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The effect of progesterone on the spontaneous interictal spike evoked by the application of penicillin to the cat's cerebral cortex
Journal of the Neurological Sciences, 1978, 36:119-133 ~ Elsevier/North-HollandBiomedicalPress
119
THE EFFECT OF PROGESTERONE ON THE SPONTANEOUS INTERICTAL SPIKE EVOKED BY THE APPLICATION OF PENICILLIN TO THE CAT'S CEREBRAL CORTEX
S. LANDGREN, T. B.~,CKSTRt~Mand G. KALISTRATOV* Department of Physiology, University of Umed, S-901 87 Umed (Sweden)
(Received 26 September, 1977)
SUMMARY The effect of intravenous infusion of progesterone and Mebumal (ACO, Sweden; pentobarbital, INN) on the frequency and amplitude of the spontaneous interictal spikes from a penicillin focus in the cerebral cortex was investigated in ovariectomized cats anaesthetized with chloralose. The plasma concentration of progesterone was determined. It was found that the generation of penicillin spikes was depressed by progesterone in plasma concentrations as low as 40 ng/ml. Equimolar doses of Mebumal were less effective.
INTRODUCTION Several investigations demonstrate that steroid hormones can influence the activity of the cerebral cortex. Progesterone in large doses (>60 mg/kg body weight) has anaesthetic effects and some related steroids (21-hydroxy-5 fl-pregnane-3,20-dione; 5fl-pregnane-3,20-dione) are effective in lower doses (6-12 mg/kg) (cf., Selye 1942; P'an and Laubach 1964; Gyermek, Genther and Fleming 1967). These effects may be mediated via the reticular activating system, as pointed out by several authors who observed EEG synchronization, spindle discharges, increase in the arousal threshold and sleep behaviour after injection of progesterone (cf., Heuser, Ling and Kliiver 1967; Kawakami and Sawyer 1967; Endr6czi 1969; Lincoln 1969). Progesterone effects on single unit responses in the cerebral cortex, thalamus and hypothalamus have been reported (Komisaruk, McDonald, Whitmoyer and Sawyer 1967; Teresawa and Sawyer 1970). Generally, but not always, the activation and depression of the single unit * Present address: Bizuzovstz. 43-164, Moskow 123060, U.S.S.R.
120 discharge occurred in parallel with desynchronization and synchronization of the EEG, respectively. Estrogen and progesterone have opposite effects on the excitability of the central nervous system as reported by Spiegel and Wycis (1945), Woolley and Timiras (1962). Marcus, Watson and Goldman (1966) and by Stitt and Kinnard (1968). The threshold current necessary to evoke convulsions in rats was thus decreased by estrogen and increased by progesterone. The steroid hormones may also influence the frequency of epileptic discharges as evidenced by Logothetis, Harner, Morrell and Torres (1959) and Laidlaw (1965) in their studies on catamenial epilepsy. These observations were recently confirmed and extended by Bfickstr6m (1976) who found that the frequency of seizures in a group of female epileptic patients was positively correlated to the quotient of estrogen/progesterone concentrations in their plasma. When the plasma concentration of progesterone was high, the seizure frequency was always low. The clinical observations indicate that steroid hormones in physiological plasma concentrations may influence the excitability of the central nervous system. The available experimental evidence was, however, obtained with pharmacological doses. We have therefore considered it of interest to study the effect of intravenous progesterone infusions of low concentrations on the spontaneous discharge of a penicillin focus in the cerebral cortex of the ovariectomized, non-estrogen-primed cat. METHODS
Experimental animals. Anaesthesia Fourteen ovariectomized female cats were used for the experiments. The method of anaesthesia during ovariectomy was Halothan (Hoechst) vaporized in a Fluotec apparatus by a gas mixture (2: 1) of Oz and N20. In the experiments with penicillin foci Halothan was used for induction and anaesthesia was then continued with chloralose (70 mg/kg, i.v.).
Surgical procedures The animals were ovariectomized and a period of at least 4 weeks was allowed for recovery. Shorter periods were avoided, because it was noted that serum obtained 7 days after the ovariectomy could itself increase the frequency of the spontaneous interictal penicillin spike. A catheter for the infusion was introduced into one of the femoral veins, and intra-arterial catheters into both femoral arteries. One of them was connected to a pressure transducer (Elema EMT 35) for continuous recording of the blood pressure. The other artery was used for the blood sampling. A tracheal cannula with a side tube for air sampling was mounted and connected to a pCO2 meter (Beckman medical gas analyzer LB 2) for continuous recording. Calibrated thermocouples were placed in the rectum and in the paraffin pools for the recording of temperatures. The right superficial and deep radial nerves were dissected and mounted for electrical stimulation in a paraffin pool. A craniectomy was made over the left sensorimotor cortex, and a paraffin pool was arranged to cover the cortex. The body tempera-
121 ture of the cat and the pool temperatures were kept constant by radiating heat lamps.
Recording procedures The electrocorticogram (ECoG) was recorded by spring mounted silver ball electrodes applied to the cortical surface. The cortical potentials evoked by electrical stimulation of the radial nerves were used for the selection of the recording points. Five cortical electrodes were mounted, but only the one at the penicillin focus will be considered in the present report. The focus was applied near the post-cruciate dimple (Pcd), where the electrical stimulation of the low threshold contralateral deep radial afferents evoked a surface response of maximal amplitude (cf., Oscarsson and Ros6n 1963). The ECoGs were recorded on an 8-channel standard Siemens-Elema Mingograf. Three of the channels were used for the continuous recording of the blood pressure, pCO2 and rectal temperature. The respiratory cycles were read on the pCOz record.
The penicillin focus and the standard plan of the experiment Pieces of filterpaper, 2 mm x 2 mm, soaked in Na-benzylpenicillin (Kabi) 100,000 1U/ml physiological saline, were placed on the cortical surface at the selected point near Pcd. The first piece was left in position for 5 min, and was then replaced by another similar piece, which remained in position during the experiment (60-100 min). The first spontaneous interictal penicillin spike generally appeared about 1 min after the first application. A quasi-stable spike frequency was established 5-10 min later. The following standard plan was adopted for the infusion experiments. Time (min)
Experimental procedure
0 5 10-15 15 15-45 45-75
First application of penicillin Second application of penicillin Control records Priming dose of progesterone (10-15 sec) Infusion of progesterone Recovery period
Blood sample
Second sample Samples every 10 min Samples every 10 min
The first blood sample was taken at the beginning of the surgical procedures, as soon as the catheter was mounted. A certain variation of the length of infusion and recovery periods was allowed (cf., Table 2).
Progesterone and Mebumal administration Progesterone was dissolved in ethanol (absolute alcohol, AB Vin- and Spritcentralen, Stockholm). The solution was dried down under nitrogen, and then dissolved again in serum from an ovariectomized cat. The solution was prepared within 1-3 hr before the experiment. The administration of the progesterone solution was standardized. A rapidly injected (10-15 sec) priming dose (1.2-1.5 ml) was first given in the femoral vein. The
122 priming dose was immediately followed by a slow intravenous infusion, I1 ml/hr, given by a standard apparatus with adjustable constant infusion speed. The priming dose and the concentration of perfused solution were initially calculated to give a plasma concentration of approximately 5000 ng/ml, which is 100 • maximal plasma concentration of progesterone observed at the preovulatory maximum of the rat (46 ~: 7 ng/ml, cf. Butcher, Collins and Fugo 1974). A blood volume of 5 ',~il of the body weight (hematocrit 37 ~o) and a total body water of 700 ml/kg was assumed according to the UFAW Handbook (1972) values for cat and dog. The concentration in the body water was assumed to be 1/10 of that in plasma. The progesterone concentration used in the first experiments was 0.6 mg/ml. The observed plasma concentrations in these experiments were around 500 ng/ml, thus only l/10 of the value expected from our calculations. This observation was interpreted as due to accumulation of progesterone in liver, kidneys and brain which invalidated our assumption concerning the concentration in body water. The uptake of progesterone in these organs is described by Bengtsson, Ullberg, Wiqvist and Diczfalusy (1964), Laumas and Farooq (1966), Seiki, Miyamoto, Yamashita and Kotani (1969) and by Wade, Harding and Feder (1973). In our series of experiments the concentration of the progesterone solutions was reduced to 0.3 and 0.15 mg/ml, which resulted in the plasma concentrations reported in Table 2. The observed mean plasma concentration was 103 ng/ml -~ SD 48, range 41-210, in 11 experiments using priming dose (1.5 mi) and infusion of progesterone 0.15 mg/ml. The amount of progesterone supplied by the selected constant infusion speed of a solution of 0.15 mg progesterone/ml is 20 mg/kg body wt/24 hr which corresponds to the production of progesterone in the pregnant rat (4.55 mg/24 hr/220 g rat) according to Pepe and Rothchild (1973). The concentrations of Mebumal solutions used were 1, 2 or 10 times the equimolar concentration of the 0.15 mg/ml progesterone solution. The Mebumal (ACO, Sweden; pentobarbital, INN) was diluted with physiological saline. The concentrations were 0.11, 0.22 and 1.1 mg/ml. The administration was similar to that of progesterone with a priming dose (1.5 ml) followed by a constant infusion (1 t ml/hr). The plasma concentration of barbiturate was not determined.
The plasma progesterone assay The plasma progesterone was assayed using a direct radioimmunoassay method as described by Furuyama and Nugent (1971), Carstensen and B~ckstr6m (1976) and B~ickstr6m and Carstensen (1974). RESULTS
The stability of the penicillin focus Spontaneous interictal spikes are discharged at a relatively stable frequency from a penicillin focus on the cerebral cortex of the cat. In control experiments, when the focus is allowed to develop under stable conditions, the frequency of the spontaneous spikes reaches a maximum within 5-10 min after the application of penicillin. The
123 TABLE 1 THE STABILITY AND DECLINE OF THE FREQUENCY OF THE SPONTANEOUS INTERICI'AL SPIKES IN THE CONTROL EXPERIMENTS Exp.
Maximal Frequency per minute. Means over 5 min and SD frequency 10-15 min 35~,0 rain 60-65 min (spikes/min) after the application of penicillin Spikes/min
750610 750619 750821 760330 760504 a 760506 a 760514 a 760519 a
30 24 24 17 20 40 17 14
Mean
SD
25.8 14.8 16.8 15.5 17.0 31.2 13.8 11.3
3.27 1.92 1.30 1.38 3.16 2.17 1.83 0.49
~
100 100 100 100 100 100 100 100
Spikes/min Mean
SD
28.5 12.6 14.8 14.2 15.3 30.8 13.2 10.8
5.45 0.89 1.30 0.84 1.86 2.39 0.84 1.30
~o
110 85 88 92 90 99 96 96 = 95.5 SD 7.8
Spikes/min Mean
SD
25.0 13.2 16.2 13.0 -29.4 13.7 10.1
4.12 1.30 1.30 1.26 -1.52 1.51 0.69
~o
97 89 96 84 -94 99 99 .~ = 94.0 SD 9.7
a Control experiments with infusion of normal ovariectomized cat serum. frequency then declines slowly d u r i n g a p e r i o d o f 1-2 hr. The effects o f p r o g e s t e r o n e infusion m u s t be r e a d on this slowly decreasing b a c k g r o u n d . The decline o f the frequency after the penicillin a p p l i c a t i o n was studied in c o n t r o l experiments with all exp e r i m e n t a l animals. The observations are s u m m a r i z e d in Table 1. The m a x i m a l frequency o f the s p o n t a n e o u s interictal penicillin spikes c o u l d reach 40/min b u t in m o s t foci was closer to 20. Relative stability was observed f r o m 10 min after the a p p l i c a t i o n o f the penicillin. The m e a n a n d s t a n d a r d deviation o f the frequency per m i n u t e averaged over a p e r i o d o f 5 min are given for the p e r i o d s 10-15, 35-40 a n d 60-65 min after the application. Using the frequency at 10-15 min as a reference the average r e d u c t i o n was 5 ~ at 35-40 min a n d 6 ~ at 60-65 min. The greatest observed r e d u c t i o n was 15 a n d 16~. The second p a r a m e t e r o f the penicillin focus, which was studied in this series o f experiments, was the g r a d u a l decrease in a m p l i t u d e o f the s p o n t a n e o u s interictal spike. The m a i n features o f the s p o n t a n e o u s discharge r e c o r d e d f r o m the cortical surface over the penicillin focus was described by W a l k e r a n d J o h n s o n (1946) a n d b y R a l s t o n (1958). The relations between the surface p o t e n t i a l a n d the n e u r o n a l responses in the u n d e r l y i n g cortex were f u r t h e r analyzed by M a t s u m o t o a n d A j m o n e M a r s a n (1964), Prince (1966) a n d D i c h t e r a n d Spencer (1969). However, less a t t e n t i o n has been p a i d to the changes in time course of the s p o n t a n e o u s surface discharge over the longer period, d u r i n g which the focal discharge slowly decreases. Such i n f o r m a t i o n is necessary for the e v a l u a t i o n o f the effect o f p r o g e s t e r o n e on the penicillin spike. A typical s p o n t a n e o u s discharge starts with a surface positive wave o f low a m plitude (Fig. 1B, c o m p o n e n t a) followed by a negative wave o f low a n d slowly increasing a m p l i t u d e ( c o m p o n e n t b). The b wave is i n t e r r u p t e d by a sharp positive wave o f
124
cf A ~
a B
c b d ~
f .....
d
L Fig. 1. Records of spontaneous interictal discharges recorded from the cortical surface over the penicillin focus. A and B were obtained 10 min after the application of the focus. Note the different time scales. C, 38 min, D, 47 min, E, 64 min after the application. A-E, from a control experiment with infusion of normal cat serum. F was obtained 66 rain after the application of the focus in an experiment with infusion of progesterone, a-f components of the action potential described in the text. Positivity upwards. Time scale in sec for A and in 100 msec for B-F. Voltage scale in mV. relatively low amplitude (component c). From the maximum of component c the record develops into a large negative spike with an amplitude of 3-5 mV and a duration of 70-100 msec (component d). This is the most conspicuous part of the response and we have referred to it as the spontaneous penicillin spike, the amplitude of which we have studied. The spike is followed by a negative wave of approximately 300 msec duration (component e) and then finally by a positive wave of 1-2 sec duration (component f, Fig. 1A and B). When studied in samples of records obtained at regular intervals during the first hour after penicillin application, it was observed that component a gradually decreased in amplitude, and component b first increased but later slowly decreased. Component c generally showed a gradual increase in amplitude. Component d displayed the most constant pattern of development. Its amplitude decreased gradually during the first hour and a notch was seen on its rising phase. It remained negative during the first hour, the decrease in amplitude progressing to about 20-30 % of its maximal amplitude. Later it changed phase into a positive spike of low amplitude before finally disappearing. The negative component e increased gradually reaching an amplitude of about 1 mV at the end of the first hour. It then gradually declined. The slow positive component f was largest (0.2 mV), when the negative spike (component d) was large. Component f decreased gradually apparently in parallel with component d. The change in time course of the focal response is shown in Fig. 1B-F.
The effect of changes in blood pressure on the frequency of the spontaneous interietal penicillin spike A slow fall in mean blood pressure (MBP) was sometimes observed during the infusion of progesterone, and the rapidly injected priming dose did occasionally induce transient blood pressure changes of moderate amplitude. It was therefore considered
125 B
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Fig. 2. The effect of a rapid (A) and a gradual (B) fall in mean systemac blood pressure (MBP) on the frequency of the spontaneous interictal penicillin spikes. The change in blood pressure was induced by arterial bleeding and retransfusion indicated by the signal.
necessary to investigate the effect of similar changes in blood pressure on the stability of the penicillin focus. Fig. 2A shows the effect of a drop in blood pressure induced by arterial bleeding through the femoral artery of 10 ml during 1 min. The blood was then reinjected during 30 sec. The bleeding led to a drop in MBP from 94 to 30 mm Hg and the retransfusion to a rise to 106 mm Hg. The corresponding effect on the frequency of the penicillin spike was a drop from 23 to 17 spikes/rain and a rise to 25/min during the retransfusion. The effect was transient and did not exceed 25 ~ of the control frequency. Fig. 2B shows a slow decrease in MBP similar to the maximal change observed during progesterone infusion. The blood pressure fell from the level of 90-60 mm Hg during the arterial bleeding of 11 ml during 10 rain. The blood was then retransfused during 5 min causing an increase in MBP to about 100 mm Hg. As shown in the diagram there is a gradual decrease in the frequency of the penicillin spike from 17-14/min during the period of 25 min of this experiment. The reduction does not exceed 20 and is of the same order of magnitude as the maximal slow decrease in frequency observed in the control foci (16 ~). It was thus concluded that transient sharp changes in MBP could give moderate and transient changes in the frequency of the spontaneous interictal penicillin spike. Slow changes in MBP of the type seen during progesterone infusion do not affect spike frequency in a manner that could explain the observed effects attributed to progesterone.
The plasma concentration, and half-life of progesterone in the ovariectomized cat We have found no reports in the literature concerning the plasma concentration of progesterone in the ovariectomized cat. Our assays of the blood samples obtained before the application of progesterone gave a mean value of 7.2 ng/ml, SD 4.9, n ~- 26. There was no significant difference between the assays of the first and the second blood samples which were obtained at the beginning of experiment and respectively 3-4 hr later after surgical procedures and other preparations for the experiment.
126 Some information was obtained concerning the half-life of progesterone from our observations of the decline in plasma concentration of progesterone during the recovery periods after the end of the infusions. In 11 experiments we found a decline to 5 0 ~ of the maximal concentration during infusion to take place within 8 min (mean 7.8 rain, SD 5.3, range 3-21). There was no obvious correlation between the maximal concentration and the half-life.
The effect of progesterone on the frequency of the spontaneous interictal penicillin spike The effect of intravenous progesterone infusion on the frequency of the spontaneous interictal penicillin spike is shown in Fig. 3A. The concentration of progesterone in the blood plasma was raised from 12 to 130 ng/ml by the priming dose. There was a further rise to 146 ng/ml during the infusion, which lasted for 16.5 min, and then a gradual decrease in the concentration. The progesterone concentration was 40 ng/ml at 30 min and 20 ng/ml at 90 min after the end of the infusion. A .E
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Fig. 3. Columns of graphs (A-C) showing from above downwards: the frequency of interictal penicillin spikes, the plasma concentration of progesterone, the mean systemic b l ~ pressure, pCO,4 respiratory rate and rectal temperature. A, intravenous infusion of progesterone; B, control infusion of normal cat serum; C, infusion of Mebumal 0.11 mg/ml. The graphs were obtained from 3 different loci A, B and C in the same animal. The chronological order was A B C. The arrows mark the beginning of the priming dose and the end of the infusion.
127 The effect on the frequency of the penicillin spike was a gradually developing decrease to about 50 % of the control level. This level was reached within 10-15 min. There was a gradual increase in frequency over the first 15 rain after the end of the infusion. Simultaneous records of MBP, pCO2, respiratory rate, and rectal temperature are displayed in the lower 4 graphs of Fig. 3A. No major changes of these parameters occurred during the infusion. A transient drop in MBP and apnoea followed by tachypnoea may have been induced by the priming dose. The corresponding records from the control infusion of cat serum are shown in Fig. 3B. The priming injection and infusion of serum lasted for 20 min, and the progesterone concentration remained low (23-27 ng/ml) during this period. The mean frequency of the spontaneous interictal penicillin spike was 16.2/rain during a period of 5 min immediately before the injection. It decreased to 16.0/min during the last 5 min of the infusion, and was 15.6/min between 5 and 10 min after the end of the infusion. The reduction in frequency in the control experiment was thus only 4%. No major changes were indicated by the records of MBP, pCO2, respiratory rate and rectal temperature. The records of Fig. 3C were obtained from an experiment on the same animal with a priming dose (1.5 ml) and infusion (15 min, 11 ml/hr) of Mebumal 0.11 mg/ml which is equimolar to a progesterone concentration of 0.15 mg/ml. The control frequency was 15/min and the frequency observed between 5 and 10 min after the end of the infusion was 12/min. The reduction was thus 20 %. The effect of Mebumal was thus smaller than that of progesterone. The results of all progesterone experiments are summarized in Table 2. They are arranged with decreasing serum concentrations of progesterone from above downwards. The figures in the second column give lowest and highest observed values during the plateau of increased concentration induced by the priming dose and the infusion of progesterone. The length of the infusions varied from 10 to 53 rain but were generally about 30 rain (column 3). The effect of progesterone is born out by a comparison of the interictal spike frequency during the control period of 5 min just before the priming dose and the frequencies observed 20-25, 60-65 and 90-95 min after the priming dose. The control frequency is given in spikes/rain (column 4) and referred to as 100 %. The frequencies during the following periods are given in per cent of the control. Further information may be obtained from a comparison of columns 6 and 7 with values from the corresponding periods after progesterone injection (6) and after injection of normal cat serum (7) in the same experimental animal. The frequency at the end of the infusion (column 8) is relevant to the assessment of the experiments with short infusion periods. The results show that progesterone in 10 out of 11 experiments caused a decrease in the frequency of the penicillin spike which exceeded the greatest observed decrease at the corresponding time in any of the control experiments (15 %, cf. Table 1). In one of the experiments the decrease was insignificant. The frequency generally increased again within 15-20 min from the end of the progesterone infusion. The dose-response relationship observed in Table 2 is very crude. The largest doses certainly gave more obvious effects, and when tested in the same animal the
128
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129 larger dose was more effective t h a n the smaller. Considerable effects were, however, also seen with c o n c e n t r a t i o n s between 40 a n d 70 n g progesterone/ml. The effect of M e b u m a l a n d progesterone was c o m p a r e d in 5 experiments. It was f o u n d that the effect of M e b u m a l was smaller t h a n that of progesterone when equim o l a r solutions ( M e b u m a l 0.11 mg/ml, progesterone 0.15 mg/ml) were a d m i n i s t e r e d similarly. This was also so with a twice e q u i m o l a r dose of M e b u m a l (0.22 mg/ml) b u t the 10 times e q u i m o l a r dose (1.1 mg/ml) depressed the frequency a n d amplitude of the interictal spike more t h a n did progesterone (cf. Fig. 4B).
The effect of progesterone on the amplitude of the spontaneous interictal penieillin spike The amplitude of the s p o n t a n e o u s interictal penicillin spike was more rapidly reduced d u r i n g the progesterone i n f u s i o n t h a n d u r i n g the c o n t r o l infusion of n o r m a l serum. The difference between the two curves may be seen in Fig. 4A. The a m p l i t u d e
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Fig. 4. Columns of graphs showing the effect of progesterone and Mebumal on the amplitude of the spontaneous interictal penicillin spike (component d in Fig. 1). A, upper graph: - - × - - , control infusion of normal cat serum; - - • --, infusion of progesterone 0.15 mg/ml. Each symbol gives mean amplitude of consecutive spikes in a minute. 95 ~ confidence intervals are given for every other symbol. The amplitudes are expressed in per cent of the mean amplitude during the minute just before the injection of the priming dose, which started at origo. Downward arrows indicate end of the infusion. Lower graph: Plasma concentration of progesterone. The symbols correspond to those of the upper graph. B, upper graph: - - × --, control infusion of normal cat serum (the dip in the curve was due to a slip of the recording electrode away from the cortical focus, which was corrected at 20 min); - - + --, infusion of progesterone 0.15 mg/ml; - - • --, infusion of progesterone 0.30 mg/ml; - - • --, infusion of Mebumal 1.1 mg/ml. Lower graph: as in A.
130 of the spike fell to zero and the spike reversed polarity within 25 rain after the start ,~l an infusion, which increased the progesterone concentration to 66 ng/ml. During the control infusion with serum the amplitude was significantly less reduced. Twenty-five minutes after the beginning of the infusion it was still 35 0/,~of the control amplitude. The gradual decay in amplitude of the penicillin spike was a more sensitive index of the progesterone effect than the frequency of the spikes. An obvious dose-response relationship was seen in the amplitude plots. In Fig. 4B the difference in amplitude decay is compared between infusions of progesterone reaching a plasma concentration of 69 and 126 ng/ml, respectively. From 10 min after the priming dose and onwards the difference between the curves is significant at the 95 o//oconfidence level. The effect of progesterone is also compared with that of Mebumal in Fig. 4B. The curve showing the reduction of spike amplitude during the infusion of Mebumal in a concentration of 1.1 mg/ml falls between the two progesterone curves. Compared on an equimolar basis the Mebumal concentration is 10 times higher than that of the progesterone solution used to raise the plasma level to 69 ng/ml (0.15 mg progesterone/ ml). The curves were obtained from the same animal and the volume of priming dose and infusion rates were identical. DISCUSSION The present observations indicate that the spontaneous activity of a penicillin focus in the cat's cerebral cortex is significantly influenced by intravenous progesterone infusion resulting in physiological plasma concentrations. In previous studies doses have been generally large, sometimes in the range of the anaesthetic effect of progesterone. The actual plasma concentration of progesterone was never determined. As we have not found any reports on the plasma concentrations of progesterone during the non-pregnant or pregnant state of the cat, we have to judge the physiological range from observations made on other species, mainly rat and guinea pig. In these animals maximal plasma concentration of 50 ng/ml was observed during the cycle and values of 300 ng/ml in pregnancy (Butcher et al. 1974; Heap and Deanesley 1966; Greep 1973). In 10 out of 1l of our experiments the observed plasma concentrations fell within this range. Clear-cut effects were found also in the range of 40-70 ng/ml. The time course of the progesterone effect is characteristic. It builds up slowly during 10-15 min and remains increased for a similar time after the end of the infusion, resembling the slow development of the anaesthesia induced by progesterone and related steroids (Merryman, Boiman, Barnes and Rothchild 1954; Howland, Boyan and Wang 1956; Laborit, Huguenard, Douzon, Weber and Guittard 1956; Taylor and Shearer 1956; P'an and Laubach 1964). Further discussion of the mechanism behind the time course of the progesterone effect must he postponed until more is known about the site of action of the hormone. It is difficult to compare the depressant effects of progesterone and Mebumal on the excitability of the cerebral cortex as the mechanism of their action is unknown. A comparison on equimolar basis does, however, emphasize the efficiency of the steroid hormone.
131 No conclusions can be drawn from the present experiments concerning the site or mode of action of progesterone, whether cortical or subcortical, whether on synaptic excitability, transmitter function or neuronal protein synthesis. It is, however, relevant to note that B~ickstr6m, Carstensen and S6derghrd (1976) observed a clear correlation between the concentration of progesterone in the plasma and in the CSF of human patients. The hormone level in the CSF corresponded approximately to the calculated levels of unbound steroids in the plasma. Intravenously infused progesterone will therefore penetrate the blood-brain barrier. An uptake of tritiated progesterone in the cerebral cortex as well as in several other parts of the central nervous system was reported by Seiki et al. (1969) and by Wade et al. (1973). We may also mention the observation that local application of progesterone to the cortical surface effectively modifies the evoked discharges from a penicillin focus (Landgren, unpublished observations). An effect of progesterone at the cortical level is thus a possibility. Localized effects in other parts of the brain have, however, been reported, i.e., the induction of sleep behaviour and synchronized EEG by the inplantation of progesterone crystals in the preoptic region (Heuser et al. 1967). The effect of penicillin on the neuronal membrane (Ayala, Lin and Vasconetto 1970) and on synaptic events (Ayala, Spencer and Gumnit 1971 ; Curtis, Game, Johnston, McCulloch and Maclachlan 1972) has been investigated, and membrane depolarization, increase in synaptic excitability and blocking of GABA inhibition described. The fact that progesterone affects the cortical penicillin discharge suggests the necessity of further investigations on the effect of progesterone on synaptic events. The possibility that the synchronizing effect of progesterone on EEG could be due to a rise in bloodpressure was raised by Beyer, Ramirez, Whitmoyer and Sawyer (1967). Synchronizing effects of baroreceptor stimulation are known from the investigations of Bonvallet, Dell and Hiebel (1954). In our series of experiments a rise in blood pressure was never observed. Transient drops in blood pressure, presumably related to the priming dose and more slowly developing reductions in pressure during the infusion were, however, seen but did not occur regularly. Blood pressure drops were also reported by Howland et al. (1956) following rapid intravenous injection of the progesterone-like steroid hydroxydione, which was used as an anaesthetic agent in man. We conclude that our observations cannot be explained as secondary to changes in blood pressure or baroreceptor activity. ACKNOWLEDGEMENTS This work was supported by Gunvor och Josef An6rs Stiftelse and by the Swedish Research Council (Proj. B78-04X-05177-01).
REFERENCES Ayala, G. F., S. Lin and C. Vasconetto (1970) Penicillin as epileptic agent: its effect on an isolated neuron, Science, 167: 1257-1260. Ayala, G. F., W. A. Spencer and R. F. Gumnit (1971) Penicillin as an epileptogenic agent: effect on an isolated synapse, Science, 171 : 915-917.
132 B~ickstr6m, T. (1976) Epileptic seizures in women related to plasma estrogen and progesterone during the menstrual cycle, Aeta neurol, seand., 54:321 347. B~ickstr6m, T. and H. Carstensen (1974) Estrogen and progesterone in plasma in relation to premenstrual tension, J. Steroid Biochem., 5: 257-260. B~.ckstr6m, T., H. Carstensen and R. SOderg~trd (1976) Concentration of estradiol, testosterone anti progesterone in cerebrospinal fluid compared to plasma unbound and total concentrations, J. Steroid Biochem., 7: 469-472. Bengtsson, G., S. Ullberg, N. Wiqvist and E. Diczfalusy (1964) Autoradiographic studies on previable human foetuses perfused with radioactive steroids, Acta endocr. (Kbh.), 46: 544-551. Beyer, C., V. D. Ramirez, D. J. Whitmoyer and C. H. Sawyer (1967) Effects of hormones on the electrical activity of the brain in the rat and rabbit, Exp. Neurol., 18: 313-326. Bonvallet, M., P. Dell and G. Hiebel (1954) Tonus sympathique et activit6 61ectrique cortical, Electroenceph, elin. Neurophysiol., 6:119-144. Butcher, R. L., W. E. Collins and N. N. Fugo (1974) Plasma concentration of LH, FSH, prolactin, progesterone and estradiol 17~3throughout the 4-day estrous cycle of the rat, Endocrinology, 94: 1704-1708. Carstensen, H. and T. B~ickstr6m (1976) A paper chromatographic saturation analysis method for measuring estradiol, testosterone and 5a-dihydrotestosterone from the same sample, J. Steroid Biochem., 7: 145-149. Curtis, D. R., C. J. A. Game, G. A. R. Johnston, R. M. McCulloch and R. W. Maelaehlan (1972) Convulsive action of penicillin, Brain Res., 43: 242-245. Dichter, M. and W. A. Spencer (1969) Penicillin-induced interictal discharges from the cat hippocampus. I. Characteristics and topographical features, J. Neurophysiol., 32: 649-662. Endr~Sczi, E. (1969) Electrophysiological studies on the mechanism of progesterone action in the rat, Aeta physiol. Acad. Sci. hung., 36: 83-93. Furuyama, S. and C. A. Nugent (1971) A radioimmunoassay for plasma progesterone, Steroids, 17: 663-674. Greep, R. O. (Ed.) (1973) Handbook of Physiology (Endocrinology Vol. 11), Amer. Physiol. Soc., Washington, D.C. Gyermek, L., G. Genther and N. Fleming (1967) Some effects of progesterone and related steroids on the central nervous system, Int. J. NeuropharmacoL, 6: 191-198. Heap, R. B. and R. Deanestey (1966) Progesterone in systemic blood and placentae of intact and ovariectomized pregnant guinea pigs, J. Endocr., 34: 417-423. Heuser, G., G. M. Ling and M. Kliiver (1967) Sleep induction by progesterone in the preoptic area in cats, Electroenceph. elin. Neurophysiol., 22: 122-127. Howland, W. S., P. Boyan and K.-C. Wang (1956) The use of a steroid (Viadril) as an anaesthetic agent, Anesthesiology, 17 : 1-8. Kawakami, M. and C. H. Sawyer (1967) Effects of sex hormones and antifertility steroids on brain thresholds in rabbit, Endocrinology, 80: 857-871. Komisaruk, B. R., P. G. McDonald, D. I. Whitmoyer and C. H. Sawyer (1967) Effects of progesterone and sensory stimulation on EEG and neuronal activity in the rat, Exp. Neurol., 19: 494-507. Laborit, H., P. Huguenard, C. Douzon, B. Weber and R. Guittard (1956) Etudes physiologiques et cliniques d'un steroide anesth~sique: le succinate sodique de 21-hydroxypregnandione, Arch. int. Pharmacodyn., 107 : 159-177. Laidlaw, J. (1965) Catamenial epilepsy, Lancet, 271 : 1235-1237. Laumas, K. R. and A. Farooq 0966) The uptake in vivo of [l,2-3H]progesterone by the brain and genital tract of the rat, J. Endocr., 36: 95-96. Lincoln, D. W, (1969) Effects of progesterone on the electrical activity of the forebrain, J. Endocr., 45 : 585-596. Logothetis, J., R. Harner, F. Morrell and F. Torres (1959) The role of estrogens in catamenial exacerbation of epilepsy, Neurology (Minneap.), 9: 352-360. Marcus, E. M., C. W. Watson and P. L. Goldman (1966) Effects of steroids on cerebral electrical activity, Arch. Neurol. (Chic.), 15: 521-532. Matsumoto, H. and C. Ajmone Marsan (1964) Cortical cellular phenomena in experimental epilepsy: "inter-ictal" manifestations, Exp. NeuroL, 9: 286-304. Merryman, W., R. Boiman, L. Barnes and I. Rothchild (1954) Progesterone "anesthesia" in human subjects, J. clin. Endocr., 14: 1567-1569. Oscarsson, O. and I. Ros6n (1963) Projection to cerebral cortex of large muscle spindle afferents in forelimb nerves of the cat, J. Physiol. (Lond.), 169: 924-945.
133 Wan, S. Y. and G. D. Laubach (1964) Steroid central depressants. In: R. Dorfman (Ed.), Methods in Hormonal Research, Vol. llI, Academic Press, New York, pp. 415475. Pepe, G. J. and I. Rothchild (1973) Metabolic clearance rate of progesterone: comparison between ovariectomized, pregnant, pseudopregnant and deciduoma-bearing pseudopregnant rats, Endocrinology, 93 : 1200-1205. Prince, D. A. (1966) Modification of focal cortical epileptogenic discharge by afferent influences, Epilepsia (Amst.), 7: 181-201. Ralston, B. L. (1958) The mechanism of transition of interictal spiking foci into ictal seizure discharges, Eleetroeneeph. clin. NeurophysioL, 10:217 232. Seiki, K., M. Miyamoto, A. Yamashita and M. Kotani (1969) Further studies on the uptake of labelled progesterone by the hypothalamus and pituitary of rats, J. Endoer., 43: 129-130. Selye, H. (1942) Correlations between the chemical structure and the pharmacological action of tke steroids, Endocrinology, 30: 437453. Spiegel, E. and H. Wycis (1945) Anticonvulsant effects of steroids, J. Lab. clin. Med., 30: 947-953. Stitt, S. L. and W. J. Kinnard (1968) The effect of certain progestins and estrogens on the threshold of electrically induced seizure patterns, Neurology (Minneap.), 18 : 213-216. Taylor, N. and W. M. Shearer (1956) The anaesthetic properties of 21-hydroxypregnandione sodium hemisuccinate (Hydroxydione), Brit. J. Anaesth., 28: 67-76. Teresawa, E. and C. H. Sawyer (1970) Diurnal variation in the effects of progesterone on multiple unit activity in the rat hypothalamus, Exp. Neurol., 27: 359-374. UF,4 W Handbook on the Care and Management of Laboratory Animals, 4th ed. (1972) Churchill-Livingstone, London. Wade, G. N., C. F. Harding and H. H. Feder (1973) Neural uptake of [1,2-SH]progesterone in ovariectomized rats, guinea pigs and hamsters: correlation with species differences in behavioral responsiveness, Brain Res., 61 : 357-367. Walker, A. E. and H. C. Johnson (1946) Penicillin in Neurology, Thomas, Springfield, Ill., pp. 78-101. Woolley, D. E. and P. S. Timiras (1962) The gonad brain relationship: effects of female sex hormones on electroshock convulsion in the rat, Endocrinology, 70: 196~209.
119
THE EFFECT OF PROGESTERONE ON THE SPONTANEOUS INTERICTAL SPIKE EVOKED BY THE APPLICATION OF PENICILLIN TO THE CAT'S CEREBRAL CORTEX
S. LANDGREN, T. B.~,CKSTRt~Mand G. KALISTRATOV* Department of Physiology, University of Umed, S-901 87 Umed (Sweden)
(Received 26 September, 1977)
SUMMARY The effect of intravenous infusion of progesterone and Mebumal (ACO, Sweden; pentobarbital, INN) on the frequency and amplitude of the spontaneous interictal spikes from a penicillin focus in the cerebral cortex was investigated in ovariectomized cats anaesthetized with chloralose. The plasma concentration of progesterone was determined. It was found that the generation of penicillin spikes was depressed by progesterone in plasma concentrations as low as 40 ng/ml. Equimolar doses of Mebumal were less effective.
INTRODUCTION Several investigations demonstrate that steroid hormones can influence the activity of the cerebral cortex. Progesterone in large doses (>60 mg/kg body weight) has anaesthetic effects and some related steroids (21-hydroxy-5 fl-pregnane-3,20-dione; 5fl-pregnane-3,20-dione) are effective in lower doses (6-12 mg/kg) (cf., Selye 1942; P'an and Laubach 1964; Gyermek, Genther and Fleming 1967). These effects may be mediated via the reticular activating system, as pointed out by several authors who observed EEG synchronization, spindle discharges, increase in the arousal threshold and sleep behaviour after injection of progesterone (cf., Heuser, Ling and Kliiver 1967; Kawakami and Sawyer 1967; Endr6czi 1969; Lincoln 1969). Progesterone effects on single unit responses in the cerebral cortex, thalamus and hypothalamus have been reported (Komisaruk, McDonald, Whitmoyer and Sawyer 1967; Teresawa and Sawyer 1970). Generally, but not always, the activation and depression of the single unit * Present address: Bizuzovstz. 43-164, Moskow 123060, U.S.S.R.
120 discharge occurred in parallel with desynchronization and synchronization of the EEG, respectively. Estrogen and progesterone have opposite effects on the excitability of the central nervous system as reported by Spiegel and Wycis (1945), Woolley and Timiras (1962). Marcus, Watson and Goldman (1966) and by Stitt and Kinnard (1968). The threshold current necessary to evoke convulsions in rats was thus decreased by estrogen and increased by progesterone. The steroid hormones may also influence the frequency of epileptic discharges as evidenced by Logothetis, Harner, Morrell and Torres (1959) and Laidlaw (1965) in their studies on catamenial epilepsy. These observations were recently confirmed and extended by Bfickstr6m (1976) who found that the frequency of seizures in a group of female epileptic patients was positively correlated to the quotient of estrogen/progesterone concentrations in their plasma. When the plasma concentration of progesterone was high, the seizure frequency was always low. The clinical observations indicate that steroid hormones in physiological plasma concentrations may influence the excitability of the central nervous system. The available experimental evidence was, however, obtained with pharmacological doses. We have therefore considered it of interest to study the effect of intravenous progesterone infusions of low concentrations on the spontaneous discharge of a penicillin focus in the cerebral cortex of the ovariectomized, non-estrogen-primed cat. METHODS
Experimental animals. Anaesthesia Fourteen ovariectomized female cats were used for the experiments. The method of anaesthesia during ovariectomy was Halothan (Hoechst) vaporized in a Fluotec apparatus by a gas mixture (2: 1) of Oz and N20. In the experiments with penicillin foci Halothan was used for induction and anaesthesia was then continued with chloralose (70 mg/kg, i.v.).
Surgical procedures The animals were ovariectomized and a period of at least 4 weeks was allowed for recovery. Shorter periods were avoided, because it was noted that serum obtained 7 days after the ovariectomy could itself increase the frequency of the spontaneous interictal penicillin spike. A catheter for the infusion was introduced into one of the femoral veins, and intra-arterial catheters into both femoral arteries. One of them was connected to a pressure transducer (Elema EMT 35) for continuous recording of the blood pressure. The other artery was used for the blood sampling. A tracheal cannula with a side tube for air sampling was mounted and connected to a pCO2 meter (Beckman medical gas analyzer LB 2) for continuous recording. Calibrated thermocouples were placed in the rectum and in the paraffin pools for the recording of temperatures. The right superficial and deep radial nerves were dissected and mounted for electrical stimulation in a paraffin pool. A craniectomy was made over the left sensorimotor cortex, and a paraffin pool was arranged to cover the cortex. The body tempera-
121 ture of the cat and the pool temperatures were kept constant by radiating heat lamps.
Recording procedures The electrocorticogram (ECoG) was recorded by spring mounted silver ball electrodes applied to the cortical surface. The cortical potentials evoked by electrical stimulation of the radial nerves were used for the selection of the recording points. Five cortical electrodes were mounted, but only the one at the penicillin focus will be considered in the present report. The focus was applied near the post-cruciate dimple (Pcd), where the electrical stimulation of the low threshold contralateral deep radial afferents evoked a surface response of maximal amplitude (cf., Oscarsson and Ros6n 1963). The ECoGs were recorded on an 8-channel standard Siemens-Elema Mingograf. Three of the channels were used for the continuous recording of the blood pressure, pCO2 and rectal temperature. The respiratory cycles were read on the pCOz record.
The penicillin focus and the standard plan of the experiment Pieces of filterpaper, 2 mm x 2 mm, soaked in Na-benzylpenicillin (Kabi) 100,000 1U/ml physiological saline, were placed on the cortical surface at the selected point near Pcd. The first piece was left in position for 5 min, and was then replaced by another similar piece, which remained in position during the experiment (60-100 min). The first spontaneous interictal penicillin spike generally appeared about 1 min after the first application. A quasi-stable spike frequency was established 5-10 min later. The following standard plan was adopted for the infusion experiments. Time (min)
Experimental procedure
0 5 10-15 15 15-45 45-75
First application of penicillin Second application of penicillin Control records Priming dose of progesterone (10-15 sec) Infusion of progesterone Recovery period
Blood sample
Second sample Samples every 10 min Samples every 10 min
The first blood sample was taken at the beginning of the surgical procedures, as soon as the catheter was mounted. A certain variation of the length of infusion and recovery periods was allowed (cf., Table 2).
Progesterone and Mebumal administration Progesterone was dissolved in ethanol (absolute alcohol, AB Vin- and Spritcentralen, Stockholm). The solution was dried down under nitrogen, and then dissolved again in serum from an ovariectomized cat. The solution was prepared within 1-3 hr before the experiment. The administration of the progesterone solution was standardized. A rapidly injected (10-15 sec) priming dose (1.2-1.5 ml) was first given in the femoral vein. The
122 priming dose was immediately followed by a slow intravenous infusion, I1 ml/hr, given by a standard apparatus with adjustable constant infusion speed. The priming dose and the concentration of perfused solution were initially calculated to give a plasma concentration of approximately 5000 ng/ml, which is 100 • maximal plasma concentration of progesterone observed at the preovulatory maximum of the rat (46 ~: 7 ng/ml, cf. Butcher, Collins and Fugo 1974). A blood volume of 5 ',~il of the body weight (hematocrit 37 ~o) and a total body water of 700 ml/kg was assumed according to the UFAW Handbook (1972) values for cat and dog. The concentration in the body water was assumed to be 1/10 of that in plasma. The progesterone concentration used in the first experiments was 0.6 mg/ml. The observed plasma concentrations in these experiments were around 500 ng/ml, thus only l/10 of the value expected from our calculations. This observation was interpreted as due to accumulation of progesterone in liver, kidneys and brain which invalidated our assumption concerning the concentration in body water. The uptake of progesterone in these organs is described by Bengtsson, Ullberg, Wiqvist and Diczfalusy (1964), Laumas and Farooq (1966), Seiki, Miyamoto, Yamashita and Kotani (1969) and by Wade, Harding and Feder (1973). In our series of experiments the concentration of the progesterone solutions was reduced to 0.3 and 0.15 mg/ml, which resulted in the plasma concentrations reported in Table 2. The observed mean plasma concentration was 103 ng/ml -~ SD 48, range 41-210, in 11 experiments using priming dose (1.5 mi) and infusion of progesterone 0.15 mg/ml. The amount of progesterone supplied by the selected constant infusion speed of a solution of 0.15 mg progesterone/ml is 20 mg/kg body wt/24 hr which corresponds to the production of progesterone in the pregnant rat (4.55 mg/24 hr/220 g rat) according to Pepe and Rothchild (1973). The concentrations of Mebumal solutions used were 1, 2 or 10 times the equimolar concentration of the 0.15 mg/ml progesterone solution. The Mebumal (ACO, Sweden; pentobarbital, INN) was diluted with physiological saline. The concentrations were 0.11, 0.22 and 1.1 mg/ml. The administration was similar to that of progesterone with a priming dose (1.5 ml) followed by a constant infusion (1 t ml/hr). The plasma concentration of barbiturate was not determined.
The plasma progesterone assay The plasma progesterone was assayed using a direct radioimmunoassay method as described by Furuyama and Nugent (1971), Carstensen and B~ckstr6m (1976) and B~ickstr6m and Carstensen (1974). RESULTS
The stability of the penicillin focus Spontaneous interictal spikes are discharged at a relatively stable frequency from a penicillin focus on the cerebral cortex of the cat. In control experiments, when the focus is allowed to develop under stable conditions, the frequency of the spontaneous spikes reaches a maximum within 5-10 min after the application of penicillin. The
123 TABLE 1 THE STABILITY AND DECLINE OF THE FREQUENCY OF THE SPONTANEOUS INTERICI'AL SPIKES IN THE CONTROL EXPERIMENTS Exp.
Maximal Frequency per minute. Means over 5 min and SD frequency 10-15 min 35~,0 rain 60-65 min (spikes/min) after the application of penicillin Spikes/min
750610 750619 750821 760330 760504 a 760506 a 760514 a 760519 a
30 24 24 17 20 40 17 14
Mean
SD
25.8 14.8 16.8 15.5 17.0 31.2 13.8 11.3
3.27 1.92 1.30 1.38 3.16 2.17 1.83 0.49
~
100 100 100 100 100 100 100 100
Spikes/min Mean
SD
28.5 12.6 14.8 14.2 15.3 30.8 13.2 10.8
5.45 0.89 1.30 0.84 1.86 2.39 0.84 1.30
~o
110 85 88 92 90 99 96 96 = 95.5 SD 7.8
Spikes/min Mean
SD
25.0 13.2 16.2 13.0 -29.4 13.7 10.1
4.12 1.30 1.30 1.26 -1.52 1.51 0.69
~o
97 89 96 84 -94 99 99 .~ = 94.0 SD 9.7
a Control experiments with infusion of normal ovariectomized cat serum. frequency then declines slowly d u r i n g a p e r i o d o f 1-2 hr. The effects o f p r o g e s t e r o n e infusion m u s t be r e a d on this slowly decreasing b a c k g r o u n d . The decline o f the frequency after the penicillin a p p l i c a t i o n was studied in c o n t r o l experiments with all exp e r i m e n t a l animals. The observations are s u m m a r i z e d in Table 1. The m a x i m a l frequency o f the s p o n t a n e o u s interictal penicillin spikes c o u l d reach 40/min b u t in m o s t foci was closer to 20. Relative stability was observed f r o m 10 min after the a p p l i c a t i o n o f the penicillin. The m e a n a n d s t a n d a r d deviation o f the frequency per m i n u t e averaged over a p e r i o d o f 5 min are given for the p e r i o d s 10-15, 35-40 a n d 60-65 min after the application. Using the frequency at 10-15 min as a reference the average r e d u c t i o n was 5 ~ at 35-40 min a n d 6 ~ at 60-65 min. The greatest observed r e d u c t i o n was 15 a n d 16~. The second p a r a m e t e r o f the penicillin focus, which was studied in this series o f experiments, was the g r a d u a l decrease in a m p l i t u d e o f the s p o n t a n e o u s interictal spike. The m a i n features o f the s p o n t a n e o u s discharge r e c o r d e d f r o m the cortical surface over the penicillin focus was described by W a l k e r a n d J o h n s o n (1946) a n d b y R a l s t o n (1958). The relations between the surface p o t e n t i a l a n d the n e u r o n a l responses in the u n d e r l y i n g cortex were f u r t h e r analyzed by M a t s u m o t o a n d A j m o n e M a r s a n (1964), Prince (1966) a n d D i c h t e r a n d Spencer (1969). However, less a t t e n t i o n has been p a i d to the changes in time course of the s p o n t a n e o u s surface discharge over the longer period, d u r i n g which the focal discharge slowly decreases. Such i n f o r m a t i o n is necessary for the e v a l u a t i o n o f the effect o f p r o g e s t e r o n e on the penicillin spike. A typical s p o n t a n e o u s discharge starts with a surface positive wave o f low a m plitude (Fig. 1B, c o m p o n e n t a) followed by a negative wave o f low a n d slowly increasing a m p l i t u d e ( c o m p o n e n t b). The b wave is i n t e r r u p t e d by a sharp positive wave o f
124
cf A ~
a B
c b d ~
f .....
d
L Fig. 1. Records of spontaneous interictal discharges recorded from the cortical surface over the penicillin focus. A and B were obtained 10 min after the application of the focus. Note the different time scales. C, 38 min, D, 47 min, E, 64 min after the application. A-E, from a control experiment with infusion of normal cat serum. F was obtained 66 rain after the application of the focus in an experiment with infusion of progesterone, a-f components of the action potential described in the text. Positivity upwards. Time scale in sec for A and in 100 msec for B-F. Voltage scale in mV. relatively low amplitude (component c). From the maximum of component c the record develops into a large negative spike with an amplitude of 3-5 mV and a duration of 70-100 msec (component d). This is the most conspicuous part of the response and we have referred to it as the spontaneous penicillin spike, the amplitude of which we have studied. The spike is followed by a negative wave of approximately 300 msec duration (component e) and then finally by a positive wave of 1-2 sec duration (component f, Fig. 1A and B). When studied in samples of records obtained at regular intervals during the first hour after penicillin application, it was observed that component a gradually decreased in amplitude, and component b first increased but later slowly decreased. Component c generally showed a gradual increase in amplitude. Component d displayed the most constant pattern of development. Its amplitude decreased gradually during the first hour and a notch was seen on its rising phase. It remained negative during the first hour, the decrease in amplitude progressing to about 20-30 % of its maximal amplitude. Later it changed phase into a positive spike of low amplitude before finally disappearing. The negative component e increased gradually reaching an amplitude of about 1 mV at the end of the first hour. It then gradually declined. The slow positive component f was largest (0.2 mV), when the negative spike (component d) was large. Component f decreased gradually apparently in parallel with component d. The change in time course of the focal response is shown in Fig. 1B-F.
The effect of changes in blood pressure on the frequency of the spontaneous interietal penicillin spike A slow fall in mean blood pressure (MBP) was sometimes observed during the infusion of progesterone, and the rapidly injected priming dose did occasionally induce transient blood pressure changes of moderate amplitude. It was therefore considered
125 B
A Spikes/min.
Spikes/rain. 30-
m m Hg
30t
.,oo
"
F
1 10-
~0 "60
90 P
0
• 20
0 -
0
,
10 -
U
-
-
rain.
" ~
I
10
,
I
, 20
,
I" 5 0
rain.
Fig. 2. The effect of a rapid (A) and a gradual (B) fall in mean systemac blood pressure (MBP) on the frequency of the spontaneous interictal penicillin spikes. The change in blood pressure was induced by arterial bleeding and retransfusion indicated by the signal.
necessary to investigate the effect of similar changes in blood pressure on the stability of the penicillin focus. Fig. 2A shows the effect of a drop in blood pressure induced by arterial bleeding through the femoral artery of 10 ml during 1 min. The blood was then reinjected during 30 sec. The bleeding led to a drop in MBP from 94 to 30 mm Hg and the retransfusion to a rise to 106 mm Hg. The corresponding effect on the frequency of the penicillin spike was a drop from 23 to 17 spikes/rain and a rise to 25/min during the retransfusion. The effect was transient and did not exceed 25 ~ of the control frequency. Fig. 2B shows a slow decrease in MBP similar to the maximal change observed during progesterone infusion. The blood pressure fell from the level of 90-60 mm Hg during the arterial bleeding of 11 ml during 10 rain. The blood was then retransfused during 5 min causing an increase in MBP to about 100 mm Hg. As shown in the diagram there is a gradual decrease in the frequency of the penicillin spike from 17-14/min during the period of 25 min of this experiment. The reduction does not exceed 20 and is of the same order of magnitude as the maximal slow decrease in frequency observed in the control foci (16 ~). It was thus concluded that transient sharp changes in MBP could give moderate and transient changes in the frequency of the spontaneous interictal penicillin spike. Slow changes in MBP of the type seen during progesterone infusion do not affect spike frequency in a manner that could explain the observed effects attributed to progesterone.
The plasma concentration, and half-life of progesterone in the ovariectomized cat We have found no reports in the literature concerning the plasma concentration of progesterone in the ovariectomized cat. Our assays of the blood samples obtained before the application of progesterone gave a mean value of 7.2 ng/ml, SD 4.9, n ~- 26. There was no significant difference between the assays of the first and the second blood samples which were obtained at the beginning of experiment and respectively 3-4 hr later after surgical procedures and other preparations for the experiment.
126 Some information was obtained concerning the half-life of progesterone from our observations of the decline in plasma concentration of progesterone during the recovery periods after the end of the infusions. In 11 experiments we found a decline to 5 0 ~ of the maximal concentration during infusion to take place within 8 min (mean 7.8 rain, SD 5.3, range 3-21). There was no obvious correlation between the maximal concentration and the half-life.
The effect of progesterone on the frequency of the spontaneous interictal penicillin spike The effect of intravenous progesterone infusion on the frequency of the spontaneous interictal penicillin spike is shown in Fig. 3A. The concentration of progesterone in the blood plasma was raised from 12 to 130 ng/ml by the priming dose. There was a further rise to 146 ng/ml during the infusion, which lasted for 16.5 min, and then a gradual decrease in the concentration. The progesterone concentration was 40 ng/ml at 30 min and 20 ng/ml at 90 min after the end of the infusion. A .E
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127 The effect on the frequency of the penicillin spike was a gradually developing decrease to about 50 % of the control level. This level was reached within 10-15 min. There was a gradual increase in frequency over the first 15 rain after the end of the infusion. Simultaneous records of MBP, pCO2, respiratory rate, and rectal temperature are displayed in the lower 4 graphs of Fig. 3A. No major changes of these parameters occurred during the infusion. A transient drop in MBP and apnoea followed by tachypnoea may have been induced by the priming dose. The corresponding records from the control infusion of cat serum are shown in Fig. 3B. The priming injection and infusion of serum lasted for 20 min, and the progesterone concentration remained low (23-27 ng/ml) during this period. The mean frequency of the spontaneous interictal penicillin spike was 16.2/rain during a period of 5 min immediately before the injection. It decreased to 16.0/min during the last 5 min of the infusion, and was 15.6/min between 5 and 10 min after the end of the infusion. The reduction in frequency in the control experiment was thus only 4%. No major changes were indicated by the records of MBP, pCO2, respiratory rate and rectal temperature. The records of Fig. 3C were obtained from an experiment on the same animal with a priming dose (1.5 ml) and infusion (15 min, 11 ml/hr) of Mebumal 0.11 mg/ml which is equimolar to a progesterone concentration of 0.15 mg/ml. The control frequency was 15/min and the frequency observed between 5 and 10 min after the end of the infusion was 12/min. The reduction was thus 20 %. The effect of Mebumal was thus smaller than that of progesterone. The results of all progesterone experiments are summarized in Table 2. They are arranged with decreasing serum concentrations of progesterone from above downwards. The figures in the second column give lowest and highest observed values during the plateau of increased concentration induced by the priming dose and the infusion of progesterone. The length of the infusions varied from 10 to 53 rain but were generally about 30 rain (column 3). The effect of progesterone is born out by a comparison of the interictal spike frequency during the control period of 5 min just before the priming dose and the frequencies observed 20-25, 60-65 and 90-95 min after the priming dose. The control frequency is given in spikes/rain (column 4) and referred to as 100 %. The frequencies during the following periods are given in per cent of the control. Further information may be obtained from a comparison of columns 6 and 7 with values from the corresponding periods after progesterone injection (6) and after injection of normal cat serum (7) in the same experimental animal. The frequency at the end of the infusion (column 8) is relevant to the assessment of the experiments with short infusion periods. The results show that progesterone in 10 out of 11 experiments caused a decrease in the frequency of the penicillin spike which exceeded the greatest observed decrease at the corresponding time in any of the control experiments (15 %, cf. Table 1). In one of the experiments the decrease was insignificant. The frequency generally increased again within 15-20 min from the end of the progesterone infusion. The dose-response relationship observed in Table 2 is very crude. The largest doses certainly gave more obvious effects, and when tested in the same animal the
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129 larger dose was more effective t h a n the smaller. Considerable effects were, however, also seen with c o n c e n t r a t i o n s between 40 a n d 70 n g progesterone/ml. The effect of M e b u m a l a n d progesterone was c o m p a r e d in 5 experiments. It was f o u n d that the effect of M e b u m a l was smaller t h a n that of progesterone when equim o l a r solutions ( M e b u m a l 0.11 mg/ml, progesterone 0.15 mg/ml) were a d m i n i s t e r e d similarly. This was also so with a twice e q u i m o l a r dose of M e b u m a l (0.22 mg/ml) b u t the 10 times e q u i m o l a r dose (1.1 mg/ml) depressed the frequency a n d amplitude of the interictal spike more t h a n did progesterone (cf. Fig. 4B).
The effect of progesterone on the amplitude of the spontaneous interictal penieillin spike The amplitude of the s p o n t a n e o u s interictal penicillin spike was more rapidly reduced d u r i n g the progesterone i n f u s i o n t h a n d u r i n g the c o n t r o l infusion of n o r m a l serum. The difference between the two curves may be seen in Fig. 4A. The a m p l i t u d e
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Fig. 4. Columns of graphs showing the effect of progesterone and Mebumal on the amplitude of the spontaneous interictal penicillin spike (component d in Fig. 1). A, upper graph: - - × - - , control infusion of normal cat serum; - - • --, infusion of progesterone 0.15 mg/ml. Each symbol gives mean amplitude of consecutive spikes in a minute. 95 ~ confidence intervals are given for every other symbol. The amplitudes are expressed in per cent of the mean amplitude during the minute just before the injection of the priming dose, which started at origo. Downward arrows indicate end of the infusion. Lower graph: Plasma concentration of progesterone. The symbols correspond to those of the upper graph. B, upper graph: - - × --, control infusion of normal cat serum (the dip in the curve was due to a slip of the recording electrode away from the cortical focus, which was corrected at 20 min); - - + --, infusion of progesterone 0.15 mg/ml; - - • --, infusion of progesterone 0.30 mg/ml; - - • --, infusion of Mebumal 1.1 mg/ml. Lower graph: as in A.
130 of the spike fell to zero and the spike reversed polarity within 25 rain after the start ,~l an infusion, which increased the progesterone concentration to 66 ng/ml. During the control infusion with serum the amplitude was significantly less reduced. Twenty-five minutes after the beginning of the infusion it was still 35 0/,~of the control amplitude. The gradual decay in amplitude of the penicillin spike was a more sensitive index of the progesterone effect than the frequency of the spikes. An obvious dose-response relationship was seen in the amplitude plots. In Fig. 4B the difference in amplitude decay is compared between infusions of progesterone reaching a plasma concentration of 69 and 126 ng/ml, respectively. From 10 min after the priming dose and onwards the difference between the curves is significant at the 95 o//oconfidence level. The effect of progesterone is also compared with that of Mebumal in Fig. 4B. The curve showing the reduction of spike amplitude during the infusion of Mebumal in a concentration of 1.1 mg/ml falls between the two progesterone curves. Compared on an equimolar basis the Mebumal concentration is 10 times higher than that of the progesterone solution used to raise the plasma level to 69 ng/ml (0.15 mg progesterone/ ml). The curves were obtained from the same animal and the volume of priming dose and infusion rates were identical. DISCUSSION The present observations indicate that the spontaneous activity of a penicillin focus in the cat's cerebral cortex is significantly influenced by intravenous progesterone infusion resulting in physiological plasma concentrations. In previous studies doses have been generally large, sometimes in the range of the anaesthetic effect of progesterone. The actual plasma concentration of progesterone was never determined. As we have not found any reports on the plasma concentrations of progesterone during the non-pregnant or pregnant state of the cat, we have to judge the physiological range from observations made on other species, mainly rat and guinea pig. In these animals maximal plasma concentration of 50 ng/ml was observed during the cycle and values of 300 ng/ml in pregnancy (Butcher et al. 1974; Heap and Deanesley 1966; Greep 1973). In 10 out of 1l of our experiments the observed plasma concentrations fell within this range. Clear-cut effects were found also in the range of 40-70 ng/ml. The time course of the progesterone effect is characteristic. It builds up slowly during 10-15 min and remains increased for a similar time after the end of the infusion, resembling the slow development of the anaesthesia induced by progesterone and related steroids (Merryman, Boiman, Barnes and Rothchild 1954; Howland, Boyan and Wang 1956; Laborit, Huguenard, Douzon, Weber and Guittard 1956; Taylor and Shearer 1956; P'an and Laubach 1964). Further discussion of the mechanism behind the time course of the progesterone effect must he postponed until more is known about the site of action of the hormone. It is difficult to compare the depressant effects of progesterone and Mebumal on the excitability of the cerebral cortex as the mechanism of their action is unknown. A comparison on equimolar basis does, however, emphasize the efficiency of the steroid hormone.
131 No conclusions can be drawn from the present experiments concerning the site or mode of action of progesterone, whether cortical or subcortical, whether on synaptic excitability, transmitter function or neuronal protein synthesis. It is, however, relevant to note that B~ickstr6m, Carstensen and S6derghrd (1976) observed a clear correlation between the concentration of progesterone in the plasma and in the CSF of human patients. The hormone level in the CSF corresponded approximately to the calculated levels of unbound steroids in the plasma. Intravenously infused progesterone will therefore penetrate the blood-brain barrier. An uptake of tritiated progesterone in the cerebral cortex as well as in several other parts of the central nervous system was reported by Seiki et al. (1969) and by Wade et al. (1973). We may also mention the observation that local application of progesterone to the cortical surface effectively modifies the evoked discharges from a penicillin focus (Landgren, unpublished observations). An effect of progesterone at the cortical level is thus a possibility. Localized effects in other parts of the brain have, however, been reported, i.e., the induction of sleep behaviour and synchronized EEG by the inplantation of progesterone crystals in the preoptic region (Heuser et al. 1967). The effect of penicillin on the neuronal membrane (Ayala, Lin and Vasconetto 1970) and on synaptic events (Ayala, Spencer and Gumnit 1971 ; Curtis, Game, Johnston, McCulloch and Maclachlan 1972) has been investigated, and membrane depolarization, increase in synaptic excitability and blocking of GABA inhibition described. The fact that progesterone affects the cortical penicillin discharge suggests the necessity of further investigations on the effect of progesterone on synaptic events. The possibility that the synchronizing effect of progesterone on EEG could be due to a rise in bloodpressure was raised by Beyer, Ramirez, Whitmoyer and Sawyer (1967). Synchronizing effects of baroreceptor stimulation are known from the investigations of Bonvallet, Dell and Hiebel (1954). In our series of experiments a rise in blood pressure was never observed. Transient drops in blood pressure, presumably related to the priming dose and more slowly developing reductions in pressure during the infusion were, however, seen but did not occur regularly. Blood pressure drops were also reported by Howland et al. (1956) following rapid intravenous injection of the progesterone-like steroid hydroxydione, which was used as an anaesthetic agent in man. We conclude that our observations cannot be explained as secondary to changes in blood pressure or baroreceptor activity. ACKNOWLEDGEMENTS This work was supported by Gunvor och Josef An6rs Stiftelse and by the Swedish Research Council (Proj. B78-04X-05177-01).
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