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Electroencephalographic and behavioural correlates of seizures development in rats in response to hyperbaric exposure
EpilepsyRes., 7 (1990) 65-71
65
Elsevier EPIRES 00343
Electroencephalographic and behavioural correlates of seizure development in rats in response to hyperbaric exposure
I. G a r c i a - C a b r e r a a, N . W . M i l g r a m b and O . - G . B e r g e a aDepartmentof Physiology, Universityof Bergen, Bergen (Norway), and bLife SciencesDivision, ScarboroughCampus, Universityof Toronto, Toronto (Canada) (Received 15 January 1990; accepted 9 April 1990)
Key words: EEG; High pressure nervous syndrome (HPNS); High pressure epilepsy; Rats
Electroencephalographic (EEG) activity was continuously monitored from the hippocampus, amygdala, reticular formation and frontal cortex in freely moving Sprague-Dawley rats exposed to 91 atmospheres absolute pressure (ATA) using compression rates of 1 or 3 ATA/min. Videotape recordings were made for subsequent behavioural analysis. Tremor, myoclonic jerks and tonic extensions of the tail were observed in all animals but did not appear to correlate with epileptiform activity. Convulsions occurred between 66.5 and 91 ATA in all subjects compressed at 3 ATA/min, but in only 1 rat (at 91 ATA) in the 1 ATA/min group. Tonic-clonic motor seizures developed explosively and involved the entire body. EEG records showed continuous spiking at all sites during the generalized ceavulsive state. There was no evidence of differential susceptibility of the various brain regions examined to the epileptogenic effects of high pressure. The behavioural and EEG data indicate that hyperbarically induced seizures differ from the classical limbic type.
INTRODUCTION Exposure to high ambient pressure induces a number of neurological symptoms in animals and humans; these are known as the high pressure nervous syndrome (HPNS) e'5'13. This syndrome is characterized by tremor, myoclonic jerks and alterations in the electroencephalogram (EEG). In animals, further increases in pressure produce tonic or tonic-clonic generalized convulsions, which may be followed by coma 5'13. While the cortical EEG during HPNS in humans and animals has been extensively studied, relativeCorrespondence to: I. Garcia-Cabrera, M.D., Dept. of Physiology, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway.
ly few studies deal with subcortical electrical activity6'7A4'15'2°.In these studies, electrodes were implanted in various cortical and subcortical structures in restrained rats. The origin of the HPNS seizures is still unclear, although there is evidence that some components of HPNS seizures can arise in the spinal cord 9'16. In animal models of limbic seizure, epileptic activity originates in limbic structures and its development correlates closely with characteristic patterns of behaviour which precede the occurrence of generalized convulsions, 1A7'19. For example, in rats to which kainic acid has been administered, the earliest electrical seizures are restricted to the ventral hippocampus and are accompanied by spells of immobility and staring. Subsequently, repetitive stereotyped head and facial motor activ-
66 ities and brief myoclonic jerks can be observed. At this time the electrical seizures become synchronized in the entorhinal cortex, amygdala and hippocampus. Finally, convulsions occur accompanied by deep and cortical paroxysmal activity 17. Animals exposed to high pressure show similar patterns of behaviour 5"11'13,which raises the possibility that these animals develop epilepsy of the limbic type. To date, the correlations between the preconvulsive behavioural signs of HPNS and the subcorticai EEG have not been thoroughly studied in freely moving animals. Likewise, no EEG registration in the temporal region has been made in animals exposed to high pressure. The aim of this study was to test whether there is a pattern of EEG discharges in several brain regions preceding the appearance of motor seizures in rats exposed to high ambient pressure; the involvement of these structures in the genesis of the motor seizures was consequently examined. Electrodes were implanted in the hippocampus and amygdala, frontal cortex and the midbrain reticular formation. Behaviour and EEG activity of the freely moving rats were recorded continuously during hyperbasic exposure. METHODS
Animals and surgery Male Sprague-Dawley rats weighing 300-400 g were housed individually in a climate-controlled room with a 12 h light-dark cycle and had access to food and water ad libitum. Rats (n -- 15) were implanted under chloral hydrate/pentobarbital anaesthesia (17 mg/kg and 3.9 mg/kg i.p., respectively) with electrodes in the frontal cortex, hippocampus, amygdala and reticular formation. The electrodes were made from twisted lengths of 120 l~m diameter Nichrome wire with a tip separation of 0.5 ram. The coordinates for the frontal cortex elect~ ~de were 1.7 mm anterior, 3.0 mm lateral and 0.5 mm below the dura mater. The hippocampal coordinates were 2.8 mm posterior, 3.0 mm and 3.0 mm belo~v dura. The amygdala coordinates were 3.3 mm posterior, 4.6 mm lateral and 7.5 mm below dura while the reticular formation coordinates were 7.8 mm posterior, 1.8 mm lateral and 5.7 mm below dura. All anterior/posterior and
lateral coordinates are relative to bregma.
Hyperbaric exposure and EEG registration The experiments were performed in a 24.5 litre steel chamber equipped with monitors for temperature and pressure, a fan, CO 2 scrubber and heating system, as described previously 12. A window at one end of the chamber permitted videotaping of the animals' behaviour. On the basis of previously obtained results 3'21 the chamber temperature was adjusted to 34 + 1 °C in order to offset helium-induced hypothermia. The partial pressure of oxygen was maintained at between 0.2 and 0.4 ATA. Testing followed a recovery period of at least 1 week after surgery. In brief, the animals were placed in a cage inside the pressure chamber where they were free to move within an area of 21 x 22 cm. The recording leads were suspended from a movable ring which traversed a bar running along the ceiling of the cage. The leads were connected to the recording equipment via a socket in the chamber. Differential EEG recordings were made by means of a 6-channel Grass model 7D polygraph. Baseline EEG was monitored in the chamber for 10 rain before compression began. The chamber was initially purged for 4 min with a mixture of 80% helium and 20% oxygen (heliox) to remove nitrogen. Nine animals were pressurized with helium at a rate of 3 ATA/min and a further six at 1 ATA/min. Pressurization continued until either a final pressure of 91 ATA was reached or motor convulsions occurred. All animals remained at the stable pressure for 30 min. Throughout the experiments, the partial pressure of 0 2 was maintained between 0.2 and 0.4 ATA. After completion of the experiment, the rats were anaesthetized with N20 and sacrificed by rapid decompression. Brains were removed and subsequently cut and stained with cresyI Aolet for verification of the recording sites. Behaviourai observations Videotape recordings were made continuously and were subsequently analysed by an observer unaware of the experimental conditions using a computer program that allowed scoring of the cumulative time and number of occurrences of the
67 between thresholds for the behavioural categories (F (4, 52) = 15.61, P < 0.00001) and a significant interaction between the 2 factors (F (4, 52) = 8.15, P < 0.0005). All subjects showed both fine and coarse tremor, but at different pressure thresholds m with fine tremor occurring at lower levels of pressure (Table I). Tremor thresholds varied with compression rate, being significantly lower in the animals exposed to the 3 ATA/min rate. Tonic extension of the tail consistently followed tremor in the 3 ATA/ min group, while th,~ reverse was the case in all but 1 animal of the 1 ATA/min group. There was no significant difference between the fast and slow compression groups with regard to the pressure threshold for the tonic extension of the tail. When pressure was further increased, tonic extension of the tail and tremor in the whole body occurred simultaneously. The slow compression group had a significantly higher threshold for this measure. Myoclonic jerks appeared between 14.3 and 61.6 ATA in the 3 ATA/min group and between 20.4 and 63 ATA in the 1 ATA/min group; no statistical differences were found between the two groups with regard to pressure thresholds. Other motor signs during compression included 'wetdog' shaking, twitching of the vibrissae, blinking, chewing, yawning and head nodding. Tonic-clor.lc motor seizures developed explosively with rapid recruitment of the entire body, in most cases starting with a strong myoclonic jerk. All the 3 ATA/min animals reached the convulsion stage between 66.5 and 91 ATA (Table II). The animal that had the longest seizure (80 sec) died immediately afterwards. Only 1 animal in the 1 ATA/min group convulsed (at 91 ATA). There was
TABLE I
Threshold pressures (ATA) for fine tremor (P~), coarse tremor ('act), myocloaic jerks (Pmj), tonic extension of the tail (Ptn) and tonic extensioi~ of the tail accompanied by coarse tremor (Ptet+ a)
Croup
Pp
P.
P.j
P,~,
P,~ ÷c,
39.0 2.7
3 ATA/min (N = 9)
Mean S.E.M.
32.3 3.1
36.6 6.4
44.8 3.4
52.5 2.0
l ATA/min (N = 6)
Mean S.E.M.
59.0* 70.5* 43.0 4.5 4.9 5.9
53.3 5.5
76.2* 4.1
*P < 0.001, significantly different from corresponding 3 ATA/ min group (Student's t test).
following: (1) fine tremor - - tremor in face and/or forelimbs; (2) coarse t r e m o r - tremor in the whole body; (3) myoclonic jerks; (4) tonic extension of the tail; (5) tonic extension of the tail accompanied by coarse tremor. Statistics
Behavioural activity data were analyzed by 2way analysis of variance for repeated measures as detailed in the results. Comparisons of group means were performed by Student's t test. RESULTS Behaviour
Compression rate affected the pressure thresholds at which rats showed preconvulsive HPNS signs. The threshold values of the scored behavioural categories were analysed by means of a 2 compression rates x 5 pressure thresholds ANOVA. This demonstrated significant differences between compression rates (F (1, 13) = 15.39, P < 0.005),
TABLE II
Seizure characteristics of rats pressurized at 3 ATA/min Data given as medians with ranges in parentheses.
Pressure threshold
First seizure EEG (sec)
(ATA ) 79.2(66.5-91)
38.0(15-80) (N=9)
Motor convul. sion (sec)
Frequency
27.0(7-41)
1-7
(Hz)
Interictal period
(sec) 10.5(4-44)
Second seizure EEG (sec)
Frequency
(Hz) 37.5(31-110)
(N-6)
1-5
68
A
B
C
23 ATA
48 ATA
72 ATA
Hipp.
I4°° v
Ft. Cx.
I, oo.
Amyg. - , ...,.
,.%-. -,~...,
,,~
,,'.,,, ,,~. '~'~,..'.~
R,F,
200nV
Fig. 1. EEGs from hippocampus (Hipp.), frontal cortex (Fr. Cx.), amygdala (Amyg.) and reticular formation (R.F.). Time marker 1 sec. The animalwas activein periodA, had fine tremor in B and coarse tremor in C. The arrowindicateswhen a myoclonicjerk occurred. electrophysiological evidence of progressive seizure development. The only abnormal electrical activity was the occasional occurrence of spiking in one or more channels during the preconvulsive stage in 4 animals• During the generalized convulsive state, E E G records showed continuous spiking (1-7 Hz) in all channels (Table II). In every case, the spiking frequency increased to a peak (5-7 Hz) in the middle of the episode and then decreased. Fig. 2 shows a typical E E G recording of
considerable variation in the durations of the convulsions (Table II). The motor seizure was followed by a period of inactivity, hyperventilation and recurrent strong myoclonic jerks.
Electroencephalography A comparison of the E E G records with the corresponding video recordings did not reveal any correlation between the EEG and the preconvulsive behavioural measures (Fig. 1). Nor was there
R1 86 ATA "
.
Fr. Cx.
Amyg.
' , ,,'
', .
.
.
,,
, ,
RE
'
' !!'
~
'''' ~
.
,,, V'
*'
' ~'~'"
r'
'
p
P
.
~ ' ' '
,
,
~ !1 , t
, ~
"
' e
:,; ' ~ ' ( ' i
I; .
T..O0~ v
1"
" I"T'~oodv
/ T2OO~v 1
Fig. 2. Electricalcorrelates of high pressure-induced motor seizures. Time marker 1 sec. Abbreviations as in Fig. 1. This record was typical of those cases where the EEG dischargeappeared simultaneouslyat all locations.
69 R9 69 ATA
Fr cx
i! ,e
,'t, 'iill;:, 'i
.
.
.
.
Fig. 3. Typical sample of an EEG hyperbaric motor seizure. Abbreviations as in Fig. 1. Note that in this case, the spiking activity started first in the reticular formation.
seizure activity during a convulsion episode. In 4 animals, spike activity started simultaneously in all 4 channels. In 3 animals, spike activity in the reticular formation preceded seizure activity in the other sites by a short interval (Fig. 3), but there was no systematic order of appearance of activity in these 3 structures. In 2 animals, seizure activity started simultaneously in 3 sites; in one animal, the amygdala and, in the other, the frontal cortex were recruited somewhat later. After a variable interictal period, a second EEG seizure (1-5 Hz) occurred in 6 subjects. These seizures were accompanied by severe myoclonic jerks but not convulsions. DISCUSSION These experiments dealt with the relationship between the development of HPNS and EEG activity in several brain regions. Our experimental model provided a suitable tool to correlate behaviour with EEG changes in freely moving animals exposed to high pressure. No significant correlation was found between behavioural preconvulsive symptoms and epileptiform activity in the EEG. This observation in freely moving animals confirms and extends previous findings in restrained Wistar rats 7. However, the possibility of
more subtle EEG changes, such as alterations in frequency, cannot be discarded. Motor seizure development in this preparation contrasts markedly with development seen in most models of epilepsy. We observed no EEG seizure activity during the preconvulsion phase. In contrast, in the kindling and KA-induced seizure models, epileptic activity originate in the temporal region or the hippocampus and there is a typical growth in the duration of the afterdischarges which precede the motor seizures ~'17'~9.Therefore, in spite of some preconvulsive behavioural similarities to limbic seizure development (such as sniffing, masticatory movements, head nodding, forelimb tremor and jerks), these hyperbarically induced seizures do not seem to originate in limbic structures. However, the fact that discharges in hippocampus and amygdala were consistently observed during motor convulsions suggests that these structures contribute to the development of these seizures. A hypothesis consistent with such findings is that there are several sites which act synergistically in initiating seizures in response to high pressure. Simultaneous EEG seizure activity occurred consistently in the hippoc~,mpus, amygdala, frontal cortex and reticular formation during motor convulsions. Other investigators have also re-
70 ported EEG paroxysmal activity in cortical and subcortical areas, including the hippocampus and reticular formation 6'7a4'~5. The higher convulsion thresholds found in these studies (mean values between 98.8 and 113 ATA), may be due to slower rates of compression or to strain differences. However, in another study, hyperbaric motor seizures in Sprague-Dawley rats were only occasionally accompanied by hippocampal seizures 8 and it was suggested that the hippocampus is not necessarily involved in the HPNS seizures. Further experiments using a wider range of compression schedules are necessary to clarify this apparent contradiction. In agreement with previous findings "~'~3,seizure development was found to vary with compression rate in this study; convulsions occurred in all animals exposed to a compression rate of 3 ATA/min but in only 1 animal in the 1 ATA/min group. The pressure threshold for certain of the HPNS symptoms, such as fine and coarse tremor, and tonic extension of the tail accompanied by tremor, was significantly lower in the animals which were pressurized at the higher rate. However, there was no significant effect of the rate of compression on the pressure thresholds for myoclonic jerks and tonic extension of the tail. This suggests that ,~remor and convulsions on the one hand, and r.ayoclonic jerks and tonic extension of the tail on the other, have different underlying mechanisms. The effects of compression rate on tonic extension of the tail and on myoclonic jerks have not been systematically examined in the rat. Once again, further experiments using a wider range of compression rates are necessary in order to ascertain .if these measures are independent of rate. It has been suggested that HPNS seizures may result from selective changes in GABA transmission. Drugs that increase GABA transmission elevate the threshold pressures for HPNS tremor and convulsions 4. Moreover, these effects correlated with the ability of the drugs to raise the threshold concentrations at which bicuculline (~ selective GABA blocker) caused convulsions, One of these drugs, sodium valproate, has recently been reported to reduce the severity of HPNS signs in freely moving baboons ~8. Finally, there is direct evidence that helium at high pressure depresses
the GABA-mediated inhibition of CA1 pyramidal cells 22. Other investigators, however, have reported that drugs which enhance GABA transmission are more effective in suppressing EEG changes induced by hyperbaric exposure, than in ameliorating the behavioural signs of HPNS 2°. They suggested that excitatory transmitters may be more important than GABA in contributing to the behavioural symptoms of HPNS. In our study, the motor seizures induced by high pressure share some features with those produced by bicuculline. Systemic administration of this drug produces a grand real tonic-clonic convulsion accompanied by a fully generalized paroxysmal activity in deep and cortical electrodes without any obvious developmental phase I. This bchavioura! similarity provides additional support for the role of GABA in HPNS seizures. The suggestion that HPNS seizure development reflects a decrease in GABAergic transmission appears to be inconsistent with the finding that hippocampal excitatory synaptic transmission decreases under hyperbaric conditions a'l°. One possibility is that all neural transmission is depressed, but to different degrees, the effects on inhibition being greater. A similar proposal of a general reduction in the efficiency of both excitatory and inhibitory transmission has previously been made 22. In conclusion, the experiments reported here indicate no differential susceptibility of the rat limbic system, frontal cortex or midbrain to the epileptogenic effects of high pressure. The similarities of the seizures reported here to those induced by administration of bicuculline are compatible with the hypothesis that changes in GABA transmission are involved in the genesis of HPNS seizures. ACKNOWLEDGEMENTS This work was supported by the Norwegian Research Council for Science and the Humanities, Hyperbaric Medical Research Program. We thank Reidun Ursin and Bolek Srebro for the use of technical facilities and for helpful discussions. Expert technical assistance was provided by Torhild Fjordheim Sunde. We thank Hugh M. Allen for editorial assistance.
71 REFERENCES 1 Ben-Ari, Y., Tremblay, E., Riche, D., Ghilini, G. and Na-
quet, R., Electrographic, clinical and pathological alterations following systemic administration of kainic acid, bicuculline or pentetrazole: metabolic mapping using the deoxyglucose method with special reference to pathology of epilepsy, Neuroscience, 6 (1981) 1361-1391. 2 Bennett, P.B., The high pressure nervous syndrome in man. In: P.B. Bennett and D.H. Elliott (Eds.), The Physiology and Medicine of Diving, Bailli~re, Tindall, London, 1982, pp. 262-296. 3 Berge,O.-G. and Garcia-Cabrera, I., Combined effects of ethanol and high pressure on body temperature in rats, Fur. Undersea Biomed. Soc. Proc., 16 (1988) 274-281. 4 Bichard, A.R. and Little, H.J., Drugs that increase yaminobutyric acid transmission protect against the high pressare neurological syndrome, Br. J. Pha~macol., 76 (1982) 447-452. 5 Brauer, R.W., The high pressure nervous syndrome: animals. In: P.B. Bennett and D.H. Elliott (Eds.), The Physiology and Medicine of Diving, Bailli~re, Tindall, London, 1975, pp. 231-247. 6 Cromer, J.A., Bennett, P.B., Hunter, W.L. and Zinn, D., Effect of helium/nitrogen/oxygen mixtures on HPNS convulsion threshold in euthermic rats, Undersea Biomed. Res., 6 (1979) 367-377. 7 Cromer, J.A., Hunter, W.L. and Bennett, P.B., Alteration of high pressure nervous syndrome in rats by alteration of colonic temperature, Undersea Biomed. Res., 3 (1976) 139-150. 8 Fagni, L., Soumireu-Mourat, B., Carlier, E. and Hugon, M., A study of spontaneous and evoked activity in the rat hippocampus under helium-oxygen high pressure, Electroenceph. Clin. Neurophysiol., 60 (1985) 267-275. 9 Fagni, L., Weiss, M., Pellet, J. and Hugon, M., The possible mechanisms of the high pressure-induced motor disturbances in the cat, Electroenceph. Clin. Neurophysiol., 53 (1982) 590-601. 10 Fagni, L., Zinebi, F. and Hugon, M., Evoked potential changes in rat hippocampal slices under helium pressure, Exp. Brain Res., 65 (1987) 513-519.
11 Garcia-Cabrera, I. and Berge, O.-G., Effects of hyperbaric exposure on spontaneous motor activity in rats, Neurosci. Left,, 26, Suppl. (1986) $63. 12 Garcia-Cabrera, I. and Berge, O.-G., Pressure reversal of the depressant effect of ethanol on spontaneous behavior in rats, Pharmacol. Biochem. Behav., 29 (1988) 133-141. 13 Halsey, M.J., Effects of high pressure on the central nervous system, Physiol. Rev., 62 (1982) 1341-1377. 14 Kaufmann, P.G., Bennett, P.B. and Farmer, J.C., Cerebellar and cerebral electroencephalogram during the high pressure nervous syndrome (HPNS) in rats, Undersea Bio. reed. Res., 4 (1977) 391-402. 15 Kaufmann, P.G., Bennett, P.B. and Farmer, J.C., Effect of cerebellar ablation on the high pressure nervous syndrome in rats, Undersea Biomed. Res., 5 (1978) 63-70. 16 Kaufmann, P.G., Finley, C.C., Bennett, P.B. and Farmer, J.C., Spinal cord seizures elicited by high pressures of helium, Electroenceph. Clin. Neurophysiol., 47 (1979) 31-40. 17 Lothman, E.W. and Collins, R.C., Kainic acid induced iimbic seizures: metabolic, behavioral, electroencephalographic and neuropathological correlates, Brain Res., 218 (1981) 299-318. 18 Pearce, P.C., Clarke, D., Dor6, C.J., Halsey, M.J., Luff, N.P. and Maclean, C.J., Sodium valproate interactions with the HPNS: EEG and behavioral observations, Undersea Biomed. Res., 16 (1989) 99-113. 19 Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroenceph. Clin. Neurophysiol., 32 (1972) 281-294. 20 Rostain, J.C., Wardley-Smith, B., Forni, C. and Halsey, M.J., Gamma-aminobutyric acid and the high pressure neurological syndrome, Neuropharmacology, 25 (1986) 545-554. 21 Stetzner, L.C. and De Boer, B. iThermai balance in the rat during exposure to helium-oxygen from 1 to 41 atmospheres, Aerospace Med., 43 (1972) 306-309. 22 Zinebi, F., Fagni, L. and Hugon, M., The influence of helium pressure on reduction induced in ficd potentials by various amino acids on the GABA-mediated inhibition in the CA1 region of hippocampal slices in the rat, Neuropharmacology, 27 (1~3) 57-65.
65
Elsevier EPIRES 00343
Electroencephalographic and behavioural correlates of seizure development in rats in response to hyperbaric exposure
I. G a r c i a - C a b r e r a a, N . W . M i l g r a m b and O . - G . B e r g e a aDepartmentof Physiology, Universityof Bergen, Bergen (Norway), and bLife SciencesDivision, ScarboroughCampus, Universityof Toronto, Toronto (Canada) (Received 15 January 1990; accepted 9 April 1990)
Key words: EEG; High pressure nervous syndrome (HPNS); High pressure epilepsy; Rats
Electroencephalographic (EEG) activity was continuously monitored from the hippocampus, amygdala, reticular formation and frontal cortex in freely moving Sprague-Dawley rats exposed to 91 atmospheres absolute pressure (ATA) using compression rates of 1 or 3 ATA/min. Videotape recordings were made for subsequent behavioural analysis. Tremor, myoclonic jerks and tonic extensions of the tail were observed in all animals but did not appear to correlate with epileptiform activity. Convulsions occurred between 66.5 and 91 ATA in all subjects compressed at 3 ATA/min, but in only 1 rat (at 91 ATA) in the 1 ATA/min group. Tonic-clonic motor seizures developed explosively and involved the entire body. EEG records showed continuous spiking at all sites during the generalized ceavulsive state. There was no evidence of differential susceptibility of the various brain regions examined to the epileptogenic effects of high pressure. The behavioural and EEG data indicate that hyperbarically induced seizures differ from the classical limbic type.
INTRODUCTION Exposure to high ambient pressure induces a number of neurological symptoms in animals and humans; these are known as the high pressure nervous syndrome (HPNS) e'5'13. This syndrome is characterized by tremor, myoclonic jerks and alterations in the electroencephalogram (EEG). In animals, further increases in pressure produce tonic or tonic-clonic generalized convulsions, which may be followed by coma 5'13. While the cortical EEG during HPNS in humans and animals has been extensively studied, relativeCorrespondence to: I. Garcia-Cabrera, M.D., Dept. of Physiology, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway.
ly few studies deal with subcortical electrical activity6'7A4'15'2°.In these studies, electrodes were implanted in various cortical and subcortical structures in restrained rats. The origin of the HPNS seizures is still unclear, although there is evidence that some components of HPNS seizures can arise in the spinal cord 9'16. In animal models of limbic seizure, epileptic activity originates in limbic structures and its development correlates closely with characteristic patterns of behaviour which precede the occurrence of generalized convulsions, 1A7'19. For example, in rats to which kainic acid has been administered, the earliest electrical seizures are restricted to the ventral hippocampus and are accompanied by spells of immobility and staring. Subsequently, repetitive stereotyped head and facial motor activ-
66 ities and brief myoclonic jerks can be observed. At this time the electrical seizures become synchronized in the entorhinal cortex, amygdala and hippocampus. Finally, convulsions occur accompanied by deep and cortical paroxysmal activity 17. Animals exposed to high pressure show similar patterns of behaviour 5"11'13,which raises the possibility that these animals develop epilepsy of the limbic type. To date, the correlations between the preconvulsive behavioural signs of HPNS and the subcorticai EEG have not been thoroughly studied in freely moving animals. Likewise, no EEG registration in the temporal region has been made in animals exposed to high pressure. The aim of this study was to test whether there is a pattern of EEG discharges in several brain regions preceding the appearance of motor seizures in rats exposed to high ambient pressure; the involvement of these structures in the genesis of the motor seizures was consequently examined. Electrodes were implanted in the hippocampus and amygdala, frontal cortex and the midbrain reticular formation. Behaviour and EEG activity of the freely moving rats were recorded continuously during hyperbasic exposure. METHODS
Animals and surgery Male Sprague-Dawley rats weighing 300-400 g were housed individually in a climate-controlled room with a 12 h light-dark cycle and had access to food and water ad libitum. Rats (n -- 15) were implanted under chloral hydrate/pentobarbital anaesthesia (17 mg/kg and 3.9 mg/kg i.p., respectively) with electrodes in the frontal cortex, hippocampus, amygdala and reticular formation. The electrodes were made from twisted lengths of 120 l~m diameter Nichrome wire with a tip separation of 0.5 ram. The coordinates for the frontal cortex elect~ ~de were 1.7 mm anterior, 3.0 mm lateral and 0.5 mm below the dura mater. The hippocampal coordinates were 2.8 mm posterior, 3.0 mm and 3.0 mm belo~v dura. The amygdala coordinates were 3.3 mm posterior, 4.6 mm lateral and 7.5 mm below dura while the reticular formation coordinates were 7.8 mm posterior, 1.8 mm lateral and 5.7 mm below dura. All anterior/posterior and
lateral coordinates are relative to bregma.
Hyperbaric exposure and EEG registration The experiments were performed in a 24.5 litre steel chamber equipped with monitors for temperature and pressure, a fan, CO 2 scrubber and heating system, as described previously 12. A window at one end of the chamber permitted videotaping of the animals' behaviour. On the basis of previously obtained results 3'21 the chamber temperature was adjusted to 34 + 1 °C in order to offset helium-induced hypothermia. The partial pressure of oxygen was maintained at between 0.2 and 0.4 ATA. Testing followed a recovery period of at least 1 week after surgery. In brief, the animals were placed in a cage inside the pressure chamber where they were free to move within an area of 21 x 22 cm. The recording leads were suspended from a movable ring which traversed a bar running along the ceiling of the cage. The leads were connected to the recording equipment via a socket in the chamber. Differential EEG recordings were made by means of a 6-channel Grass model 7D polygraph. Baseline EEG was monitored in the chamber for 10 rain before compression began. The chamber was initially purged for 4 min with a mixture of 80% helium and 20% oxygen (heliox) to remove nitrogen. Nine animals were pressurized with helium at a rate of 3 ATA/min and a further six at 1 ATA/min. Pressurization continued until either a final pressure of 91 ATA was reached or motor convulsions occurred. All animals remained at the stable pressure for 30 min. Throughout the experiments, the partial pressure of 0 2 was maintained between 0.2 and 0.4 ATA. After completion of the experiment, the rats were anaesthetized with N20 and sacrificed by rapid decompression. Brains were removed and subsequently cut and stained with cresyI Aolet for verification of the recording sites. Behaviourai observations Videotape recordings were made continuously and were subsequently analysed by an observer unaware of the experimental conditions using a computer program that allowed scoring of the cumulative time and number of occurrences of the
67 between thresholds for the behavioural categories (F (4, 52) = 15.61, P < 0.00001) and a significant interaction between the 2 factors (F (4, 52) = 8.15, P < 0.0005). All subjects showed both fine and coarse tremor, but at different pressure thresholds m with fine tremor occurring at lower levels of pressure (Table I). Tremor thresholds varied with compression rate, being significantly lower in the animals exposed to the 3 ATA/min rate. Tonic extension of the tail consistently followed tremor in the 3 ATA/ min group, while th,~ reverse was the case in all but 1 animal of the 1 ATA/min group. There was no significant difference between the fast and slow compression groups with regard to the pressure threshold for the tonic extension of the tail. When pressure was further increased, tonic extension of the tail and tremor in the whole body occurred simultaneously. The slow compression group had a significantly higher threshold for this measure. Myoclonic jerks appeared between 14.3 and 61.6 ATA in the 3 ATA/min group and between 20.4 and 63 ATA in the 1 ATA/min group; no statistical differences were found between the two groups with regard to pressure thresholds. Other motor signs during compression included 'wetdog' shaking, twitching of the vibrissae, blinking, chewing, yawning and head nodding. Tonic-clor.lc motor seizures developed explosively with rapid recruitment of the entire body, in most cases starting with a strong myoclonic jerk. All the 3 ATA/min animals reached the convulsion stage between 66.5 and 91 ATA (Table II). The animal that had the longest seizure (80 sec) died immediately afterwards. Only 1 animal in the 1 ATA/min group convulsed (at 91 ATA). There was
TABLE I
Threshold pressures (ATA) for fine tremor (P~), coarse tremor ('act), myocloaic jerks (Pmj), tonic extension of the tail (Ptn) and tonic extensioi~ of the tail accompanied by coarse tremor (Ptet+ a)
Croup
Pp
P.
P.j
P,~,
P,~ ÷c,
39.0 2.7
3 ATA/min (N = 9)
Mean S.E.M.
32.3 3.1
36.6 6.4
44.8 3.4
52.5 2.0
l ATA/min (N = 6)
Mean S.E.M.
59.0* 70.5* 43.0 4.5 4.9 5.9
53.3 5.5
76.2* 4.1
*P < 0.001, significantly different from corresponding 3 ATA/ min group (Student's t test).
following: (1) fine tremor - - tremor in face and/or forelimbs; (2) coarse t r e m o r - tremor in the whole body; (3) myoclonic jerks; (4) tonic extension of the tail; (5) tonic extension of the tail accompanied by coarse tremor. Statistics
Behavioural activity data were analyzed by 2way analysis of variance for repeated measures as detailed in the results. Comparisons of group means were performed by Student's t test. RESULTS Behaviour
Compression rate affected the pressure thresholds at which rats showed preconvulsive HPNS signs. The threshold values of the scored behavioural categories were analysed by means of a 2 compression rates x 5 pressure thresholds ANOVA. This demonstrated significant differences between compression rates (F (1, 13) = 15.39, P < 0.005),
TABLE II
Seizure characteristics of rats pressurized at 3 ATA/min Data given as medians with ranges in parentheses.
Pressure threshold
First seizure EEG (sec)
(ATA ) 79.2(66.5-91)
38.0(15-80) (N=9)
Motor convul. sion (sec)
Frequency
27.0(7-41)
1-7
(Hz)
Interictal period
(sec) 10.5(4-44)
Second seizure EEG (sec)
Frequency
(Hz) 37.5(31-110)
(N-6)
1-5
68
A
B
C
23 ATA
48 ATA
72 ATA
Hipp.
I4°° v
Ft. Cx.
I, oo.
Amyg. - , ...,.
,.%-. -,~...,
,,~
,,'.,,, ,,~. '~'~,..'.~
R,F,
200nV
Fig. 1. EEGs from hippocampus (Hipp.), frontal cortex (Fr. Cx.), amygdala (Amyg.) and reticular formation (R.F.). Time marker 1 sec. The animalwas activein periodA, had fine tremor in B and coarse tremor in C. The arrowindicateswhen a myoclonicjerk occurred. electrophysiological evidence of progressive seizure development. The only abnormal electrical activity was the occasional occurrence of spiking in one or more channels during the preconvulsive stage in 4 animals• During the generalized convulsive state, E E G records showed continuous spiking (1-7 Hz) in all channels (Table II). In every case, the spiking frequency increased to a peak (5-7 Hz) in the middle of the episode and then decreased. Fig. 2 shows a typical E E G recording of
considerable variation in the durations of the convulsions (Table II). The motor seizure was followed by a period of inactivity, hyperventilation and recurrent strong myoclonic jerks.
Electroencephalography A comparison of the E E G records with the corresponding video recordings did not reveal any correlation between the EEG and the preconvulsive behavioural measures (Fig. 1). Nor was there
R1 86 ATA "
.
Fr. Cx.
Amyg.
' , ,,'
', .
.
.
,,
, ,
RE
'
' !!'
~
'''' ~
.
,,, V'
*'
' ~'~'"
r'
'
p
P
.
~ ' ' '
,
,
~ !1 , t
, ~
"
' e
:,; ' ~ ' ( ' i
I; .
T..O0~ v
1"
" I"T'~oodv
/ T2OO~v 1
Fig. 2. Electricalcorrelates of high pressure-induced motor seizures. Time marker 1 sec. Abbreviations as in Fig. 1. This record was typical of those cases where the EEG dischargeappeared simultaneouslyat all locations.
69 R9 69 ATA
Fr cx
i! ,e
,'t, 'iill;:, 'i
.
.
.
.
Fig. 3. Typical sample of an EEG hyperbaric motor seizure. Abbreviations as in Fig. 1. Note that in this case, the spiking activity started first in the reticular formation.
seizure activity during a convulsion episode. In 4 animals, spike activity started simultaneously in all 4 channels. In 3 animals, spike activity in the reticular formation preceded seizure activity in the other sites by a short interval (Fig. 3), but there was no systematic order of appearance of activity in these 3 structures. In 2 animals, seizure activity started simultaneously in 3 sites; in one animal, the amygdala and, in the other, the frontal cortex were recruited somewhat later. After a variable interictal period, a second EEG seizure (1-5 Hz) occurred in 6 subjects. These seizures were accompanied by severe myoclonic jerks but not convulsions. DISCUSSION These experiments dealt with the relationship between the development of HPNS and EEG activity in several brain regions. Our experimental model provided a suitable tool to correlate behaviour with EEG changes in freely moving animals exposed to high pressure. No significant correlation was found between behavioural preconvulsive symptoms and epileptiform activity in the EEG. This observation in freely moving animals confirms and extends previous findings in restrained Wistar rats 7. However, the possibility of
more subtle EEG changes, such as alterations in frequency, cannot be discarded. Motor seizure development in this preparation contrasts markedly with development seen in most models of epilepsy. We observed no EEG seizure activity during the preconvulsion phase. In contrast, in the kindling and KA-induced seizure models, epileptic activity originate in the temporal region or the hippocampus and there is a typical growth in the duration of the afterdischarges which precede the motor seizures ~'17'~9.Therefore, in spite of some preconvulsive behavioural similarities to limbic seizure development (such as sniffing, masticatory movements, head nodding, forelimb tremor and jerks), these hyperbarically induced seizures do not seem to originate in limbic structures. However, the fact that discharges in hippocampus and amygdala were consistently observed during motor convulsions suggests that these structures contribute to the development of these seizures. A hypothesis consistent with such findings is that there are several sites which act synergistically in initiating seizures in response to high pressure. Simultaneous EEG seizure activity occurred consistently in the hippoc~,mpus, amygdala, frontal cortex and reticular formation during motor convulsions. Other investigators have also re-
70 ported EEG paroxysmal activity in cortical and subcortical areas, including the hippocampus and reticular formation 6'7a4'~5. The higher convulsion thresholds found in these studies (mean values between 98.8 and 113 ATA), may be due to slower rates of compression or to strain differences. However, in another study, hyperbaric motor seizures in Sprague-Dawley rats were only occasionally accompanied by hippocampal seizures 8 and it was suggested that the hippocampus is not necessarily involved in the HPNS seizures. Further experiments using a wider range of compression schedules are necessary to clarify this apparent contradiction. In agreement with previous findings "~'~3,seizure development was found to vary with compression rate in this study; convulsions occurred in all animals exposed to a compression rate of 3 ATA/min but in only 1 animal in the 1 ATA/min group. The pressure threshold for certain of the HPNS symptoms, such as fine and coarse tremor, and tonic extension of the tail accompanied by tremor, was significantly lower in the animals which were pressurized at the higher rate. However, there was no significant effect of the rate of compression on the pressure thresholds for myoclonic jerks and tonic extension of the tail. This suggests that ,~remor and convulsions on the one hand, and r.ayoclonic jerks and tonic extension of the tail on the other, have different underlying mechanisms. The effects of compression rate on tonic extension of the tail and on myoclonic jerks have not been systematically examined in the rat. Once again, further experiments using a wider range of compression rates are necessary in order to ascertain .if these measures are independent of rate. It has been suggested that HPNS seizures may result from selective changes in GABA transmission. Drugs that increase GABA transmission elevate the threshold pressures for HPNS tremor and convulsions 4. Moreover, these effects correlated with the ability of the drugs to raise the threshold concentrations at which bicuculline (~ selective GABA blocker) caused convulsions, One of these drugs, sodium valproate, has recently been reported to reduce the severity of HPNS signs in freely moving baboons ~8. Finally, there is direct evidence that helium at high pressure depresses
the GABA-mediated inhibition of CA1 pyramidal cells 22. Other investigators, however, have reported that drugs which enhance GABA transmission are more effective in suppressing EEG changes induced by hyperbaric exposure, than in ameliorating the behavioural signs of HPNS 2°. They suggested that excitatory transmitters may be more important than GABA in contributing to the behavioural symptoms of HPNS. In our study, the motor seizures induced by high pressure share some features with those produced by bicuculline. Systemic administration of this drug produces a grand real tonic-clonic convulsion accompanied by a fully generalized paroxysmal activity in deep and cortical electrodes without any obvious developmental phase I. This bchavioura! similarity provides additional support for the role of GABA in HPNS seizures. The suggestion that HPNS seizure development reflects a decrease in GABAergic transmission appears to be inconsistent with the finding that hippocampal excitatory synaptic transmission decreases under hyperbaric conditions a'l°. One possibility is that all neural transmission is depressed, but to different degrees, the effects on inhibition being greater. A similar proposal of a general reduction in the efficiency of both excitatory and inhibitory transmission has previously been made 22. In conclusion, the experiments reported here indicate no differential susceptibility of the rat limbic system, frontal cortex or midbrain to the epileptogenic effects of high pressure. The similarities of the seizures reported here to those induced by administration of bicuculline are compatible with the hypothesis that changes in GABA transmission are involved in the genesis of HPNS seizures. ACKNOWLEDGEMENTS This work was supported by the Norwegian Research Council for Science and the Humanities, Hyperbaric Medical Research Program. We thank Reidun Ursin and Bolek Srebro for the use of technical facilities and for helpful discussions. Expert technical assistance was provided by Torhild Fjordheim Sunde. We thank Hugh M. Allen for editorial assistance.
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