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Effects of dizocilpine (MK-801) and ethanol on the eeg and event-related potentials (erps) in rats
Neuropharmacology Vol. 31, No. 4, pp. 369-378, 1992
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002%3908/92 $5.00 + 0.00 1992 Pergamon Press plc
EFFECTS OF DIZOCILPINE (MK-801) AND ETHANOL ON THE EEG AND EVENT-RELATED POTENTIALS (ERPS) IN RATS C. L. EHLERS,* W. M. KANEKO, T. L. WALL and R. I. CHAPLIN Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037, U.S.A. (Accepted 28 Augusr 1991) Summary-Recent neurophysiological data have suggested an interaction of ethanol (EtOH) with the glutamate-NMDA receptor complex. For instance, low levels of alcohol have been found to inhibit the ion current, activated by NMDA in in vitro preparations. The present study extends these paradigms in order to evaluate the electrophysiological effects of ethanol and the nonspecific NMDA receptor antagonist, dizocilpine (MK-801) in awake, conscious rats. Twenty Wistar rats were stereotaxically implanted with electrodes, aimed at dorsal hippocampus, amygdala, thalamus and frontal cortex. Rats received the following drugs: saline (s.c.), 0.01 and 0.1 mg/kg MK-801 (s.c.); EtOH, 0.75 g/kg (i.p.); 0.75 kg EtOH plus 0.01 mg/kg MK-801; 0.75g/kg EtOH plus O.lOmg/kg MK-801. Five minutes of EEG was collected and event-related potentials (ERPs) recorded in response to an auditory “oddball” paradigm. Spectral analysis revealed that MK-801 (0.1 m/kg) produced significant increases in low frequency EEG components, at all sites (14 Hz) and decreases in higher frequencies (16-32 Hz). Whereas ethanol (0.75 g/kg) produced decreases in power in all frequency bands. The combined administration of EtOH and MK-801 produced some antagonistic effects on the EEG in the low frequency range. Evaluation of ERPs revealed that MK-801 (0.1 mg/kg) produced significant decreases in amplitude of the Nl and P2 components in the cortex, decreases in the Pl and N2 in the thalamus and a profound decrease in the P3 components in hippocampus and amygdala. Ethanol was also found to produce decreases in the Nl component in cortex. The administration of MK-801 and ethanol together did not produce significant interactions on ERPs. These studies suggest that antagonism of the NMDA receptor by MK-801 may produce some effects similar to those of ethanol, however, their combined administration did not produce synergistic effects within these dose ranges. Key words-ethanol,
NMDA receptor, EEG, ERPs.
Several lines of investigation support a role for I-glutamate as an excitatory neurotransmitter in the mammalian brain. The glutamate-N-methyl-Daspartate (NMDA) receptor has been a specific target for electrophysiological, biochemical and behavioral studies, due to its potential role in synaptic plasticity and memory processes, as well as glutamate-mediated epileptogenic and neurotoxic actions (Lynch and Baudry, 1984; Collingridge and Bliss, 1987; Cotman and Iversen, 1987; Mayer and Westbrook, 1987). Recent studies have provided evidence to suggest that some of the effects of small to moderate doses of ethanol may be mediated by the NMDA subtype of glutamate receptor (see Hoffman, Rabe, Grant, Valverius, Hudspith and Tabakoff, 1990). In biochemical and electrophysiological experiments, ethanol, at small concentrations, has been shown to selectively inhibit NMDA receptor-mediated events in vitro (Hoffman, Rabe, Moses and Tabakoff, 1989; Lovinger, White and Weight, 1989; Lima-Landman and Albuquerque, 1989; Rabe and Tabakoff, 1990), *Address correspondence to: C. L. Ehlers, BCR-1, TSRI, 10666 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
while other glutamate receptors may be less sensitive to ethanol-induced inhibition. Further evidence that the effects of small doses of ethanol may be mediated by NMDA receptors, comes from studies utilizing the noncompetitive NMDA receptor antagonist ( + )-5methyl-lo,1 1 dihydro-SH-dibenzo [a,d] cyclohepten5, lo-imine maleate (MK-80 1). In behavioral studies, MK-801 has been shown to enhance the hypnotic (Wilson, Boxy and Ruth, 1990), anticonvulsant (Shrinivas and Ticku, 1989) and anxiolytic (Goldberg, Salama, Pate1 and Malick, 1983) effects of ethanol in rodents. Also, MK-801 has been demonstrated to attenuate the seizure score in animals, during withdrawal of alcohol (Grant, Valverius, Hudspith and Tabakoff, 1990; Morrisett, Rezvani, Overstreet, Janowsky, Wilson and Sqartzwelder, 1989). However, MK-801 has also been shown to have potent anticonvulsant and behavioral actions of its own (see Troupin, Mendius, Cheng and Risinger, 1986; Wozniak, Olney, Kettinger, Price and Miller, 1990). For instance, at larger doses (0.25-10 mg/kg), MK-801 produced somnolence, akinesia, impaired food consumption, locomotion and swimming, whereas small doses (0.05-0.1 mg/kg) impaired performance in a place learning paradigm (Whishaw and
369
C. L. EHLERS et al.
370
Auer, 1989). Since the behavioral effects of ethanol may result from the simultaneous action of several neurotransmitter systems the actions of which may be mediated through several different receptor-gated ion channels (Gonzales and Hoffman, 1991), it is hard to predict, in behavioral experiments, whether the MK801-induced enhancements of the effects of ethanol are non-specifically additive or enacted selectively, through NMDA receptor-mediated events. In previous studies, in uiuo electrophysiological experiments have been useful in discriminating the actions of ethanol from other classes of drugs, with similar properties, such as benzodiazepines and opiates (Ehlers and Reed, 1987; Ehlers, 1988, 1989). However, in uiuo electrophysiological experiments, which have simultaneously evaluated the effects of ethanol and MK-801 in awake animals have not been accomplished. Thus, the purpose of the present study was to utilize two in uiuo electrophysiological techniques in rats in order to explore the effects of small doses of MK-801 (0.01, 0.1 mg/kg s.c.) alone and in combination with a small dose of ethanol (0.75 g/kg ip.). The two electrophysiological paradigms utilized were analysis of spectral components of the electroencephalogram (EEG) and an auditory event-related potential (ERP) paradigm, both of which have been shown to be highly sensitive to the effects of ethanol (Ehlers and Chaplin, 1991; Ehlers, Chaplin, Lumeng and Li, 1991a). METHOD
The experimental subjects were 20 experimentally naive, male Wistar rats, weighing 280-300 g. The rats were housed individually and maintained in a temperature- and light- (12: 12 hr, LD) controlled room. Food and water were given ad libitum. At least 2 weeks prior to the experimental procedures, the rats were surgically prepared with recording electrodes. Animals were anesthetized (Nembutal, 50mg/kg i.p.), placed in a stereotaxic apparatus and stainless steel unipolar electrodes were aimed at hippocampus (AP -3.0, ML + 3.0, DV - 3.1), the ventral thalamus (AP -3.4, ML &-1.7, DV -7.8) and amygdala (AP + 1.0, ML k6.0, DV -9.5). Stainless steel screw electrodes were also placed over the frontal cortex and in the bony calvarium, 3 mm posterior to lambda which lies tangential to the cerebellum, which was grounded. Because these are complex regions of the brain, it is unlikely that the electrodes were placed in a specific subregion of the structure. In all animals, electrode attachments were made to a multipin (Amphenol) connector and the entire assembly was anchored to the skull with dental acrylic. Three sets of administrations of drugs, spaced 2 weeks apart, were given to the rats. In the first set, one half of the rats was given 0.1 mg/kg MK-801 and the other half was given saline, subcutaneously (s.c.). Two weeks later the groups were reversed; the
MK-801-injected group received saline and the saline-treated group received MK-801 (0.1 mg/kg). In the second set of administrations of drugs one half of the rats received saline (s.c.) and 15 min later received ethanol (0.75 g/kg i.p.). The other half of the rats received MK-801 (0.1 mg/kg s.c.) plus ethanol (0.75 g/kg i.p.), 15 min later. In the third set of experiments, one half of the rats received MK-801 (0.01 mg/kg s.c.) and the other half received MK-801 (0.01 mg/kg s.c.) plus ethanol (0.75 g/kg i.p.) 15 min later. The threshold for the appearance of behavioral effects of MK-801 in the rat lies between 0.05 and 0.1 mg/kg (Whishaw and Auer, 1989). Larger doses of MK-801 are known to produce gross behavioral effects and somnolence, thus making the assessment of the background EEG, as well as the response to sensory stimuli, difficult to interpret. Likewise the dose of ethanol (0.75 g/kg) was chosen, as prior studies have shown that this dose produces mild behavioral intoxication, without inducing sedation (Ehlers et al., 1991a). Thus, the combined administration of alcohol and MK-801, in these dose ranges, allowed for the assessment of potential synergistic or antagonistic effects on electrophysiological parameters within a range of awake, active behavior. For electrophysiological recordings, the animals were placed in a Naugahyde sling, which comfortably supported the animal in an awake state but prevented movement-induced artifact. The sling was placed in an electrically shielded light-, sound- and temperature-controlled BRS/LVE recording chamber. All animals were adapted to the chamber, prior to exposure to drug. On a test day, the rats were placed singly in the chamber and a connector, attached to a microdot cable, was used to transfer the monopolar (referred to the lambda ground screw) EEG signals to a polygraph. The bandpass for recordings was set at 0.3-75 Hz, with a 60 Hz notch filter in. The signals were amplified (50% gain) and the EEG, ERPs, as well as calibration signals, were transferred from the polygraph on-line to a DEC (LSI 11-2) computer, which also controlled the presentation of the auditory stimuli. The EEG recordings were collected 15-25 min after administration of drug. Five minutes of EEG recordings of unipolar signals (to ground) were obtained from the cortex, thalamus, amygdala and dorsal hippocampus from the six drug conditions. The EEG signals were then transferred to a Vetter Model D recorder, for off-line analysis. For quantification of the EEG, 5 min of EEG were digitized (128 Hz) and the power spectra of a continuous 4 set epoch were determined for the range 0.2564 Hz. The Fourier-transformed data were then further compressed into seven frequency bands (l-2, 2-4, 4-6, 6-8, 8-16, 16-32, 32-64Hz). Mean power density was calculated for each band, for each of the six conditions, as described previously (Ehlers and Havstad, 1982).
MK-801 and ethanol on EEG and ERPs Immediately after the EEG recordings, ERPs were collected. Free field auditory stimuli were presented through a small loudspeaker, centered approx 20 cm above the head of the rat. The ERPs were elicited by an acoustic “oddball” paradigm. The tones were generated with a programmable multiple-tone generator, the characteristics of which have been described previously (Polich, Fischer and Starr, 1983). The acoustic parameters for this paradigm were two square-wave tones (rise/fall times < 1 msec): a standard (soft) tone (20 msec, 1 kHz, 70 dB SPL) presented on 84% of the trials and a rare (louder) tone (20 msec, 2 kHz, 80 dB SPL), presented on 16% of the trials. Rare tones were interspersed with standards, such that no two rare tones occurred successively. The digitizing epoch for each trial was 1 set and a 0.5-l set intertrial interval was used (to reduce habitation). The total number of trials in a recording session was 150. The ERP recordings were analyzed for the six drug conditions. The ERP trials were digitized at a rate of 256 Hz. Trials containing excessive movement artifact were eliminated prior to averaging (< 5% of the trials). An artifact rejection program was utilized to eliminate individual trials in which the EEG exceeded &250 pV. The components of the ERP were quantified by computer, by identifying a peak amplitude (baseline-to-peak) within a standard latency range. The baseline was determined by averaging the 100 msec of pre-stimulus activity, obtained for each trial. The latency of a component was defined as the time of occurrence of the peak amplitude, within a latency window. The latency windows were initially determined by visual inspection of the data and then standardized to allow for computerautomated peak dete~inations. Components were labeled solely by their polarities and latency positions, relative to each other. The latency windows for cortex were: Pl, O-50 msec; NlA, 2.5-80 msec; NlB, 50-100 msec and P2, 90-250 msec. The windows for dorsal hippocampus were: Pl, 0-50msec; Nl, 25-80 msec; P2, 150-250 msec and P3,25&350 msec. The latency windows for ventral thalamus were: Ni, O-50 msec; PlA, 25-50 msec; PlB, 50-100 msec and N2, 100-250msec. The latency windows for amygdala were: Pl, l@-50msec; NlA, SO-75 msec; P2, 150-250 msec and P3, 250400 msec. These ERP analyses have been described previously (Ehlers, 1988). For statistical analysis, mean power values in each EEG frequency band and amplitude and latencies of ERP components were compared within subjects, for the six conditions, using analysis of variance (ANOVA). RESULTS
Administration of MK-801 was found to produce dose-related changes in the EEG, which were significantly different to those produced by injections of NP 3114-E
371
saline. At the smallest dose (0.01 mg/kg), MK-801 was found to produce a decrease in slow frequencies in the 24 Hz range, which reached significance in the hipp~~pal leads (P < 0.04). No overt behavioral findings were observed at this dose. At larger doses, as seen in Fig. 1, much more significant EEG findings were observed, when compared to saline. Large increases were found in the low frequencies (l-6 Hz) in cortex, dorsal hippocampus (2-6 Hz) and amygdala (1-4 Hz). These increases in low frequencies were found to occur concomitantly with decreases in the higher frequencies (16-32 Hz) in all electrode sites. At this dose, the rats were observed to be mildly intoxicated. Ethanol (0.75g/kg) was also found to produce significant effects on EEG spectral components. Overall, ethanol was found to produce si~ificant decreases in spectral power, primarily in the midrange theta frequencies (6-8 Hz) in the cortex, hippocampus and thalamus as also seen in Fig. 1. This finding of reduced 6-8 Hz activity in the hippocampus, after ethanol, was similar to the effect observed after admi~stration of MK-801 (0.1 mgjkg). However, this dose of ethanol was not found to produce significant increases in slower waves (1-6 Hz), as was observed after MK-801. However, ethanol, at this dose (0.75g/kg), like MK-801 (0.1 mg/kg), was observed to produce significant decreases in higher frequency bands in the cortex, dorsal hippocampus and thalamus. Administration of ethanol (0.75 g/kg) together with small doses of MK-801 (0.01 mg/kg) did not produce many significant interactions on EEG spectra, when compared to the administration of either drug alone. Some reversal of the MK-8OI-induced increases in slow waves (2-4Hz) was found in the thalamus when ethanol was administered together with small doses of MK-801 (P < 0.05). However, administration of ethanol (0.75 g/kg) with larger doses of MK-801 (0.1 mg/kg) was found to produce a more significant reversal of the MK-801-induced increases in slow waves in the thalamus, as well as in amygdala, as seen in Fig. 1. Administration of MK-801 was also found to produce significant dose-dependent changes in eventrelated potentials (ERPs). As seen in Fig. 2, a series of waves could be averaged from the EEG, following pre~ntations of the two-tone stimulus paradigm, under saline conditions, for the four leads recorded from: cortex, ventral thalamus, dorsal hippocampus and amygdala. The response of the auditory cortex to the frequent, (soft) tone consisted of any early positive component (Pi), with a latency of 25msec, followed by a broad negative component, designated the NI wave (first negative peak) which had a peak latency of 60-80 msec. In response to the infrequent (louder) tone, Nl could also be more clearly differentiated into two separate waves: the NlA with a latency of 50-70 msec and an NIB with a latency of 80-100 msec.
2-4
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Fig. 1. Effects of MK-801 and ethanol, alone and in combination, on the EEG spectral power in rats. Data are presented for 4 different electrode locations: cortex, dorsal hippocampus, thalamus and amygdala. In each graph 4 conditions are presented: saline (SC); MK-801 (0.1 mg/kg s.c.); ethanol (0.75 g/kg i.p.); and MK-801 (0.1 mg/kg s.c.) plus ethanol (0.75 g/kg i.p.). Note that MK-801 produced significant increases in the low frequencies (14 Hz) and decreases in the higher frequencies (6-8 Hz, 16-32 Hz). Ethanol, in general, was found to produce overall reductions in spectral power. The combined administration of the two drugs tended to cancel each other out. SAL = saline; ETOH = ethanol.
1.2
&a
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CORTEX
0
MK-801 and ethanol on EEG and ERPs
373
SALINE (RARE
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100 200 300 Time lmsecl
400 500
-100
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0
100 200 300 Time (msecl
400 500
Fig. 2. Grand averages of components of the ERP, generated by a two-tone auditory “oddball” paradigm in 18 rats. Data are presented for 4 different electrode locations: cortex, dorsal hippocampus, thalamus and amygdala. The frequent (standard) tone is represented by the solid line and the infrequent (rare) tone by the dashed line. Components of the ERP used in statistical analysis are identified. Note a component of P3 was identifi~ in the dorsal ~pp~mpus and amygdala. THAL = thalamus; DHPC = dorsal
hippo~mpus; AMYG = amygdala. Figure 2 also displays the response of the ventral thalamus to the two-tone auditory paradigm. The ERP response to the standard (soft) tone consisted of an early negative wave (Nl) with a latency of 10 msec, followed by a two-peaked positive wave, the PlA (25-50 msec) and PlB (SO-80 msec) and a broad negative wave, designated the N2 (second negative wave) with a peak latency of 120-170msec. The ERPs after the rare (louder) tone were different from the standard (softer) tone, in that a PlB wave could also be identified. The response of the dorsal hippocampus (DHPC) to the standard (soft) tone consisted of an early
positive potential (Pl), which occurred at about 25mseq followed by a large amplitude negative wave, (Nl) with a latency of about 40-6Omsec and a later positive wave (P2), with a latency of lo&200 msec. Hippocampal ERPs from the rare (louder) tone differed from the standard (soft) tone in that a third positive wave (P3), which occurred at a latency of 250-3OOmsec, could clearly be identified, as seen in Fig. 2. The response of the amygdaia (AMYG) to the standard (soft) tone consisted of an early positive potential Pl, followed by a complex group of negative waves (Nls), with a latency of 50-l 50 msec and
C. L. EHLERS et al.
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a later positive complex waves (P2-P3). After the rare (louder) tone, a third positive wave (P3), which occurred at a latency of 250-400 msec, could also be identified as seen in Fig. 2. These components were similar to those reported previously in other studies which have used this same ERP paradigm (see Ehlers, Wall and Chaplin, 1989a; Ehlers, Wall and Chaplin, 1991b). These components have also been demonstrated to be sensitive to stimulus factors, as probability and loudness, in a manner similar to that seen in human studies. Small doses of MK-801 (0.01 mg/kg) were not found to have significant effects on components of the ERP, produced by the standard tone, whereas com-
ponents of the ERP, generated in response to the rare tones were affected. A significant decrease in the amplitude of the NlA and N2 components in the cortex was observed after this dose. Larger doses of MK-801 (0.1 mg/kg s.c.) produced more dramatic reductions in the amplitude of several ERP components, as illustrated in Fig. 3. A decrease in the amplitude and an increase in the latency of ERP components, in response to both the standard and rare tones, was observed. In response to the standard tone, a decrease in the amplitude (P c 0.001) and an increase in the latency (P < 0.0001) of the Pl component and a decrease in the amplitude of Nl (P < 0.001) were found in cortical leads. Reductions
MK 801 0.1 MC/KG
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100 200 300 Time (msecl
400 500
Fig. 3. Grand averages of components of the ERP observed after administration of MK-801 (0.1 mg/kg s.c.). In each graph, data are presented for response to the rare tone in the “saline” condition (-) n = 18 and in the “MK-801” condition (---) n = 20. Note that MK-801 produced a significant and profound reduction in the P3 component in the amygdala and dorsal hippocampus. Abbreviations as in Fig. 2.
MK-801
and ethanol on EEG and ERPs
Table 1I ERP amplitudesto the
rate tone (mean + SEM) (F, p)
Saline
MEL-801 (0.01 mg/kg)
Cortex PI
25.7 f 4.16
NS
NlA
91.1 f 17.0
73.0 f 9.0 (F = 5.88, P i 0.04) NS
P2
28.23 f 5.2
N2
15.2 k 5.0
5.5 + 2.0 (F = 5.3, P < 0.05)
Thalamus PIG
128.72 k 23.3
NS
N2
79.68 k 16.6
NS
256.47 f 28.0
NS
72.9 k 10.5
NS
75.6 i 11.7
NS
Amygdala PI
53.48 i 12.3
NS
P3
75.88 + 14.9
NS
Dorsal hippocampus Nl
P3B
in the amplitude (P < 0.001) and increases in the latency (P < 0.04) of the N2 component, to the standard tone in the thalamus, were also noted. A reduction in the amplitude of the P2 response (P < O-03), to the standard tone, was observed in the amygdala. Reductions in the amplitudes of several components of the ERP to the rare tone were also seen, as outlined in Table 1. In cortex, significant reductions in the amplitudes of the Pl, Nl A, P2 and N2 components were observed. In the thalamus, significant reductions in amplitude in the PlA and N2 were seen. The prominent effects of MK-801, at this dose, were the profound reductions observed in the amplitudes of the P3 components, recorded in the hippocampus and the amygdala. Ethanol (0.75 g/kg) was also observed to produce significant reductions in the amplitudes and increases in the latencies of several components of the ERP. A reduction in the amplitude of the NlA component in the amygdala (P < 0.02) and an increase in the latency of the Pl (P c 0.05) and P2 (P < 0.01) components in cortex, were found following the standard tone. Further reductions in amplitude and increases in the latencies of several components were also found after ethanol (0.75 g/kg), when responses to the rare tone were evaluated. As seen in Fig. 3, ethanol produced increases in the latency of the Pl (P < 0.05) and NlA (P c 0.002), and decreases in the amplitude of the NlA and P2 components in the cortex. In addition, decreases in the amplitude of the PlA component were observed in the thalamus and amygdala after this dose of ethanol. Ethanol, at this dose, unlike MK-801, did not produce significant decreases in the amplitude of the P3 component in the hippocampus and amygdala, although a trend in that direction was noted in the amygdala (P < 0,06).
375
MK-801
(0.1 mgjkg)
8.2 + 2.38 (F = 20.2, P < 0.001~ 63.2 f 1.2 IF = 4.6. P c 0.04\ 1.9’*0.9 (F = 27.4, P < 0.001) 27.3 k 4.3 (F = 8.8, P < 0.009)
ETOH(0.75g/kg) NS 66.4 f 11.3 (F = 7.73, P c 0.03) 22.7 t 3.4 (F = 6.4,-P < 0.04) NS
67.91 k 12.9 (F=7.15, P.zO.02) 37.75 f 6.6 (F = 6.97, P < 0.02)
103.4 f 21.6 (F = 6.4. P
175.63 f 29.7 CF = 6.9. P < 0.02) 24.9’* 5.2 ’ (F = 24.4, P < 0.0001) 39.7 f 16.0 (F = 7.55, P < 0.05)
NS
21.34k5.8 (F = 5.6, P c 0.03) 33.1 f 9.4 (F = 9.61, P c 0.006)
NS
NS NS
49.9 f 11.25 (~=4.19, P <0.06)
The combination of MK-801 and ethanol on components of the ERP was found to produce conflicting results. Ethanol potentiated the reduction in the amplitude of NlA in the amygdala, when combined with small doses of MK-801 (0.01 mg/kg) (P c 0.01). However, ethanol was not found to produce significant potentiation amplitudes or latencies of components when combined with the larger dose of MK-801 (0.1 mg/kg) and, in fact, was observed to shorten the latency of P3 in hippocampus (P c 0.01). DISCUSSION
Electrophysiological measures have been used effectively to investigate several aspects of the actions of ethanol in the CNS (see Porjesz and Begleiter, 1985). Studies evaluating the acute effects of small to moderate doses of ethanol on the spontaneous cortical EEG in awake human subjects have reported increases in theta activity, increases in slow alpha activity and a slowing of the dominant alpha frequency (Lucas, Mendelson, Benedikt and Jones, 1986; Ehlers, Wall and Schuckit, 1989b). In the present study, small doses of ethanol (0.75 g/kg), given to rats were, in general, found to produce reductions in power in many frequency bands, including decreases in theta activity (6-8 Hz) in hippocampal leads. It has been suggested, based on in vitro electrophysiological studies, that these small “intoxicating” doses of ethanol may selectively inhibit the function of the NMDA receptor (Lima-Landman and Albuquerque, 1989; Lovinger ef al., 1989). Thus, it seems that an NMDA receptor antagonist should mimic and/or potentiate the actions of ethanol. The non-competitive NMDA receptor antagonist, MK801 has been shown, in some behavioral tests, to potentiate the actions of ethanol, For instance,
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MK-801 has been shown to potentiate the hypnotic (Wilson et al., 1990) effects of ethanol in rats and to inhibit alcohol withdrawal-induced seizures (Morrisett et al., 1989), at doses which produce behavioral somnolence (0.33 mg/kg). However, in tests of locomotion, MK-801 at smaller doses (0.1 mg/kg), has been reported to produce increases in locomotion, whereas ethanol, in moderate doses (0.75 g/kg), has a depressant effect on locomotion (Robledo, Kaneko and Ehlers, 1991). In addition, administration of small doses of ethanol, together with small doses of MK-801, has not been found to produce synergism in locomotor experiments. Instead, their effects tended to cancel each other out (Robledo et al., 1991). Few studies have evaluated the effects of MK-801 in in uioo electrophysiological systems, in unanesthetized animals. Whishaw and Auer (1989) have demonstrated that no “gross” differences in the EEG of rats were noted after administration of 0.1 mg/kg MK801, with only a slight reduction in hippocampal rhythmic slow activity, observed after the subsequent administration of atropine sulfate. However, EEG activity was not quantified in that study. The effects of MK-801 on the EEG in the present study were dose-dependent and were found to parallel the findings observed in previous locomotor studies. The MK-801 tended to produce an opposite EEG spectral profile to that observed after small doses of ethanol, with MK-801 (0.1 mg/kg) producing significant increases in slow waves (14 Hz). However, both ethanol and MK-801 were found to produce decreases in the 68 Hz frequencies in the hippocampus and reductions in power in several leads in the high frequency ranges (16-32 Hz). Also, like the locomotor findings, administration of ethanol and MK801, together did not produce prominent synergistic effects on the EEG and, in general, the combined effects of the two drugs tended to cancel each other out. The development of event-related potential paradigms has provided additional in viuo electrophysiological measures, whereby effects of drugs can be assessed. In these electrophysiological paradigms, subjects are typically required to detect the presence of certain, infrequently-presented “target” stimuli, which are embedded within a series of frequently stimuli. However, P300 presented “non-target” waves have also been reported to occur in “passive paradigms”, where subjects are presented with different sets of stimuli but are not required to respond to them (Polich, 1987). In such paradigms, when the EEG is averaged in a time-locked fashion to the target or infrequent stimuli, a series of waves can be identified, one of which is positive-going and occurs at a latency of about 300msec in young adults (see Donchin, Karis, Bashore, Coles and Gratton, 1986). This P3 or P300 wave has been suggested to be a measure of stimulus evaluation and may possibly be related to memory processes (Donchin et al., 1986). Depth recordings in human subjects suggest that the
P300 may actually be generated in the hippocampus or amygdala (Halgren, Squires, Wilson, Rohrbaugh, Babb and Crandall, 1980), although cortical sites may also be important (Knight, Scabin, Woods and Clayworth, 1989). These event-related potential paradigms have also been attempted in animal studies, where “P3-like” waves have also been identified. Lesion studies in cats also support a role for the hippocampus in the generation of the P3 in that species (Harrison, Buchwald, Kaga, Woolf and Bucher, 1988) and in rats P3 waves have been recorded from depth electrodes placed in both hippocampus and amygdala (Ehlers et al., 1991b). After small doses of ethanol in human subjects, decreases in the amplitude of the Nl and P3 components of ERPs have generally been found (Pfefferbaum, Horvath, Roth, Clifford and Kopell, 1980; Campbell and Lowick, 1987); however, task difficulty (Roth, Tinkle&erg and Kopell, 1977) may modify responsivity. As in the human, acute administration of small doses of ethanol has been found to produce decreases in Nl and larger doses of ethanol, which cause some sedation, caused reductions in the P3 components, recorded in a passive auditory ERP paradigm in both monkeys (Ehlers, 1988, 1989) and rats (Ehlers and Chaplin, 1991; Ehlers et al., 1991a). In the present study, a reduction in the Nl component of the ERP was also observed after the administration of a small dose of ethanol (0.75 g/kg) in rats, with a trend towards the lowering of the P3 in amygdala. Decreases in the amplitude of the Nl component have also been found following the administration of other anxiolytics. For instance, modest doses of diazepam (2.5mg/kg) were found to produce profound reductions in the amplitude of Nl without having significant effects on the P3 component (Ehlers, 1988). Also, MK-801 was found to produce decreases in several components of the ERP, which were dose-dependent. Both doses of MK-801 produced significant decreases in the amplitude of the Nl component in the cortex. Profound reductions in the P3 component in the hippocampus and amygdala were also prominent after administration of the somewhat larger dose of MK-801 (0.1 mg/kg). However, administration of ethanol and MK-801 together was not found to produce significant synergistic effects on ERP recordings. The finding of a highly significant reduction in P3 in the rat in the hippocampus and amygdala after administration of small doses of MK-801 (0.1 mg/kg), suggests that the glutamate-NMDA receptor system may modulate the generation of that component. While the cognitive concomitants of the P3 component are unknown, some studies in human subjects have suggested that it may be associated with memory processes (Donchin et al., 1986). Several studies have also linked the NMDA receptor with some mechanisms which underlie memory, such as long-term potentiation (Lynch and Baudry, 1984). In addition, MK-801 has been demonstrated to disrupt
MK-801 and ethanol on EEG and ERPs
learning in animals, particularly spatial learning (Whishaw and Auer, 1989; Wozniak, 1990). Thus, the profound reduction in the amplitude of P3, found in the present study, after administration of MK-801, lends further support for a relationship between the P3, the NMDA receptor and memory processes. Some of the behavioral effects of MK-801, particularly its locomotor-stimulating effects, have been suggested to be produced through blockade of NMDA receptors which subsequently causes release of catecholamines, particularly dopamine (Clineschmidt, Martin, Bunting and Papp, 1982; Carlsson and Svensson, 1990). Thus, it is possible that the reductions in P3 observed in the present study, may have occurred secondarily, through modulation by NMDA of catecholamines. However, in previous studies, no reductions in the amplitude of P3 were noted in rats, which had neurochemically verified significant reductions in dopamine in brain, produced by 6-hydroxydopamine (6-OHDA)induced lesions to the ventral tegmental area (Ehlers et al., 1991b). In addition, no reduction in the components of P3 in the rat were noted in animals with significant depletions of serotonin produced by p-chlorophenylalanine (PCPA) (Ehlers et al., 1991b). Some changes in morphology of P3 have been noted following destruction of noradrenergic neurons. For instance, Pineda, Foote and Neville (1989) found that electrolytic lesions of the locus coeruleus, followed by knife cuts of the ascending fibers, altered “P3004ike” potentials in the monkey recorded at the cortical surface. In rats, 6-OHDA-induced lesions to the dorsal noradrenergic bundle, which caused significant depletion of norepinephrine in brain were also found to produce some reduction in the components of P3 (Ehlers et al., 1989a). The reductions in the amplitude of P3, observed in dorsal bundle-lesioned rats however, were not as profound as those observed in the present study after small doses of MK-801. In addition, MK-801 is thought to increase catecholaminergic tone, which should theoretically result in increases in the amplitude of P3. Thus, the reductions in the amplitude of P3, observed in the present study, were most likely not to be the result of catecholaminergic modulation. In summary, MK-801 was found to produce slow waves in the EEG and dose-dependent reductions in the amplitude of several components of the ERP and profound reductions in the P3 potential, recorded in the hippocampus and amygdala. Ethanol was also found to produce reductions in some components of the ERP but also produced reductions in the overall EEG spectral power. These studies suggest that antagonism of the NMDA receptor may produce some effects similar to those of ethanol, however, their combined administration did not produce synergistic effects within these dose ranges. There are several factors which should be considered when interpreting these results. First, is the potential nonpharmacological specificity of MK-801. For instance, some of the
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EEG and behavioral effects of MK-801 have been shown to resemble those of phencyclidine (see Koek, Woods and Gail, 1988; Mattia and Moreton, 1986). However, phencyclidine is known to affect the function of other receptor systems, in addition to NMDA (Manallack, Beartand and Gundlach, 1986). Thus, MK-801 may also have actions at other receptor sites. This would make ethanol-MK-801 interactions in any model system, where both receptors were present, difficult to interpret. Conversely, not all the actions of ethanol are also enacted through NMDA receptors (see Gonzales and Hoffman, 1991). Thus, ethanol may be producing effects on systems in the brain, other than NMDA, which could possibly reverse the effects of MK-801. Further studies will be necessary to specifically interpret these findings. Acknowledgements-The
authors would like to thank MS Susan Lopez for help with statistical analysis and typing and editing of the manuscript. The computer programs were written by Dr James Havstad. The work was supported by grants AA00098 and AA06059 from the National Institute of Alcoholism and Alcohol Abuse to C.L.E. The MK-801 was a gift of Merck Pharmaceuticals. REFERENCES Campbell K. B. and Lowick B. M. (1987) Ethanol and event-related potentials: The influence of distractor stimuli. Alcohol 4: 257-263. Carlsson A. and Svensson A. (1990) Interfering with glutamate& neurotransmission by means of NMDA antagonist administration discloses the locomotor stimulatory potential of other transmitter systems. Pharmac. Biochem. Behav. 36: 45-50.
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EFFECTS OF DIZOCILPINE (MK-801) AND ETHANOL ON THE EEG AND EVENT-RELATED POTENTIALS (ERPS) IN RATS C. L. EHLERS,* W. M. KANEKO, T. L. WALL and R. I. CHAPLIN Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037, U.S.A. (Accepted 28 Augusr 1991) Summary-Recent neurophysiological data have suggested an interaction of ethanol (EtOH) with the glutamate-NMDA receptor complex. For instance, low levels of alcohol have been found to inhibit the ion current, activated by NMDA in in vitro preparations. The present study extends these paradigms in order to evaluate the electrophysiological effects of ethanol and the nonspecific NMDA receptor antagonist, dizocilpine (MK-801) in awake, conscious rats. Twenty Wistar rats were stereotaxically implanted with electrodes, aimed at dorsal hippocampus, amygdala, thalamus and frontal cortex. Rats received the following drugs: saline (s.c.), 0.01 and 0.1 mg/kg MK-801 (s.c.); EtOH, 0.75 g/kg (i.p.); 0.75 kg EtOH plus 0.01 mg/kg MK-801; 0.75g/kg EtOH plus O.lOmg/kg MK-801. Five minutes of EEG was collected and event-related potentials (ERPs) recorded in response to an auditory “oddball” paradigm. Spectral analysis revealed that MK-801 (0.1 m/kg) produced significant increases in low frequency EEG components, at all sites (14 Hz) and decreases in higher frequencies (16-32 Hz). Whereas ethanol (0.75 g/kg) produced decreases in power in all frequency bands. The combined administration of EtOH and MK-801 produced some antagonistic effects on the EEG in the low frequency range. Evaluation of ERPs revealed that MK-801 (0.1 mg/kg) produced significant decreases in amplitude of the Nl and P2 components in the cortex, decreases in the Pl and N2 in the thalamus and a profound decrease in the P3 components in hippocampus and amygdala. Ethanol was also found to produce decreases in the Nl component in cortex. The administration of MK-801 and ethanol together did not produce significant interactions on ERPs. These studies suggest that antagonism of the NMDA receptor by MK-801 may produce some effects similar to those of ethanol, however, their combined administration did not produce synergistic effects within these dose ranges. Key words-ethanol,
NMDA receptor, EEG, ERPs.
Several lines of investigation support a role for I-glutamate as an excitatory neurotransmitter in the mammalian brain. The glutamate-N-methyl-Daspartate (NMDA) receptor has been a specific target for electrophysiological, biochemical and behavioral studies, due to its potential role in synaptic plasticity and memory processes, as well as glutamate-mediated epileptogenic and neurotoxic actions (Lynch and Baudry, 1984; Collingridge and Bliss, 1987; Cotman and Iversen, 1987; Mayer and Westbrook, 1987). Recent studies have provided evidence to suggest that some of the effects of small to moderate doses of ethanol may be mediated by the NMDA subtype of glutamate receptor (see Hoffman, Rabe, Grant, Valverius, Hudspith and Tabakoff, 1990). In biochemical and electrophysiological experiments, ethanol, at small concentrations, has been shown to selectively inhibit NMDA receptor-mediated events in vitro (Hoffman, Rabe, Moses and Tabakoff, 1989; Lovinger, White and Weight, 1989; Lima-Landman and Albuquerque, 1989; Rabe and Tabakoff, 1990), *Address correspondence to: C. L. Ehlers, BCR-1, TSRI, 10666 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
while other glutamate receptors may be less sensitive to ethanol-induced inhibition. Further evidence that the effects of small doses of ethanol may be mediated by NMDA receptors, comes from studies utilizing the noncompetitive NMDA receptor antagonist ( + )-5methyl-lo,1 1 dihydro-SH-dibenzo [a,d] cyclohepten5, lo-imine maleate (MK-80 1). In behavioral studies, MK-801 has been shown to enhance the hypnotic (Wilson, Boxy and Ruth, 1990), anticonvulsant (Shrinivas and Ticku, 1989) and anxiolytic (Goldberg, Salama, Pate1 and Malick, 1983) effects of ethanol in rodents. Also, MK-801 has been demonstrated to attenuate the seizure score in animals, during withdrawal of alcohol (Grant, Valverius, Hudspith and Tabakoff, 1990; Morrisett, Rezvani, Overstreet, Janowsky, Wilson and Sqartzwelder, 1989). However, MK-801 has also been shown to have potent anticonvulsant and behavioral actions of its own (see Troupin, Mendius, Cheng and Risinger, 1986; Wozniak, Olney, Kettinger, Price and Miller, 1990). For instance, at larger doses (0.25-10 mg/kg), MK-801 produced somnolence, akinesia, impaired food consumption, locomotion and swimming, whereas small doses (0.05-0.1 mg/kg) impaired performance in a place learning paradigm (Whishaw and
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Auer, 1989). Since the behavioral effects of ethanol may result from the simultaneous action of several neurotransmitter systems the actions of which may be mediated through several different receptor-gated ion channels (Gonzales and Hoffman, 1991), it is hard to predict, in behavioral experiments, whether the MK801-induced enhancements of the effects of ethanol are non-specifically additive or enacted selectively, through NMDA receptor-mediated events. In previous studies, in uiuo electrophysiological experiments have been useful in discriminating the actions of ethanol from other classes of drugs, with similar properties, such as benzodiazepines and opiates (Ehlers and Reed, 1987; Ehlers, 1988, 1989). However, in uiuo electrophysiological experiments, which have simultaneously evaluated the effects of ethanol and MK-801 in awake animals have not been accomplished. Thus, the purpose of the present study was to utilize two in uiuo electrophysiological techniques in rats in order to explore the effects of small doses of MK-801 (0.01, 0.1 mg/kg s.c.) alone and in combination with a small dose of ethanol (0.75 g/kg ip.). The two electrophysiological paradigms utilized were analysis of spectral components of the electroencephalogram (EEG) and an auditory event-related potential (ERP) paradigm, both of which have been shown to be highly sensitive to the effects of ethanol (Ehlers and Chaplin, 1991; Ehlers, Chaplin, Lumeng and Li, 1991a). METHOD
The experimental subjects were 20 experimentally naive, male Wistar rats, weighing 280-300 g. The rats were housed individually and maintained in a temperature- and light- (12: 12 hr, LD) controlled room. Food and water were given ad libitum. At least 2 weeks prior to the experimental procedures, the rats were surgically prepared with recording electrodes. Animals were anesthetized (Nembutal, 50mg/kg i.p.), placed in a stereotaxic apparatus and stainless steel unipolar electrodes were aimed at hippocampus (AP -3.0, ML + 3.0, DV - 3.1), the ventral thalamus (AP -3.4, ML &-1.7, DV -7.8) and amygdala (AP + 1.0, ML k6.0, DV -9.5). Stainless steel screw electrodes were also placed over the frontal cortex and in the bony calvarium, 3 mm posterior to lambda which lies tangential to the cerebellum, which was grounded. Because these are complex regions of the brain, it is unlikely that the electrodes were placed in a specific subregion of the structure. In all animals, electrode attachments were made to a multipin (Amphenol) connector and the entire assembly was anchored to the skull with dental acrylic. Three sets of administrations of drugs, spaced 2 weeks apart, were given to the rats. In the first set, one half of the rats was given 0.1 mg/kg MK-801 and the other half was given saline, subcutaneously (s.c.). Two weeks later the groups were reversed; the
MK-801-injected group received saline and the saline-treated group received MK-801 (0.1 mg/kg). In the second set of administrations of drugs one half of the rats received saline (s.c.) and 15 min later received ethanol (0.75 g/kg i.p.). The other half of the rats received MK-801 (0.1 mg/kg s.c.) plus ethanol (0.75 g/kg i.p.), 15 min later. In the third set of experiments, one half of the rats received MK-801 (0.01 mg/kg s.c.) and the other half received MK-801 (0.01 mg/kg s.c.) plus ethanol (0.75 g/kg i.p.) 15 min later. The threshold for the appearance of behavioral effects of MK-801 in the rat lies between 0.05 and 0.1 mg/kg (Whishaw and Auer, 1989). Larger doses of MK-801 are known to produce gross behavioral effects and somnolence, thus making the assessment of the background EEG, as well as the response to sensory stimuli, difficult to interpret. Likewise the dose of ethanol (0.75 g/kg) was chosen, as prior studies have shown that this dose produces mild behavioral intoxication, without inducing sedation (Ehlers et al., 1991a). Thus, the combined administration of alcohol and MK-801, in these dose ranges, allowed for the assessment of potential synergistic or antagonistic effects on electrophysiological parameters within a range of awake, active behavior. For electrophysiological recordings, the animals were placed in a Naugahyde sling, which comfortably supported the animal in an awake state but prevented movement-induced artifact. The sling was placed in an electrically shielded light-, sound- and temperature-controlled BRS/LVE recording chamber. All animals were adapted to the chamber, prior to exposure to drug. On a test day, the rats were placed singly in the chamber and a connector, attached to a microdot cable, was used to transfer the monopolar (referred to the lambda ground screw) EEG signals to a polygraph. The bandpass for recordings was set at 0.3-75 Hz, with a 60 Hz notch filter in. The signals were amplified (50% gain) and the EEG, ERPs, as well as calibration signals, were transferred from the polygraph on-line to a DEC (LSI 11-2) computer, which also controlled the presentation of the auditory stimuli. The EEG recordings were collected 15-25 min after administration of drug. Five minutes of EEG recordings of unipolar signals (to ground) were obtained from the cortex, thalamus, amygdala and dorsal hippocampus from the six drug conditions. The EEG signals were then transferred to a Vetter Model D recorder, for off-line analysis. For quantification of the EEG, 5 min of EEG were digitized (128 Hz) and the power spectra of a continuous 4 set epoch were determined for the range 0.2564 Hz. The Fourier-transformed data were then further compressed into seven frequency bands (l-2, 2-4, 4-6, 6-8, 8-16, 16-32, 32-64Hz). Mean power density was calculated for each band, for each of the six conditions, as described previously (Ehlers and Havstad, 1982).
MK-801 and ethanol on EEG and ERPs Immediately after the EEG recordings, ERPs were collected. Free field auditory stimuli were presented through a small loudspeaker, centered approx 20 cm above the head of the rat. The ERPs were elicited by an acoustic “oddball” paradigm. The tones were generated with a programmable multiple-tone generator, the characteristics of which have been described previously (Polich, Fischer and Starr, 1983). The acoustic parameters for this paradigm were two square-wave tones (rise/fall times < 1 msec): a standard (soft) tone (20 msec, 1 kHz, 70 dB SPL) presented on 84% of the trials and a rare (louder) tone (20 msec, 2 kHz, 80 dB SPL), presented on 16% of the trials. Rare tones were interspersed with standards, such that no two rare tones occurred successively. The digitizing epoch for each trial was 1 set and a 0.5-l set intertrial interval was used (to reduce habitation). The total number of trials in a recording session was 150. The ERP recordings were analyzed for the six drug conditions. The ERP trials were digitized at a rate of 256 Hz. Trials containing excessive movement artifact were eliminated prior to averaging (< 5% of the trials). An artifact rejection program was utilized to eliminate individual trials in which the EEG exceeded &250 pV. The components of the ERP were quantified by computer, by identifying a peak amplitude (baseline-to-peak) within a standard latency range. The baseline was determined by averaging the 100 msec of pre-stimulus activity, obtained for each trial. The latency of a component was defined as the time of occurrence of the peak amplitude, within a latency window. The latency windows were initially determined by visual inspection of the data and then standardized to allow for computerautomated peak dete~inations. Components were labeled solely by their polarities and latency positions, relative to each other. The latency windows for cortex were: Pl, O-50 msec; NlA, 2.5-80 msec; NlB, 50-100 msec and P2, 90-250 msec. The windows for dorsal hippocampus were: Pl, 0-50msec; Nl, 25-80 msec; P2, 150-250 msec and P3,25&350 msec. The latency windows for ventral thalamus were: Ni, O-50 msec; PlA, 25-50 msec; PlB, 50-100 msec and N2, 100-250msec. The latency windows for amygdala were: Pl, l@-50msec; NlA, SO-75 msec; P2, 150-250 msec and P3, 250400 msec. These ERP analyses have been described previously (Ehlers, 1988). For statistical analysis, mean power values in each EEG frequency band and amplitude and latencies of ERP components were compared within subjects, for the six conditions, using analysis of variance (ANOVA). RESULTS
Administration of MK-801 was found to produce dose-related changes in the EEG, which were significantly different to those produced by injections of NP 3114-E
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saline. At the smallest dose (0.01 mg/kg), MK-801 was found to produce a decrease in slow frequencies in the 24 Hz range, which reached significance in the hipp~~pal leads (P < 0.04). No overt behavioral findings were observed at this dose. At larger doses, as seen in Fig. 1, much more significant EEG findings were observed, when compared to saline. Large increases were found in the low frequencies (l-6 Hz) in cortex, dorsal hippocampus (2-6 Hz) and amygdala (1-4 Hz). These increases in low frequencies were found to occur concomitantly with decreases in the higher frequencies (16-32 Hz) in all electrode sites. At this dose, the rats were observed to be mildly intoxicated. Ethanol (0.75g/kg) was also found to produce significant effects on EEG spectral components. Overall, ethanol was found to produce si~ificant decreases in spectral power, primarily in the midrange theta frequencies (6-8 Hz) in the cortex, hippocampus and thalamus as also seen in Fig. 1. This finding of reduced 6-8 Hz activity in the hippocampus, after ethanol, was similar to the effect observed after admi~stration of MK-801 (0.1 mgjkg). However, this dose of ethanol was not found to produce significant increases in slower waves (1-6 Hz), as was observed after MK-801. However, ethanol, at this dose (0.75g/kg), like MK-801 (0.1 mg/kg), was observed to produce significant decreases in higher frequency bands in the cortex, dorsal hippocampus and thalamus. Administration of ethanol (0.75 g/kg) together with small doses of MK-801 (0.01 mg/kg) did not produce many significant interactions on EEG spectra, when compared to the administration of either drug alone. Some reversal of the MK-8OI-induced increases in slow waves (2-4Hz) was found in the thalamus when ethanol was administered together with small doses of MK-801 (P < 0.05). However, administration of ethanol (0.75 g/kg) with larger doses of MK-801 (0.1 mg/kg) was found to produce a more significant reversal of the MK-801-induced increases in slow waves in the thalamus, as well as in amygdala, as seen in Fig. 1. Administration of MK-801 was also found to produce significant dose-dependent changes in eventrelated potentials (ERPs). As seen in Fig. 2, a series of waves could be averaged from the EEG, following pre~ntations of the two-tone stimulus paradigm, under saline conditions, for the four leads recorded from: cortex, ventral thalamus, dorsal hippocampus and amygdala. The response of the auditory cortex to the frequent, (soft) tone consisted of any early positive component (Pi), with a latency of 25msec, followed by a broad negative component, designated the NI wave (first negative peak) which had a peak latency of 60-80 msec. In response to the infrequent (louder) tone, Nl could also be more clearly differentiated into two separate waves: the NlA with a latency of 50-70 msec and an NIB with a latency of 80-100 msec.
2-4
Z-4
S-8 FRKWEM&Y BANDS
4-6
T~ALAMUS
FR=@iz-
S-16
16-32
l-2
m
2-4
2-4
6.8
HIPPOCAMPUS
WI
FREOUEMCV
4-6
AMYGDALA
(Hrk
BAlDB
6-6
FREWEMCY MMJS
4.6
DORSAL
~
6-18
(L-16
16.32
16-32
. * .
Fig. 1. Effects of MK-801 and ethanol, alone and in combination, on the EEG spectral power in rats. Data are presented for 4 different electrode locations: cortex, dorsal hippocampus, thalamus and amygdala. In each graph 4 conditions are presented: saline (SC); MK-801 (0.1 mg/kg s.c.); ethanol (0.75 g/kg i.p.); and MK-801 (0.1 mg/kg s.c.) plus ethanol (0.75 g/kg i.p.). Note that MK-801 produced significant increases in the low frequencies (14 Hz) and decreases in the higher frequencies (6-8 Hz, 16-32 Hz). Ethanol, in general, was found to produce overall reductions in spectral power. The combined administration of the two drugs tended to cancel each other out. SAL = saline; ETOH = ethanol.
1.2
&a
l-2
k
CORTEX
0
MK-801 and ethanol on EEG and ERPs
373
SALINE (RARE
VS STANDARD)
CORTEX -STANDARD
I
_______ RARE
_ _
N’
N2 I’-\ \
‘~.___..__.---* P3
,““I
,,,,,““,,‘,‘,l”‘,““,
100 200 300 Time lmsecl
400 500
-100
IIIl,l”‘,““,“l’,‘.,,,
0
100 200 300 Time (msecl
400 500
Fig. 2. Grand averages of components of the ERP, generated by a two-tone auditory “oddball” paradigm in 18 rats. Data are presented for 4 different electrode locations: cortex, dorsal hippocampus, thalamus and amygdala. The frequent (standard) tone is represented by the solid line and the infrequent (rare) tone by the dashed line. Components of the ERP used in statistical analysis are identified. Note a component of P3 was identifi~ in the dorsal ~pp~mpus and amygdala. THAL = thalamus; DHPC = dorsal
hippo~mpus; AMYG = amygdala. Figure 2 also displays the response of the ventral thalamus to the two-tone auditory paradigm. The ERP response to the standard (soft) tone consisted of an early negative wave (Nl) with a latency of 10 msec, followed by a two-peaked positive wave, the PlA (25-50 msec) and PlB (SO-80 msec) and a broad negative wave, designated the N2 (second negative wave) with a peak latency of 120-170msec. The ERPs after the rare (louder) tone were different from the standard (softer) tone, in that a PlB wave could also be identified. The response of the dorsal hippocampus (DHPC) to the standard (soft) tone consisted of an early
positive potential (Pl), which occurred at about 25mseq followed by a large amplitude negative wave, (Nl) with a latency of about 40-6Omsec and a later positive wave (P2), with a latency of lo&200 msec. Hippocampal ERPs from the rare (louder) tone differed from the standard (soft) tone in that a third positive wave (P3), which occurred at a latency of 250-3OOmsec, could clearly be identified, as seen in Fig. 2. The response of the amygdaia (AMYG) to the standard (soft) tone consisted of an early positive potential Pl, followed by a complex group of negative waves (Nls), with a latency of 50-l 50 msec and
C. L. EHLERS et al.
314
a later positive complex waves (P2-P3). After the rare (louder) tone, a third positive wave (P3), which occurred at a latency of 250-400 msec, could also be identified as seen in Fig. 2. These components were similar to those reported previously in other studies which have used this same ERP paradigm (see Ehlers, Wall and Chaplin, 1989a; Ehlers, Wall and Chaplin, 1991b). These components have also been demonstrated to be sensitive to stimulus factors, as probability and loudness, in a manner similar to that seen in human studies. Small doses of MK-801 (0.01 mg/kg) were not found to have significant effects on components of the ERP, produced by the standard tone, whereas com-
ponents of the ERP, generated in response to the rare tones were affected. A significant decrease in the amplitude of the NlA and N2 components in the cortex was observed after this dose. Larger doses of MK-801 (0.1 mg/kg s.c.) produced more dramatic reductions in the amplitude of several ERP components, as illustrated in Fig. 3. A decrease in the amplitude and an increase in the latency of ERP components, in response to both the standard and rare tones, was observed. In response to the standard tone, a decrease in the amplitude (P c 0.001) and an increase in the latency (P < 0.0001) of the Pl component and a decrease in the amplitude of Nl (P < 0.001) were found in cortical leads. Reductions
MK 801 0.1 MC/KG
* L---(RARE
CORTEX
-100 -
TONE)
-SALINE -------b/K 801
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8’ .,
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:
‘.
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---__ --.\ -_
__
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-200 -
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200
1 1’ ’
-100
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0
100
IIII,““,““,‘l”,““,
200
300
Time (msecl
400
500
100 200 300 Time (msecl
400 500
Fig. 3. Grand averages of components of the ERP observed after administration of MK-801 (0.1 mg/kg s.c.). In each graph, data are presented for response to the rare tone in the “saline” condition (-) n = 18 and in the “MK-801” condition (---) n = 20. Note that MK-801 produced a significant and profound reduction in the P3 component in the amygdala and dorsal hippocampus. Abbreviations as in Fig. 2.
MK-801
and ethanol on EEG and ERPs
Table 1I ERP amplitudesto the
rate tone (mean + SEM) (F, p)
Saline
MEL-801 (0.01 mg/kg)
Cortex PI
25.7 f 4.16
NS
NlA
91.1 f 17.0
73.0 f 9.0 (F = 5.88, P i 0.04) NS
P2
28.23 f 5.2
N2
15.2 k 5.0
5.5 + 2.0 (F = 5.3, P < 0.05)
Thalamus PIG
128.72 k 23.3
NS
N2
79.68 k 16.6
NS
256.47 f 28.0
NS
72.9 k 10.5
NS
75.6 i 11.7
NS
Amygdala PI
53.48 i 12.3
NS
P3
75.88 + 14.9
NS
Dorsal hippocampus Nl
P3B
in the amplitude (P < 0.001) and increases in the latency (P < 0.04) of the N2 component, to the standard tone in the thalamus, were also noted. A reduction in the amplitude of the P2 response (P < O-03), to the standard tone, was observed in the amygdala. Reductions in the amplitudes of several components of the ERP to the rare tone were also seen, as outlined in Table 1. In cortex, significant reductions in the amplitudes of the Pl, Nl A, P2 and N2 components were observed. In the thalamus, significant reductions in amplitude in the PlA and N2 were seen. The prominent effects of MK-801, at this dose, were the profound reductions observed in the amplitudes of the P3 components, recorded in the hippocampus and the amygdala. Ethanol (0.75 g/kg) was also observed to produce significant reductions in the amplitudes and increases in the latencies of several components of the ERP. A reduction in the amplitude of the NlA component in the amygdala (P < 0.02) and an increase in the latency of the Pl (P c 0.05) and P2 (P < 0.01) components in cortex, were found following the standard tone. Further reductions in amplitude and increases in the latencies of several components were also found after ethanol (0.75 g/kg), when responses to the rare tone were evaluated. As seen in Fig. 3, ethanol produced increases in the latency of the Pl (P < 0.05) and NlA (P c 0.002), and decreases in the amplitude of the NlA and P2 components in the cortex. In addition, decreases in the amplitude of the PlA component were observed in the thalamus and amygdala after this dose of ethanol. Ethanol, at this dose, unlike MK-801, did not produce significant decreases in the amplitude of the P3 component in the hippocampus and amygdala, although a trend in that direction was noted in the amygdala (P < 0,06).
375
MK-801
(0.1 mgjkg)
8.2 + 2.38 (F = 20.2, P < 0.001~ 63.2 f 1.2 IF = 4.6. P c 0.04\ 1.9’*0.9 (F = 27.4, P < 0.001) 27.3 k 4.3 (F = 8.8, P < 0.009)
ETOH(0.75g/kg) NS 66.4 f 11.3 (F = 7.73, P c 0.03) 22.7 t 3.4 (F = 6.4,-P < 0.04) NS
67.91 k 12.9 (F=7.15, P.zO.02) 37.75 f 6.6 (F = 6.97, P < 0.02)
103.4 f 21.6 (F = 6.4. P
175.63 f 29.7 CF = 6.9. P < 0.02) 24.9’* 5.2 ’ (F = 24.4, P < 0.0001) 39.7 f 16.0 (F = 7.55, P < 0.05)
NS
21.34k5.8 (F = 5.6, P c 0.03) 33.1 f 9.4 (F = 9.61, P c 0.006)
NS
NS NS
49.9 f 11.25 (~=4.19, P <0.06)
The combination of MK-801 and ethanol on components of the ERP was found to produce conflicting results. Ethanol potentiated the reduction in the amplitude of NlA in the amygdala, when combined with small doses of MK-801 (0.01 mg/kg) (P c 0.01). However, ethanol was not found to produce significant potentiation amplitudes or latencies of components when combined with the larger dose of MK-801 (0.1 mg/kg) and, in fact, was observed to shorten the latency of P3 in hippocampus (P c 0.01). DISCUSSION
Electrophysiological measures have been used effectively to investigate several aspects of the actions of ethanol in the CNS (see Porjesz and Begleiter, 1985). Studies evaluating the acute effects of small to moderate doses of ethanol on the spontaneous cortical EEG in awake human subjects have reported increases in theta activity, increases in slow alpha activity and a slowing of the dominant alpha frequency (Lucas, Mendelson, Benedikt and Jones, 1986; Ehlers, Wall and Schuckit, 1989b). In the present study, small doses of ethanol (0.75 g/kg), given to rats were, in general, found to produce reductions in power in many frequency bands, including decreases in theta activity (6-8 Hz) in hippocampal leads. It has been suggested, based on in vitro electrophysiological studies, that these small “intoxicating” doses of ethanol may selectively inhibit the function of the NMDA receptor (Lima-Landman and Albuquerque, 1989; Lovinger ef al., 1989). Thus, it seems that an NMDA receptor antagonist should mimic and/or potentiate the actions of ethanol. The non-competitive NMDA receptor antagonist, MK801 has been shown, in some behavioral tests, to potentiate the actions of ethanol, For instance,
316
C. L. EHLERS et al.
MK-801 has been shown to potentiate the hypnotic (Wilson et al., 1990) effects of ethanol in rats and to inhibit alcohol withdrawal-induced seizures (Morrisett et al., 1989), at doses which produce behavioral somnolence (0.33 mg/kg). However, in tests of locomotion, MK-801 at smaller doses (0.1 mg/kg), has been reported to produce increases in locomotion, whereas ethanol, in moderate doses (0.75 g/kg), has a depressant effect on locomotion (Robledo, Kaneko and Ehlers, 1991). In addition, administration of small doses of ethanol, together with small doses of MK-801, has not been found to produce synergism in locomotor experiments. Instead, their effects tended to cancel each other out (Robledo et al., 1991). Few studies have evaluated the effects of MK-801 in in uioo electrophysiological systems, in unanesthetized animals. Whishaw and Auer (1989) have demonstrated that no “gross” differences in the EEG of rats were noted after administration of 0.1 mg/kg MK801, with only a slight reduction in hippocampal rhythmic slow activity, observed after the subsequent administration of atropine sulfate. However, EEG activity was not quantified in that study. The effects of MK-801 on the EEG in the present study were dose-dependent and were found to parallel the findings observed in previous locomotor studies. The MK-801 tended to produce an opposite EEG spectral profile to that observed after small doses of ethanol, with MK-801 (0.1 mg/kg) producing significant increases in slow waves (14 Hz). However, both ethanol and MK-801 were found to produce decreases in the 68 Hz frequencies in the hippocampus and reductions in power in several leads in the high frequency ranges (16-32 Hz). Also, like the locomotor findings, administration of ethanol and MK801, together did not produce prominent synergistic effects on the EEG and, in general, the combined effects of the two drugs tended to cancel each other out. The development of event-related potential paradigms has provided additional in viuo electrophysiological measures, whereby effects of drugs can be assessed. In these electrophysiological paradigms, subjects are typically required to detect the presence of certain, infrequently-presented “target” stimuli, which are embedded within a series of frequently stimuli. However, P300 presented “non-target” waves have also been reported to occur in “passive paradigms”, where subjects are presented with different sets of stimuli but are not required to respond to them (Polich, 1987). In such paradigms, when the EEG is averaged in a time-locked fashion to the target or infrequent stimuli, a series of waves can be identified, one of which is positive-going and occurs at a latency of about 300msec in young adults (see Donchin, Karis, Bashore, Coles and Gratton, 1986). This P3 or P300 wave has been suggested to be a measure of stimulus evaluation and may possibly be related to memory processes (Donchin et al., 1986). Depth recordings in human subjects suggest that the
P300 may actually be generated in the hippocampus or amygdala (Halgren, Squires, Wilson, Rohrbaugh, Babb and Crandall, 1980), although cortical sites may also be important (Knight, Scabin, Woods and Clayworth, 1989). These event-related potential paradigms have also been attempted in animal studies, where “P3-like” waves have also been identified. Lesion studies in cats also support a role for the hippocampus in the generation of the P3 in that species (Harrison, Buchwald, Kaga, Woolf and Bucher, 1988) and in rats P3 waves have been recorded from depth electrodes placed in both hippocampus and amygdala (Ehlers et al., 1991b). After small doses of ethanol in human subjects, decreases in the amplitude of the Nl and P3 components of ERPs have generally been found (Pfefferbaum, Horvath, Roth, Clifford and Kopell, 1980; Campbell and Lowick, 1987); however, task difficulty (Roth, Tinkle&erg and Kopell, 1977) may modify responsivity. As in the human, acute administration of small doses of ethanol has been found to produce decreases in Nl and larger doses of ethanol, which cause some sedation, caused reductions in the P3 components, recorded in a passive auditory ERP paradigm in both monkeys (Ehlers, 1988, 1989) and rats (Ehlers and Chaplin, 1991; Ehlers et al., 1991a). In the present study, a reduction in the Nl component of the ERP was also observed after the administration of a small dose of ethanol (0.75 g/kg) in rats, with a trend towards the lowering of the P3 in amygdala. Decreases in the amplitude of the Nl component have also been found following the administration of other anxiolytics. For instance, modest doses of diazepam (2.5mg/kg) were found to produce profound reductions in the amplitude of Nl without having significant effects on the P3 component (Ehlers, 1988). Also, MK-801 was found to produce decreases in several components of the ERP, which were dose-dependent. Both doses of MK-801 produced significant decreases in the amplitude of the Nl component in the cortex. Profound reductions in the P3 component in the hippocampus and amygdala were also prominent after administration of the somewhat larger dose of MK-801 (0.1 mg/kg). However, administration of ethanol and MK-801 together was not found to produce significant synergistic effects on ERP recordings. The finding of a highly significant reduction in P3 in the rat in the hippocampus and amygdala after administration of small doses of MK-801 (0.1 mg/kg), suggests that the glutamate-NMDA receptor system may modulate the generation of that component. While the cognitive concomitants of the P3 component are unknown, some studies in human subjects have suggested that it may be associated with memory processes (Donchin et al., 1986). Several studies have also linked the NMDA receptor with some mechanisms which underlie memory, such as long-term potentiation (Lynch and Baudry, 1984). In addition, MK-801 has been demonstrated to disrupt
MK-801 and ethanol on EEG and ERPs
learning in animals, particularly spatial learning (Whishaw and Auer, 1989; Wozniak, 1990). Thus, the profound reduction in the amplitude of P3, found in the present study, after administration of MK-801, lends further support for a relationship between the P3, the NMDA receptor and memory processes. Some of the behavioral effects of MK-801, particularly its locomotor-stimulating effects, have been suggested to be produced through blockade of NMDA receptors which subsequently causes release of catecholamines, particularly dopamine (Clineschmidt, Martin, Bunting and Papp, 1982; Carlsson and Svensson, 1990). Thus, it is possible that the reductions in P3 observed in the present study, may have occurred secondarily, through modulation by NMDA of catecholamines. However, in previous studies, no reductions in the amplitude of P3 were noted in rats, which had neurochemically verified significant reductions in dopamine in brain, produced by 6-hydroxydopamine (6-OHDA)induced lesions to the ventral tegmental area (Ehlers et al., 1991b). In addition, no reduction in the components of P3 in the rat were noted in animals with significant depletions of serotonin produced by p-chlorophenylalanine (PCPA) (Ehlers et al., 1991b). Some changes in morphology of P3 have been noted following destruction of noradrenergic neurons. For instance, Pineda, Foote and Neville (1989) found that electrolytic lesions of the locus coeruleus, followed by knife cuts of the ascending fibers, altered “P3004ike” potentials in the monkey recorded at the cortical surface. In rats, 6-OHDA-induced lesions to the dorsal noradrenergic bundle, which caused significant depletion of norepinephrine in brain were also found to produce some reduction in the components of P3 (Ehlers et al., 1989a). The reductions in the amplitude of P3, observed in dorsal bundle-lesioned rats however, were not as profound as those observed in the present study after small doses of MK-801. In addition, MK-801 is thought to increase catecholaminergic tone, which should theoretically result in increases in the amplitude of P3. Thus, the reductions in the amplitude of P3, observed in the present study, were most likely not to be the result of catecholaminergic modulation. In summary, MK-801 was found to produce slow waves in the EEG and dose-dependent reductions in the amplitude of several components of the ERP and profound reductions in the P3 potential, recorded in the hippocampus and amygdala. Ethanol was also found to produce reductions in some components of the ERP but also produced reductions in the overall EEG spectral power. These studies suggest that antagonism of the NMDA receptor may produce some effects similar to those of ethanol, however, their combined administration did not produce synergistic effects within these dose ranges. There are several factors which should be considered when interpreting these results. First, is the potential nonpharmacological specificity of MK-801. For instance, some of the
317
EEG and behavioral effects of MK-801 have been shown to resemble those of phencyclidine (see Koek, Woods and Gail, 1988; Mattia and Moreton, 1986). However, phencyclidine is known to affect the function of other receptor systems, in addition to NMDA (Manallack, Beartand and Gundlach, 1986). Thus, MK-801 may also have actions at other receptor sites. This would make ethanol-MK-801 interactions in any model system, where both receptors were present, difficult to interpret. Conversely, not all the actions of ethanol are also enacted through NMDA receptors (see Gonzales and Hoffman, 1991). Thus, ethanol may be producing effects on systems in the brain, other than NMDA, which could possibly reverse the effects of MK-801. Further studies will be necessary to specifically interpret these findings. Acknowledgements-The
authors would like to thank MS Susan Lopez for help with statistical analysis and typing and editing of the manuscript. The computer programs were written by Dr James Havstad. The work was supported by grants AA00098 and AA06059 from the National Institute of Alcoholism and Alcohol Abuse to C.L.E. The MK-801 was a gift of Merck Pharmaceuticals. REFERENCES Campbell K. B. and Lowick B. M. (1987) Ethanol and event-related potentials: The influence of distractor stimuli. Alcohol 4: 257-263. Carlsson A. and Svensson A. (1990) Interfering with glutamate& neurotransmission by means of NMDA antagonist administration discloses the locomotor stimulatory potential of other transmitter systems. Pharmac. Biochem. Behav. 36: 45-50.
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