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Glucocorticoid treatment increases the ability of CRH to induce seizures
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~
~,
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ELSEVIER
Neuroscience Letters 174 (1994) 113-116
NEUROSCIENCE lITTERS
Glucocorticoid treatment increases the ability of CRH to induce seizures Jeffrey B. Rosen a'*, Shervin K. Pishevar b, Susan R.B. Weiss a, Mark A. Smith a, Mitchel A. Kling b, Philip W. Gold b, Jay Schulkin b aBiological Psychiatry Branch, Bldg. 10, Ran. 3N212 and bClinical Neuroendocrinology Branch, National Institute of Mental Health, Bethesda, MD 20892, USA
Received 14 October 1993; Revisedversionreceived 17 February 1994; Accepted 12 April 1994
Abstract We examined whether glucocorticoids could enhance the ability of corticotropin-releasing hormone (CRH) to induce seizures. Rats were treated with systemicglucocorticoids (dexamethasone, 100/lg) or vehicle for either 3 days (chronic) or 2 h (acute) before intracerebroventricular CRH (3 or 10gg) or saline injectionsand then monitored for 8 h followingeach injection. Our results suggest that chronic, but not acute, glucocorticoid pretreatment increases the likelihood of CRH-induced seizures. Key words." Glucocorticoid; Corticotropin-releasing hormone; Seizure; Amygdala
Intracerebrally applied corticotropin-releasing hormone (CRH) has been shown to produce arousal, fear and anxiety related behaviors, and, at higher doses, to induce seizures [3,6,7,14,15]. Glucocorticoids are known to influence CRH in brain; they inhibit the expression of CRH and CRH mRNA in the paraventricular nucleus of the hypothalamus, while increasing CRH mRNA in the central nucleus of the amygdala [8,11]. Glucocorticoids also facilitate cocaine-induced kindling and amphetamine-induced self-administration [5,10]. Elimination of glucocorticoids by adrenalectomy does not seem to markedly affect amygdala-kindled seizures [1,9,12]. However, this does not imply that glucocorticoids cannot play some role in the modulation of seizures. For example, Lewis rats which have low levels of central CRH and low systemic corticosterone (Cort) take longer to kindle through stage II seizures. Moreover, treatment with Cort accelerated the rate of kindling in these rats[13]. One prediction is that glucocorticoids might reduce CRH-induced seizures by restraining CRH synthesis in the paraventricular nucleus of the hypothalamus (PVN). However, because glucocorticoids can increase kindled
*Correspondingauthor. Fax: (1) (301) 402-0052. 0304-3940/94l$7.00 © 1994ElsevierScienceIreland Ltd. All rights reserved
seizures induced by cocaine, accelerate amygdala kindling in Lewis rats, and increase CRH mRNA levels in the central nucleus of the amygdala, we thought that glucocorticoids might also facilitate the ability of CRH to induce seizures. Whether chronic administration (three days) of glucocorticoids is necessary for an effect or acute administration (2 h before CRH) is sufficient to increase CRH's ability to induce seizures was also examined. Male Sprague-Dawley rats weighing about 350 g at the beginning of the experiment were used. They were housed in group cages until surgery, and then individually throughout the remainder of the experiment. Rats were kept on a 12/12 h day/night cycle and tested during the day. Rats were given Purina Rat Chow and water ad libitum throughout the experiment. Rats were anesthetized with 5 ml/kg, i.p., of 8% chloral hydrate. A 25-gauge stainless steel cannula was stereotaxically implanted into the right lateral ventricle. Rats were then returned to their individual cages and allowed one week to recover before the onset of the experiment. In experiment 1, eight rats received 100/tg s.c./day of dexamethasone (Dex; Sigma Chemicals), suspended in sesame oil, for 3 days before testing, and 2 h before each intracerebral injection of CRH. Vehicle pretreated rats (n = 7) received 0.1 ml s.c. sesame oil on the same regimen as the Dex groups.
114
J.B. Rosen et aL INeuroscience Letters 174 (1994) 113 116
lOO.
80. 0 ¢f) J:
80.
m. al n.
40.
[]
Vehlcle-CRH
[]
DEX-CRH
0 a.
Day 1
Day 2
100.
80 O (n
6O []
m I ri-
Vehlcle-CRH
I~J. DEX-CRH
b O ¢ O O O.
2O
0 Day 1
Day 2
Fig. 1. The percent of rats having seizures following C R H either with or without Dex. Data shown are from days 1 and 2 of the first (upper graph) and second experiment (lower graph). *Denotes that Dex-CRH treatment induced significantly more seizures than the Vehicle-CRH treatment.
ioral observation. These injections were given for two consecutive days. In experiment 2, the Dex-CRH regimen was the same as in experiment 1. However, since all of the Veh-CRH rats in the first experiment exhibited seizures on day 1, we decided to lower the dose of CRH in some animals to potentiate our ability to detect an enhancement of CRH's effects by Dex. Two doses of CRH were given: three Dex-treated and two vehicle-treated rats were given 3/~g (0.6/lg//ll) CRH i.c.v. Six other Dex-treated rats and six vehicle-treated rats were given 10/lg (2/Jg//ll) of CRH i.c.v. Two Dex-treated and two vehicle-treated rats were injected with 10/~1 saline i.c.v. In experiment 3, rats were pretreated for three days with vehicle (0.1 ml s.c. sesame oil), then received 100/~g/rat/day of Dex (n = 8) or Veh (n = 8) 2 h before each 3 ~tg (0.6/1g//11) injection of i.c.v. CRH. At least two trained observers, blind to which rats received CRH, rated CRH-induced behaviors for 8 h. Seizures were monitored by noting unilateral or bilateral forepaw clonus; and bilateral forelimb clonus with rearing and falling. Other behaviors monitored were the number of wet shakes and aggressive behavior occurrences. Wet shakes were monitored because they are consistently observed following i.c.v, administration of C R H at the doses used in the present study. Two different forms of defensive aggression were measured: the first being conspecific aggression and the second being resistance to capture. Conspecific aggression is fighting between a pair of animals, where the aggressor stands with its hind limbs and dominates the second animal which lies on its back on the cage bottom. The second form of aggression consisted of picking up the animal every hour over the 8 h period and noting its level of resistance to capture on a 4-point scale: 0 = lack of resistance; 1 = animal shies away when 40m Q .lg m tt~
Previous studies have shown that moderate to high doses (10-100 Ftg) of CRH, injected intracerebroventricularly, can induce late-onset seizures [3,15]. Thus, to investigate whether Dex would increase the likelihood of CRH-induced seizures, low to moderate doses of C R H were used. Following 3 days of Dex pretreatment, 10/zg (2 ~tg/Ftl) of rat CRH (Pennisula Laboratories) were injected into the ventricle (i.c.v.) through PE 20 tubing attached to the cannula in four of the Dex-treated and three of the vehicle-treated rats. To be certain that all of the CRH entered the ventricle, the cannula was flushed with 5/tl of saline. The four other Dex-treated and four vehicle-treated rats received 10/~1 of saline i.c.v. Two rats were then placed together in a plexiglas cage for behav-
30.
| "°
20
[] Vehide
c
~
lO
I! o Saline
CRH
Fig. 2. Median number of wet shakes on the first day (experiment l) following saline or C R H either with or without Dex. C R H treatment significantly increased the number of wet shakes in both the vehicle and dexamethasone pretreated rats (n = 9.5, P < 0.03).
J.B. Rosen et al./Neuroscience Letters 174 (1994) 113-116 3.
8~
0
,~- O 0 I= 7-, L. m
I"1 Vehicle
[]OEX
Eo
I
I Saline
CRH
Fig. 3. Median number of 3 and 4 ratings of resistance to capture on the first day (experiment 1) following saline or CRH either with or without Dex. C R H treatment significantly increased the number o f high resistance to capture ratings in both the vehicle and dexamethasone pretreated rats (n = 7, P < 0.02).
grasped; 2 = animal avoids hand by running, or struggles when captured, or both; 3 = animal leaps to avoid capture and struggles vigorously; 4 = animal leaps, struggles and bites. Ratings of 3 and 4 were considered to be greater than normal aggressive responses. The number of wet shakes and resistance to capture ratings of 3 and 4 were counted for each rat and used to perform statistical analyses with Kruskal-Wallis and Mann-Whitney U-tests. In experiment 1 on day 1, most of the rats that received C R H (10 ¢tg) had seizures irrespective of their pretreatment (3 out of 4 in the Dex-CRH group, 4 out of 4 in the vehicle-CRH group). The latencies to seizures were also not different. However, on day 2 all the rats that received Dex had seizures, while only one of 4 vehicletreated rats had seizures 0(2 = 8.4, P < 0.004) (Fig. 1). None of saline-injected rats that received vehicle or Dex had seizures on either day. C R H treated rats had significantly greater numbers of wet shakes than saline injected rats on each day of C R H treatment. However, there were no differences between the Dex and vehicle treated rats that were given CRH. The results from the first day of testing are presented in Fig. 2. A similar pattern was revealed for one of the measures of aggression: C R H injected rats had more resistance to capture ratings of 3 and 4 than saline injected rats on the first day of testing (n = 7, P < 0.02), whereas the Dex and vehicle treated rats given C R H were not different on these measures (Fig. 3). On the second day of C R H administration, the C R H injected rats' levels of resistance to capture diminished and did not differ from saline injected controls (data not shown). For conspecific
115
aggression, no differences were observed between any of the groups on any day. The above results of CRH-induced seizures were extended by looking at a new group of rats (experiment 2). In this replication on day 1, 5 out of 9 (4 out of 6 at 10 ¢tg and 1 out of 3 at 3/tg), and on day 2, 5 out o f 8 (3 out of 5 at 10 p g and 2 out of 3 at 3 ~tg) Dex-CRH treated rats had seizures, while none of the vehicle-CRH treated rats had seizures (0 out o f 6), Z 2 = 5.0, P < 0.025 for day 1, a n d z 2 = 5.1, P < 0.025 for day 2 (Fig. 1). No dose- related difference was found; both 3 and 10 fig o f C R H had the same effect. None of the saline alone or Dex alone treated rats had seizures. Reasons for the increased sensitivity of the rats to C R H in the first set of animals compared to the second set are unclear. One possible explanation for this discrepancy is that rats in the second study were moved to a different housing room just prior to their first Dex injection. However, despite the variability in C R H convulsive responsivity, the effect of Dex was to enhance the frequency of seizures in both studies. The same pattern of results in experiment 2 was seen for wet shakes and aggression as described for experiment 1; that is, C R H increased the number of wet shakes and resistance to capture responses without an effect of Dex pretreatment (data not shown). There were no differences between 3 and 10 ¢tg CRH. In experiment 3, there were no differences in the responses to C R H on any of the measures between rats given acute administration of Dex (2 h before C R H injections) and vehicle-treated groups. Only 1 out of 8 Dextreated rats and 1 out of 8 vehicle-treated rats had seizures. Therefore, chronic pretreatment (3 days) with Dex seemed to be necessary to increase the likelihood of CRH-induced seizures. Results of our first experiment suggested that Dex increased CRH's ability to induce seizures insofar as only the Dex pretreated rats had seizures on the second day. Rapid tolerance to repeated C R H was also found with the resistance to capture measure and has been shown before [15]. However, pretreatment with Dex may have slowed or inhibited tolerance development to CRHinduced seizures. Experiment 2 demonstrated that Dex pretreatment increased C R H seizures on both day 1 and day 2. Taken together these results suggest that Dex increases the ability of C R H to induce seizures. Our inability to replicate previously published CRHinduced conspecific aggression [15] may be due to the lower doses of C R H used in this study. Nevertheless, the fact that Dex increased CRH's ability to induce seizures but not CRH-induced wet shakes and resistance to capture suggests that the Dex effect may be specific to CRHinduced seizures and not to some other CRH-induced behaviors. Thus, the facilitatory interaction between Dex and C R H must be considered cautiously, and may not be generalizable to other behavioral or physiological ef-
116
J.B. Rosen et al./Neuroscience Letters 174 (1994) 113 116
fects. However, recent work demonstrates that glucocorticoids facilitate the effects of CRH on acoustic startle (Lee, Davis, Gold, Schulkin, in preparation), suggesting that glucocorticoids may have more general facilitatory effects on CRH-induced behaviors, and not only on seizures. Similar to our results, kindling with cocaine occurred more rapidly with chronic glucocorticoid treatment [5]. Recently, glucocorticoids have been shown to increase CRH mRNA levels in the central nucleus of the amygdala after 3 days, but not one day, of pretreatment [8]. The amygdala plays a central role in kindled and other types of seizures [4]. The enhanced seizures by 3 days of glucocorticoid pretreatment in the present study may reflect increased CRH synthesis in the central nucleus of the amygdala. Finally, these results are contrary to the prevalent notion that glucocorticoids inhibit CRH regulated functions [2]; we demonstrate that they may enhance the ability of CRH to induce seizures. [1] Cottrell, G.A., Nyakas, C., DeKloet, E.R. and Bohus, B., Hippocampal kindling: corticosterone modulation of induced seizures, Brain Res., 309 (1984) 377-381. [2] Dallman, M.F., Akana, S.F., Cascio, C.S., Darlington, D.N.. Jacobson, L. and Levin, N., Regulation of ACTH secretion: variations on a theme of B, Recent Prog. Horm. Res., 43 (1987) 113-173. [3] Ehlers, C.L., Henriksen, S.J., Wang, M., Rivier, J., Vale, W. and Bloom, F.E., Corticotropin releasing factor produces increases in brain excitability and convulsive seizures in rats, Brain Res., 278 (1983) 332-336. [4] Goddard, L.S., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. [5] Kling, M.A., Glowa, J.R., Pluznik, D., Demas, J., DeBellis, M.D., Gold, P.W. and Schulkin, J., Facilitation of cocaine-kindling by glucocorticoids in rats, Brain Res., 629 (1993) 163 166.
[6] Koob, G.F., Heinrichs, S.C., Pich, E.M., Menzaghi, F., Baldwin, H., Miczek, K. and Britton, K.T., The role of corticotropinreleasing factor in behavioural responses to stress, Ciba Found. Symp., 172 (1993) 277-89. [7] Liang, K.C., Melia, K.R., Miserendino, M.J., Falls, W.A., Campeau, S. and Davis, M., Corticotropin-releasing factor: long-lasting facilitation of the acoustic startle reflex, J. Neurosci., 12 (1992) 2303-12. [8] Makino, S., Gold, P.W. and Schulkin, J., Corticosterone effects on CRF mRNA in the central nucleus of the amygdala and the paraventricular nucleus of the hypothalamus, Soc. Neurosci. Abstr., 19 (1993). [9] McIntyre, D.C. and Wann, P.D., Adrenalectomy: protection from kindled convulsion induced dissociation in rats, Physiol. Behav., 20 (1978) 469-474. [10] Piazza, P.V., Maccari, S., Demniere, J.-M., Le Moak M., Mormede, P. and Simon, H., Corticosterone levels determine individual vulnerability to amphetamine self-administration, Proc. Natl. Acad. Sci. USA, 88 (1991) 2088-2092. [11] Swanson, L.W. and Simmons, D.M., Differential steroid hormone and neural influences on peptide mRNA CRH cell of the paraventricular nucleus: a hybridization histochemical study in the rat, J. Comp. Neurol., 285 (1989) 413-425. [12] Weiss, G., Lucero, K., Fernandez, M., Karnaze, D. and Castillo, N., The effect of adrenalectomy on the circadian variation in the rate of kindled seizure development, Brain Res., 612 (1993) 354 356. [13] Weiss, G.K., Castillo, N. and Fernandez, M., Amygdala kindling rate is altered in rats with a deficit in the responsiveness of the hypothalamo-pituitary-adrenal axis, Neurosci. Lett., 157 (1993) 91-94. [14] Weiss, S.R.B., Nierenberg, J., Lewis, R. and Post, R.M., Corticotropin-releasing hormone: Potentiation of cocaine-kindled seizures and lethality, Epilepsia, 33 (1992) 248-254. [15] Weiss, S.R.B., Post, R.M., Gold, P.W., Chrousos, G., Sullivan, T.L., Walker, D. and Pert, A., CRF-induced seizures and behavior: interactions with amygdala kindling, Brain Res., 372 (1986) 345 351.
~
~,
'
~
ELSEVIER
Neuroscience Letters 174 (1994) 113-116
NEUROSCIENCE lITTERS
Glucocorticoid treatment increases the ability of CRH to induce seizures Jeffrey B. Rosen a'*, Shervin K. Pishevar b, Susan R.B. Weiss a, Mark A. Smith a, Mitchel A. Kling b, Philip W. Gold b, Jay Schulkin b aBiological Psychiatry Branch, Bldg. 10, Ran. 3N212 and bClinical Neuroendocrinology Branch, National Institute of Mental Health, Bethesda, MD 20892, USA
Received 14 October 1993; Revisedversionreceived 17 February 1994; Accepted 12 April 1994
Abstract We examined whether glucocorticoids could enhance the ability of corticotropin-releasing hormone (CRH) to induce seizures. Rats were treated with systemicglucocorticoids (dexamethasone, 100/lg) or vehicle for either 3 days (chronic) or 2 h (acute) before intracerebroventricular CRH (3 or 10gg) or saline injectionsand then monitored for 8 h followingeach injection. Our results suggest that chronic, but not acute, glucocorticoid pretreatment increases the likelihood of CRH-induced seizures. Key words." Glucocorticoid; Corticotropin-releasing hormone; Seizure; Amygdala
Intracerebrally applied corticotropin-releasing hormone (CRH) has been shown to produce arousal, fear and anxiety related behaviors, and, at higher doses, to induce seizures [3,6,7,14,15]. Glucocorticoids are known to influence CRH in brain; they inhibit the expression of CRH and CRH mRNA in the paraventricular nucleus of the hypothalamus, while increasing CRH mRNA in the central nucleus of the amygdala [8,11]. Glucocorticoids also facilitate cocaine-induced kindling and amphetamine-induced self-administration [5,10]. Elimination of glucocorticoids by adrenalectomy does not seem to markedly affect amygdala-kindled seizures [1,9,12]. However, this does not imply that glucocorticoids cannot play some role in the modulation of seizures. For example, Lewis rats which have low levels of central CRH and low systemic corticosterone (Cort) take longer to kindle through stage II seizures. Moreover, treatment with Cort accelerated the rate of kindling in these rats[13]. One prediction is that glucocorticoids might reduce CRH-induced seizures by restraining CRH synthesis in the paraventricular nucleus of the hypothalamus (PVN). However, because glucocorticoids can increase kindled
*Correspondingauthor. Fax: (1) (301) 402-0052. 0304-3940/94l$7.00 © 1994ElsevierScienceIreland Ltd. All rights reserved
seizures induced by cocaine, accelerate amygdala kindling in Lewis rats, and increase CRH mRNA levels in the central nucleus of the amygdala, we thought that glucocorticoids might also facilitate the ability of CRH to induce seizures. Whether chronic administration (three days) of glucocorticoids is necessary for an effect or acute administration (2 h before CRH) is sufficient to increase CRH's ability to induce seizures was also examined. Male Sprague-Dawley rats weighing about 350 g at the beginning of the experiment were used. They were housed in group cages until surgery, and then individually throughout the remainder of the experiment. Rats were kept on a 12/12 h day/night cycle and tested during the day. Rats were given Purina Rat Chow and water ad libitum throughout the experiment. Rats were anesthetized with 5 ml/kg, i.p., of 8% chloral hydrate. A 25-gauge stainless steel cannula was stereotaxically implanted into the right lateral ventricle. Rats were then returned to their individual cages and allowed one week to recover before the onset of the experiment. In experiment 1, eight rats received 100/tg s.c./day of dexamethasone (Dex; Sigma Chemicals), suspended in sesame oil, for 3 days before testing, and 2 h before each intracerebral injection of CRH. Vehicle pretreated rats (n = 7) received 0.1 ml s.c. sesame oil on the same regimen as the Dex groups.
114
J.B. Rosen et aL INeuroscience Letters 174 (1994) 113 116
lOO.
80. 0 ¢f) J:
80.
m. al n.
40.
[]
Vehlcle-CRH
[]
DEX-CRH
0 a.
Day 1
Day 2
100.
80 O (n
6O []
m I ri-
Vehlcle-CRH
I~J. DEX-CRH
b O ¢ O O O.
2O
0 Day 1
Day 2
Fig. 1. The percent of rats having seizures following C R H either with or without Dex. Data shown are from days 1 and 2 of the first (upper graph) and second experiment (lower graph). *Denotes that Dex-CRH treatment induced significantly more seizures than the Vehicle-CRH treatment.
ioral observation. These injections were given for two consecutive days. In experiment 2, the Dex-CRH regimen was the same as in experiment 1. However, since all of the Veh-CRH rats in the first experiment exhibited seizures on day 1, we decided to lower the dose of CRH in some animals to potentiate our ability to detect an enhancement of CRH's effects by Dex. Two doses of CRH were given: three Dex-treated and two vehicle-treated rats were given 3/~g (0.6/lg//ll) CRH i.c.v. Six other Dex-treated rats and six vehicle-treated rats were given 10/lg (2/Jg//ll) of CRH i.c.v. Two Dex-treated and two vehicle-treated rats were injected with 10/~1 saline i.c.v. In experiment 3, rats were pretreated for three days with vehicle (0.1 ml s.c. sesame oil), then received 100/~g/rat/day of Dex (n = 8) or Veh (n = 8) 2 h before each 3 ~tg (0.6/1g//11) injection of i.c.v. CRH. At least two trained observers, blind to which rats received CRH, rated CRH-induced behaviors for 8 h. Seizures were monitored by noting unilateral or bilateral forepaw clonus; and bilateral forelimb clonus with rearing and falling. Other behaviors monitored were the number of wet shakes and aggressive behavior occurrences. Wet shakes were monitored because they are consistently observed following i.c.v, administration of C R H at the doses used in the present study. Two different forms of defensive aggression were measured: the first being conspecific aggression and the second being resistance to capture. Conspecific aggression is fighting between a pair of animals, where the aggressor stands with its hind limbs and dominates the second animal which lies on its back on the cage bottom. The second form of aggression consisted of picking up the animal every hour over the 8 h period and noting its level of resistance to capture on a 4-point scale: 0 = lack of resistance; 1 = animal shies away when 40m Q .lg m tt~
Previous studies have shown that moderate to high doses (10-100 Ftg) of CRH, injected intracerebroventricularly, can induce late-onset seizures [3,15]. Thus, to investigate whether Dex would increase the likelihood of CRH-induced seizures, low to moderate doses of C R H were used. Following 3 days of Dex pretreatment, 10/zg (2 ~tg/Ftl) of rat CRH (Pennisula Laboratories) were injected into the ventricle (i.c.v.) through PE 20 tubing attached to the cannula in four of the Dex-treated and three of the vehicle-treated rats. To be certain that all of the CRH entered the ventricle, the cannula was flushed with 5/tl of saline. The four other Dex-treated and four vehicle-treated rats received 10/~1 of saline i.c.v. Two rats were then placed together in a plexiglas cage for behav-
30.
| "°
20
[] Vehide
c
~
lO
I! o Saline
CRH
Fig. 2. Median number of wet shakes on the first day (experiment l) following saline or C R H either with or without Dex. C R H treatment significantly increased the number of wet shakes in both the vehicle and dexamethasone pretreated rats (n = 9.5, P < 0.03).
J.B. Rosen et al./Neuroscience Letters 174 (1994) 113-116 3.
8~
0
,~- O 0 I= 7-, L. m
I"1 Vehicle
[]OEX
Eo
I
I Saline
CRH
Fig. 3. Median number of 3 and 4 ratings of resistance to capture on the first day (experiment 1) following saline or CRH either with or without Dex. C R H treatment significantly increased the number o f high resistance to capture ratings in both the vehicle and dexamethasone pretreated rats (n = 7, P < 0.02).
grasped; 2 = animal avoids hand by running, or struggles when captured, or both; 3 = animal leaps to avoid capture and struggles vigorously; 4 = animal leaps, struggles and bites. Ratings of 3 and 4 were considered to be greater than normal aggressive responses. The number of wet shakes and resistance to capture ratings of 3 and 4 were counted for each rat and used to perform statistical analyses with Kruskal-Wallis and Mann-Whitney U-tests. In experiment 1 on day 1, most of the rats that received C R H (10 ¢tg) had seizures irrespective of their pretreatment (3 out of 4 in the Dex-CRH group, 4 out of 4 in the vehicle-CRH group). The latencies to seizures were also not different. However, on day 2 all the rats that received Dex had seizures, while only one of 4 vehicletreated rats had seizures 0(2 = 8.4, P < 0.004) (Fig. 1). None of saline-injected rats that received vehicle or Dex had seizures on either day. C R H treated rats had significantly greater numbers of wet shakes than saline injected rats on each day of C R H treatment. However, there were no differences between the Dex and vehicle treated rats that were given CRH. The results from the first day of testing are presented in Fig. 2. A similar pattern was revealed for one of the measures of aggression: C R H injected rats had more resistance to capture ratings of 3 and 4 than saline injected rats on the first day of testing (n = 7, P < 0.02), whereas the Dex and vehicle treated rats given C R H were not different on these measures (Fig. 3). On the second day of C R H administration, the C R H injected rats' levels of resistance to capture diminished and did not differ from saline injected controls (data not shown). For conspecific
115
aggression, no differences were observed between any of the groups on any day. The above results of CRH-induced seizures were extended by looking at a new group of rats (experiment 2). In this replication on day 1, 5 out of 9 (4 out of 6 at 10 ¢tg and 1 out of 3 at 3/tg), and on day 2, 5 out o f 8 (3 out of 5 at 10 p g and 2 out of 3 at 3 ~tg) Dex-CRH treated rats had seizures, while none of the vehicle-CRH treated rats had seizures (0 out o f 6), Z 2 = 5.0, P < 0.025 for day 1, a n d z 2 = 5.1, P < 0.025 for day 2 (Fig. 1). No dose- related difference was found; both 3 and 10 fig o f C R H had the same effect. None of the saline alone or Dex alone treated rats had seizures. Reasons for the increased sensitivity of the rats to C R H in the first set of animals compared to the second set are unclear. One possible explanation for this discrepancy is that rats in the second study were moved to a different housing room just prior to their first Dex injection. However, despite the variability in C R H convulsive responsivity, the effect of Dex was to enhance the frequency of seizures in both studies. The same pattern of results in experiment 2 was seen for wet shakes and aggression as described for experiment 1; that is, C R H increased the number of wet shakes and resistance to capture responses without an effect of Dex pretreatment (data not shown). There were no differences between 3 and 10 ¢tg CRH. In experiment 3, there were no differences in the responses to C R H on any of the measures between rats given acute administration of Dex (2 h before C R H injections) and vehicle-treated groups. Only 1 out of 8 Dextreated rats and 1 out of 8 vehicle-treated rats had seizures. Therefore, chronic pretreatment (3 days) with Dex seemed to be necessary to increase the likelihood of CRH-induced seizures. Results of our first experiment suggested that Dex increased CRH's ability to induce seizures insofar as only the Dex pretreated rats had seizures on the second day. Rapid tolerance to repeated C R H was also found with the resistance to capture measure and has been shown before [15]. However, pretreatment with Dex may have slowed or inhibited tolerance development to CRHinduced seizures. Experiment 2 demonstrated that Dex pretreatment increased C R H seizures on both day 1 and day 2. Taken together these results suggest that Dex increases the ability of C R H to induce seizures. Our inability to replicate previously published CRHinduced conspecific aggression [15] may be due to the lower doses of C R H used in this study. Nevertheless, the fact that Dex increased CRH's ability to induce seizures but not CRH-induced wet shakes and resistance to capture suggests that the Dex effect may be specific to CRHinduced seizures and not to some other CRH-induced behaviors. Thus, the facilitatory interaction between Dex and C R H must be considered cautiously, and may not be generalizable to other behavioral or physiological ef-
116
J.B. Rosen et al./Neuroscience Letters 174 (1994) 113 116
fects. However, recent work demonstrates that glucocorticoids facilitate the effects of CRH on acoustic startle (Lee, Davis, Gold, Schulkin, in preparation), suggesting that glucocorticoids may have more general facilitatory effects on CRH-induced behaviors, and not only on seizures. Similar to our results, kindling with cocaine occurred more rapidly with chronic glucocorticoid treatment [5]. Recently, glucocorticoids have been shown to increase CRH mRNA levels in the central nucleus of the amygdala after 3 days, but not one day, of pretreatment [8]. The amygdala plays a central role in kindled and other types of seizures [4]. The enhanced seizures by 3 days of glucocorticoid pretreatment in the present study may reflect increased CRH synthesis in the central nucleus of the amygdala. Finally, these results are contrary to the prevalent notion that glucocorticoids inhibit CRH regulated functions [2]; we demonstrate that they may enhance the ability of CRH to induce seizures. [1] Cottrell, G.A., Nyakas, C., DeKloet, E.R. and Bohus, B., Hippocampal kindling: corticosterone modulation of induced seizures, Brain Res., 309 (1984) 377-381. [2] Dallman, M.F., Akana, S.F., Cascio, C.S., Darlington, D.N.. Jacobson, L. and Levin, N., Regulation of ACTH secretion: variations on a theme of B, Recent Prog. Horm. Res., 43 (1987) 113-173. [3] Ehlers, C.L., Henriksen, S.J., Wang, M., Rivier, J., Vale, W. and Bloom, F.E., Corticotropin releasing factor produces increases in brain excitability and convulsive seizures in rats, Brain Res., 278 (1983) 332-336. [4] Goddard, L.S., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. [5] Kling, M.A., Glowa, J.R., Pluznik, D., Demas, J., DeBellis, M.D., Gold, P.W. and Schulkin, J., Facilitation of cocaine-kindling by glucocorticoids in rats, Brain Res., 629 (1993) 163 166.
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