Harvard Medical School and Brockton Veterans Affairs Medical Center, Department of Psychiatry, Neuroscience Laboratory 151C, Brockton, Massachusetts 02301
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ABSTRACT |
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Rainnie, Donald G..
Serotonergic Modulation of Neurotransmission in the Rat
Basolateral Amygdala.
J. Neurophysiol. 82: 69-85, 1999.
Whole cell patch-clamp recordings were obtained
from projection neurons and interneurons of the rat basolateral
amygdala (BLA) to understand local network interactions in
morphologically identified neurons and their modulation by serotonin.
Projection neurons and interneurons were characterized morphologically
and electrophysiologically according to their intrinsic membrane
properties and synaptic characteristics. Synaptic activity in
projection neurons was dominated by spontaneous inhibitory postsynaptic
currents (IPSCs) that were multiphasic, reached 181 ± 38 pA in
amplitude, lasted 296 ± 27 mS, and were blocked by the
GABAA receptor antagonist, bicuculline methiodide (30 µM). In interneurons, spontaneous synaptic activity was characterized
by a burst-firing discharge patterns (200 ± 40 Hz) that
correlated with the occurrence of
6-cyano-7-nitroquinoxaline-2,3-dione-sensitive, high-amplitude
(260 ± 42 pA), long-duration (139 ± 19 mS) inward excitatory postsynaptic currents (EPSCs). The interevent interval of
831 ± 344 mS for compound inhibitory postsynaptic potentials (IPSPs), and 916 ± 270 mS for EPSC bursts, suggested that
spontaneous IPSP/Cs in projection neurons are driven by burst of action
potentials in interneurons. Hence, BLA interneurons may regulate the
excitability of projection neurons and thus determine the degree of
synchrony within ensembles of BLA neurons. In interneurons
5-hydroxytryptamine oxalate (5-HT) evoked a direct, dose-dependent,
membrane depolarization mediated by a 45 ± 6.9 pA inward current,
which had a reversal potential of 90 mV. The effect of 5-HT was
mimicked by the 5-HT2 receptor agonist,
-methyl-5-hydroxytryptamine (
-methyl-5-HT), but not by the
5-HT1A receptor agonist, (±)
8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), or the
5-HT1B agonist, CGS 12066A. In projection neurons, 5-HT
evoked an indirect membrane hyperpolarization (~2 mV) that was
associated with a 75 ± 42 pA outward current and had a reversal
potential of
70 mV. The response was independent of 5-HT
concentration, blocked by TTX, mimicked by
-methyl-5-HT but not by
8-OH-DPAT. In interneurons, 5-HT reduced the amplitude of the evoked
EPSC and in the presence of TTX (0.6 µM) reduced the frequency of
miniature EPSCs but not their quantal content. In projection neurons,
5-HT also caused a dose-dependent reduction in the amplitude of
stimulus evoked EPSCs and IPSCs. These results suggest that acute
serotonin release would directly activate GABAergic interneurons of the
BLA, via an activation of 5-HT2 receptors, and increase the
frequency of inhibitory synaptic events in projection neurons. Chronic
serotonin release, or high levels of serotonin, would reduce the
excitatory drive onto interneurons and may act as a feedback mechanism
to prevent excess inhibition within the nucleus.
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INTRODUCTION |
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Intracellular recordings from morphologically
identified neurons of the basolateral complex of the amygdala (BLA)
have demonstrated that projection neurons and interneurons can be
distinguished according to their electrophysiological properties
(Rainnie et al. 1993; Washburn and Moises
1992
). Furthermore several intracellular current-clamp studies
have examined synaptic transmission occurring in the basolateral
complex in response to activation of intrinsic and/or extrinsic
afferent inputs (Gean and Chang 1992
; Rainnie et
al. 1991a
,b
; Sugita et al. 1993
; Washburn
and Moises 1992
). Irrespective of the point of stimulation, the
most prominent synaptic potentials evoked are a glutamatergic
excitatory postsynaptic potential (EPSP) and two GABAergic inhibitory
postsynaptic potentials (IPSPs), one fast and one slow. The EPSP is
mediated by activation of both
N-methyl-D-aspartate (NMDA) and AMPA/kainate
subtypes of glutamate receptor (Gean and Chang 1992
;
Rainnie et al. 1991a
). The fast IPSP is mediated by
activation of GABAA receptors, whereas the slow IPSP is
mediated by activation of GABAB receptors (Rainnie et al. 1991b
).
These in vitro observations have been substantiated further by
experiments that have demonstrated that similar excitatory and
inhibitory processes occur in the BLA in vivo (Brothers and Finch 1985; Fi et al. 1996
; Lang and
Paré 1997a
,b
; Mello et al. 1992
;
Paré et al. 1995
). However, the relatively high
resistance of the microelectrodes used in these studies introduced
undesirable noise into voltage-clamp records and caused
"space-clamp" problems. Whole cell patch-clamp recording has
overcome these problems, and this technique now has been applied to the
amygdala slice preparation (Keele et al. 1997
;
Neugebauer et al. 1997
; Rainnie 1995
;
Smith and Dudek 1996
). Using this technique,
Smith and Dudek (1996)
have confirmed the predominance
of a glutamatergic and a GABAergic drive onto BLA neurons in juvenile
rats. Moreover on the basis of their observations of spontaneous
activity in the BLA, these authors have proposed that local feedforward
and/or feedback circuitry regulates neuronal activity within this complex.
The dynamic interaction between the relative expression of EPSPs and
IPSPs within the basolateral complex can directly influence the
input/output relationship for the amygdala. Hence pharmacological manipulations of either glutamatergic or GABAergic transmission in the
BLA can markedly alter behavioral responses in vivo (Davis et
al. 1994). Consequently if monoamine neuromodulators, such as
serotonin, regulate the normal balance between excitatory and inhibitory transmission, they may be expected to alter signal processing within the amygdala and hence modify any behavioral responses dependent on this processing. Support for this hypothesis comes from the observation that the amygdala receives a prominent innervation from serotonergic terminals originating from neurons of the
dorsal raphé nucleus (Bobillier et al. 1976
;
Ma et al. 1991
; Sadikot and Parent 1990
),
stimulation of the dorsal raphé results in the facilitation of
firing in a subset of amygdala neurons (Jacobs 1972
),
iontophoretic application of 5-hydroxytryptamine (5-HT) into the rat
amygdala reduced glutamate-induced cell firing in vivo (Mah and
Cunningham 1993
, Stutzman et al. 1998
), and
serotonin release in the amygdala is increased during states of stress
and anxiety (Fernandes et al. 1994
; Gargiulo et
al. 1996
; Kawahara et al. 1993
; Kirby et
al. 1995
; Rueter and Jacobs 1996
).
Serotonin receptors can be grouped into seven classes of receptor,
named 5-HT1 through 5-HT7 (see Hoyer
1996). In addition to there being multiple receptor subtypes,
individual 5-HT receptors also show a differential distribution within
the CNS. Hence the amygdala has moderate to high levels of binding
sites for 5-HT1A, 5-HT2, 5-HT3,
5-HT4, and 5-HT6 receptors (Barnes et
al. 1989
; Eglen et al. 1995
; Morales et
al. 1996
; Morilak et al. 1993
; Radja et
al. 1991
; Ward et al. 1995
). Even within the
amygdala regional differences exist. Hence 5-HT1A receptors
are found predominantly in the central nucleus, whereas
5-HT2, 5-HT3, and 5-HT6 receptors are found predominantly in the basolateral complex.
In many areas of the CNS, the principal receptor mediating postsynaptic
5-HT-induced inhibition is the 5-HT1A receptor subtype (see
Saxena 1995). However, the low levels of
5-HT1A receptor expression observed in the BLA suggested
that the 5-HT-mediated inhibition of BLA cell firing (see preceding
text) may not result from a direct activation of postsynaptic
5-HT1A receptors but, rather, by an indirect inhibition of
glutamate release via activation of presynaptic 5-HT1A
receptors (Bobker and Williams 1989
). Moreover, expression of 5-HT3 receptors on interneurons of the BLA
(Morales et al. 1996
) and 5-HT2 receptors
suggested that inhibition of cell firing also may be facilitated by a
direct excitation of GABAergic interneurons (Alreja
1996
; Sheldon and Aghajanian 1991
).
These hypothesis were tested using whole cell patch-clamp recording from morphologically, and electrophysiologically, identified neurons of BLA in vitro. Spontaneous synaptic currents were examined to establish if an intrinsic connectivity exists between projection neurons and interneurons of the BLA slice preparation. Identified neurons then were examined for their intrinsic membrane response to application of serotonin. The effects of serotonin on spontaneous and evoked synaptic inputs to these same neurons also was examined. Finally the response of neurons to specific 5-HT receptor agonists and antagonists were then examined to isolate the 5-HT receptor subtypes mediating these responses.
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METHODS |
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The basolateral complex, composed of the lateral and basolateral nuclei, was visualized easily in the coronal slice in vitro as it was outlined laterally by the white matter tract of the external capsule (corpus callosum) and medially by the white matter tract of the longitudinal association bundle. Neuronal responses reported in this study were obtained only from neurons located in the basolateral subdivision of the basolateral complex.
Slice preparation
Slices (500-µm thick) containing the basolateral complex were
obtained from isoflurane anesthetized 24- to 56-day-old male Long-Evans
rats and prepared using procedures as previously described (see
Rainnie et al. 1991a). Slices were maintained fully
submerged in a tissue chamber and continuously perfused with artificial cerebrospinal fluid (ACSF) of the following composition: (in mM) 124 NaCl, 3.75 KCl, 1.25 KH2PO4, 1.3 MgCl2, 3.5 CaCl2, 26 NaHCO3, and 10 glucose. The ACSF was heated to 32 ± 2°C and gassed with a
95%-5% oxygen/carbon dioxide mixture. Whole cell patch-clamp recordings were obtained using the technique of Blanton et al. (1989)
. Briefly, borosilicate glass patch electrodes
(resistance 6-8 M
) were pulled on a Flaming/Brown micropipette
puller (Model P-87) and filled with (in mM) 120 K-gluconate, 10 phosphocreatinine, 10 KCl, 10 HEPES, 3 MgCl2, 2 MgATP, and
0.2 NaGTP and biocytin 0.45%. Patch solution was buffered to pH 7.2 with KOH and filtered through a 0.2-µm filter (Altech Associates, IL)
before use. Final osmolarity of the patch solution was 280 mOsm.
Recordings were made with an Axopatch-1D amplifier (Axon Instruments,
Burlingame, CA) using pClamp 6.02 software and a Digidata 1200 A-D
interface (Axon Instruments). For patch electrodes, a seal resistance
was considered acceptable if it was >1.5 G
and had a series
resistance of <20 M
. Afferent fibers were stimulated with a
concentric bipolar stimulating electrode (MCE-100; Kopf Instruments),
which was placed on the central nucleus of the amygdala through which
course the fibers of the stria terminalis.
Data acquisition
Whole cell patch-clamp records initially were established in
current-clamp mode and then switched to voltage clamp. This procedure allowed the viability of the cell to be determined before voltage-clamp experiments were performed. Only those BLA neurons that showed a stable
resting membrane potential more negative than 55 mV for >5 min and
an action potential that overshot +5 mV were considered acceptable for
further analysis.
All current- and voltage-clamp paradigms were computer controlled using
either clampex or fetchex data acquisition programs (Axon Instruments),
and data were sampled at a frequency determined by the speed of the
response to be measured. Voltage excursions in response to
hyperpolarizing current injection were used to determine the membrane
input resistance and were sampled at 10 kHz. Voltage excursions in the
depolarizing direction were sampled at 20 kHz. This was done to ensure
an accurate evaluation of action potential height and duration. Data
were filtered at 5 kHz in current clamp and at 2 kHz in voltage clamp.
For acquisition of spontaneous PSP/Cs, data were sampled at 20 kHz.
Spontaneous PSP/Cs were sampled continuously for 30 s. Miniature
spontaneous PSCs were recorded in the presence of TTX (0.6 µM) and
also sampled at 20 kHz for 30 s. Only those miniature events that
displayed a rise time of 0.8 mS were included in the analysis to
reduce contamination by events originating in electrotonically distant regions the amplitude of which may not reflect the true synaptic current. Synaptic events were analyzed semi-automatically using the
Fetchan analysis package of the pClamp software bundle, and the
amplitude and time constant of decay (
) for EPSCs and IPSCs was
calculated for each neuron examined during one continuous 30-s sweep.
The accuracy of the analysis was confirmed with an off-line examination
of marked traces in Clampfit. Events were averaged, and only those
sweeps containing >100 events were included in the analysis. In
projection neurons, miniature IPSCs could not be clearly discerned in
the presence of TTX, consequently the effects of 5-HT on presynaptic
GABA release was not examined. To examine the voltage dependency of the
effects of applied drugs, cells were held at
60 mV in voltage clamp
and then "ramped" from
100 to
40 mV during a 6-s period. The
ramp then was repeated in the presence of the drug, and subtraction of
the control current from the drug-induced current allowed the voltage
dependency of the pure drug-induced current to be determined.
Statistical analysis
Means, SD, and SE were calculated for each treatment and two-tailed, paired and unpaired Student's t-tests or a single factor ANOVA test were performed as necessary. Data are expressed as means ± SE and significance was accepted if P < 0.05.
Drug application
Drugs were applied directly to the ACSF using a continuous
gravity-fed bath application. This application allowed accurate dose-response relationships to be constructed because the extracellular fluid reached equilibrium with the concentration of applied drug in
~2 min. Drugs applied were: 5-HT (5-100 µM);
-methyl-5-hydroxytryptamine (
-methyl-5-HT, 50-100 µM); (±)
8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT, 1-10 µM);
CGS-12066B maleate (20-50 µM); TTX (0.6-1.2 µM);
[2-[4-(2-methoxyphenyl)-1-piperazinyl[ethyl]-N-2-pyridinyl-cyclohexane-carboxamide maleate (WAY 100635, 20-40 nM); 6-cyano-7-nitroquinoxaline-2,3,-dione (CNQX, 10 µM); bicuculline methiodide (Bic, 20-50 µM); and
2-hydroxy-saclofen (2-OH-Sac, 100 µM) from Research Biochemicals
International (Natick, MA).
Histochemical visualization
During the course of each experiment, biocytin diffused from the
patch electrode into the recorded cell. No current injection protocol
was necessary to load the cells with biocytin. At the termination of
each experiment, the 500-µm slice containing the loaded cell was
removed from the recording chamber and placed in 4% paraformaldehyde
overnight. For rapid identification of cell types, slices were washed
with 0.1 M phosphate buffered saline (PBS) and then incubated for 1 h
in PBS containing 0.5% Triton X-100. Slices then were incubated for
2 h in PBS-Triton X-100 supplemented with 40 µL/ml Texas
Red-Avidin D conjugate (Vector Laboratories) to visualize the neurons.
The slices were washed in PBS and mounted on the chuck of a Vibroslice
(Campden Instruments) for further resectioning to 80 µm. Resectioned
slices were mounted on gelatin-subbed microscope slides, air dried
overnight, mounted in AM 100 fluorescence-free, permanent mounting
media (Chemicon; Temecula, CA), and visualized using a Nikon Diaphot
microscope equipped with epifluorescence. In addition, 44 cells were
characterized electrophysiologically and then shipped to the University
of South Carolina School of Medicine for independent morphological
identification and determination of location by Dr. A. J. McDonald
(see Fig. 2A, 1-3).
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RESULTS |
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Stable whole cell records were obtained from 205 neurons of the
basolateral nucleus, 13% of which were considered to be interneurons because of their morphological and electrophysiological
characteristics. Namely, a spine-sparse dendritic arborization, a
nonpyramiform cell soma, a high-input resistance (~121 M), and
expression of a high-frequency train of action potentials in response
to depolarizing current injection (Fig.
1B) (see also Rainnie
et al. 1993
). Of the remaining neurons, 86% resembled
pyramidal projection neurons (Fig. 1A) with a prominent
apical dendrite, a spine dense dendritic arbor, and low input
resistance (~57 M
). One neuron had a neurogliaform-like appearance
and firing properties similar to that of a projection neuron. All of
the neurons the position of which could be verified anatomically (86)
were located in the anterior and posterior subdivisions of the
basolateral nucleus between
2.3 and
3.3 mm Bregma, a representative
sample of which is shown in Fig.
2A, 1-3. The
majority of neurons (80%) were located in the rostral BLA between
2.3 and
2.8 mm Bregma, the remaining 20% were located caudal to
Bregma
3.14. Moreover, 76% were located in the anterior subdivision. However, no differences were observed in the electrophysiological characteristics of neurons recorded either from the rostrocaudal aspect
or from the anterior or posterior subdivisions of the nucleus.
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Spontaneous synaptic currents
PROJECTION NEURONS.
The majority of neurons in this study (80%) displayed spontaneous
synaptic activity in which, at a membrane potential close to rest (60
mV), the predominant synaptic activity in projection neurons was
low-frequency, long-duration (250-500 ms) IPSP/Cs and high-frequency,
short-duration (15-25 mS) EPSP/Cs (Fig.
3A). These results are in
agreement with previous sharp electrode studies (see Rainnie et
al. 1991b
). In current clamp, the waveform of the long-duration
spontaneous IPSPs fell into two categories: those that appeared to be
"pure" IPSPs (47%) and those IPSPs in which the initial rising
phase was corrupted by a barrage of EPSPs (53%). The decay rate of the
spontaneous pure IPSPs could be fit by a single exponential with time
constants (
) of 105 ± 36 mS (n = 8). This time
constant is more than three times as long as those reported for
monosynaptic GABAA-mediated IPSPs in other areas of the CNS
(see Buhl et al. 1995
; Ropert and Guy
1991
), suggesting that the spontaneous IPSPs observed in the
BLA were compound IPSPs. Projection neurons also revealed spontaneous
"unitary," or monosynaptic, IPSPs with a peak amplitude of
0.76 ± 0.1 mV (n = 4) at the resting membrane
potential.
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INTERNEURONS.
In interneurons (Fig. 4), the spontaneous
activity consisted primarily of fast high-frequency EPSPs (Fig.
4B1) that were interspersed with bursts of action potentials
riding on a depolarizing wave of summated EPSPs (Fig. 4B2).
Within each burst, action potentials fired at a frequency of 200 ± 40 Hz (n = 8). In voltage clamp, the bursts of
action potentials were seen to correlate with the occurrence of
high-amplitude (260 ± 42 pA, n = 11),
long-duration (139 ± 19 mS) inward currents (Fig. 4C)
that had characteristics similar to a synchronous burst of EPSCs (Fig.
3D). Individual EPSCs had a mean amplitude of 46 ± 5 pA and a rapid (2.8 ± 0.2 mS; n = 4). Both
the fast EPSCs and the synchronous bursts of EPSCs were blocked fully
by CNQX (10 µM, not shown).
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Network properties
The low frequency and long duration of the spontaneous, compound IPSP/Cs suggested that they may arise from intrinsic network activity within the amygdaloid complex. Support for this hypothesis came from the observations that raising the external K+ concentration from 5 to 8.5 mM caused a significant increase in the frequency of occurrence of spontaneous IPSCs (Fig. 5A, n = 4) from 0.8 to 1.3 Hz, that compound IPSP/Cs were abolished by exogenous CNQX (10µM, not shown), and that a dorsoventral transection of the slice at the level of the external capsule, to remove reciprocal connectivity of the BLA with the entorhinal and piriform cortex (Fig. 2B), failed to prevent spontaneous inhibitory activity. Furthermore an apparent correlation was observed between the frequency of spontaneous bursts in interneurons and the frequency of spontaneous IPSPs in projection neurons. Interevent-interval histograms generated for either burst firing in interneurons (n = 6) or spontaneous IPSPs in projection neurons (n = 10), supported this hypothesis (Fig. 5B). When a curve was fit to the data using a Marquardt least-squares analysis fitting protocol, the modal inter-IPSP interval was 831 ± 344 mS, whereas the modal interburst interval was 916 ± 270 mS. These data suggest that the occurrence of IPSPs in projection neurons probably is driven by the occurrence of bursts in interneurons. Furthermore because burst firing rarely is observed in projection neurons, this implies that a synchronized input from two or more projection neurons would be required to drive a single interneuron. Conversely this data also suggests that a single interneuron may innervate multiple projection neurons.
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Serotonergic modulation of BLA neuronal properties
Once the electrophysiological characteristics of a cell had been
ascertained, the effect of 5-HT on its intrinsic membrane properties
was examined. In all cases, exogenous 5-HT was applied to neurons held
at 60 mV with DC current injection in current clamp or when
voltage-clamped at
60 mV.
PROJECTION NEURONS.
The effect of 5-HT on the intrinsic membrane properties of projection
neurons was small, but variable, and appeared independent of the dose
applied (5-100 µM). Hence 5-HT evoked a reversible, membrane
hyperpolarization in 54% of the neurons tested (n = 23) and had no effect in the remaining 46% of the neurons
(n = 19). The effects of increasing the concentration
of 5-HT in those neurons that did respond is shown in Fig.
6A1 (solid bars). Here,
increasing concentrations of 5-HT appear to increase the membrane
response in projection neurons; however, the observed difference was
not statistically significant (ANOVA, P = 0.66). For
example, 20 µM 5-HT evoked a mean hyperpolarization of 2 ± 0.4 mV (n = 4), whereas 100 µM 5-HT evoked a
hyperpolarization of only
3 ± 0.5 mV (n = 4).
In those neurons that did respond to 5-HT with a membrane hyperpolarization, the membrane input resistance was reduced by 7 ± 3 M
(n = 6). However, the reduction of input
resistance did not alter the firing properties of BLA projection
neurons in response to depolarizing current injection.
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INTERNEURONS.
Unlike the variable response found in projection neurons, the effects
of 5-HT on interneurons was more consistent. Hence, 5-HT evoked a
membrane depolarization in 10 of 13 neurons examined and had no effect
in the remaining 3 neurons. Moreover the response to 5-HT was
unaffected by prior application of either TTX or CNQX (data not shown),
and interneurons showed a dose-response relationship with increasing
concentrations of 5-HT applied (see Fig. 6A1, stippled
bars). At a concentration of 20 µM, 5-HT evoked a depolarization of
4.0 ± 0.8 mV (n = 4), whereas at 50 µM 5-HT
evoked a significantly larger depolarization of 9.0 ± 1.3 mV
(n = 6). ANOVA, at the 95% confidence level used for
significance in this study, revealed a statistical significance between
responses to 5-HT in interneurons (P = 0.09). A typical
response to 5-HT (50 µM) is shown in Fig. 6B. In current
clamp, the depolarization always was associated with a concomitant
increase in membrane input resistance. -Methyl-5-HT (100 µM,
n = 5) mimicked the effects of 5-HT in the majority of interneurons: evoking a depolarization (8-10 mV) and an associated increase in membrane input resistance in two neurons and an inward current (59 pA) in one neuron and having no effect in the remaining two
neurons. In those interneurons that were responsive to 5-HT, neither
8-OH-DPAT (n = 2) nor CGS 12066A, a specific
5-HT1B-receptor agonist (n = 2), had any
effect on the resting membrane potential. In voltage clamp, 5-HT (50 µM) evoked an inward current 45 ± 6.9 pA in four of five
interneurons examined, and a comparison of the membrane conductance
before and during 5-HT application revealed a reversal potential close
to
90 mV (Fig. 6C2). These data suggest that one of the
primary functions of serotonergic transmission within the BLA is to
increase the excitability of inhibitory interneurons possibly via
activation of 5-HT2 receptors, which, in conjunction with
the small inhibition of projection neurons, would result in a dampening
of the input-output response of the BLA.
Serotonergic modulation of synaptic transmission
REGULATION OF NETWORK ACTIVITY. During 5-HT application the most consistent observation was a marked alteration in the occurrence of spontaneous synaptic potentials (n = 47). Irrespective of the intrinsic response of individual BLA neurons, 5-HT caused an alteration in either the frequency, and/or the amplitude, of spontaneous synaptic potentials/currents in all neurons examined. Moreover the effect of 5-HT was both time and concentration dependent.
In all projection neurons examined, continuous bath application of 5-HT (50 µM) evoked a marked reduction in the amplitude of the GABAA-mediated IPSP/Cs that was independent of the effects of 5-HT on membrane potential. Hence in those neurons in which 5-HT evoked a small hyperpolarization, DC current injection to the predrug membrane potential (
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REGULATION OF TRANSMITTER RELEASE. In both projection neurons and interneurons application of 5-HT caused a reduction in the amplitude of stimulus evoked EPSP/Cs and IPSP/Cs (see Fig. 10). In projection neurons, 5-HT caused a dose-dependent reduction of the evoked EPSC, whereby the EPSC amplitude was reduced 75 ± 9% by 100 µM 5-HT (n = 8), 43 ± 16% by 50 µM 5-HT (n = 4), and 34 ± 13% by 20 µM 5-HT (n = 4). In contrast, the effect of 5-HT on the evoked IPSP/C was more profound and also more sensitive to lower concentrations of 5-HT. Hence, 100 µM 5-HT reduced the IPSP/C amplitude by 90 ± 10% (n = 8), 50 µM 5-HT by 92 ± 3% (n = 4), and 20 µM 5-HT by 56 ± 23% (n = 4). In addition, in those interneurons in which a distinct EPSC could be evoked (n = 3), 50 µM 5-HT reduced the amplitude by 65% (Fig. 10B). In those neurons in which a biphasic IPSC was observed, 5-HT (50 µM) caused a reduction in the amplitude of both the evoked and spontaneous fast and slow IPSC (Fig. 10C).
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DISCUSSION |
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The results of this study demonstrate that neuronal activity in the BLA is regulated by a complex reciprocal interaction between projection neurons and interneurons, whereby synaptically driven burst firing in interneurons may result in the concomitant expression of compound IPSCs in projection neurons. This feedback inhibition could result in synchronous activity in ensembles of BLA projection neurons. Moreover this interaction, and hence the input-output relationship of the BLA, can be modulated by 5-HT in two distinct ways.
The primary action of exogenous 5-HT was a dose-dependent excitation of interneurons probably via activation of postsynaptic 5-HT2 receptors. This excitation resulted in a concomitant indirect inhibition of projection neurons via an increased release of GABA and subsequent activation of postsynaptic GABAA receptors. In a minority of projection neurons (<12%), 5-HT also had a direct inhibitory action via an activation of postsynaptic 5-HT1A-receptors.
With higher concentrations (>50 µM) or prolonged application, 5-HT had an additional indirect effect on neuronal excitability by reducing the probability of glutamate release from presynaptic glutamatergic terminals. Although the 5-HT receptor subtype mediating this response remains to be determined, it is not mediated by either 5-HT1A or 5-HT1B receptors.
It is proposed that a target-specific, serotonergic input from the brain stem raphé nuclei may regulate interneuronal activity and hence create synchronized ensembles of projection neurons. Consequently, serotonergic input to the BLA may act at a subnuclear level to modulate behavioral responses in which the amygdala has been implicated.
Spontaneous synaptic activity
In the adult rat BLA, spontaneous synaptic activity in projection
neurons was characterized by fast EPSCs and IPSCs that were interspersed by long-duration, bicuculline-sensitive, compound IPSCs.
In contrast in interneurons, spontaneous synaptic activity primarily
was characterized by fast EPSCs that were themselves interspersed with
bursts of summated EPSCs. However, in a recent BLA study by
Smith and Dudek (1996), no compound IPSCs were observed at the resting membrane potential (about
60 mV) of principal neurons.
This discrepancy is most probably due to the age difference in the
populations of animals used in these two studies. In this study, fully
mature animals were used (mean 41 ± 1 days, n = 74), compared with those 8- to 25-days old in the study of Smith and Dudek. Indeed, spontaneous EPSCs also predominate in BLA projection neurons from immature (12 days) Long-Evans rats (personal observation). Hence strain differences are unlikely to account for the discrepancy, and the compound IPSP/C may not be expressed in BLA neurons until the
network has reached maturity. Support for this hypothesis comes from
the observation that application of the GABAA antagonist bicuculline onto BLA slices from mature animals induces spontaneous epileptiform burst firing activity in all cells tested (see Gean and Chang 1991
; Rainnie et al. 1991b
, 1992
). In
contrast, no bicuculline-induced epileptiform activity was reported in
the study of Smith and Dudek. Therefore it would appear that
disinhibition is much more debilitating in mature BLA circuits than it
is in immature circuits. Whether this reflects an alteration in
synaptic connectivity, an alteration in GABAA receptor
subunit composition, or an alteration of glutamate receptor subunit
composition remains to be determined.
Support for an age-related alteration in the strength of synaptic
contacts comes from a comparison of the amplitude and time constants of
decay () of the mean EPSCs and IPSCs in mature and immature BLA
neurons. At a holding of
60 mV, the mean EPSC and IPSC amplitudes
recorded from projection neurons were similar to those reported by
Smith and Dudek, (
12 pA and 25 pA, respectively). However, the
for the spontaneous IPSC was four times longer in mature projection
neurons (21 vs. 5.8 mS) and the EPSC
was almost five times longer
(12.5 vs. 2.7 mS) than those measured in immature neurons. Several
possibilities may account for these differences: developmental
alterations in the gating kinetics of the GABAergic and glutamatergic
receptor-channels, developmental alterations in transmitter re-uptake
kinetics, and the longer
s may reflect frequency-dependent
attenuation of the synaptic current due to an imperfect space clamp at
electrotonically more distant locations on the dendritic arbor.
However, attenuation is unlikely because the rise time of the current
also would be prolonged (see Spruston et al. 1994
), and
PSCs only were accepted with a rise time of ~ 0.8 mS, which is
similar to the rise time of 1.0 mS for EPSCs reported in immature neurons.
In addition, the mean EPSC recorded from interneurons had a larger
amplitude and a shorter than that recorded from projection neurons.
These results suggest that even within the same nucleus projection
neurons and interneurons may express different glutamate receptor
subtypes. In agreement with Smith and Dudek, the EPSC recorded from
both cell types was abolished by CNQX at a holding potential of
60
mV, indicating that they were mediated by AMPA receptor activation. The
AMPA receptor is a heterologous receptor-channel complex that is
assembled from a combination of four subunits (GluR1-4). Thus the
difference in amplitude and
may reflect a difference in the AMPA
receptor subunit composition. Interestingly, GluR2/3 AMPA receptor
subunits are expressed in projection neurons, whereas interneurons
express the GluR1 subunit but not the GluR2/3 subunits (McDonald
1994
, 1996
). Indeed the interneuronal EPSC amplitude and
reported here are similar to those reported by Mahanty and Sah
(1998)
, who recently have demonstrated that EPSCs, recorded in
"putative" BLA interneurons, have no NMDA component and are
mediated by an inwardly rectifying calcium-permeable AMPA current that
is sensitive to external polyamines.
Network properties
The existence of a reciprocal interaction between projection
neurons and interneurons has been discussed in earlier studies (see
Gean and Chang 1992; Rainnie et al.
1991a
; Smith and Dudek 1996
). However, no direct
correlation had been established between spontaneous synaptic events
occurring in projection neurons with those occurring in interneurons.
This study has demonstrated, for the first time, that the occurrence of
spontaneous compound IPSCs in projection neurons has a similar
interevent interval to the occurrence of spontaneous bursts of EPSCs in
interneurons. It is possible, therefore, that compound IPSPs observed
in projection neurons may be driven by the occurrence of bursts of
action potentials in local interneurons. The local nature of this
connectivity was supported by the observation that depolarizing neurons
within the slice, by raising external concentrations of potassium in the ACSF, increased the frequency of spontaneous potentials. In addition, spontaneous bursts were blocked by both TTX and CNQX; this
implied that an action potential-dependent, glutamatergic drive was
necessary to trigger the network. However, spontaneous action
potentials rarely are observed at the resting membrane potential of
projection neurons in vitro. Consequently it is unlikely that these
neurons drive the network. Moreover a dorsoventral transection of the
slice that severed the reciprocal connectivity between the basolateral
nucleus and the piriform and entorhinal cortex (Krettek and
Price 1978
; Ottersen 1982
) also failed to abolish spontaneous IPSCs or bursts of EPSCs. It is possible, that the
excitatory input comes from afferent inputs from the lateral nucleus
(see Pitkänen et al. 1997
), projection neurons of
which demonstrate burst firing, and oscillating firing patterns, both
in vitro and in vivo (Pape et al. 1998
;
Paré and Gaudreau 1996
).
Irrespective of the source of the excitatory input, the membrane time
constant (m = 18 mS) in projection neurons and the frequency of action potential firing in BLA interneurons (200 Hz)
suggests that one interneuron could generate the compound IPSP/C
observed in projection neurons. Even assuming a single release site and
a 50% failure rate of any given action potential in an interneuron to
evoke transmitter release (see Allen and Stevens 1994
),
it still would be possible for multiple IPSCs to summate during a
150-mS burst. If each interneuron makes multiple contacts with a single
projection neuron (Buhl et al. 1995
; Tamás et al. 1997
; Thomson et al. 1996
), this would
increase further the potential for compound IPSPs. If each interneuron
innervates more than one projection neuron, this then would facilitate
synchronous activity in a micronetwork, or ensemble, of BLA projection neurons.
Two recent studies have reported a slow inhibitory synaptic potential
in projection neurons of the lateral nucleus (Danober and Pape
1998; Lang and Paré 1997a
) that was
bicuculline insensitive and mediated by activation of an intrinsic
calcium-dependent potassium conductance. In contrast, bicuculline
blocked both the fast and slow components of the spontaneous compound
IPSP/C. In a recent dual-cell recording study, a long-duration (>200
mS) IPSP was observed in projection neurons of the hippocampus after
activation of a subtype GABAergic interneuron (Thomson et al.
1996
). Interestingly, >20 action potentials at >100 Hz were
required before the slow IPSP was observed. The similarity between the
spontaneous burst firing rate of interneurons in this study and that
required to evoked the slow IPSP in the hippocampus suggests that these
slow postsynaptic responses may be mediated by a common mechanism.
Serotonergic modulation of intrinsic neuronal properties
Extracellular studies in the amygdala have reported apparently
contradictory observations after serotonin application in vivo. Using a
strategy of post hoc morphological identification of recorded neurons,
this study has revealed that the primary action of exogenous 5-HT
within the BLA was a direct excitation of interneurons. Additional indirect evidence came from the observation that 5-HT also caused an
increase in the frequency of spontaneous IPSP/Cs in BLA projection neurons. This excitation probably is mediated by activation of 5-HT2 receptors as both the direct and indirect effects
could be mimicked by the 5-HT2-receptor agonists,
-methyl-5-HT. However, unlike 5-HT,
-methyl-5-HT did not cause a
depolarization in all interneurons examined. These results support the
notion that subpopulations of interneurons within the BLA may express
different 5-HT receptor subtypes (see Morales and Bloom
1997
) and that no one 5-HT receptor subtype is expressed in all interneurons.
The 5-HT-induced depolarization of BLA interneurons was associated with
an increase in membrane input resistance and had a reversal potential
of 90 mV, suggesting modulation of a potassium conductance. These
data are in agreement with previous observations of a
5-HT2-receptor-mediated increase in interneuronal activity in other areas of the CNS (Alreja 1996
; Gellman
and Aghajanian 1994
; McCormick and Wang 1991
;
Sheldon and Aghajanian 1991
). In the piriform cortex and
medial septum, this excitation is mediated by activation of
5-HT2A receptors but the underlying ionic mechanism is
unclear (Alreja 1996
; Marek and Aghajanian
1995
). In contrast, the 5-HT-induced depolarization of thalamic
reticular neurons appears to be mediated by a decrease in a
"leak" potassium conductance (IKL) but the
5-HT receptor subtype has yet to be identified (McCormick and
Wang 1991
).
Projection neurons of the BLA appear to be particularly unresponsive to
5-HT application. Indeed the small 5-HT-induced hyperpolarization was
found to be independent of the concentration of 5-HT applied, blocked
by TTX, and has a reversal potential close to 70 mV. These data
suggest an indirect response mediated, in part, by an increase in
presynaptic GABA release concomitant to the increased excitability of
BLA interneurons. The majority of projection neurons do not respond to
the 5-HT1A receptor agonist 8-OH-DPAT, and the 5-HT-induced
response was insensitive to the 5-HT1A antagonist, WAY
100635. These data support the observation that the BLA has only a low
density of 5-HT1A receptors, the highest levels being found
in the central nucleus (Pazos and Palacios 1985
;
Radja et al. 1991
).
Serotonergic modulation of synaptic transmission
High concentrations or prolonged application of 5-HT reduced the
frequency and amplitude of spontaneous IPSP/Cs. I have shown previously
that, in the BLA, evoked and spontaneous IPSPs are reduced by glutamate
receptor antagonists (Rainnie et al. 1991b). The
reduction of the IPSP/C amplitude by 5-HT provided indirect evidence
that an additional action of 5-HT may be to reduce the excitatory drive
within the amygdaloid network. This was substantiated by the
observation that 5-HT reduced the amplitude of the stimulus evoked
EPSP/Cs and IPSP/Cs in projection neurons and reduced the frequency of
spontaneous mEPSCs in interneurons. In other areas of the brain, a
reduction in glutamatergic and/or GABAergic transmission has been
reported to result from activation of presynaptic 5-HT1A and 5-HT1B receptors (Bobker and Williams
1989
; Johnson et al. 1992
; Stanford and
Lacey 1996
). However, the lack of effect of 8-OH-DPAT and
CGS-12066A on either spontaneous or evoked EPSP/Cs and IPSP/Cs would
suggest that these receptors do not contribute to the presynaptic
modulation of transmitter release in the BLA. In contrast, while this
manuscript was in preparation, Cheng et al. (1998)
,
using sharp microelectrode recording in the BLA, have reported that the
EPSP reduction is probably mediated by 5-HT1A receptors
because it can be mimicked partially by 10 µM 8-OH-DPAT. This
concentration is ~1,000 times higher than its receptor binding affinity (pKi) for the 5-HT1A receptor, and the effect
should not be considered specific. Experiments are in progress to
determine the pharmacological profile of the 5-HT receptor involved in
the reduction of glutamate release.
At first glance there is an apparent paradox in the response of the BLA network to 5-HT application. Why would a circuit decrease excitatory drive onto interneurons at a time when the membrane potential of these same interneurons is shifted toward threshold for action potential generation? The answer may lie in the dose-response relationship for each response. As noted above, there is a delay between the expression of increased interneuron excitability and the decreased glutamatergic drive in response to bath applied 5-HT. This could reflect differing affinities of the 5-HT receptors mediating these two responses. Bath application does not instantaneously raise the extracellular concentration of a drug to the desired concentration. Consequently neurons in the slice will receive a time-dependent concentration gradient. If 5-HT receptors located on the postsynaptic membrane of inhibitory interneurons have a high affinity for 5-HT, they may be activated at low 5-HT concentrations, whereas presynaptic 5-HT receptors located on glutamatergic terminals may have a low affinity for 5-HT and hence only may be activated at higher concentrations. This may explain why an increase in spontaneous IPSP/Cs was observed only at 5-HT concentrations <100 µM; above this concentration, the reduction of glutamatergic transmission may occlude the increased excitability of the interneurons. This mechanism could act as a feedback loop to prevent excess inhibition in the presence of prolonged 5-HT release. Moreover the reduced glutamatergic drive would reduce synaptic "noise" and ensure that the nucleus responds only to inputs above a particular intensity.
An alternative explanation for the time-dependent decrease in
spontaneous IPSP frequency in response to prolonged 5-HT application may be an activation and subsequent desensitization of
5-HT3 receptors on BLA interneurons. The 5-HT3
receptor, unlike the other serotonin receptors, is a ligand gated ion
channel and rapidly desensitizes with prolonged activation
(Derkach et al. 1989). Moreover, Morales and
Bloom (1997)
have reported that a small subpopulation of BLA interneurons express 5-HT3 receptors. Two observations
suggest that although 5-HT3 receptors may contribute to the
5-HT-mediated response, the time-dependent response probably involves
activation of more that one 5-HT receptor subtype: the response always
is observed with 5-HT application and the specific 5-HT3
receptor agonist, m-chlorophenylbiguanide,
(m-CPBG, 500 nM), evoked a brief membrane depolarization in
only one of three interneurons examined (personal observation).
Functional implications
Regulation of glutamatergic and GABAergic transmission in
the amygdala has been implicated in aspects of fear, anxiety, memory consolidation, and stimulus-reward associations (Brioni et al. 1989; Cador et al. 1989
; Davis et al.
1994
; Dickinson-Anson and McGaugh 1997
;
Ferry and Di Scala 1997
; Salinas and McGaugh
1996
; Wan and Swerdlow 1996
). The results of
this study suggest that pharmacological manipulations that modulate
serotonergic transmission in the BLA would have possible repercussions
in each of these behavioral responses. Indeed, Deakin and Graeff
(Graeff et al. 1996
) have postulated that an ascending
5-HT pathway from the dorsal raphé, which innervates the amygdala
and frontal cortex, facilitates conditioned fear. Moreover local
infusion of the 5-HT3 antagonist, BRL 46470A, into the
amygdala produces an anxiogenic effect (Gargiulo et al.
1996
), and acute treatment with the serotonin uptake inhibitor,
citalopram, reduces the acquisition and expression of conditioned fear
(Inoue et al. 1996
). The data reported here would
suggest that intra-amygdaloid infusion of specific 5-HT2 receptor agonists and/or antagonists also would affect these behaviors. In contrast, infusion of 5-HT1A receptor agonists would be
expected to have minimal effects on these behaviors. It is interesting, therefore, that a recent study has reported that local activation of
5-HT1A receptors in the BLA may produce anxiogenic effects (Gonzalez et al. 1996
). It is possible that the
anxiogenic response observed in this study results from activation
5-HT1A in the adjacent central nucleus due to drug
spill-over from the infusion site.
Further experiments are needed to fully elucidate the pharmacological and ionic profiles of the serotonin responses reported here. Moreover, the BLA also has a relatively high density of 5-HT4 and 5-HT6 receptors, and additional experiments are in progress to determine the effects of activation of these receptors on the network properties of the BLA.
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ACKNOWLEDGMENTS |
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The author is indebted to Dr. A. J. McDonald of the University of South Carolina for agreeing to independently determine the morphological characteristics of a representative sample of filled neurons. The author also thanks K. E. McLaughlin and M. J. Mudrick for skillful assistance in preparing this manuscript and Drs. R. W. McCarley, M. Patil, and R. Bergeron for helpful discussion.
This work was supported by National Institute of Mental Health Grant R29 MH-57016-01 and National Alliance for Research on Schizophrenia and Depression Grant NAR97RAIN704-01 to D. G. Rainnie and by the Brockton Veterans Affairs Schizophrenia Center.
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FOOTNOTES |
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Address for reprint requests: Harvard Medical School and Brockton VAMC, Dept. of Psychiatry, Neuroscience Laboratory 151C, 940 Belmont St., Brockton, MA 02301.
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 9 December 1998; accepted in final form 26 February 1999.
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REFERENCES |
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