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A middle-latency auditory-evoked potential in the rat
BrainResearchBulletin,Vol. 37, No. 3, pp. 247-255, 1995 Copyright© 1995ElsevierScienceLtd Printedin the USA.All rightsreserved 0361-9230/95 $9.50 + .00
Pergamon 0361-9230(95)00003-8
A Middle-latency Auditory-evoked Potential in the Rat H. MIYAZATO,* R. D. SKINNER,.1 N. B. REESE,~ F. A. BOOP1- AND E. GARCIA-RILL*
Departments of *Anatomy and l~Neurosurgery, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205 ~:Department of Physical Therapy, University of Central Arkansas, Conway, AR [Received 3 August 1994; Accepted 22 December 1994] ABSTRACT: Previous studies have established the Wesence of a middle-latency audltmy-evoked poten1~ that is charactedzed by a) sleep-stere dependem:, b) iow following f m ~ m ~ y (i~., rapid
hab,wUo, to ~ = ; ; , . J ~ . ) , and c) bk)ckede by the choUner~c aotagonist, sco~lamine. A vertex-recorded evoked potential having t h e w characteristics was described in humans at a 5080 ms letency (termed ti'm P1 or PSO potmtial) and in the cat at a 20-25 ms latency (ten'ned wave A). ~ atudies wera undertaken w ~ If a c l c k sh~mulm-evoked p o t ~ , t ~ havk',g U',e same chamcll~i~k:s was preeent in 'lhe ~lact rat, Vertex and aud~ory cortex recordings in intact rats studied in a sound-attenuating chamber and exposed to free-field click sUmuii showed a) the presence of a verlex racorded potenUal at a 11-15 ms letency, termed P13, and of an auditory cortex recorded potential at a 7-11 ms latency, termed Pa; b) the P13 was prasent during waldng and paredoxical s:~.~pbut al~ent in slow-wave sleep, whSe Pa was prNent in all sleep-wake stat~; c) the P13 habituated markedly at stknulaUon r a t ~ above I Hz while Pa did not; and d) the P13 was blocked by low _(t~___ of scopolamine while Pa was not. These atudles demonstrate tlhe ~ of a Pl-Iike potential in the rat at a 13 ± 2 ms letency. KEY WORDS: Auditory-evoked potentials, Middle-latency potentials, Pl Potential, Intact rat.
INTRODUCTION Evoked potentials recorded in the first 10 ms following an auditory click stimulus, the brain stem auditory evoked responses (BAERs), have been well characterized [ 14,21,27]. However, the middle-latency potentials, those in the 10-100 ms range, have not been characterized as well. In humans, auditory-evoked potentials in this latency range can be recorded at the vertex, one at a latency of 25-35 ms and termed Pa [8,21,22], and another at a latency of 5 0 - 8 0 ms and termed P1 [8,22], Pb [20,25], or P50 [1]. When these potentials were recorded across states of wakefulness and sleep, Pa was shown to remain constant in amplitude across all states while P1 disappeared during slow-wave sleep (SWS) and reappeared at its waking amplitude during rapid eye movement (REM) sleep [8,9]. When rates of auditory stimulation were increased gradually from 0.5 Hz to 10 Hz, Pa remained at a constant amplitude, whereas the amplitude of P1 decreased significantly as stimulation rates exceeded 1 Hz [5,9]. Intravenous injections of the cholinergic antagonist scopolamine resulted in a decrease in amplitude and ultimate disappearance of the P1
potential, an effect that was reversed by subsequent injections of the cholinergic agonist physostigmine. Injections of scopolamine and physostigmine had an opposite and much smaller effect on the Pa potential [5]. Auditory-evoked potentials that have been equated to the human middle-latencyPa and P1 responses also have been recorded in a variety of nonhuman species including monkey [3], cat [4,6,15,30,32), rat [11,13,16,19], and guinea pig [17,18,28]. Studies in the cat revealed the presence of an auditory-evoked potential recorded at a latency of 10-15 ms following an auditory click stimulus. It was present during all stages of sleep, was not abolished by high stimulation rates, and was resistant to barbiturate anesthesia [4,6,30]. This potential was termed ER1 [30] and, more recently, wave 7 [4]. On the other hand, an auditoryevoked potential, which seems by its response characteristics to be analogous to the P1 potential in the human, has been recorded from the vertex in the cat at a latency of 17-25 ms [4,30]. Like the human P1, this potential was present during waking and REM sleep but disappeared during SWS, decreased in amplitude as stimulus presentation rates exceeded 1 Hz, and could not be recorded when the animal was anesthetized [4,6,30]. Subsequent pharmacological studies in the cat demonstrated that this potential, termed wave A [4], could be abolished by administration of scopolamine in a dose-dependent fashion [7]. Subsequent lesion studies showed that wave A was reduced or abolished by lesions of the mesopontine cholinergic pedunculopontinenucleus (PPN), and that the degree of cholinergic cell loss correlated with the reduction in amplitude of wave A [10,12]. Thus, an auditory-evoked potential in the middle-latency range can be recorded both in the human and the cat, and has the characteristics of sleep-state dependence, low following frequency (rapid habituation to repetitive stimulation), and sensitivity to scopolamine, a muscarinic antagonist. The purpose of this study was to determine if an auditory-evoked potential recorded in the intact rat showed similar characteristics as the P1 potential in the human and wave A in the cat, and, therefore, may represent the rat equivalent of these potentials. Preliminary reports of this study have been published [19]. METHOD
Surgical Procedures Adult male Sprague-Dawley rats (n = 22, 250 to 350 g) were anesthetized with ketamine HCI (60 mg/kg, IM) and sodium barbiturate (20 mg/kg, IP). Anesthetic levels were maintained such
~To whom requests for reprints should be addressed. 247
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that the withdrawal reflex to paw pinch was absent. The animal was placed in a rat stereotaxic instrument using hollow ear bars, which protected the middle ear from injury. After reflection of the scalp, stainless steel screws for recording auditory-evoked potentials were installed epidurally at the vertex, 5.5 mm anterior to the interaural line, 1.0 mm lateral to the midline, midway between bregma and lambda bilaterally. Also, a screw was placed over the right auditory cortex, 4.5 mm anterior to the interaural line and 4.5 mm ventral to the dorsal surface of the skull. A reference screw was inserted into the frontal sinus. Pairs of wires were inserted into the dorsal nucchal muscles bilaterally for electromyographic (EMG) recordings. A wire placed in the dorsal neck served as ground. Wires from all electrodes were led to a nine-pin receptacle (Amphenol), which was cemented to the skull. Penicillin G was given IM and the animals were placed in a warm environment during recovery. Rats were housed individually in a vivarium with a 14 L:I0 D schedule, and food and water were available ad lib. Recordings began after a 7-day recovery period.
Recording Procedures Rats were placed in a sound-attenuating chamber (2 × 2 × 2 r ) with a fan that provided both continuous air flow and white noise background at a level of 65 dB. The chamber was lit by a 12 V DC bulb and a window and peep hole viewer allowed undetected observation of the animal. Wires from a swivel commutator were connected to a plug that mated with the receptacle on the rat's head. Following a 15-rain acclimation period in the chamber, auditory-evoked potentials were recorded from unrestrained rats. In all trials, evoked potentials recorded from the vertex and auditory cortex were amplified (20,000×) and filtered at 3 Hz to 1 kHz. The EMG signal was amplified (5000×) and filtered at 30 Hz to 3 kHz. All recordings were stored on magnetic tape and digitized at a rate of 10 kHz, averaged using SuperScope II software (G. W. Instruments) and the averages stored on disk. Superscope allows a computer and monitor to function as a storage oscilloscope and display individual responses, running averages, rectified, normalized and averaged EMG, frequency analysis of EEG, etc. online. All subjects were tested between 0900 and 1300 h to control for possible time-of-day effects.
Stimulation Procedures Auditory stimuli were delivered by a Grass Click-Tone module (model S10CTCMA) and led to speakers in opposite walls of the chamber. Rarefied clicks having a rise time of 100 #s were routinely presented at 4 0 - 1 0 3 dB, and could be graded in 1-dB increments to determine thresholds. Trains of clicks (5 or 10 clicks at 1 kHz) at a train repetition rate of 0.2 Hz also were used to test auditory-evoked responses. Averaged responses were based on 64 individual evoked responses. Threshold responses were obtained with the white noise background turned off. Although threshold levels for middle-latency responses were determined from averages made at the rate of 0.2 Hz, B A E R threshold levels were obtained from averages of 2048 trials evoked at 10 Hz by a single-click stimulus. In order to establish state dependence of the auditory-evoked potentials, continuous recordings displayed on a paper chart recorder were utilized. Blocks of trials were averaged only during waking (low amplitude, high frequency EEG on the vertex vs. frontal leads, muscle tone background on EMG and a visually alert, orienting animal in the chamber), slow-wave sleep (high amplitude, low frequency EEG, decrease of muscle tone in the EMG, eyes closed, inactive animal), and desynchronized sleep
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(low amplitude, high frequency EEG, absence of EMG activity, eyes closed, inactive animal). In order to determine the response characteristics of the auditory-evoked potentials, different stimulation paradigms were used. To test continuous habituation, the sensitivity of these potentials was determined by averaging sequential trials at a specific interstimulus interval for the entire block of 64 trials. This was used to determine the following frequency of the respective responses. Frequencies of 0.2, 0.5, l, 2, 3, 5, and 10 Hz were used. To test instantaneous habituation, a two-stimulus paradigm was used in which the interstimulus interval was either 0.1, 0.2, 0.3, 0.5, 0.7, or 1 s, but the pairs of stimuli were presented every 5 s. This was used to determined the habituation properties of the respective responses.
Injection Procedures In seven male rats, 0.5 ml of scopolamine hydrobromide (Sigma Chemical Co.) in physiological saline was injected (IP) in concentrations of 0.2, 1.0, or 5.0 mg/kg. Equal amounts of physiological saline were injected in control experiments. Preceding each injection, the evoked potentials were recorded and the average of two blocks of averaged potentials was used as the control level for that injection. Control potentials were normalized to 100% for comparison with postinjection values. Injections were made in random order, not less than 7 days apart.
WaveJbrm Measurements During the first 6 ms following an auditory stimulus, BAERs were evident. The threshold level of the BAERs was based on the first discernible presence of wave 4 [26]. To determine the threshold for BAERs and for middle-latency auditory-evoked potentials, the background white noise was turned off. Likewise, the threshold of the P13 auditory-evoked potential was determined to be the decibel (dB) level at which this wave was first discernible. Measurement of the amplitude of auditory-evoked potentials was made from the beginning of the wave to its peak.
RESULTS After acclimation to the recording chamber, the animals were tbund to shift from periods of quiet to periods of exploratory behavior. On occasion, averaging of evoked potentials was stopped if the animals became too active, as evidenced by excessive EMG activity. Recordings were not carried out under restraint (e.g., a body bag) due to any possible deleterious effects on arousal induced by restraint. Using a free-field approach, recordings could be carried out consistently during quiet periods. It, therefore, became important to determine the arousal state of each animal during averaging. Prolonged periods of quiet alertness could lead to drowsiness and slow-wave sleep, as evidenced by slowing of the EEG, and could ultimately result in the induction of periods of paradoxical sleep. The degree of desynchronization of the EEG and the level of muscle tone reflected in the EMG thus were monitored. The first section of the results describes the behavior of vertex (Vx) and auditory cortex (ACx)recorded middle-latency potentials during various sleep-wake states. Subsequent sections describe the response characteristics of these potentials and the effects of scopolamine administration. The recordings shown in these later sections all were obtained in the awake, quietly alert preparation (desynchronized EEG, muscle tone evident in EMG). The Vx-recorded potentials described below were present in 20 out of 22 animals studied. In two animals, a potential at or around a latency of 13 ms was not evident in the Vx recording. It is not clear if a procedural (surgical and/or record-
RAT P13 P O T E N T I A L
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FIG. I. Sleep-state dependence of the Pi3 potential. Top row. Brief periods of EEG and EMG recordings representative of the sleep-wake state of the rat during the period in which the averages in the bottom row were obtained. Note low amplitude, high frequency activity in the EEG and muscle tone in the EMG during waking (AWK). During slow-wave sleep (SWS), high amplitude, low frequency activity was evident in the EEG along with a reduction in EMG activity. During desynchronized or paradoxical sleep (DS), low amplitude, high frequency EEG again was present, but no muscle tone or activity was evident in the EMG. Bottom row. Vertex (Vx) and auditory cortex (ACx) recordings during each of the states described. Averages of the Vx electrode showed a positive potential at 13 ms (PI 3), a negative potential at 20 ms (N20), and a positive potential at 25 ms (P25) latency. In the ACx, there was a positive response at 9 ms (Pa) and a negative potential at 15 ms (NI5). During SWS, no P13 potential was present, while all the other potentials were present in the Vx and ACx recordings. During DS, the PI3 potential returned at its waking amplitude in the Vx average, while the ACx-recorded Pa potential remained the same across all states. Calibration bars--vertical bar 20/~V for averages, horizontal bar 1 s for EEG and EMG recordings. ing) problem was responsible for the lack of P13 potential in these animals. However, it does appear that wave A is not manifested in a small proportion of intact cats [4]. Following auditory click stimuli (103dB), BAERs were evident during the first 6 ms period. These were followed by the middle-latency potentials in the 1 0 - 2 5 ms range. In the rat, the averaged responses recorded at the vertex (Vx) showed peaks near 13 ms (termed P13), 20 ms (termed N20), and 25 ms (termed P25) (Fig. 1). In contrast, recordings over the auditory cortex (ACx) showed a different set of potentials. These peaks occurred at 6 - 9 ms (termed Pa) and 15 ms (termed N15). Thus, in temporal presentation, the middle-latency potentials recorded at the Vx were distinct from those recorded over the ACx. As in other species, the sleep-state dependence and response characteristics of Vx-recorded potentials were found to differ from those of ACx-recorded potentials.
Sleep-state Dependence of the P13 One characteristic of the P13 potential was its sleep-state dependence. Similar to what has been reported for the P1 potential
in the human and wave A in the cat, the rat P13 potential was present during waking, absent during slow-wave sleep, but reappeared at its waking amplitude during paradoxical sleep. Figure 1 shows averages obtained from the Vx and ACx electrodes across s l e e p - w a k e states. During waking, high frequency, low amplitude EEG, orienting responses, and active E M G recordings were evident. During such periods, the Vx-recorded average showed P13, N20, and P25 potentials, while the ACx average showed Pa and N 15 potentials. During slow-wave sleep, slowing of the EEG and high amplitude spindle activity was present along with a reduction, but not total absence, of muscle tone. During slow-wave sleep, no P13 potential was evident, while other Vx and ACx potentials were manifested as in waking. During paradoxical sleep, the EEG once again became desynchronized and of lower amplitude than in slow wave sleep, no orienting or startie-like responses were evident, and there was a total absence of muscle tone. During paradoxical sleep, the PI3 potential recorded from the Vx returned at its waking amplitude, while Pa was present as usual in the ACx recording. Some attenuation of N20, P25, and N15 was evident during paradoxical sleep, making the reappearance of P13 potential at the Vx and the maintenance
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M I Y A Z A T O ET AL.
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FIG. 2. Following frequency of the Vx-recorded PI3 potential and the ACx-recorded Pa potential. Evoked potential amplitudes observed at a stimulation rate of 0.2/s was considered control for both potentials. Some attenuation of the Vx-recorded PI 3 potential was evident at 0.5Is and l/s, followed by a disappearance of the potential at higher (->2/s) stimulation rates. In contrast, the ACx-recorded Pa potential was present at all frequencies tested, with some attenuation evident at stimulation rates of 5/s and 10/s. Calibration bar--vertical bar 20/~V.
of Pa at the ACx even more significant. Such effects were observed consistently in all four animals tested for sleep dependence of auditory middle-latency potentials. It should be noted that recording sessions typically lasted 1 h, during which time little attenuation of the P13 potential was evident. However, when testing for prolonged periods, the P13 potential was found to decrease in amplitude after 90 min (82.4 ± 26.8% of control). Such results led to setting a maximum recording time of 60 min for all sessions (see below, saline control, Fig. 5).
Response Characteristics Following frequency of the P13 potential. Figure 2 shows the following f r e q u e n c y characteristics of the Vx- and ACxrecorded potentials used to test continuous habituation. At stimulation frequencies of 0.2/s, the P13 and Pa potentials were d e t e r m i n e d to be 100%, or control. As stimulation frequencies were increased to 0.5/s and l/s, the amplitude of the V x - r e c o r d e d P13 potential was 70.1 ± 34.2% and 42.7 _+ 24.7% of control, respectively. On the other hand, the ACxrecorded Pa potential was 100.4 ± 19.3% and 91.5 _+ 23.5% of control, respectively, for stimulation rates of 0.5/s and l/s. No P13 potential was evident in the Vx recording at stimulation rates -> 2Is. In contrast, the A C x - r e c o r d e d Pa potential was found to habituate s o m e w h a t (53.6 _+ 17.0% of control), but only at stimulation rates of 10Is.
These results demonstrated the rapid habituation to continuous stimulation exhibited by the P I 3 potential contrasted with the resistance of the Pa potential. The longer latency potentials (N20, P25, and N15) all showed some habituation, but only at high stimulation rates. The effects described above were observed in every animal tested with this paradigm (n = 5). Habituation of the P13. Figure 3 shows representative recordings from the Vx electrode in response to pairs of stimuli of identical amplitude (103 dB) used to test instantaneous habituation. The intertrial interval for all pairs of stimuli was 5 s. The averaged responses to the first stimulus of a pair are shown on the left (each representing 100%, or control). The averaged responses to the second stimulus of a pair are shown on the right. The P13 potential in response to the second stimulus was 34.9 + 17.0% of control when the interstimulus interval was 1 s, while it was 36.6 ± 15.5 % and 27.5 ± 20.2% of control at intervals of 0.7 and 0.5 s, respectively. The response to the second stimulus was absent at shorter interstimulus intervals (6.6 ± 10.7% at 0.2 s and 1.0 -+ 2.4% at 0.1 s). In contrast, Fig. 4 shows representative recordings of the Pa potential obtained from the ACx electrode in response to the paired stimulus paradigm. No decrement in responsiveness was evident at interstimulus intervals ->0.2 s. Only at the shortest interstimulus interval (0.1 s) was there a moderate reduction of the Pa response to the second stimulus compared to the response to the first stimulus (52.7 ± 15.7% of control). All of the animals
R A T P13 P O T E N T I A L
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Vx(P13) ISl 1.0sec
0.7sec
plitudes of 70 dB. For the group of animals tested for threshold (n = 12), the m e a n threshold for the P13 potential was 72.7 +_ 2.6 dB. By comparison, the amplitude of the ACx-recorded Pa potential was 77.6 _+ 21.2% of control at 75 dB, 45.9 _+ 29.7% at 60 dB, and 33.0 __ 9.8% at 50 dB. As with the P13 potential, a slight increase in latency was evident as stimulus amplitude decreased. No detectable Pa potential was evident at < 4 5 dB. For the group of animals tested for threshold, the mean threshold for the Pa potential was 51.7 + 2.9 dB. The threshold for the B A E R s (not shown) at 50.4 +_ 1.6 dB was similar to that o f the Pa potential. The B A E R and Pa thresholds were not significantly different from each other, while the Pa threshold was significantly lower than the threshold of the P13 potential ( M a n n - W h i t n e y U-test, one-tailed p-value 0.01). The B A E R averages were generated using 10 Hz stimulation and
0.Ssec
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ct.l~lt FIG. 3. Habituation of the Vx-reeorded PI3 potential in the two-click paradigm. The column on the left shows the averaged Vx recording following the first stimulus of a pair, while the column on the right shows the averaged Vx recording following the second stimulus of a pair. The interstimulus interval is shown between the columns. The intertrial interval (between pairs of stimuli) was the same for all averages at 5 s. At interstimulus intervals of 1 s, some attenuation of the PI3 potential already was evident, with further attenuation and disappearance at 0.7 s and 0.5 s interstimulus intervals. Calibration bars--vertical bar 20/~V, horizontal bar 10 ms.
tested in the two-stimulus paradigm (n = 5) showed habituation properties for the P13 and Pa potentials as described above. Threshold. A final element of the response characteristics tested was threshold. Figure 5 shows averaged recordings from the Vx and ACx electrodes obtained at various stimulus amplitudes. All averages were recorded at 0.2 Hz stimulation frequency. At 103 dB, the averaged P13 and Pa potentials were assumed to be 100%, or control. As stimulus amplitude was reduced to 85 dB and 75 dB, the P13 potential amplitude decreased to 50.3 + 28.7% and 31.0 _ 23.2% of control, respectively. A slight increase in latency was evident as stimulus amplitude decreased. No detectable P13 potential was evident at stimulus am-
0.2sec
~ FIR|T CLICK
0.1eec
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FIG. 4. Habituation of the ACx-recorded Pa potential in the two-stimulus paradigm. Columnar organization as in Fig. 3. Averaged Pa potentials showed little habituation at most intervals >0.2 s, with some habituation at the shortest interval tested, 0.1 s. Calibration bars as in Fig. 3.
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Vx
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85dB 75dB
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TIME (msec) FIG. 5. Thresholds of the Vx-recorded P I3 potential and the ACx-recorded Pa potential. Left column. Averages of the P I3 potential using different amplitudes of stimulation. All averages were carried out at 0.2/s stimulation rate. A maximal response was evident in the Vx-recorded PI3 potential at 103 dB; however, the potential decreased at 85 dB and 75 dB, ultimately disappearing at -<70 dB. Right column. On the other hand, the ACx-recorded Pa potential was present at 75 dB, somewhat reduced at 60 dB and 50 dB, below which it was no longer evident. These recordings also showed a small increase in latency as stimulus amplitude decreased. Calibration bar--vertical bar 20 #V. averaging 2048 trials to obtain a clear series (usually four) of peaks at latencies --<6 ms.
Scopolamine Sensitivity of the P13 Potential It was considered important to use low doses of scopolamine and to monitor the alertness state of the animal in case scopolamine were to have soporific effects and, thus, result in an unspecific effect unrelated to cholinergic blockade of this system. Slowing of the EEG and lethargy, indeed, became intermittent at doses of 5 mg/kg. These became prolonged at doses of 10 mg/ kg, and stereotypic head movements became evident in some animals. However, at lower doses, no detectable unspecific effects were evident either visually or in the EEG. Figure 6 shows the effects on the Vx-recorded P13 potential following injections of saline or of various doses of scopolamine. In this series of animals (n = 7), the amplitude of the PI3 potential was significantly lower at 15 min and 30 min following each of the doses of scopolamine (0.2, 1, and 5 mg/kg). At the highest dose of scopolamine (5 mg/kg), there was significant suppression at 45 rain after injection compared to the potential recorded at 45 rain after saline injection. Maximal suppression was evident at 30 min after injection. The P13 potential amplitude was 63.1 ~ 23.3% of control at 0.2 mg/kg, 36.1 _+ 20.6% of control at 1 mg/kg, and 18.7 _+ 24.7% of control at 5 mg/kg. Because the suppression induced by 1 mg/ kg was not statistically greater than that at 5 mg/kg, the 1 mg/kg dose may be near the maximum effective dose for suppressing
amplitude. On the other hand, the 5 mg/kg dose did have longer lasting effects as PI3 potential amplitude was significantly decreased at 45 min. However, at 60 min postinjection, the P13 potential amplitude was not different from control after injections of any of these three doses. This effect of scopolamine appears shorter lasting than that reported for wave A in the cat [7] or the PI potential in the human [5]. On the other hand, the ACx-recorded Pa potential did not change significantly at any of these doses of scopolamine. Control injections of equal amounts of saline were made to assess unspecific effects of injections on the manifestation of the Vxrecorded P13 potential. As the recordings in Fig. 6 demonstrate, repeated averaging of the P13 potential showed no changes in amplitude compared to control (presaline average) at 15, 30, 45, and 60 min after saline injection. A slight decrease in PI3 potential amplitude was evident at 90 min postinjection. Such an effect was discussed above in terms of the decrement in amplitude observed when recording sessions lasted over 60 min. DISCUSSION The main finding described herein is the identification of an auditory stimulus-evoked potential in the intact rat, which shows characteristics similar to those of the human anditory-evoked middle-latency P1 potential and to those of wave A in the cat. In the intact rat, only a potential at a 13 ms latency showed all of the following characteristics: a) sleep-state dependence; b) rapid habituation to repetitive stimulation, and c) blockade by the cho-
RAT PI3 POTENTIAL
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FIG. 6. Effects of scopolamineon the Vx-recorded PI3 potential. Left column. Recordingsbefore and after saline injection showed no changes in the P13 potential at 15, 30, 45, or 60 min postinjection, with a decrease in amplitude at 90 rain postinjection.Such a decrease was interpreted as a sign of fatigue due to the prolonged recording session and not due to an injection effect. Middle and right columns. Recordingsbefore and after injections of scopolamine of three different concentrations, 0.2, 1. and 5 mg/kg. The averages showed a significant decrement in the amplitude of the P13 potential at 15 and 30 min after the 0.2 mg/kg injection. This effect was more pronounced after the 1 mg/kg injection, while the 5 mg/kg injection produced significant reductions in the amplitude of the PI3 potential at 15, 30, and 45 min after injection. Calibration bar--vertical bar 10/~V. Student's t-test significance*p ~ 0.05, **p --<0.01. linergic antagonist, scopolamine. To determine the presence of sleep-state dependence, it was essential to monitor the level of arousal using EEG and EMG recordings. Blocks of trials were averaged only during waking (low amplitude, high frequency EEG, muscle tone background on EMG, and a visually alert, orienting animal in the chamber), slow-wave sleep (high amplitude, low frequency EEG, decrease of muscle tone in the EMG, inactive animal) and paradoxical sleep (low amplitude, high frequency EEG, absence of EMG activity, inactive animal). Only the Vx-recorded P13 potential was found to be present in waking and paradoxical sleep but absent in slow-wave sleep. This is a major defining characteristic of the P1 potential in the human and wave A in the cat, species in which this potential is thought to be a measure of facilitation of the reticular activating system [4,9,10]. That is, the potential is manifested only during episodes of cortical desynchronization. These recordings clearly demonstrated the sleep-state dependence of the Vx-recorded P13 potential compared to the constant expression of the ACx-recorded Pa potential. Although this is an important characteristic of the P1 potential in the human and wave A in the cat, it was necessary also to determine the nature of the response characteristics of the P13 and Pa potentials. In contrast, the ACx-recorded Pa potential was not affected by sleep-wake changes and appears to be a manifestation of cortical activation by specific sensory pathways. That the earlier latency Pa potential is a product of such fast-conducting, synaptically secure pathways is also evident in its ability to follow high frequencies of stimulation. Little habituation of the Pa potential was evident either following repetitive stimulation or in the two stimulus paradigm. On the other hand, the Vx-recorded P13 potential showed rapid habituation in both stimulation paradigms, a characteristic of slowly conducting, synaptically insecure reticular systems. The third characteristic of the P1 potential in the human and wave A in the cat is sensitivity to the cholinergic antagonist scopolamine [5,7]. The dose-dependent blockade observed in the
present study suggests that the Vx-recorded P13 potential may be mediated by cholinergic mechanisms. Lesions of the mesopontine cholinergic cell group known as the pedunculopontine nucleus (PPN) in the cat, have been found to reduce or block the manifestation of wave A [11]. Thus, the PPN may be at least partly responsible for the manifestation of wave A in the cat and, perhaps, the P1 potential in the human [5,10,11]. The PPN appears to be responsible for generating ponto-geniculo-occipital (PGO) waves [29], which, in turn, can be facilitated by auditory stimulation [2]. The relationship between the P1 potential and PGO waves needs to be determined, as both appear to be generated by the same neurological substrate and follow similar response characteristics. It is tempting to speculate that the P1 potential is a form of PGO wave-like activation. Current efforts are directed at investigating such a possibility. Considerable caution, however, needs to be exercised when interpreting the manifestation at the vertex of an evoked potential presumably generated by subcortical structures. Volume conduction of such potentials would be expected to be considerably reduced at the vertex compared to cortically generated potentials. On the other hand, consideration also needs to be given to the nature of the neurological substrates generating either type of potential. For example, the Pa potential was best observed by recording electrodes located over the auditory cortex and only in a few animals could the Pa potential be observed in the nearby vertex electrode. That is, the auditory cortex is a small region and the Pa potential generated there was fairly localized. On the other hand, the P13 potential appears to result, at least in part, from the effects of mesopontine cholinergic action at its targets. That is, the evoked potential and single-unit studies described in the subsequent articles suggest that it is the activity of cholinergic efferents at its targets that perhaps generates these potentials [23,24]. These efferents terminate in all thalamic nuclei and various basal ganglia structures [31] which, in turn, send massive projections to other cortical and subcortical structures. When the mesopontine cholinergic system is activated, then, a wide array
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(msec)
FIG. 7. Recordings across species. Top. Averaged Vx recordings in the intact rat showing a P13 potential with sleepstate dependence, rapid habituation, and scopolamine sensitivity. Middle. Averaged potential in the cat showing wave A at a 25 ms latency with the same characteristics as the rat P13 potential. Bottom. Averaged P1 potential in the human at a 50-60 ms latency also showing the same characteristics. Calibration bar--vertical bar 10/zV for rat and cat, 2/~V for humans. of neurological substrates subsequently will be activated, making it likely that a vertex recording electrode will pick up this widespread, volume conducted activation. Further evidence that these potentials are mediated at least in part by the PPN is the nature of the threshold of these potentials.
The Vx-recorded PI3 potential exhibited a threshold in the 7 0 80 dB range compared to the thresholds of the BAER and the ACx-recorded Pa potential, which were in the 5 0 - 6 0 dB range. The succeeding articles demonstrate that a potential recorded in the area of the PPN in the rat (at a 12.9 _ 1.5 ms latency) and
RAT P13 POTENTIAL
in the cat (at a latency of 22.6 _+ 2.9 ms) both showed thresholds in the 7 0 - 8 0 dB range [23], and that a significant proportion of single neurons recorded in the area of the PPN in the cat responded to auditory input at thresholds in the 7 0 - 8 0 dB range [241. Although additional studies are needed to establish the Vxrecorded PI3 potential in the intact rat as the equivalent of wave A in the cat, at least they both appear to possess the essential characteristics of the PI potential in the human (i.e., sleep-state dependence, rapid habituation, scopolamine sensitivity). These three potentials are shown in Fig. 7 for comparison. In terms of brain size vs. latency, it would be expected that the equivalent evoked potential in the cat would have a shorter latency compared to that in the human, and that the equivalent potential in the rat would have a shorter latency compared to that in the cat. Moreover, all of these potentials should have longer latencies than the BAERs (-<10 ms in all species) and the Pa ( 6 - 9 ms in the rat, 12-15 ms in the cat, and 2 0 - 3 0 ms in the human) in their respective species. On the other hand, selective lesions of the PPN in the rat will need to be carried out to determine the effects of these lesions on the manifestation of the Vx-recorded P13 potential. In addition, further pharmacological testing of the putative mediation of the P13 potential by cholinergic mechanisms is required. For example, its abolition by other (than scopolamine) cholinergic antagonists and its reinstatement by cholinergic agonists (e.g., physostigmine) need to be tested. In summary, suggestive evidence is provided herein for the presence in the intact rat of an auditory-evoked potential with characteristics similar to those of the P1 potential in the human and wave A in the cat, namely, sleep-state dependence, rapid habituation, and blockade by the cholinergic antagonist scopolamine. ACKNOWLEDGEMENT This work was supported by USPHS Grant NS20246.
REFERENCES 1. Adler, L. E.; Pachtman, E.; Franks, R. D.; Pecevich, M.; Waldo, M. C.; Freedman, R. Neurophysioiogical evidence for a defect in neuronal mechanisms involved in sensory gating in schizophrenia. Biol. Psychiatry 17:639-654; 1982. 2. Arankowsky-Sandoval, G.; Prospero-Garcia, O.; Aguilar-Roblero, R.; Drucker-Colin, R. Cholinergic reduction of REM sleep duration is reversed by auditory stimulation. Brain Res. 365:377380; 1986. 3. Arezzo, J.; Picoff, A.; Vaughan, H. G., Jr. The sources and intracerebral distribution of auditory evoked potentials in the alert rhesus monkey. Brain Res. 90:57-73; 1975. 4. Buchwald, J. S.; Hinman, C.; Norman, R. J.; Huang, C. M.; Brown, K. A. Middle- and long-latency auditory evoked responses recorded from the vertex of normal and chronically lesioned cats. Brain Res. 205:91 - 109; 1981. 5. Buchwald, J. S.; Rubinstein, E. H.; Schwafel, J.; Strandburg, R. J. Midlatency auditory evoked responses: Differential effects of a cholinergic agonist and antagonist. Electroencephalogr. Clin. Neurophysiol. 80:303-309; 1991. 6. Chen, B. M.; Buchwald, J. S. Midlatency auditory evoked responses: Differential effects of sleep in the cat. Eiectroencephalogr. Clin. Neurophysiol. 65:373-382; 1986. 7. Dickerson, L. W.; Buchwald, J. S. Midlatency anditory-evoked responses: Effect of scopolamine in the cat and implications for brain stem cholinergic mechanisms. Exp. Neurol. 112:229-239; 1991.
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8. Erwin, R. J.; Buchwald, J. S. Midlatency auditory evoked responses: Differential recovery cycle characteristics. Electroencephalogr. Clin. Neurophysiol. 64:417-423; 1986. 9. Erwin, R. J.; Buchwald, J. S. Midlatency auditory evoked responses: Differential effects of sleep in the human. Electroencephalogr. Clin. Neurophysiol. 65:383-392; 1986. 10. Erwin, R. J.; Buchwald, J. S. Midlatency auditory evoked responses in the human and the cat model. In: Johnson, R., Jr.; Rohrbaugh, J. W.; Parasuraman, R., eds. Current trends in event-related potential research. Electroencephalogr. Clin. Neurophysiol., Suppl. 40: 461467; 1987. l 1. Hall, R. D.; Borbely, A. A. Acoustically evoked potentials in the rat during sleep and waking. Exp. Brain Res. 11:93-110; 1970. 12. Harrison, J. B.; Woolf, N. J.; Buchwald, J. S. Cholinergic neurons of the feline pontomesencephaion. I. Essential role in "Wave A" generation. Brain Res. 520:43-54; 1990. 13. lwasa, H.; Potsic, W. P. Maturational change of early, middle, and late components of the auditory evoked responses in rats. Otolaryngol. Head Neck Surg. 90:95-102; 1982. 14. Jewett, D. L.; Romano, M. N.; Williston, I. S. Human auditory evoked potentials: Possible brainstem components detected on the scalp. Science 167:1517-1518; 1970. 15. Kaga, K.; Hink, R. F.; Shinoda, Y.; Suzuki, J. Evidence for a primary cortical origin of a middle latency auditory evoked potential in cats. Electroencephalogr. Clin. Neurophysiol. 50:254-266; 1980. 16. Knight, R. T.; Brailowsky, S.; Scabini, D.; Simpson, G. V. Surface auditory evoked potentials in the unrestrained rat: Component definition. Electroencephalogr. Clin. Neurophysiol. 61:430-439; 1985. 17. Kraus, N.; Smith, D. I.; McGee, T. Midline and temporal lobe MLRs in the guinea pig originate from different generator systems: A conceptual framework for new and existing data. Electroencephalogr. Clin. Neurophysiol. 70:541-558; 1988. 18. McGee, T. J.; Ozdamar, O.; Kraus, N. Auditory middle latency responses in the guinea pig. Am. J. Otolaryngol. 4:116-122; 1983. 19. Miyazato, H.; Skinner, R. D.; Garcia-Rill, E.; Reese, N.; Hendricks, B. The rat Pl auditory middle latency evoked potential. Soc. Neurosci. Abstr. 19:1422; 1993. 20. Ozdamar, O.; Kraus, N. Auditory middle-latency responses in humans. Audiology 22:34-49; 1983. 21. Picton, T. W.; Hillyard, S. A. Human auditory evoked potentials. II: Effects of attention. Electroencephalogr. Clin. Neurophysiol. 36:191-199; 1974. 22. Picton, T. W.; Hillyard, S. A.; Krausz, H. I.; Galambos, R. Human auditory evoked potentials. I: Evaluation of components. Electroencephalogr. Clin. Neurophysiol. 36:179-190; 1974. 23. Reese, N. B.; Garcia-Rill, E.; Skinner, R. D. Auditory input to the pedunculopontine nucleus. I. Evoked potentials. Brain Res. Bull. (submitted). 24. Reese, N. B.; Garcia-Rill, E.; Skinner, R. D. Auditory input to the peduncuiopontine nucleus. II. Unit responses. Brain Res. Bull. (submired). 25. Shallop, J. K. Electric response audiometry: The morphology of normal responses. Adv. Otorhinolaryngol. 29:124-139; 1983. 26. Simpson, G. V.; Knight, R. T.; Brailowsky, S., Prospero-Garcia, O.; Scabini, D. Altered peripheral and brainstem auditory function in aged rats. Brain Res. 348:28-35; 1985. 27. Skinner, P.; Glattke, T. J. Electrophysiologic response audiometry: State of the art. J. Speech Hear. Dis. 42:179-198; 1977. 28. Smith, D. I.; Kraus, N. Intracranial and extracranial recordings of the auditory middle latency response. Electroencephaiogr. Clin. Neurophysioi. 71:296-303; 1988. 29. Steriade, M.; McCarley, R. Brain stem control of wakefulness and sleep. New York: Plenum Press; 1990. 30. Teas, D. C.; Kiang, N. Y. Evoked responses from the auditory cortex. Exp. Neurol. 10:91-119; 1964. 31. Woolf, N. J.; Butcher, L. L. Cholinergic systems in the rat brain. III. Projections from the pontomesencephalic tegmentum to the thalamus, tectum, basal ganglia and basal forebrain. Brain Res. Bull. 16:603-637; 1986. 32. Yokoyama, T.; Uemura, K. Study of neural activities of the primary auditory cortex and middle latency auditory evoked potentials. J. Clin. Neurophysiol. 10(2):217-225; 1993.
Pergamon 0361-9230(95)00003-8
A Middle-latency Auditory-evoked Potential in the Rat H. MIYAZATO,* R. D. SKINNER,.1 N. B. REESE,~ F. A. BOOP1- AND E. GARCIA-RILL*
Departments of *Anatomy and l~Neurosurgery, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205 ~:Department of Physical Therapy, University of Central Arkansas, Conway, AR [Received 3 August 1994; Accepted 22 December 1994] ABSTRACT: Previous studies have established the Wesence of a middle-latency audltmy-evoked poten1~ that is charactedzed by a) sleep-stere dependem:, b) iow following f m ~ m ~ y (i~., rapid
hab,wUo, to ~ = ; ; , . J ~ . ) , and c) bk)ckede by the choUner~c aotagonist, sco~lamine. A vertex-recorded evoked potential having t h e w characteristics was described in humans at a 5080 ms letency (termed ti'm P1 or PSO potmtial) and in the cat at a 20-25 ms latency (ten'ned wave A). ~ atudies wera undertaken w ~ If a c l c k sh~mulm-evoked p o t ~ , t ~ havk',g U',e same chamcll~i~k:s was preeent in 'lhe ~lact rat, Vertex and aud~ory cortex recordings in intact rats studied in a sound-attenuating chamber and exposed to free-field click sUmuii showed a) the presence of a verlex racorded potenUal at a 11-15 ms letency, termed P13, and of an auditory cortex recorded potential at a 7-11 ms latency, termed Pa; b) the P13 was prasent during waldng and paredoxical s:~.~pbut al~ent in slow-wave sleep, whSe Pa was prNent in all sleep-wake stat~; c) the P13 habituated markedly at stknulaUon r a t ~ above I Hz while Pa did not; and d) the P13 was blocked by low _(t~___ of scopolamine while Pa was not. These atudles demonstrate tlhe ~ of a Pl-Iike potential in the rat at a 13 ± 2 ms letency. KEY WORDS: Auditory-evoked potentials, Middle-latency potentials, Pl Potential, Intact rat.
INTRODUCTION Evoked potentials recorded in the first 10 ms following an auditory click stimulus, the brain stem auditory evoked responses (BAERs), have been well characterized [ 14,21,27]. However, the middle-latency potentials, those in the 10-100 ms range, have not been characterized as well. In humans, auditory-evoked potentials in this latency range can be recorded at the vertex, one at a latency of 25-35 ms and termed Pa [8,21,22], and another at a latency of 5 0 - 8 0 ms and termed P1 [8,22], Pb [20,25], or P50 [1]. When these potentials were recorded across states of wakefulness and sleep, Pa was shown to remain constant in amplitude across all states while P1 disappeared during slow-wave sleep (SWS) and reappeared at its waking amplitude during rapid eye movement (REM) sleep [8,9]. When rates of auditory stimulation were increased gradually from 0.5 Hz to 10 Hz, Pa remained at a constant amplitude, whereas the amplitude of P1 decreased significantly as stimulation rates exceeded 1 Hz [5,9]. Intravenous injections of the cholinergic antagonist scopolamine resulted in a decrease in amplitude and ultimate disappearance of the P1
potential, an effect that was reversed by subsequent injections of the cholinergic agonist physostigmine. Injections of scopolamine and physostigmine had an opposite and much smaller effect on the Pa potential [5]. Auditory-evoked potentials that have been equated to the human middle-latencyPa and P1 responses also have been recorded in a variety of nonhuman species including monkey [3], cat [4,6,15,30,32), rat [11,13,16,19], and guinea pig [17,18,28]. Studies in the cat revealed the presence of an auditory-evoked potential recorded at a latency of 10-15 ms following an auditory click stimulus. It was present during all stages of sleep, was not abolished by high stimulation rates, and was resistant to barbiturate anesthesia [4,6,30]. This potential was termed ER1 [30] and, more recently, wave 7 [4]. On the other hand, an auditoryevoked potential, which seems by its response characteristics to be analogous to the P1 potential in the human, has been recorded from the vertex in the cat at a latency of 17-25 ms [4,30]. Like the human P1, this potential was present during waking and REM sleep but disappeared during SWS, decreased in amplitude as stimulus presentation rates exceeded 1 Hz, and could not be recorded when the animal was anesthetized [4,6,30]. Subsequent pharmacological studies in the cat demonstrated that this potential, termed wave A [4], could be abolished by administration of scopolamine in a dose-dependent fashion [7]. Subsequent lesion studies showed that wave A was reduced or abolished by lesions of the mesopontine cholinergic pedunculopontinenucleus (PPN), and that the degree of cholinergic cell loss correlated with the reduction in amplitude of wave A [10,12]. Thus, an auditory-evoked potential in the middle-latency range can be recorded both in the human and the cat, and has the characteristics of sleep-state dependence, low following frequency (rapid habituation to repetitive stimulation), and sensitivity to scopolamine, a muscarinic antagonist. The purpose of this study was to determine if an auditory-evoked potential recorded in the intact rat showed similar characteristics as the P1 potential in the human and wave A in the cat, and, therefore, may represent the rat equivalent of these potentials. Preliminary reports of this study have been published [19]. METHOD
Surgical Procedures Adult male Sprague-Dawley rats (n = 22, 250 to 350 g) were anesthetized with ketamine HCI (60 mg/kg, IM) and sodium barbiturate (20 mg/kg, IP). Anesthetic levels were maintained such
~To whom requests for reprints should be addressed. 247
248
that the withdrawal reflex to paw pinch was absent. The animal was placed in a rat stereotaxic instrument using hollow ear bars, which protected the middle ear from injury. After reflection of the scalp, stainless steel screws for recording auditory-evoked potentials were installed epidurally at the vertex, 5.5 mm anterior to the interaural line, 1.0 mm lateral to the midline, midway between bregma and lambda bilaterally. Also, a screw was placed over the right auditory cortex, 4.5 mm anterior to the interaural line and 4.5 mm ventral to the dorsal surface of the skull. A reference screw was inserted into the frontal sinus. Pairs of wires were inserted into the dorsal nucchal muscles bilaterally for electromyographic (EMG) recordings. A wire placed in the dorsal neck served as ground. Wires from all electrodes were led to a nine-pin receptacle (Amphenol), which was cemented to the skull. Penicillin G was given IM and the animals were placed in a warm environment during recovery. Rats were housed individually in a vivarium with a 14 L:I0 D schedule, and food and water were available ad lib. Recordings began after a 7-day recovery period.
Recording Procedures Rats were placed in a sound-attenuating chamber (2 × 2 × 2 r ) with a fan that provided both continuous air flow and white noise background at a level of 65 dB. The chamber was lit by a 12 V DC bulb and a window and peep hole viewer allowed undetected observation of the animal. Wires from a swivel commutator were connected to a plug that mated with the receptacle on the rat's head. Following a 15-rain acclimation period in the chamber, auditory-evoked potentials were recorded from unrestrained rats. In all trials, evoked potentials recorded from the vertex and auditory cortex were amplified (20,000×) and filtered at 3 Hz to 1 kHz. The EMG signal was amplified (5000×) and filtered at 30 Hz to 3 kHz. All recordings were stored on magnetic tape and digitized at a rate of 10 kHz, averaged using SuperScope II software (G. W. Instruments) and the averages stored on disk. Superscope allows a computer and monitor to function as a storage oscilloscope and display individual responses, running averages, rectified, normalized and averaged EMG, frequency analysis of EEG, etc. online. All subjects were tested between 0900 and 1300 h to control for possible time-of-day effects.
Stimulation Procedures Auditory stimuli were delivered by a Grass Click-Tone module (model S10CTCMA) and led to speakers in opposite walls of the chamber. Rarefied clicks having a rise time of 100 #s were routinely presented at 4 0 - 1 0 3 dB, and could be graded in 1-dB increments to determine thresholds. Trains of clicks (5 or 10 clicks at 1 kHz) at a train repetition rate of 0.2 Hz also were used to test auditory-evoked responses. Averaged responses were based on 64 individual evoked responses. Threshold responses were obtained with the white noise background turned off. Although threshold levels for middle-latency responses were determined from averages made at the rate of 0.2 Hz, B A E R threshold levels were obtained from averages of 2048 trials evoked at 10 Hz by a single-click stimulus. In order to establish state dependence of the auditory-evoked potentials, continuous recordings displayed on a paper chart recorder were utilized. Blocks of trials were averaged only during waking (low amplitude, high frequency EEG on the vertex vs. frontal leads, muscle tone background on EMG and a visually alert, orienting animal in the chamber), slow-wave sleep (high amplitude, low frequency EEG, decrease of muscle tone in the EMG, eyes closed, inactive animal), and desynchronized sleep
M I Y A Z A T O ET AL.
(low amplitude, high frequency EEG, absence of EMG activity, eyes closed, inactive animal). In order to determine the response characteristics of the auditory-evoked potentials, different stimulation paradigms were used. To test continuous habituation, the sensitivity of these potentials was determined by averaging sequential trials at a specific interstimulus interval for the entire block of 64 trials. This was used to determine the following frequency of the respective responses. Frequencies of 0.2, 0.5, l, 2, 3, 5, and 10 Hz were used. To test instantaneous habituation, a two-stimulus paradigm was used in which the interstimulus interval was either 0.1, 0.2, 0.3, 0.5, 0.7, or 1 s, but the pairs of stimuli were presented every 5 s. This was used to determined the habituation properties of the respective responses.
Injection Procedures In seven male rats, 0.5 ml of scopolamine hydrobromide (Sigma Chemical Co.) in physiological saline was injected (IP) in concentrations of 0.2, 1.0, or 5.0 mg/kg. Equal amounts of physiological saline were injected in control experiments. Preceding each injection, the evoked potentials were recorded and the average of two blocks of averaged potentials was used as the control level for that injection. Control potentials were normalized to 100% for comparison with postinjection values. Injections were made in random order, not less than 7 days apart.
WaveJbrm Measurements During the first 6 ms following an auditory stimulus, BAERs were evident. The threshold level of the BAERs was based on the first discernible presence of wave 4 [26]. To determine the threshold for BAERs and for middle-latency auditory-evoked potentials, the background white noise was turned off. Likewise, the threshold of the P13 auditory-evoked potential was determined to be the decibel (dB) level at which this wave was first discernible. Measurement of the amplitude of auditory-evoked potentials was made from the beginning of the wave to its peak.
RESULTS After acclimation to the recording chamber, the animals were tbund to shift from periods of quiet to periods of exploratory behavior. On occasion, averaging of evoked potentials was stopped if the animals became too active, as evidenced by excessive EMG activity. Recordings were not carried out under restraint (e.g., a body bag) due to any possible deleterious effects on arousal induced by restraint. Using a free-field approach, recordings could be carried out consistently during quiet periods. It, therefore, became important to determine the arousal state of each animal during averaging. Prolonged periods of quiet alertness could lead to drowsiness and slow-wave sleep, as evidenced by slowing of the EEG, and could ultimately result in the induction of periods of paradoxical sleep. The degree of desynchronization of the EEG and the level of muscle tone reflected in the EMG thus were monitored. The first section of the results describes the behavior of vertex (Vx) and auditory cortex (ACx)recorded middle-latency potentials during various sleep-wake states. Subsequent sections describe the response characteristics of these potentials and the effects of scopolamine administration. The recordings shown in these later sections all were obtained in the awake, quietly alert preparation (desynchronized EEG, muscle tone evident in EMG). The Vx-recorded potentials described below were present in 20 out of 22 animals studied. In two animals, a potential at or around a latency of 13 ms was not evident in the Vx recording. It is not clear if a procedural (surgical and/or record-
RAT P13 P O T E N T I A L
249
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FIG. I. Sleep-state dependence of the Pi3 potential. Top row. Brief periods of EEG and EMG recordings representative of the sleep-wake state of the rat during the period in which the averages in the bottom row were obtained. Note low amplitude, high frequency activity in the EEG and muscle tone in the EMG during waking (AWK). During slow-wave sleep (SWS), high amplitude, low frequency activity was evident in the EEG along with a reduction in EMG activity. During desynchronized or paradoxical sleep (DS), low amplitude, high frequency EEG again was present, but no muscle tone or activity was evident in the EMG. Bottom row. Vertex (Vx) and auditory cortex (ACx) recordings during each of the states described. Averages of the Vx electrode showed a positive potential at 13 ms (PI 3), a negative potential at 20 ms (N20), and a positive potential at 25 ms (P25) latency. In the ACx, there was a positive response at 9 ms (Pa) and a negative potential at 15 ms (NI5). During SWS, no P13 potential was present, while all the other potentials were present in the Vx and ACx recordings. During DS, the PI3 potential returned at its waking amplitude in the Vx average, while the ACx-recorded Pa potential remained the same across all states. Calibration bars--vertical bar 20/~V for averages, horizontal bar 1 s for EEG and EMG recordings. ing) problem was responsible for the lack of P13 potential in these animals. However, it does appear that wave A is not manifested in a small proportion of intact cats [4]. Following auditory click stimuli (103dB), BAERs were evident during the first 6 ms period. These were followed by the middle-latency potentials in the 1 0 - 2 5 ms range. In the rat, the averaged responses recorded at the vertex (Vx) showed peaks near 13 ms (termed P13), 20 ms (termed N20), and 25 ms (termed P25) (Fig. 1). In contrast, recordings over the auditory cortex (ACx) showed a different set of potentials. These peaks occurred at 6 - 9 ms (termed Pa) and 15 ms (termed N15). Thus, in temporal presentation, the middle-latency potentials recorded at the Vx were distinct from those recorded over the ACx. As in other species, the sleep-state dependence and response characteristics of Vx-recorded potentials were found to differ from those of ACx-recorded potentials.
Sleep-state Dependence of the P13 One characteristic of the P13 potential was its sleep-state dependence. Similar to what has been reported for the P1 potential
in the human and wave A in the cat, the rat P13 potential was present during waking, absent during slow-wave sleep, but reappeared at its waking amplitude during paradoxical sleep. Figure 1 shows averages obtained from the Vx and ACx electrodes across s l e e p - w a k e states. During waking, high frequency, low amplitude EEG, orienting responses, and active E M G recordings were evident. During such periods, the Vx-recorded average showed P13, N20, and P25 potentials, while the ACx average showed Pa and N 15 potentials. During slow-wave sleep, slowing of the EEG and high amplitude spindle activity was present along with a reduction, but not total absence, of muscle tone. During slow-wave sleep, no P13 potential was evident, while other Vx and ACx potentials were manifested as in waking. During paradoxical sleep, the EEG once again became desynchronized and of lower amplitude than in slow wave sleep, no orienting or startie-like responses were evident, and there was a total absence of muscle tone. During paradoxical sleep, the PI3 potential recorded from the Vx returned at its waking amplitude, while Pa was present as usual in the ACx recording. Some attenuation of N20, P25, and N15 was evident during paradoxical sleep, making the reappearance of P13 potential at the Vx and the maintenance
250
M I Y A Z A T O ET AL.
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FIG. 2. Following frequency of the Vx-recorded PI3 potential and the ACx-recorded Pa potential. Evoked potential amplitudes observed at a stimulation rate of 0.2/s was considered control for both potentials. Some attenuation of the Vx-recorded PI 3 potential was evident at 0.5Is and l/s, followed by a disappearance of the potential at higher (->2/s) stimulation rates. In contrast, the ACx-recorded Pa potential was present at all frequencies tested, with some attenuation evident at stimulation rates of 5/s and 10/s. Calibration bar--vertical bar 20/~V.
of Pa at the ACx even more significant. Such effects were observed consistently in all four animals tested for sleep dependence of auditory middle-latency potentials. It should be noted that recording sessions typically lasted 1 h, during which time little attenuation of the P13 potential was evident. However, when testing for prolonged periods, the P13 potential was found to decrease in amplitude after 90 min (82.4 ± 26.8% of control). Such results led to setting a maximum recording time of 60 min for all sessions (see below, saline control, Fig. 5).
Response Characteristics Following frequency of the P13 potential. Figure 2 shows the following f r e q u e n c y characteristics of the Vx- and ACxrecorded potentials used to test continuous habituation. At stimulation frequencies of 0.2/s, the P13 and Pa potentials were d e t e r m i n e d to be 100%, or control. As stimulation frequencies were increased to 0.5/s and l/s, the amplitude of the V x - r e c o r d e d P13 potential was 70.1 ± 34.2% and 42.7 _+ 24.7% of control, respectively. On the other hand, the ACxrecorded Pa potential was 100.4 ± 19.3% and 91.5 _+ 23.5% of control, respectively, for stimulation rates of 0.5/s and l/s. No P13 potential was evident in the Vx recording at stimulation rates -> 2Is. In contrast, the A C x - r e c o r d e d Pa potential was found to habituate s o m e w h a t (53.6 _+ 17.0% of control), but only at stimulation rates of 10Is.
These results demonstrated the rapid habituation to continuous stimulation exhibited by the P I 3 potential contrasted with the resistance of the Pa potential. The longer latency potentials (N20, P25, and N15) all showed some habituation, but only at high stimulation rates. The effects described above were observed in every animal tested with this paradigm (n = 5). Habituation of the P13. Figure 3 shows representative recordings from the Vx electrode in response to pairs of stimuli of identical amplitude (103 dB) used to test instantaneous habituation. The intertrial interval for all pairs of stimuli was 5 s. The averaged responses to the first stimulus of a pair are shown on the left (each representing 100%, or control). The averaged responses to the second stimulus of a pair are shown on the right. The P13 potential in response to the second stimulus was 34.9 + 17.0% of control when the interstimulus interval was 1 s, while it was 36.6 ± 15.5 % and 27.5 ± 20.2% of control at intervals of 0.7 and 0.5 s, respectively. The response to the second stimulus was absent at shorter interstimulus intervals (6.6 ± 10.7% at 0.2 s and 1.0 -+ 2.4% at 0.1 s). In contrast, Fig. 4 shows representative recordings of the Pa potential obtained from the ACx electrode in response to the paired stimulus paradigm. No decrement in responsiveness was evident at interstimulus intervals ->0.2 s. Only at the shortest interstimulus interval (0.1 s) was there a moderate reduction of the Pa response to the second stimulus compared to the response to the first stimulus (52.7 ± 15.7% of control). All of the animals
R A T P13 P O T E N T I A L
251
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plitudes of 70 dB. For the group of animals tested for threshold (n = 12), the m e a n threshold for the P13 potential was 72.7 +_ 2.6 dB. By comparison, the amplitude of the ACx-recorded Pa potential was 77.6 _+ 21.2% of control at 75 dB, 45.9 _+ 29.7% at 60 dB, and 33.0 __ 9.8% at 50 dB. As with the P13 potential, a slight increase in latency was evident as stimulus amplitude decreased. No detectable Pa potential was evident at < 4 5 dB. For the group of animals tested for threshold, the mean threshold for the Pa potential was 51.7 + 2.9 dB. The threshold for the B A E R s (not shown) at 50.4 +_ 1.6 dB was similar to that o f the Pa potential. The B A E R and Pa thresholds were not significantly different from each other, while the Pa threshold was significantly lower than the threshold of the P13 potential ( M a n n - W h i t n e y U-test, one-tailed p-value 0.01). The B A E R averages were generated using 10 Hz stimulation and
0.Ssec
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ct.l~lt FIG. 3. Habituation of the Vx-reeorded PI3 potential in the two-click paradigm. The column on the left shows the averaged Vx recording following the first stimulus of a pair, while the column on the right shows the averaged Vx recording following the second stimulus of a pair. The interstimulus interval is shown between the columns. The intertrial interval (between pairs of stimuli) was the same for all averages at 5 s. At interstimulus intervals of 1 s, some attenuation of the PI3 potential already was evident, with further attenuation and disappearance at 0.7 s and 0.5 s interstimulus intervals. Calibration bars--vertical bar 20/~V, horizontal bar 10 ms.
tested in the two-stimulus paradigm (n = 5) showed habituation properties for the P13 and Pa potentials as described above. Threshold. A final element of the response characteristics tested was threshold. Figure 5 shows averaged recordings from the Vx and ACx electrodes obtained at various stimulus amplitudes. All averages were recorded at 0.2 Hz stimulation frequency. At 103 dB, the averaged P13 and Pa potentials were assumed to be 100%, or control. As stimulus amplitude was reduced to 85 dB and 75 dB, the P13 potential amplitude decreased to 50.3 + 28.7% and 31.0 _ 23.2% of control, respectively. A slight increase in latency was evident as stimulus amplitude decreased. No detectable P13 potential was evident at stimulus am-
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FIG. 4. Habituation of the ACx-recorded Pa potential in the two-stimulus paradigm. Columnar organization as in Fig. 3. Averaged Pa potentials showed little habituation at most intervals >0.2 s, with some habituation at the shortest interval tested, 0.1 s. Calibration bars as in Fig. 3.
252
M I Y A Z A T O ET AL.
Vx
ACx
P13
/F
103dB
85dB 75dB
60dB 75dB 70dB
50dB 45dB ~
~
~
TIME (msec) FIG. 5. Thresholds of the Vx-recorded P I3 potential and the ACx-recorded Pa potential. Left column. Averages of the P I3 potential using different amplitudes of stimulation. All averages were carried out at 0.2/s stimulation rate. A maximal response was evident in the Vx-recorded PI3 potential at 103 dB; however, the potential decreased at 85 dB and 75 dB, ultimately disappearing at -<70 dB. Right column. On the other hand, the ACx-recorded Pa potential was present at 75 dB, somewhat reduced at 60 dB and 50 dB, below which it was no longer evident. These recordings also showed a small increase in latency as stimulus amplitude decreased. Calibration bar--vertical bar 20 #V. averaging 2048 trials to obtain a clear series (usually four) of peaks at latencies --<6 ms.
Scopolamine Sensitivity of the P13 Potential It was considered important to use low doses of scopolamine and to monitor the alertness state of the animal in case scopolamine were to have soporific effects and, thus, result in an unspecific effect unrelated to cholinergic blockade of this system. Slowing of the EEG and lethargy, indeed, became intermittent at doses of 5 mg/kg. These became prolonged at doses of 10 mg/ kg, and stereotypic head movements became evident in some animals. However, at lower doses, no detectable unspecific effects were evident either visually or in the EEG. Figure 6 shows the effects on the Vx-recorded P13 potential following injections of saline or of various doses of scopolamine. In this series of animals (n = 7), the amplitude of the PI3 potential was significantly lower at 15 min and 30 min following each of the doses of scopolamine (0.2, 1, and 5 mg/kg). At the highest dose of scopolamine (5 mg/kg), there was significant suppression at 45 rain after injection compared to the potential recorded at 45 rain after saline injection. Maximal suppression was evident at 30 min after injection. The P13 potential amplitude was 63.1 ~ 23.3% of control at 0.2 mg/kg, 36.1 _+ 20.6% of control at 1 mg/kg, and 18.7 _+ 24.7% of control at 5 mg/kg. Because the suppression induced by 1 mg/ kg was not statistically greater than that at 5 mg/kg, the 1 mg/kg dose may be near the maximum effective dose for suppressing
amplitude. On the other hand, the 5 mg/kg dose did have longer lasting effects as PI3 potential amplitude was significantly decreased at 45 min. However, at 60 min postinjection, the P13 potential amplitude was not different from control after injections of any of these three doses. This effect of scopolamine appears shorter lasting than that reported for wave A in the cat [7] or the PI potential in the human [5]. On the other hand, the ACx-recorded Pa potential did not change significantly at any of these doses of scopolamine. Control injections of equal amounts of saline were made to assess unspecific effects of injections on the manifestation of the Vxrecorded P13 potential. As the recordings in Fig. 6 demonstrate, repeated averaging of the P13 potential showed no changes in amplitude compared to control (presaline average) at 15, 30, 45, and 60 min after saline injection. A slight decrease in PI3 potential amplitude was evident at 90 min postinjection. Such an effect was discussed above in terms of the decrement in amplitude observed when recording sessions lasted over 60 min. DISCUSSION The main finding described herein is the identification of an auditory stimulus-evoked potential in the intact rat, which shows characteristics similar to those of the human anditory-evoked middle-latency P1 potential and to those of wave A in the cat. In the intact rat, only a potential at a 13 ms latency showed all of the following characteristics: a) sleep-state dependence; b) rapid habituation to repetitive stimulation, and c) blockade by the cho-
RAT PI3 POTENTIAL
253
Scopolamine Saline VX
Img/kg
0.2mg/Ikg
5mg/kg
P13
Pr.-,o,.o,on Post-injection 15m in
." /~~ /~/ ~\/ ~ / / ~ ~,~
30mini/
\\\-/i~
TIME (meec)
FIG. 6. Effects of scopolamineon the Vx-recorded PI3 potential. Left column. Recordingsbefore and after saline injection showed no changes in the P13 potential at 15, 30, 45, or 60 min postinjection, with a decrease in amplitude at 90 rain postinjection.Such a decrease was interpreted as a sign of fatigue due to the prolonged recording session and not due to an injection effect. Middle and right columns. Recordingsbefore and after injections of scopolamine of three different concentrations, 0.2, 1. and 5 mg/kg. The averages showed a significant decrement in the amplitude of the P13 potential at 15 and 30 min after the 0.2 mg/kg injection. This effect was more pronounced after the 1 mg/kg injection, while the 5 mg/kg injection produced significant reductions in the amplitude of the PI3 potential at 15, 30, and 45 min after injection. Calibration bar--vertical bar 10/~V. Student's t-test significance*p ~ 0.05, **p --<0.01. linergic antagonist, scopolamine. To determine the presence of sleep-state dependence, it was essential to monitor the level of arousal using EEG and EMG recordings. Blocks of trials were averaged only during waking (low amplitude, high frequency EEG, muscle tone background on EMG, and a visually alert, orienting animal in the chamber), slow-wave sleep (high amplitude, low frequency EEG, decrease of muscle tone in the EMG, inactive animal) and paradoxical sleep (low amplitude, high frequency EEG, absence of EMG activity, inactive animal). Only the Vx-recorded P13 potential was found to be present in waking and paradoxical sleep but absent in slow-wave sleep. This is a major defining characteristic of the P1 potential in the human and wave A in the cat, species in which this potential is thought to be a measure of facilitation of the reticular activating system [4,9,10]. That is, the potential is manifested only during episodes of cortical desynchronization. These recordings clearly demonstrated the sleep-state dependence of the Vx-recorded P13 potential compared to the constant expression of the ACx-recorded Pa potential. Although this is an important characteristic of the P1 potential in the human and wave A in the cat, it was necessary also to determine the nature of the response characteristics of the P13 and Pa potentials. In contrast, the ACx-recorded Pa potential was not affected by sleep-wake changes and appears to be a manifestation of cortical activation by specific sensory pathways. That the earlier latency Pa potential is a product of such fast-conducting, synaptically secure pathways is also evident in its ability to follow high frequencies of stimulation. Little habituation of the Pa potential was evident either following repetitive stimulation or in the two stimulus paradigm. On the other hand, the Vx-recorded P13 potential showed rapid habituation in both stimulation paradigms, a characteristic of slowly conducting, synaptically insecure reticular systems. The third characteristic of the P1 potential in the human and wave A in the cat is sensitivity to the cholinergic antagonist scopolamine [5,7]. The dose-dependent blockade observed in the
present study suggests that the Vx-recorded P13 potential may be mediated by cholinergic mechanisms. Lesions of the mesopontine cholinergic cell group known as the pedunculopontine nucleus (PPN) in the cat, have been found to reduce or block the manifestation of wave A [11]. Thus, the PPN may be at least partly responsible for the manifestation of wave A in the cat and, perhaps, the P1 potential in the human [5,10,11]. The PPN appears to be responsible for generating ponto-geniculo-occipital (PGO) waves [29], which, in turn, can be facilitated by auditory stimulation [2]. The relationship between the P1 potential and PGO waves needs to be determined, as both appear to be generated by the same neurological substrate and follow similar response characteristics. It is tempting to speculate that the P1 potential is a form of PGO wave-like activation. Current efforts are directed at investigating such a possibility. Considerable caution, however, needs to be exercised when interpreting the manifestation at the vertex of an evoked potential presumably generated by subcortical structures. Volume conduction of such potentials would be expected to be considerably reduced at the vertex compared to cortically generated potentials. On the other hand, consideration also needs to be given to the nature of the neurological substrates generating either type of potential. For example, the Pa potential was best observed by recording electrodes located over the auditory cortex and only in a few animals could the Pa potential be observed in the nearby vertex electrode. That is, the auditory cortex is a small region and the Pa potential generated there was fairly localized. On the other hand, the P13 potential appears to result, at least in part, from the effects of mesopontine cholinergic action at its targets. That is, the evoked potential and single-unit studies described in the subsequent articles suggest that it is the activity of cholinergic efferents at its targets that perhaps generates these potentials [23,24]. These efferents terminate in all thalamic nuclei and various basal ganglia structures [31] which, in turn, send massive projections to other cortical and subcortical structures. When the mesopontine cholinergic system is activated, then, a wide array
254
M I Y A Z A T O ET AL.
WAVE
A
c,T
P1
HUMAN
I
i i
~
t'e
l's
2'0
2'~
3'~
3's
4'0
TIME
4'5 s'e s~
6~
6~
z~
r~
(msec)
FIG. 7. Recordings across species. Top. Averaged Vx recordings in the intact rat showing a P13 potential with sleepstate dependence, rapid habituation, and scopolamine sensitivity. Middle. Averaged potential in the cat showing wave A at a 25 ms latency with the same characteristics as the rat P13 potential. Bottom. Averaged P1 potential in the human at a 50-60 ms latency also showing the same characteristics. Calibration bar--vertical bar 10/zV for rat and cat, 2/~V for humans. of neurological substrates subsequently will be activated, making it likely that a vertex recording electrode will pick up this widespread, volume conducted activation. Further evidence that these potentials are mediated at least in part by the PPN is the nature of the threshold of these potentials.
The Vx-recorded PI3 potential exhibited a threshold in the 7 0 80 dB range compared to the thresholds of the BAER and the ACx-recorded Pa potential, which were in the 5 0 - 6 0 dB range. The succeeding articles demonstrate that a potential recorded in the area of the PPN in the rat (at a 12.9 _ 1.5 ms latency) and
RAT P13 POTENTIAL
in the cat (at a latency of 22.6 _+ 2.9 ms) both showed thresholds in the 7 0 - 8 0 dB range [23], and that a significant proportion of single neurons recorded in the area of the PPN in the cat responded to auditory input at thresholds in the 7 0 - 8 0 dB range [241. Although additional studies are needed to establish the Vxrecorded PI3 potential in the intact rat as the equivalent of wave A in the cat, at least they both appear to possess the essential characteristics of the PI potential in the human (i.e., sleep-state dependence, rapid habituation, scopolamine sensitivity). These three potentials are shown in Fig. 7 for comparison. In terms of brain size vs. latency, it would be expected that the equivalent evoked potential in the cat would have a shorter latency compared to that in the human, and that the equivalent potential in the rat would have a shorter latency compared to that in the cat. Moreover, all of these potentials should have longer latencies than the BAERs (-<10 ms in all species) and the Pa ( 6 - 9 ms in the rat, 12-15 ms in the cat, and 2 0 - 3 0 ms in the human) in their respective species. On the other hand, selective lesions of the PPN in the rat will need to be carried out to determine the effects of these lesions on the manifestation of the Vx-recorded P13 potential. In addition, further pharmacological testing of the putative mediation of the P13 potential by cholinergic mechanisms is required. For example, its abolition by other (than scopolamine) cholinergic antagonists and its reinstatement by cholinergic agonists (e.g., physostigmine) need to be tested. In summary, suggestive evidence is provided herein for the presence in the intact rat of an auditory-evoked potential with characteristics similar to those of the P1 potential in the human and wave A in the cat, namely, sleep-state dependence, rapid habituation, and blockade by the cholinergic antagonist scopolamine. ACKNOWLEDGEMENT This work was supported by USPHS Grant NS20246.
REFERENCES 1. Adler, L. E.; Pachtman, E.; Franks, R. D.; Pecevich, M.; Waldo, M. C.; Freedman, R. Neurophysioiogical evidence for a defect in neuronal mechanisms involved in sensory gating in schizophrenia. Biol. Psychiatry 17:639-654; 1982. 2. Arankowsky-Sandoval, G.; Prospero-Garcia, O.; Aguilar-Roblero, R.; Drucker-Colin, R. Cholinergic reduction of REM sleep duration is reversed by auditory stimulation. Brain Res. 365:377380; 1986. 3. Arezzo, J.; Picoff, A.; Vaughan, H. G., Jr. The sources and intracerebral distribution of auditory evoked potentials in the alert rhesus monkey. Brain Res. 90:57-73; 1975. 4. Buchwald, J. S.; Hinman, C.; Norman, R. J.; Huang, C. M.; Brown, K. A. Middle- and long-latency auditory evoked responses recorded from the vertex of normal and chronically lesioned cats. Brain Res. 205:91 - 109; 1981. 5. Buchwald, J. S.; Rubinstein, E. H.; Schwafel, J.; Strandburg, R. J. Midlatency auditory evoked responses: Differential effects of a cholinergic agonist and antagonist. Electroencephalogr. Clin. Neurophysiol. 80:303-309; 1991. 6. Chen, B. M.; Buchwald, J. S. Midlatency auditory evoked responses: Differential effects of sleep in the cat. Eiectroencephalogr. Clin. Neurophysiol. 65:373-382; 1986. 7. Dickerson, L. W.; Buchwald, J. S. Midlatency anditory-evoked responses: Effect of scopolamine in the cat and implications for brain stem cholinergic mechanisms. Exp. Neurol. 112:229-239; 1991.
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8. Erwin, R. J.; Buchwald, J. S. Midlatency auditory evoked responses: Differential recovery cycle characteristics. Electroencephalogr. Clin. Neurophysiol. 64:417-423; 1986. 9. Erwin, R. J.; Buchwald, J. S. Midlatency auditory evoked responses: Differential effects of sleep in the human. Electroencephalogr. Clin. Neurophysiol. 65:383-392; 1986. 10. Erwin, R. J.; Buchwald, J. S. Midlatency auditory evoked responses in the human and the cat model. In: Johnson, R., Jr.; Rohrbaugh, J. W.; Parasuraman, R., eds. Current trends in event-related potential research. Electroencephalogr. Clin. Neurophysiol., Suppl. 40: 461467; 1987. l 1. Hall, R. D.; Borbely, A. A. Acoustically evoked potentials in the rat during sleep and waking. Exp. Brain Res. 11:93-110; 1970. 12. Harrison, J. B.; Woolf, N. J.; Buchwald, J. S. Cholinergic neurons of the feline pontomesencephaion. I. Essential role in "Wave A" generation. Brain Res. 520:43-54; 1990. 13. lwasa, H.; Potsic, W. P. Maturational change of early, middle, and late components of the auditory evoked responses in rats. Otolaryngol. Head Neck Surg. 90:95-102; 1982. 14. Jewett, D. L.; Romano, M. N.; Williston, I. S. Human auditory evoked potentials: Possible brainstem components detected on the scalp. Science 167:1517-1518; 1970. 15. Kaga, K.; Hink, R. F.; Shinoda, Y.; Suzuki, J. Evidence for a primary cortical origin of a middle latency auditory evoked potential in cats. Electroencephalogr. Clin. Neurophysiol. 50:254-266; 1980. 16. Knight, R. T.; Brailowsky, S.; Scabini, D.; Simpson, G. V. Surface auditory evoked potentials in the unrestrained rat: Component definition. Electroencephalogr. Clin. Neurophysiol. 61:430-439; 1985. 17. Kraus, N.; Smith, D. I.; McGee, T. Midline and temporal lobe MLRs in the guinea pig originate from different generator systems: A conceptual framework for new and existing data. Electroencephalogr. Clin. Neurophysiol. 70:541-558; 1988. 18. McGee, T. J.; Ozdamar, O.; Kraus, N. Auditory middle latency responses in the guinea pig. Am. J. Otolaryngol. 4:116-122; 1983. 19. Miyazato, H.; Skinner, R. D.; Garcia-Rill, E.; Reese, N.; Hendricks, B. The rat Pl auditory middle latency evoked potential. Soc. Neurosci. Abstr. 19:1422; 1993. 20. Ozdamar, O.; Kraus, N. Auditory middle-latency responses in humans. Audiology 22:34-49; 1983. 21. Picton, T. W.; Hillyard, S. A. Human auditory evoked potentials. II: Effects of attention. Electroencephalogr. Clin. Neurophysiol. 36:191-199; 1974. 22. Picton, T. W.; Hillyard, S. A.; Krausz, H. I.; Galambos, R. Human auditory evoked potentials. I: Evaluation of components. Electroencephalogr. Clin. Neurophysiol. 36:179-190; 1974. 23. Reese, N. B.; Garcia-Rill, E.; Skinner, R. D. Auditory input to the pedunculopontine nucleus. I. Evoked potentials. Brain Res. Bull. (submitted). 24. Reese, N. B.; Garcia-Rill, E.; Skinner, R. D. Auditory input to the peduncuiopontine nucleus. II. Unit responses. Brain Res. Bull. (submired). 25. Shallop, J. K. Electric response audiometry: The morphology of normal responses. Adv. Otorhinolaryngol. 29:124-139; 1983. 26. Simpson, G. V.; Knight, R. T.; Brailowsky, S., Prospero-Garcia, O.; Scabini, D. Altered peripheral and brainstem auditory function in aged rats. Brain Res. 348:28-35; 1985. 27. Skinner, P.; Glattke, T. J. Electrophysiologic response audiometry: State of the art. J. Speech Hear. Dis. 42:179-198; 1977. 28. Smith, D. I.; Kraus, N. Intracranial and extracranial recordings of the auditory middle latency response. Electroencephaiogr. Clin. Neurophysioi. 71:296-303; 1988. 29. Steriade, M.; McCarley, R. Brain stem control of wakefulness and sleep. New York: Plenum Press; 1990. 30. Teas, D. C.; Kiang, N. Y. Evoked responses from the auditory cortex. Exp. Neurol. 10:91-119; 1964. 31. Woolf, N. J.; Butcher, L. L. Cholinergic systems in the rat brain. III. Projections from the pontomesencephalic tegmentum to the thalamus, tectum, basal ganglia and basal forebrain. Brain Res. Bull. 16:603-637; 1986. 32. Yokoyama, T.; Uemura, K. Study of neural activities of the primary auditory cortex and middle latency auditory evoked potentials. J. Clin. Neurophysiol. 10(2):217-225; 1993.