Laboratory of Sensory Neuroscience, Institute of Neuroscience and Department of Psychology, Carleton University, Ottawa, Ontario K1S 5B6, Canada
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ABSTRACT |
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Kelly, Jack B. and
Sean A. Kidd.
NMDA and AMPA Receptors in the Dorsal Nucleus of the Lateral
Lemniscus Shape Binaural Responses in Rat Inferior Colliculus.
J. Neurophysiol. 83: 1403-1414, 2000.
Binaural responses of single neurons in the rat's central nucleus of
the inferior colliculus (ICC) were recorded before and after local
injection of excitatory amino acid receptor antagonists (either
1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium [NBQX], (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid [CPP], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX], or
(±)-2amino-5-phosphonovaleric acid [APV]) into the dorsal
nucleus of the lateral lemniscus (DNLL). Responses were evoked by
clicks delivered separately to the two ears at interaural time delays
between 1.0 and +30 ms (positive values referring to ipsilateral
leading contralateral click pairs). The neurons in our sample were
excited by contralateral stimulation and inhibited by ipsilateral
stimulation, and the probability of action potentials was reduced as
the ipsilateral stimulus was advanced. Binaural inhibition resulted in
response suppression that lasted up to 30 ms. Injection of excitatory
amino acid antagonists into the DNLL contralateral to the recording
site reduced the strength of binaural inhibition in the ICC. The
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor
antagonist NBQX preferentially affected responses at small interaural
time intervals (0-1.0 ms), whereas the
N-methyl-D-aspartate (NMDA) antagonist CPP
preferentially affected responses at longer intervals (1-30 ms). Both
CNQX and APV produced a release from binaural inhibition, but neither
drug was selective for specific intervals. The data support the idea
that binaural inhibition in the rat ICC is influenced by both AMPA and
NMDA receptor-mediated excitatory events in the contralateral DNLL.
The results suggest that the AMPA receptors contribute selectively to
the initial component of binaural inhibition and the NMDA receptors to
a longer lasting component.
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INTRODUCTION |
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Previous studies have shown that the dorsal
nucleus of the lateral lemniscus (DNLL) exerts an inhibitory influence
on the contralateral central nucleus of the inferior colliculus (ICC) and that blockage of synaptic activity in the DNLL by local injection of the excitatory amino acid antagonist kynurenic acid or other pharmacological agents reduces the strength of the binaural inhibition imposed on ICC neurons (Faingold et al. 1993;
Kelly and Li 1997
; Kidd and Kelly 1996
;
Li and Kelly 1992
). Injection of kynurenic acid affects
responses to dichotically presented sounds with either binaural time or
intensity differences and reduces the strength of inhibition in the
contralateral ICC of rats at either short (0-1 ms) or long (1-30 ms)
interaural time delays (Kidd and Kelly 1996
; Li
and Kelly 1992
). No changes have been found in binaural responses in the ICC ipsilateral to the injection site. These results
indicate that the excitatory responses in the DNLL make an important
contribution to binaural responses in the contralateral ICC.
Brain slice studies have shown that both
N-methyl-D-aspartate (NMDA) and
non-NMDA receptors are involved in the synaptic excitation evoked in
the DNLL by electrical stimulation of the lateral lemniscus (Fu
et al. 1997; Wu and Kelly 1996
). A
single-current pulse delivered to the lateral lemniscus elicits both
early and late excitatory postsynaptic potentials (EPSPs) and
excitatory postsynaptic currents (EPSCs), which can then be blocked
respectively by non-NMDA and NMDA receptor antagonists. The early
component of the synaptic response is thought to be mediated primarily
by
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptors and the longer lasting component by NMDA receptors in the DNLL.
Given that most neurons in the DNLL are GABAergic (Adams
and Mugnaini 1984; Glendenning and Baker 1988
;
González-Hernández et al. 1996
; Moore
and Moore 1987
; Roberts and Ribak 1987
;
Shneiderman et al. 1988
; Thompson et al.
1985
; Vater et al. 1992
; Winer et al.
1995
; Zhang et al. 1998
) and that most of these
neurons (70% in the rat) project directly to the contralateral ICC and
DNLL (Adams 1979
; Bajo et al. 1993
;
Beyerl 1978
; Brunso-Bechtold et al. 1981
;
Coleman and Clerici 1987
; Covey and Casseday
1995
; Hutson et al. 1991
; Ito et al.
1996
; Kudo 1981
; Merchán et al.
1994
; Oliver and Shneiderman 1989
; Ross
et al. 1988
; Shneiderman and Oliver 1989
;
Shneiderman et al. 1988
; Tanaka et al.
1985
; van Adel et al. 1999
; Zook and
Casseday 1982
), it seems likely that both NMDA and AMPA
receptors contribute to the release of GABA in the contralateral
auditory midbrain. The time course and duration of the resulting
inhibition would be shaped by the pattern of activation of the NMDA and
non-NMDA receptors in the DNLL.
The purpose of the present study was to examine the relative influence of NMDA and non-NMDA receptors in the DNLL on binaural inhibitory responses in the rat ICC. To achieve this objective, NMDA and AMPA receptor antagonists were pressure injected locally into the DNLL, and binaural responses to paired clicks at various interaural time delays were recorded from neurons in the contralateral ICC before and after blocking the receptors.
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METHODS |
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Physiological procedures
The methods of recording responses and injecting drugs were
similar to those reported previously by Li and Kelly
(1992) and Kidd and Kelly (1996)
. Briefly,
binaural responses to paired clicks were recorded from single neurons
in the ICC before and after pressure injection of AMPA antagonists
(either
1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium [NBQX] or 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) or
NMDA antagonists (either
[±])-3-[2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid [CPP]
or [±]-2-amino-5-phosphonovaleric acid [APV]) into the
contralateral DNLL (Fig. 1). The response
probability of ICC neurons was determined by manipulating the
interaural time difference (ITD) between the clicks over the range from
1.0 ms to +30 ms, where positive values refer to ipsilateral leading
contralateral ITD intervals. The laterality of the stimulus
(contralateral and ipsilateral) refers to the position of the ear
relative to the recording site in ICC.
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Successful experiments were performed on 51 male Wistar albino rats
(250-450 g) from Charles River, St. Constant, Quebec, Canada. The
animals were initially anesthetized with pentobarbital sodium (60 mg/kg, ip) and subsequently maintained in an areflexive state by
injections of Equithesin [0.5 ml/kg, ip; see Sally and Kelly (1992) for preparation]. The animals were placed in a
head holder that left the external ear canals free for insertion of earphone drivers. A midline incision was made in the scalp, and the
tissue was retracted to expose the skull. Two craniotomies were made to
allow penetration of recording and injection pipettes into the inferior
colliculus and the DNLL, respectively.
The stereotaxic coordinates for positioning the recording and injection
pipettes were referenced from lambda with the skull flat
(Paxinos and Watson 1997). For placements in the DNLL,
the injection pipette was tilted 30° in the sagittal plane and
lowered into the brain to a depth of 7.8 mm from a point 6.7 mm lateral and 0.3-0.4 mm rostral to lambda. The injection pipette doubled as a
recording electrode to monitor neural activity in the DNLL as described
previously by Li and Kelly (1992)
, and the position of
the pipette was fine-tuned to give the best acoustically driven response.
Recording pipettes were pulled from single-barrel glass tubing (Sutter,
1.0 mm OD, 0.5 mm ID) to a tip diameter of approximately 2 µm. They
were back-filled with 2 M saline and had impedances between 1.5 and 2.5 M.
The recording pipettes were inserted into the inferior colliculus with
the use of a Kopf Model 650 micropositioner. The final position was
adjusted to obtain short-latency, acoustically driven responses that
exhibited narrow-frequency tuning and a clearly defined characteristic
frequency (CF, the frequency to which a neuron responded at the lowest
sound pressure level). As previously reported (Kelly et al.
1991; Syka et al. 1981
), the CFs of neurons in
the inferior colliculus showed a reliable progression from low to high
frequencies as the recording pipette was lowered through the central
nucleus. Histological reconstructions confirmed the location of all
electrode placements in the ICC.
Injection pipettes were pulled from single-barrel glass tubing (Sutter, 1.0 mm OD, 0.5 mm ID) to a tip diameter of 20-40 µm. The pipettes were back-filled with excitatory amino acid receptor antagonists in normal saline. They were connected by a short length of flexible tubing to a small chamber constructed from two disposable hypodermic needles positioned back to back. A thin wire was fitted and glued in place between the two needles with one end extending within and along the shaft of the needle and into the tubing so that it could make contact with the solution in the pipette. The other end of the wire emerged from the junction between the two needles and was connected to a preamplifier for electrical recordings. The end of the second needle was connected by a longer length of flexible tubing to a 5-ml syringe for pressure injections. The volume of the injection (1.5-2.0 µl) was controlled by monitoring the progression of the solution along the length of the pipette, and the flow was stopped by releasing pressure through a three-way stopcock.
The drugs used were NBQX, CNQX, CPP, and APV. All were obtained from Research Biochemicals International (catalog numbers N-183, C-127, C-104, and A-110, respectively). The drugs were applied at various concentrations to determine the limits of their effectiveness under the conditions of our experiment. The vehicle for delivery of the drug was physiological saline. To avoid possible accumulated effects associated with multiple injections, only one drug at one concentration was tested per animal. The single neurons tested with different pharmacological agents were recorded from separate rats.
Physiological potentials were amplified by a Dagan EX4-400 amplifier, displayed on oscilloscopes, and monitored acoustically over a loudspeaker. Neural responses were digitized and processed by MALab 881, a data acquisition system designed and produced by Steve Kaiser (Department of Neurobiology, University of California, Irvine; Kaiser Instruments) for use with Macintosh computers (in our case, a Quadra 700). The program provided a digital window discriminator for selection of action potentials and displayed poststimulus time histograms on-line. Physiological responses were stored on optical disk and processed later with standard database and graphics software.
All procedures were approved by the Carleton University Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care.
Stimulus parameters
Sounds were presented separately to the two ears through sealed headphones (Pioneer SE-50D) coupled to hollow specula that were inserted into the rat's external ears. Sounds were generated digitally by a Kaiser Instruments DA interface controlled by MALab 881 to produce either tone pulses (100 ms with 10-ms rise and fall times) or clicks (50 µs square waves). The clicks had a broad spectrum from 0.1 to 25 kHz, essentially flat up to 4.0 kHz and rolling off at higher frequencies. The sound pressure of tone pulses was referenced to a cell's threshold at CF, and the sound pressure of clicks was calibrated in dB sound pressure level (SPL; re 0.0002 dynes/cm2) using a 0.5-in. B&K microphone with the headphone speculum inserted into a Tygon enclosure that served as an artificial ear. For most experiments the sound pressure level of clicks delivered to either ear was fixed at 20 dB above the threshold for eliciting a contralateral excitatory response.
All recordings were obtained from well-isolated single units defined by
action potentials of constant amplitude and waveform. Before
investigating responses to binaural time differences, we examined the
neural response to tone pulses. First, the CF was determined with
monaural stimulation of the contralateral ear. Then the binaural
response pattern to either tone or click stimulation was determined by
setting the contralateral stimulus level at 20 dB above threshold and
presenting ipsilateral sounds simultaneously in steps of increasing
intensity. In some cases, both ipsilateral and contralateral
stimulation produced excitation, and combined stimulation resulted in
facilitation. The majority of cells, however, showed binaural
suppressioni.e., the contralateral response was strongly inhibited by
simultaneous ipsilateral stimulation. Among the neurons that exhibited
binaural suppression, some showed a slight response facilitation at low
levels of ipsilateral stimulation, but strong suppression as the
ipsilateral level was increased. All the recordings in the present
study were made from neurons showing strong binaural suppression.
The rat's range of hearing is restricted primarily to high
frequencies, and most of the neurons in its central auditory pathway have CFs above 1 kHz (Kelly and Masterton 1977;
Kelly and Phillips 1991
; Kelly et al.
1991
; Sally and Kelly 1988
). Because
phase-locking is not secure at these frequencies, most of the neurons
in the rat ICC are insensitive to the ongoing time (i.e., phase)
differences between tones presented to the two ears. Therefore, after
the responses to tone bursts had been recorded, ICC neurons were tested with transients (clicks) to determine their response to binaural time
differences. The clicks were delivered to the left and right ears in
pairs at various ITD intervals, and each click pair was repeated 30 times at a rate of one per second. The sound pressure level of the
clicks was the same in both ears. The probability of a spike was
determined for a wide range of ITDs from
1.0 to +30 ms (positive
values representing ipsilateral leading contralateral time
differences). The data were plotted separately for small ITDs (+1.0 to
1.0 ms) and large ITDs (1.0-30 ms). The smaller intervals were
chosen to span the range of ITDs produced by a single sound source
located at various free-field positions in the azimuthal plane and to
bracket the dynamic range of responses of neurons to small ITD
intervals. The larger intervals were explored to determine the duration
of the inhibition produced by stimulation of the ipsilateral ear. For
each neuron, responses to both short and long binaural time intervals
were examined before and after injection of pharmacological agents into
the DNLL.
Quantitative and statistical analysis
The effects of drug injection on each neuron were expressed as indices of response change for short and long binaural time intervals separately. For the short intervals the index was calculated by measuring the difference in spike counts before and after drug injection for ITDs of 0.0, 0.25, 0.50, 0.75, and 1.0 ms and averaging over the five ITD periods to give a single measure of response change, with positive values indicating an increase in the number of spikes. For the long time intervals the index was calculated by measuring the differences in spike counts for 2.0, 4.0, 6.0, 8.0, 10, and 12 ms and averaging over the intervals to obtain a single mean value. A positive index reflects an increase in spikes. The effects of antagonists were evaluated by plotting the indices of response change as a function of drug concentration. Statistical comparisons were made using the Kruskal-Wallis nonparametric ANOVA to determine the effect of drug concentration. Further comparisons between short and long binaural time intervals were made using the Mann-Whitney U test.
Histology
The positions of recording and injection pipettes were marked by passing positive current through the electrode to produce a small lesion. In preparation for histology the animals were given an injection of pentobarbital (120 mg/kg ip) and perfused through the heart with normal saline followed by 10% formalin. The brains were removed, stored in 20% sucrose-10% formalin and cut serially at 40 µm in the frontal plane on a freezing microtome. The location of pipette tracks and lesions at the pipette tips were determined microscopically from cresyl violet-stained sections. All data presented here were obtained from cases in which the injection and recording pipettes were clearly in the DNLL and ICC, respectively.
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RESULTS |
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NBQX
Injection of the AMPA antagonist NBQX produced a consistent
release from binaural inhibition as shown in Fig.
2. Responses to click
pairs are plotted separately for short (1.0-1.0 ms) and long (1-30
ms) ITD intervals. The CFs as determined by the response to
contralateral tone bursts are shown at the top of each pair of graphs.
The binaural response pattern for each of these cells was contralateral
excitatory and ipsilateral inhibitory (EI). Before drug injection, the
cells were strongly inhibited by acoustic stimulation of the
ipsilateral ear. Click pairs with contralateral lead times usually
evoked action potentials after each stimulus presentation, but as the
binaural time difference was shifted in favor of the ipsilateral ear,
the probability of an action potential progressively decreased. The
duration of the inhibitory effect varied from cell to cell but in most
cases lasted for 15-20 ms.
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At concentrations of 5.0 and 2.5 mM, NBQX reduced the strength of the binaural inhibition imposed on cells in the contralateral ICC. At both concentrations the effect of NBQX was most evident at short time delays with relatively little effect at longer ITD intervals. Release from inhibition was seen for cells with widely varying CFs ranging from 3.5 to 22.6 kHz. At a concentration of 1.25 mM NBQX had an inconsistent effect on binaural responses. In two cells (1.2 mM, Fig. 2, A and C) there was a release from inhibition that was most pronounced at short interaural time delays, but in two other cells (B and D) there was no effect.
As shown in Fig. 3, A and B, a 0.75-mM concentration of NBQX had no systematic effect on binaural responses of neurons in the contralateral ICC. The results indicate that the injection procedure itself was without effect and that the action of NBQX was concentration dependent.
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CPP
Injection of a 10 mM concentration of the NMDA antagonist, CPP, produced a consistent release from binaural inhibition in each of the five cells tested (Fig. 4). The effect was most apparent at long time intervals. The strength of inhibition produced by stimulation of the ipsilateral ear was reduced at intervals between 1 and 30 ms in all five cases (Fig. 4, A-E). In three of these cases (A, B, and E) there was no corresponding release at shorter time intervals, although some effect was apparent in two other cases (C and D). Injections of CPP at lower concentrations (5.0, 2.5, and 1.25 mM) had no consistent effect on binaural responses (see Table 1 for summary).
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CNQX and APV
Injection of 10 mM CNQX into the DNLL resulted in a release from binaural inhibition at both long and short ITD intervals (Fig. 5). Binaural responses recorded from the contralateral ICC were less strongly suppressed after the drug injection, but there was no indication of a selective effect on short versus long ITD intervals. There was a less pronounced release from inhibition after injection of 5.0 mM CNQX. Little or no effect was recorded with concentrations of either 2.5 or 1.25 mM (see Table 1 for summary).
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Injection of 30 mM APV resulted in a release from binaural inhibition that was apparent at both short and long ITD intervals (Fig. 6). A less pronounced release from inhibition was seen after injection of 15 mM APV. At this concentration the effect was most evident at long ITD intervals, but there was also some release at short ITD intervals. Lower concentrations (7.5 or 3.7 mM) were without effect on binaural responses of neurons in ICC (see Table 1).
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The mean response change produced by each of the four excitatory amino acid antagonists is shown in Fig. 7. Response change is plotted as a function of drug concentration for both long and short binaural time delays. For each of the drugs the magnitude of the release from binaural inhibition increased with concentration. The AMPA receptor antagonist NBQX had a greater effect at short time intervals, whereas the NMDA antagonist CPP had a greater effect at long time intervals. CNQX and APV had effects at both long and short ITD intervals, although there was some tendency for selectivity at moderate concentrations.
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Statistical results
ANOVA (Kruskal-Wallis) showed a significant effect of drug concentration for each of the four excitatory amino acid antagonists used in this study (CNQX, NBQX, APV, and CPP). For CNQX there was an increased release from inhibition at both short and long interaural time intervals (H = 8.36, P < 0.01 and 7.72, P < 0.01 respectively). For NBQX the increase was significant for the short intervals only (H = 6.24, P < 0.05); no differences were found for the longer time intervals (H = 0.76). For both APV and CPP significant differences were found for long intervals (H = 6.72, P < 0.05 and H = 6.76, P < 0.05, respectively), but the differences for short intervals were not statistically significant (H = 3.95 and H = 1.45 respectively).
The magnitude of response change produced at the highest drug concentrations was compared for long and short ITD intervals using the Mann-Whitney U test. Injection of NBQX at 5.0 and 2.5 mM concentrations was found to have a significantly greater effect on responses to short time intervals (U = 6, P < 0.002). A significant difference was also found for 5.0 mM CPP (U = 1, P < 0.008) with the greater change occurring at long time intervals. There were no statistically significant differences between long and short intervals for either APV or CNQX at the concentrations used in this study.
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DISCUSSION |
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The DNLL-ICC circuit
The results of our study show that the DNLL plays an important role in the regulation of binaural responses in the contralateral ICC. Pharmacological block of excitatory activity in the DNLL by local injection of either AMPA or NMDA receptor antagonists results in a release from binaural inhibition in the contralateral ICC. In our sample of EI neurons, binaural responses were altered in every case provided that a sufficient concentration of drug was injected into the DNLL. After pharmacological blockage, the ICC neurons were less strongly inhibited by stimulation of the ipsilateral ear, and their binaural response curves were shifted relative to normal.
These data confirm the results of earlier studies in which kynurenic
acid was injected into the DNLL (Kelly 1997;
Kelly and Kidd 1997
; Kidd and Kelly 1996
;
Li and Kelly 1992
). Local injection of kynurenic acid, a
nonspecific excitatory amino acid antagonist, resulted in a consistent
release from binaural inhibition and a consequent shift in binaural
response curves of ICC neurons located contralateral to the injection
site. The reduction in inhibition was apparent over a wide range of
interaural time delays and affected the sensitivity of cells to either
binaural time or intensity differences (Kidd and Kelly
1996
; Li and Kelly 1992
). Evidence of
disinhibition was found in both the contralateral ICC and DNLL, but no
effect was seen when the recordings were made in the ICC ipsilateral to
the injection site (Kelly and Kidd 1997
; Li and
Kelly 1992
).
Our interpretation of the contribution made by the DNLL to binaural
responses in the ICC is presented in Fig.
8. First, we recognize that binaural
responses in the mammalian auditory system are established in the
superior olivary complex through neural circuits in the lateral and
medial superior olivary nuclei (LSO and MSO, respectively)
(Irvine 1992). Neurons within the LSO are typically
excited by stimulation of the ipsilateral ear and inhibited by
stimulation of the contralateral ear (Boudreau and Tsuchitani 1968
; Caird and Klinke 1983
; Finlayson
and Caspary 1989
; Tsuchitani 1988
). The
contralateral inhibition of LSO neurons is imposed through a
glycinergic projection from the medial nucleus of the trapezoid body
(MNTB), which itself receives an excitatory projection from the
cochlear nucleus on the opposite side of the brain (Sanes 1990
; Wu and Kelly 1991
). The neurons in LSO
then project either ipsilaterally or contralaterally to the auditory
midbrain through the lateral lemniscus. The contralaterally projecting
LSO neurons are considered to be excitatory, and the ipsilaterally
projecting neurons are predominantly glycinergic and presumably
inhibitory (Glendenning et al. 1992
; St. Marie et
al. 1989
). Therefore, preferential stimulation of one ear over
the other, due to either more intense or earlier acoustic input, would
cause a net excitation of midbrain structures contralateral to the
preferred ear. A net inhibition of structures ipsilateral to the
preferred ear would also be expected, because of the glycinergic
projection from LSO (see Fig. 8A). In addition, the MSO
contributes to early binaural processing through converging projections
from the left and right cochlear nuclei (Kuwada et al.
1997a
; Yin and Chan 1990
). The neurons in MSO
are sensitive to binaural phase differences and are maximally excited
by sounds that favor the contralateral ear. Because of the circuitry
within the superior olivary complex (SOC), a free field sound
that results in earlier and/or more intense stimulation of the ear
closer to the sound source would be expected to excite neurons
primarily in the contralateral auditory midbrain (DNLL and ICC).
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The DNLL provides a second level of binaural processing that reinforces
the contralateral bias already established by the SOC (Fig.
8B). The neurons in the DNLL are GABAergic and project heavily to the DNLL and ICC on the opposite side of the brain through
the commissure of Probst (CP). A separate population of DNLL neurons
projects to the ipsilateral ICC (Adams and Mugnaini 1984; Glendenning and Baker 1988
;
González-Hernández et al. 1996
; Moore
and Moore 1987
; Roberts and Ribak 1987
;
Shneiderman et al. 1988
; Thompson et al.
1985
; Vater et al. 1992
; Winer et al.
1995
; Zhang et al. 1998
). In the rat retrograde
tract tracing shows that 70% of the neurons in the DNLL that project
to the ICC have crossed connections, and the remaining 30% have
uncrossed connections (Ito et al. 1996
;
Merchán et al. 1994
). Also, nearly 70% of the
neurons in the DNLL degenerate after cutting the decussating fibers in
the rat CP (van Adel et al. 1999
). Many of the DNLL neurons with crossed projections to ICC have collateral connections with neurons in the contralateral DNLL (van Adel and Kelly
1998
). Thus, the DNLL is in an excellent position to impart a
GABAergic inhibitory influence on the DNLL and ICC on the opposite side of the brain. This contralateral inhibition would have the effect of
magnifying any difference in neural activity that might arise between
auditory structures on the left and right sides of the brain. The
laterality of responses in the central auditory system first
established by circuits within the superior olive would be enhanced by
the contralateral inhibitory projections of the CP. This role of the CP
has been demonstrated recently by recording binaural evoked responses
in the rat ICC before and after surgical transection of the decussating
fibers as they pass the midline just below the medial longitudinal
fasciculus (van Adel et al. 1999
). The result is a
reduction in the strength of the inhibition produced by stimulation of
the ipsilateral ear as reflected in the amplitude of binaural evoked
responses. Pharmacological blockage of excitatory activity in the DNLL
by local injection of the excitatory amino acid antagonist, kynurenic
acid, produces a similar effect, presumably through disruption of the
crossed projections in the CP. Thus, the DNLL and its efferent fibers
in the CP play a role in maintaining the laterality of responses in the
central auditory system and can be considered a part of the "acoustic
chiasm" described previously by Glendenning and Masterton
(1983)
(see also Glendenning et al. 1985
, 1992
).
The concept of a progressive refinement of binaural responses in the
ascending auditory pathway is consistent with the recent results of
Spitzer and Semple (1998) showing emergent properties in
the ICC of the gerbil. The response of ICC neurons to time-varying phase disparities between the two ears is strongly influenced by
dynamic aspects of the stimulus, whereas responses of binaural neurons
in the SOC are largely predictable on the basis of static stimulus
properties. One possible source of this transformation in binaural
response characteristics may be the GABAergic projection from the DNLL.
Furthermore, the neurons in the gerbil ICC show lasting changes in
their response to interaural level differences (ILDs) depending on
prior exposure to binaural stimulation. Dynamic changes in the balance
of binaural stimulation can produce a "conditioned enhancement" or
a "conditioned suppression" of subsequent binaural responses.
Comparable conditioning is not found in the gerbil superior olive
(Spitzer and Semple 1993
, 1995
). The dynamic
conditioning of responses in the ICC can also be induced
pharmacologically by the release of GABA-glycine from the recording
pipette, which shows the importance of inhibition in shaping the
emerging properties of binaural responses in ICC (Sanes et al.
1998
). The crossed GABAergic projection from DNLL is a possible
source of inhibition for generating these dynamic binaural response properties.
The effects of blocking activity in the DNLL are also consistent with
the finding that binaural responses in ICC can be altered by release of
the GABAA antagonist, bicuculline, at the recording site
(Klug et al. 1995; Park 1998
; Park
and Pollak 1993
, 1994
). Bicuculline results in a release from
inhibition that is probably due in part to a reduction in the influence
of crossed GABAergic projections from the DNLL. Park
(1998)
has recently suggested on the basis of his comparison of
responses in the SOC and ICC that binaural inhibitory responses can be
formed locally in the auditory midbrain, a conclusion that is supported
by intracellular recordings from the ICC (Covey et al.
1996
; Kuwada et al. 1997a
,b
; Nelson and Erulkar
1963
; Pedemonte et al. 1997
) and extracellular recordings of binaural responses following SOC lesions (Kelly and Sally 1993
; Li and Kelly 1992b
; Sally
and Kelly 1992
). One possible source of local binaural
inhibition in the ICC is the crossed GABAergic projection from the
DNLL through the CP.
NMDA and AMPA receptors
The present results show that injection of the receptor specific
excitatory amino acid antagonists, NBQX and CPP, into the DNLL alters
the response of ICC neurons to sounds with short and long ITDs
selectively. The AMPA receptor antagonist NBQX results in a
preferential release from binaural inhibition at short ITD intervals
(0-1 ms) and has little effect at longer intervals (1-30 ms). In
contrast, injection of the NMDA antagonist CPP produces a release from
inhibition at the long intervals and has little or no effect at the
shorter intervals. Under the same experimental conditions, neurons
tested with kynurenic acid show a generalized release from inhibition
at both short and long ITD intervals with no indication of selectivity
for specific time delays (Kidd and Kelly 1996).
We attribute the effects of NBQX and CPP to their selective action on
AMPA and NMDA receptors in the DNLL. Both receptor types are present,
and their role in generating postsynaptic responses has been shown by
intracellular recordings in brain slice preparations (Fu et al.
1997; Wu and Kelly 1996
). Excitatory potentials
evoked in the DNLL by electrical stimulation of the lateral lemniscus have two distinct components: an early response that can be blocked by
AMPA antagonists and a later response that can be blocked by NMDA
antagonists (Fu et al. 1997
; Wu and Kelly
1996
). The AMPA antagonists selectively eliminate short latency
action potentials, whereas NMDA antagonists selectively block longer
latency action potentials. Because most of the neurons in the DNLL are
GABAergic, their excitation would be expected to result in the
inhibition of structures to which they project. The early and late
components of excitatory responses in the DNLL would be translated into
an early and late inhibitory action on target neurons in the ICC and
contralateral DNLL. Thus, blockage of AMPA and NMDA receptors by
injection of receptor specific antagonists into the DNLL would be
expected to have selective effects on binaural responses of ICC neurons
to short and long ITD intervals. The results of the present study
confirm this expectation.
Unexpectedly, CNQX was not highly selective in its effect on
binaural responses at specific ITD intervals. At concentrations of 5.0 and 10 mM, it caused a release from binaural inhibition at both short
and long ITDs. One explanation for this apparent nonselectivity may be
that CNQX exerted an indirect effect on NMDA as well as AMPA receptors
at the concentrations used in this study. It is well known that
activation of the NMDA receptors in the hippocampus and other CNS
structures is dependent on a concomitant depolarization mediated by
AMPA receptors. In DNLL, as in these other structures, the NMDA
component of excitatory responses is voltage dependent and subject to a
magnesium block that can be overcome by membrane depolarization
(Fu et al. 1997). Thus, block of AMPA receptors may have
resulted in the elimination of both NMDA and non-NMDA
receptor-mediated excitation in the DNLL. On the other hand, two
observations mitigate against this possibility. First, our brain slice
studies of the DNLL show that AMPA antagonists can block the early
phase of excitatory responses without eliminating the longer lasting,
NMDA receptor-mediated responses. These data indicate that the NMDA
receptors in the DNLL are normally available at or near resting
potential and do not require depolarization through AMPA
receptors for their activation (Fu et al. 1997
;
Wu and Kelly 1996
). Second, the injection of the AMPA
antagonist NBQX into the DNLL in fact produced a selective release from binaural inhibition at short ITD intervals. Thus, even
though some interaction between NMDA and non-NMDA receptor-mediated events almost certainly occurs, this mechanism alone cannot explain why
the effects of CNQX are not selective, whereas those of NBQX under
similar experimental conditions are.
A more likely explanation of the nonselective effect of CNQX is its
relative nonspecificity as a receptor channel antagonist compared with
NBQX. At high concentrations CNQX exerts an antagonistic action at
glycine receptors as well as AMPA receptors (Lester et al.
1989). Because the activation of the NMDA receptor requires the
presence of both glycine and glutamate (Asher and Johnson 1989
), an inadvertent block of the glycine binding site by CNQX would eliminate both NMDA and non-NMDA receptor-mediated responses. In
contrast, NBQX does not block the glycine receptor at high concentrations. Thus, for our studies, which require a relatively high
concentration of drug to produce an effect, NBQX is a superior antagonist and a better choice than CNQX for selectively blocking AMPA receptors.
Injection of the NMDA antagonist APV into the DNLL was also relatively
nonselective in its effect on binaural responses in the contralateral
ICC. At the concentrations used in this study the drug resulted in a
release from binaural inhibition at both long and short ITD intervals.
Although there was some indication of a larger release at long ITDs,
the effect was not statistically significant. It is not known why APV
was less selective than CPP in producing a shift in binaural responses.
However, another investigator has reported greater selectivity
with CPP than APV in studies of other CNS structures (Kita
1996).
Functional implications
The ability to localize sounds in space is dependent on central
processing of small binaural time and intensity cues (Heffner and Masterton 1990). For mammals with small heads, the
interaural time delays produced by sound sources at various positions
in the horizontal plane are considerably <1 ms. For the rat, with an
interaural distance of 3.5 cm, the maximum ITD associated with a sound
positioned on the left or right is at most ±130 µs (Kelly and
Phillips 1991
). In the present study, injection of the AMPA antagonist NBQX into the DNLL primarily affected binaural responses to
ITDs within the range that is useful for localization of a single sound
source (±1.0 ms). The release from inhibition produced by blocking the
AMPA receptors in the DNLL resulted in a shift in the binaural response
of neurons in ICC that would likely degrade the ability to localize a
single sound source. Indeed, our behavioral studies have shown that the
crossed projection from the DNLL is important for maintaining accurate
sound localization. Either surgical transection of the crossed fibers
in the CP or kainic acid lesions of the DNLL result in elevated minimum
audible angles for localization of a brief noise burst (Ito et
al. 1996
; Kelly et al. 1996
). The acuity for
sound localization in the horizontal plane is significantly reduced by
either lesion.
Binaural time differences >1.0 ms are well beyond the range that is
useful for localization of single sound sources. However, several
investigators have suggested that binaural interactions at ITDs >1 ms
may play an important role in localization of multiple sound sources or
the suppression of reflected sounds as demonstrated by the
"precedence" effect (Carney and Yin 1989;
Fitzpatrick et al. 1995
; Kelly and Kidd
1997
; Kidd and Kelly 1996
; Yang and
Pollak 1994a
,b
; Yin 1994
). The results of the present
study indicate that responses in ICC to binaural stimuli with ITDs in
the range of 1-20 ms are selectively influenced by activation of NMDA
receptors in the DNLL. Pharmacological blockage of the NMDA receptor in the DNLL reduces the strength of long-lasting binaural inhibition in
the contralateral ICC, but has relatively little effect on responses to
binaural stimuli with ITDs in the range of 0-1 ms. These observations
suggest that the NMDA receptor in the DNLL plays a role in sensory
processing and serves to regulate the period of GABAergic inhibition
imposed on neurons in the ICC. We suggest that one functional
contribution of the NMDA receptor in the auditory midbrain is to extend
the period of excitation in the DNLL and thus prolong the period of
inhibition in the contralateral ICC, providing a neural mechanism for
echo suppression.
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ACKNOWLEDGMENTS |
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The authors thank B. van Adel for many helpful contributions and Prof. Shu Hui Wu for comments on an earlier version of the paper.
This research was supported by a grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada.
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FOOTNOTES |
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Address for reprint requests: J. B. Kelly, Laboratory of Sensory Neuroscience, 329 Life Science Building, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada.
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 2 August 1999; accepted in final form 19 October 1999.
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REFERENCES |
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