture for
21 days
(Scholz and Miller 1995
). Given that the lower
concentration of this toxin had little effect on EPSC size, the
reduction in EPSC size seen after application of the higher
concentration of toxin must be due to blockade of Q-type channels.
After application of 0.5-1 µM
-agatoxin TK, application of 1 µM
WIN55,212-2- blocked EPSC size by 64.8 ± 7.0% (n = 6; Fig. 5). The
WIN55,212-2-mediated reduction in EPSC size that persists after
blockade of Q-type calcium channels indicates either that WIN55,212-2
does not mediate its effects through these channels, or that
WIN55,212-2 mediates its effects through the combined inhibition of
Q-type channels and another subtype of calcium channel that also
supports release in these neurons.
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EFFECT OF N-TYPE CALCIUM CHANNEL BLOCKADE ON WIN55,212-2-MEDIATED
REDUCTION OF EPSCS.
To test the possible contribution of N-type calcium channel inhibition
to WIN55,212-2-mediated reduction of EPSCs, I investigated the
effects of 1 µM -conotoxin GVIA.
-Conotoxin GVIA selectively blocks N-type Ca2+ channels (Kasai et al.
1987
; Williams et al. 1992
). Application of 1 µM
-conotoxin GVIA reduced EPSC size by 39.8 ± 2.7%
(n = 6), comparable with results reported previously
for cultured hippocampal neurons (Reid et al. 1998
;
Scholz and Miller 1995
). After application of 1 µM
-conotoxin GVIA, application of 1 µM WIN55,212-2 blocked EPSC
size by 62.2 ± 3.0% (n = 6; Fig. 5). The
WIN55,212-2-mediated reduction in EPSC size that persists after
blockade of N-type calcium channels indicates either that WIN55,212-2
does not mediate its effects through these channels or that
WIN55,212-2 mediates its effects through the combined inhibition of
N-type channels and another subtype of calcium channel that also
supports release in these neurons.
EFFECT OF COMBINED Q- AND N-TYPE CALCIUM CHANNEL BLOCKADE ON
WIN55,212-2-MEDIATED REDUCTION OF EPSCS.
To test the possibility that WIN55,212-2 mediates its effects through
the combined inhibition of Q- and N-type calcium channels, the effects
of WIN55,212-2 were tested before and after the combined application
of -agatoxin TK and
-conotoxin GVIA. Combined application of
0.5-1 µM
-agatoxin TK and 1 µM
-conotoxin GVIA reduced EPSC size by 87.2 ± 3.4% (n = 12), an effect
comparable with that reported for hippocampal neurons maintained in
culture for
21 days (Scholz and Miller 1995
). After
application of 0.5-1 µM
-agatoxin TK and 1 µM
-conotoxin
GVIA, application of 1 µM WIN55,212-2 reduced EPSC size by only
13.8 ± 8.5% (n = 12; P < 0.001, paired t-test; Fig. 5). In 7 of 12 neurons, all of which
responded to WIN55,212-2 before coapplication of
-agatoxin TK and
-conotoxin GVIA, WIN55,212-2 had no inhibitory effect at all after
the coapplication of these toxins. Linear regression analysis
(least-squares method) revealed no significant correlation between the
reduction of EPSC size by coapplication of
-agatoxin TK and
-conotoxin GVIA and the reduction of EPSC size by WIN55,212-2 after
toxin application (regression slope =
0.11 ± 0.12;
r2 = 0.08, not significantly different
from 0). Taken together, these results indicate that the
WIN55,212-2-mediated reduction of EPSCs is mediated primarily by
inhibiting both Q- and N-type calcium channels supporting glutamate
release in hippocampal neurons.
EFFECT OF LOW EXTRACELLULAR CALCIUM ON WIN55,212-2-MEDIATED
REDUCTION OF EPSCS.
WIN55,212-2's effects on EPSC size were tested in low
extracellular calcium to rule out the possibility that inhibition of these effects by combined application of -agatoxin TK and
-conotoxin GVIA is due either to inaccurate measurement of small
EPSCs or to a minimal requirement for calcium in the presynaptic
terminal. EPSCs first were recorded in normal (2.5 mM) extracellular
calcium. The extracellular Ca2+ concentration was
then lowered to 0.6-0.75 mM (3.25-3.4 mM Mg2+).
This decrease in extracellular Ca2+ reduced EPSC
size by 89.0 ± 3.4% (n = 4), comparable with the reduction after coapplication of
-agatoxin TK and
-conotoxin GVIA. Under these low-Ca2+ conditions,
application of 1 µM WIN55,212-2 further reduced the EPSC size by
76.0 ± 7.1% in these same cells (data not shown). Simply
reducing intracellular calcium therefore does not inhibit WIN55,212-2's effects on EPSC size. These results indicate that the
inhibition of WIN55,212-2's effects on EPSC size after toxin blockade
of Q- and N-type calcium channels was due to the selective occlusion of
the molecular targets of cannabinoid receptor activation.
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DISCUSSION |
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Activation of cannabinoid receptors by WIN55,212-2 decreased the size of EPSCs by ~50%. This effect was entirely presynaptic: there was no change in the amplitude of spontaneous mEPSCs, indicating that there was no change in postsynaptic responsiveness to glutamate. The cannabinoid-receptor-mediated decrease in EPSC size was accompanied by an increase in PPF (or by a decrease in PPD), indicating that the decrease in EPSC size was due to a decrease in the probability that neurotransmitter would be released in response to an action potential. This decrease in the probability of release was not due to a change in size of the pool of readily releasable vesicles but was instead the result of cannabinoid-receptor-mediated inhibition of presynaptic Q- and N-type calcium currents supporting glutamate release in hippocampal neurons.
Cannabinoid receptor activation recently has been found to inhibit N-
and P- and/or Q-type calcium currents measured at the somas of cultured
rat hippocampal neurons (Shen and Thayer 1998; Twitchell et al. 1997
). These studies did not, however,
establish that this inhibition was responsible for the
cannabinoid-receptor-mediated change in neurotransmitter release. The
finding that WIN55,212-2-mediated inhibition dropped from 50 down to
14% after exposure to Q- and N-type calcium channel blockers is
evidence that inhibition of these channels is the primary mechanism of
cannabinoid-receptor-mediated reduction in the probability of
transmitter release. If there were other major mechanisms underlying
cannabinoid-receptor-mediated inhibition of synaptic transmission that
were downstream of calcium entry, one would not expect to find a lack
of effect of WIN55,212-2 on the majority of cells tested after
blockade of Q- and N-type calcium channels. The formal possibility
remains that cannabinoid receptor activation modulates a protein that
mediates release and is associated selectively with Q- and N-type
calcium channels. However, the degree of inhibition of synaptic
currents is very close to what would be expected given the degree of
cannabinoid-receptor-mediated inhibition of Q- and N-type calcium
currents reported previously (Shen and Thayer 1998
;
Twitchell et al. 1997
). This correlation argues strongly
for Q- and N-type calcium channel inhibition as the primary mechanism
for the observed effects on EPSC size. In addition, the ability of
WIN55,212-2 to inhibit EPSC size normally in low extracellular
calcium, as well as the lack of correlation between the inhibition of
EPSC size by combined application of
-agatoxin TK and
-conotoxin
GVIA and the inhibition of EPSC size by WIN55,212-2 after toxin
blockade, argues against the possibility that
cannabinoid-receptor-mediated inhibition is dependent on a critical
concentration of calcium in the presynaptic terminal. The complex
pattern of effects of WIN55,212-2 on EPSC size after the separate and
combined application of Q- and N-type calcium channel blockers is
likely due to the complicated consequences of inhibiting channels that
share the responsibility of supporting release by controlling the entry
of a second messenger (Ca2+) that exerts its
effect on release with a fourth-order cooperativity (see Scholz
and Miller 1996
; Wheeler et al. 1996
; Wu
and Saggau 1994
for related discussion). As low concentrations
of
-agatoxin TK had little effect on EPSC size and therefore no
obvious effect on neurotransmitter release, inhibition of P-type
calcium channels does not contribute significantly to the effects of
WIN55,212-2.
Although the combined application of -agatoxin TK and
-conotoxin
GVIA completely blocked the effects of WIN55,212-2 in 7 of 12 neurons,
some reduction in EPSC size was still observed in 5 of 12 cells. What
might explain this persistent effect? First, the residual reduction
could be due to incomplete blockade of Q- and N-type channels. A second
possibility is that after blockade of N-, P-, and Q-type calcium
channels, there are still two or more subtypes of calcium channel that
support the small amount of transmitter release that remains. If only
some of those subtypes of channel are susceptible to
cannabinoid-receptor-mediated inhibition, then the persistence of
WIN55,212-2 effects could be explained by a cell-to-cell variability
in the proportion of cannabinoid-receptor-sensitive calcium channels
supporting release after Q- and N-type channel blockade. A third
possibility is that the residual effects of WIN55,212-2 after
-agatoxin TK and
-conotoxin GVIA application were due to
cannabinoid-receptor-mediated activation of potassium channels
(Deadwyler et al. 1993
; Henry and Chavkin
1995
; Mackie et al. 1995
).
Cannabinoid receptor activation previously has been found to activate
inwardly rectifying potassium channels (Henry and Chavkin 1995; Mackie et al. 1995
) and transient A-type
potassium channels in cultured hippocampal neurons (Deadwyler et
al. 1993
). Activation of potassium channels could reduce EPSC
size in three different ways: by shortening the duration of the action
potential and decreasing calcium entry into the presynaptic terminal;
by shunting postsynaptic currents; and by influencing excitability.
However, the lack of effect of cannabinoid receptor activation on
action potential duration and amplitude in cultured hippocampal neurons
(Shen et al. 1996
) suggests that changes in A-type
potassium currents do not play a role in WIN55,212-2-mediated effects
in these cells. A cannabinoid-receptor-mediated increase in the
inwardly rectifying potassium channel (Henry and Chavkin
1995
; Mackie et al. 1995
) could reduce the size
of EPSCs by shunting synaptic current, but the absence of a change in
mEPSC amplitude (shown here; Levenes et al. 1998
), as
well as the lack of effect of WIN55,212-2 on resting membrane
potential or action potential threshold (Shen et al.
1996
), argues against this possibility. Cannabinoid-mediated changes in spike firing patterns may contribute to marijuana's effects
in vivo but would not be expected to contribute to the effects observed
here in cells under voltage-clamp control. Assessing the contribution
of these potential changes in excitability will require further
experiments beyond the scope of this study. Finally, it remains
possible that cannabinoids modulate potassium channels that are
expressed in vivo, but not in the cultured cells used for the
experiments described here. In summary, cannabinoid-receptor-mediated reduction of EPSCs is due primarily to inhibition of N- and Q-type calcium channels; inhibition of other calcium channels and perhaps activation of potassium channels also may play a role in these effects,
albeit a less significant one.
The observation that WIN55,212-2 reduced the frequency of mEPSCs
suggests that cannabinoid receptor activation has effects in addition
to its inhibition of calcium channels. A decrease in mEPSC frequency
previously has been observed in response to several other agonists of
presynaptic receptors that inhibit synaptic transmission, such as
adenosine and baclofen (Scanziani et al. 1992;
Thompson et al. 1993
). Because concentrations of calcium channel blockers sufficient to abolish all evoked transmitter release
have no effect on mEPSC frequency, changes in mEPSC frequency are not
likely to be the result of changes in Ca2+ influx
at resting membrane potentials (Scanziani et al. 1992
). The cannabinoid-receptor-mediated decrease in mEPSC frequency therefore may reflect a direct effect on proteins involved in vesicular
release. These effects may contribute to the residual inhibition of
release after combined application of
-agatoxin TK and
-conotoxin GVIA.
What is the mechanism of this cannabinoid-receptor-mediated inhibition
of calcium channels? Almost certainly it occurs via activation of a
pertussis-toxin-sensitive inhibitory G protein (Howlett
1995). Pertussis toxin treatment blocked the effects of
cannabinoid receptor activation on inhibition of calcium channels (Mackie and Hille 1992
; Mackie et al.
1995
; Pan et al. 1996
; Twitchell et al.
1997
) and activation of potassium channels (Deadwyler et al. 1993
; Mackie et al. 1995
). Pertussis toxin
treatment previously was found to block WIN55,212-2-mediated
inhibition of synaptic transmission in cultured hippocampal neurons
(Shen et al. 1996
), a result confirmed here by the
finding that pertussis toxin treatment blocked the effects of
cannabinoid receptor activation on EPSC size. Taken together, these
data provide strong evidence supporting activation of an inhibitory G
protein as the mechanism for cannabinoid-receptor-mediated calcium
channel inhibition and consequent reduction in the probability of
neurotransmitter release. Inhibition of neurotransmitter release is
likely to play a role in the learning and memory deficits produced by
marijuana and also may underlie some of the potential therapeutic benefits of marijuana.
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ACKNOWLEDGMENTS |
---|
I am indebted to Dr. C. F. Stevens for providing generous support. I also thank Dr. A. K. McAllister and Dr. A. M. Zador for critical reading of the manuscript, Dr. E. P. Huang for editorial assistance, M. A. Pilla for excellent technical assistance, and Dr. K. Mackie for a gift of SR141716A.
This work was supported by grants from the National Alliance for Research on Schizophrenia and Depression and the National Institute on Drug Abuse (DA-11736).
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FOOTNOTES |
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Address for reprint requests: Molecular Neurobiology Laboratory, The Salk Institute, 10010 N. Torrey Pines Rd., La Jolla, CA 92037.
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 10 February 1999; accepted in final form 21 May 1999.
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REFERENCES |
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