Department of Neurobiology, University of Alabama, Birmingham, Alabama 35294
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ABSTRACT |
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Zhou, Fu-Ming and John J. Hablitz. Activation of Serotonin Receptors Modulates Synaptic Transmission in Rat Cerebral Cortex. J. Neurophysiol. 82: 2989-2999, 1999. The cerebral cortex receives an extensive serotonergic (5-hydroxytryptamine, 5-HT) input. Immunohistochemical studies suggest that inhibitory neurons are the main target of 5-HT innervation. In vivo extracellular recordings have shown that 5-HT generally inhibited cortical pyramidal neurons, whereas in vitro studies have shown an excitatory action. To determine the cellular mechanisms underlying the diverse actions of 5-HT in the cortex, we examined its effects on cortical inhibitory interneurons and pyramidal neurons. We found that 5-HT, through activation of 5-HT2A receptors, induced a massive enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs) in pyramidal neurons, lasting for ~6 min. In interneurons, this 5-HT-induced enhancement of sIPSCs was much weaker. Activation of 5-HT2A receptors also increased spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons. This response desensitized less and at a slower rate. In contrast, 5-HT slightly decreased evoked IPSCs (eIPSCs) and eEPSCs. In addition, 5-HT via 5-HT3 receptors evoked a large and rapidly desensitizing inward current in a subset of interneurons and induced a transient enhancement of sIPSCs. Our results suggest that 5-HT has widespread effects on both interneurons and pyramidal neurons and that a short pulse of 5-HT is likely to induce inhibition whereas the prolonged presence of 5-HT may result in excitation.
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INTRODUCTION |
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Serotonin (5-hydroxytryptamine, 5-HT) is an
important endogenous neuromodulator in the CNS (Baumgarten and
Grozdanovic 1997; Jacobs and Azmitia 1992
).
Since the discovery of brain 5-HT pathways in the 1960s
(Dahlstrom and Fuxe 1964
), 5-HT has been implicated in
drug-induced psychoses and a number of psychiatric disorders, major
depression, and schizophrenia in particular (Abi-Dargham et al.
1997
; Blier and De Montigny 1997
). Histochemical
studies have shown that the mammalian cerebral cortex receives an
extensive 5-HT input originating from midbrain raphe 5-HT neurons
(Jacobs and Azmitia 1992
; Tork 1990
).
GABAergic inhibitory neurons appear to be the principal cortical target
of 5-HT fibers (DeFelipe et al. 1991
; Hornung and
Celio 1992
; Smiley and Goldman-Rakic 1996
), suggesting a potential role of GABAergic neurons in functioning of the
5-HT system.
5-HT receptors comprise a complex family. On the basis of their
pharmacology, signal transduction mechanisms and molecular structure,
more than a dozen types of 5-HT receptors have been identified
(Hoyer et al. 1994). Most of these receptors are coupled to various G proteins with the exception of the
5-HT3 receptor, which is a ligand gated cation
channel (Derkach et al. 1989
; Jackson and Yakel
1995
; Maricq et al. 1991
). Multiple 5-HT
receptor subtypes are expressed in the cerebral cortex (Mengod
et al. 1997
). In cerebral cortex, 5-HT3 receptors are only
expressed in inhibitory neurons (Morales and Bloom 1997
)
whereas 5-HT2A receptors are heavily expressed in
pyramidal cells and to a lesser extent in inhibitory neurons
(Hamada et al. 1998
; Jakab and Goldman-Rakic 1998
; Willins et al. 1997
).
Since the 1960s, many experiments using in vivo microiontophoretic
methods have characterized how 5-HT affects neuronal behavior. The
predominant effect of 5-HT on cerebral cortical pyramidal neurons is an
inhibition of spontaneous spiking, but the underlying mechanism is not
clear (see Jacobs and Azmitia 1992; Phillis
1984
; Reader and Jasper 1984
for review).
However, intracellular studies in rat cortical slices suggested that
5-HT induces depolarization and action potential firing in pyramidal
cells (Araneda and Andrade 1991
; Davies et al.
1987
; Tanaka and North 1993
). Furthermore, Aghajanian and Marek (1997)
reported that 5-HT enhances
spontaneous excitatory postsynaptic currents (sEPSCs) without
significantly changing spontaneous inhibitory postsynaptic currents
(sIPSCs) in frontal pyramidal neurons. These in vitro results suggest
that 5-HT is mainly excitatory in cortical neuronal circuitry.
The present study directly compares the effects of 5-HT on EPSCs and
IPSCs in cortical inhibitory neurons and pyramidal cells. We paid
particular attention to layer I neurons because these neurons are
mostly GABAergic and layer I receives a dense 5-HT innervation
(DeFelipe et al. 1991; Gabbott and Somogyi
1986
; Hornung and Celio 1992
; Hornung et
al. 1990
; Lidov et al. 1980
; Mulligan and
Tork 1988
; Smiley and Goldman-Rakic
1996
). We found that in pyramidal neurons 5-HT induces a robust
and desensitizing enhancement of sIPSCs and a weaker and
longer-lasting enhancement of sEPSCs through 5-HT2A
receptor activation. 5-HT also induced a rapidly desensitizing direct
inward current through activation of 5-HT3 receptors in a
small subset of cortical interneurons. Our results suggest that a short
pulse of 5-HT released under physiological conditions is likely to have
an inhibitory effect on cortical neuronal circuitry.
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METHODS |
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Slice preparation
Brain slices were prepared according to methods described
previously (Zhou and Hablitz 1996a). Briefly, 9- to
28-day-old Sprague-Dawley rats were decapitated, and the brains were
dissected out in <1 min. The isolated brain was immersed immediately
in ice-cold saline. Brain slices (300-µm thick) were cut from frontal
and anterior cingulate areas (Paxinos and Watson 1986
)
on a Vibratome. Slices were placed in a storage chamber at room
temperature (22-24°C). During recordings individual slices were
transferred to a microscope mounted chamber where they were perfused at
a rate of ~3 ml/min. The normal bathing solution contained (in mM)
125 NaCl, 3.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 26 NaHCO3, and 10 D-glucose. The bathing solution was continuously bubbled
with 95% O2-5% CO2 to
maintain pH around 7.4.
Whole cell recording
Individual neurons were visualized using an Olympus BX50WI
upright microscope equipped with Nomarski optics, a ×40 water
immersion lens and a Hamamatsu Newvicon video camera. Layer I neurons
were identified reliably during recording by their location below the pial surface (Zhou and Hablitz 1996a). Fast-spiking
interneurons in layers II/III were identified by their nonpyramidal
appearance and fast-spiking characteristics. Pyramidal neurons (layers
II-VI) were identified by their pyramidal shape, prominent apical
dendrites, and regular spiking properties. All recordings were made at
room temperature. Patch electrodes were prepared from Garner KG-33 glass tubing using a Narishige PP-83 puller. Electrodes were coated with silicone elastomer (Sylgard). Series resistance
(Rs) was estimated according to
Rs = 10 mV/I, where
I was the current (filtered
10 kHz) evoked by a 10-mV
pulse when the pipette capacitance was fully compensated. During
recordings, Rs was 4-15 M
among different cells and was not compensated. Care was exercised to monitor
the constancy of the series resistance, and recordings were terminated
whenever Rs was >15 M
or a
significant increase (>20%) occurred. The intracellular solutions
contained (in mM) 135 CsCl or KCl, 10 HEPES, 2 Mg-ATP, 0.2 Na-GTP, and
0.5 EGTA. pH and osmolarity were adjusted to 7.3 and 280 mOsm,
respectively. About 50% of voltage-clamp experiments were performed
using the CsCl-based intracellular solution, and the rest were
conducted with the KCl-based intracellular solution. No difference was
observed in 5-HT effects with the use of these two intracellular
solutions. Current-clamp experiments used only the KCl-based
intracellular solution. Tight seals (~5 G
before breaking into
whole cell mode) were achieved without cleaning the cell. Before
forming tight seals, all offsets were nulled using the offset feature
of the patch amplifier. To examine evoked synaptic responses in layers IV and V pyramidal neurons, a stainless steel bipolar electrode was
placed ~200 µm lateral to the recorded cells. Electrical stimuli were controlled by a Grass stimulator-isolator system.
Electrical signals were recorded using an Axopatch-200A amplifier (Axon
Instruments), stored on videotape, and analyzed off-line. Digitization
and analysis of the records were achieved using SCAN software (courtesy
of J. Dempster, University of Strathclyde, Glasgow, UK). Spontaneous
and miniature synaptic events were defined as those recorded in the
absence and presence of 0.3 µM tetrodotoxin (TTX), respectively.
Frequencies of synaptic events were calculated as the reciprocals of
interevent intervals. The decay of synaptic currents was fitted to
double exponential functions. Statistical comparisons of the frequency
and amplitude of synaptic currents before, during, and after 5-HT were
made using the Kolmogorov-Smirnov (K-S) test. P < 0.01 was considered significant. Numerical values are expressed as mean ± SD. About two-thirds of the cells were recorded in the shoulder
region or Fr2 region of the frontal cortex and the rest were in the
anterior cingulate cortex (Paxinos and Watson 1986).
Because no difference was observed among cells from the two areas, data
were pooled.
Bicuculline methiodide (Bic) (10 µM) was used to block
GABAA receptor-mediated synaptic events whereas
ionotropic glutamate receptors were blocked by
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 µM) and
D(-)2-amino-5-phosphonovaleric acid (D-APV) (20 µM). 5-HT, -methyl-5-HT, 1-(m-chlorophenyl)-biguanide (mCPBG), and
risperidone were obtained from Research Biochemical International (Natick, MA).
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RESULTS |
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5-HT induces an enhancement of sIPSCs in neocortical pyramidal cells
Bath application of 40 µM 5-HT induced an enhancement of
sIPSCs recorded in pyramidal cells. Specimen records under control conditions and during application of 5-HT are shown in Fig.
1, A and B,
respectively. The effects of 5-HT lasted for ~6 min (range 3-10
min), as shown in Fig. 1C. During the 2-min period of peak 5-HT enhancement, sIPSC frequency and amplitude were increased by
810 ± 260% and 110 ± 45%, respectively (n = 38). The kinetics of sIPSCs were not altered by 5-HT. Depending on
recording conditions, the 10-90% rise time was 0.5-1 ms, and the
double exponential decay had a fast time constant of 2-4 ms and a
slower time constant of 10-20 ms, similar to those described
previously (Zhou and Hablitz 1997b). In the presence of
5-HT, there were more large-amplitude events although small-amplitude
events also were increased. The effect of 5-HT declined in the
continued presence of the agonist. Dose-response relations were not
studied due to the long time needed for a full recovery. However, 10 µM 5-HT was not able to reliably induce a large sIPSC enhancement,
whereas the responses induced by 40 and 100 µM 5-HT were
indistinguishable, indicating that 40 µM was a nearly saturation
concentration. Events recorded under our recording conditions (20 µM
D-APV, 10 µM CNQX, up to 100 µM 5-HT, symmetric
Cl
and a holding potential of
70 mV) were
sIPSCs because all events were blocked by 10 µM Bic in three cells
tested (see also Zhou and Hablitz 1997b
).
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5-HT2A receptors enhance sIPSCs in pyramidal cells
5-HT2A receptors are expressed
abundantly in the cortex (Wright et al. 1995). To
examine if activation of these receptors could increase sIPSC frequency
in pyramidal cells, the 5-HT2-specific agonist
-methyl-5-HT (Baxter et al. 1995
; Sheldon and
Aghajanian 1990
) was bath applied. In all pyramidal neurons
tested (n = 25), 20 µM
-methyl-5-HT produced an
enhancement of sIPSCs similar to that induced by 5-HT (Fig.
2). The mean frequency and amplitude of
sIPSCs were increased by 820 ± 180% and 115 ± 42% over
control levels, respectively. The kinetics of sIPSCswere not
altered by
-methyl-5-HT. The enhancing effect of
-methyl-5-HT
also declined over time, lasting from 3 to 10 min among different
cells. We also tested the selective 5-HT2A
antagonist, risperidone (Baxter et al. 1995
). Because of
the desensitization, experiments attempting to apply risperidone (10 µM) after an
-methyl-5-HT-induced enhancement of sIPSCs were
inconclusive. Therefore we preincubated brain slices with 5 or 10 µM
risperidone. With this treatment,
-methyl-5-HT failed to induce any
significant change of sIPSCs in six pyramidal neurons from six slices.
In two additional pyramidal neurons, pretreatment with risperidone also
rendered 40 µM 5-HT ineffective in enhancing sIPSCs. These results
indicate that 5-HT enhancement of sIPSCs in pyramidal cells was
mediated by activation of 5-HT2A receptors on
GABAergic interneurons.
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We sought to determine the mechanism underlying the
5-HT2A receptor enhancement of sIPSCs. Under the
somatic recording conditions employed, -methyl-5-HT induced no
detectable direct inward current in layer I neurons (n = 20) or layer II/III fast-spiking interneurons (n = 3). In current-clamp recordings,
-methyl-5-HT failed to induce a
significant depolarization or increase in spontaneous firing in seven
layer I neurons tested.
-methyl-5-HT also failed to alter input
resistance under either voltage- or current-clamp conditions. No
hyperpolarization or outward current was induced by 5-HT or
-methyl-5-HT in any of the cells recorded in this study. Furthermore
despite the enhancement of sIPSCs, bath application of 5-HT
(n = 4) or
-methyl-5-HT (n = 2) did
not induce any detectable change in the frequency or amplitude of
miniature IPSCs (mISPCs) recorded in the presence of 0.3 µM TTX (Fig.
3).
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Activation of 5-HT2A receptors enhances sIPSCs in interneurons
Layer I neurons receive GABAergic inputs (Zhou and
Hablitz 1997b). Our results presented in the preceding section
suggest that serotonergic activation may enhance this GABAergic input to layer I neurons. To test this idea, we studied the effects of 5-HT
on sIPSCs recorded in layer I neurons. 5-HT (40-100 µM) produced an
enhancement of sIPSCs in 37/52 layer I neurons. As shown in Fig.
4,
-methyl-5-HT (20 µM) also
increased sIPSCs in layer I neurons (15/20). The sIPSC enhancement in
layer I neurons was also desensitizing. However, the percentage
increases in frequency (90 ± 48%) and amplitude (46 ± 37%) in layer I neurons were lower than those in pyramidal neurons.
Similarly,
-methyl-5-HT did not induce any significant enhancement
of sIPSCs in four layer I neurons from four slices preincubated with 10 µM risperidone, indicating an involvement of
5-HT2A receptors.
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Contribution of 5-HT3 receptors
In other cell systems, 5-HT3
receptors are coupled to cation channels conducting inward currents
under physiological conditions (Jackson and Yakel 1995;
Kawa 1994
; McMahon and Kauer 1997
). A recent immunohistochemical study indicated that
5-HT3 receptors are expressed only in a subset of
inhibitory neurons in cerebral cortex (Morales and Bloom
1997
). Therefore we reasoned that activation of these
5-HT3 receptors might induce a transient
enhancement of sIPSCs through a direct excitation of inhibitory
neurons. Indeed, in 4 of 52 layer I cells, bath application of 40-100
µM 5-HT induced a burst-like enhancement of sIPSCs,lasting for
5-20 s. The rapid desensitization of this enhancement is indicative of
5-HT3 receptor involvement. Therefore we tested
the specific 5-HT3 receptor agonist, mCPBG. In 2 of 21 layer I cells and in 2 of 19 pyramidal neurons, bath application
of 40 or 100 µM mCPBG also induced a brief enhancement of sIPSCs in
the absence TTX (Fig. 5). The similarity
of 5-HT and mCPBG responses suggests that the transient enhancement of sIPSCs was mediated by activation of 5-HT3
receptors in inhibitory neurons.
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Activation of 5-HT3 receptors induces a desensitizing inward current in interneurons
Interneurons were examined for a
5-HT3-receptor-mediated inward current. Indeed,
as shown in Fig. 6A, bath
application of 5-HT produced an inward current in the absence or
presence of 0.3 µM TTX. Similar currents were observed in response to
mCPBG (Fig. 6B). In layer I neurons, 40-100 µM 5-HT (10 of 108) and 40-100 µM mCPBG (4 of 43 cells) induced a direct inward
current (315 ± 319 pA, ranging from 30 to 1,000 pA) at a holding
potential of 70 mV (Fig. 6, A and B). In the
remainder of the layer I cells, 5-HT and mCPBG did not induce
detectable direct inward currents or changes in input resistance. On
application of 5-HT or mCPBG to responding cells, the inward current
developed rapidly. In the continued presence of 5-HT or mCPBG, the
current declined, presumably due to desensitization. The exact kinetics
of both activation and desensitization were not examined because bath application of drugs was employed. In the responding cells, 5-HT induced a decrease in input resistance, ranging from 10 to 75% (Fig.
6, A and B). The decrease in input resistance was
correlated with the magnitude of the inward current. The relatively
large amplitude of the observed
5-HT3-receptor-mediated current suggests that the
low number of responsive cells was due to a paucity of 5-HT3-receptor-expressing cells not a problem in
current detection. Furthermore neither 5-HT nor mCPBG was able to
induce any direct inward current in the pyramidal neurons tested
(n = 82).
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The reversal potential of the
5-HT3-receptor-mediated current was examined by
voltage-ramp experiments. A CsCl-based intracellular pipette solution
was used to partially block voltage-dependent K+ currents in these experiments.
Currents were evoked by ramping cells from 35 to 65 mV in 10 ms in
the absence and presence of 100 µM 5-HT. The difference current,
obtained by subtracting the control current from the current evoked in
the presence of 5-HT, represents the current induced by 5-HT. The
current reversed polarity between
5 and
2 mV in the three layer I
cells tested (Fig. 7). An inward
rectification was evident in the current-voltage relation in the
voltage range
65 to 35 mV. A similar inward rectification of
5-HT3-receptor-mediated currents has been
observed in other cell types (Kawa 1994
; Yang et
al. 1992
).
|
We also tested both mCPBG and -methyl-5-HT in 10 pyramidal neurons.
In each of these cells, bath application of 40 or 80 µM mCPBG for
5
min failed to induce any visible change in sIPSCs, whereas subsequent
application of 20 µM
-methyl-5-HT was able to induce a strong
enhancement of sIPSCs (Fig. 8). These
results suggest that there are more interneurons expressing
5-HT2A than 5-HT3 receptor
in cerebral cortex, as indicated by recent anatomic studies
(Jakab and Goldman-Rakic 1998
; Morales and Bloom
1997
; Willins et al. 1998
).
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5-HT enhances sEPSCs but not mEPSCs in pyramidal cells
It has been reported that 5-HT can increase pyramidal cell
excitability (Araneda and Andrade 1991; Sheldon
and Aghajanian 1991
). Accordingly, we reasoned that 5-HT might
induce an increase in sEPSCs. To test this idea, we recorded sEPSCs
from pyramidal neurons after blockade of GABAergic inhibition with 10 µM Bic. Bath application of 5-HT (50-100 µM, n = 7) or
-methyl-5-HT (20 µM, n = 8) enhanced sEPSCs
in each of these cells (Fig. 9). The mean
frequency of sEPSCs was increased to 880 ± 230% of control levels. Most of the increase in frequency was due to an augmentation in
the number of small- and medium-amplitude events. The mean amplitude of
sEPSCs was not or only minimally enhanced (
20%). The 5-HT- and
-methyl-5-HT-induced enhancement of sEPSC frequency also displayed
desensitization. However, this process was slower than that observed
with sIPSCs: the frequency started to decline
10 min after applying
5-HT or
-methyl-5-HT. Further, this desensitization was not
complete. The frequency of sEPSCs was still higher in the prolonged
presence of 5-HT or
-methyl-5-HT than that under control conditions
and was sustained as long as the recording condition was stable.
Preincubation of brain slices with 10 µM risperidone prevented
-methyl-5-HT from having any effect on sEPSCs in two pyramidal
neurons from two slices, suggesting an involvement of
5-HT2A receptors.
|
In the presence of 0.3 µM TTX, neither 5-HT nor -methyl-5-HT
induced any change in mEPSCs (n = 6). These results
suggested that 5-HT and
-methyl-5-HT might induce action potential
firing in pyramidal neurons. However, bath application of 5-HT or
-methyl-5-HT did not induce any detectable direct inward current in
the absence (n = 53) or presence (n = 6) of TTX or depolarization (n = 4) in pyramidal
neurons. Input resistance of these pyramidal neurons was also not
changed by 5-HT or
-methyl-5-HT.
5-HT did not enhance sEPSCs in layer I neurons
To further characterize the effects of 5-HT on cortical neuronal
circuits, we studied the modulation of excitatory synaptic inputs to
layer I neurons, a type of cortical interneuron. In contrast to
pyramidal neurons, 5-HT (50 or 100 µM, n = 6) and -methyl-5-HT (20 µM, n = 5) were unable to induce
any detectable change in the frequency or amplitude of sEPSCs (Fig.
10). Kinetics of these sEPSCs were also
not altered by 5-HT or
-methyl-5-HT and were similar to those
reported previously (Zhou and Hablitz 1997a
).
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5-HT decreased eIPSCs and eEPSCs in pyramidal neurons
In the nucleus accumbens, Nicola and Malenka (1997)
reported that dopamine inhibited evoked IPSCs by depressing
Ca2+ influx through voltage-dependent
Ca2+ channels. Therefore we reasoned that the
enhancement of sIPSCs and sEPSCs induced by
5-HT2A receptor activation might mediated by
increased Ca2+ influx through voltage-dependent
Ca2+ channels. To test this idea, we examined the
effects of 5-HT on evoked IPSCs (eIPSCs) and eEPSCs in pyramidal
neurons. As shown in Fig. 11,
monosynaptic eIPSCs were reliably evoked in the presence of 10 µM
CNQX and 40 µM D-APV. A modest paired-pulse synaptic depression was observed at intervals of 100-200 ms. Bath application of 40 µM 5-HT induced a robust increase in sIPSCs each of the 5 pyramidal neurons tested (Fig. 11). However, 5-HT simultaneously decreased the amplitude of eIPSCs to ~80% of control level in these
cells. Paired-pulse depression was not significantly changed. Similarly, eEPSCs in pyramidal neurons were reduced, whereas sEPSCs were enhanced in frequency (n = 4 cells).
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The strong enhancement of sIPSCs and sEPSCs induced by
5-HT2A receptor activation does not appear to be
mediated by increased Ca2+ influx. Instead, a
decrease in eEPSCs and eIPSCs suggests that 5-HT might modulate the
availability of transmitter vesicles (Wang and Zucker
1998). Similarly, Kondo and Marty (1998)
reported that norepinephrine increased sIPSCs but decreased eIPSCs in
cerebellar stellate neurons.
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DISCUSSION |
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The main findings of this study are that 5-HT induces a large, desensitizing enhancement of sIPSCs and a weaker longer-lasting enhancement of sEPSCs through 5-HT2A receptors and that 5-HT3 receptors appear to be present exclusively on interneurons. These results may provide a cellular explanation to the predominantly inhibitory 5-HT effect in cerebral cortex.
Activation of 5-HT2A receptors induces a desensitizing enhancement of sIPSCs in pyramidal neurons
In the present study, carried out in a neocortical in vitro slice
preparation, the most prominent effect of 5-HT was a strong enhancement
of sIPSCs in pyramidal neurons. This effect appeared to be mediated
predominantly by 5-HT2A receptors because it was mimicked by the 5-HT2-agonist -methyl-5-HT and
blocked by the specific 5-HT2A-antagonist
risperidone. This is consistent with the fact that
5-HT2A receptors are the most abundant 5-HT
receptors in the cerebral cortex (Burnet et al. 1995
;
Jakab and Goldman-Rakic 1998
; Mengod et al.
1997
; Willins et al. 1997
; Wright
et al. 1995
). A similar action of 5-HT2A receptors
has been reported in the hippocampus (Piguet and Galvan
1994
; Shen and Andrade 1998
). This 5-HT
enhancement of sIPSCs fits well with the generally inhibitory effects
of 5-HT repeatedly observed in cerebral cortex in vivo (Reader
and Jasper 1984
). However, Aghajanian and Marek
(1997)
recently reported that, in rat neocortical pyramidal
neurons in vitro, 5-HT2A receptor activation induced only a
minimal enhancement of sIPSCs with a strong enhancement of sEPSCs,
indicating an excitatory 5-HT effect on cerebral cortex. The reason(s)
for this discrepancy is unknown.
How 5-HT2A receptor activation leads to an enhancement of
sIPSCs is not clear. The increase in both frequency and amplitude of
sIPSCs and its TTX sensitivity suggested an enhanced excitability of
inhibitory neurons. However, our somatic recordings failed to reveal a
5-HT2A-receptor-mediated direct inward current or depolarization. This is similar to the situation with 5-HT-induced enhancement of sEPSCs (Aghajanian and Marek 1997;
present study). However, we cannot rule out the possibility that a
subset of GABAergic neurons were depolarized by 5-HT but might have
evaded our detection, especially most of our interneurons were from
layer I and laminar differences may exist. Cox et al.
(1998)
recently reported that glutamate locally activates
dendritic terminals and induces dendritic transmitter release, which is
not detected by somatic recording in thalamic interneurons. 5-HT may
act on axon and/or dendritic terminals in a similar fashion.
Furthermore, because we used relatively large recording pipettes,
intracellular factor(s) might be dialyzed such that certain 5-HT
responses including the 5-HT-induced depolarization (Araneda and
Andrade 1991
) were washed out in the recorded cells (Yakel et al. 1988
). On the other hand, we found that
5-HT decreased the amplitude of eIPSCs, indicating that 5-HT did not
increase action potential-evoked Ca2+ influx. Therefore it
seems that 5-HT2A receptor activation may instead affect
transmitter vesicles through other signaling pathways. This is
consistent with studies showing that 5-HT2A receptor
activation increases intracellular Ca2+ concentration by
releasing Ca2+ from intracellular stores in nonneuronal
cell types (Hagberg et al. 1998
; Ozaki et al.
1997
; Saucier and Albert 1997
) and that 5-HT
might increase the number of transmitter vesicles available for release
(Wang and Zucker 1998
).
The mechanism(s) underlying the desensitization of
5-HT2A-receptor-mediated enhancement of sIPSCs was not
examined in this paper. However, studies in nonneuronal cell systems
have indicated that agonist-induced desensitization was associated with
decreased numbers of 5-HT2A receptors (Berry et al.
1996; Roth et al. 1995
; Saucier and
Albert 1997
). Because our observed desensitization of
5-HT2A-receptor-mediated enhancement of sIPSCs showed a
time course comparable with the agonist-induced internalization of 5-HT2A receptors in a cell line (Berry et al.
1996
), a similar mechanism might be operational in cortical
interneurons. A tonic desensitization may exist in cerebral cortex due
to the physiological release of 5-HT, which may underlie 5-HT
supersensitivity of cortical pyramidal neurons after 5-HT denervation
(Ferron et al. 1982
).
We also found that 5-HT enhancement of sIPSCs in layer I neurons was much weaker than in pyramidal neurons, indicating that GABAergic neurons innervating interneurons versus pyramidal neurons are differentially modulated by 5-HT. Therefore our results that 5-HT can greatly increase GABAergic inhibitory inputs to pyramidal neurons are consistent with the large body of in vivo studies showing that 5-HT inhibits pyramidal neurons.
Activation of 5-HT2A receptors enhances sEPSC frequency in pyramidal neurons
5-HT and -methyl-5-HT greatly increased the frequency but not
the amplitude of sEPSCs in every pyramidal neuron tested, as reported
by Aghajanian and Marek (1997)
. This is consistent with Jakab and Goldman-Rakic (1998)
that
5-HT2A receptors are expressed uniformly in all
cortical pyramidal neurons. The enhancing effect was TTX sensitive. In
a large sample of pyramidal neurons, no detectable direct inward
current or input conductance change was produced by 5-HT or
-methyl-5-HT. 5-HT also decreased the amplitude of eEPSCs. This
situation is quite similar to that of 5-HT-induced enhancement of
sIPSCs. Therefore the possibilities envisioned for sIPSC enhancement
are also applicable to sEPSCs.
Recent data have shown that 5-HT2A receptors are
highly expressed in cortical pyramidal neurons, the proximal apical
dendrites in particular (Hamada et al. 1998;
Jakab and Goldman-Rakic 1998
; Roth et al.
1997
). This suggests that dendrites, the major postsynaptic membrane, are the major site of 5-HT action. However, the strong enhancement of sEPSC frequency suggests that 5-HT was acting
presynaptically. This apparent contradiction may be reconciled by the
following possibilities. First, dendritic 5-HT2A
receptors might induce a local depolarization (Cox et al.
1998
). Such local effects can cause either dendritic
transmitter release or a release of retrograde messengers with
subsequent effects on presynaptic axon terminals (Maletic-Savatic and Malinow 1998
; Morishita et
al. 1998
). Second, 5-HT2A receptors have
been detected in a minority of axon terminals (Jakab and
Goldman-Rakic 1998
) and activation of these terminal 5-HT
receptors may be responsible for the observed sEPSC enhancement.
5-HT does not affect sEPSCs in layer I neurons
5-HT and -methyl-5-HT had no effect on sEPSCs in layer I
neurons. Even though sampling bias might have contributed to this observation, the fact that activation of 5-HT2A
receptors induced robust enhancement of sEPSCs in all pyramidal neurons
tested suggests that this differential modulation of sEPSCs in the two
cell types was real. 5-HT2A receptor expression
is high in pyramidal neuron proximal apical dendrites and low in distal
parts (Jakab and Goldman-Rakic 1998
; Willins et
al. 1997
). It is possible that activation of dendritic
5-HT2A receptors may induce dendritic transmitter release and/or release of retrograde messenger(s). Therefore a lack of proximity of layer I neurons (Zhou and Hablitz 1996b
) to
pyramidal proximal apical dendrites may lead to a lack of 5-HT effect
on sEPSCs in layer I neurons.
5-HT3 receptors were observed only in interneurons
Our present results show that 5-HT and mCPBG induced a large
desensitizing inward current in fast spiking interneurons. This current
had a reversal potential near 0 mV and showed a modest inward
rectification. The induction of this current was accompanied by a large
increase in input conductance, indicating that 5-HT was opening ion
channels. These biophysical and pharmacological properties suggest that
this inward current was mediated by 5-HT3 receptors (Derkach et al. 1989; Jackson and Yakel
1995
; Kawa 1994
; McMahon and Kauer
1997
; Yang et al. 1992
). Consistent with a
recent immunohistochemical study (Morales and Bloom
1997
), this 5-HT3-receptor-mediated current was recorded exclusively in a small number of cortical interneurons. However, it is possible that our relatively slow drug
application system might have caused a certain amount of underestimation of 5-HT3 receptor expression in
cortical neurons.
The functional significance of such a highly selective expression of
5-HT3 receptors remains to be established.
However, it appears certain that a short pulse of 5-HT released from
5-HT nerve terminals can result in a transient excitation of
fast-spiking interneurons and a subsequent transient inhibition of
targets innervated by these interneurons. It will be important to
determine if GABAergic neurons receiving 5-HT baskets (Hornug
and Celio 1992) express 5-HT3 receptors.
If that is the case, then 5-HT baskets are suited ideally to rapidly
deliver large amounts of 5-HT to 5-HT3 receptors
on these neurons.
Functional considerations
Our data clearly show that
5-HT2A-receptor-mediated effects are widespread
in cerebral cortex. Because of the low density of 5-HT terminals and
synapses, our results also suggest that nerve terminal-released 5-HT
may spread and act on nonsynaptic 5-HT receptors (Audet et al.
1989; Descarries and Umbriaco 1995
; Seguela et al. 1989
). Further, our data indicate that in
cerebral cortex, the predominantly inhibitory effect of 5-HT might be
affected by multiple factors; i.e., precise spatial and temporal
location and quantity of 5-HT released, the rate of 5-HT clearance, and the state of the 5-HT receptors. As shown by Blier and De
Montigny (1997)
, most antidepressants increase brain 5-HT
levels, whereas our results suggest that such treatments may alter the
sensitivity of 5-HT receptors on GABAergic neurons and thereby the
excitation-inhibition balance in cortical neuronal circuitry. Therefore
5-HT modulation of GABAergic neurons may play an important role in the
pathophysiology and treatment of major psychiatric disorders
(Abi-Dargham et al. 1997
).
In conclusion, our study demonstrated that 5-HT preferentially induced a large desensitizing enhancement of sIPSCs in cortical pyramidal neurons; 5-HT also induced a weaker but longer-lasting enhancement of sEPSCs in pyramidal neurons. Therefore short 5-HT pulses may inhibit cortical circuitry, whereas prolonged 5-HT presence may result in excitation.
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ACKNOWLEDGMENTS |
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We thank A. Margolis for excellent technical help and Dr. Lori McMahon for commenting on an early version of this paper.
This work was supported by National Institutes of Health grants to J. J. Hablitz and a Young Investigator Award from National Alliance for Research on Schizophrenia and Depression to F.-M. Zhou.
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FOOTNOTES |
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Present address and address for reprint requests: F.-M. Zhou, Division of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
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 16 February 1999; accepted in final form 15 July 1999.
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REFERENCES |
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