1Department of Pharmacology and 2Department of Psychiatry, Duke University Medical Center; and 3Division of Neurology Research and 4Division of Psychiatry, Durham Veterans Administration Medical Center, Durham, North Carolina 27705
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ABSTRACT |
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Wang, C., W. A. Wilson, and S. D. Moore. Role of NMDA, Non-NMDA, and GABA Receptors in Signal Propagation in the Amygdala Formation. J. Neurophysiol. 86: 1422-1429, 2001. Although the synaptic physiology of the amygdala has been studied with single neuron recordings, the properties of the networks between the various nuclei have resisted characterization because of the limitations of field recording in a neuronally diffuse structure. We addressed this issue in the rat amygdala complex in vitro by using a photodiode array coupled with a voltage-sensitive dye. Low-intensity single pulse stimulation of the lateral amygdala nucleus produced a complex multi-phasic potential. This signal propagated to the basolateral nucleus and the amygdalostriatal transition zone but not to the central nucleus. The local potential, which depended on both synaptic responses and activation of voltage-dependent ion channels, was reduced in amplitude by the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist 6,7-dinitroquinoxaline (DNQX) and reduced to a lesser extent by the NMDA glutamate receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV). We next characterized the less complex signals that propagated to more distal regions with or without the addition of the GABA receptor antagonist bicuculline (BIC). BIC alone greatly increased the signal propagation and permitted activation of previously silent areas within the amygdala. DNQX blocked signal propagation to amygdala regions outside of La, even in the presence of BIC, whereas D-APV had minimal effects on these distal signals. These data represent several novel findings: the characterization of the multi-component potential near the site of stimulation, the gating of signal propagation within the amygdala by GABAergic inhibition, the critical role of non-NMDA receptor-mediated depolarization in signal propagation, and the lack of a role for NMDA receptors in maintaining propagation.
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INTRODUCTION |
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The amygdaloid formation
consists of a group of temporal lobe nuclei that are involved in
multiple functions including memory, emotion, attention, and autonomic
control (Davis 1992). Animal models of experimental fear
and anxiety have indicated a critical role for the amygdala in the
acquisition and expression of fear-conditioned behaviors (Davis
1992
; Davis et al. 1994
). The rat amygdala has essentially the same extrinsic and intrinsic connections as in the
human and nonhuman primate (Davis et al. 1994
;
McDonald 1998
) and has served as a model for detailing
physiological mechanisms underlying functions served by the human amygdala.
The amygdala has extensive reciprocal intranuclear and internuclear
connections (Davis et al. 1994; Rainnie et al.
1993
). The synaptic physiology of the basolateral amygdala has
been previously characterized using intracellular recordings
(Rainnie et al. 1991a
,b
). Individual neurons in the
basolateral complex receive input from multiple sites; excitatory
postsynaptic potentials (EPSPs) can be elicited in pyramiform neurons
by stimulation of stria terminalis, external capsule, or lateral
amygdala (Chapman et al. 1990
; Rainnie et al.
1991a
,b
). These EPSPs consist of a fast component, mediated by
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors, and a slow component mediated by
N-methyl-D-aspartate (NMDA) receptors
(Calton et al. 2000
; Rainnie et al.
1991a
; Weisskopf and LeDoux 1999
). The EPSPs are
generally followed by a fast, GABAA
receptor-mediated inhibitory postsynaptic potential (IPSP), or by a
combined fast IPSP and slow (possibly GABAB
mediated) IPSP (Rainnie et al. 1991b
; Washburn
and Moises 1992
). In disinhibited preparations, synaptic
excitation may also elicit calcium spikes mediated by voltage-dependent
Ca2+ channels (Calton et al.
2000
). Synaptic responses may also be evoked in central nucleus
neurons by stimulation of the basolateral complex (Nose et al.
1991
). These responses have been characterized as complex,
multi-component EPSPs and IPSPs. The compound EPSP has components
mediated by both NMDA and non-NMDA excitatory amino acid receptors.
Several distinct IPSPs have been characterized as mediated by
GABAA receptors, GABAB
receptors (Rainnie et al. 1991b
),
GABAC-like receptors (Delaney and Sah
1999
), and possibly glycine receptors (Nose et al.
1991
).
We examined components of the evoked responses in the amygdala
using voltage-sensitive dye imaging with a photodiode array. This
relatively new technique has been successfully utilized to characterize
spatiotemporal aspects of evoked and spontaneous activity in neocortex
(Tsau et al. 1999; Wu et al. 1999b
),
olfactory bulb (Keller et al. 1998
), piriform cortex
(Demir et al. 1998
), and thalamocortical pathways
(Laaris et al. 2000
). As the sampling rate of the diodes
is considerably faster than that of charge-coupled device (CCD)
cameras, it may be used to record rapid electrophysiological events in
brain slices stained with voltage-sensitive dyes. The resulting
composite images retain a high degree of spatial resolution (Lowe 1999
; Wu et al. 1999a
). These two
features of the photodiode array make it feasible to study the signal
propagation along network pathways.
We used this system to characterize the signal propagation and synaptic pharmacology of the in vitro amygdala formation. We found that the response proximal to the stimulation site was composed of both synaptic and nonsynaptic elements that could be identified pharmacologically. In addition, we found that the distal responses contained almost entirely synaptically mediated components. We were able to simultaneously record the temporal phases of the synaptic responses and the anatomic distribution of these same responses. We then characterized the effects of neurotransmitter blockade on the synaptic responses and thus were able to uncover key aspects of the function of the amygdala network.
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METHODS |
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Coronal brain slices (500 µm) were prepared from 21- to 35-day-old male Sprague-Dawley rats using a vibratome (Series 1000; Technical Products International, St. Louis, MO). Rats were decapitated under halothane anesthesia, and the brains were quickly removed. Coronal slices containing the amygdala formation were identified and incubated in standard artificial cerebrospinal fluid (ACSF) containing (in mM): 124 NaCl, 10 dextrose, 26 NaHCO3, 2 KCl, 1.25 KH2PO4, 2 CaCl, and 1 MgSO4, equilibrated with 95% O2-5% CO2.
After at least 1 h of incubation, slices were transferred into
separate incubation chamber containing ACSF and the voltage-sensitive dye JPW1131 (also known as RH479; from Dr. L. Loew, University of
Connecticut, Farmington, CT) at 0.02 mg/ml for 40-60 min. The stained
slices were then transferred to the recording chamber and perfused (3 ml/min) with dye-free ACSF at 29°C for at least 30 min before
recording to wash out unbound dye. The optical signal was obtained with
a Zeiss microscope (Axioskop 2FS; Carl Zeiss) using a water immersion
lens (×10), coupled with a 464 hexagonal photodiode array (WuTech
Instruments). Light from a HAL halogen lamp (12 V 100 W) was passed
through a 705 ± 50 nm filter (D705/50; Chroma Technology)
to the slice where it was focused. The image of the slice was then
projected to the 464-element photodiode array through a ×10 water
immersion objective lens and a zoom port. The zoom was set at about
0.83 so that the photodiode array maximally covered the objective
field. With this setting, each diode received light from a 80 × 80 µm2 area of the objective field. The
objective plane was also calibrated with a CCD camera (SensiCam; Cooke)
and each pixel received 3.125 × 3.125 µm2
area of light (with a ×10 objective lens). The double calibration allowed location of a specified area on the slice to a corresponding diode. The output from each diode was then amplified, multiplexed, digitized, and stored in the computer (Wu et al. 1999a).
Single stimuli were delivered through a tungsten microelectrode (0.02 in., 5 M, 8 Degree; A-M System, Carlsborg, WA) placed in the lateral
nucleus of amygdala. The intensity of stimulation was 70-100 µA/200
µs. Glass microelectrodes were made from TW150F-4 glass (thin wall
glass with filement, 1.5 mm diam; World Precision Instruments) using a
micropipette puller (P-87; Sutter Instrument). These electrodes were
filled with NaCl (150 mM; 3-5 M
) and were used to monitor field
potentials to monitor viability of the slice. The signal from the
microelectrode was amplified with an Axopatch 200B amplifier (Axon
Instruments) and then stored concurrently with the optical images.
Data acquisition and analysis was performed using NeuroPlex software (RedShirt Imaging, LLC) on a Pentium PC. A vibration isolation system (250WS-1; Minus-k Technology) was used to minimize the vibration noise.
After at least 30 min of washing of the stained slice in the recording chamber, a 2-s data acquisition (at a sample rate of 0.944 ms per frame) was triggered manually every 5 min. The stimulus was delivered during the acquisition with a 0.5-s delay. Averaged responses from a 240 × 240 µm2 area (3 by 3 diodes) close to the stimulating electrode was constantly observed until the responses were stabilized for half an hour before applying any drug. With this recording method, the responses remained stable for at least 2 h. Once the responses were stabilized for 30 min (6 recordings), the drugs were washed in for 30 min (6 recordings) followed by a 60-min wash out (12 recordings). The stabilized recordings from each condition (or the last 6 from wash out) were then averaged for comparison. An averaged response of six diodes immediately adjacent to the stimulating electrode (within the lateral nucleus of the amygdala complex) was used for analysis of temporal responses. With the evoked response, the very tip of the stimulating electrode generated an artifact revealed by a single diode that had a significantly larger response than other diodes (Fig. 1B). This diode was excluded from analysis but was used as an indicator for anatomically correlating diodes within specific areas of the slice.
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The response of voltage-sensitive dye (JPW1131) has submillisecond
kinetics and a linear dependence on voltage change within ±100 mV
(Lowe 1999) with a specific light absorption at 705 ± 50 nm. The optical imaging was made of pseudocolor intensity scaling in which the warm color corresponded to depolarization. A fixed scale
was used to compare the images between the control and drug application. The term "fixed scale" refers to scaling signals of
all diodes to a set maximum and minimum value, as opposed to "variable scale" in which the response of each diode is relative to
its own individual maximum and minimum of response. The variable scale
is best used for displaying the dynamic spreading of the peak signal;
the fixed scale is best used for demonstrating the pattern of the
spreading and relative intensity of the signal in different regions. In
addition, fixed scale is used so that the changes before and after drug
can be determined. Data are expressed as means ± SE.
All pharmacological agents were obtained from Sigma, except for D-2-amino-5-phosphonovaleric acid (D-APV; Acros) and TTX (Calbiochem, La Jolla, CA). Pharmacological agents were prepared from stock solutions and dissolved in ACSF before use. All stock solutions were made using distilled water except for 6,7-dinitroquinoxaline (DNQX), nifedipine [with dimethyl sulfoxide (DSMO)], and DL-threo-beta-hydroxyaspartic acid (THA; with 300 mM of NaOH).
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RESULTS |
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Characterization of the evoked signal in the lateral amygdala
Results are reported from a total of 45 slices. To examine the
network properties of the amygdala complex (Fig. 1A), we
stimulated the lateral nucleus (La) and observed the propagation of the
signal toward the basolateral nucleus (BL) and the amygdalostriatal
transition zone (AStr; Fig. 1C). Under normal conditions,
the evoked signal did not propagate to the central nucleus (Ce). These
results are consistent with the anatomical findings of direct
connections from La to AStr but not to the core of Ce
(Pitkanen et al. 1995). Figure 1B
shows the diode array with each short line representing a 2-s trace
from a single diode following a single stimulus. Since the dye binds to
all cell membranes and changes its absorbance (at 705 ± 50 nm)
according to membrane potential, the signal we observed represented
integrative changes in membrane potential from both neuronal and
nonneuronal elements. The diode located directly over the tip of the
stimulating electrode always produced a markedly larger signal than the
other diodes (Fig. 1B) and served as a reference point to
locate different portions of the slice.
A typical local response in the amygdala following stimulation is shown
in Fig. 2A. The trace was
produced by averaging the response of the six diodes surrounding, but
excluding, the tip of the stimulating electrode (the averaging did not
alter the characteristics of the response curve but served as a mean to reduce the background noise). The optical response included three components: a fast depolarizing peak (phase 1), a valley (phase 2), and
a slow depolarizing peak (phase 3). These three components were all
wavelength specific (Fig. 2A). When using very high stimulus intensities, a delayed response was occasionally observed following phase 3, which involved a wavelength nonspecific intrinsic response, presumably due to morphologic changes in the brain slice in response to
the pathway activation (Sato et al. 1997). As this
response did not appear to be related to membrane potential changes and was only present when high stimulation intensities were used, we did
not investigate it further. The traces in Fig. 2B indicate that the response resulted completely from physiological actions, as
TTX totally blocked the response from diodes around the tip of the
stimulating electrode (n = 3 slices). In TTX, the
diodes immediately adjacent to the tip of the stimulating electrode did not did not show any of the stimulus artifact seen in the single diode
over the stimulus electrode, demonstrating lack of "cross-talk" between diodes.
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The characteristics of the evoked optical signal were first examined using the local (La) response (Fig. 1A). Phase 1 was composed of two kinetically distinguishable components (solid arrow, Fig. 3, A and B). The phase 1 fast component was presumably a fast sodium potential, since the peak was not changed by DNQX (20 µM), bicuculline (BIC; 20 µM), or removal of Ca2+ from the medium, but it was blocked completely by TTX (0.5 µM; Figs. 3 and 4; n = 3 slices). The slower component of phase 1 was reduced by application of DNQX by 31 ± 6%, and addition of BIC to the DNQX perfused slice had little effect on this phase (Fig. 3A; n = 4 slices; Fig. 3A). However, D-APV did not change the slope or amplitude of the phase 1 slow component even in the presence of BIC (n = 5 slices; in Fig. 3B, arrowhead indicates the peak of the phase 1 slow component). Therefore we concluded that the phase 1 slow component was mainly composed of a synaptically activated AMPA receptor-mediated potential. Note that BIC alone increased both the slope and amplitude of the phase 1 slow component (Fig. 3B), indicating that BIC facilitated neurotransmitter release.
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As DNQX greatly deepened the phase 2 valley (Fig. 3A), the AMPA receptor-mediated potential was also a significant component of this phase. GABAA inhibition also played an important role in forming the phase 2 valley as BIC suppressed the valley (Figs. 3B and 6, A and C). The inhibition by D-APV started at phase 2 (Fig. 3B), indicating a small composition of the NMDA component in this phase. Therefore phase 2 was primarily a combination of rapid decay of AMPA potentials, GABAergic inhibition, and subsequent activation of phase 3.
Phase 3 was less sensitive to DNQX (Fig. 3A), and the
residual potential after DNQX was only observed in diodes proximal to the stimulation site (within 400 µm), suggesting that it was composed of nonsynaptically activated components. DNQX inhibition of phase 3 was
only 13 ± 2%, significantly smaller compared with inhibition of
phase 1 (n = 5, P < 0.01; Fig. 7). On
the other hand, D-APV inhibited phase 3 by 17 ± 3%
but had no significant effect on phase 1 (n = 5; Fig.
7). To determine the composition of phase 3, we applied several calcium
channel blockers including nifedipine (100 µM, an L-type channel
blocker), Ni2+ (50 µM, a T-type channel
blocker), agatoxin-TK (50 nM, a P/Q-type channel blocker),
-conotoxin MVIIA (100 nM, a N-type channel blocker) in the presence
of DNQX, D-APV, and BIC (n = 3 slices). Phase 3 was sensitive to nickel and
-conotoxin MVIIA (Fig. 4), while
nifedipine and agatoxin had little effect (data not shown). After
addition of Ca2+-free medium, we observed a
calcium-independent component remaining in phase 3. This component was
blocked by TTX (0.5 µM), indicating a slow sodium-dependent potential
in phase 3.
We also attempted to remove the contribution from active glutamate
reuptake to the phase 3 depolarization using THA (500 mM, reportedly an
inhibitor of glutamate uptake following trains of stimulation)
(Momose-Sato et al. 1998; Tong and Jahr
1994
). However, we found that THA appeared to affect the signal
in a complex manner. In control conditions, it depressed both phase 1 and 3 (data not shown), which is consistent with a previous finding
that THA inhibited glutamate transmission in the amygdala (Wang
et al. 1995
). However, based on the observation that there was
little response left in BL after treatment with DNQX and
D-APV (Fig. 6), we did not consider that glutamate reuptake
significantly contributed to the residual signal. Adding BIC into
slices perfused with DNQX significantly enhanced phase 3 over its level
in the presence of DNQX alone (Figs. 3A and 7), indicating
that GABA blockade facilitates activation of voltage-dependent channels.
Effects of DNQX, D-APV, and BIC on the propagation of the signal
Stimulation of La generated optical signals that propagated to BL and AStr (Figs. 1C and 5B). The traces of BL responses were generated by averaging a cluster of 7 diodes at BL centered 0.5 mm away from the tip of the stimulating electrode. Applying DNQX (20 µM) caused a significant block of the signal spreading toward both BL and AStr (Fig. 5B), and the optical signal was limited to La. This residual distal trace consisted of a fast sodium spike and slow potential as shown by the optical traces (Fig. 5A). In comparison to traces from the La local response, traces from distal areas appeared to be less complex, as the phases (see above) were much less distinct. All effects of DNQX, D-APV, and BIC on the traces recorded in AStr were qualitatively similar to those of BL (data not shown).
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On the other hand, D-APV (50 µM) did not significantly change the area of spreading but caused a small reduction of the optical signal in phase 3 (Fig. 5, C and D). Comparing the reduction caused by D-APV in local (La) and distal (BL) with or without BIC, we found that NMDA component did not play a significant role in gating the signal propagation (Figs. 5D and 6D).
Addition of BIC (20 µM) greatly enhanced both the intensity of responses (Fig. 6, A and C) and the area of propagation (Fig. 6, B and D) to involve areas that were previously silent, in particular portions of the central amygdala nucleus. Addition of DNQX still significantly blocked the propagation and reduced the intensity of the response (Fig. 6, A and B), while D-APV still did not block signal propagation and only caused a reduction in the late (phase 3) depolarization (Fig. 6, C and D).
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Analysis of peak responses showed that the percentage inhibition by DNQX in BL (81 ± 3%) was significantly greater than in La (31 ± 6%; n = 5; P < 0.01; Fig. 7). In contrast, inhibition of responses by D-APV was similar in La (17 ± 3% for phase 3 of La) and BL (15 ± 1%; n = 5; Fig. 7). In the presence of BIC, the enhancement of the peak responses was 28 ± 5% for phase 1 of La and 88 ± 10% for BL peak, demonstrating a significantly greater effect in the distal region (BL; n = 5; P < 0.01; Fig. 7). In the presence of BIC, the peak of phase 1 was measured at the inflection of the initial response as indicated by the open arrowhead in Fig. 3B. This determination of the peak of the evoked response was used to minimize possible contamination by epileptiform activity during the late phase of the response in the presence of BIC.
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The latency to peak was also examined. The latency to peak was 5.5 ± 0.2 ms for phase 1 of La and 10.6 ± 0.3 ms for BL. Given the distance between the two measured areas was about 400 µm, an estimated velocity of 78.4 ± 6.9 mm/s (n = 5) was obtained for the evoked signal transmission. Because the peaks indicated the maximum synaptic activation, the velocity reflected the propagation of synaptic transmission. When the synaptic transmission was blocked with DNQX, the latency to peak was 2.5 ± 0.2 ms for phase 1 of La and 4.0 ± 0.2 ms for BL, respectively, an approximate velocity of 266.7 ± 61.6 mm/s (n = 5). Such a velocity reflected nonsynaptic conduction through the neural fibers, which is considerably faster than the velocity for synaptic transmission. BIC and D-APV had no significant effects on the latencies (or the velocity) of signal propagation.
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DISCUSSION |
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Clinical studies suggest that the amygdala is necessary for
processing various types of emotional information (Adolphs et al. 1994, 1998
; Halgren and Chauvel
1993
; LaBar et al. 1998
). This idea is supported
by animal models of experimental anxiety that indicate a critical role
for the amygdala in the acquisition and expression of fear-conditioned
behaviors (Davis 1992
; Davis et al. 1994
;
Fanselow and LeDoux 1999
). In addition, other workers have emphasized the importance of the amygdala in memory and
attentional processes (Gallagher and Holland 1994
;
McGaugh et al. 1993
), which, along with emotional
processing and autonomic regulation, may demonstrate a continuum of
functionality within the temporal lobe. The amygdala subserves these
functions through a unique set of external connections and intrinsic
networks (Pitkanen et al. 1997
).
Previous studies using visualization of voltage-sensitive dyes with a
photodiode array have focused on evoked and spontaneous activity in
neocortex (Tsau et al. 1999; Wu et al.
1999b
), olfactory bulb (Keller et al. 1998
),
piriform cortex (Demir et al. 1998
), and thalamocortical
pathways (Laaris et al. 2000
). In neocortex, spread of
epileptiform activity was visualized following superfusion with either
Mg2+-free medium or normal medium containing BIC
(Tsau et al. 1999
; Wu et al. 1999b
).
These studies indicated that populations of neocortical neurons form
dynamic ensembles that propagate waves of excitation that are distinct
from epileptiform activity. In the above studies, the spatiotemporal
aspects of the activities could be examined with much greater
resolution than with conventional field electrode recording. A previous
study using optical imaging showed that signal propagated to the medial
and central amygdala nuclei following stria terminalis stimulation
(Nakanishi et al. 1998
). However, this study did not
provide optical traces to allow comparison with our data.
We observed several novel findings with this technique in the in vitro
amygdala preparation. First, our data indicate that the local optical
signal produced by electrical stimulation in the La contains multiple
physiological components that may be isolated pharmacologically. A
significant portion of this signal was dependent on synaptic
transmission and included responses to both excitatory and inhibitory
neurotransmitters. However, in the region near the stimulating
electrode, the stimulus also produced a large signal that was
independent of glutamatergic and GABAergic synaptic transmission. This
signal appeared to be due to activation of voltage-dependent ion
channels and was significantly attenuated by blockers of
voltage-dependent Ca2+ channels, although we
cannot as yet rule out an additional minor component due to active
reuptake of glutamate or other transmitters (Tong and Jahr
1994). The depolarization associated with the
Ca2+ channels was of slower onset but
significantly greater duration than the fast synaptic depolarization
mediated by AMPA receptors. These voltage-dependent cation channels in
the vicinity of the stimulus electrode contribute significantly to the
total depolarization at the site of origin of the efferent fibers and
may regulate patterns of efferent fiber spike activity. However,
addition of DNQX reduced the amplitude of all phases of the optical
signal, including this late depolarization, suggesting that local
activation of voltage-dependent channels is dependent in part on
excitatory synaptic activity.
Second, the signals propagated to areas away from the stimulus site appeared to be almost entirely dependent on synaptic activity, and specifically AMPA receptor-mediated depolarization. The AMPA receptor antagonist DNQX could effectively block the signal to all target areas outside the La, and this effect persisted when the preparation was disinhibited with BIC. However, regardless of whether BIC was present, D-APV had minimal effect on the propagation of the signal, suggesting that NMDA receptors have little role in mediating the extent of synaptic activation.
GABAergic inhibition also regulated the extent of signal
propagation, as seen by the effect of disinhibition with BIC. Under this condition, depolarization was observed in several previously silent areas of the amygdala, including portions of Ce. Such gating of
inputs to Ce by GABAergic interneurons has previously been suggested on
the basis of anatomic and electrophysiological data (Royer et
al. 1999). An interesting observation was that BIC produced a
significantly larger enhancement of phase 3 or the late depolarizing response in distal areas. The enhancement of phase 3 by BIC is probably
mediated by multiple mechanisms. First, BIC should potentiate glutamate
release, thereby activating more glutamate receptors. Second, BIC may
also block Ca2+-dependent
K+ channels (Khawaled et al.
1999
), which may subsequently cause prolonged activation of the
voltage-gated Ca2+ or Na+
channels that are major components of phase 3. In fact, our previous study demonstrated an increased Ca2+ conductance
in the presence of BIC (Calton et al. 2000
). Third, the
evoked epileptiform burst activity in the presence of BIC (Gean
and Chang 1991
) may also contribute to phase 3. Therefore BIC,
even in the presence of DNQX and D-APV, may enhance the
voltage-gated components of phase 3 to an extent greater that the
combined voltage-gated and NMDA components in the control condition
(Fig. 6C). We also occasionally observed spontaneous,
nonevoked synchronized activities in the presence of BIC. These
spontaneous activities always arose from the lateral side of the BL
(corresponding to the bottom left corner of the photodiode
frame) and did not propagate to other areas of the amygdala. The
amplitude was much smaller than that of the evoked response, and the
duration was also shorter (about 150 ms) than that of the evoked
response (beyond 300 ms).
Optical recordings of excitatory synaptic responses in the rat
olfactory bulb appear qualitatively similar to our findings in the
amygdala, as CNQX significantly attenuated the signal, while APV
reduced a small residual component (Keller et al. 1998). However, in contrast to the amygdala, BIC had little effect on the
optical signal, suggesting minimal endogenous inhibitory tone mediated
by GABAA receptors. In mouse barrel cortex, BIC
increased the amplitude of the optical signal evoked by thalamic
stimulation, without affecting the spatial propagation, while
superfusion of Mg2+-free medium produces an
APV-sensitive spread of excitation to adjacent cortical areas
(Laaris et al. 2000
).
We observed that all residual components of the optical signal, including the initial fast transient, were eliminated by addition of TTX. Under these conditions, the only detectable signal was the stimulus artifact that was confined to the single diode overlaying the tip of the stimulating electrode. Thus current spread from the stimulus did not appear to be a factor in any component of the physiological signal, even from diodes immediately adjacent to the stimulus site. We have also shown that the optical signal was not contaminated by nonspecific artifacts, as filtering out the signal at the wavelength of the dye response completely eliminated the signal.
Our observations of transmitter-dependent activation of the basolateral
complex is consistent with the data obtained from single neurons using
intracellular recordings (Calton et al. 2000; Rainnie et al. 1991a
,b
; Washburn and Moises
1992
) and appears to accurately reflect the time course of
monosynaptic activation observed in those studies. However, we have
also demonstrated the extent to which the spatial distribution of the
synaptic response is dependent on glutamatergic and GABAergic
transmission. We hypothesize that the small signal remaining after
blockade of glutamatergic and GABAergic neurotransmission may be due to
the depolarization of afferent fibers and terminals projecting into the
BL, antidromic activation of neuronal elements in the BL, or due to a
synaptic response produced by other transmitters in this pathway (e.g., neuropeptides).
We suggest that use of the photodiode array in conjuction with
voltage-sensitive dyes should greatly enhance our understanding of
network functioning in the amygdala. In the amygdala formation, we have
so far demonstrated that internuclear signal propagation is determined
by non-NMDA and GABA receptors, while NMDA receptors participate to a
much lesser extent. Given the apparent critical role of the amygdala in
generating fear and anxiety responses (Davis 1992;
Davis et al. 1994
), we speculate that this gating action
may account for the potent anti-anxiety effect of drugs that facilitate
GABAA receptor-mediated activity (Davis
et al. 1994
; Sanders and Shekhar 1995
). We also
suggest that modulation of AMPA receptors may have similar anxiolytic effects.
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ACKNOWLEDGMENTS |
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This work was supported by a Veterans Affairs Merit Review and National Institutes of Health Grants R29 AA-10994 (to S. D. Moore) and R01-NS-32125 (to W. A. Wilson).
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FOOTNOTES |
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Address for reprint requests: C. Wang, Rm. 31, Bldg. 16, DVAMC, 508 Fulton St., Durham, NC 27705 (E-mail: joewang{at}duke.edu).
Received 4 December 2000; accepted in final form 24 April 2001.
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REFERENCES |
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