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Effects of pentylenetetrazole-induced status epilepticus on behavior, emotional memory and learning in immature rats
Epilepsy & Behavior 6 (2005) 537–542 www.elsevier.com/locate/yebeh
Effects of pentylenetetrazole-induced status epilepticus on behavior, emotional memory and learning in immature rats Fu¨sun Erdog˘an a,*, Asuman Go¨lgeli b, Aysßegu¨l Ku¨c¸u¨k b, Fehim Arman a, Yahya Karaman a, Ali Ersoy a a b
Neurology Department, Erciyes University, 38039 Kayseri, Turkey Physiology Department, Erciyes University, 38039 Kayseri, Turkey
Received 27 December 2004; revised 28 February 2005; accepted 1 March 2005 Available online 26 April 2005
Abstract Status epilepticus (SE) can be harmful to the developing brain. Our knowledge of the emotional and behavioral consequences of generalized SE in developing animals remains limited. Therefore, we investigated the short- and long-term effects of pentylenetetrazole (PTZ)-induced SE on emotional memory and learning and behavioral parameters in immature rats. SE was induced in 16- to 20-day-old rats (P16–P20) using intraperitoneal injections of PTZ (n = 21); control rats received saline (n = 10). All animals were tested using an elevated T-maze and open-field test 2, 14, 30, and 180 days after SE, to evaluate emotional memory and learning and behavior. Anxiety levels decreased 2 and 14 days after SE, and conditioned learning of PTZ-treated immature rats was better than that of the control rats. These results indicate that a decreased anxiety level facilitates conditioned learning. Behavioral changes are transient, and no emotional memory or learning deficits occur following PTZ-induced SE in immature rats. 2005 Elsevier Inc. All rights reserved. Keywords: Status epilepticus; Immature brain; Pentylenetetrazole; Emotional memory; Learning; Behavior
1. Introduction Status epilepticus (SE) is more prevalent in children than in adults.The high incidence of seizures in the first decade of life and the propensity of children for febrile seizures and SE are reflective of increased susceptibility of the immature brain to seizures. However, there is ongoing clinical discussion of the possible damage induced by epileptic activity in the brains of infants and children [1,2]. It is known that the immature brain is less vulnerable than the mature brain to seizure-related damage [1,3]. Animal experiments unequivocally demonstrate that electrically or chemically (e.g., kainic acid, pilocarpine, lithium–pilocarpine) induced SE results in im*
Corresponding author. Fax: +90 352 4375285. E-mail address: [email protected] (F. Erdog˘an).
1525-5050/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2005.03.002
paired spatial and emotional learning and memory [4,5]. In these experiments, the model most commonly used was that of temporal lobe epilepsy, and the most commonly tested functions were spatial memory and learning functions [6–18]. In the literature, there is not sufficient information on the effects of generalized SE on emotional memory and learning functions in immature rats. We demonstrated that pentylenetetrazole (PTZ)-induced generalized SE causes transient emotional memory dysfunction in mature rats [19]. As we knew that the effects of seizures may differ between the mature and immature brain, we aimed to investigate the effects of PTZ-induced SE on emotional memory and learning and behavior over short and long periods in immature rats in this follow-up study. PTZ was used because PTZ-induced seizure is a model of primary generalized epilepsy excluding that of genetic origin [20–22].
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To evaluate anxiety and behavioral changes, an openfield test was used. The open-field test is based on the conflict between exploration of a new environment and aversion to open spaces from which escape is prevented by a surrounding wall [23]. This test assesses mainly exploration and fear behaviors [9]. Two paradigms that have been used extensively in studying learning are the conditioned fear task and the Morris water task. In fear conditioning, animals learn to associate neutral stimuli with an aversive stimulus, such as a foot shock. In the spatial learning version of the Morris water task, animals learn to locate a hidden escape platform in a pool of water using various distal visual cues [24]. Memories formed during emotional experiences are stored for future use in similar situations. The expression of emotional response in the presence of the appropriate stimulus serves as a measure of the emotional memory created during the learning experience. Fear conditioning is a powerful procedure for studying the neural basis of emotional memory and of memory in general, because the learning occurs rapidly and the memory is extremely persistent [25]. The elevated T-maze test permits the simultaneous assessment of memory and learning for both conditioned and unconditioned emotional behaviors in the same subject [26,27], so we used the elevated T-maze test to investigate emotional memory and learning functions. As for conditioned learning related to anticipatory anxiety, rats refrain from making certain experimenter-defined responses, and failure to refrain results in punishment. The rats can learn to remain immobile to avoid punishment. Unconditioned learning means learning actively to avoid places or items that are harmful.Learning of this active behavior occurs as a result of innate threats or fears [28].
intraperitoneal injections of PTZ [29,30]. The rats were first injected with 40 mg/kg PTZ, followed 10 minutes later by 20 mg/kg and, subsequently, 10 mg/kg every 10 minutes until SE occurred, a point characterized by a loss of postural control and tonic–clonic seizures. This procedure lasted at least 30 minutes and consisted of prolonged episodes of seizures, interrupted by postictal depression phases with no return to the quadruped posture or to consciousness. Control animals received the same number of saline injections as their paired PTZ-exposed congeners. Open-field and elevated T-maze tests were run on the 2nd, 14th, 30th, and 180th days after SE in both groups. On test days, animals were tested between 8:00 AM and 12:00 noon. 2.1. Open-field test The floor of a square plastic board (100 · 100 cm) with plastic sides (30 cm high) is divided into 16 squares. The rats are individually placed in one corner of the open field and allowed to explore the area freely. The activity level is expressed as the total number of squares crossed, whereas exploratory activity is expressed as the total number of rearings and fear is expressed as the total number of fecal boli during a 5-minute testing period [9,31,29,32]. To interpret open-field test results, data related to these three parameters are combined. Increases in locomotor response and rearings with a decrease in defecation and decreases in locomotor response and rearings with an increase in defecation can be interpreted as less emotional activity and more emotional activity, respectively, in rats [33,34]. The open-field apparatus is cleaned using alcohol before the next animal is introduced, to preclude the possible cuing effects of odors left by previous subjects [35]. 2.2. Elevated T-maze test
2. Methods The experiments were carried out on male Wistar rats, 16–20 days old, weighing 23–50 g. The rats were obtained from the Erciyes University Experimental and Clinical Research Center. The animals were kept on a 12-hour light–dark cycle and allowed free access to food and water. Following SE, the animals were replaced with their mother in their normal environment, and paired controls from the same litter were kept under the same breeding conditions. All experiments were performed between 10:00 AM and 12:00 noon in a silent room at temperatures between 22 and 24 C. The animals were randomly divided into two experimental groups: a control group (n = 10) and a SE group (n = 23). All animals were intact and had no previous experimental history. In the SE group, PTZ was used as the convulsant. To reach SE of controlled intensity and sufficient duration, the animals received repeated
An elevated T-maze consists of three plastic arms of equal dimensions (50 · 12 cm) elevated 50 cm from the floor. One of these arms is enclosed by plastic lateral walls (40 cm high) and is positioned perpendicularly to two opposed open arms. When a rat is placed at the end of the enclosed arm, the open arms are not visible until the rat pokes its head beyond the walls of the closed arm. The time taken to leave this arm with all four paws is then recorded (baseline latency). The animal is then removed from the maze and the same measurement is repeated twice more at 30-second intervals; each timed event occurs over a period of 300 seconds (passive avoidance 1 and 2, inhibitory avoidance, or conditioned learning). This allows the animal, as it explores a maze, to learn inhibitory avoidance if repeatedly placed inside the enclosed arm. Being on an open arm seems to cause a fear-averse experience, as rats have an innate fear of heights and open spaces. After another
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30 seconds, the rat is placed at the end of the right open arm, and it can move toward the enclosed arm (one-way escape, active avoidance, or unconditioned learning). The time taken to leave this arm with all four paws is then recorded (escape latency) [27,26,36]. The animals are reexposed to the situation after an interval and memory of these emotionally related behaviors is assessed [27]. 2.3. Statistical analysis For statistical analysis of the data from both groups, the Mann–Whitney U test was used, and for analysis of the data from repeated tests in the given periods in each group, FriedmannÕs two-way analysis of variance was used. Values are expressed as means ± SEM and significance is defined at P < 0.05 for all tests.
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the number of squares crossed in the SE group on the second day after SE (Mann–Whitney U = 15.5, P = 0.005), and in the number of rearings on the 14th day after SE (Mann–Whitney U = 15, P = 0.004) (Fig. 1). In addition, there was a significant change in the number of fecal boli; the number of fecal boli decreased on the second day after SE (Mann–Whitney U = 24.5, P = 0.02) (Fig. 2). The effects of reexposure to the open field test were evaluated in each group. Significant differences in rearings were noted in the control group with time. The decrease on the 14th day after SE and the increases on the 30th and 180th days were statistically significant. Decreases on the 14th and 30th days after SE and an increase on the 180th day were observed in the SE group, although these were not statistically significant (Fig. 3).
3. Results 3.1. Behavioral features of PTZ-induced seizures Convulsive behaviors were observed with intraperitoneal injection of a 40 mg/kg dose of PTZ in nine rats and of 60 mg/kg in 14 rats. Inhibition of motor activity in a ‘‘no response stage’’ was observed in all rats. With additional successive doses of PTZ, myoclonic jerks were observed, involving predominantly hind limbs; in addition, rearing with clonic movements of forelimbs without loss of postural control was observed in all rats. This kind of seizure occurred one to three times, and duration ranged between 0.5 and 15 minutes. After this stage, rapid backward walking in the presence of myoclonia in the forelimbs was observed in every rat in the PTZ group. During these periods, excessive motor activity and hyperreaction to external stimuli were observed. All rats were very active and irritable. They startled when touched or exposed to noise, and all ran wildly. Subsequently, the rats reached generalized tonic contractions that were characterized by the loss of postural control and strong contractions in the extensor muscles of the forelimbs and hind limbs with cyanosis. All deaths occurred in the tonic stage. After this stage, generalized SE occurred. SE was provoked by intraperitoneal injection of 60–100 mg/kg PTZ. SE occurred at 60 mg/kg in 9 rats, 70 mg/kg in 7 rats, 80 mg/kg in 5 rats, 90 mg/kg in 1 rat, and 100 mg/kg in 1 rat. In all rats, the duration of SE ranged between 30 and 70 minutes, and the 11 surviving rats spontaneously returned to baseline activities. The total mortality rate in PTZ-treated rats was 52%. 3.2. Open-field test Squares crossed, rearings, and fecal boli were counted in 5-minute periods. There was a significant increase in
Fig. 1. Comparison of exploratory activity, motor activity, and fear score on the second day after SE. Each column represents the mean ± SEM; n = 10 for control group and n = 11 for SE group. Rearings: exploratory activity, squares crossed: motor activity, defecation: fear score. Motor activity significantly increased (Mann– Whitney U = 15.5, P = 0.005) and fear score significantly decreased (Mann–Whitney U = 24.5, P = 0.02) in the SE group.
Fig. 2. Comparison of exploratory activity, motor activity, and fear score on the 14th day after SE. Each column represents the mean ± SEM; n = 10 for control and n = 11 for SE group. Rearings: exploratory activity, squares crossed: motor activity, defecation: fear score. Exploratory activity significantly increased in the SE group (Mann–Whitney U = 15, P = 0.004).
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Fig. 3. Change in the number of rearings over short and long periods after SE in the control and SE groups. The decrease on the 14th day after SE and increases on the 30th and 180th days were statistically significant (Friedmann test, x2 = 15). Decreases on the 14th and 30th days after SE and an increase on the 180th day were seen in the SE Group although it was not statistically significant.
Fig. 4. Comparison of elevated T-maze baseline latencies in the control and SE groups on the 2nd, 14th, 30th, and 180th days after SE. Each column represents the mean ± SEM; n = 10 for the control group and n = 11 for the SE group. Baseline latency significantly decreased in the SE group 2 days after SE (Mann–Whitney U = 27.5, P = 0.026).
3.3. Elevated T-maze test Inhibitory avoidance latencies (passive avoidance or conditioned learning) and one-way escape latency (active avoidance or unconditioned learning) were evaluated with the elevated T-maze test. The only significant difference between the two groups was in baseline latencies, which were significantly short in the SE group 2 days after SE (Mann–Whitney U = 27.5, P = 0.026) (Fig. 4). The effects of reexposure to the T-maze were evaluated in each group. There were no statistically significant changes on reexposure.
4. Discussion The purpose of the present study was to investigate the long- and short-term effects of PTZ-induced SE on behavior and emotional memory and learning functions in the immature brain. The behavioral changes of rats
were evaluated using an open-field test. Emotional activity was found to be significantly different in the SE group a short time after SE. The number of squares crossed increased on the second day after SE and the number of rearings increased on the 14th day after SE in the open-field test. Significant differences in the number of fecal boli were also noted between the two groups, and the number of fecal boli decreased 2 days after SE in the SE group. These parameters indicate that in PTZtreated immature rats, fear and anxiety are significantly reduced a short time after SE. It was observed that the control rats were more hesitant to explore in the open-field test, which is an adaptive response, while PTZ-treated rats lacked this restraint [9]. An open field is a novel environment that induces anxiety and exploratory activity in normal rats [9,33,37]. The behavior of an animal in the open field involves integration of complex polymodal sensory information into motor output and must be mediated at several levels. The hippocampus is essential in several aspects of open-field behavior [9,37]. Hippocampal damage may release natural inhibitory tendencies, which may be an adaptive response in normal rats [9,33]. It has been demonstrated that open-field exploratory activity increases after kainic acid-induced SE as a result of hippocampal damage in both mature and immature rats [9,7]. Lithium chloride–pilocarpine-induced seizures cause hyperactivity in P12 rats [38]. Furthermore, it has been demonstrated that hippocampal neuronal loss and open-field motor activity decrease after PTZ kindling in mature rats over long periods [22]. Electrical kindling in mature rats, [37] and recurrent PTZ-induced seizures [31] in immature rats do not have an effect on a open-field exploratory activity. In genetically epilepsy-prone rats, frequent and brief seizures cause less motor activity in the open-field test [39]. No difference, in terms of motor and exploratory activity, was noted between PTZ-treated and control mature rats in the open-field test [19]. All these studies show that behavioral changes are related to the age of rats and type of seizure. Our findings suggest that generalized SE causes transient hyperactivity and reduced anxiety and, possibly, transitory hippocampal effects in developing brains, whereas such effects are not seen in mature rats. We observed that there were significant changes with respect to rearings in the control group with time. The decrease on the 14th day after SE and increases on the 30th and 180th days were statistically significant. Decreases on the 14th and 30th days after SE and an increase on the 180th day, although not statistically significant, were observed in the SE group. In the control group, repeated trials caused a decrease in open-field activity directed toward searching the environment. This is considered an adaptive response [9]. It seems that PTZ-induced SE impairs this adaptive response in immature rats, but not in mature rats [19].
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We subjected immature rats to the elevated T-maze test to evaluate emotional memory and learning performance. This experimental model allows the parallel measurement of responses related to both conditioned and unconditioned fear in the same subject. It also permits simultaneous assessment of memory and learning of these behaviors [35,40]. There was only one significant difference in baseline latencies between the two groups: baseline latencies were significantly short in the SE group 2 days after SE. PTZ-treated rats discovered the exit from the closed arm more easily in the elevated T-maze. Open-field test results indicated that PTZ-treated rats were less fearful 2 days after SE in the open-field test, suggesting that their decreased anxiety causes high motor activity and a reduction in baseline latency in the elevated T-maze test 2 days after SE. Our results demonstrate that immature rats are hyperactive and disinhibited from exhibiting increased exploration in the open field and also have a shortened baseline latency 2 days after SE. There are no emotional memory or learning functions in the immature rats. We demonstrated that PTZ-induced SE causes transient emotional memory deficit in mature rats in the previous study [19]. Thus, we conclude that PTZ-induced SE affects mature and immature brain differently, and the effects of PTZ-induced generalized SE are age dependent. SE-related learning impairments, mostly those related to spatial learning, have been widely investigated. Studies in healthy adult animals indicate that SE causes longterm deficits in spatial learning and memory. Studies of spatial memory after SE in immature animals have produced controversial data [9,10,16,38]. In these studies, models in which SE is induced by kainic acid and lithium chloride–pilocarpine were used. Kubova et al. concluded that the severity of SE-induced impairment in hippocampus-dependent spatial learning increases with maturation of the brain [38]. It has been shown that PTZ kindling in mature rats and repeated seizures in immature rats cause behavioral changes and spatial learning and memory impairments. These changes are permanent when they are accompanied by hippocampal damage [22,23,31,41– 44]. No histological abnormality or behavioral changes were detected by de Feo et al. [45] after PTZ-induced SE in P10 rats. Nehlig et al. [30] also reported low metabolic and blood flow activity in the cortical, hippocampal, and sensory areas, although there is no neuronal death in P21 rats after PTZ-induced SE. Holmes et al. [21] indicated that during development, recurrent PTZinduced seizures result in granule cell neurogenesis without loss of principal neurons, and recurrent seizures can result in significant alterations in cell number and axonal growth. They pointed out that the relationship between these morphologic changes after seizures during development and subsequent cognitive impairment is not yet clear. Huang et al. [31] demonstrated that PTZ-induced recurrent seizures in the immature brain cause neuronal
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circuit abnormalities in the hippocampus, in addition to long-term cognitive deficits. There have been no studies on emotional memory and learning after PTZ-induced SE in immature rats; however, a lack of histologic aspects is a limitation of the present study. The neuronal circuit underlying fear conditioning has been implicated in emotional disorders in humans [25]. Therefore, studies including SE-related behavior and emotional memory and learning functions lead to an understanding of behavioral and emotional consequences of SE. The present study demonstrates that PTZ-induced SE inhibits natural anxiety reactions and, in turn, causes hyperactivity and disinhibition in a short period. The behavioral changes are mostly transient, with no long-term emotional memory or learning deficits after PTZ-induced SE in the developing brain.
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[15] Balakrishnan S, Nidhi G, Pandhi P. Effect of duration of pilocarpine-induced status epilepticus on subsequent cognitive function in rats. Methods Find Exp Clin Pharmacol 2001;23:85–7. [16] Wu CL, Huang LT, Liou CW, et al. Lithium–pilocarpineinduced status epilepticus in immature rats results in long-term deficits in spatial learning and hippocampal cell loss. Neurosci Lett 2001;312:113–7. [17] Dube C, da Silva Fernandes MJ, Nehlig A. Age-dependent consequences of seizures and the development of temporal lobe epilepsy in the rat. Dev Neurosci 2001;23:219–23. [18] Rutten A, Van Albada M, Silveira DC, et al. Memory impairment following status epilepticus in immature rats: time-course and environmental effects. Eur J Neurosci 2002;16:501–13. [19] Erdog˘an F, Go¨lgeli A, Arman F, Ersoy A. The effects of pentylenetetrazole-induced status epilepticus on behavior, emotional memory, and learning in rats. Epilepsy Behav 2004;5:388–93. [20] Veliskova J, Velisek L, Moshe S. Age-specific effects of baclofen on pentylenetetrazol-induced seizures in developing rats. Epilepsia 1996;37:718–22. [21] Holmes GL, Sarkısıan M, Ben-Arı Y, Chevassus-Au-Louıs N. Mossy fiber sprouting after recurrent seizures during early development in rats. J Comp Neurol 1999;404:537–53. [22] Franke H, Kittner H. Morphological alterations of neurons and astrocytes and changes in emotional behavior in pentylenetetrazol-kindled rats. Pharmacol Biochem Behav 2001;70:291–303. [23] Grecksch G, Becker A, Rauca C. Effect of age on the pentylenetetrazol-kindling and kindling-induced impairments of learning performance. Pharmacol Biochem Behav 1997;56:595–601. [24] Selcher JC, Atkins CM, Trzaskos JM, Paylor R, Sweatt J. A necessity for MAP kinase activation in mammalian spatial learning. Learn Mem 1999;6:478–90. [25] Rodrigues SM, Schafe GE, LeDoux JE. Molecular mechanisms underlying emotional learning and memory in the lateral amygdala. Neuron 2004;44:75–91. [26] Graef FG, Viana MB, Tomaz C. The elevated T maze, a new experimental model of anxiety and memory: effect of diazepam. Braz J Med Biol Res 1993;26:67–70. [27] Conde CA, Costa V, Tomaz C. Measuring emotional memory in the elevated T-maze using a training-to-criterion procedure. Pharmacol Biochem Behav 1999;63:63–9. [28] OÕKeefe J, Nadel L. The hippocampus as a cognitive map. Oxford: Clarendon Press; 1978. [29] Pineau N, Charriaut-Marlangue C, Motte J, Nehlig A. Pentylenetetrazol seizures induce cell suffering but not death in the immature rat brain. Dev Brain Res 1999;112:139–44. [30] Nehlig A, Vasconcelos AP. The model of pentylenetetrazolinduced status epilepticus in the immature rat: short-and longterm effects. Epilepsy Res 1996;26:93–103.
[31] Huang LT, Yang SN, Liou CW, et al. Pentylenetetrazol-induced recurrent seizures in rat pups: time course on spatial learning and long-term effects. Epilepsia 2002;43:567–73. [32] Volke V, Soosaar A, Koks S, Bourin M, Mannisto P, Vasar E. 7Nitroindazole, a nitric oxide synthase inhibitor, has anxiolyticlike properties in exploratory models of anxiety. Psychopharmacology 1997;131:399–405. [33] Denenberg V. Open field behavior in the rat: what does it mean? Ann NY Acad Sci 1969;159:852–9. [34] Mechan A, Moran P, Elliot J, Young A, Joseph M, Green A. A study of the effect of a single neurotoxic dose of 3,4-methylenedioxymethamphetamine (MDMA; ÔecstasyÕ) on the subsequent long-term behaviour of rats in the plus maze and open field. Psychopharmacology 2002;159:167–75. [35] Harro J, Haidkind R, Harro M, et al. Chronic mild unpredictable stress after noradrenergic denervation: attenuation of behavioural and biochemical effects of DSP-4 treatment. Eur Neuropsychopharmacol 1999;10:5–16. [36] Conde C, Costa V, Tomaz C. Effects of emotional reactivity on inhibitory avoidance in the elevated T-maze. Braz J Med Biol Res 2000;33:233–6. [37] Holmes GL, Chronopoulos A, Stafstrom CE, et al. Effects of kindling on subsequent learning, memory, behavior, and seizure susceptibility. Dev Brain Res 1993;73:71–7. [38] Kubova H, Mares P, Suchomelova L, Brozek G, Druga R, Pitkanen A. Status epilepticus in immature rats leads to behavioural and cognitive impairment and epileptogenesis. Eur J Neurosci 2004;19:3255–65. [39] Holmes GL, Thompson JL, Marchi TA, et al. Effects of seizures on learning, memory, and behavior in the genetically epilepsyprone rat. Ann Neurol 1990;27:24–32. [40] Gargiulo PA, Viana MB, Graeff FG, Silva MA, Tomaz C. Effects of anxiety and memory of systemic and intra-amygdala injection of 5-HT3 receptor antagonist BRL 46470A. Neuropsychobiology 1996;33:189–95. [41] Voigt JP, Morgenstern E. Pentylenetetrazole kindling impairs learning in mice. Biomed Biochim Acta 1990;1:143–5. [42] Grecksch G, Becker A, Gadau C, Matthies H. Gangliosides improve a memory deficits in pentylenetetrazol-kindled rats. Pharmacol Biochem Behav 1990;39:825–8. [43] Becker A, Grecksch GA, Ru¨thrich HL, Pohle W, Marx B, Matthies H. Kindling and its consequences on learning. Behav Neural Biol 1992;7:37–43. [44] Becker A, Tiedge AG. Diazepam: its effects on the development of pentylenetetrazol kindling, related learning impairments, and neuronal cell loss. Pharmacol Res 1997;35:27–32. [45] de Feo MR, Mecarelli O, Palladini G, Ricci GF. Long-term effects of early status epilepticus on the acquisition of conditioned avoidance behavior in rats. Epilepsia 1986;27:476–82.
Effects of pentylenetetrazole-induced status epilepticus on behavior, emotional memory and learning in immature rats Fu¨sun Erdog˘an a,*, Asuman Go¨lgeli b, Aysßegu¨l Ku¨c¸u¨k b, Fehim Arman a, Yahya Karaman a, Ali Ersoy a a b
Neurology Department, Erciyes University, 38039 Kayseri, Turkey Physiology Department, Erciyes University, 38039 Kayseri, Turkey
Received 27 December 2004; revised 28 February 2005; accepted 1 March 2005 Available online 26 April 2005
Abstract Status epilepticus (SE) can be harmful to the developing brain. Our knowledge of the emotional and behavioral consequences of generalized SE in developing animals remains limited. Therefore, we investigated the short- and long-term effects of pentylenetetrazole (PTZ)-induced SE on emotional memory and learning and behavioral parameters in immature rats. SE was induced in 16- to 20-day-old rats (P16–P20) using intraperitoneal injections of PTZ (n = 21); control rats received saline (n = 10). All animals were tested using an elevated T-maze and open-field test 2, 14, 30, and 180 days after SE, to evaluate emotional memory and learning and behavior. Anxiety levels decreased 2 and 14 days after SE, and conditioned learning of PTZ-treated immature rats was better than that of the control rats. These results indicate that a decreased anxiety level facilitates conditioned learning. Behavioral changes are transient, and no emotional memory or learning deficits occur following PTZ-induced SE in immature rats. 2005 Elsevier Inc. All rights reserved. Keywords: Status epilepticus; Immature brain; Pentylenetetrazole; Emotional memory; Learning; Behavior
1. Introduction Status epilepticus (SE) is more prevalent in children than in adults.The high incidence of seizures in the first decade of life and the propensity of children for febrile seizures and SE are reflective of increased susceptibility of the immature brain to seizures. However, there is ongoing clinical discussion of the possible damage induced by epileptic activity in the brains of infants and children [1,2]. It is known that the immature brain is less vulnerable than the mature brain to seizure-related damage [1,3]. Animal experiments unequivocally demonstrate that electrically or chemically (e.g., kainic acid, pilocarpine, lithium–pilocarpine) induced SE results in im*
Corresponding author. Fax: +90 352 4375285. E-mail address: [email protected] (F. Erdog˘an).
1525-5050/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2005.03.002
paired spatial and emotional learning and memory [4,5]. In these experiments, the model most commonly used was that of temporal lobe epilepsy, and the most commonly tested functions were spatial memory and learning functions [6–18]. In the literature, there is not sufficient information on the effects of generalized SE on emotional memory and learning functions in immature rats. We demonstrated that pentylenetetrazole (PTZ)-induced generalized SE causes transient emotional memory dysfunction in mature rats [19]. As we knew that the effects of seizures may differ between the mature and immature brain, we aimed to investigate the effects of PTZ-induced SE on emotional memory and learning and behavior over short and long periods in immature rats in this follow-up study. PTZ was used because PTZ-induced seizure is a model of primary generalized epilepsy excluding that of genetic origin [20–22].
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To evaluate anxiety and behavioral changes, an openfield test was used. The open-field test is based on the conflict between exploration of a new environment and aversion to open spaces from which escape is prevented by a surrounding wall [23]. This test assesses mainly exploration and fear behaviors [9]. Two paradigms that have been used extensively in studying learning are the conditioned fear task and the Morris water task. In fear conditioning, animals learn to associate neutral stimuli with an aversive stimulus, such as a foot shock. In the spatial learning version of the Morris water task, animals learn to locate a hidden escape platform in a pool of water using various distal visual cues [24]. Memories formed during emotional experiences are stored for future use in similar situations. The expression of emotional response in the presence of the appropriate stimulus serves as a measure of the emotional memory created during the learning experience. Fear conditioning is a powerful procedure for studying the neural basis of emotional memory and of memory in general, because the learning occurs rapidly and the memory is extremely persistent [25]. The elevated T-maze test permits the simultaneous assessment of memory and learning for both conditioned and unconditioned emotional behaviors in the same subject [26,27], so we used the elevated T-maze test to investigate emotional memory and learning functions. As for conditioned learning related to anticipatory anxiety, rats refrain from making certain experimenter-defined responses, and failure to refrain results in punishment. The rats can learn to remain immobile to avoid punishment. Unconditioned learning means learning actively to avoid places or items that are harmful.Learning of this active behavior occurs as a result of innate threats or fears [28].
intraperitoneal injections of PTZ [29,30]. The rats were first injected with 40 mg/kg PTZ, followed 10 minutes later by 20 mg/kg and, subsequently, 10 mg/kg every 10 minutes until SE occurred, a point characterized by a loss of postural control and tonic–clonic seizures. This procedure lasted at least 30 minutes and consisted of prolonged episodes of seizures, interrupted by postictal depression phases with no return to the quadruped posture or to consciousness. Control animals received the same number of saline injections as their paired PTZ-exposed congeners. Open-field and elevated T-maze tests were run on the 2nd, 14th, 30th, and 180th days after SE in both groups. On test days, animals were tested between 8:00 AM and 12:00 noon. 2.1. Open-field test The floor of a square plastic board (100 · 100 cm) with plastic sides (30 cm high) is divided into 16 squares. The rats are individually placed in one corner of the open field and allowed to explore the area freely. The activity level is expressed as the total number of squares crossed, whereas exploratory activity is expressed as the total number of rearings and fear is expressed as the total number of fecal boli during a 5-minute testing period [9,31,29,32]. To interpret open-field test results, data related to these three parameters are combined. Increases in locomotor response and rearings with a decrease in defecation and decreases in locomotor response and rearings with an increase in defecation can be interpreted as less emotional activity and more emotional activity, respectively, in rats [33,34]. The open-field apparatus is cleaned using alcohol before the next animal is introduced, to preclude the possible cuing effects of odors left by previous subjects [35]. 2.2. Elevated T-maze test
2. Methods The experiments were carried out on male Wistar rats, 16–20 days old, weighing 23–50 g. The rats were obtained from the Erciyes University Experimental and Clinical Research Center. The animals were kept on a 12-hour light–dark cycle and allowed free access to food and water. Following SE, the animals were replaced with their mother in their normal environment, and paired controls from the same litter were kept under the same breeding conditions. All experiments were performed between 10:00 AM and 12:00 noon in a silent room at temperatures between 22 and 24 C. The animals were randomly divided into two experimental groups: a control group (n = 10) and a SE group (n = 23). All animals were intact and had no previous experimental history. In the SE group, PTZ was used as the convulsant. To reach SE of controlled intensity and sufficient duration, the animals received repeated
An elevated T-maze consists of three plastic arms of equal dimensions (50 · 12 cm) elevated 50 cm from the floor. One of these arms is enclosed by plastic lateral walls (40 cm high) and is positioned perpendicularly to two opposed open arms. When a rat is placed at the end of the enclosed arm, the open arms are not visible until the rat pokes its head beyond the walls of the closed arm. The time taken to leave this arm with all four paws is then recorded (baseline latency). The animal is then removed from the maze and the same measurement is repeated twice more at 30-second intervals; each timed event occurs over a period of 300 seconds (passive avoidance 1 and 2, inhibitory avoidance, or conditioned learning). This allows the animal, as it explores a maze, to learn inhibitory avoidance if repeatedly placed inside the enclosed arm. Being on an open arm seems to cause a fear-averse experience, as rats have an innate fear of heights and open spaces. After another
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30 seconds, the rat is placed at the end of the right open arm, and it can move toward the enclosed arm (one-way escape, active avoidance, or unconditioned learning). The time taken to leave this arm with all four paws is then recorded (escape latency) [27,26,36]. The animals are reexposed to the situation after an interval and memory of these emotionally related behaviors is assessed [27]. 2.3. Statistical analysis For statistical analysis of the data from both groups, the Mann–Whitney U test was used, and for analysis of the data from repeated tests in the given periods in each group, FriedmannÕs two-way analysis of variance was used. Values are expressed as means ± SEM and significance is defined at P < 0.05 for all tests.
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the number of squares crossed in the SE group on the second day after SE (Mann–Whitney U = 15.5, P = 0.005), and in the number of rearings on the 14th day after SE (Mann–Whitney U = 15, P = 0.004) (Fig. 1). In addition, there was a significant change in the number of fecal boli; the number of fecal boli decreased on the second day after SE (Mann–Whitney U = 24.5, P = 0.02) (Fig. 2). The effects of reexposure to the open field test were evaluated in each group. Significant differences in rearings were noted in the control group with time. The decrease on the 14th day after SE and the increases on the 30th and 180th days were statistically significant. Decreases on the 14th and 30th days after SE and an increase on the 180th day were observed in the SE group, although these were not statistically significant (Fig. 3).
3. Results 3.1. Behavioral features of PTZ-induced seizures Convulsive behaviors were observed with intraperitoneal injection of a 40 mg/kg dose of PTZ in nine rats and of 60 mg/kg in 14 rats. Inhibition of motor activity in a ‘‘no response stage’’ was observed in all rats. With additional successive doses of PTZ, myoclonic jerks were observed, involving predominantly hind limbs; in addition, rearing with clonic movements of forelimbs without loss of postural control was observed in all rats. This kind of seizure occurred one to three times, and duration ranged between 0.5 and 15 minutes. After this stage, rapid backward walking in the presence of myoclonia in the forelimbs was observed in every rat in the PTZ group. During these periods, excessive motor activity and hyperreaction to external stimuli were observed. All rats were very active and irritable. They startled when touched or exposed to noise, and all ran wildly. Subsequently, the rats reached generalized tonic contractions that were characterized by the loss of postural control and strong contractions in the extensor muscles of the forelimbs and hind limbs with cyanosis. All deaths occurred in the tonic stage. After this stage, generalized SE occurred. SE was provoked by intraperitoneal injection of 60–100 mg/kg PTZ. SE occurred at 60 mg/kg in 9 rats, 70 mg/kg in 7 rats, 80 mg/kg in 5 rats, 90 mg/kg in 1 rat, and 100 mg/kg in 1 rat. In all rats, the duration of SE ranged between 30 and 70 minutes, and the 11 surviving rats spontaneously returned to baseline activities. The total mortality rate in PTZ-treated rats was 52%. 3.2. Open-field test Squares crossed, rearings, and fecal boli were counted in 5-minute periods. There was a significant increase in
Fig. 1. Comparison of exploratory activity, motor activity, and fear score on the second day after SE. Each column represents the mean ± SEM; n = 10 for control group and n = 11 for SE group. Rearings: exploratory activity, squares crossed: motor activity, defecation: fear score. Motor activity significantly increased (Mann– Whitney U = 15.5, P = 0.005) and fear score significantly decreased (Mann–Whitney U = 24.5, P = 0.02) in the SE group.
Fig. 2. Comparison of exploratory activity, motor activity, and fear score on the 14th day after SE. Each column represents the mean ± SEM; n = 10 for control and n = 11 for SE group. Rearings: exploratory activity, squares crossed: motor activity, defecation: fear score. Exploratory activity significantly increased in the SE group (Mann–Whitney U = 15, P = 0.004).
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Fig. 3. Change in the number of rearings over short and long periods after SE in the control and SE groups. The decrease on the 14th day after SE and increases on the 30th and 180th days were statistically significant (Friedmann test, x2 = 15). Decreases on the 14th and 30th days after SE and an increase on the 180th day were seen in the SE Group although it was not statistically significant.
Fig. 4. Comparison of elevated T-maze baseline latencies in the control and SE groups on the 2nd, 14th, 30th, and 180th days after SE. Each column represents the mean ± SEM; n = 10 for the control group and n = 11 for the SE group. Baseline latency significantly decreased in the SE group 2 days after SE (Mann–Whitney U = 27.5, P = 0.026).
3.3. Elevated T-maze test Inhibitory avoidance latencies (passive avoidance or conditioned learning) and one-way escape latency (active avoidance or unconditioned learning) were evaluated with the elevated T-maze test. The only significant difference between the two groups was in baseline latencies, which were significantly short in the SE group 2 days after SE (Mann–Whitney U = 27.5, P = 0.026) (Fig. 4). The effects of reexposure to the T-maze were evaluated in each group. There were no statistically significant changes on reexposure.
4. Discussion The purpose of the present study was to investigate the long- and short-term effects of PTZ-induced SE on behavior and emotional memory and learning functions in the immature brain. The behavioral changes of rats
were evaluated using an open-field test. Emotional activity was found to be significantly different in the SE group a short time after SE. The number of squares crossed increased on the second day after SE and the number of rearings increased on the 14th day after SE in the open-field test. Significant differences in the number of fecal boli were also noted between the two groups, and the number of fecal boli decreased 2 days after SE in the SE group. These parameters indicate that in PTZtreated immature rats, fear and anxiety are significantly reduced a short time after SE. It was observed that the control rats were more hesitant to explore in the open-field test, which is an adaptive response, while PTZ-treated rats lacked this restraint [9]. An open field is a novel environment that induces anxiety and exploratory activity in normal rats [9,33,37]. The behavior of an animal in the open field involves integration of complex polymodal sensory information into motor output and must be mediated at several levels. The hippocampus is essential in several aspects of open-field behavior [9,37]. Hippocampal damage may release natural inhibitory tendencies, which may be an adaptive response in normal rats [9,33]. It has been demonstrated that open-field exploratory activity increases after kainic acid-induced SE as a result of hippocampal damage in both mature and immature rats [9,7]. Lithium chloride–pilocarpine-induced seizures cause hyperactivity in P12 rats [38]. Furthermore, it has been demonstrated that hippocampal neuronal loss and open-field motor activity decrease after PTZ kindling in mature rats over long periods [22]. Electrical kindling in mature rats, [37] and recurrent PTZ-induced seizures [31] in immature rats do not have an effect on a open-field exploratory activity. In genetically epilepsy-prone rats, frequent and brief seizures cause less motor activity in the open-field test [39]. No difference, in terms of motor and exploratory activity, was noted between PTZ-treated and control mature rats in the open-field test [19]. All these studies show that behavioral changes are related to the age of rats and type of seizure. Our findings suggest that generalized SE causes transient hyperactivity and reduced anxiety and, possibly, transitory hippocampal effects in developing brains, whereas such effects are not seen in mature rats. We observed that there were significant changes with respect to rearings in the control group with time. The decrease on the 14th day after SE and increases on the 30th and 180th days were statistically significant. Decreases on the 14th and 30th days after SE and an increase on the 180th day, although not statistically significant, were observed in the SE group. In the control group, repeated trials caused a decrease in open-field activity directed toward searching the environment. This is considered an adaptive response [9]. It seems that PTZ-induced SE impairs this adaptive response in immature rats, but not in mature rats [19].
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We subjected immature rats to the elevated T-maze test to evaluate emotional memory and learning performance. This experimental model allows the parallel measurement of responses related to both conditioned and unconditioned fear in the same subject. It also permits simultaneous assessment of memory and learning of these behaviors [35,40]. There was only one significant difference in baseline latencies between the two groups: baseline latencies were significantly short in the SE group 2 days after SE. PTZ-treated rats discovered the exit from the closed arm more easily in the elevated T-maze. Open-field test results indicated that PTZ-treated rats were less fearful 2 days after SE in the open-field test, suggesting that their decreased anxiety causes high motor activity and a reduction in baseline latency in the elevated T-maze test 2 days after SE. Our results demonstrate that immature rats are hyperactive and disinhibited from exhibiting increased exploration in the open field and also have a shortened baseline latency 2 days after SE. There are no emotional memory or learning functions in the immature rats. We demonstrated that PTZ-induced SE causes transient emotional memory deficit in mature rats in the previous study [19]. Thus, we conclude that PTZ-induced SE affects mature and immature brain differently, and the effects of PTZ-induced generalized SE are age dependent. SE-related learning impairments, mostly those related to spatial learning, have been widely investigated. Studies in healthy adult animals indicate that SE causes longterm deficits in spatial learning and memory. Studies of spatial memory after SE in immature animals have produced controversial data [9,10,16,38]. In these studies, models in which SE is induced by kainic acid and lithium chloride–pilocarpine were used. Kubova et al. concluded that the severity of SE-induced impairment in hippocampus-dependent spatial learning increases with maturation of the brain [38]. It has been shown that PTZ kindling in mature rats and repeated seizures in immature rats cause behavioral changes and spatial learning and memory impairments. These changes are permanent when they are accompanied by hippocampal damage [22,23,31,41– 44]. No histological abnormality or behavioral changes were detected by de Feo et al. [45] after PTZ-induced SE in P10 rats. Nehlig et al. [30] also reported low metabolic and blood flow activity in the cortical, hippocampal, and sensory areas, although there is no neuronal death in P21 rats after PTZ-induced SE. Holmes et al. [21] indicated that during development, recurrent PTZinduced seizures result in granule cell neurogenesis without loss of principal neurons, and recurrent seizures can result in significant alterations in cell number and axonal growth. They pointed out that the relationship between these morphologic changes after seizures during development and subsequent cognitive impairment is not yet clear. Huang et al. [31] demonstrated that PTZ-induced recurrent seizures in the immature brain cause neuronal
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circuit abnormalities in the hippocampus, in addition to long-term cognitive deficits. There have been no studies on emotional memory and learning after PTZ-induced SE in immature rats; however, a lack of histologic aspects is a limitation of the present study. The neuronal circuit underlying fear conditioning has been implicated in emotional disorders in humans [25]. Therefore, studies including SE-related behavior and emotional memory and learning functions lead to an understanding of behavioral and emotional consequences of SE. The present study demonstrates that PTZ-induced SE inhibits natural anxiety reactions and, in turn, causes hyperactivity and disinhibition in a short period. The behavioral changes are mostly transient, with no long-term emotional memory or learning deficits after PTZ-induced SE in the developing brain.
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