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Regional effects of ethanol on limbic afterdischarge activity
Brain
Research
Ed/din,
0361-9230/84 $3.00 + .OO
Vol. 13, pp. 519-525, 1984. 0 Ankho International Inc. Printed in the U.S.A.
Regional Effects of Ethanol on Limbic Afterdischarge Activity HENRY Department
LESSE’
AND REBECCA
K. HARPER
of Psychiatry and Biobehavioral Sciences, The Neuropsychiatric and Brain Research University of California, Los Angeles, CA 90024 Received
Institutes
11 June 1984
LESSE, H. AND R. K. HARPER. Regional effects of ethanol on limbic afterdischarge activity. BRAIN RES BULL 13(4) 519-525, 1984.-This study,assessed the effects of acute administration of ethanol on afterdischarge (AD) activity evoked by electrical stimulation of the amygdala, septum and hippocampus. Limbic AD thresholds, duration and propagation were determined in cats following intravenous infusions of saline or ethanol (0.4, 0.8 and 1.6 g/kg). Ethanol administrations significantly increased septal and amygdalar, but not hippocampal AD thresholds. This effect was dose-related and most pronounced at the septum. Reductions in AD duration followed ethanol treatment and demonstrated similar regional differences. In addition, projected discharges were reduced, propagation of AD from stimulation sites to limbic and neocortical projections sites were suppressed and ictal episodes were attenuated following ethanol treatment. Afterdischarge activity was affected even by the 0.4 g/kg dose which produced no observable change in behavior. These findings indicate that ethanol reduces the responsiveness of limbic structures to electrical stimulation-suppressing the initiation, maintenance and propagation of limbic afterdischarges. The amygdala, septum and hippocampus proved differentially
sensitive to ethanol. Ethanol Epilepsy
Limbic system
Brain stimulation
Amygdala
Hippocampus
Septum
Afterdischarges
METHOD
THERE is increasing clinical and experimental evidence suggesting that ethanol has important actions on electrical
Subjects
events in the limbic system. Ethanol has been reported to activate EEG abnormalities in temporal lobe epileptics [23], attenuate and potentiate seizures produced by amygdalar stimulation in kindled rats [24], and to change limbic system EEG, single and multiple unit activity during acute and chronic treatment [3, 11, 12, 25, 26, 27, 281. It has been suggested that progressive limbic system dysfunction produced by repeated episodes of intoxication and withdrawal is important in mediating the neurobehavioral changes characteristic of chronic alcoholism [2]. There are, however, conflicting reports about the sensitivity of various limbic structures to ethanol and it is not known whether ethanol has preferential actions on major components of the limbic system [3, 14, 15, 261. The present study compared the effects of ethanol on afterdischarge activity evoked by electrical stimulation of the amygdala, septum and hippocampus. The present results indicate that these structures are differentially sensitive to ethanol and that ethanol has potent inhibitory effects on the electrical excitability of limbic structures-elevating afterdischarge thresholds, and reducing afterdischarge duration and propagation. These findings provide new information about regional changes in limbic activity during acute ethanol intoxication and provide clues for understanding the mechanism of ethanol’s anticonvulsant effect. A preliminary report of the work has appeared [17].
Subjects were twelve adult cats prepared surgically under pentobarbital anesthesia (35-45 mg/kg) with a jugular cannula and indwelling stainless steel electrodes. Arrays of 0.25 mm bipolar needle and concentric electrodes with 1 mm separation between exposed tips were implanted bilaterally in the dorsal hippocampus, basolateral amygdala and septal area under stereotaxic guidance. Epidural electrodes were placed over the occipital cortex, a stainless steel screw in the frontal sinus served as a reference electrode and interconnected screws provided a ground. An acrylic pedestal, fixed to the skull, supported both a luer lock terminus for the jugular cannula and a miniature electrode connector. At least three weeks were allowed for post-operative recovery before experiments were initiated. Apparatus subjects were placed in an During test sessions, electrically-shielded, sound-attenuating chamber equipped with a one-way mirror, bar-press and milk delivery apparatus. Recordings of subcortical and neocortical activity were obtained with a 14 channel polygraph and stored on magnetic tape for subsequent analysis. Brain stimulation was provided by a square wave stimulator and constant current unit under micro-computer control. A dual beam oscilloscope was em-
‘Requests for reprints should be addressed to H. Lesse, Department of Psychiatry, UCLA Neuropsychiatric Los Angeles, CA 90024.
519
Institute, 760 Westwood Plaza,
ployed to measure current and voltage. corder registered bar-pressing responses.
A cumulative
re-
The cats were trained to bar-press for milk reinforcement. A 24 hour period of food deprivation preceded ail test sessions, which were conducted while the subjects were barpressing. This procedure provided both a stable level of arousal and an activated EEG pattern during the induction of afterdischarges by focal limbic stimulation. Afterdischarge threshold determinations were conducted employing a previously described method [20]. In brief, two pairs of bipolar electrodes in each limbic structure were employed for simultaneous brain stimulation and recording. Focal electrical stimulation (3 Hz biphasic, 0.5 msec, rectangular pulses; 30 set trains) was employed to evoke amygdalar and hippocampal AD. Since AD usually were not induced by 3 Hz septal stimulation at currents below 3 mA, the stimulation frequency was adjusted to 69 Hz for the septum to provide current thresholds comparable to those of the hippocampus and amygdala. Utilizing low frequency stimulation, ele~trophysiological responses were recorded during the intervals between pulses from a site in the stimulated structure adjacent to the stimulating electrode and from projection sites in related structures. Thus, the initiation of AD was detected while brain stimulation was occurring and drug effects could be studied when only brief localized limhic seizure patterns were evoked (see Figs. 3 and 4). Electrophysiological recordings from multiple cortical and limbic sites were obtained between both the bipolar electrodes and individual electrode tips and the common sinus reference. Stimulation was applied for 30 seconds at one minute intervals. Current was increased in steps of 10% or less until self-sustaining AD were evoked. Stimulation was terminated when focal limbic afterdischarges were elicited. The AD threshold was defined as the minimal current required to evoke self-sustaining afterdischarges in the stimulated structure, persisting for at least one second after stimulation was terminated. These determinations continued at 48 hour intervals until stable threshold values were obtained (i.e., there was no more than a 10% variation for three successive test sessions). Additional stimulations were applied to 15 of the 20 limbic test sites to obtain diffuse AD prorogation before drug testing. After stable AD threshold levels were obtained, a series of alternating physiological saline and ethanol tests was initiated. Sessions were separated by at least 48 hours and ethanol tests by 96 hours. Based on pilot experiments, slow intravenous infusions of 0.4, 0.8, and I A g/kg (15% ethanol) were selected to represent a wide range of intoxication levels. An infusion pump was employed to administer the drug at a constant 3 ml/min rate. The AD thresholds were determined 10 minutes after infusion of either ethanol or saline. The drug testing began with initial current intenstiy set 50% below the previously determined threshold for each stimulation site. Low, medium and high doses were tested in a varied order, counterbalanced across subjects. When continued patency of the jugular cannula permitted, drug testing was completed at all three stimulation sites. Most animals, however, were tested at one or two limbic structures. The structure receiving initial focal brain stimulation was varied across subjects. Seven subjects were tested with amygdalar and hippocampal stimulation and six with septal stimulation. Thus. the procedure yielded a total of 60 paired saline and
1 SFPTUM
i
l
AMYCDALA
:)
HIPPOCAMPUS
;r
0.4
0.8
1.6
DOSE FIG. I. Dose-related effects of ethanol on limhic afterdischarne threshold. Drug effects are expressed as the difference (in p.k means_tSEM) followine t&red ethanol and saline tests. The ethnnnt -_.._. effect differed among-&e three limbic regions threshold elevations at the septum and the smallest pus following each of the test doses.
with the largest at the hippocam-
ethanol tests. At the conclusion of experiments. an overdose of pentobarbit~ anesthesia was administered and brains were perfused with saline and 15% formalin. The location of electrode tips was identified microscopically in stained serial sections. Histological studies could not be completed in two subjects who died after ethanol testing was completed but before perfusions could be performed and in three animals subsequently used in other experiments. In these cases, the presence of characteristic sub~o~ical electrographic patterns (hippocampal theta activity, amygdalar 40 Hz rhythmic patterns and septal low amplitude activity) and characteristic propagation patterns evoked by focal stimulation served as guides to the accuracy of the stereotaxic electrode placements. The results of afterdischarge threshold and duration tests were each evaluated initially with a three way analysis of variance with two repeated measures. The three main effects were site, drug, and drug dose; the latter two being repeated measures. The difference scores between paired ethanol and saline tests subsequently were evaluated with a two way analysis of variance. Significant F values were folfowed by post hoc comparisons using paired t-tests with the appropriate error variances derived from the co~esponding ANUVA [301.
ETHANOL
521
AND LIMBIC AFTERDISCHARGES TABLE 2
I
TABLE
AFTERDISCHARGE THRESHOLD (MA) FOLLOWING ETHANOL AND SALINE ADMINISTRATION*
PAIRED
AFTERDISCHARGE DURATION (SEC) FOLLOWING PAIRED ETHANOL AND SALINE ADMINISTRATIONS*
Hippocampus
Amygdala
Septum
Low Dose Saline
69.2 + 16.7 73.3 +- 16.2
45.9 + t1.5 85.3 + 10.3
61.3 t 17.9 67.9 -c 19.5
1.24 ir 0.24 1.16 rt: 0.23
Medium Saline
54.1 rt 12.8 72.6 L 14.9
31.1 t 14.7 96.8 zt 16.9
55.1 It 18.2 67.6 L 19.8
2.66 t 0.43 1.53 i 0.22
1.30 r 0.23 I .18 2 0.22
High Saline
33.8 t 6.0 68.4 + It.3
14.6 t 3.3 70.5 _i_14.3
46.7 r 15.8 68.0 t 17.0
2.18 -+ 0.20 1.54 2 0.12
1.25 * 0.13 1.19 t 0.12
Mean Ethanol Mean Saline
52.4 I 7.7 71.4 z!z 8.0
30.5 i 84.2 it
54.4 t 9.6 67.9 Y?Z 10.3
Amygdala
Septum
Low Dose Saline
0.94 + 0.20 0.87 !I 0.25
1.78‘-t 0.27 1.56 2 0.20
I.19 -r 0.23 1.21 ir 0.22
Medium Saline
I .09 + 0.31 0.87 lr 0.26
2.11 + 0.30 1.54 + 0.22
High Saline
1.30 + 0.37 0.74 i 0.18
Mean Ethanol Mean Saline
I.11 i- 0.18 0.83 -c 0.13
6.7 8.1
Hippocampus
*Means 2 SEM.
“Means ‘_ SEM.
RESULTS
Table 1 summarizes the results of saline and ethanol tests on AD threshold for all subjects; Fig. 1 illustrates regional, dose-related effects of ethanol. An analysis of variance comparing all limbic afterdischarge threshold tests indicated significant values for drug effect, F( 1,17)=21.72,p
In addition to these alterations in threshold, dose-related reductions in AD duration followed ethanol administrations (see Table 2). There was a significant drug effect at all limbic areas, F(1,17)=41 Sl, p
d P
T
0.4
0.11
1.6
DOSE
FIG. 2. Dose-related effects of ethanol on limbic afterdischarge duration. Drug effects are expressed as the difference (in seconds, means?SEM) following paired ethanol and saline tests. Symbols representing the septum, amygdaia and hippocampus are the same as in Fig. 1. There were dose-related reductions in afterdischarge duration at all areas. The largest effects were found at the septal area.
of the three ethanol doses (see Fig. 2). This effect was significantly dose-related at all limbic sites, F(2,34)=5.53, p
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FIG. 3. Effects of ethanol on afterdischarges evoked by left basolateral amygdalar stimulation. Under saline control conditions (upper tracings), afterdischarges initiated in the left amygdala spread to other limbic sites and then to the neocortex. Following the 0.8 g/kg ethanol dose (left lower tracings), AD duration decreased and propagation to the right hippocampus and neocortex was blocked. Following the I .6 g/kg dose, there was a further abbreviation of the ictal episode. Amygdalar afterdischarges were attenuated and propagation of seizure discharges to all other projection sites was suppressed. Amygdalar stimulation induced fully developed tonic-clonic seizures after saline treatment. Following ethanol, behavioral seizures were limited to brief automatisms. To display entire ictal events, recording speed was reduced to 6 mm/set; standard speed for AD duration measurements was 30 mm.
largest change was found at the septal area (mean reduction 53.7k6.8 set) and the smallest at the hippocampus (13.5+4.3), with an intermediate decrease in amygdalar AD duration (19.125.7). A large reduction in septal AD duration (39.4%) followed even the lowest ethanol dose and the drug
effect was substantially greater at the septal area than at the amygdala or hippocampus for each test dose (see Fig. 2). Effects of Ethanol on Afterdischarge
Propagation
Ethanol, in addition, diminished the spread of seizure discharges from the stimulated structure to distant limbic and neocortical projection sites. These effects are illustrated in Figs. 3 and 4. Figure 3 displays a characteristic sequence of electrophysiological changes following saline, 0.8 g/kg and 1.6 g/kg ethanol during amygdalar AD threshold tests. The initiation of afterdischarges is evident during 3 Hz electrical stimulations of the left basolateral amygdala at threshold intensity. In the upper tracings, obtained after saline treatment, stimulation evoked afterdischarges propagating sequentially to the right amygdala, left and right hippocampus and finally to the neocortex. Ictal episodes were progressively abbreviated as the ethanol dose increased. After 0.8 g/kg of ethanol, the duration of afterdischarges recorded from the stimulated structure, the contralateral amygdala and ipsilateral hippocampal projection sites was reduced. Propagation of seizure discharges to both the contralateral hippocampus and to the neocortex was blocked and afterdischarge amplitudes diminished. Following the 1.6 g/kg dose, there was a further attenuation of the amygdalar AD and propagation of discharges to alI other sites was suppressed. During saline control tests, amygdalar stimulation consis-
tently evoked fully developed tonic-clonic motor convulsions. Following ethanol administrations, the behavioral response to stimulation was limited to the automatisms characteristic of focal limbic seizures (after the 0.8 g/kg dose, an arrest reaction, head turning, mouth movements and salivation; following the 1.6 g/kg dose. only brief mouth movemerits) . Figure 4 illustrates a different series of electrophysiological events typically evoked by hippocampal stimulation. Following ethanol, afterdischarge duration decreased, propagation of seizure discharges to the amygdala and neocortex was blocked and the ictal episode was abbreviated. The fully developed, generalized motor convulsion evoked by hippocampal stimulation under saline control conditions was replaced by minimal mouth movements and a transient arrest reaction. Similar drug effects followed septal stimulations. Paroxysmal seizure discharges spread from amygdalar, septal and hippocampal stimulation sites to the neocortex during 46 of the 60’saline control tests. The high dose of ethanol effectively suppressed the propagation of limbic afterdischarges to the neocortex in 13 of 15 (86.7%) paired saline and ethanol tests. This effect occurred in 12 of 15 (80%) medium dose tests and in 7 of 16 (43.8%) low dose tests. Signs of acute alcohol intoxication such as marked sedation, ataxia, and suppression of bar pressing were observed regularly following the 1.6 g/kg dose. Following the 0.8 g/kg dose, bar press rates decreased markedly in most subjects and usually some ataxia was evident. Following the 0.4 g/kg dose, there was no gross behavior change and no observable alteration in the state of arousal. Cats generally continue to bar press for milk at their normal rate. Thus, the limbic elec-
ETHANOL
523
AND LIMBIC AFTERDISCHARGES SALINE
L. HIPPO.
STIY. 0.8
01110
CORTLX
K.
ETHANOL
KIPPO.
L. HIPPO.
FIG. 4. Effects of alternate saline and ethanol administrations on afterdischarges evoked by left dorsal hippocampai stimulation. Following ethanol treatment, ~p~c~pa~ AD duration decreased and the propa~tion of seizure discharges to the amygdala and neocortex was suppressed. The generalized tonic-clonic convuision evoked by hippocampal stimulation under control conditions was replaced by brief mouth movements and a transient arrest reaction.
changes at the lowest ethanol dose occurred in the absence of observable behavioral changes or alterations in task performance.
trophysiological
DISCUSSION
The present findings indicate that ethanol has a powerful protective action against seizure activity initiated in major components of the limbic system. A sequence of electrophysiological changes contributed to this anticonvulsant effect. There were significant dose-related increases in the current required to evoke afterdischarges, as well as doserelated reductions in AD duration at the amygdala, septum and hip~c~pus. In addition, the duration of projected limbic discharges was reduced and the spread of seizure discharges from their site of origin to distant limbic and neocortical projection sites was restricted. Consequently, ictal episodes were abbreviated and their behavioral manifestations were markedly attenuated. Thus, several mutually reinforcing mechanisms appear responsible for ethanol’s acute anticonvuls~t effect. Ethanol suppresses the initiation and maintenance of self-sustaining afterdischarges by reducing the responsiveness of limbic seizure foci to stimulation. In addition, the drug suppresses the propagation of seizure discharges to limbic and neocortical projection sites. The effectiveness of the 0.4 g/kg dose (which had no observable behavioral effect) in altering afterdischarge activity indicates ethanol’s potency in maiming brain functions that are not manifest in overt behavior and also demonstrates the sensitivity of limbic afterdischarge activity as an indicator of ethanol actions. The present findings provide clear evidence that ethanol,
in doses ranging from 0.4-1.6 g/kg, differenti~y affects the sensitivity of closely interconnected limbic structures to electrical stimulation. The largest dose-related threshold increase was found following septal stimulation and a smaller, but significant, elevation followed amygdalar stimulation. Changes in hippocampal thresholds, however, proved insignificant. ~terdischarge duration changes reflected similar differences in the sensitivity of the three limbic structures to ethanol. It is noteworthy that the hippocampus, which has been the most frequent brain target for alcohol research [27], proved substantially less sensitive to ethanol than either the septum or amygdala. It is natural to expect ethanol, a depressant drug, to decrease seizure sensitivity (as in the present experiments). Surprisingly, many similar changes in limbic afterdischarge activity are induced by cocaine, a central nervous system stimulant. In parallel experiments, cocaine also significantly reduces the duration of self-sustaining afterdischarges evoked by electrical stimulation of the arnygdala, septum and hippocampus 1201. In addition, both drugs decrease projected rhythmic discharges, suppress the spread of paroxysmal activity to distant limbic and neocortical projection sites and attenuate ictal episodes originating in limbic structures [ 191. Limbic afterdischarge thresholds are modified by both drugs, although cocaine has a dual, frequency-related effect at the same limbic sites [21]. The limbic structures studied receive impulses from multiple sensory systems and have an important role in evaluating the emotional and motivational aspects of environmental stimuli [8, 9, 161. Characteristic electrographic rhythmic patterns have been identified in the amygdala and hippocampus during exposure to environmental stimuli with motivational relevance [ 1, 10, 18,
221. Thus, similar drug effects attenuating the maintenance and propagation of rhythmic afterdischarges suggest the possibility that both ethanol and cocaine (drugs with contrasting central nervous system sedative and stimulant properties) may change the excitability of limbic structures, altering their propensity for sustained rhythmic responses to motivationally relevant environmental stimuli. An electrophysiological change of this nature may be one of the important factors associated with the altered emotional responses to sensory experiences characteristic of both alcohol and cocaine intoxication. There have been very few studies reporting ethanoiinduced changes in septal bioelectrical activity. The present findings are consistent with a report that ethanol-induced depression of spontaneous multiple unit activity is greater in the septum than in the hippocampus [14]-but at variance with a conflicting observation from the same laboratory that septal units are relatively insensitive to alcohol [IS]. Our results are consistent with reports that ethanol decreases seizure duration and increases thresholds for motor seizure produced by stimulation of the amygdala in kindled rats [ 131; and decreases amygdaiar afterdischarge duration 1241. The present results are at variance with another conflicting report that alcohol fails to alter AD duration in kindled rats and that hippocampal threshold elevations exceed amygdalar elevations [6].
The present results have implications I’Lr rhr j,tudq oi temporal lobe epilepsy in man. Ethanol has been reported II) activate EEG abnormalities in certain temporal lobe epilep tics [23]. The activation of temporal area EMi spikes may reflect differential ethanol-induced changes in limbic clcctri cal excitability similar to those found in the present cxpcrimental seizures. This possibility is consistent with reccm reports that limbic spike discharges are correlated with in. hibitory phenomena and lowered seizure susceptibility 14, 5. 71. Thus, the present finding that limbic structures are differentially sensitive to the inhibitory effects of ethanol suggests further research reevaluating the alcohol activation proccadure as a potential aid in distinguishing hippocampal from amygdalar and septal epileptic foci in seizure disorder-5 nf fimbic origin.
ACKNOWLEDGEMENTS
The authors wish to thank J. P. Collins, Ph.D. who helped in initiating this work, Mary Mettler for technical assistance, William Bergerson for expert electronic assistance and Michael Levine. Ph.D. for statistical consultations. This reserach was supported in part by Grants 03.513from the NIAAA. RR 5756from NIMH and by 25 UCLA Donor’s Fund.
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Hippocampal waves; distribution and phase relations in the course of approach learning. Arch ~eur~~l3: 74-90, 1960. 2. BaIlenger, J. C. and R. M. Post. Kindling as a model for alcohol withdrawal syndromes. Br J Psy~hiairy 133: l-14, 1978. 3. Dolce, G. and I-I. Decker. The effects of ethanol on cortical and subcortical electrical activity in cats. RFS Commcm C&m Parhal Pharrnacol3:
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4. Engel, J., Jr. and R. F. Ackermann. Interictal EEG spikes correlated with decreased, rather than increased, epileptogenicity in amygdaloid kindled rats. Brain Res 190: 541-548, 1980. s. Engle, J., Jr., R. F. Ackermann, S. Caldecott-Hazard and D. E. Kuhl. Epileptic activation and antagonistic systems may explain paradoxical features of experimental and human epilepsy: a review and hypothesis. In: Kindling 2, edited by J. A. Wada. New York: Raven Press, 1981, pp. 193-217. 6, Freeman, F. G. Effects of alcohol on kindled seizure thresholds in rats. Pharmacuf B~~~hem Behav 8: 641-644, 1978. 7. Fuji@ Y. and M. Sakuranaga. Spontaneous hy~r~la~zations in pyramidal cells of chronically stimulated rabbit hippocampus. Jpn J.physiol 31: 879-889, 1981. 8. Fuster, J. M. and A. A. Uyeda. Reactivity of limbic neurons of the monkey to appetitive and aversive signals. Elecfroencephalogr Clin Neurophysiol30: 281-293, 1971. 9. Gloor, P. Amygdala. In: Handbook of Physiology, Sect. 1, vol 3, edited by J. Fields et al. Washington, DC: American Physiological Society, 1960, pp. 1395-1420. 10. Grastyan, E., G. Karmas, L. Vereczkey and L. Kellemyi. The hippocampal electrical correlates of the homeostatic regulation of motivation. Electruencephalogr Clin Neurophysiol21: 34-53, 1966. 11. Grupp, L. A. and E. Perlanski. Ethanol induced changes in the spontaneous activity of single units in the hi~~~pus of the awake rat: a dose response study. ~eur#pharrna~~~lf~~y 18: 63-70,
i 979.
12. Hunter, B. E., C. A. Boast, D. W. Walker and S. F. Zornetzer.
Alcohol withdrawal syndrome in rats: Neural and behavioral correlates. fhurma~~~~ B~~~~h~,rn Behrw 1: 719-725, 19’73. 13. Kemble, E. D., T. J. Skoglund and V. A. Davies. Effects of ethanol on threshold and duration of amygdalo;d kindled seizures. Bult Psychonomic Sttc 16: 299-300, 1980. 14. Klemm, W. F., L. R. Dreyfus, E. Forney and M. A. Mayfield. Differential effects of low doses of ethanol ORthe impulse activity in various regions of the limbic system. Psvch,‘phm”Nc”/ogy (Berlin) 50: 131-138, 1976. 15. Klemm, W. F., C. G. Mallari, L. R. Dreyfus, J. C. Fiske, E. Forney and J. A. Mikaska. Ethanol-induced regional and dose-response differences in multiple-unit activity in rabbits. Psyc,hr)pharmac~oi~y (Berlin) 4% 235-244,
1976.
16. Kluver, H. and P. C. Bucy. Preliminary analysis offunctions of the temporal lobe in monkeys. Arch Neuroi P.~.whiaf 42: 9791000, 1939. 17. Lesse, H. Acute effects of ethanol on limbic afterdischarges. Sot Neurosci Abstr 8: 54.8, 1981. 18. Lesse, H. Rhinenceph~ic electrophysiolo~cal activity during emotional behavior in cats. In: ~xpi(~ruf~~~nin the Physioiwy r:l Emotions. edited bv L. J. West and M. Greenblatt. Washington, DC: Lord Baltimore Press, Psyrhiafry RPS Rep 12: 224-238, 1960. 19. Lesse, H. and J. P. Collins. Effects of cocaine on the propagation of limbic seizure activity. Pharmacol Riochem Behal, 11: 689-694, 1979. 20. Lesse, H. and J. P. Collins. Differential effects of cocaine on limbic excitability. Pharmarol B&hem BehalS IJ: 695-703, 1980. 21. Lesse, H. and R. K. Harper. Frequency-related,. bidirectional
limbic responses to cocaine: Comparisons and lidocaine. Brain RPS, in press, 1984.
with amphetamine
ETHANOL
AND LIMBIC AFTERDISCHARGES
22. Lesse, H., R. G. Heath, W. A. Mickle, R. R. Monroe and W. H. Miller. Rhinencephalic activity during thought. J Nerv Ment Dis 122: 433-440, 1955. 23. Marinacci, A. A. and K. 0. Von Hagen. Alcohol and temporal lobe dysfunction: some of its psychomotor equivalents. Behav Neuropsychiatry 30: 2-l 1, 1972. 24. Mucha, R. F. and J. P. Pinel. Increased susceptibility to kindled seizures in rats following a single injection of alcohol. J Stud A/coho/ 40: 258, 1979. 25. Newlin, S. A., J. Mantillas-Trevino and F. E. Bloom. Ethanol causes increases in excitation and inhibition in area CA3 of the dorsal hippocampus. Brain Res 209: 113-128, 1981.
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26. Perrin, R. G., C. H. Hockman, H. Kalant and K. E. Livingston. Acute effects of ethanol on spontaneous and auditory evoked electrical activity in cat brain. EEG Clin Nrurophysiol Xi: 19-31, 1974. 27. S&ins, G. R. and F. E. Bloom. Alcohol-related electrophysiology Pharmacol Biochem Behav 13: 203-211, 1980. 28. Walker. D. W. and S. F. Zometzer. Alcohol withdrawal in mice: electroencephalographic and behavioral correlates. EEG Clin Neurophysiol 36: 233-234, 1974. 29. Winer, B. J. Statistical Prinicples in Experimental Design. New
York: McGraw-Hill.
1962.
Research
Ed/din,
0361-9230/84 $3.00 + .OO
Vol. 13, pp. 519-525, 1984. 0 Ankho International Inc. Printed in the U.S.A.
Regional Effects of Ethanol on Limbic Afterdischarge Activity HENRY Department
LESSE’
AND REBECCA
K. HARPER
of Psychiatry and Biobehavioral Sciences, The Neuropsychiatric and Brain Research University of California, Los Angeles, CA 90024 Received
Institutes
11 June 1984
LESSE, H. AND R. K. HARPER. Regional effects of ethanol on limbic afterdischarge activity. BRAIN RES BULL 13(4) 519-525, 1984.-This study,assessed the effects of acute administration of ethanol on afterdischarge (AD) activity evoked by electrical stimulation of the amygdala, septum and hippocampus. Limbic AD thresholds, duration and propagation were determined in cats following intravenous infusions of saline or ethanol (0.4, 0.8 and 1.6 g/kg). Ethanol administrations significantly increased septal and amygdalar, but not hippocampal AD thresholds. This effect was dose-related and most pronounced at the septum. Reductions in AD duration followed ethanol treatment and demonstrated similar regional differences. In addition, projected discharges were reduced, propagation of AD from stimulation sites to limbic and neocortical projections sites were suppressed and ictal episodes were attenuated following ethanol treatment. Afterdischarge activity was affected even by the 0.4 g/kg dose which produced no observable change in behavior. These findings indicate that ethanol reduces the responsiveness of limbic structures to electrical stimulation-suppressing the initiation, maintenance and propagation of limbic afterdischarges. The amygdala, septum and hippocampus proved differentially
sensitive to ethanol. Ethanol Epilepsy
Limbic system
Brain stimulation
Amygdala
Hippocampus
Septum
Afterdischarges
METHOD
THERE is increasing clinical and experimental evidence suggesting that ethanol has important actions on electrical
Subjects
events in the limbic system. Ethanol has been reported to activate EEG abnormalities in temporal lobe epileptics [23], attenuate and potentiate seizures produced by amygdalar stimulation in kindled rats [24], and to change limbic system EEG, single and multiple unit activity during acute and chronic treatment [3, 11, 12, 25, 26, 27, 281. It has been suggested that progressive limbic system dysfunction produced by repeated episodes of intoxication and withdrawal is important in mediating the neurobehavioral changes characteristic of chronic alcoholism [2]. There are, however, conflicting reports about the sensitivity of various limbic structures to ethanol and it is not known whether ethanol has preferential actions on major components of the limbic system [3, 14, 15, 261. The present study compared the effects of ethanol on afterdischarge activity evoked by electrical stimulation of the amygdala, septum and hippocampus. The present results indicate that these structures are differentially sensitive to ethanol and that ethanol has potent inhibitory effects on the electrical excitability of limbic structures-elevating afterdischarge thresholds, and reducing afterdischarge duration and propagation. These findings provide new information about regional changes in limbic activity during acute ethanol intoxication and provide clues for understanding the mechanism of ethanol’s anticonvulsant effect. A preliminary report of the work has appeared [17].
Subjects were twelve adult cats prepared surgically under pentobarbital anesthesia (35-45 mg/kg) with a jugular cannula and indwelling stainless steel electrodes. Arrays of 0.25 mm bipolar needle and concentric electrodes with 1 mm separation between exposed tips were implanted bilaterally in the dorsal hippocampus, basolateral amygdala and septal area under stereotaxic guidance. Epidural electrodes were placed over the occipital cortex, a stainless steel screw in the frontal sinus served as a reference electrode and interconnected screws provided a ground. An acrylic pedestal, fixed to the skull, supported both a luer lock terminus for the jugular cannula and a miniature electrode connector. At least three weeks were allowed for post-operative recovery before experiments were initiated. Apparatus subjects were placed in an During test sessions, electrically-shielded, sound-attenuating chamber equipped with a one-way mirror, bar-press and milk delivery apparatus. Recordings of subcortical and neocortical activity were obtained with a 14 channel polygraph and stored on magnetic tape for subsequent analysis. Brain stimulation was provided by a square wave stimulator and constant current unit under micro-computer control. A dual beam oscilloscope was em-
‘Requests for reprints should be addressed to H. Lesse, Department of Psychiatry, UCLA Neuropsychiatric Los Angeles, CA 90024.
519
Institute, 760 Westwood Plaza,
ployed to measure current and voltage. corder registered bar-pressing responses.
A cumulative
re-
The cats were trained to bar-press for milk reinforcement. A 24 hour period of food deprivation preceded ail test sessions, which were conducted while the subjects were barpressing. This procedure provided both a stable level of arousal and an activated EEG pattern during the induction of afterdischarges by focal limbic stimulation. Afterdischarge threshold determinations were conducted employing a previously described method [20]. In brief, two pairs of bipolar electrodes in each limbic structure were employed for simultaneous brain stimulation and recording. Focal electrical stimulation (3 Hz biphasic, 0.5 msec, rectangular pulses; 30 set trains) was employed to evoke amygdalar and hippocampal AD. Since AD usually were not induced by 3 Hz septal stimulation at currents below 3 mA, the stimulation frequency was adjusted to 69 Hz for the septum to provide current thresholds comparable to those of the hippocampus and amygdala. Utilizing low frequency stimulation, ele~trophysiological responses were recorded during the intervals between pulses from a site in the stimulated structure adjacent to the stimulating electrode and from projection sites in related structures. Thus, the initiation of AD was detected while brain stimulation was occurring and drug effects could be studied when only brief localized limhic seizure patterns were evoked (see Figs. 3 and 4). Electrophysiological recordings from multiple cortical and limbic sites were obtained between both the bipolar electrodes and individual electrode tips and the common sinus reference. Stimulation was applied for 30 seconds at one minute intervals. Current was increased in steps of 10% or less until self-sustaining AD were evoked. Stimulation was terminated when focal limbic afterdischarges were elicited. The AD threshold was defined as the minimal current required to evoke self-sustaining afterdischarges in the stimulated structure, persisting for at least one second after stimulation was terminated. These determinations continued at 48 hour intervals until stable threshold values were obtained (i.e., there was no more than a 10% variation for three successive test sessions). Additional stimulations were applied to 15 of the 20 limbic test sites to obtain diffuse AD prorogation before drug testing. After stable AD threshold levels were obtained, a series of alternating physiological saline and ethanol tests was initiated. Sessions were separated by at least 48 hours and ethanol tests by 96 hours. Based on pilot experiments, slow intravenous infusions of 0.4, 0.8, and I A g/kg (15% ethanol) were selected to represent a wide range of intoxication levels. An infusion pump was employed to administer the drug at a constant 3 ml/min rate. The AD thresholds were determined 10 minutes after infusion of either ethanol or saline. The drug testing began with initial current intenstiy set 50% below the previously determined threshold for each stimulation site. Low, medium and high doses were tested in a varied order, counterbalanced across subjects. When continued patency of the jugular cannula permitted, drug testing was completed at all three stimulation sites. Most animals, however, were tested at one or two limbic structures. The structure receiving initial focal brain stimulation was varied across subjects. Seven subjects were tested with amygdalar and hippocampal stimulation and six with septal stimulation. Thus. the procedure yielded a total of 60 paired saline and
1 SFPTUM
i
l
AMYCDALA
:)
HIPPOCAMPUS
;r
0.4
0.8
1.6
DOSE FIG. I. Dose-related effects of ethanol on limhic afterdischarne threshold. Drug effects are expressed as the difference (in p.k means_tSEM) followine t&red ethanol and saline tests. The ethnnnt -_.._. effect differed among-&e three limbic regions threshold elevations at the septum and the smallest pus following each of the test doses.
with the largest at the hippocam-
ethanol tests. At the conclusion of experiments. an overdose of pentobarbit~ anesthesia was administered and brains were perfused with saline and 15% formalin. The location of electrode tips was identified microscopically in stained serial sections. Histological studies could not be completed in two subjects who died after ethanol testing was completed but before perfusions could be performed and in three animals subsequently used in other experiments. In these cases, the presence of characteristic sub~o~ical electrographic patterns (hippocampal theta activity, amygdalar 40 Hz rhythmic patterns and septal low amplitude activity) and characteristic propagation patterns evoked by focal stimulation served as guides to the accuracy of the stereotaxic electrode placements. The results of afterdischarge threshold and duration tests were each evaluated initially with a three way analysis of variance with two repeated measures. The three main effects were site, drug, and drug dose; the latter two being repeated measures. The difference scores between paired ethanol and saline tests subsequently were evaluated with a two way analysis of variance. Significant F values were folfowed by post hoc comparisons using paired t-tests with the appropriate error variances derived from the co~esponding ANUVA [301.
ETHANOL
521
AND LIMBIC AFTERDISCHARGES TABLE 2
I
TABLE
AFTERDISCHARGE THRESHOLD (MA) FOLLOWING ETHANOL AND SALINE ADMINISTRATION*
PAIRED
AFTERDISCHARGE DURATION (SEC) FOLLOWING PAIRED ETHANOL AND SALINE ADMINISTRATIONS*
Hippocampus
Amygdala
Septum
Low Dose Saline
69.2 + 16.7 73.3 +- 16.2
45.9 + t1.5 85.3 + 10.3
61.3 t 17.9 67.9 -c 19.5
1.24 ir 0.24 1.16 rt: 0.23
Medium Saline
54.1 rt 12.8 72.6 L 14.9
31.1 t 14.7 96.8 zt 16.9
55.1 It 18.2 67.6 L 19.8
2.66 t 0.43 1.53 i 0.22
1.30 r 0.23 I .18 2 0.22
High Saline
33.8 t 6.0 68.4 + It.3
14.6 t 3.3 70.5 _i_14.3
46.7 r 15.8 68.0 t 17.0
2.18 -+ 0.20 1.54 2 0.12
1.25 * 0.13 1.19 t 0.12
Mean Ethanol Mean Saline
52.4 I 7.7 71.4 z!z 8.0
30.5 i 84.2 it
54.4 t 9.6 67.9 Y?Z 10.3
Amygdala
Septum
Low Dose Saline
0.94 + 0.20 0.87 !I 0.25
1.78‘-t 0.27 1.56 2 0.20
I.19 -r 0.23 1.21 ir 0.22
Medium Saline
I .09 + 0.31 0.87 lr 0.26
2.11 + 0.30 1.54 + 0.22
High Saline
1.30 + 0.37 0.74 i 0.18
Mean Ethanol Mean Saline
I.11 i- 0.18 0.83 -c 0.13
6.7 8.1
Hippocampus
*Means 2 SEM.
“Means ‘_ SEM.
RESULTS
Table 1 summarizes the results of saline and ethanol tests on AD threshold for all subjects; Fig. 1 illustrates regional, dose-related effects of ethanol. An analysis of variance comparing all limbic afterdischarge threshold tests indicated significant values for drug effect, F( 1,17)=21.72,p
In addition to these alterations in threshold, dose-related reductions in AD duration followed ethanol administrations (see Table 2). There was a significant drug effect at all limbic areas, F(1,17)=41 Sl, p
d P
T
0.4
0.11
1.6
DOSE
FIG. 2. Dose-related effects of ethanol on limbic afterdischarge duration. Drug effects are expressed as the difference (in seconds, means?SEM) following paired ethanol and saline tests. Symbols representing the septum, amygdaia and hippocampus are the same as in Fig. 1. There were dose-related reductions in afterdischarge duration at all areas. The largest effects were found at the septal area.
of the three ethanol doses (see Fig. 2). This effect was significantly dose-related at all limbic sites, F(2,34)=5.53, p
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FIG. 3. Effects of ethanol on afterdischarges evoked by left basolateral amygdalar stimulation. Under saline control conditions (upper tracings), afterdischarges initiated in the left amygdala spread to other limbic sites and then to the neocortex. Following the 0.8 g/kg ethanol dose (left lower tracings), AD duration decreased and propagation to the right hippocampus and neocortex was blocked. Following the I .6 g/kg dose, there was a further abbreviation of the ictal episode. Amygdalar afterdischarges were attenuated and propagation of seizure discharges to all other projection sites was suppressed. Amygdalar stimulation induced fully developed tonic-clonic seizures after saline treatment. Following ethanol, behavioral seizures were limited to brief automatisms. To display entire ictal events, recording speed was reduced to 6 mm/set; standard speed for AD duration measurements was 30 mm.
largest change was found at the septal area (mean reduction 53.7k6.8 set) and the smallest at the hippocampus (13.5+4.3), with an intermediate decrease in amygdalar AD duration (19.125.7). A large reduction in septal AD duration (39.4%) followed even the lowest ethanol dose and the drug
effect was substantially greater at the septal area than at the amygdala or hippocampus for each test dose (see Fig. 2). Effects of Ethanol on Afterdischarge
Propagation
Ethanol, in addition, diminished the spread of seizure discharges from the stimulated structure to distant limbic and neocortical projection sites. These effects are illustrated in Figs. 3 and 4. Figure 3 displays a characteristic sequence of electrophysiological changes following saline, 0.8 g/kg and 1.6 g/kg ethanol during amygdalar AD threshold tests. The initiation of afterdischarges is evident during 3 Hz electrical stimulations of the left basolateral amygdala at threshold intensity. In the upper tracings, obtained after saline treatment, stimulation evoked afterdischarges propagating sequentially to the right amygdala, left and right hippocampus and finally to the neocortex. Ictal episodes were progressively abbreviated as the ethanol dose increased. After 0.8 g/kg of ethanol, the duration of afterdischarges recorded from the stimulated structure, the contralateral amygdala and ipsilateral hippocampal projection sites was reduced. Propagation of seizure discharges to both the contralateral hippocampus and to the neocortex was blocked and afterdischarge amplitudes diminished. Following the 1.6 g/kg dose, there was a further attenuation of the amygdalar AD and propagation of discharges to alI other sites was suppressed. During saline control tests, amygdalar stimulation consis-
tently evoked fully developed tonic-clonic motor convulsions. Following ethanol administrations, the behavioral response to stimulation was limited to the automatisms characteristic of focal limbic seizures (after the 0.8 g/kg dose, an arrest reaction, head turning, mouth movements and salivation; following the 1.6 g/kg dose. only brief mouth movemerits) . Figure 4 illustrates a different series of electrophysiological events typically evoked by hippocampal stimulation. Following ethanol, afterdischarge duration decreased, propagation of seizure discharges to the amygdala and neocortex was blocked and the ictal episode was abbreviated. The fully developed, generalized motor convulsion evoked by hippocampal stimulation under saline control conditions was replaced by minimal mouth movements and a transient arrest reaction. Similar drug effects followed septal stimulations. Paroxysmal seizure discharges spread from amygdalar, septal and hippocampal stimulation sites to the neocortex during 46 of the 60’saline control tests. The high dose of ethanol effectively suppressed the propagation of limbic afterdischarges to the neocortex in 13 of 15 (86.7%) paired saline and ethanol tests. This effect occurred in 12 of 15 (80%) medium dose tests and in 7 of 16 (43.8%) low dose tests. Signs of acute alcohol intoxication such as marked sedation, ataxia, and suppression of bar pressing were observed regularly following the 1.6 g/kg dose. Following the 0.8 g/kg dose, bar press rates decreased markedly in most subjects and usually some ataxia was evident. Following the 0.4 g/kg dose, there was no gross behavior change and no observable alteration in the state of arousal. Cats generally continue to bar press for milk at their normal rate. Thus, the limbic elec-
ETHANOL
523
AND LIMBIC AFTERDISCHARGES SALINE
L. HIPPO.
STIY. 0.8
01110
CORTLX
K.
ETHANOL
KIPPO.
L. HIPPO.
FIG. 4. Effects of alternate saline and ethanol administrations on afterdischarges evoked by left dorsal hippocampai stimulation. Following ethanol treatment, ~p~c~pa~ AD duration decreased and the propa~tion of seizure discharges to the amygdala and neocortex was suppressed. The generalized tonic-clonic convuision evoked by hippocampal stimulation under control conditions was replaced by brief mouth movements and a transient arrest reaction.
changes at the lowest ethanol dose occurred in the absence of observable behavioral changes or alterations in task performance.
trophysiological
DISCUSSION
The present findings indicate that ethanol has a powerful protective action against seizure activity initiated in major components of the limbic system. A sequence of electrophysiological changes contributed to this anticonvulsant effect. There were significant dose-related increases in the current required to evoke afterdischarges, as well as doserelated reductions in AD duration at the amygdala, septum and hip~c~pus. In addition, the duration of projected limbic discharges was reduced and the spread of seizure discharges from their site of origin to distant limbic and neocortical projection sites was restricted. Consequently, ictal episodes were abbreviated and their behavioral manifestations were markedly attenuated. Thus, several mutually reinforcing mechanisms appear responsible for ethanol’s acute anticonvuls~t effect. Ethanol suppresses the initiation and maintenance of self-sustaining afterdischarges by reducing the responsiveness of limbic seizure foci to stimulation. In addition, the drug suppresses the propagation of seizure discharges to limbic and neocortical projection sites. The effectiveness of the 0.4 g/kg dose (which had no observable behavioral effect) in altering afterdischarge activity indicates ethanol’s potency in maiming brain functions that are not manifest in overt behavior and also demonstrates the sensitivity of limbic afterdischarge activity as an indicator of ethanol actions. The present findings provide clear evidence that ethanol,
in doses ranging from 0.4-1.6 g/kg, differenti~y affects the sensitivity of closely interconnected limbic structures to electrical stimulation. The largest dose-related threshold increase was found following septal stimulation and a smaller, but significant, elevation followed amygdalar stimulation. Changes in hippocampal thresholds, however, proved insignificant. ~terdischarge duration changes reflected similar differences in the sensitivity of the three limbic structures to ethanol. It is noteworthy that the hippocampus, which has been the most frequent brain target for alcohol research [27], proved substantially less sensitive to ethanol than either the septum or amygdala. It is natural to expect ethanol, a depressant drug, to decrease seizure sensitivity (as in the present experiments). Surprisingly, many similar changes in limbic afterdischarge activity are induced by cocaine, a central nervous system stimulant. In parallel experiments, cocaine also significantly reduces the duration of self-sustaining afterdischarges evoked by electrical stimulation of the arnygdala, septum and hippocampus 1201. In addition, both drugs decrease projected rhythmic discharges, suppress the spread of paroxysmal activity to distant limbic and neocortical projection sites and attenuate ictal episodes originating in limbic structures [ 191. Limbic afterdischarge thresholds are modified by both drugs, although cocaine has a dual, frequency-related effect at the same limbic sites [21]. The limbic structures studied receive impulses from multiple sensory systems and have an important role in evaluating the emotional and motivational aspects of environmental stimuli [8, 9, 161. Characteristic electrographic rhythmic patterns have been identified in the amygdala and hippocampus during exposure to environmental stimuli with motivational relevance [ 1, 10, 18,
221. Thus, similar drug effects attenuating the maintenance and propagation of rhythmic afterdischarges suggest the possibility that both ethanol and cocaine (drugs with contrasting central nervous system sedative and stimulant properties) may change the excitability of limbic structures, altering their propensity for sustained rhythmic responses to motivationally relevant environmental stimuli. An electrophysiological change of this nature may be one of the important factors associated with the altered emotional responses to sensory experiences characteristic of both alcohol and cocaine intoxication. There have been very few studies reporting ethanoiinduced changes in septal bioelectrical activity. The present findings are consistent with a report that ethanol-induced depression of spontaneous multiple unit activity is greater in the septum than in the hippocampus [14]-but at variance with a conflicting observation from the same laboratory that septal units are relatively insensitive to alcohol [IS]. Our results are consistent with reports that ethanol decreases seizure duration and increases thresholds for motor seizure produced by stimulation of the amygdala in kindled rats [ 131; and decreases amygdaiar afterdischarge duration 1241. The present results are at variance with another conflicting report that alcohol fails to alter AD duration in kindled rats and that hippocampal threshold elevations exceed amygdalar elevations [6].
The present results have implications I’Lr rhr j,tudq oi temporal lobe epilepsy in man. Ethanol has been reported II) activate EEG abnormalities in certain temporal lobe epilep tics [23]. The activation of temporal area EMi spikes may reflect differential ethanol-induced changes in limbic clcctri cal excitability similar to those found in the present cxpcrimental seizures. This possibility is consistent with reccm reports that limbic spike discharges are correlated with in. hibitory phenomena and lowered seizure susceptibility 14, 5. 71. Thus, the present finding that limbic structures are differentially sensitive to the inhibitory effects of ethanol suggests further research reevaluating the alcohol activation proccadure as a potential aid in distinguishing hippocampal from amygdalar and septal epileptic foci in seizure disorder-5 nf fimbic origin.
ACKNOWLEDGEMENTS
The authors wish to thank J. P. Collins, Ph.D. who helped in initiating this work, Mary Mettler for technical assistance, William Bergerson for expert electronic assistance and Michael Levine. Ph.D. for statistical consultations. This reserach was supported in part by Grants 03.513from the NIAAA. RR 5756from NIMH and by 25 UCLA Donor’s Fund.
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ETHANOL
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