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Midlatency auditory-evoked potentials in the rat: effects of interventions that modulate arousal
Brain Research Bulletin, Vol. 48, No. 5, pp. 545–553, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/99/$–see front matter
PII S0361-9230(99)00034-9
Midlatency auditory-evoked potentials in the rat: Effects of interventions that modulate arousal H. Miyazato,1 R. D. Skinner,1 M. Cobb,2 B. Andersen2 and E. Garcia-Rill1* Departments of 1Anatomy and 2Neurosurgery, University of Arkansas for Medical Sciences, Little Rock, AR, USA [Received 22 September 1998; Accepted 22 January 1999] ABSTRACT: The vertex-recorded P13 midlatency auditoryevoked potential in the rat shows the same characteristics as the P1 potential in the human, namely, sleep-state dependence, rapid habituation and blockade by the cholinergic antagonist scopolamine. The P13 potential appears to be generated, at least in part, by projections of the pedunculopontine nucleus, the cholinergic arm of the reticular activating system. On the other hand, the auditory cortex-recorded P7 potential appears to be of primary cortical origin. Simultaneous recordings from the vertex and the auditory cortex showed that (1) the P13 potential was suppressed by administration of the anesthetics ketamine, pentobarbital or halothane in a dose-dependent manner, but the P7 potential was not; (2) the P13 potential was suppressed by intragastric injections of ethanol in a dose-dependent manner, but the P7 potential was not; (3) the amplitude of the P13 potential was negatively correlated with blood ethanol levels; (4) both the P13 and P7 potentials were still present following injections of the neuromuscular blocker pancuronium bromide; and (5) both the P13 and P7 potentials were decreased by diffuse brain injury induced by a weight-drop device in a weight-dependent manner. These findings suggest that the P13 potential is more sensitive than the P7 potential to changes in arousal and that the P13 and P7 potentials are not of myogenic but of neural origin. © 1999 Elsevier Science Inc.
be suppressed by the cholinergic antagonist scopolamine in a dose-dependent manner, (4) be blocked by pentobarbital; (5) be suppressed in a dose-dependent manner by microinjections of neuroactive compounds into the pedunculopontine nucleus (PPN); and (6) be unaffected by bilateral auditory cortex ablation [30,31,42]. The P13 potential appears to be the rodent equivalent of the P1 (or P50) midlatency auditory-evoked potential in the human, because they share the three main characteristics of sleep-state dependence, rapid habituation and blockade by scopolamine [7,14,15,31]. These characteristics suggest that these potentials are manifestations, at least in part, of the reticular activating system (RAS). On the other hand, the P7 (or Pa) potential at a latency of 6 –9 ms is (1) highly localized at the ACx, (2) not affected by sleep-wake changes, (3) stable at high stimulation rates up to 10/s, (4) resistant to scopolamine or pentobarbital, (5) not affected by microinjections of neuroactive compounds into the PPN, and (6) abolished by bilateral auditory cortex ablation [31,42]. These characteristics suggest that this potential is a manifestation of primary auditory pathway activity. The overall aim of the present research was to test the effects of various interventions that modulate arousal on the manifestation of the P13 potential compared to the P7 potential. Auditory-evoked potentials have been used to assess functional activity after some of these interventions. For instance, the effects on the BAEPs of anesthetics [4,23,37,38], ethanol [9,10,43] or head injury [49] have been investigated in the rat. Based on the known sites of origin of the BAEPs, these effects appear to reflect functional changes in areas between the acoustic nerve and the lateral lemniscus [39]. In contrast, the effects on subsequent midlatency potentials are relatively unknown. Thus, a specific aim of the present study was to explore the effects on the P13 and P7 potentials of anesthetics, ethanol and head injury by recording these potentials simultaneously. The other aim was to determine, using a neuromuscular blocker, if muscle activity elicited by auditory stimulation contributes to the morphology of the P13 and/or P7 potentials. It is important to clarify this issue because the auditory startle response exhibits a peak at a latency of 8 –11 ms, which is close to the peak latency of the P13 potential and could overlap with that of the P7 potential. Preliminary findings have been reported [2,29].
KEY WORDS: P13 potential, Ketamine, Pentobarbital, Halothane, Ethanol, Head injury.
INTRODUCTION The evoked potentials recorded in the first 5 ms following auditory stimulation, the brain stem auditory-evoked potentials (BAEPs), have been well characterized in the rat [18,23,39], but subsequent potentials have not been as well characterized. Recently, however, it was demonstrated that there are two sets of midlatency components in the rat, one set maximal at the vertex (Vx) and another set maximal laterally over the auditory cortex (ACx). Among Vx-recorded potentials, the P13 potential at a latency of 11–15 ms has been found to: (1) be sleep-state dependent, that is, present during waking and rapid eye movement sleep, but absent during slow-wave sleep (i.e., present during desynchronized electroencephalographic states); (2) undergo rapid habituation at stimulation rates of more than 1/s; (3)
* Address for correspondence: Edgar Garcia-Rill, Department of Anatomy, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA. Fax: (501) 686-6382; E-mail: [email protected]
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546 MATERIALS AND METHODS Surgical Procedures Adult male Sprague-Dawley rats (250 to 350 g; Charles River, Wilmington, MA, USA) were anesthetized with ketamine HCl (60 mg/kg, i.m.) and sodium pentobarbital (20 mg/kg, i.p.). Anesthetic levels were maintained such that the withdrawal reflex to paw pinch was absent. The animal was placed in a stereotaxic instrument using hollow ear bars that protected the middle ear from injury. After reflection of the scalp, stainless steel screws for recording auditory-evoked potentials were inserted epidurally at the Vx, 5.5 mm anterior to the interaural line, 1.0 mm lateral to the midline, bilaterally. Also, a screw was placed over the right ACx through the lateral skull, that is, 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. All Vx and ACx potentials were recorded with reference to this electrode. Pairs of wires were inserted into the dorsal nuchal muscles bilaterally for electromyographic (EMG) recordings. A wire placed in the dorsal neck served as a ground. A 9-pin receptacle, to which wires from all electrodes were led, was cemented to the skull. A chronic gastric tube (flexible polyethylene tube, 0.38 mm internal diameter [I.D.]) was implanted for ethanol injection studies. Modified procedures were used for head injury studies. Instead of the screws, thin wire electrodes, the ends of which were exposed, were inserted epidurally over the Vx and ACx, and a metallic helmet (a stainless-steel disc 10 mm in diameter and 3 mm thick) was fixed with dental cement to the central portion of the skull vault between the coronal and lambdoid sutures. Penicillin G was given i.m., and the animals were placed in a warm environment during recovery. Rats were housed individually in a vivarium with a 12:12 light/ dark schedule (lights on 0600 h), and food and water were available ad libitum. Recordings began after a 2-week recovery period. Recording and Stimulation Procedures Evoked potentials were recorded from the Vx and the ACx, and EMG from neck muscles. Neck muscle EMGs were recorded to monitor startle responses elicited by auditory stimuli. Rats were placed in a sound-attenuating chamber (2 3 2 3 2 ft) with a fan that provided both continuous air flow and white noise background at a level of 60 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. For head injury studies, each wire was connected to a clip that mated with the individual wire electrodes. Following a 15-min acclimation period in the chamber, auditory-evoked potentials were recorded before and after administration of a compound or of a weight-drop. In all trials, evoked potentials from the Vx and the ACx were amplified (10,000 3) and filtered at 3 Hz–1 KHz. Measurement of the amplitude of auditory-evoked potentials was made from the beginning of the wave to its peak. The EMG signal was amplified (10,000 3), filtered at 30 Hz–3KHz and rectified. All recordings were amplified by a Grass A.C. Pre-Amplifier (Model P511K), digitized at a rate of 10 KHz, averaged, stored on VHS videotape and analyzed off-line using SuperScope II (GW Instruments, Cambridge, MA, USA) software. Averaged potentials consisted of 32 individual evoked potentials. All subjects were tested between 0900 h and 1300 h to control for possible time-of-day effects. Auditory stimuli were delivered by a Grass Instruments ClickTone module (Model S10ASCM) and led to speakers on opposite walls of the chamber. Click stimuli having a duration of 0.1 ms (square wave pulse, broad band) were presented at 103 dB in trains of 5 clicks at 1 kHz. Trains of clicks were delivered once every 5 s until 32 evoked potentials were acquired for averaging.
MIYAZATO ET AL. Testing Procedures Control recordings of auditory-evoked potentials were performed prior to administration of each compound or a weight-drop, while the animal was alert but resting quietly. Procedures of each study after these control recordings were as follows: 1. Ketamine and pentobarbital: For dose-response studies, ketamine 2, 7.5, 30 or 120 mg/kg i.m. (n 5 4 rats) or pentobarbital 1.5, 5, 20 or 40 mg/kg i.p. (n 5 4 rats) was injected, and recordings were performed thereafter. Physiological saline was injected alone as a control. 2. Halothane: Animals (n 5 4 rats) were placed in a 26 3 16 3 13 cm closed Plexiglast chamber, and halothane (1, 2 or 4%) was administered at a flow rate of 3 l/min using compressed oxygen. The administration of oxygen (3 l/min) alone was used as a control (halothane 0%) condition. After exposure to halothane for 15 min, animals were returned to the recording chamber and evoked potentials were recorded. The time when the halothane was discontinued was designated as 0 min of recording time, and recordings were made at 3, 10 and 20 min thereafter. 3. Ethanol: Ethanol 1, 3 or 5 g/kg (40% in saline; n 5 6 rats) was injected through an intragastric tube, and post-injection recordings were performed thereafter. In addition, blood ethanol concentration was measured in a separate experiment (n 5 16 rats) in which rats were administered ethanol in a 40% solution in saline by gavage in amounts up to 4 ml such that doses of 1, 3 or 5 g/kg resulted. Each dosage group and a control group (4 ml saline only) had 4 animals each. Thirty minutes after gavage, anesthesia was induced by injecting pentobarbital (100 mg/kg, i.p.), and blood samples were taken via cardiac puncture. Ethanol blood levels were determined using an enzymatic method (Sigma Diagnosticst #332-UV, St. Louis, MO, USA) kit and photometry at 340 nm in comparison to standard ethanol solutions. In all dose-response studies for anesthetics and ethanol, the administration of an agent or saline was made in random order, and no animal was injected more than once per week or more than six times overall. 4. Pancuronium bromide (n 5 4 rats): Under halothane anesthesia, the trachea was intubated for artificial ventilation, and an intraperitoneal tube was inserted for drug injection. Animals were then placed in the recording chamber, and recordings were made before and after the injection of pancuronium bromide (0.5 mg/kg) through the intraperitoneal tube. Halothane (1%) anesthesia was performed at a flow rate of 0.5 l/min with compressed oxygen such that the withdrawal reflex was absent. This level was established before the injection of pancuronium bromide and maintained during post-injection recordings. At the conclusion of post-injection recordings, rats were sacrificed by an overdose of pentobarbital. 5. Head injury: Animals (n 5 13 rats) were first anesthetized with 3% halothane by the same procedure used in halothane doseresponse studies as described above. After 15 min of exposure to halothane, animals were returned to the recording chamber, and the withdrawal reflex was tested every 15 s. The time when the reflex first reappeared was designated as 0 min of recording time, then recordings were made at 5, 15, 30, 60, 180 min and 24 h. Three days after this halothane control study (sham injury), head injury was induced by a weight-drop device consisting of a weight falling through a Plexiglas guide tube (19 mm I.D.) [16,28]. After control recordings were performed, animals were again anesthetized with halothane (3%). Fifteen minutes after exposure, the halothane was discontinued and animals were placed on a foam bed beneath the head injury device. When the withdrawal reflex first reappeared (the 0-min
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recording time), the injury was delivered by dropping a 450-g weight from a predetermined height (1 m, mild injury, n 5 4 rats; or 2 m, severe injury, n 5 9 rats) onto the metallic disc helmet fixed on the skull of the rat. Immediately after the impact, animals were placed into the recording chamber, and post-impact recordings were made at 5, 15, 30, 60, 180 min and 24 h. All animal procedures were approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee. Statistics To determine the effects of compounds (or weights) on the amplitude of the P13 potential, the pre-injection (pre-impact) amplitude was designated as 100%, and subsequent post-injection (post-impact) amplitudes were calculated as a percent amplitude of this control recording. Evoked potential data were evaluated using two-factor repeated-measures analysis of variance (ANOVA) with within-subject factors of dose for each compound (ketamine, pentobarbital, halothane or ethanol) and time after exposure. For head injury data, weight and time were used as within-subject factors. In addition, blood ethanol concentration data were analyzed using one-factor ANOVA. Significant main effects were followed with post hoc within-subject contrasts with significance adjusted for multiple comparisons using the Scheffe´ test. Differences were considered to be significant when a p-value , 0.05 was found. RESULTS Effects of Anesthetics on P13 Potential Amplitude Ketamine. The effects of ketamine injections on P13 potential amplitude are shown in Fig. 1A (n 5 4 rats). The ANOVA performed on these data revealed significant effects of dose, F(4,79) 5 36.28, p , 0.0001, and time, F(3,79) 5 13.82, p 5 0.0003, and there was a Dose 3 Time interaction, F(12,79) 5 5.22, p , 0.0001. The dose effects of ketamine, evaluated with post hoc within-subject comparisons, indicated that: (1) the 7.5mg/kg ketamine dose effect was greater than both the saline effect, t 5 8.85, p , 0.02, and the 2-mg/kg dose effect, t 5 10.46, p 5 0.0072; (2) the 30-mg/kg ketamine dose effect was greater than both the saline effect, t 5 40.89, p , 0.0001, and the 2-mg/kg dose effect, t 5 45.48, p , 0.0001, and tended to be greater than the 7.5-mg/kg dose effect, t 5 3.59, p 5 0.082; (3) the 120-mg/kg ketamine dose effect was greater than the saline effect, t 5 87.97, p , 0.0001, the 2-mg/kg dose effect, t 5 135.74, p , 0.0001, the 7.5-mg/kg dose effect, t 5 61.75, p , 0.0001, and the 30-mg/kg dose effect, t 5 39.02, p , 0.0001; and (4) the 2-mg/kg ketamine dose effect was not significantly different from the saline effect, t 5 0.20, n.s. Therefore, ketamine decreased the amplitude of the P13 potential in a dose-dependent manner. Pentobarbital. The effects of pentobarbital on P13 potential amplitude are shown in Fig. 1B (n 5 4 rats). Pentobarbital also decreased the amplitude of the P13 potential in a dose-dependent manner. Analysis of variance testing revealed significant effects of dose, F(3,63) 5 78.33, p , 0.0001, and time, F(3,63) 5 7.57, p 5 0.0042, and there was a Dose 3 Time interaction, F(9,63) 5 4.32, p 5 0.0007. The dose effects of pentobarbital were evaluated with post hoc within-subject comparisons, which indicated that: (1) the 5-mg/kg pentobarbital dose effect was greater than both the saline effect, t 5 49.66, p , 0.0001, and the 1.5-mg/kg dose effect, t 5 17.90, p 5 0.0012; (2) the 20-mg/kg pentobarbital dose effect was greater than the saline effect, t 5 236.08, p , 0.0001, the 1.5mg/kg dose effect, t 5 67.07, p , 0.0001, and the 5-mg/kg dose effect, t 5 60.62, p , 0.0001; and (3) the 1.5-mg/kg pentobarbital
FIG. 1. Effects on P13 potential amplitude of ketamine (A), pentobarbital (B) and halothane (C). The P13 potential was averaged over 32 trials before and after injection of ketamine (KET: 2, 7.5, 30 or 120 mg/kg, i.m.), pentobarbital (PB: 1.5, 5 or 20 mg/kg, i.p.) or saline (SAL), or the inhalation of halothane with oxygen (HAL: 1, 2 or 4%) or oxygen only (HAL 0). Each point is the mean 6 standard error of the P13 potential amplitude as a percent of the pre-administration P13 potential amplitude. Note that the time scale for halothane is shorter than that for ketamine or pentobarbital. Scheffe´ post hoc test significance: 1p , 0.05 and *p , 0.01 compared to saline (A and B) or oxygen only (C).
dose effect was not significantly different from the saline effect, t 5 0.54, n.s. At the 40-mg/kg pentobarbital dose (Fig. 2), the Vx-recorded P13 potential was completely suppressed for about 2 h whereas the ACx-recorded P7 potential was stable. At 1 day after the injection, however, the amplitude of the P13 potential recovered to the pre-injection level. Halothane. The effects of halothane on P13 potential amplitude are shown in Fig. 1C (n 5 4 rats). The ANOVA performed on these data revealed significant effects of dose, F(3,63) 5 21.56, p , 0.0001, and time, F(3,63) 5 13.25, p 5 0.0004, and there was a Dose 3 Time interaction, F(9,63) 5 4.30, p 5 0.0008. The dose effects of halothane were evaluated with post hoc within-subject comparisons, which indicated that: (1) the 1% halothane dose effect was greater than the oxygen-only effect, t 5 5.91, p 5 0.032; (2) the 2% halothane dose effect was greater than the oxygen-only effect, t 5 14.44, p 5 0.0025, but not than the 1%
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FIG. 2. Effects of pentobarbital (40 mg/kg, i.p.) on the amplitudes of the vertex (Vx)-recorded P13 potential and the auditory cortex (ACx)-recorded P7 potential. Left column: pre-injection control (CTL) recordings show the averaged P13 and P7 potentials (32 trials) induced by auditory stimulation. Middle columns: 15- and 30-min post-injection recordings showed the disappearance of the P13 potential (significant decrease in amplitude is noted by a filled circle in this and subsequent figures) but the presence of the P7 potential. Right column: 1-day post-injection recordings showed the recovery of the amplitude of the P13 potential. Calibration bars: vertical 5 20 mV; horizontal 5 10 ms. Arrow indicates onset of auditory stimulus for this and subsequent figures.
effect, t 5 1.03, n.s.; and (3) the 4% halothane dose effect was greater than the oxygen-only effect, t 5 50.10, p , 0.0001, the 1% dose effect, t 5 35.06, p , 0.0001, and the 2% dose effect, t 5 41.73, p , 0.0001. Hence, halothane decreased the amplitude of the P13 potential in a dose-dependent manner. Effects of Ethanol on P13 Potential Amplitude The effects of intragastric injections of ethanol or saline are shown in Fig. 3A (n 5 6 rats). Analysis of variance revealed significant effects of dose, F(3,167) 5 54.44, p , 0.0001, and time, F(6,167) 5 10.56, p , 0.0001, and there was a significant Dose 3 Time interaction, F(18,167) 5 2.14, p , 0.009. Post hoc within-subject comparisons indicated that: (1) the 3-g/kg ethanol dose effect was greater than both the saline effect, t 5 67.57, p , 0.0001, and the 1-g/kg dose effect, t 5 50.04, p , 0.0001; (2) the 5-g/kg ethanol dose effect was greater than the saline effect, t 5 157.96, p , 0.0001, the 1-g/kg dose effect, t 5 73.20, p , 0.0001, and the 3-g/kg dose effect, t 5 9.94, p 5 0.0033; and (3) the 1-g/kg ethanol dose effect was not significantly different from the saline effect, t 5 0.42, n.s. Thus, ethanol also decreased the amplitude of the P13 potential in a dose-dependent manner. In 16 other male rats (250 –350 g), blood ethanol concentrations were measured 30 min after ethanol (or saline) injection. In animals given saline (n 5 4) or ethanol 1 g/kg (n 5 4), 3 g/kg (n 5 4) or 5 g/kg (n 5 4), the blood ethanol level was 0.0113 6 0.0037, 0.0433 6 0.0268, 0.1123 6 0.0293 or 0.1201 6 0.0262% (mean 6 SD), respectively. A one-factor ANOVA revealed a significant main effect of ethanol doses on blood levels, F(3,15) 5 25.76, p , 0.0001, and a Sheffe´ post hoc test showed increased blood ethanol levels at higher ethanol dosages (df 5 3 for each comparison: 1 g/kg vs. saline, t 5 1.57, n.s.; 3 g/kg vs. saline, t 5 15.57, p , 0.01; 3 g/kg vs. 1 g/kg, t 5 7.25, p , 0.01; 5 g/kg vs. saline, t 5 18.05, p , 0.01; 5 g/kg vs. 1 g/kg, t 5 8.98, p , 0.01; and 5 g/kg vs. 3 g/kg, t 5 0.092, n.s.). Fig. 3B shows the negative correlation between P13 amplitude and blood ethanol level at 30 min after
FIG. 3. (A) Effects of ethanol on P13 potential amplitude. The P13 potential was averaged over 32 trials before and after intragastric injection of ethanol (EtOH: 1, 3 or 5 g/kg) or saline (SAL). Each point is the mean 6 standard error of the P13 potential amplitude as a percent of pre-injection P13 potential amplitude at various times after administration. Scheffe´ post hoc test significance: 1p , 0.05 and *p , 0.01 compared to saline. (B) Correlation between the mean values of P13 potential amplitude and of blood ethanol level. P13 potentials, averaged at 30 min after intragastric injection of ethanol (1, 3 or 5 g/kg) or saline, are plotted as a function of blood ethanol levels. These were determined in a different group of rats at 30 min after gavage of the same doses of ethanol. The coefficient of correlation was r 5 20.97, p , 0.03.
administering 0 (saline only), 1, 3 or 5 g/kg of ethanol. It should be noted that the amplitude of the P13 potential in the saline control (SAL) condition is not 100% in Fig. 3B because this denotes (1) the non-statistically significant effect of making an i.p. injection compared to the undisturbed, non-injected condition (100%) and (2) fluctuations within the normal range of amplitude for the P13 potential. It is important to test for unspecific effects of vehicle/control injections. Effects of Neuromuscular Blocker on P13 and P7 Potential Amplitudes Figure 4 shows the effects of injections of pancuronium bromide on the amplitudes of auditory-evoked potentials in lightly anesthetized animals (n 5 4 rats). During quiet waking, auditory stimulation elicited the P13 potential, the P7 potential
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FIG. 5. Effects of weight-drop-induced head injury on P13 potential amplitude. The P13 potential was averaged over 32 trials before and after a weight-drop (450 g at 1 m, mild injury; or 450 g at 2 m, severe injury). Because weight-drop impact was delivered under halothane anesthesia, recordings from animals exposed to halothane without being subjected to head trauma served as sham injury (SHAM). Note a decrease in P13 potential amplitude at 5 min in sham injury because of the suppressive effects of halothane. Scheffe´ post hoc test significance: 1p , 0.05 and *p , 0.01 compared to sham injury.
FIG. 4. Averaged recordings (32 trials) during quiet waking (A), during artificial ventilation with 1% halothane (B), and 15 min after the injection of pancuronium bromide (0.5 mg/kg, i.p.) under 1% halothane anesthesia (C). Note that muscle activity, such as the startle response (SR), was absent but that the vertex (Vx)-recorded P13 and the auditory cortex (ACx)recorded P7 potentials were present after pancuronium bromide injection. Note the dramatic attenuation of background muscle activity and the lack of SR (filled circles) under anesthesia (B) and/or neuromuscular blockade (C). EMG, electromyogram. Calibration bars: vertical 5 20 mV; horizontal 5 5 ms.
and the startle response in the Vx, ACx and EMG, respectively (Fig. 4A). During artificial ventilation with 1% halothane, the P13 and P7 potentials were still present while the startle response had disappeared (Fig. 4B). Fifteen minutes after an injection of pancuronium bromide (0.5 mg/kg, i.p.) in the presence of continued 1% halothane, the amplitudes of the P13 and P7 potentials did not change significantly compared to pre-injection values (Fig. 4C). The mean amplitude of the P13 potential before injection was 20 6 7 mV, and after injection it
was 20 6 2 mV (t 5 2.1, p 5 0.92, n.s.). Likewise, the mean peak latency was 14.8 6 0.4 ms before and 14.4 6 0.7 ms after neuromuscular blockade (t 5 0.98, p 5 0.35, n.s.). The amplitude and latency of the P7 potential were also unchanged by treatment (amplitude before 5 15 6 4 mV, after 5 14 6 3 mV, t 5 0.41, p 5 0.7, n.s.; latency before 5 8.2 6 0.2 ms, after 5 7.8 6 0.3 ms, t 5 2.28, p 5 0.06, n.s.). Effects of Diffuse Head Injury on P13 Potential Amplitude Every rat (4 of 4) that received the 450-g, 1-m weight-drop impact and 6 of 9 rats that received the 450-g, 2-m impact survived, compatible with previous findings [16,28]. The effects of head injury on P13 potential amplitude are shown in Fig. 5, and averaged recordings are shown in Fig. 6. Weight-drop impact decreased the amplitude of the P13 potential in a weight-dependent manner. The ANOVA performed on these data revealed significant effects of weight, F(2,194) 5 114.27, p , 0.0001, and time, F(4,194) 5 137.23, p , 0.0001, and there was a significant Weight 3 Time interaction, F(8,194) 5 14.08, p , 0.0001. The weight effects were explored with post hoc within-subject comparisons, which indicated that (1) the 450-g, 1-m weight effect was greater than the sham effect, t 5 21.67, p , 0.0001, and (2) the 450-g, 2-m weight effect was greater than both the sham effect, F(1) 5 297.3, p , 0.0001, and the 450-g, 1-m effect, t 5 19.44, p 5 0.0002.
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FIG. 6. Effects on P13 potential amplitude of the 450-g, 1-m mild injury (left column), the 450-g, 2-m severe injury (middle column) and sham injury (right column). Note that the amplitude of the P13 potential was reduced by impact in a weight-dependent manner. A filled circle indicates that mild injury reduced the P13 potential at 5 min (p , 0.05) and that severe injury reduced it at 5, 15 and 30 min (p , 0.01) compared to sham injury. Calibration bar: vertical bar 5 20 mV.
Effects of Anesthetics, Ethanol or Head Injury on P7 Potential Amplitude The effects of various interventions on the amplitude of the P7 potential were investigated. Analysis of variance revealed that administrations of ketamine (2, 7.5, 30 or 120 mg/kg i.m., n 5 4 rats), pentobarbital (1.5, 5 or 20 mg/kg i.p., n 5 4 rats), halothane (1, 2 or 4%, n 5 4 rats) or ethanol (1, 3 or 5 g/kg i.p., n 5 6 rats) failed to change statistically the amplitude of the ACx-recorded P7 potential. In contrast, the ANOVA performed on head injury data revealed significant effects on P7 potential amplitude of impact, F(2,134) 5 66.29, p , 0.0001, and time, F(4,134) 5 5.84, p 5 0.0008, and there was a significant Impact 3 Time interaction, F(8,134) 5 9.14, p , 0.0001. The impact effects were evaluated with post hoc withinsubject comparisons, which indicated that (1) the 450-g, 1-m effect was greater than the sham effect, t 5 5.85, p 5 0.020, and (2) the 450-g, 2-m effect was greater than both the sham effect, t 5 84.89, p , 0.0001, and the 450-g, 1-m effect, t 5 16.58, p 5 0.001. Therefore, the amplitude of the P7 potential, as well as that of the P13 potential, was decreased by weight-drop impact in a weight-dependent manner. DISCUSSION Anesthesia and Ethanol Ketamine, pentobarbital, halothane and ethanol suppressed the P13 potential in a dose-dependent manner but not the P7 potential,
indicating a clear difference in susceptibility between the P13 and P7 potentials to these compounds. This dissociation is most likely due to the differences in mechanisms of the generation of each potential. The P7 potential now appears to be well established as a primary auditory cortical potential in the rat [3,5,21,31,39,40,42]. On the other hand, data have accumulated as to the source(s) of the P13 potential. The Vx-recorded P13 potential was not eliminated by bilateral lesions of the auditory cortex, implying that this potential did not represent primary cortical events [42]. In addition, an auditory-evoked potential at a latency of 11–15 ms was recorded in and around the PPN in the decerebrate rat, that is, in the absence of the cerebral cortex, basal ganglia and hippocampus, emphasizing the possibility of a subcortical origin for the P13 potential [34]. The PPN, the cholinergic arm of the RAS, has been implicated in various functions such as sleep–wake mechanisms, arousal and locomotion [35,44]. More recently, it was shown that the P13 potential was suppressed by activation of known inhibitory synapses, both GABAergic [30] and noradrenergic (Miyazato et al., in press), at the level of the PPN. Furthermore, in PPN-lesioned rats, there was a positive correlation between the amplitude of the P13 potential and the number of NADPH diaphorase-positive (cholinergic) neurons in the PPN (unpublished data). These findings also indicate that the PPN is involved, at least in part, in the generation of the P13 potential. The results of the present study are consistent with this hypothesis, because it has been shown that the multisynaptic, barbiturate-sensitive extralemniscal pathways, such
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551 Pancuronium Bromide Paralysis The injection of the neuromuscular blocker, pancuronium bromide, did not affect the morphology of the P13 potential or the P7 potential, indicating that muscle potentials elicited by auditory stimulation, typically the startle response, were not the sources of the P13 and P7 potentials. To our knowledge, this is the first direct evidence excluding the possibility of a myogenic contribution to surface-recorded midlatency potentials in the rat. This finding is consistent with previous findings showing that, although response characteristics bear some similarities, the P13 potential and the startle response subserve different, although linked, functions and that the P7 potential has properties different from those of the startle response [31,32]. It should be noted, however, that the animals were anesthetized with halothane (1%) throughout the pancuronium bromide experiments. It has been shown that, in the paralyzed non-anesthetized cat, the midlatency auditory-evoked wave A and wave 7 potentials were unchanged from their appearance in the awake, restrained preparation [6]. Wave A and wave 7 are considered to be the feline equivalent of the P1 potential in the human and the primary auditory cortical potential, respectively [6,8,13]. It has also been shown that the P1 and Pa potentials in the human were not eliminated by a neuromuscular blocking agent, indicating that these midlatency potentials are not of myogenic origin [22,24]. It should also be noted that the P13 potential is present during paradoxical sleep [31,32], when atonia is maximal, lending further evidence to the suggestion that the P13 potential is not of myogenic origin. Head Injury
FIG. 7. Recordings across species. (A) Averaged vertex (Vx) and auditory cortex (ACx) recordings in the intact rat showing P13 and P7 potentials, respectively. In this case, a potential (the latency of which was similar to the ACx-recorded P7 potential) was also recorded at the Vx. (B) An averaged P1 potential in the human at a 50-ms latency along with a Pa potential at a 25-ms latency. This particular recording is from a 30-year-old man. Although this recording has not been published previously, it is similar to those obtained in similar studies in humans [45]. Note that the latencies of the human Pa, P1 and P2 potentials are approximately four times as long as those of the rat P7, P13 and P25 potentials, respectively. Calibration bar: 15 mV for rat and 1 mV for human.
as the RAS, are more susceptible to anesthetic suppression than the barbiturate-insensitive lemniscal pathways consisting of synaptically secure afferent neurons, such as primary auditory projections [11,17,19,20,41]. Unit recordings have demonstrated that reticular formation neurons are also sensitive to ethanol, whereas cells of the cerebral cortex appear to be relatively less sensitive [25,50]. The present results clearly indicate that the P13 potential is related to arousal and blocked by various anesthetics and ethanol in a dose-dependent manner, whereas the primary auditory P7 potential is not significantly affected by the dosages that do block the P13 potential. The synaptic security of the lemniscal pathways is assumed to be the reason for the maintenance of the P7 potential following exposure to these agents, whereas the multi-synaptic reticular pathways responsible for P13 potential manifestation appear greatly affected.
Weight-drop impact suppressed both the P13 and P7 potentials, indicating that the effects of weight-drop impact appeared to be non-specific compared to those of anesthetics or ethanol. In agreement with this interpretation is the fact that the weight-drop procedure used in the present study has been shown to produce graded diffuse axonal injury similar to that described in man. That is, when applying the definition of diffuse axonal injury [1], mild (450-g, 1-m) injury is consistent with Grade 1 (axonal injury involved the cerebral hemisphere and brain stem without focal lesions in the corpus callosum or the brain stem). On the other hand, severe (450-g, 2-m) injury can be categorized as Grade 3 (more global axonal abnormalities involving the cerebellum with petechial hemorrhages in the brain stem) [16]. It has been demonstrated that BAEPs failed to differentiate between these two grades of injury, because BAEPs were unaffected by mild or severe injury [49]. Based on this finding, these authors concluded that brain-stem function was not severely compromised in the weight-drop closed-head injury model. In contrast, the present study demonstrated that both the P13 and P7 potentials decreased in a weight-dependent (or injury grade) manner. Because the P13 potential is most likely of posterior midbrain origin, this impact model appears to produce sustained dysfunction not only at the cortical but also at the brain-stem level. The P13 and P7 potentials, thus, could be used as more sensitive monitors in head injury studies. Most incidences of coma are caused by posterior midbrain damage due to head trauma/injury. Recovery from coma is essentially a re-establishment of consciousness, that is, of cortical desynchronization and arousal. The P1 potential in the human and the P13 potential in the rat may some day be useful in determining and, perhaps, predicting recovery from coma. What these preliminary studies show is that there is some recovery of the P13 potential from mild (full recovery within hours) and severe (partial recovery within a day) head trauma/injury.
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Comparison with Midlatency Potentials in the Human In the human, the Pa potential (25- to 40-ms latency), which precedes the P1 potential (50 –70 ms), has been shown to be an auditory cortical potential [12,14,15,26,27]. The Pa potential, as well as the BAEPs, has been used clinically for monitoring the depth of general anesthesia during surgery, because the amplitude of the Pa potential was shown to be suppressed by volatile anesthetic agents in a dose-related manner [33,46 – 48]. In contrast the rat P7 primary auditory cortical potential was not suppressed by halothane in the present study. It should be noted, however, that, in the present halothane dose-response study, the P7 potential was recorded after the discontinuation of halothane, whereas, in the human study, the Pa potential was recorded while anesthesia was maintained. Recording the P7 potential during anesthesia would be required to compare the findings across the species. In contrast the effects of general anesthetic agents on the human P1 potential are not as well investigated. Schwender et al. [36] reported that the amplitude of the P1 potential, as well as that of the Pa potential, was suppressed in a dose-dependent manner by general anesthesia. As a comparison, Fig. 7 shows midlatency auditory-evoked potentials in the rodent and the human [45]. It should be noted that, except for the differences in latency, the morphology of the waveform is similar in the two species. Based on available evidence, it appears that the P13 and P7 potentials are the rodent equivalents of the P1 and Pa potentials in the human, respectively. The present study describes the basic characteristics of the effects on the P13 and P7 potentials of various interventions that modulate arousal, such as anesthetics, ethanol and diffuse brain injury. The findings discussed provide additional evidence suggesting that the P13 potential is a manifestation of the RAS and may be a useful tool for assessing pharmacological, pathological and physiological changes in arousal. ACKNOWLEDGEMENT
This work was supported by USPHS grant NS20246.
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553 43. Squires, K. C.; Chu, N.-S.; Starr, A. Auditory brain stem potentials with alcohol. Electroenceph. Clin. Neurophysiol. 45:577–584; 1978. 44. Steriade, M.; McCarley, R. W. Brainstem control of wakefulness and sleep. New York: Plenum Press; 1990. 45. Teo, C.; Rasco, L.; Al-Mefty, K.; Skinner, R. D.; Boop, F. A.; Garcia-Rill, E. Decreased habituation of midlatency auditory evoked responses in Parkinson’s disease. Mov. Disord. 12:655– 664; 1997. 46. Thornton, C.; Barrowcliffe, M. P.; Konieczko, K. M.; Ventham, P.; Dore, C. J.; Newton, D. E. F.; Jones, J. G. The auditory evoked response as an indicator of awareness. Br. J. Anaesth. 63:113–115; 1989. 47. Thornton, C.; Catley, D. M.; Jordan, C.; Lehane, J. R.; Royston, D.; Jones, J. G. Enflurane anaesthesia causes graded changes in the brainstem and early cortical auditory evoked response in man. Br. J. Anaesth. 55:479 – 486; 1983. 48. Thornton, C.; Heneghan, C. P. H.; James, M. F. M.; Jones, J. G. Effects of halothane or enflurane with controlled ventilation on auditory evoked potentials. Br. J. Anaesth. 56:315–323; 1984. 49. van den Brink, W. A.; Slomka, W.; Marmarou, A.; Avezaat, C. J. J. Neurophysiological evidence of preservation of brain stem function in experimental closed head injury in the rat. In: Avezaat, C.; van Eijndhoven, J.; Maas, A.; et al., eds. Intracranial pressure VII. Berlin: Springer-Verlag; 1993:221–226. 50. Wayner, M. J.; Ono, T.; Nolley, D. Effects of ethyl alcohol on central neurons. Pharmacol. Biochem. Behav. 3:499 –506; 1975.
PII S0361-9230(99)00034-9
Midlatency auditory-evoked potentials in the rat: Effects of interventions that modulate arousal H. Miyazato,1 R. D. Skinner,1 M. Cobb,2 B. Andersen2 and E. Garcia-Rill1* Departments of 1Anatomy and 2Neurosurgery, University of Arkansas for Medical Sciences, Little Rock, AR, USA [Received 22 September 1998; Accepted 22 January 1999] ABSTRACT: The vertex-recorded P13 midlatency auditoryevoked potential in the rat shows the same characteristics as the P1 potential in the human, namely, sleep-state dependence, rapid habituation and blockade by the cholinergic antagonist scopolamine. The P13 potential appears to be generated, at least in part, by projections of the pedunculopontine nucleus, the cholinergic arm of the reticular activating system. On the other hand, the auditory cortex-recorded P7 potential appears to be of primary cortical origin. Simultaneous recordings from the vertex and the auditory cortex showed that (1) the P13 potential was suppressed by administration of the anesthetics ketamine, pentobarbital or halothane in a dose-dependent manner, but the P7 potential was not; (2) the P13 potential was suppressed by intragastric injections of ethanol in a dose-dependent manner, but the P7 potential was not; (3) the amplitude of the P13 potential was negatively correlated with blood ethanol levels; (4) both the P13 and P7 potentials were still present following injections of the neuromuscular blocker pancuronium bromide; and (5) both the P13 and P7 potentials were decreased by diffuse brain injury induced by a weight-drop device in a weight-dependent manner. These findings suggest that the P13 potential is more sensitive than the P7 potential to changes in arousal and that the P13 and P7 potentials are not of myogenic but of neural origin. © 1999 Elsevier Science Inc.
be suppressed by the cholinergic antagonist scopolamine in a dose-dependent manner, (4) be blocked by pentobarbital; (5) be suppressed in a dose-dependent manner by microinjections of neuroactive compounds into the pedunculopontine nucleus (PPN); and (6) be unaffected by bilateral auditory cortex ablation [30,31,42]. The P13 potential appears to be the rodent equivalent of the P1 (or P50) midlatency auditory-evoked potential in the human, because they share the three main characteristics of sleep-state dependence, rapid habituation and blockade by scopolamine [7,14,15,31]. These characteristics suggest that these potentials are manifestations, at least in part, of the reticular activating system (RAS). On the other hand, the P7 (or Pa) potential at a latency of 6 –9 ms is (1) highly localized at the ACx, (2) not affected by sleep-wake changes, (3) stable at high stimulation rates up to 10/s, (4) resistant to scopolamine or pentobarbital, (5) not affected by microinjections of neuroactive compounds into the PPN, and (6) abolished by bilateral auditory cortex ablation [31,42]. These characteristics suggest that this potential is a manifestation of primary auditory pathway activity. The overall aim of the present research was to test the effects of various interventions that modulate arousal on the manifestation of the P13 potential compared to the P7 potential. Auditory-evoked potentials have been used to assess functional activity after some of these interventions. For instance, the effects on the BAEPs of anesthetics [4,23,37,38], ethanol [9,10,43] or head injury [49] have been investigated in the rat. Based on the known sites of origin of the BAEPs, these effects appear to reflect functional changes in areas between the acoustic nerve and the lateral lemniscus [39]. In contrast, the effects on subsequent midlatency potentials are relatively unknown. Thus, a specific aim of the present study was to explore the effects on the P13 and P7 potentials of anesthetics, ethanol and head injury by recording these potentials simultaneously. The other aim was to determine, using a neuromuscular blocker, if muscle activity elicited by auditory stimulation contributes to the morphology of the P13 and/or P7 potentials. It is important to clarify this issue because the auditory startle response exhibits a peak at a latency of 8 –11 ms, which is close to the peak latency of the P13 potential and could overlap with that of the P7 potential. Preliminary findings have been reported [2,29].
KEY WORDS: P13 potential, Ketamine, Pentobarbital, Halothane, Ethanol, Head injury.
INTRODUCTION The evoked potentials recorded in the first 5 ms following auditory stimulation, the brain stem auditory-evoked potentials (BAEPs), have been well characterized in the rat [18,23,39], but subsequent potentials have not been as well characterized. Recently, however, it was demonstrated that there are two sets of midlatency components in the rat, one set maximal at the vertex (Vx) and another set maximal laterally over the auditory cortex (ACx). Among Vx-recorded potentials, the P13 potential at a latency of 11–15 ms has been found to: (1) be sleep-state dependent, that is, present during waking and rapid eye movement sleep, but absent during slow-wave sleep (i.e., present during desynchronized electroencephalographic states); (2) undergo rapid habituation at stimulation rates of more than 1/s; (3)
* Address for correspondence: Edgar Garcia-Rill, Department of Anatomy, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA. Fax: (501) 686-6382; E-mail: [email protected]
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546 MATERIALS AND METHODS Surgical Procedures Adult male Sprague-Dawley rats (250 to 350 g; Charles River, Wilmington, MA, USA) were anesthetized with ketamine HCl (60 mg/kg, i.m.) and sodium pentobarbital (20 mg/kg, i.p.). Anesthetic levels were maintained such that the withdrawal reflex to paw pinch was absent. The animal was placed in a stereotaxic instrument using hollow ear bars that protected the middle ear from injury. After reflection of the scalp, stainless steel screws for recording auditory-evoked potentials were inserted epidurally at the Vx, 5.5 mm anterior to the interaural line, 1.0 mm lateral to the midline, bilaterally. Also, a screw was placed over the right ACx through the lateral skull, that is, 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. All Vx and ACx potentials were recorded with reference to this electrode. Pairs of wires were inserted into the dorsal nuchal muscles bilaterally for electromyographic (EMG) recordings. A wire placed in the dorsal neck served as a ground. A 9-pin receptacle, to which wires from all electrodes were led, was cemented to the skull. A chronic gastric tube (flexible polyethylene tube, 0.38 mm internal diameter [I.D.]) was implanted for ethanol injection studies. Modified procedures were used for head injury studies. Instead of the screws, thin wire electrodes, the ends of which were exposed, were inserted epidurally over the Vx and ACx, and a metallic helmet (a stainless-steel disc 10 mm in diameter and 3 mm thick) was fixed with dental cement to the central portion of the skull vault between the coronal and lambdoid sutures. Penicillin G was given i.m., and the animals were placed in a warm environment during recovery. Rats were housed individually in a vivarium with a 12:12 light/ dark schedule (lights on 0600 h), and food and water were available ad libitum. Recordings began after a 2-week recovery period. Recording and Stimulation Procedures Evoked potentials were recorded from the Vx and the ACx, and EMG from neck muscles. Neck muscle EMGs were recorded to monitor startle responses elicited by auditory stimuli. Rats were placed in a sound-attenuating chamber (2 3 2 3 2 ft) with a fan that provided both continuous air flow and white noise background at a level of 60 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. For head injury studies, each wire was connected to a clip that mated with the individual wire electrodes. Following a 15-min acclimation period in the chamber, auditory-evoked potentials were recorded before and after administration of a compound or of a weight-drop. In all trials, evoked potentials from the Vx and the ACx were amplified (10,000 3) and filtered at 3 Hz–1 KHz. Measurement of the amplitude of auditory-evoked potentials was made from the beginning of the wave to its peak. The EMG signal was amplified (10,000 3), filtered at 30 Hz–3KHz and rectified. All recordings were amplified by a Grass A.C. Pre-Amplifier (Model P511K), digitized at a rate of 10 KHz, averaged, stored on VHS videotape and analyzed off-line using SuperScope II (GW Instruments, Cambridge, MA, USA) software. Averaged potentials consisted of 32 individual evoked potentials. All subjects were tested between 0900 h and 1300 h to control for possible time-of-day effects. Auditory stimuli were delivered by a Grass Instruments ClickTone module (Model S10ASCM) and led to speakers on opposite walls of the chamber. Click stimuli having a duration of 0.1 ms (square wave pulse, broad band) were presented at 103 dB in trains of 5 clicks at 1 kHz. Trains of clicks were delivered once every 5 s until 32 evoked potentials were acquired for averaging.
MIYAZATO ET AL. Testing Procedures Control recordings of auditory-evoked potentials were performed prior to administration of each compound or a weight-drop, while the animal was alert but resting quietly. Procedures of each study after these control recordings were as follows: 1. Ketamine and pentobarbital: For dose-response studies, ketamine 2, 7.5, 30 or 120 mg/kg i.m. (n 5 4 rats) or pentobarbital 1.5, 5, 20 or 40 mg/kg i.p. (n 5 4 rats) was injected, and recordings were performed thereafter. Physiological saline was injected alone as a control. 2. Halothane: Animals (n 5 4 rats) were placed in a 26 3 16 3 13 cm closed Plexiglast chamber, and halothane (1, 2 or 4%) was administered at a flow rate of 3 l/min using compressed oxygen. The administration of oxygen (3 l/min) alone was used as a control (halothane 0%) condition. After exposure to halothane for 15 min, animals were returned to the recording chamber and evoked potentials were recorded. The time when the halothane was discontinued was designated as 0 min of recording time, and recordings were made at 3, 10 and 20 min thereafter. 3. Ethanol: Ethanol 1, 3 or 5 g/kg (40% in saline; n 5 6 rats) was injected through an intragastric tube, and post-injection recordings were performed thereafter. In addition, blood ethanol concentration was measured in a separate experiment (n 5 16 rats) in which rats were administered ethanol in a 40% solution in saline by gavage in amounts up to 4 ml such that doses of 1, 3 or 5 g/kg resulted. Each dosage group and a control group (4 ml saline only) had 4 animals each. Thirty minutes after gavage, anesthesia was induced by injecting pentobarbital (100 mg/kg, i.p.), and blood samples were taken via cardiac puncture. Ethanol blood levels were determined using an enzymatic method (Sigma Diagnosticst #332-UV, St. Louis, MO, USA) kit and photometry at 340 nm in comparison to standard ethanol solutions. In all dose-response studies for anesthetics and ethanol, the administration of an agent or saline was made in random order, and no animal was injected more than once per week or more than six times overall. 4. Pancuronium bromide (n 5 4 rats): Under halothane anesthesia, the trachea was intubated for artificial ventilation, and an intraperitoneal tube was inserted for drug injection. Animals were then placed in the recording chamber, and recordings were made before and after the injection of pancuronium bromide (0.5 mg/kg) through the intraperitoneal tube. Halothane (1%) anesthesia was performed at a flow rate of 0.5 l/min with compressed oxygen such that the withdrawal reflex was absent. This level was established before the injection of pancuronium bromide and maintained during post-injection recordings. At the conclusion of post-injection recordings, rats were sacrificed by an overdose of pentobarbital. 5. Head injury: Animals (n 5 13 rats) were first anesthetized with 3% halothane by the same procedure used in halothane doseresponse studies as described above. After 15 min of exposure to halothane, animals were returned to the recording chamber, and the withdrawal reflex was tested every 15 s. The time when the reflex first reappeared was designated as 0 min of recording time, then recordings were made at 5, 15, 30, 60, 180 min and 24 h. Three days after this halothane control study (sham injury), head injury was induced by a weight-drop device consisting of a weight falling through a Plexiglas guide tube (19 mm I.D.) [16,28]. After control recordings were performed, animals were again anesthetized with halothane (3%). Fifteen minutes after exposure, the halothane was discontinued and animals were placed on a foam bed beneath the head injury device. When the withdrawal reflex first reappeared (the 0-min
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recording time), the injury was delivered by dropping a 450-g weight from a predetermined height (1 m, mild injury, n 5 4 rats; or 2 m, severe injury, n 5 9 rats) onto the metallic disc helmet fixed on the skull of the rat. Immediately after the impact, animals were placed into the recording chamber, and post-impact recordings were made at 5, 15, 30, 60, 180 min and 24 h. All animal procedures were approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee. Statistics To determine the effects of compounds (or weights) on the amplitude of the P13 potential, the pre-injection (pre-impact) amplitude was designated as 100%, and subsequent post-injection (post-impact) amplitudes were calculated as a percent amplitude of this control recording. Evoked potential data were evaluated using two-factor repeated-measures analysis of variance (ANOVA) with within-subject factors of dose for each compound (ketamine, pentobarbital, halothane or ethanol) and time after exposure. For head injury data, weight and time were used as within-subject factors. In addition, blood ethanol concentration data were analyzed using one-factor ANOVA. Significant main effects were followed with post hoc within-subject contrasts with significance adjusted for multiple comparisons using the Scheffe´ test. Differences were considered to be significant when a p-value , 0.05 was found. RESULTS Effects of Anesthetics on P13 Potential Amplitude Ketamine. The effects of ketamine injections on P13 potential amplitude are shown in Fig. 1A (n 5 4 rats). The ANOVA performed on these data revealed significant effects of dose, F(4,79) 5 36.28, p , 0.0001, and time, F(3,79) 5 13.82, p 5 0.0003, and there was a Dose 3 Time interaction, F(12,79) 5 5.22, p , 0.0001. The dose effects of ketamine, evaluated with post hoc within-subject comparisons, indicated that: (1) the 7.5mg/kg ketamine dose effect was greater than both the saline effect, t 5 8.85, p , 0.02, and the 2-mg/kg dose effect, t 5 10.46, p 5 0.0072; (2) the 30-mg/kg ketamine dose effect was greater than both the saline effect, t 5 40.89, p , 0.0001, and the 2-mg/kg dose effect, t 5 45.48, p , 0.0001, and tended to be greater than the 7.5-mg/kg dose effect, t 5 3.59, p 5 0.082; (3) the 120-mg/kg ketamine dose effect was greater than the saline effect, t 5 87.97, p , 0.0001, the 2-mg/kg dose effect, t 5 135.74, p , 0.0001, the 7.5-mg/kg dose effect, t 5 61.75, p , 0.0001, and the 30-mg/kg dose effect, t 5 39.02, p , 0.0001; and (4) the 2-mg/kg ketamine dose effect was not significantly different from the saline effect, t 5 0.20, n.s. Therefore, ketamine decreased the amplitude of the P13 potential in a dose-dependent manner. Pentobarbital. The effects of pentobarbital on P13 potential amplitude are shown in Fig. 1B (n 5 4 rats). Pentobarbital also decreased the amplitude of the P13 potential in a dose-dependent manner. Analysis of variance testing revealed significant effects of dose, F(3,63) 5 78.33, p , 0.0001, and time, F(3,63) 5 7.57, p 5 0.0042, and there was a Dose 3 Time interaction, F(9,63) 5 4.32, p 5 0.0007. The dose effects of pentobarbital were evaluated with post hoc within-subject comparisons, which indicated that: (1) the 5-mg/kg pentobarbital dose effect was greater than both the saline effect, t 5 49.66, p , 0.0001, and the 1.5-mg/kg dose effect, t 5 17.90, p 5 0.0012; (2) the 20-mg/kg pentobarbital dose effect was greater than the saline effect, t 5 236.08, p , 0.0001, the 1.5mg/kg dose effect, t 5 67.07, p , 0.0001, and the 5-mg/kg dose effect, t 5 60.62, p , 0.0001; and (3) the 1.5-mg/kg pentobarbital
FIG. 1. Effects on P13 potential amplitude of ketamine (A), pentobarbital (B) and halothane (C). The P13 potential was averaged over 32 trials before and after injection of ketamine (KET: 2, 7.5, 30 or 120 mg/kg, i.m.), pentobarbital (PB: 1.5, 5 or 20 mg/kg, i.p.) or saline (SAL), or the inhalation of halothane with oxygen (HAL: 1, 2 or 4%) or oxygen only (HAL 0). Each point is the mean 6 standard error of the P13 potential amplitude as a percent of the pre-administration P13 potential amplitude. Note that the time scale for halothane is shorter than that for ketamine or pentobarbital. Scheffe´ post hoc test significance: 1p , 0.05 and *p , 0.01 compared to saline (A and B) or oxygen only (C).
dose effect was not significantly different from the saline effect, t 5 0.54, n.s. At the 40-mg/kg pentobarbital dose (Fig. 2), the Vx-recorded P13 potential was completely suppressed for about 2 h whereas the ACx-recorded P7 potential was stable. At 1 day after the injection, however, the amplitude of the P13 potential recovered to the pre-injection level. Halothane. The effects of halothane on P13 potential amplitude are shown in Fig. 1C (n 5 4 rats). The ANOVA performed on these data revealed significant effects of dose, F(3,63) 5 21.56, p , 0.0001, and time, F(3,63) 5 13.25, p 5 0.0004, and there was a Dose 3 Time interaction, F(9,63) 5 4.30, p 5 0.0008. The dose effects of halothane were evaluated with post hoc within-subject comparisons, which indicated that: (1) the 1% halothane dose effect was greater than the oxygen-only effect, t 5 5.91, p 5 0.032; (2) the 2% halothane dose effect was greater than the oxygen-only effect, t 5 14.44, p 5 0.0025, but not than the 1%
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FIG. 2. Effects of pentobarbital (40 mg/kg, i.p.) on the amplitudes of the vertex (Vx)-recorded P13 potential and the auditory cortex (ACx)-recorded P7 potential. Left column: pre-injection control (CTL) recordings show the averaged P13 and P7 potentials (32 trials) induced by auditory stimulation. Middle columns: 15- and 30-min post-injection recordings showed the disappearance of the P13 potential (significant decrease in amplitude is noted by a filled circle in this and subsequent figures) but the presence of the P7 potential. Right column: 1-day post-injection recordings showed the recovery of the amplitude of the P13 potential. Calibration bars: vertical 5 20 mV; horizontal 5 10 ms. Arrow indicates onset of auditory stimulus for this and subsequent figures.
effect, t 5 1.03, n.s.; and (3) the 4% halothane dose effect was greater than the oxygen-only effect, t 5 50.10, p , 0.0001, the 1% dose effect, t 5 35.06, p , 0.0001, and the 2% dose effect, t 5 41.73, p , 0.0001. Hence, halothane decreased the amplitude of the P13 potential in a dose-dependent manner. Effects of Ethanol on P13 Potential Amplitude The effects of intragastric injections of ethanol or saline are shown in Fig. 3A (n 5 6 rats). Analysis of variance revealed significant effects of dose, F(3,167) 5 54.44, p , 0.0001, and time, F(6,167) 5 10.56, p , 0.0001, and there was a significant Dose 3 Time interaction, F(18,167) 5 2.14, p , 0.009. Post hoc within-subject comparisons indicated that: (1) the 3-g/kg ethanol dose effect was greater than both the saline effect, t 5 67.57, p , 0.0001, and the 1-g/kg dose effect, t 5 50.04, p , 0.0001; (2) the 5-g/kg ethanol dose effect was greater than the saline effect, t 5 157.96, p , 0.0001, the 1-g/kg dose effect, t 5 73.20, p , 0.0001, and the 3-g/kg dose effect, t 5 9.94, p 5 0.0033; and (3) the 1-g/kg ethanol dose effect was not significantly different from the saline effect, t 5 0.42, n.s. Thus, ethanol also decreased the amplitude of the P13 potential in a dose-dependent manner. In 16 other male rats (250 –350 g), blood ethanol concentrations were measured 30 min after ethanol (or saline) injection. In animals given saline (n 5 4) or ethanol 1 g/kg (n 5 4), 3 g/kg (n 5 4) or 5 g/kg (n 5 4), the blood ethanol level was 0.0113 6 0.0037, 0.0433 6 0.0268, 0.1123 6 0.0293 or 0.1201 6 0.0262% (mean 6 SD), respectively. A one-factor ANOVA revealed a significant main effect of ethanol doses on blood levels, F(3,15) 5 25.76, p , 0.0001, and a Sheffe´ post hoc test showed increased blood ethanol levels at higher ethanol dosages (df 5 3 for each comparison: 1 g/kg vs. saline, t 5 1.57, n.s.; 3 g/kg vs. saline, t 5 15.57, p , 0.01; 3 g/kg vs. 1 g/kg, t 5 7.25, p , 0.01; 5 g/kg vs. saline, t 5 18.05, p , 0.01; 5 g/kg vs. 1 g/kg, t 5 8.98, p , 0.01; and 5 g/kg vs. 3 g/kg, t 5 0.092, n.s.). Fig. 3B shows the negative correlation between P13 amplitude and blood ethanol level at 30 min after
FIG. 3. (A) Effects of ethanol on P13 potential amplitude. The P13 potential was averaged over 32 trials before and after intragastric injection of ethanol (EtOH: 1, 3 or 5 g/kg) or saline (SAL). Each point is the mean 6 standard error of the P13 potential amplitude as a percent of pre-injection P13 potential amplitude at various times after administration. Scheffe´ post hoc test significance: 1p , 0.05 and *p , 0.01 compared to saline. (B) Correlation between the mean values of P13 potential amplitude and of blood ethanol level. P13 potentials, averaged at 30 min after intragastric injection of ethanol (1, 3 or 5 g/kg) or saline, are plotted as a function of blood ethanol levels. These were determined in a different group of rats at 30 min after gavage of the same doses of ethanol. The coefficient of correlation was r 5 20.97, p , 0.03.
administering 0 (saline only), 1, 3 or 5 g/kg of ethanol. It should be noted that the amplitude of the P13 potential in the saline control (SAL) condition is not 100% in Fig. 3B because this denotes (1) the non-statistically significant effect of making an i.p. injection compared to the undisturbed, non-injected condition (100%) and (2) fluctuations within the normal range of amplitude for the P13 potential. It is important to test for unspecific effects of vehicle/control injections. Effects of Neuromuscular Blocker on P13 and P7 Potential Amplitudes Figure 4 shows the effects of injections of pancuronium bromide on the amplitudes of auditory-evoked potentials in lightly anesthetized animals (n 5 4 rats). During quiet waking, auditory stimulation elicited the P13 potential, the P7 potential
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FIG. 5. Effects of weight-drop-induced head injury on P13 potential amplitude. The P13 potential was averaged over 32 trials before and after a weight-drop (450 g at 1 m, mild injury; or 450 g at 2 m, severe injury). Because weight-drop impact was delivered under halothane anesthesia, recordings from animals exposed to halothane without being subjected to head trauma served as sham injury (SHAM). Note a decrease in P13 potential amplitude at 5 min in sham injury because of the suppressive effects of halothane. Scheffe´ post hoc test significance: 1p , 0.05 and *p , 0.01 compared to sham injury.
FIG. 4. Averaged recordings (32 trials) during quiet waking (A), during artificial ventilation with 1% halothane (B), and 15 min after the injection of pancuronium bromide (0.5 mg/kg, i.p.) under 1% halothane anesthesia (C). Note that muscle activity, such as the startle response (SR), was absent but that the vertex (Vx)-recorded P13 and the auditory cortex (ACx)recorded P7 potentials were present after pancuronium bromide injection. Note the dramatic attenuation of background muscle activity and the lack of SR (filled circles) under anesthesia (B) and/or neuromuscular blockade (C). EMG, electromyogram. Calibration bars: vertical 5 20 mV; horizontal 5 5 ms.
and the startle response in the Vx, ACx and EMG, respectively (Fig. 4A). During artificial ventilation with 1% halothane, the P13 and P7 potentials were still present while the startle response had disappeared (Fig. 4B). Fifteen minutes after an injection of pancuronium bromide (0.5 mg/kg, i.p.) in the presence of continued 1% halothane, the amplitudes of the P13 and P7 potentials did not change significantly compared to pre-injection values (Fig. 4C). The mean amplitude of the P13 potential before injection was 20 6 7 mV, and after injection it
was 20 6 2 mV (t 5 2.1, p 5 0.92, n.s.). Likewise, the mean peak latency was 14.8 6 0.4 ms before and 14.4 6 0.7 ms after neuromuscular blockade (t 5 0.98, p 5 0.35, n.s.). The amplitude and latency of the P7 potential were also unchanged by treatment (amplitude before 5 15 6 4 mV, after 5 14 6 3 mV, t 5 0.41, p 5 0.7, n.s.; latency before 5 8.2 6 0.2 ms, after 5 7.8 6 0.3 ms, t 5 2.28, p 5 0.06, n.s.). Effects of Diffuse Head Injury on P13 Potential Amplitude Every rat (4 of 4) that received the 450-g, 1-m weight-drop impact and 6 of 9 rats that received the 450-g, 2-m impact survived, compatible with previous findings [16,28]. The effects of head injury on P13 potential amplitude are shown in Fig. 5, and averaged recordings are shown in Fig. 6. Weight-drop impact decreased the amplitude of the P13 potential in a weight-dependent manner. The ANOVA performed on these data revealed significant effects of weight, F(2,194) 5 114.27, p , 0.0001, and time, F(4,194) 5 137.23, p , 0.0001, and there was a significant Weight 3 Time interaction, F(8,194) 5 14.08, p , 0.0001. The weight effects were explored with post hoc within-subject comparisons, which indicated that (1) the 450-g, 1-m weight effect was greater than the sham effect, t 5 21.67, p , 0.0001, and (2) the 450-g, 2-m weight effect was greater than both the sham effect, F(1) 5 297.3, p , 0.0001, and the 450-g, 1-m effect, t 5 19.44, p 5 0.0002.
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FIG. 6. Effects on P13 potential amplitude of the 450-g, 1-m mild injury (left column), the 450-g, 2-m severe injury (middle column) and sham injury (right column). Note that the amplitude of the P13 potential was reduced by impact in a weight-dependent manner. A filled circle indicates that mild injury reduced the P13 potential at 5 min (p , 0.05) and that severe injury reduced it at 5, 15 and 30 min (p , 0.01) compared to sham injury. Calibration bar: vertical bar 5 20 mV.
Effects of Anesthetics, Ethanol or Head Injury on P7 Potential Amplitude The effects of various interventions on the amplitude of the P7 potential were investigated. Analysis of variance revealed that administrations of ketamine (2, 7.5, 30 or 120 mg/kg i.m., n 5 4 rats), pentobarbital (1.5, 5 or 20 mg/kg i.p., n 5 4 rats), halothane (1, 2 or 4%, n 5 4 rats) or ethanol (1, 3 or 5 g/kg i.p., n 5 6 rats) failed to change statistically the amplitude of the ACx-recorded P7 potential. In contrast, the ANOVA performed on head injury data revealed significant effects on P7 potential amplitude of impact, F(2,134) 5 66.29, p , 0.0001, and time, F(4,134) 5 5.84, p 5 0.0008, and there was a significant Impact 3 Time interaction, F(8,134) 5 9.14, p , 0.0001. The impact effects were evaluated with post hoc withinsubject comparisons, which indicated that (1) the 450-g, 1-m effect was greater than the sham effect, t 5 5.85, p 5 0.020, and (2) the 450-g, 2-m effect was greater than both the sham effect, t 5 84.89, p , 0.0001, and the 450-g, 1-m effect, t 5 16.58, p 5 0.001. Therefore, the amplitude of the P7 potential, as well as that of the P13 potential, was decreased by weight-drop impact in a weight-dependent manner. DISCUSSION Anesthesia and Ethanol Ketamine, pentobarbital, halothane and ethanol suppressed the P13 potential in a dose-dependent manner but not the P7 potential,
indicating a clear difference in susceptibility between the P13 and P7 potentials to these compounds. This dissociation is most likely due to the differences in mechanisms of the generation of each potential. The P7 potential now appears to be well established as a primary auditory cortical potential in the rat [3,5,21,31,39,40,42]. On the other hand, data have accumulated as to the source(s) of the P13 potential. The Vx-recorded P13 potential was not eliminated by bilateral lesions of the auditory cortex, implying that this potential did not represent primary cortical events [42]. In addition, an auditory-evoked potential at a latency of 11–15 ms was recorded in and around the PPN in the decerebrate rat, that is, in the absence of the cerebral cortex, basal ganglia and hippocampus, emphasizing the possibility of a subcortical origin for the P13 potential [34]. The PPN, the cholinergic arm of the RAS, has been implicated in various functions such as sleep–wake mechanisms, arousal and locomotion [35,44]. More recently, it was shown that the P13 potential was suppressed by activation of known inhibitory synapses, both GABAergic [30] and noradrenergic (Miyazato et al., in press), at the level of the PPN. Furthermore, in PPN-lesioned rats, there was a positive correlation between the amplitude of the P13 potential and the number of NADPH diaphorase-positive (cholinergic) neurons in the PPN (unpublished data). These findings also indicate that the PPN is involved, at least in part, in the generation of the P13 potential. The results of the present study are consistent with this hypothesis, because it has been shown that the multisynaptic, barbiturate-sensitive extralemniscal pathways, such
MODULATION OF P13 POTENTIAL
551 Pancuronium Bromide Paralysis The injection of the neuromuscular blocker, pancuronium bromide, did not affect the morphology of the P13 potential or the P7 potential, indicating that muscle potentials elicited by auditory stimulation, typically the startle response, were not the sources of the P13 and P7 potentials. To our knowledge, this is the first direct evidence excluding the possibility of a myogenic contribution to surface-recorded midlatency potentials in the rat. This finding is consistent with previous findings showing that, although response characteristics bear some similarities, the P13 potential and the startle response subserve different, although linked, functions and that the P7 potential has properties different from those of the startle response [31,32]. It should be noted, however, that the animals were anesthetized with halothane (1%) throughout the pancuronium bromide experiments. It has been shown that, in the paralyzed non-anesthetized cat, the midlatency auditory-evoked wave A and wave 7 potentials were unchanged from their appearance in the awake, restrained preparation [6]. Wave A and wave 7 are considered to be the feline equivalent of the P1 potential in the human and the primary auditory cortical potential, respectively [6,8,13]. It has also been shown that the P1 and Pa potentials in the human were not eliminated by a neuromuscular blocking agent, indicating that these midlatency potentials are not of myogenic origin [22,24]. It should also be noted that the P13 potential is present during paradoxical sleep [31,32], when atonia is maximal, lending further evidence to the suggestion that the P13 potential is not of myogenic origin. Head Injury
FIG. 7. Recordings across species. (A) Averaged vertex (Vx) and auditory cortex (ACx) recordings in the intact rat showing P13 and P7 potentials, respectively. In this case, a potential (the latency of which was similar to the ACx-recorded P7 potential) was also recorded at the Vx. (B) An averaged P1 potential in the human at a 50-ms latency along with a Pa potential at a 25-ms latency. This particular recording is from a 30-year-old man. Although this recording has not been published previously, it is similar to those obtained in similar studies in humans [45]. Note that the latencies of the human Pa, P1 and P2 potentials are approximately four times as long as those of the rat P7, P13 and P25 potentials, respectively. Calibration bar: 15 mV for rat and 1 mV for human.
as the RAS, are more susceptible to anesthetic suppression than the barbiturate-insensitive lemniscal pathways consisting of synaptically secure afferent neurons, such as primary auditory projections [11,17,19,20,41]. Unit recordings have demonstrated that reticular formation neurons are also sensitive to ethanol, whereas cells of the cerebral cortex appear to be relatively less sensitive [25,50]. The present results clearly indicate that the P13 potential is related to arousal and blocked by various anesthetics and ethanol in a dose-dependent manner, whereas the primary auditory P7 potential is not significantly affected by the dosages that do block the P13 potential. The synaptic security of the lemniscal pathways is assumed to be the reason for the maintenance of the P7 potential following exposure to these agents, whereas the multi-synaptic reticular pathways responsible for P13 potential manifestation appear greatly affected.
Weight-drop impact suppressed both the P13 and P7 potentials, indicating that the effects of weight-drop impact appeared to be non-specific compared to those of anesthetics or ethanol. In agreement with this interpretation is the fact that the weight-drop procedure used in the present study has been shown to produce graded diffuse axonal injury similar to that described in man. That is, when applying the definition of diffuse axonal injury [1], mild (450-g, 1-m) injury is consistent with Grade 1 (axonal injury involved the cerebral hemisphere and brain stem without focal lesions in the corpus callosum or the brain stem). On the other hand, severe (450-g, 2-m) injury can be categorized as Grade 3 (more global axonal abnormalities involving the cerebellum with petechial hemorrhages in the brain stem) [16]. It has been demonstrated that BAEPs failed to differentiate between these two grades of injury, because BAEPs were unaffected by mild or severe injury [49]. Based on this finding, these authors concluded that brain-stem function was not severely compromised in the weight-drop closed-head injury model. In contrast, the present study demonstrated that both the P13 and P7 potentials decreased in a weight-dependent (or injury grade) manner. Because the P13 potential is most likely of posterior midbrain origin, this impact model appears to produce sustained dysfunction not only at the cortical but also at the brain-stem level. The P13 and P7 potentials, thus, could be used as more sensitive monitors in head injury studies. Most incidences of coma are caused by posterior midbrain damage due to head trauma/injury. Recovery from coma is essentially a re-establishment of consciousness, that is, of cortical desynchronization and arousal. The P1 potential in the human and the P13 potential in the rat may some day be useful in determining and, perhaps, predicting recovery from coma. What these preliminary studies show is that there is some recovery of the P13 potential from mild (full recovery within hours) and severe (partial recovery within a day) head trauma/injury.
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Comparison with Midlatency Potentials in the Human In the human, the Pa potential (25- to 40-ms latency), which precedes the P1 potential (50 –70 ms), has been shown to be an auditory cortical potential [12,14,15,26,27]. The Pa potential, as well as the BAEPs, has been used clinically for monitoring the depth of general anesthesia during surgery, because the amplitude of the Pa potential was shown to be suppressed by volatile anesthetic agents in a dose-related manner [33,46 – 48]. In contrast the rat P7 primary auditory cortical potential was not suppressed by halothane in the present study. It should be noted, however, that, in the present halothane dose-response study, the P7 potential was recorded after the discontinuation of halothane, whereas, in the human study, the Pa potential was recorded while anesthesia was maintained. Recording the P7 potential during anesthesia would be required to compare the findings across the species. In contrast the effects of general anesthetic agents on the human P1 potential are not as well investigated. Schwender et al. [36] reported that the amplitude of the P1 potential, as well as that of the Pa potential, was suppressed in a dose-dependent manner by general anesthesia. As a comparison, Fig. 7 shows midlatency auditory-evoked potentials in the rodent and the human [45]. It should be noted that, except for the differences in latency, the morphology of the waveform is similar in the two species. Based on available evidence, it appears that the P13 and P7 potentials are the rodent equivalents of the P1 and Pa potentials in the human, respectively. The present study describes the basic characteristics of the effects on the P13 and P7 potentials of various interventions that modulate arousal, such as anesthetics, ethanol and diffuse brain injury. The findings discussed provide additional evidence suggesting that the P13 potential is a manifestation of the RAS and may be a useful tool for assessing pharmacological, pathological and physiological changes in arousal. ACKNOWLEDGEMENT
This work was supported by USPHS grant NS20246.
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