Synergistic action of corticosterone on kainic acid-induced electrophysiological alterations in the hippocampus

BRAIN RESEARCH ELSEVIER

Brain Research 704 (1995) 97- 102

Short communication

Synergistic action of corticosterone on kainic acid-induced electrophysiological alterations in the hippocampus Mustapha Talmi a,c,*, Edmond Carlier b, Wahil Bengelloun

‘, Bernard Soumireu-Mourat

a

aLaboratoire de Neurobiologie des Comportements, CNRS URA 372, Uniuersit5 de Prouence, Marseille, France b Laboratoire de Neurobiologie des canaax ioniques, INSERM U 374, Faculti? de Mt!decine, Secteur Nerd, Marseille, France ’ Department de Biologie, Uniuersiti Mohamed V, Rabat, Maroc Accepted 22 August 1995

Abstract The present study investigates the effect of overexposure to high doses of the stress hormone corticosterone (CORT) on the electrophysiological changes produced in the hippocampus after local microinjection of KA. Extracellular recordings were performed in the CA1 area of mouse hippocampal slices prepared after a 7-day recovery period following KA microinfusion alone or combined with 3 days overexposure to CORT. The results showed that CORT shifts the KA response profile approximately 40-fold, since animals treated with a non-toxic dose of 0.01 pg KA and CORT exhibited epileptic activity and a shift on the paired-pulse response similar to that observed in animals treated with high doses of KA (0.4 pg). This synergistic action of CORT on the electrophysiological changes induced by KA was antagonized by the antiglucocorticoid RU486 whereas the antimineralocorticoid spironolactone was ineffective. These results suggest that CORT may play an important role in modulating the severity of KA-induced seizures in the hippocampal structure Keywords:

probably

by GR-receptor

Corticosterone;

Glucocorticoids,

mediated

action.

Kainic acid; Hippocampus;

the steroid

hormones

Epileptic activity;

secreted

by the

adrenal gland during stress, have an important modulatory effect on hippocampal activity [3,8,20]. However, prolonged overexposure to CORT-the naturally occurring

glucocorticoid in rodents-either by exogenous CORT administration or via the chronic stress paradigm, appears to be capable of damaging hippocampal neurons [6,21,24,28]. CORT also seems to control the state of hippocampal vulnerability to a variety of neurological insults produced by antimetabolites and epileptogenic excitotoxins such as kainic acid &A), a structural analog of the excitatory amino-acid glutamate [5,17,22,23]. Indeed, several studies have shown that CORT overexposure exacerbates, while adrenalectomy attenuates, the KA-induced hippocampal neuron loss [22,26]. In the same context, glucocorticoids have also been shown to potentiate KA-induced wet-dog shakes, convulsions and motor seizures [131.

* Corresponding author. Laboratoire de Neurobiologie ments, URA CNRS 372, IBHOP - Traverse Charles Marseille Cedex 13, France. Fax: (33) 91 98 26 97.

des ComporteSusini, 13388

0006-8993/95/$09.50 0 1995 Elsevier Science B.V. AI1 rights reserved SSDI 0006-8993(95)01123-4

RU486; GR-receptor

Previous investigations from Sapolsky and co-workers have shown that energy supplementation and NMDA receptor blockers could prevent CORT potentiation of KAinduced damage in the hippocampus [1,23]. Other studies showed that this CORT effect seems to act directly through CORT interaction with hippocampal glucocorticoidreceptors [16,25]. Nevertheless, a direct effect of CORT on KA-induced electrophysiological alterations in the hippocampus has not yet been established. The present study was conducted to investigate the effect of CORT overexposure on KA-induced electrophysiopathological changes in the hippocampus, in order to characterize the electrophysiological synergic action of CORT and the nature of the type I (MR, mineralocorticoid-receptor) or type II (GR, glucocorticoid-receptor) CORT-receptors (which are co-existing in hippocampal neuron [19]> that is involved. For this purpose, we used extracellular recordings from the CA1 area of hippocampal slices taken from BALB/c mice. This hippocampal area has been characterized by the fact that an epileptiform burst of population spikes develops in response to orthodromic stimulation after KA-induced damage [2,1.5]. Hippocampal slices were taken from mice treated with an

M. Talmi et al. /Brain

98

Research 704 ClYY5) 97-102

intrahippocampal microinfusion either of different doses of KA or of a non-toxic dose of KA combined with an overexposure to CORT with or without an antiglucocorticoid (RU486) or an antimineralocorticoid (spironolactone). Application of toxin and CORT. Kainic acid (KA, Sigma) was dissolved in an acidified saline solution (300 pg of ascorbic acid/l ml of saline solution [22]). Four doses of KA were used : 0.01, 0.04, 0.1 and 0.4 pg. Mice were anesthetized and stereotaxically prepared. KA mi-

croinfusion was made unilaterally into the hippocampus. KA was injected with a Hamilton syringe in a volume of 0.3 ~1 for 1 min, with 1 min allowed for diffusion of toxin before withdrawal. KA control animals were microinfused with 0.3 ~1 of vehicle. CORT/KA animals were assigned to a microinfusion of 0.01 pg KA combined with CORT (Sigma) overexposure for 3 days. The mice were injected subcutaneously (s.c.) daily (24 h before, the same day, and 24 h after KA

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Fig. 1. Effect of CORT overexposure on KA-induced epileptogenic activity in the hippocampus. Examples of field potentials evoked in slices taken from control animals (Al, Bl, Cl) and from 0.4 pg K&treated animals (A2, B2, C2) by different stimulus intensities: 100 /.LA (Al, A2), 50 I.LA (Bl, B2) and 30 PA (Cl, C2). The number of epileptic slices was positively correlated with the dose of KA used (D). The number of slices used was 14 for the control group (8 mice), 18 for the 0.01 pg KA group (9 mice), 16 for the 0.04 pg KA group (9 mice), 15 for the 0.1 pg KA group (8 mice) and 18 for the 0.4 wg KA group (10 mice). E shows that the presence of a high dose of CORT (20 mg/kg/day for 3 days) in mice when 0.01 pg KA was microinfused (CORT/O.Ol Kg KA, n = 17 slices, 10 mice) significantly increased the number of epileptic slices obtained for these mice. This synergistic action of CORT was antagonized by antiglucocorticoid RU486 treatment (RU486/CORT/0.01 yg KA group, n = 16 slices, 9 mice), whereas antimineralocorticoid treatment was ineffective (SPI/CORT/O.Ol pg KA, n = 12 slices, 8 mice). * P < 0.05, * * P < 0.001, * * * P < 0.0001 Significant difference from controls by one-tailed z-test. a P < 0.02, h P < 0.0001 Significant difference by one-tailed z-test between two proportions.

M. Talk

et al. /Brain

99

Research 704 (1995) 97-102

microinfusion) with a dose of 20 mg/kg of CORT suspended in 0.1 ml of sesame oil. Such an injection in rodents produces prolonged elevation in circulating CORT levels equivalent to those observed during stress [21]. CORT control animals were injected with CORT (20 mg/kg) only. No significant difference was observed between the ISA control and CORT control groups (data not shown), so these two groups of mice were pooled into one group of control animals. In addition to CORT/O.Ol pg KA treatment, the last group of mice used in our experiments received a daily administration of RU486 (Roussel Uclaf) or spironolactone (Sigma), at a dose of 20 mg/kg, for 3 days. Electrophysiological methods. After a 7-day recovery

period following the KA microinfusion, the animals were decapitated and slices were prepared from the injected hippocampal side. For a given mouse, at most two slices were examined. The methods used for slice preparation and the standard electrophysiological techniques have already been described in detail [20]. Briefly, the slices were fully submerged in an artificial cerebrospinal fluid aerated with a gas mixture of 95% 0, and 5% CO, (pH 7.35) and maintained at 34°C. The ACSF composition was in mM: 124 NaCl, 4.96 KCl, 1.25 Kh,Po,, 1.5 MgSo,, 25.7 NaHCo,, 2.5 CaCl,, 10 p-glucose. The stratum radiatum stimulation lasted 0.15 ms and consisted of constant current pulses at 0.3 Hz with intensities ranging from 30 to

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Fig. 2. Effect of CORT overexposure on KAinduced shift on the paired-pulse response. Examples of paired-pulse responses observed in control mice (A) and in 0.4 pg KA-treated mice (B). C shows variation of the paired-pulse response [(At - Ac)/Ac, 9’ D1 as a function of stimulus intensity in the control and in the 0.4 pg KA-treated animals, with an important shift of the response from inhibition to facilitation at a stimulus intensity of 30 PA. Dose-effect of KA on the paired-pulse response recorded at 30 FA (D). The paired-pulse inhibition observed in slices taken from 0.01 pg KA-treated animals (similar to the one recorded in control mice) shifted to paired-pulse facilitation when this non-toxic dose of KA was microinfused in mice overexposed to a high dose of CORT (E). This phenomenon was reversed by RU 486 treatment, whereas spironolactone had no effect. a P < 0.05; ’ P < 0.001 Significant difference from controls by Student’s unpaired f-test. * P < 0.05, * * P < 0.01, * * * P < 0.001 Significant difference of At vs. AC by Student’s paired f-test. (Y P < 0.001 Significant difference from 0.01 pg KAtreated animals by Student’s unpaired r-test.

100

M. Talmi et d/Brain

100 PA. Paired-pulse stimuli were presented at a 20 ms interpulse interval. At this interpulse interval, a paired-pulse inhibition or facilitation, depending on stimulus intensity and on cell excitability [ll], could be observed. Extracellular recordings were made into the cell body layer. The response recorded correspond to a negative field potential, classically termed population spike (PS) which is superimposed to a positive field EPSP. Ten evoked responses were averaged and quantified by measuring the PS amplitude determined by the voltage difference between the negative peak and the positive waveform maximum just after the appearance of the spike. For paired-pulse stimulation, the PS amplitude was measured for the conditioning stimulus (AC) and for the test stimulus (At). The paired-pulse response was defined as the percentage of variation in the test response amplitude vs the conditioning response amplitude [(At - Ac)/Ac, %I. Statistics. A one - tailed z-test for the significance of the difference between two proportions, and paired and Student’s unpaired t-tests were used for statistical analysis. The one-tailed z-test was used for pairwise comparisons of the percentages of epileptic slices observed in the different experimental groups. Student’s paired t-test was used for the comparison of the test response amplitude (At) vs. the conditioning response amplitude (AC) recorded in the same slices. The unpaired Student’s t-test allowed to compare the paired-pulse response [(At - Ac)/Ac] obtained in slices taken from the different experimental groups. Effects were considered significant when P < 0.05. Dose-related effect of KA microinfusions on hippocampal slices activity. The results obtained shows that in slices taken from 0.4 pg KA-treated mice stimulation of the Schaffer-commissural afferents evoked a response which often (17 out of 18 slices were epileptic, 94%) triggered multiple action potentials corresponding to multiple extracellular PS (Fig. 1). This response was defined in our experiments as an ‘epileptic’ activity (epileptic slice). Slices taken from control mice never developed status epilepticus (14 slices tested). The number of bursts observed in the epileptic slices depended on the stimulus intensity used, and varied between 0 and 4 after-discharges. The maximal number (4 bursts) appeared for a stimulation around 3 times the threshold intensity (SO-100 PA: Fig. 1, A2). The ability to induce an epileptic activity, assessed in our experimental conditions by the number of epileptic slices, was positively correlated with the dose of KA used (Fig. 1D). As a matter of fact, in 0.01 pg KA-treated mice, only one slice out of 18 tested (5%, z (in comparison to controls) = 0.83, ns) showed a bursting discharge, whereas in 0.04 pg and 0.1 pg KA-treated animals, 25% (4/16 of the slices tested, z = 2.08, P < 0.04) and 66% (lo/15 of the slices tested, z = 3.75, P < 0.001) respectively, were epileptic. Finally, in 0.4 pg KA-treated mice, nearly all slices (17/18 of the slices tested, 94%, z = 5.28, P < 0.000) exhibited an epileptiform discharge. The effect of KA treatment was also manifested on slice

Research 704 (1995) 97-102

electrical activity by an important shift on the paired-pulse response evoked by stimulation at an intensity around the threshold (30 PA) (Fig. 2A, B, C). The shift observed here on the paired-pulse response from inhibition in control mice to facilitation in KA-treated mice also seems to be dose-dependent (Fig. 2D). Indeed, paired-pulse facilitation also appeared at 30 PA, but moderately, in slices taken from 0.04 and 0.1 pg KA-treated animals ( + 98%, P < 0.05; + 118%, P < 0.03, respectively). In contrast, recordings on slices taken from 0.01 Fug KA-treated animals showed an inhibition of 22% (P < 0.05) which was not different than that obtained in slices taken from control mice (- 18%, t,, (control X 0.01 pg KA) = 1.28, ns). Effect of CORT overexposure on KA-induced electrophysiological changes. The lowest dose of KA used (0.01 pg) seems to be non-toxic, since animals treated with this dose did not exhibit any significant differences from the control group (Fig. 1D and Fig. 2D). Interestingly, the results described in Fig. 1E and Fig. 2E show that overexposure to a high dose of CORT (20 mg/kg) over a 3-day period severely increased hippocampal sensitivity to this 0.01 pg KA infusion. Indeed, slices taken from mice treated with a dose of 0.01 kg of KA combined with overexposure to CORT exhibited almost the same electrophysiological perturbations as mice treated with high doses of KA (0.1 or 0.4 wg). In fact, 70% (12/17) of the slices taken from CORT/O.Ol pg KA-treated mice developed epileptic activity (vs. 5% in 0.01 pg KA-treated mice, z = 3.89, P < 0.0001). Furthermore, the paired-pulse response observed in the slices taken from these CORT/O.Ol pg KA-treated mice ( + 179%, t,, (At X AC) = 3.03, P < 0.01) corresponded to as strong a facilitation as the one observed in mice treated with the high dose of 0.4 pg IL4 (t3, (CORT/O.Ol pg KA X 0.4 pg KA) = 1.58, ns). The electrophysiological synergy demonstrated here between KA and CORT was then investigated under the same experimental conditions, but in the co-presence of an antiglucocorticoid receptor (RU486) or an antimineralocorticoid receptor (spironolactone:SPI). This investigation showed that RU486 administration reduced the synergistic ability of CORT to potentiate the development of the electrophysiopathological effect of KA. Thus, the percentage of epileptic slices (26%, 4/16) observed in the RU486/CORT/0.01 pg KA-treated mice was not significantly different from the one observed in 0.01 pg KAtreated mice (z = 1.68, ns). Furthermore, the paired-pulse response observed in these mice (inhibition of - 20%) was not significantly different from the response obtained in the 0.01 pg KA group or in the control group (t,, (vs. control) = 1.08, ns). However, in the SPI/CORT/O.Ol pg KA-treated mice, treatment with spironolactone did not lead to any important difference relative to the electrophysiological perturbation observed in CORT/O.Ol pg KAtreated animals. Accordingly, 75% (9/12) of the slices taken from this group (SPI/CORT/O.Ol pg KA) showed

M. Talk et al./Brain

epileptiform activity and the paired-pulse response recorded on these slices (facilitation of + 170%) was similar to the one already reported in the CORT/O.Ol pg KA-group. The present study shows that overexposure to CORT heightens the electrophysiopathological effects, including the development of epileptogenic activity, induced after KA administration. Our results also showed that this synergistic action of CORT can be blocked by RU 486, suggesting substantial involvement of type II GR-receptor mediation. The epileptiform activity described here in response to orthodromic activation in KA-treated mice has often been reported in the literature, both in vivo and in vitro, in KA-lesioned hippocampus after systemic or intracerebral application of KA [2,15]. Furthermore, the ability to induce this abnormal activity, as assessed by the number of epiletiic slices in each group of KA-treated animals, seems to be positively correlated with the dose of KA microinfused. This result appears to be consistent with other studies showing a dose-relationship of KA-induced histopathological, neurochemical, and behavioral changes [ 14,271. Elevation of CORT concentrations for 24 h before and after the IL4 microinfusion significantly potentiated the abnormal activity produced by KA. Thus, in animals overexposed to high levels of CORT, the non-toxic 0.01 pg dose of KA produced the same amount of epileptogenic activity as that observed in animals microinfused with a high dose of KA (0.4 pg). Interestingly, the CORT-augmented KA-induced epileptic activity was also associated with a shift in paired-pulse inhibition and a manifestation of paired-pulse facilitation indicating a loss of the intrinsic mechanism of inhibition [ll]. This observation suggests that CORT increases the sensitivity of hippocampal neurons to the specific action mechanism of KA. The described synergistic action of CORT on the electrophysiological changes induced by KA could be related to the results obtained in the recent work by Lee and co-workers [13], who showed that glucocorticoids potentiate KA-induced seizures and wet-dog shakes. These authors used systemic injection of KA. However, the seizures described in their experiment originated within the hippocampus. Our experiment using intrahippocampal microinfusion of KA could provide evidence for specific and direct synergistic action of CORT on KA-induced abnormal electric activity within the hippocampus. CORT is unlikely to potentiate KA-induced pathological effects by influencing IL4 distribution or receptor affinity [22]. Recent reports have shown that CORT-induced impairment of calcium regulation in hippocampal neurons may be the prominent mechanism underlaying neuronal vulnerability and toxicity following CORT treatment and stress [4]. CORT enhances and prolongs the elevation of intracellular calcium concentration in response to IL4 in hippocampal neurons. CORT may act through two mechanisms, either by exacerbating the pathogenic

Research 704 (1995) 97-102

101

glutamate/calcium cascade principally via NMDA receptors [l] and/or by enhancing voltage-dependent Ca2+ conductance [lo]. Critically, in all of these instances, the CORT effect seems to be energy-dependent since CORT could inhibit glucose utilisation and transport in the hippocampus [7,9], in that CORT effects can be reversed by supplementation with excess sugars [23]. Our results show that the synergistic action of CORT on the KA-induced electrophysiopathological changes described here can be reversed by the antiglucocorticoid RU 486. Indeed, the increase in abnormal electrical activity (epileptogenic activity) produced by CORT in the KAtreated mice was antagonized by RU 486 treatment. However, under the same conditions, antimineralocorticoid spinolactone was ineffective. These observations suggest substantial GR-receptor-mediated action in this synergistic effect of CORT. Previous histological studies have shown that glucocorticoid removal by surgical adrenalectomy or chemical metyrapone treatment reduces CORT-induced hippocampal vulnerability and neurotoxicity [12,24,26]. The present report shows that a specific blockade of the GR-receptor-mediated effect was protective, and reduced CORT-induced hippocampal sensitivity to the electrophysiopathological changes produced by KA. Indeed, it has been well established that the type of RU486 administration used here blocks the effects of pharmacological doses of glucocorticoid, mediated by GR-receptors, both at the peripheral and central levels [18]. These results are in line with our previous study showing that chronic RU486 treatment from mid-age to senescence may prevent age-related CORT neurotoxicity and electrophysiological alterations in the hippocampus [29]. In summary, our results show that hippocampal seizure and epileptogenic activity induced by neurotoxins are under the control of the pituitary-adrenal function. Excessive CORT secretion may potentiate this abnormal hippocampal activity, whereas RU486 may prevent such potentiation. Thus, RU486 used efficiently in other clinical contexts might prove to have neuroprotective effects on the brain following epileptogenic neurotoxin insults.

Acknowledgements We are grateful to Michelle BAUGET for her secretarial assistance, and to Vivian WALTZ for her helpful English language assistance. This work was supported by a grant from the INSERM, CRE 910813. Our thanks to Roussel Uclaf, Romainville, France, for providing the RU486.

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