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The Journal of Neuroscience, January 15, 1999, 19(2):570-577
Cross-Modulation of Synaptic Plasticity by  -Adrenergic and
5-HT1A Receptors in the Rat Basolateral Amygdala
Su-Jane
Wang,
Li-Liang
Cheng, and
Po-Wu
Gean
Department of Pharmacology, College of Medicine, National
Cheng-Kung University, Tainan City, Taiwan 701
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ABSTRACT |
Neurotransmitter receptors are often colocalized in a neuron with
other receptors, and activation of one receptor can either amplify or
antagonize the response to a colocalized receptor. The aim of this
study was to investigate the cross-regulation of synaptic transmission
by  -adrenergic and serotonin 1A (5-HT1A) receptors and to elucidate their underlying mechanisms. Stimulation of
presynaptic -adrenergic receptors with isoproterenol (Iso) in the
basolateral amygdala resulted in a long-lasting increase in synaptic
transmission. This effect was mimicked by forskolin, an activator for
adenylyl cyclase and a cAMP analog. In addition, the effect of
forskolin was blocked by catalytic and regulatory site antagonists for
cAMP-dependent protein kinase (PKA), indicating a PKA-mediated
mechanism. Application of 5-HT depressed the synaptic transmission and
blocked Iso- and forskolin-induced potentiation. The effect of 5-HT was
mimicked by the selective 5-HT1A agonist 8-hydroxy-dipropylaminotetralin and was blocked by the selective 5-HT1A antagonist
1-(2-methoxyphenyl)-4[4-(2-phthalimido)butyl]piperazine, indicating its mediation by 5-HT1A receptors. To
determine the locus of interaction, Sp-cAMPS, a membrane-permeable
activator of PKA, was applied, and the potentiation produced by
Sp-cAMPS was completely blocked in slices pretreated with 5-HT. These
results suggest that the interaction between the intracellular
signaling pathways activated by 5-HT1A and -adrenergic
receptors occurs at a step downstream from cAMP production.
Key words:
serotonin; isoproterenol; cAMP; protein kinase A; calcium
channel; long-term potentiation; amygdala
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INTRODUCTION |
Serotonin 1A
(5-HT1A) receptor belongs to a family of
neurotransmitter receptors that act through G-proteins of the
Gi/Go class to inhibit adenylyl cyclase
(Andrade et al., 1986 ; Taussig et al., 1993). In the amygdala,
application of 5-HT and 5-HT1A receptor agonists caused a
depression of EPSP with no concomitant changes in the resting membrane
potential or neuronal input resistance (Rainnie, 1995; Cheng et al.,
1998). In addition, postsynaptic depolarization evoked by glutamate
receptor agonists was unaltered in the presence of 5-HT, indicating an
effect on the excitatory synapses rather than a change in the
excitability of basolateral amygdala neurons (Cheng et al., 1998). On
the other hand, activation of -adrenergic receptors that are coupled
positively to adenylyl cyclase through Gs proteins induced
long-term enhancement of synaptic transmission in these same neurons
(Huang et al., 1996).
Cross-talk between G-protein-coupled receptors has been demonstrated in
a number of systems. For example, stimulation of  -adrenoceptors in
hippocampal CA1 neurons strongly upregulated the effect of -adrenoceptors on afterhyperpolarzing currents
(IAHP) (Pedarzani and Storm, 1996). Heterologous
regulation has also been reported between GABAB
and -adrenoceptors in the hippocampus in which agonist stimulation
of Gi-linked receptors may upregulate or downregulate -adrenergic responses (Andrade, 1993; Gerber and Gahwiler, 1994). Similarly, in ileal and tracheal smooth muscles, antagonism of M2 muscarinic receptors leads to an increase in the
relaxant potency of agonists (Fernandes et al., 1992). In the
basolateral amygdala, functional 5-HT1A and -adrenergic
receptors are present in the excitatory nerve endings (Huang et al.,
1996; Cheng et al., 1998), which represent a useful system to
investigate cross-talk between these receptors. Therefore, in this
study, we investigated the effect of Gi-linked receptors on
-adrenoceptor-induced synaptic plasticity and elucidated their
underlying mechanisms.
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MATERIALS AND METHODS |
Male Sprague Dawley rats, 4-6 weeks of age, were decapitated,
and their brains were rapidly removed and placed in cold oxygenated artificial CSF (ACSF) solution. Subsequently, the brain was
hemisected and cut transversely posterior to the first branch and
anterior to the last branch of the superior cerebral vein. The
resulting section was glued to the chuck of a Vibroslice tissue slicer
(Campden Instruments, Silbey, UK). Transverse slices of 500 µm
thickness were cut, and the appropriate slices were placed in a beaker
of oxygenated ACSF at room temperature for at least 1 hr before
recording. ACSF solution had the following composition (in
mM): NaCl 117, KCl 4.7, CaCl2 2.5, MgCl2 1.2, NaHCO3 25, NaH2PO4 1.2, and glucose 11. The ACSF was
bubbled with 95% O2 and 5% CO2 and had the pH of 7.4.
A single slice was transferred to the recording chamber, in which it
was held submerged between two nylon nets and maintained at 32 ± 1°C. The chamber consisted of a circular well of a low volume (1-2
ml) and was perfused constantly at a rate of 2-3 ml/min. Intracellular
recording microelectrodes were pulled from 1.0 mm microfiber capillary
tubing on a Flaming-Brown electrode puller (Sutter Instruments, San
Rafael, CA). The electrodes were filled with 4 M potassium
acetate with resistance ranging from 70 to 130 M . For chelating
intracellular Ca2+, the electrodes were filled with
50 mM BAPTA in addition to 3 M potassium
acetate. When BAPTA-containing electrodes were used, loading of the
cells with BAPTA was assayed by the blockade of Ca2+-activated afterhyperpolarization and
spike-frequency accommodation. The microelectrode tips were positioned
into the basolateral subdivision of amygdala (BLA). Monosynaptic EPSPs
were evoked in BLA neurons by electrical stimulation of afferents from
the lateral nucleus of amygdala with a concentric bipolar stimulating
electrode (SNE-100; Kopf Instruments, Bern, Germany). Electrical
stimuli (150 µsec) were delivered at a frequency of 0.05 Hz.
Extracellular recordings of field potentials were obtained using
microelectrodes filled with 3 M NaCl (3-8 M). The
stimulus intensity was adjusted individually for each experiment to
produce field potential amplitude that was 40-50% of the maximal
response. Experimental treatments were not initiated until the response
had been stable for at least 20 min. The strength of synaptic
transmission was quantified by measuring the amplitude of field
potentials. The amplitude of field potential was measured as the
difference between negative peak and the average value of the following
positive peak. Electrical signals were amplified by using an
Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) and recorded
on a Gould 3200 chart recorder. All data were expressed as mean ± SE. Statistical analysis was performed using the Student's
t test, and p < 0.05 was considered statistically significant.
5-HT and forskolin were obtained from Sigma (St. Louis, MO),
and other chemicals were purchased from Research Biochemicals (Natick, MA).
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RESULTS |
PKA mediates isoprotenol-induced synaptic potentiation
Previous studies from this laboratory have demonstrated that
administration of the -adrenergic agonist isoproterenol (Iso) to the BLA neurons resulted in long-term enhancement of
synaptic transmission (Huang et al., 1996). We tested whether
-adrenergic response is mediated by stimulation of adenylyl cyclase,
resulting in an increase in intracellular cAMP by application of
forskolin, a direct activator of adenylyl cyclase. Figure
1A shows that forskolin (25 µM) produced an effect similar to that of Iso. In
seven cells, the amplitude of EPSP was increased to 188% of baseline,
which remained potentiated for at least 30 min after washout of the drug (control, 4.8 ± 0.6 mV; 30 min after treatment with
forskolin, 9.0 ± 0.9 mV, n = 7, p < 0.001). The sustained enhancement of EPSP could be caused by a slow
washout of Iso or forskolin. To examine this possibility, a
-receptor blocker propranolol was applied during the washing period.
In three neurons tested, propranolol (1 µM) did not
affect Iso-induced potentiation (data not shown), suggesting that the
long-term effect was not caused by a continued activation of receptors.
Moreover, extracellular field recordings were made because it could be
maintained for a long period of time. Electrical stimulation of the
lateral nucleus resulted in a biphasic response, presumably a
presynaptic fiber volley followed by a postsynaptic potential. Bath
application of the ionotropic glutamate receptor antagonist
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM)
abolished the latter component, demonstrating that this component was
mediated by excitatory synaptic transmission (Fig.
1B). Figure 1C shows that forskolin
induced a long-term enhancement of field potentials. The amplitude of
field potentials was 191 ± 15% (n = 7) of
control 105 min after application of forskolin. To test the involvement
of cAMP-independent action of forskolin, 1,9-dideoxy-forskolin (25 µM), which has no effect on adenylyl cyclase but does
mimic many cAMP-independent actions of forskolin (Laurenza et al.,
1989), was applied. In four neurons, 1,9-dideoxy-forskolin did not
significantly affect the amplitude of field potentials (104 ± 8%).
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Figure 1.
Long-term potentiation of EPSP induced by
forskolin in BLA neurons. A, The amplitude of EPSP was
plotted as a function of time. Bar denotes period of
application of forskolin (25 µM). Inset
shows superimposed records taken at different times as indicated. The
EPSP was preceded by a transient hyperpolarizing current pulse (0.2 nA,
50 msec) passed through the recording electrode to monitor input
resistance. B, Extracellular recordings of a typical
biphasic potential, demonstrating that the latter event was sensitive
to CNQX (10 µM). C, LTP of extracellularly
recorded potentials induced by forskolin (25 µM).
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As a further test of the involvement of cAMP, the effect of
membrane-permeable cAMP analog Sp-cAMPS (25 µM) on the
EPSP was investigated. Figure
2A shows that
superfusion of Sp-cAMPS produced a reversible depression of EPSP,
presumably caused by an action at adenosine A1 receptors
(Dunwiddie and Hoffer, 1980; Pockett et al., 1993), and no LTP was seen
after washout of the drug. Sp-cAMPS was therefore coapplied with
3-isobutyl-1-methylxanthine (IBMX, 50 µM), an adenosine
A1 receptor antagonist and phosphodiesterase inhibitor.
IBMX alone increased the amplitude of EPSP to 146 ± 10%
(n = 6) of control, which was completely reversible on
washout of the IBMX. As shown in Figure 2B,
concurrent application of Sp-cAMPS and IBMX did not result in a
depression such as that caused by Sp-cAMPS alone. By contrast, they
induced a long-term enhancement of EPSP (177 ± 15% of control;
n = 7; p < 0.001).
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Figure 2.
Effect of Sp-cAMPS on the EPSP. A,
Application of Sp-cAMPS (25 µM) caused a depression of
EPSP that returned to control level after washout of the drug without
initiating LTP. B, The depression caused by Sp-cAMPS was
prevented by IBMX (50 µM) and, in the presence of IBMX,
Sp-cAMPS induced LTP.
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Finally, a catalytic site antagonist for PKA was applied to confirm the
involvement of PKA in the action of Iso. In these experiments, slices
were presoaked in 1 µM KT 5720 for at least 1 hr
before being transferred to the recording chamber where the drug was
maintained at the same concentration. Figure
3 shows that Iso-induced potentiation was
completely blocked in KT 5720-pretreated slices. In control slices, the
EPSP amplitude was increased to 194% of baseline by Iso (15 µM) (EPSP amplitude was 6.7 ± 0.6 mV before and
13.0 ± 0.9 mV 30 min after treatment with Iso, n = 7), whereas in KT 5720-pretreated slices the EPSP amplitude was
6.0 ± 1.3 mV before and 6.0 ± 1.4 mV (n = 7) in the presence of Iso. The difference in the effect of Iso between
two groups was statistically significant (p < 0.01, unpaired t test). Similar result was obtained when
slices were preincubated with 25 µM Rp-cAMPS, a PKA
regulatory site antagonist (EPSP amplitude was 6.2 ± 0.9 mV
before and 7.0 ± 1.0 mV in the presence of Iso, n = 6) (Fig. 3).
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Figure 3.
Blockade of Iso-induced potentiation by PKA
inhibitors. Slices were incubated for at least 1 hr in 25 µM Rp-cAMPS or 1 µM KT 5720 before being
transferred to the recording chamber where the same concentration of
drugs was maintained. Iso-induced potentiation normally observed in
control slices was blocked in the Rp-cAMPS- or KT 5720-treated
slices.
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Blockade of Iso- and forskolin-induced potentiation by 5-HT
We examined the influence of 5-HT on Iso-induced potentiation
because a subtype of 5-HT receptors (5-HT1A) has
been shown to reduce forskolin-stimulated cAMP formation in the
hippocampus (De Vivo and Maayani, 1986). Figure
4A shows that
superfusion of 5-HT (10 µM) depressed the EPSP amplitude
to 52% of control (control, 8.8 ± 1.4 mV; in the presence of
5-HT, 4.6 ± 0.5 mV, n = 6). Iso was then added to
the bath but failed to enhance the EPSP. In the presence of 5-HT, the
EPSP amplitude in Iso remained 100 ± 10% (n = 6), which was significantly different from those in the absence of 5-HT
(194 ± 13%; n = 7; p < 0.001).
Similarly, in the presence of 5-HT, forskolin also failed to affect the
EPSP (103 ± 7%; n = 6; Fig.
4B).
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Figure 4.
Antagonism of Iso- and forskolin-induced
potentiation by 5-HT. Application of 5-HT (10 µM) reduced
synaptic responses. Subsequent addition of Iso (15 µM)
(A) or forskolin (25 µM)
(B) in the presence of 5-HT failed to potentiate
the EPSP.
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We sought to clarify which 5-HT receptor subtype was involved in the
inhibition of synaptic transmission and forskolin-induced potentiation.
Figure 5A shows that the
depression of EPSP produced by 5-HT was blocked by the
selective 5-HT1A receptor antagonist 1-(2-methoxyphenyl)-4[4-(2-phthalimido)butyl]piperazine (NAN-190, 2 µM). In the presence of 5-HT and NAN-190, forskolin
potentiated the EPSP amplitude to 196 ± 13% (n = 6) of control, which was not significantly different from that without
5-HT + NAN-190. Furthermore, the selective 5-HT1A receptor
agonist 8-hydroxy-dipropylaminotetralin (8-OH-DPAT, 10 µM) reversibly depressed the EPSP amplitude to 53 ± 5% of baseline and, in the presence of 8-OH-DPAT, forskolin failed to
affect the EPSP amplitude (103 ± 10%; n = 6;
Fig. 5B).
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Figure 5.
The effect of 5-HT is blocked by the selective
5-HT1A receptor antagonist and is mimicked by the selective
5-HT1A agonist. A, In the presence of
NAN-190 (2 µM), 5-HT did not affect the EPSP
significantly. Subsequent application of forskolin (25 µM) induced potentiation. Inset shows
superimposed traces taken at the time points indicated.
B, Application of 8-OH-DPAT (10 µM)
mimicked 5-HT in reducing synaptic responses. Subsequent addition of
forskolin (25 µM) in the presence of 8-OH-DPAT failed to
potentiate the EPSP.
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Cross-talk mechanism between 5-HT and -adrenergic receptors
We intended to identify the level in the second messenger cascade
at which the cross-talk between 5-HT and -adrenergic receptors took
place. If the cross-talk occurred at the enzyme adenylyl cyclase, then
5-HT could not block the effect of cAMP analog. On the other hand, if
the cross-talk took place at the level downstream from cAMP production,
then 5-HT should be able to inhibit the effect of cAMP analog. These
experiments were performed in the presence of IBMX to prevent the
degradation of cAMP analog and to block adenosine A1
receptors. Figure 6 shows that Sp-cAMPS + IBMX no longer induced potentiation in slices pretreated with 5-HT;
EPSP amplitudes were 10.2 ± 0.6 mV for control and 9.6 ± 0.6 mV (n = 6) 40 min after washout of Sp-cAMPS + IBMX.
These results indicate that the action is downstream of cAMP
production; likely at the Ca2+ channels or the
release processes.
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Figure 6.
Antagonism of Sp-cAMPS-induced LTP by 5-HT.
Application of 5-HT (10 µM) depressed the EPSP.
Subsequent application of Sp-cAMPS (25 µM) + IBMX (50 µM) failed to potentiate the EPSP.
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Thus, it is possible that 5-HT may block Iso-induced potentiation by
inhibiting Ca2+ influx. This hypothesis is supported
by our recent report showing that activation of 5-HT1A
receptors in the amygdala depressed synaptic transmission primarily by
inhibiting presynaptic Ca2+ channels (Cheng et al.,
1998). If this is the case, we predicted that (1) buffering
intraterminal Ca2+ with chelator should be able to
block Iso-induced potentiation; (2) 5-HT should be able to depress
synaptic transmission in forskolin-treated slices because its action is
downstream of cAMP production; and (3) neurotransmitters that have been
shown to inhibit Ca2+ channels are expected to
reduce forskolin-induced potentiation too.
First, as demonstrated in Figure
7A, application of
membrane-permeant Ca2+ chelator BAPTA-AM, which
concentration dependently decreased the amplitude of EPSP, virtually
abolished Iso-induced potentiation. To differentiate presynaptic or
postsynaptic sites of action, we loaded the recorded postsynaptic
neuron with BAPTA salt. After impalement, the cells were allowed to
stabilize for at least 30 min to allow the cell to fill with BAPTA,
which was manifested by blockade of slow afterhyperpolarization.
Baseline responses were then obtained for a further 10 min before
superfusing Iso. As shown in Figure 7B, Iso still induced a
potentiation under this condition (control, 5.2 ± 0.5 mV; 25 min
after treatment with 15 µM Iso, 13.0 ± 1.5 mV,
n = 6, p < 0.001), suggesting a rise
in intraterminal Ca2+ is required for the action of
Iso.
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Figure 7.
A rise in intraterminal Ca2+ is
required for the Iso-induced potentiation. A,
Superfusion of BAPTA-AM concentration-dependently depressed the EPSP
and abolished Iso-induced potentiation. The amplitude of EPSP was
plotted against time. Bars denote the periods of
delivery of BAPTA-AM and 15 µM Iso. B, The
effect of Iso was not affected by intracellular BAPTA. Electrodes were
filled with BAPTA (50 mM) as described in Materials and
Methods.
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Second, as shown in Figure 8, when
forskolin (50 µM) was superfused into the bath, field
potentials increased gradually. Subsequent addition of 5-HT (30 µM) reduced the slope of field potentials by 61 ± 2% (n = 6), not significantly different from that
observed without forskolin pretreatment (63 ± 6%;
n = 6). Thus, PKA pretreatment did not affect the
action of 5-HT. Thirdly, we examined the effects of activation of
adenosine A1 and GABAB receptors, two receptors known to inhibit Ca2+ channels (Pfrieger et al.,
1994; Wu and Saggau, 1994, 1995) on the forskolin-induced potentiation.
Superfusion of N6-cyclopentyladenosine
(CPA, 0.5 µM), an adenosine A1 receptor agonist, depressed the EPSP amplitude to 62 ± 6% of baseline
(n = 6). However, it can be seen from Figure
9, the effect of forskolin (25 µM) was completely blocked in the presence of CPA.
Similar experiments were performed for baclofen, a GABAB
receptor agonist. Figure 9 shows that application of baclofen (5 µM) nearly abolished the EPSP. Subsequent addition of
forskolin (25 µM) no longer induced potentiation.
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Figure 8.
Forskolin pretreatment did not affect the action
of 5-HT. As forskolin (50 µM) was perfused into the bath,
the amplitude of field potential gradually increased. Thirty minutes
after application of forskolin, 5-HT (30 µM) was added,
which still exerted an average of 61 ± 2% inhibition.
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Figure 9.
Antagonistic effect of 5-HT on the
forskolin-induced LTP is mimicked by adenosine A1 or
GABAB receptor agonists. Application of CPA (0.5 µM) or baclofen (5 µM) depressed the EPSP.
Subsequent addition of forskolin (25 µM) in the presence
of these agonists failed to potentiate the EPSP.
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DISCUSSION |
In the present study, we intend to accomplish two goals. First, we
wish to identify the intracellular mechanism by which Iso produces
long-term enhancement of EPSP. Second, we wish to illuminate any
convergence between the 5-HT-induced EPSP inhibition and Iso-induced enhancement.
PKA-mediated synaptic enhancement
Our previous report has shown that when Iso was perfused into the
bath, the amplitude of EPSP began to increase in 2-3 min. The Iso
effect was long-lasting and was blocked by the antagonist propranolol;
besides, it did not affect the resting membrane potential or neuronal
input resistance (Huang et al., 1996). The long-latency of Iso action
and its sustained effect suggested that a chain of biochemical steps
involving second messengers might be the mechanism leading to the
enhancement. In the present study, we have demonstrated that forskolin
but not 1,9-dideoxy-forskolin mimics the effect of Iso. Similarly,
activation of PKA by Sp-cAMPS results in a potentiation.
Furthermore, the effect of forskolin was blocked by KT 5720 or
Rp-cAMPS, the respective PKA catalytic and regulatory site inhibitors.
These results suggest that PKA mediates the enhancement of EPSP by Iso
in the amygdala.
Iso-induced potentiation was blocked in Ca2+-free
solution or by specific P/Q type Ca2+ channel
blockers (Huang et al., 1996), suggesting that potentiation required
Ca2+ entry into presynaptic terminals. Of the known
adenylyl cyclases, type 1 calmodulin-sensitive adenylyl cyclase (AC1)
is stimulated directly by Ca2+ and is
synergistically stimulated by combination of Ca2+
and receptor activation. In view of the fact that AC1 is neurospecific, expressed in the amygdala (Xia et al., 1991), and is important for
synaptic plasticity (Mons and Cooper, 1995; Xia and Storm, 1997; Storm
et al., 1998; Villacres et al., 1998), it is hypothesized that entry of
Ca2+ into the presynaptic terminal activates AC1,
resulting in an increase in intracellular cAMP and activation of PKA,
which subsequently phosphorylates Ca2+ channels and
causes a persistent increase in glutamate release.
PKA-mediated synaptic potentiation has been described in several brain
areas, including hippocampal mossy fiber-CA3 synapses and cerebellar
parallel fiber-Purkinje cell synapses (Huang et al., 1994; Weisskopf
et al., 1994; Salin et al., 1996). It is suggested that modulation of
these synapses by PKA occurs via presynaptic mechanisms that do not
alter Ca2+ entry; PKA may directly modulate the
secretory processes, or the silent release sites are activated by PKA
(Trudeau et al., 1996; Chen and Regehr, 1997; Chavis et al., 1998).
This study is one of a few examples in which modulation of presynaptic
Ca2+ channels contributes to synaptic enhancement
(McGehee et al., 1995).
Blockade of -adrenergic response by 5-HT1A
5-HT1A receptors are representative of a family of
neurotransmitter receptors known to couple functionally to
Gi/o proteins to inhibit adenylyl cyclase (Andrade et al.,
1986). We have shown that activation of presynaptic 5-HT1A
receptors reduces transmitter release in the basolateral amygdala
(Cheng et al., 1998). Previous biochemical studies have shown that
activation of Gi-linked receptors inhibited
forskolin-stimulated adenylyl cyclase (Cooper et al., 1980; Fredholm et
al., 1985; Taissig et al., 1993) and, on the other hand, enhanced the
ability of -adrenergic receptors to generate cAMP (Tang et al.,
1991). It was suggested that activation of these Gi-linked
receptors led to liberation of  complex, which alone had no
effect on adenylyl cyclase but potentiated Gs -stimulated
adenylyl cyclase II activity. This hypothesis was supported recently by
the electrophysiological studies showing that 5-HT1A,
GABAB, and -adrenergic receptor agonists enhanced -adrenergic-mediated reduction in the IAHP in rat
hippocampal CA1 neurons (Andrade, 1993; Pedarzani and Storm, 1996). In
the present study, we have shown that 5-HT inhibited synaptic
transmission and completely blocked Iso-induced potentiation. Blockade
of Iso-induced potentiation by 5-HT could be prevented by NAN-190,
indicating its mediation by 5-HT1A receptors. Our data
suggest antagonistic interaction between 5-HT and -adrenergic
receptors. Interestingly, Sp-cAMPS alone depressed the EPSP without
inducing potentiation after its washout. Only in the presence of IBMX
did Sp-cAMPS induce LTP. These results suggest that Sp-cAMPS may act as
an adenosine A1 agonist and further support the
antagonistic relationship between adenosine A1 and
-adrenergic receptors. Finally, our results, although in contrast to
the hypothesis suggested by Andrade (1993), are consistent with the
report of Gerber and Gahwiler (1994), who showed that the action of Iso
on IAHP was curtailed significantly by GABAB or
adenosine A1 receptor agonists in rat hippocampal CA3 neurons.
Cross-talk mechanism
In principle, the cross-talk described in the present study might
occur at any steps involving the modulation of -adrenergic receptor,
G-proteins, adenylyl cyclase, cAMP metabolism, PKA, Ca2+ channels, or the release processes. However,
the enhancement of EPSP induced by activation of -receptor, direct
stimulation of adenylyl cyclase by forskolin, or application of cAMP
analog was completely blocked in the presence of 5-HT. These results indicate that the interaction between the intracellular signaling pathways activated by 5-HT and Iso occurs at a step downstream from
cAMP production. Indeed, several lines of evidence support this
hypothesis. First, buffering intraterminal Ca2+ with
membrane-permeant Ca2+ chelator blocked Iso-induced
potentiation. This finding coupled with our previous observation that
5-HT inhibits glutamate release via a presynaptic blockade of
Ca2+ influx suggests that signaling pathways
activated by these two receptors may converge at presynaptic
Ca2+ channels. Second, If 5-HT action depends on
adenylyl cyclase (AC) inhibition, there should be a simple summation of
agonist and AC inhibitor effects. Because forskolin produced a 88%
increase in EPSP amplitude, and 5-HT decreased EPSP amplitude by 48%,
the sum result would be an increase of ~40%. In fact, pretreatment with 5-HT completely blocked the forskolin enhancement. More
importantly, 5-HT depressed the normal and forskolin-enhanced EPSP to
the similar extent (~50%), indicating that 5-HT directly targets
Ca2+ channels. Moreover, an important note from
Figure 8 is that delayed application of 5-HT in forskolin-pretreated
slices depressed the EPSP in a reversible manner. These results
indicate that 5-HT blocks the induction but not the expression of
forskolin-induced potentiation.
Finally, adenosine A1 and GABAB receptor
agonists, which have been shown to depress transmitter release by
inhibiting presynaptic Ca2+ channels (Pfrieger et
al., 1994; Wu and Saggau, 1994, 1995), also blocked forskolin-induced potentiation.
Functional implication
The functional role of cross-talk explored here is not clear.
Because cAMP induces or enhances epileptiform activity (Yokoyama et
al., 1989; Boulton et al., 1993), and elevation of cAMP levels are
measured in amygdala-kindled animals (Mori, 1983), it is expected that
5-HT1A receptors may have physiological consequence of
reducing the response to prolonged excitatory synaptic inputs and act
as an anticonvulsant in the amygdala.
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FOOTNOTES |
Received Aug. 27, 1998; revised Oct. 26, 1998; accepted Oct. 27, 1998.
This study was supported by the National Science Council of Taiwan
(NSC86-2314-B006-002-M10).
Correspondence should be addressed to Dr. Po-Wu Gean at the above address.
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Abstract
Introduction
