Laboratory of Sensory Neuroscience, Department of Psychology, Carleton University, Ottawa, Ontario K1S 5B6, Canada
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ABSTRACT |
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Zhang, Huiming and Jack B. Kelly. AMPA and NMDA Receptors Regulate Responses of Neurons in the Rat's Inferior Colliculus. J. Neurophysiol. 86: 871-880, 2001. The contribution of N-methyl-D-aspartate (NMDA) and AMPA receptors to auditory responses in the rat's inferior colliculus was examined by recording single-unit activity before, during, and after local iontophoretic application of receptor-specific antagonists. Tone bursts and sinusoidal amplitude modulated sounds were presented to one ear, and recordings were made from the contralateral central nucleus of inferior colliculus (ICC). The receptor specific antagonists, (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) for NMDA receptors and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) for AMPA receptors, were released at the recording site through a multi-barreled pipette. For most neurons, either CPP or NBQX alone resulted in a reversible reduction in the number of action potentials evoked by tonal stimulation. For neurons with an onset response pattern, NBQX either completely eliminated or greatly reduced the number of action potentials. CPP also reduced the number of action potentials but had a less pronounced effect than NBQX. For neurons with a sustained firing pattern, NBQX reduced the total number of action potentials, but had a preferential effect on the early part (first 10-20 ms) of the response. CPP also resulted in a reduction in the total number of action potentials, but had a more pronounced effect on the later part (>20 ms) of the response. These results indicate that both AMPA and NMDA receptors contribute to sound evoked excitatory responses in the ICC. They have a selective influence on early and late components of tone-evoked responses. Both receptor types are involved in generating excitatory responses across a wide range of sound pressure levels as indicated by rate level functions obtained before and during drug application. In addition, both CPP and NBQX reduced responses to sinusoidal amplitude modulated sounds. The synchrony of firing to the modulation envelope as measured by vector strength at different rates of modulation was not greatly affected by either CPP or NBQX in spite of the decrease in firing rate.
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INTRODUCTION |
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The excitatory amino acid,
glutamate, is known to be an important neurotransmitter in the central
nucleus of the inferior colliculus (ICC). Anatomical studies have
revealed the presence of glutamate-immunoreactive neurons (Adams
and Wenthold 1979; Otterson and Storm-Mathisen
1984
) and glutamate binding sites (Cotman and Iversen
1987
; Gaza and Ribak 1997
; Greenamyre et
al. 1984
) throughout the ICC, and two types of glutamate
receptor, N-methyl-D-aspartate (NMDA) and
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) have been
identified (Cotman and Iversen 1987
; Gaza and
Ribak 1997
).
Electrophysiological studies indicate that both NMDA and AMPA receptors
are involved in the generation of excitatory responses in the auditory
midbrain, including the inferior colliculus and the dorsal nucleus of
the lateral lemniscus (DNLL) (Burger and Pollak 1998;
Faingold et al. 1989
, 1991
; Fu et al.
1997
; Wu and Kelly 1996
). In vivo recordings
have shown that iontophoretic application of the glutamate agonist NMDA
results in increased spontaneous and tone-evoked firing of neurons in
rat's ICC and that the effect of NMDA is blocked by simultaneous
application of the NMDA receptor antagonist
2-amino-5-phosphono-valerate (APV). Also, the responses of most neurons
in the ICC are greatly reduced by application of APV alone
(Faingold et al. 1989
, 1991
). These data show that the
NMDA receptor is involved in the mediation and regulation of neural
responses in the adult ICC.
Burger and Pollak (1998) examined the effects of APV on
responses of neurons in the mustache bat's ICC to sinusoidal
amplitude-modulated (SAM) sound and found a reduction in the number of
sound-evoked action potentials. Simultaneous application of
bicuculline, a gamma-aminobutyric acid (GABA) receptor antagonist,
resulted in an increase in the number of action potential, which
off-set the effect of APV. These results support the idea that the NMDA
receptor plays an important role in regulating responses in ICC through the interaction of excitatory and inhibitory synapses. On the other
hand, APV had no effect on the synchrony of discharges to the
modulation envelope or the upper limit of vector strength modulation
transfer functions.
Kelly and Kidd (2000) have shown that both AMPA and NMDA
receptors in the rat's DNLL contribute to binaural responses recorded from single neurons in the contralateral ICC. They examined the effects
of pressure injecting the selective AMPA and NMDA receptor antagonists,
1,2,3,4-tetra hydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) and
(±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP),
respectively, into the rat's DNLL on responses of single neurons in
ICC to paired clicks with binaural time delays and found that NBQX had
a greater effect on the responses to small delays, whereas CPP
preferentially affected responses to longer delays.
Also, in vitro brain slice studies have demonstrated the presence of
both NMDA and AMPA receptor-mediated excitatory responses in the
auditory midbrain. Wu and Kelly (1996) and Fu et
al. (1997)
showed that postsynaptic responses in the rat's
DNLL have early and late excitatory components mediated preferentially
by AMPA and NMDA receptors, respectively. Similar data have been
reported for neurons in the rat's inferior colliculus (Ma et
al. 2001
; Smith 1992
). Clearly distinguishable
early and late components elicited in either DNLL or ICC by electrical
stimulation of the lateral lemniscus can be selectively blocked by bath
application of AMPA and NMDA receptor antagonists, respectively
(Fu et al. 1997
; Ma et al. 2001
;
Wu and Kelly 1996
).
The present in vivo study was undertaken to examine the role of AMPA and NMDA receptors in mediating sound-evoked responses in the rat's ICC. Single-unit responses to contralaterally presented tone bursts and amplitude-modulated sounds were recorded before, during, and after iontophoretic application of the AMPA and NMDA receptor antagonists, NBQX and CPP. The effects of drugs on spike count were determined for tone bursts over a wide range of sound pressure levels including near-threshold stimuli to obtain rate level functions. Both firing rate and response synchrony (vector strength) were determined for SAM stimuli over a wide range modulation rates to generate modulation transfer functions. The results show that both AMPA and NMDA receptors are involved in processing acoustic stimuli in the adult rat's ICC.
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METHODS |
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Animal preparation
Experiments were conducted on 37 male adult Wistar albino rats (Rattus norvegicus; 300-450 g) obtained from Charles River Canada, St. Constant, Quebec. The external ears and tympanic membranes of all animals were examined with an otoscope and determined to be free of infection or other abnormalities. Surgical anesthesia was induced initially with pentobarbital sodium (60 mg/kg ip), and a state of areflexia was maintained throughout the course of an experiment by supplemental injections of ketamine hydrochloride (30 mg/kg im) or pentobarbital sodium (20 mg/kg ip) at intervals of about 1 h. No systematic differences were noticed in the effect of the anesthetic on the recordings. A midline incision was made in the scalp, the skin and muscles were retracted laterally, and a small craniotomy was made to permit insertion of a pipette assembly for recording and drug release into the ICC. Small bone screws were placed in the skull and fixed to a stainless steel rod with dental acrylic. The rod was then attached to a stereotaxic instrument holding the head firmly in place while leaving the external ear canals free for insertion of earphone drivers. Recordings were made with the rats inside a single-wall sound-attenuated booth (Eckel Industries).
All procedures were approved by the Carleton University Animal Care Committee and were in accordance with the guidelines of the Canadian Council on Animal Care.
Sound stimulation
Sounds were presented monaurally to the right ear through a sealed headphone and coupler (Beyerdynamic DT 48) connected to a hollow speculum that was inserted into the rat's external meatus. To reduce acoustic cross talk, a second headphone assembly was inserted into the left external ear, although no sound was delivered through the headphone. The acoustic waveforms were generated digitally by MaLab 881 software controlled by a Macintosh G-3 computer. The sound-generating and data-acquisition system (MaLab 881) was designed and produced by Steve Kaiser, Department of Neurobiology, University of California, Irvine (Kaiser Instruments).
Various stimuli were used in this study including noise bursts, tone bursts, and SAM sounds. Noise bursts (100-ms duration, 10-ms rise-fall time) were used as search stimuli. Tone-bursts of 100 ms with 10-ms rise-fall time were used to determine a single neuron's characteristic frequency (CF, the frequency at which the neuron showed the lowest response threshold) and rate-level function at CF. Summed spike counts were used rather than firing rate for presentation of tone burst data because of the small number of spikes elicited in neurons with onset responses. SAM stimuli (10-s duration, 10-ms rise-fall time) were used to study the synchronization of discharges to the modulation envelope at various rates of modulation from 0.5 to 1,000 Hz. The carrier frequency of a SAM stimulus was set at the cell's CF, and the depth of modulation was fixed at 100%. The summed neural responses to tone bursts were obtained by presenting 20 stimuli at a rate of 1/s, and the responses to SAM stimuli were obtained by presenting 2 stimuli at a rate of 1/12 s.
The sound-pressure level for all stimuli was calibrated over a frequency range of 100-40,000 Hz using a condenser microphone (Bruel and Kjaer 4134) with the headphone assembly fitted into an enclosed chamber constructed from tygon tubing. The acoustic response of the headphone assembly was adjusted on-line to provide a constant sound pressure over the range from 500 Hz to 30 kHz.
Recording and drug delivery
A piggy-back multi-barrel glass electrode (Havey and
Caspary 1980) was used for extracellular recording and
iontophoretic delivery of drugs. The electrode was fabricated by fixing
a single-barrel recording micropipette to a five-barrel glass pipette
with a cyanoacrylate glue (Viachem, Quebec) at an angle of about 20°.
The tip of the single-barrel pipette was 2-3 µm in diameter, and the
tip of the five-barrel pipette was 8-10 µm. The recording electrode
was filled with 3 M potassium chloride. One barrel of the five-barrel
pipette was filled with NBQX (5 mM, pH 9.0, Sigma), an AMPA receptor
antagonist, and another barrel was filled with CPP (10 mM, pH 8.0, Sigma), an NMDA receptor antagonist. The remaining three barrels were filled with 165 mM sodium chloride for maintaining an electrical balance across the injection pipette tip. We choose NBQX and CPP as
antagonists of AMPA and NMDA receptors, respectively, because these two
drugs were found to be highly effective in our previous in vivo
pharmacological studies (see Kelly and Kidd 2000
)
The electrode assembly was driven by a Kopf model 650 micropositioner. The multi-barrel pipette was connected to iontophoresis pumps of a Neurophore-BH-2 micro-iontophoresis system. Neural activity registered by the single-barrel pipette was amplified by a Dagan EX 4-400 Quad differential amplifier and monitored audiovisually. Neural responses were digitized and discriminated on the basis of amplitude and waveform using an A/D converter and MaLab 881 software. The occurrence time of spikes was recorded with 1-µs resolution, stored on computer and processed later with standard database and graphics software.
Recording procedure
During a recording session, the right ICC was approached obliquely with the electrode tilted by 30° relative to the sagittal plane and stereotaxic coordinates referenced to a point 3.8-5.0 mm lateral and 0.3-0.5 mm rostral to lambda. The electrode was then lowered into the brain to a depth between 2.3 and 6.0 mm while monitoring for responses to acoustic stimulation. As the electrode was lowered into the brain a retaining current of +10 nA was applied to the pipette barrels containing NBQX and CPP to prevent leakage of the drugs during the search for single-unit activity and during predrug recording or postdrug recovery phases of the experiment.
Single-unit activity was recognized by spikes of constant amplitude and waveform. A window discriminator was used to isolate spikes from background activity. We typically used a noise burst presented to the right ear to search for responsive neurons. After an auditory neuron had been identified, tone bursts of variable frequency and amplitude were used to determine CF on the basis of spike counts and audiovisual monitoring of driven responses. Rate-level functions were then determined by systematically varying the sound-pressure level of the tone at the cell's CF.
NBQX and CPP were delivered iontophoretically to the recording site to
determine the contribution of AMPA and NMDA receptors to unit
responses. The currents used to release the drugs were 20 to
60 nA
(typically about
30 nA) for NBQX and
5 to
15 nA (typically around
10 nA) for CPP. The effect of the drug on firing rate was monitored
by repeatedly generating a rate-level function at the cell's CF. When
a change in spike count reached a plateau, the drug release was
terminated. Recovery was observed by continuing to monitor the cell's
rate-level function after the drug release was discontinued.
For some neurons, the effect of drugs on SAM stimuli was observed. The SAM stimuli were delivered with the carrier frequency at the cell's CF and the sound-pressure level typically fixed at 10-20 dB above threshold. The effect of a drug on the response to SAM stimulation was monitored only after the response to tonal stimuli had reached a plateau, i.e., the rate-level function for the cell had stabilized. After responses to SAM stimuli had been recorded drug release was discontinued and a holding current of 10 nA was re-applied. The response to SAM stimuli was monitored after drug release to determine the time course of recovery.
Data analysis
Peri-stimulus time histograms were collected for all neurons from which recordings were made. The responses were categorized on the basis of the temporal pattern of spike discharge into two broad groups: onset and sustained. Onset responses were defined as those with a transient burst of action potentials followed by a period of inactivity for the duration of the tone (100 ms). Sustained responses were defined as those with continued firing for the duration of the tone. These included patterns in which a pause separated an initial burst of action potentials from activity that was maintained for the duration of the tone as well as those with uninterrupted activity. The effects of drugs were analyzed separately for neurons with onset and sustained responses. For neurons with sustained firing, the effect of drug application was analyzed first for the full period of the tone burst and then separately for early (up to 30 ms) and late responses.
The responses to SAM stimulation were averaged over two repetitions of a 10-s sound presentation. Spikes recorded during the first 20 ms of each stimulus presentation were excluded from the analysis to avoid contamination by onset responses. The distribution of spikes was examined for the duration of the stimulus and for each modulation cycle to determine the extent to which spike discharges were synchronized to the modulation envelope.
A measure of vector strength, as described by Goldberg and Brown
(1969), was used with SAM stimuli to characterize the synchrony of neural firing with each modulation cycle. Vector strength was defined as follows
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Vector strength was plotted versus modulation frequency to create a synchronization modulation transfer function (MTFvs). We also calculated a modulation transfer function based on the neuron's firing rate (MTFfr) to determine how the drugs affected the total amount of neural activity at each modulation frequency.
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RESULTS |
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We recorded from a total of 105 neurons in the rat's ICC. Thirty-one of these neurons exhibited an onset response pattern, and the remainder had various patterns of sustained activity including 15 with a continuous response, 47 with an onset-pause-sustained response, and 12 with other patterns of sustained activity not included in the above two categories. We examined the effects of AMPA- and NMDA-receptor antagonists on tone burst responses in 93 of the 105 neurons and on responses to SAM stimulation in 22 of these neurons. Of the 93 neurons tested with tone bursts the effect of NBQX alone was examined in 56 neurons and the effect of CPP alone was examined in 48 neurons. The effect of simultaneous application of CPP and NBQX was determined in 32 neurons.
Response to tone bursts
Application of NBQX reduced tone evoked responses by at least 20% in 50 of the 56 neurons examined in the ICC. Examples are shown in Fig. 1, A and B, for two neurons with sustained and onset firing patterns, respectively. The neuron in Fig. 1A had a response with early and late spikes separated by a distinct pause. Prior to drug application, a tone at the cell's CF (11 kHz, 60 dB SPL) resulted in 246 spikes summed over 20 stimulus presentations. During application of NBQX, the spike count was reduced to 103 for the same stimulus condition. Both early and late components of the response were affected by the drug. Eight minutes after the drug was discontinued, the spike count returned to predrug levels. The effect of NBQX on the response of a neuron with an onset firing pattern is shown in Fig. 1B. Prior to drug application, the spike count over 20 presentations of a tone burst at CF (5.5 Hz, 80 dB SPL) was 26. During application of NBQX, the spike count was reduced to 0. After cessation of the drug the response returned to baseline levels.
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Application of CPP reduced tone evoked responses by at least 20% in 41 of the 48 neurons examined. Examples of the effects of CPP on neurons with sustained and onset responses are shown in Fig. 1, C and D. The neuron illustrated in Fig. 1C had a sustained firing pattern with a pause between early and late components of the response. The total spike count over 20 presentations of a CF tone (18.0 kHz, 85 dB SPL) was 142 prior to drug release. Application of CPP reduced the spike count to 26. The effect of CPP on a cell with an onset firing pattern is shown in Fig. 1D. The drug reduced the number of spikes evoked by a CF tone burst (8.5 kHz, 60 dB SPL) from 43 to 26. The response returned to predrug levels after CPP was discontinued.
Although both NBQX and CPP greatly reduced the number of spikes evoked by contralaterally presented tones, the temporal distribution of the responses was usually unchanged, at least for those cases in which the drug did not eliminate the response completely. In other words, neurons with an onset response retained their onset pattern during drug application and neurons with a sustained response retained their sustained pattern during drug application.
In general, NBQX had a greater effect on total spike count than CPP. The spike count to a stimulus 20-40 dB above threshold was reduced by more than half in 75% of the neurons tested with NBQX. A comparable reduction in firing under the same stimulus conditions was found in 55% of the neurons tested with CPP. For both drugs the reduction in spike count was reversible. After the cessation of drug application, the responses typically returned to predrug levels as illustrated in Fig. 1.
Rate level functions
The reduction in spike count by either NBQX or CPP was evident over a wide range of stimulus levels from near threshold to the point of response saturation. Both drugs had an effect on firing rate with tone bursts just a few dB above the threshold for eliciting a response. A decrease in response at all stimulus levels was observed in all drug-sensitive neurons, regardless of their temporal response pattern.
Rate-level functions for the same neurons presented in Fig. 1 are shown in Fig. 2 to illustrate the effect of the drugs over a range of sound pressure levels. The neuron shown in Fig. 2A had a sustained response and displayed a monotonic rate-level function before drug application. During application of NBQX, there was a decrease in spike count at all sound pressure levels. The rate-level function retained its monotonic shape but reached a plateau at a lower level of response. The spike count returned to predrug levels 6 min after drug application was terminated. The neuron in Fig. 2B, which exhibited an onset response pattern, had a slightly nonmonotonic rate-level function before the drug was applied. Release of NBQX reduced the spike count to zero at all sound pressure levels. The responses rapidly returned to near normal levels after the drug was stopped.
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The effects of CPP are shown in Fig. 2, C and D, for neurons with sustained and onset responses, respectively. In both cases, application of the drug reduced spike counts over the full range of stimulus intensities, including levels very near the threshold for evoking a response. All neurons showed recovery after cessation of the drug.
Drug effects on neurons with onset responses
Under the conditions of our experiment, NBQX had a more pronounced effect than CPP on neurons with onset response patterns. Application of NBQX resulted in a decrease in spike count of at least 80% in 12 of 16 neurons tested (75%), as shown in Fig. 3, and eliminated the response completely in 9 of these cells. In contrast, application of CPP resulted in a decrease in spike count of at least 80% in only 2 of the 12 neurons tested (17%). There were no cells in which the response was entirely eliminated.
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Seven neurons with onset responses were tested with both CPP and NBQX applied at different times separated by a period of recovery. In each of these cases, there was a greater reduction in spike count during the application of NBQX than CPP. Thus NBQX was shown to be more effective than CPP in reducing the response of onset neurons.
Drug effects on neurons with sustained responses
For neurons with sustained responses, both NBQX and CPP were highly effective in reducing the overall firing rate, but the magnitude of the reduction was different for the early and late components of tone evoked responses. Generally, NBQX had a more pronounced effect on the early part of the response and CPP had a greater effect on the later part.
The effect of NBQX is shown in Fig. 4 for a neuron with a sustained response comprised of an early burst of spikes followed by a pause and intermittent firing for the duration of the tone (CF = 16 kHz). NBQX greatly reduced the early response (0-30 ms), but had little effect on the later part of the response (30-120 ms). For example, NBQX reduced the number of spikes evoked by a tone of 50 dB SPL by 74% during the early part of the response but did not reduce the spike count during the remainder of the response. The effect of the drug on the total spike count in this case could be attributed to the reduced number of action potentials during the initial part of the response.
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The effect of CPP is shown in Fig. 5 for a neuron with an early response separated by a brief pause from a later response with a regular spike discharge that lasted for the duration of the tone (CF = 17.5 kHz). CPP had relatively little effect on the early response (0-10 ms) but greatly reduced the number of spikes during the later part of the response (20-120 ms). For this neuron, the effect of the drug on the total spike count could be attributed largely to the reduction of spikes during the later part of the response.
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To quantify the relative effects of NBQX and CPP on responses to tonal stimulation, we measured the percent reduction in spike count during the early (0-20 ms) and late (20-120 ms) parts of responses to a CF tone at 20 dB above threshold for every neuron that had a sustained response pattern. Drug effects on early and late responses were then plotted together on the same graph.
The percent reduction in firing for early and late responses to tone bursts are shown in Fig. 6, A and B, for neurons under the influence of NBQX and CPP. Figure 6A summarizes the effects of NBQX; the diagonal reflects an equal effect of the drug on early and late response components. For neurons tested with NBQX, more points were distributed below the line, reflecting a greater effect on the early component. These results can be compared with those shown in Fig. 6B, which plots the effect of CPP on early and late responses. For neurons tested with CPP, the majority of data points were located above the diagonal line indicating a greater effect on the late than early component of the response.
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Table 1 shows the number of neurons
exhibiting a greater percentage reduction in spike count during the
early or late component of a sustained response under the influence of
NBQX and CPP. Statistical analysis of this distribution showed that the
effects of the two drugs on the early and late components of sustained
responses were significantly different (2
=14.5, P < 0.001).
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Combined effect of NBQX and CPP on responses to tonal stimuli
NBQX and CPP in combination produced a greater reduction in firing than either drug alone in more than 90% of the cells tested (29 of 32 neurons) excluding from the analysis those neurons in which responses were completely eliminated by application of a single drug. In other words, excitatory responses that were not completely eliminated by one receptor antagonist (AMPA or NMDA) could usually be reduced by application of the other receptor antagonist, which suggests that the excitatory activity of most neurons in ICC is influenced by both AMPA and NMDA receptors. In many cases, the combination of antagonists reduced the firing rate to zero, although in some cases, it was still possible to elicit a remnant response in the presence of both antagonists. Whether these remaining responses reflect the presence of another class of excitatory receptor or a partial block of the AMPA or NMDA receptor cannot be determined from our data.
An example of the effect of combined drug application on a neuron with an onset response is shown in Fig. 7A. This neuron displayed a nonmonotonic rate-level function prior to drug application. Release of either NBQX or CPP alone resulted in a decrease in the response at most suprathreshold levels of stimulation. NBQX had a greater effect than CPP when applied alone, but neither drug by itself completely eliminated the tone evoked response. Simultaneous injection of NBQX and CPP, however, eliminated all responses at each of the stimulus levels tested.
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The combined effect of NBQX and CPP on a neuron with a sustained response pattern is shown in Fig. 7B. Before drug application, this neuron had a nonmonotonic rate-level function. NBQX alone had very little effect on spike count, but CPP alone reduced the number of spikes substantially over a wide range of stimulus intensities. The combined effect of NBQX and CPP was greater than that of either drug alone and tone-evoked responses were further reduced at most intensity levels. Although both antagonists had a marked effect, the excitatory responses were not completely eliminated by the combined drug application in this neuron.
A summary of the combined effects of NBQX and CPP is shown in Fig. 8. The average reduction in firing rate based on the full range of sound-pressure levels tested above predrug threshold for each neuron was calculated for NBQX alone, CPP alone and both drugs in combination. The degree of spike count reduction was plotted as the proportion of cells showing an effect under each of the three drug conditions. As can be seen in Fig. 8, combined application of NBQX and CPP resulted in a larger reduction in spike count than either drug alone. The spike count was reduced by at lease 80% of the predrug levels in more than half (68%) of the neurons tested with a combination of drugs.
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Responses to SAM stimuli
We examined the response to SAM stimuli, determined the MTF, and tested the effects of drugs in 22 neurons in ICC. Most of these (19 neurons) exhibited sustained response patterns to tone bursts. Neurons with onset responses typically did not respond to modulated sounds after their initial discharge of spikes at the beginning of the stimulus. However, three neurons that did show a continued response to SAM stimuli were tested with drugs and included in our analysis. All cells exhibited peak vector strengths to modulation frequencies between 10 and 100 Hz. The modal best modulation frequency for our sample of cells was 50 Hz. All neurons had peak vector strengths >0.5, and all but two neurons had peak vector strengths greater than 0.7 prior to drug application.
The effect of NBQX alone was studied in 13 neurons and the effect of CPP alone was studied in 10 neurons with one cell yielding data under both drug conditions. Both NBQX and CPP reduced the firing rate to SAM stimulation. On the other hand, there was very little effect of either drug on vector strength. Of the 22 neurons tested, none showed a reduction in vector strength greater than 0.2 at peak modulation rates when tested with either NBQX or CPP. The modulation rate at which the cells showed maximum vector strength was unaffected. Application of NBQX resulted in a reduction in vector strength at some off-peak modulation frequencies in some cells (4 of 13 tested), but for the majority of neurons, there was no change in vector strength at any modulation frequency. The MTFvs remained stable in spite of large reductions in firing rate for most of the neurons examined.
The effect of CPP on the response of a typical ICC neuron to modulated sounds is shown in Fig. 9. Before application of CPP, this neuron responded well to SAM stimulation at all modulation rates between 0.5 and 200 Hz with a peak response at 10 Hz (Fig. 9A). The responses were well synchronized to the modulation envelope at frequencies between 0.5 and 100 Hz, and the MTFvs reached its peak at 50 Hz (Fig. 9B). Application of CPP resulted in a marked decrease in firing over a wide range of modulation frequencies with the largest effect at 10 Hz. On the other hand, there was no reduction in vector strength at any modulation frequency. Every neuron tested with CPP showed the same result, viz., a pronounced reduction in firing rate, but no change in the MTFvs.
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The effect of NBQX is shown in Fig. 10 for two representative ICC neurons. In both cases, the drug resulted in a lower firing rate over a wide range of modulation frequencies. For the neuron shown in Fig. 10A, there was a reduction in vector strength at off-peak modulation rates, but the synchrony of responses at the best rate (50 Hz) remained unchanged. Thus although the MTFvs was affected by the drug in this case, it retained its essential band-pass characteristic. Similar results were obtained in 4 of the 13 ICC neurons tested. A different result is shown in Fig. 10B. In this case, NBQX had no effect on vector strength at any modulation frequency although the firing rate was substantially reduced. This result was found in 9 of the 13 cells tested with NBQX.
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DISCUSSION |
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Responses to tone bursts
Our data show that excitatory responses of single neurons in the
rat's ICC are dependent on both AMPA and NMDA receptors. Pharmacological block of either receptor type by iontophoretic application of its respective antagonist (NBQX or CPP) reduces the
number of action potentials evoked by a contralaterally presented tone
burst. The combined application of both antagonists further reduces the
response in most ICC neurons. Several previous in vivo studies have
shown that both AMPA and NMDA receptors contribute to excitatory
responses in the auditory midbrain. First, Faingold et al.
(1989, 1991
) reported that iontophoretic application of the
NMDA antagonist, APV, greatly reduced single-unit responses to tones in
the rat's ICC and that local application of the agonists NMDA or
glutamate greatly increased neural activity. The role of the AMPA
receptor was not investigated in these experiments because adequately
specific receptor antagonists were not available at the time. More
recently, Burger and Pollak (1998)
showed that iontophoretic application of APV substantially reduced tone-evoked responses of neurons in the mustache bat's ICC, indicating that NMDA
receptors contribute to excitatory responses in the inferior colliculus. The possible contribution of AMPA receptors, however, was
not investigated.
Feldman and Knudsen (1994) examined the role of both
AMPA and NMDA receptors in regulating auditory responses in the
midbrain of the barn owl. They showed that the AMPA antagonist,
6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), and the NMDA antagonist,
APV, were both effective in reducing excitatory responses to sounds in
the external nucleus of the inferior colliculus and the lateral shell
region of the owl's auditory midbrain and that the combination of AMPA
and NMDA antagonists was more effective than application of either
antagonist alone. Their results are consistent with our data regarding
the relative contribution of AMPA and NMDA receptors to auditory
responses in the inferior colliculus and suggest that similar
mechanisms underlie excitatory responses in the mammalian ICC and its
avian homologue.
We found that both AMPA and NMDA receptors contribute to excitatory
responses at all levels of acoustic stimulation that normally elicit
action potentials. Responses were reduced at all levels by application
of either AMPA or NMDA antagonists. A similar result was reported for
neurons in the barn owl's auditory midbrain (Feldman and
Knudsen 1994). These findings imply that both AMPA and NMDA receptors are involved in sensory processing over a wide range of
stimulus intensities and that the contribution of the NMDA receptor is
not limited to high levels of stimulation as would be expected from in
vitro studies of hippocampal pyramidal cells, which require
depolarization through tetanic stimulation to bring NMDA receptors into
play (see Cotman and Iversen 1987
). Our data are
consistent with the idea that the NMDA receptor is available for
sensory processing at near-threshold levels, but that AMPA and NMDA
receptor-mediated events interact to produce suprathreshold excitatory
responses to acoustic stimuli. Although the NMDA receptor is capable of
sustained activity even after pharmacological block of the AMPA
receptor, the two receptor types would be expected to interact under
normal conditions to provide an amplification or gain control of
auditory responses as suggested by other investigators for visual and
other sensory modalities (Daw et al. 1993
; Fox et
al. 1990
). In other words, AMPA receptor-mediated
depolarization produced by increasing suprathreshold levels of acoustic
stimulation would be expected to recruit progressively larger NMDA
receptor-mediated responses by virtue of the voltage-dependent
properties of the NMDA receptor. This effect would magnify the response
to sound relative to the AMPA receptor-mediated excitation alone.
Our data show that AMPA and NMDA receptors make distinctive
contributions to tone-evoked responses in ICC. The AMPA receptor antagonist, NBQX, results in a proportionally greater reduction in
firing during the early part of a sustained response to a tone burst
(100 ms). In contrast, the NMDA receptor antagonist, CPP, produces a
larger reduction during the later part of such responses. Furthermore,
in our study, NBQX was more effective than CPP in reducing the firing
of neurons with an onset response pattern even though both antagonists
had an effect. These results indicate that the AMPA receptor is
particularly important during the initial response of an ICC neuron and
that both receptor types are involved in the maintenance of activity
for the duration of the stimulus. The differential contribution of AMPA
and NMDA receptors is consistent with known differences in the time
constants for activation of the two receptor types. The AMPA receptor
has relatively rapid onset and decay times, whereas the NMDA receptor
has a much slower time course (Fu et al. 1997; Ma
et al. 2001
; Smith 1992
). Thus one would expect
that the AMPA receptor would contribute preferentially, although not
exclusively, to the initial response of neurons in the ICC.
Furthermore, both NMDA and AMPA receptors would be expected to
contribute to the steady generation of spikes during continuous stimulation. Also the relative contribution of AMPA and NMDA receptors during the later part of a tone burst response might be influenced by
the desensitization of the AMPA receptor under conditions of prolonged
activation (Otis et al. 1995
; Zhang and Trussell
1994
).
The differential effect of AMPA and NMDA antagonists on early and late
responses in the auditory midbrain has been shown recently in the
context of binaural stimulation (Kelly and Kidd 2000). Injection of the AMPA antagonist, NBQX, into the dorsal nucleus of the
lateral lemniscus affects responses of neurons in the contralateral ICC
to sounds presented with relatively short interaural time delays,
whereas injections of the NMDA antagonist, CPP, has a proportionally
larger effect on responses to longer time delays. These results are
consistent with the idea that the AMPA receptor preferentially mediates
early excitatory responses in DNLL, whereas the NMDA receptor
contributes preferentially to the later responses.
The results of these in vivo studies are supported by in vitro brain
slice recordings from the auditory midbrain (Fu et al. 1997; Ma et al. 2001
; Smith 1992
;
Wu and Kelly 1996
). Intracellular recordings of
excitatory postsynaptic responses in the inferior colliculus or dorsal
nucleus of the lateral lemniscus to electrical stimulation of afferent
pathways have demonstrated that both AMPA and NMDA receptor-mediated
postsynaptic potentials or currents can be elicited and selectively
blocked by bath application of selective antagonists. For example, in
the rat's DNLL stimulation of the lateral lemniscus typically elicits
excitatory responses with both AMPA and NMDA receptor-mediated
components. AMPA and NMDA antagonists selectively reduce the early and
late components of the responses, respectively (Fu et al.
1997
; Wu and Kelly 1996
). The NMDA
receptor-mediated response is present near the cell's resting
potential and can still be activated after pharmacological block of the
AMPA receptor-mediated response. Thus the NMDA receptor can be brought
into play without concomitant depolarization through activation of the
AMPA receptor. On the other hand, the NMDA receptor is certainly
voltage dependent and regulated by a magnesium block that is overcome
by membrane depolarization indicating that the two receptor types would
normally interact to produce a response (Fu et al.
1997
).
A similar relation between AMPA and NMDA receptors has been reported
for neurons in the rat's ICC. Brain slice studies have shown that
excitatory responses with an early AMPA and later NMDA component can be
activated by stimulation of the lateral lemniscus. Both response
components are present at values near the cell's resting potential and
have properties resembling in every respect those described in the
preceding text for DNLL (Ma et al. 2001).
Responses to SAM stimulation
The excitatory responses of ICC neurons to continuous SAM stimulation are greatly reduced by iontophoretic application of either CPP or NBQX. Thus it can be shown that both AMPA and NMDA receptors are involved in maintaining firing of ICC neurons to dynamically changing acoustic stimuli as well as simple tone bursts. This raises the question of whether the different receptor types contribute selectively to responses at different rates of modulation as might be expected on the basis of their different time constants for activation. For example, the AMPA receptor might be selectively involved in mediating responses to very rapid rates of modulation, whereas the NMDA receptor might be more important at slower rates; or because of its relatively slow time constant the NMDA receptor might set an upper limit to the rate of modulation that a neuron in ICC can follow by synchronous discharge of action potentials.
Our results provide no compelling evidence for selective involvement of AMPA or NMDA receptors in mediating responses to different rates of modulation. Modulation transfer functions based on an index of synchronization (vector strength) showed no change under the influence of the NMDA antagonist, CPP. There was also very little change under the influence of the AMPA antagonist, NBQX, which resulted in a reduction in vector strength only at off-peak modulation rates in a small proportion of the neurons tested. Even in these cases, the modulation frequency that resulted in maximum vector strength was not substantially altered by the drug. Thus in spite of large changes in firing rate there were no significant changes in the synchrony of firing to SAM stimuli in the vast majority of neurons tested with either AMPA or NMDA receptor antagonists.
This result is consistent with a previous report by Burger and
Pollak (1998) that showed that the upper limit of synchronized responding of neurons in the mustache bat's ICC to SAM stimulation was
not altered by iontophoretic application of the NMDA antagonist, APV.
The vector strengths at high rates of SAM stimulation remained the same
after application of APV, suggesting that the NMDA receptor was not
involved in setting the upper limit of synchronized responses to
modulated sounds.
These studies suggest that the synchrony of responses in ICC to SAM
stimulation is determined by factors other than the activation of local
excitatory receptor types. For example, inhibitory receptors, such as
the GABAA receptor, might be involved. Although
GABA seems a likely candidate for shaping responses to modulated sounds
in ICC, Burger and Pollak (1998) did not find any change
in the upper limit of response synchronization to SAM stimulation
during application of the GABAA antagonist,
bicuculline, in the bat's ICC even though the firing rates of the
neurons were greatly increased.
One possible explanation of the lack of effect of pharmacological block
of local synaptic activity on the synchrony of responses to SAM
stimulation might be that the temporal properties of the response
patterns are established by circuits elsewhere in the auditory system
and that the patterns in ICC are inherited from projections from other
structures. In support of this possibility are the findings that
bicuculline can alter the synchrony of responses to SAM stimulation in
neurons located in the dorsal nucleus of the lateral lemniscus,
superior olivary complex and cochlear nucleus (Backoff et al.
1999; Grothe 1994
; Yang and Pollak
1997
). To our knowledge, however, there are no data on the
effects of AMPA or NMDA receptor antagonists on the response of neurons
to modulated sounds in structures other than the ICC.
Another possibility is that the temporal response patterns to SAM
stimulation are determined by intrinsic properties of the neurons in
ICC. The timing and upper limit of synchronous activity of neurons in
several auditory lower brain stem nuclei, viz., the cochlear nucleus,
the superior olive and the ventral nucleus of the lateral lemniscus,
are determined in part by the presence of voltage-sensitive channels
that are regulated near the cell's resting potential resulting in a
low-threshold potassium conductance activated by depolarization and a
mixed cation conductance, Ih, activated by hyperpolarization of the cell membrane (Oertel
1997). Furthermore the upper limit of synchronized firing of
neurons can be determined by the presence of combinations of
voltage-dependent potassium channels expressed by genes of the Kv1,
Kv2, Kv3, Kv4, Kv8 or Kv9 subfamilies. For example, computer
simulations show that co-expression of Kv2.1 and Kv9.1 genes can lead
to more synchronized firing of action potentials than expression of the
Kv2.1 subtype alone. The Kv9.1 and other related genes are expressed in
the rat's inferior colliculus and could shape the temporal firing patterns of neurons based on activation of specific combinations of
voltage-sensitive potassium channels (Richardson and Kaczmarek 2000
; Wang et al. 1998
). Combinations of ion
channels can determine not only the upper limits of synchronous firing
of action potentials but also the resonant properties of neural
activity at particular rates of oscillation. For example, the presence
of a low-voltage calcium current in neurons of the inferior olivary
nucleus contributes to their predominant resonance at rates around 4 Hz, and the presence of a hyperpolarization-activated cation current
contributes to the resonance of pyramidal neurons at rates around 1-2
Hz (Hutcheon and Yarom 2000
; Llinás
1988
). Thus the band-pass properties of the
MTFvs for many ICC neurons could be determined by
the activation of particular combinations of ion channels acting in
concert. The extent to which intrinsic properties of ICC neurons or the presence of specific potassium channels can determine the temporal patterns of responses to SAM stimuli has yet to be determined.
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ACKNOWLEDGMENTS |
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This work was supported by a research grant to J. B. Kelly and a postdoctoral fellowship to H. Zhang from the Natural Science and Engineering Research Council of Canada.
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FOOTNOTES |
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Address for reprint requests: J. B. Kelly, Dept. of Psychology, 329 Life Science Research Building, Carleton University, Ottawa, Ontario K1S 5B6, Canada (E-mail: jkelly{at}ccs.carleton.ca).
Received 16 February 2001; accepted in final form 7 May 2001.
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REFERENCES |
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