Brain Research, 93 (1975) 535-542 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
535
Glucocorticoids and the hippocampal theta rhythm in loosely restrained, unanesthetized rabbits
SANDRA M. MARTIN, GARY P. MOBERG ANDJOHN M. HOROWITZ Departments of Animal Science and Animal Physiology, University of California, Davis, Calif. 95616 (U.S.A.)
(Accepted May 5th, 1975)
Studies relating glucocorticoids to the hippocampus can build on the extensive electrophysiological data obtained since hippocampal theta waves (4-7 Hz in the rabbit) were first shown to be elicited by auditory, olfactory, visual and tactile stimulP 1. As this rhythm was evoked by several sensory modalities, it was postulated to reflect a non-specific 'arousal' of the animal. More recently, in reviewing studies relating theta activity to behavior in unrestained animals, Bennett 4 concluded that these waves appear when an animal directs attention towards or explores a potentially significant environmental stimulus. While there is currently debate on the precise behavioral significance of the theta rhythm in areas as diverse as memory and learning, there is general agreement that it (a) can be elicited by novel stimuli, (b) is correlated with pyramidal cells in the hippocampus, and (c) can be driven by signals from the reticular formation to the hippocampus via the septal areal0,13. More recently glucocorticoids have been related to the hippocampus. Thus, behavioral studies showed that, in response to olfactory stimulation, adrenalectomized rats alter their exploratory activity, but when treated with corticosterone intact animal behavior is restored 23. These experiments are consistent with the hypothesis that corticosterone acts to modify hippocampal activity as Bennett 4 related exploratory activity to the hippocampus. More direct evidence that the hippocampus serves as a target for glucocorticoids has come from several studies. The rat hippocampus selectively binds corticosterone and this binding involves the pyramidal cell layer 9, 15-17. Pfaff et al. 2° have shown a decrease in telemetered unit activity of pyramidal cells following corticosterone injections, while Dafny et al. 5 reported that although there was a relative lack of responsiveness of the hippocampus with respect to other brain structures following cortisol injection, there was both an increase and a decrease in firing rate. In investigations of the hippocampal role in the function of the pituitaryadrenal axis, hippocampal effects on A C T H release appeared to depend on prior stress to the animaP 4. That is, with stress hippocampal stimulation inhibits A C T H release, while without stress A C T H release is facilitated by hippocampal stimulation.
536 The concept of a modulated hippocampal responsiveness dependent on the steroid environment is supported by data indicating a limited capacity uptake system for cortico3terone operating~within normal daily concentrations of endogenous corticosteroids 16. Further evidence that the hippocampus has an effect on A T C H release is given by work where cortisol implanted in the hippocampus abolished the diurnal alterations in corticosterone levels 21. All these studies serve to further relate the interaction between the hippocampus and the pituitary-adrenal axis, and indicate that the relationship is a complex one which can be modified by a variety of factors including stress. The changes in hippocampal neural activity over time following administration of glucocorticoids may thus reflect stress to the animal at the time of injection in addition to a direct action on neural networks within the central nervous system. The intent of this study was to determine the effect, if any, of exogenous glucocorticoids on the theta rhythm in unanesthetized, loosely restrained rabbits over a period of several hours. Special care was taken to avoid stressing the rabbits during administration of the glucocorticoid so that data could be interpreted in terms of exogenous corticoid action on neural networks. However, circulating corticoid levels were not measured during the course of this study. Six rnale New Zealand white rabbits (3-4 kg) were anesthetized with sodium pentobarbital (35 mg/kg), given atropine sulfate (0.1 rag, i.m.) and implanted with bipolar stainless steel electrodes by first placing the electrodes 3 m m above the fornix and hippocampus using stereotaxic coordinates is. Final placement was determined by concurrently stimulating and lowering the electrodes until characteristic inverted waveforms were recorded (Fig. 1)13. Data in this study were obtained from these bipolar electrodes straddling the pyramidal cell layer and from screw electrodes over the neocortex.
A
I .~.~. ~ - ~ . ~ . ~ _ . , , _ . ~
I
0.2 MVOLT
I
I 40 MSEC
B ONSET OF THETA RHYTHM
1500
1PVOLT I
I
I SEC.
Fig. 1. Waveforms from electrodes straddling the hippocampal pyramidal cell layer. A: characteristic waveforms obtained from anesthetized rabbits when electrodes were positioned across the pyramidal layer. Trace 1 from electrode lateral to the layer and trace 2 from medially placed electrode. Break in traces marks shock artifact for fornix stimulation. B: bipolar EEG recording from a loosely restrained, unanesthetized rabbit with electrodes across the pyramidal cell layer (as in A).
537
HIPPOCAMPAL
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Fig. 2. Sketch showing placement of electrodes and cannula in rabbit (not to scale). Fornix electrodes (for stimulation), hippocampal electrodes (for recording), and cortical screw electrodes (not shown) were cemented to a Cannon connector (MD1-9SL1) secured with dental cement to skull. The cannula was placed in ear vein 1 week after electrode placement and 1 day prior to first trials.
The animals were maintained on 80 g of commercial rabbit pellets per day with water ad libitum. The lights were on from 0700 to 1600 h. After one week of postoperative recovery, and one day prior to initial testing, a cannula (PE 20) lz was inserted into the marginal ear vein after local subcutaneous injection of 4.0 mg of xylocaine. The chronically prepared animal, with electrodes and a cannula for infusion of drugs, is sketched in Fig. 2. On the days EEG waveforms were recorded, the rabbit was placed in a chamber designed to limit background light and sound; electrode leads and the cannula were threaded through openings at the top of the chamber. This arrangement permitted recording of E E G activity and injection of drugs without disturbing the animal. The protocol consisted of four stages: (1) a 30 min equilibration period, followed by (2) the 30 rain preinjection control period, (3) a 1 rain period over which either a drug or the carrier was infused, and (4) a 2 h postinjection period. During the last three stages EEG activity was recorded 13 and at random times auditory stimuli were given. The stimulus was either a sharp rap on the side of the chamber or a loud 1-3 sec buzz from a speaker within the chamber. During stage 3 either hydrocortisone-21sodium succinate (25 rag, Sigma) or the carrier (6.35 mg sodium succinate) was infused. The drug or just the carrier was suspended in 2.0 ml normal saline. Hippocampal EEG activity was analyzed in terms of spontaneous and evoked theta activity. To be classified as a response to the auditory stimulus, the theta activity must have been evoked within one second after stimulus presentation. The number of responses elicited divided by the number of stimuli given (the per cent evoked response) was tabulated. The duration of the evoked response was also measured. The theta rhythm, a high amplitude waveform (Fig. 1B), was easily identified and often occurred spontaneously even when no auditory stimulus was given. Each 2 h test period was divided into 30 min time intervals, and the intervals scanned to determine the per cent time the theta rhythm was present. The 30 min preinjection period was considered a control period establishing a base level of theta activity for a given
538 TABLE I RESPONSE TO ACOUSTIC STIMULI
Time (min)
Durationof response (sec)
% Response
Na succinate*
Hydrocortisone*
Na succinate*
Hydrocortisone
3.20 ± 0.54**
2.57 ± 0.38
95.2 ± 3.34
87.8 ± 4.35
3.22 ± 2.76 ~ 2.74 i 3.78 ~ 3.14 ±
2.50 ~ 2.40 ± 2.82 ± 3.41 ± 2.88 ±
93.0 ± 90.2 ~ 82.2 ± 93.4 ± 91.2 ±
94.6 ± 94.3 ± 87.6 ± 81.8 ~ 88.0 ±
Preinjection
0-30 Postinjection
0-30 30-60 60-90 90-120 0-120
0.83 0.43 0.48 1.23 0.69
0.72 0.66 0.61 1.36 0.83
4.46 5.77 11.76 2.16 3.07
3.56 3.69 0.98 7.10 5.56
* N=6. ** ±S.E.
trial. The above criteria were adopted because of the variable and labile onset and duration of theta activity in unanesthetized rabbits. To determine significance of data the Student t-test for paired samples was used. The theta response to randomly timed acoustic stimuli (Table I) was analyzed by comparing intervals in the postinjection period with the preinjection control period. Neither the per cent response nor the duration of theta activity showed a statistically significant difference (P > 0.1) between sodium succinate (the carrier) and hydrocortisone-21-sodium succinate. This data indicates that these parameters of the theta rhythm evoked following stimuli were not altered by glucocorticoid administration. On the other hand, for the spontaneous theta rhythm, specific parameters appeared to be altered by infusion of glucocorticoids. The theta occurring in consecutive E E G tracings was tabulated and expressed as per cent theta (Table II). In the hydrocortisone treated animals the average (n = 6) peak value occurring 60-90 min postinjection was significantly (P < 0.05) increased over the preinjection activity (Fig. 3). As shown in Table II, this increase is apparent in all individual animal responses with the exception of trial No. 2, where two peaks are evident (at 30-60 and 90-120 min). Significant differences were detected during no other hydrocortisone postinjection period. Sodium succinate treated animals showed no significant differences in pre- or postinjection response. Also, the duration of the bursts of theta occurring within the spontaneous E E G are not significantly different in the hydrocortisone and sodium succinate treated animals. The effect of glucocorticoids on hippocampal theta activity in the chronic animal does not appear to have been previously studied. In acute preparations Feldman and Davidson a drove hippocampal theta activity by electrical stimulation of the reticular activating system and observed no change on hydrocortisone injection. In the present experiments on unanesthetized rabbits, the theta response evoked by
539
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Fig. 3. Bar graph showing the per cent of spontaneous activity over 30 rain intervals in the loosely restrained, unanesthetized rabbit. A: shows the mean activity when only the carrier was infused. B: shows peak response during 60-90 min interval following infusion of hydrocortisone. Bars are averages for each interval, 4- S.E. auditory stimuli also remained unchanged, both in duration and per cent response. In contrast, an increase in spontaneous theta activity was seen 60-90 min after hydrocortisone infusion using techniques to avoid stress at the time of injection. The transient increase in spontaneous hippocampal theta activity is correlated with the temporal events of glucocorticoid effects on the central nervous system. McEwen et al. 16 have shown a time-dependent, limited uptake system for corticosterone in the hippocampus, while Smelik 2~ observed the inhibitory effect of corticoids on A C T H release occurring an hour or more after steroid administration. Support for the concept that the altered theta activity seen here may reflect a direct hippocampal event is the demonstration of intracellular binding of corticosterone in pyramidal cells9,15-17 and the modification of evoked potentials 40 min after corticoid administration 2°. These correlations of binding and changes in E E G activity do not, however, rule out changes elsewhere in the brain in response to glucocorticoids which could in turn alter hippocampal electrical activity by modifying signals over neural pathways linking the hippocampus. The effects of bound corticoids on hippocampal neurons is not known. However, evidence does exist that the corticoids may affect either the levels or activities of enzymes. De Vellis e t a [ . 6,7 found that glycerol phosphate dehydrogenase levels in brain and cultured glial cells are regulated by cortisol. In addition, in the developing chick retina glutamine synthetase activity can be prematurely induced by cortiso119. Recently Azmitia and his coworkers l-a have shown increased tryptophan hydroxylase activity in the midbrain and preoptic-septal area of rats 1 h after cortisol
541 injection. This latter finding is also consistent with the data presented above o n the time course of s p o n t a n e o u s theta activity in response to corticoid a d m i n i s t r a t i o n . The consequences of altered enzyme activity m a y be related to c h a n g i n g neural t r a n s m i t t e r levels which could in t u r n alter synaptic thresholds for firing or firing patterns in n e u r o n a l circuits. Regardless o f the m e c h a n i s m , in this study it appears that hydrocortisone a d m i n i s t r a t i o n is correlated with a change in s p o n t a n e o u s theta activity occurring at times consistent with changing b i n d i n g a n d altered enzyme activity. The a u t h o r s w o u l d like to t h a n k Mr. Mike K a t o v i c h a n d Mr. Piers Nye for their technical assistance in developing experimental techniques. This study was supported in part by University o f California G r a n t s D-529 a n d D-926.
1 AZMITIA,E. C., JR., ALGERI,S., AND COSTA,E., Turnover rate of in vivo conversion of tryptophan into serotonin in brain areas of adrenalectomized rats, Science, 169 (1970) 201-203. 2 AZMITIA,E. C., JR., AND MeEWEN, B. S., Corticosterone regulation of tryptophan hydroxylase in midbrain of the rat, Science, 166 (1969) 1274~1276. 3 AZMITIA,E. C., JR., AND MCEWEN, B. S., Adrenalcortical influence on rat brain tryptophan hydroxylase activity, Brain Research, 78 (1974) 291-302. 4 BENNETT,T. L., Hippocampal theta activity and behavior - - a review, Commun. Behav. Biol., 6 (1971) 37-48. 5 DAFNY,N., PHILIPS, M. I., TAYLOR,A. N., AND GILMAN,S., Dose effects of cortisol on single unit activity in hypothalamus, reticular formation and hippocampus of freely behaving rats correlated with plasma steroid levels, Brain Research, 59 (1973) 257-272. 6 DE VELLIS,J., AND INGLISH,D., Hormonal control of glycerol phosphate dehydrogenase in the rat brain, J. Neurochem., 15 (1968) 1061-1070. 7 DE VELLIS,J., IN~LISH,D., COLE, R., AND MOLSON,J., Effects of hormones on the differentiation of cloned lines of neurons and glial cells. In D. FORD(Ed.), Influence of Hormones on the Nervous System, Karger, Basel, 1971, pp. 25-39. 8 FELDMAN,S., AND DAVIDSON,J. M., Effect of hydrocortisone on electrical activity, arousal thresholds and evoked potentials in the brain of chronically implanted rabbits, J. neurol. Sci., 3 (1966) 462-472. 9 GERLACH,J. L., AND McEWEN, B. S., Rat brain binds adrenal steroid hormone: radioautography of hippocampus with corticosterone, Science, 175 (1972) 1133-1136. 10 GREEN,J. D., The hippocampus. In J. FIELD,H. W., MAGOUNANDV. E. HALL(Eds.), Handbook of Physiology, Section 1: Neurophysiology, Vol. II, Amer. Physiol. Soc., Washington, D.C., 1960, pp. 1373-1390. 11 GREEN,J. D., AND ARDUINI, m. m., Hippocampal electrical activity in arousal, J. Neurophysiol., 17 (1954) 532-557. 12 HEATLEY, N. G., AND WEEKS, J. R., Fashioning polyethylene tubing for use in physiological experiments, J. appl. Physiol., 19 (1964) 542-545. 13 HOROWITZ,J. M., SALEH, M. A., AND KAREM, R. D., Correlation of the hippocampal theta rhythm with changes in cutaneous temperature, Amer. J. Physiol., 277 (1974) 635-642. 14 KAWAKAMr,M., SETO, K., TERASAWA,E., YOSHIDA, K., MIYAMOTO, Y., SEKIGUCHI, M., AND HATTORI,Y., Influence of electrical stimulation and lesion in limbic structures upon biosynthesis of adrenocorticoid in the rabbit, Neuroendocrinology, 3 (1968) 337-348. 15 MCEWEN, B. S., AND WALLACrI,G., Corticosterone binding to hippocampus: nucleuar and cytosol binding in vitro, Brain Research, 57 (1973) 373-386. 16 MCEWEN, B. S., WALLACH, G., AND MAGNUS, C., Corticosterone binding to hippocampus: immediate and delayed influence of the absence of adrenal secretion, Brain Research, 70 (1974) 321-334.
542 17 McEWEN, B. S., WEISS, J. M., AND SCHWARTZ, L. S., Uptake of corticosterone by rat brain and its concentration by certain limbic structures, Brain Research, 16 (1969) 227-241. 18 MONNIER, M., AND GANGLOEF,H., Atlas for Stereotaxic Brain Research, Elsevier, Amsterdam, 1961, 76 pp. 19 MOSCONA,A. A., AND P1DDINGTON, R., Stimulation by hydrocortisone of premature changes in the development pattern of glutamine synthetase in embryonic retina, Biochim. biophys. Acta (Amst.), 121 (1966)409-411. 20 PFAEF, O. W., SILVA, M. T. A., AND WEISS, J. M., Telemetered recording of hormonal effects on hippocampal neurons, Science, 172 (1971) 394-395. 21 SLUSHER,M. A., Effects of cortisol implants in the brain stem and ventral hippocampus on diurnal corticosteroid levels, Exp. Brain Res., 1 0966) 184-194. 22 SMELIK,P. G., Relation between blood level of corticoids and their inhibiting effect on the hippocampal stress response, Proc. Soc. exp. Biol. (N. Y.), 113 (1963) 616-619. 23 WEZSS,J. M., MCEWEN, B. S., SILVA, M. T., AND KALKUT, M., Pituitary-adrenal alterations and fear responding, Amer. J. Physiol., 218 (1970) 864-868.
535
Glucocorticoids and the hippocampal theta rhythm in loosely restrained, unanesthetized rabbits
SANDRA M. MARTIN, GARY P. MOBERG ANDJOHN M. HOROWITZ Departments of Animal Science and Animal Physiology, University of California, Davis, Calif. 95616 (U.S.A.)
(Accepted May 5th, 1975)
Studies relating glucocorticoids to the hippocampus can build on the extensive electrophysiological data obtained since hippocampal theta waves (4-7 Hz in the rabbit) were first shown to be elicited by auditory, olfactory, visual and tactile stimulP 1. As this rhythm was evoked by several sensory modalities, it was postulated to reflect a non-specific 'arousal' of the animal. More recently, in reviewing studies relating theta activity to behavior in unrestained animals, Bennett 4 concluded that these waves appear when an animal directs attention towards or explores a potentially significant environmental stimulus. While there is currently debate on the precise behavioral significance of the theta rhythm in areas as diverse as memory and learning, there is general agreement that it (a) can be elicited by novel stimuli, (b) is correlated with pyramidal cells in the hippocampus, and (c) can be driven by signals from the reticular formation to the hippocampus via the septal areal0,13. More recently glucocorticoids have been related to the hippocampus. Thus, behavioral studies showed that, in response to olfactory stimulation, adrenalectomized rats alter their exploratory activity, but when treated with corticosterone intact animal behavior is restored 23. These experiments are consistent with the hypothesis that corticosterone acts to modify hippocampal activity as Bennett 4 related exploratory activity to the hippocampus. More direct evidence that the hippocampus serves as a target for glucocorticoids has come from several studies. The rat hippocampus selectively binds corticosterone and this binding involves the pyramidal cell layer 9, 15-17. Pfaff et al. 2° have shown a decrease in telemetered unit activity of pyramidal cells following corticosterone injections, while Dafny et al. 5 reported that although there was a relative lack of responsiveness of the hippocampus with respect to other brain structures following cortisol injection, there was both an increase and a decrease in firing rate. In investigations of the hippocampal role in the function of the pituitaryadrenal axis, hippocampal effects on A C T H release appeared to depend on prior stress to the animaP 4. That is, with stress hippocampal stimulation inhibits A C T H release, while without stress A C T H release is facilitated by hippocampal stimulation.
536 The concept of a modulated hippocampal responsiveness dependent on the steroid environment is supported by data indicating a limited capacity uptake system for cortico3terone operating~within normal daily concentrations of endogenous corticosteroids 16. Further evidence that the hippocampus has an effect on A T C H release is given by work where cortisol implanted in the hippocampus abolished the diurnal alterations in corticosterone levels 21. All these studies serve to further relate the interaction between the hippocampus and the pituitary-adrenal axis, and indicate that the relationship is a complex one which can be modified by a variety of factors including stress. The changes in hippocampal neural activity over time following administration of glucocorticoids may thus reflect stress to the animal at the time of injection in addition to a direct action on neural networks within the central nervous system. The intent of this study was to determine the effect, if any, of exogenous glucocorticoids on the theta rhythm in unanesthetized, loosely restrained rabbits over a period of several hours. Special care was taken to avoid stressing the rabbits during administration of the glucocorticoid so that data could be interpreted in terms of exogenous corticoid action on neural networks. However, circulating corticoid levels were not measured during the course of this study. Six rnale New Zealand white rabbits (3-4 kg) were anesthetized with sodium pentobarbital (35 mg/kg), given atropine sulfate (0.1 rag, i.m.) and implanted with bipolar stainless steel electrodes by first placing the electrodes 3 m m above the fornix and hippocampus using stereotaxic coordinates is. Final placement was determined by concurrently stimulating and lowering the electrodes until characteristic inverted waveforms were recorded (Fig. 1)13. Data in this study were obtained from these bipolar electrodes straddling the pyramidal cell layer and from screw electrodes over the neocortex.
A
I .~.~. ~ - ~ . ~ . ~ _ . , , _ . ~
I
0.2 MVOLT
I
I 40 MSEC
B ONSET OF THETA RHYTHM
1500
1PVOLT I
I
I SEC.
Fig. 1. Waveforms from electrodes straddling the hippocampal pyramidal cell layer. A: characteristic waveforms obtained from anesthetized rabbits when electrodes were positioned across the pyramidal layer. Trace 1 from electrode lateral to the layer and trace 2 from medially placed electrode. Break in traces marks shock artifact for fornix stimulation. B: bipolar EEG recording from a loosely restrained, unanesthetized rabbit with electrodes across the pyramidal cell layer (as in A).
537
HIPPOCAMPAL
_
ELECTRODES FORNIX
--
I ,
" ,~---,-I I
SUTURES
~
J
I~
E'ECTROOES" i i A L f .ORN,X _Jd
CANNULA M^RG . . . . .
~AR'V;I'-N
k /// I
/ L ~
~
• //
RAPHENUC.EUS RET,¢OLAR .O...T,O. ~
M
E
D
I
A
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FOREBRAIN
Fig. 2. Sketch showing placement of electrodes and cannula in rabbit (not to scale). Fornix electrodes (for stimulation), hippocampal electrodes (for recording), and cortical screw electrodes (not shown) were cemented to a Cannon connector (MD1-9SL1) secured with dental cement to skull. The cannula was placed in ear vein 1 week after electrode placement and 1 day prior to first trials.
The animals were maintained on 80 g of commercial rabbit pellets per day with water ad libitum. The lights were on from 0700 to 1600 h. After one week of postoperative recovery, and one day prior to initial testing, a cannula (PE 20) lz was inserted into the marginal ear vein after local subcutaneous injection of 4.0 mg of xylocaine. The chronically prepared animal, with electrodes and a cannula for infusion of drugs, is sketched in Fig. 2. On the days EEG waveforms were recorded, the rabbit was placed in a chamber designed to limit background light and sound; electrode leads and the cannula were threaded through openings at the top of the chamber. This arrangement permitted recording of E E G activity and injection of drugs without disturbing the animal. The protocol consisted of four stages: (1) a 30 min equilibration period, followed by (2) the 30 rain preinjection control period, (3) a 1 rain period over which either a drug or the carrier was infused, and (4) a 2 h postinjection period. During the last three stages EEG activity was recorded 13 and at random times auditory stimuli were given. The stimulus was either a sharp rap on the side of the chamber or a loud 1-3 sec buzz from a speaker within the chamber. During stage 3 either hydrocortisone-21sodium succinate (25 rag, Sigma) or the carrier (6.35 mg sodium succinate) was infused. The drug or just the carrier was suspended in 2.0 ml normal saline. Hippocampal EEG activity was analyzed in terms of spontaneous and evoked theta activity. To be classified as a response to the auditory stimulus, the theta activity must have been evoked within one second after stimulus presentation. The number of responses elicited divided by the number of stimuli given (the per cent evoked response) was tabulated. The duration of the evoked response was also measured. The theta rhythm, a high amplitude waveform (Fig. 1B), was easily identified and often occurred spontaneously even when no auditory stimulus was given. Each 2 h test period was divided into 30 min time intervals, and the intervals scanned to determine the per cent time the theta rhythm was present. The 30 min preinjection period was considered a control period establishing a base level of theta activity for a given
538 TABLE I RESPONSE TO ACOUSTIC STIMULI
Time (min)
Durationof response (sec)
% Response
Na succinate*
Hydrocortisone*
Na succinate*
Hydrocortisone
3.20 ± 0.54**
2.57 ± 0.38
95.2 ± 3.34
87.8 ± 4.35
3.22 ± 2.76 ~ 2.74 i 3.78 ~ 3.14 ±
2.50 ~ 2.40 ± 2.82 ± 3.41 ± 2.88 ±
93.0 ± 90.2 ~ 82.2 ± 93.4 ± 91.2 ±
94.6 ± 94.3 ± 87.6 ± 81.8 ~ 88.0 ±
Preinjection
0-30 Postinjection
0-30 30-60 60-90 90-120 0-120
0.83 0.43 0.48 1.23 0.69
0.72 0.66 0.61 1.36 0.83
4.46 5.77 11.76 2.16 3.07
3.56 3.69 0.98 7.10 5.56
* N=6. ** ±S.E.
trial. The above criteria were adopted because of the variable and labile onset and duration of theta activity in unanesthetized rabbits. To determine significance of data the Student t-test for paired samples was used. The theta response to randomly timed acoustic stimuli (Table I) was analyzed by comparing intervals in the postinjection period with the preinjection control period. Neither the per cent response nor the duration of theta activity showed a statistically significant difference (P > 0.1) between sodium succinate (the carrier) and hydrocortisone-21-sodium succinate. This data indicates that these parameters of the theta rhythm evoked following stimuli were not altered by glucocorticoid administration. On the other hand, for the spontaneous theta rhythm, specific parameters appeared to be altered by infusion of glucocorticoids. The theta occurring in consecutive E E G tracings was tabulated and expressed as per cent theta (Table II). In the hydrocortisone treated animals the average (n = 6) peak value occurring 60-90 min postinjection was significantly (P < 0.05) increased over the preinjection activity (Fig. 3). As shown in Table II, this increase is apparent in all individual animal responses with the exception of trial No. 2, where two peaks are evident (at 30-60 and 90-120 min). Significant differences were detected during no other hydrocortisone postinjection period. Sodium succinate treated animals showed no significant differences in pre- or postinjection response. Also, the duration of the bursts of theta occurring within the spontaneous E E G are not significantly different in the hydrocortisone and sodium succinate treated animals. The effect of glucocorticoids on hippocampal theta activity in the chronic animal does not appear to have been previously studied. In acute preparations Feldman and Davidson a drove hippocampal theta activity by electrical stimulation of the reticular activating system and observed no change on hydrocortisone injection. In the present experiments on unanesthetized rabbits, the theta response evoked by
539
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540 80
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0.
60 a.
40
ul
0
0-30 0-30 I ~ pR F'.-I I
30-60
60-90 POST
90-1;)0 I
Fig. 3. Bar graph showing the per cent of spontaneous activity over 30 rain intervals in the loosely restrained, unanesthetized rabbit. A: shows the mean activity when only the carrier was infused. B: shows peak response during 60-90 min interval following infusion of hydrocortisone. Bars are averages for each interval, 4- S.E. auditory stimuli also remained unchanged, both in duration and per cent response. In contrast, an increase in spontaneous theta activity was seen 60-90 min after hydrocortisone infusion using techniques to avoid stress at the time of injection. The transient increase in spontaneous hippocampal theta activity is correlated with the temporal events of glucocorticoid effects on the central nervous system. McEwen et al. 16 have shown a time-dependent, limited uptake system for corticosterone in the hippocampus, while Smelik 2~ observed the inhibitory effect of corticoids on A C T H release occurring an hour or more after steroid administration. Support for the concept that the altered theta activity seen here may reflect a direct hippocampal event is the demonstration of intracellular binding of corticosterone in pyramidal cells9,15-17 and the modification of evoked potentials 40 min after corticoid administration 2°. These correlations of binding and changes in E E G activity do not, however, rule out changes elsewhere in the brain in response to glucocorticoids which could in turn alter hippocampal electrical activity by modifying signals over neural pathways linking the hippocampus. The effects of bound corticoids on hippocampal neurons is not known. However, evidence does exist that the corticoids may affect either the levels or activities of enzymes. De Vellis e t a [ . 6,7 found that glycerol phosphate dehydrogenase levels in brain and cultured glial cells are regulated by cortisol. In addition, in the developing chick retina glutamine synthetase activity can be prematurely induced by cortiso119. Recently Azmitia and his coworkers l-a have shown increased tryptophan hydroxylase activity in the midbrain and preoptic-septal area of rats 1 h after cortisol
541 injection. This latter finding is also consistent with the data presented above o n the time course of s p o n t a n e o u s theta activity in response to corticoid a d m i n i s t r a t i o n . The consequences of altered enzyme activity m a y be related to c h a n g i n g neural t r a n s m i t t e r levels which could in t u r n alter synaptic thresholds for firing or firing patterns in n e u r o n a l circuits. Regardless o f the m e c h a n i s m , in this study it appears that hydrocortisone a d m i n i s t r a t i o n is correlated with a change in s p o n t a n e o u s theta activity occurring at times consistent with changing b i n d i n g a n d altered enzyme activity. The a u t h o r s w o u l d like to t h a n k Mr. Mike K a t o v i c h a n d Mr. Piers Nye for their technical assistance in developing experimental techniques. This study was supported in part by University o f California G r a n t s D-529 a n d D-926.
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