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Summary: Purpose: The aim of the study was to define sleep disturbances in pentylenetetrazole (PTZ)-kindled rats and to explore the effects of the nootropic drug piracetam (Pir; 100 mg/kg) and the noncompetitive N-methyl-d-aspartate (NMDA)-antagonist MK-801 (0.3 mg/kg), which normalized learning performance in PTZ-kindled rats, on altered sleep parameters.
Methods: This is the first report showing a significant reduction in paradoxical sleep (PS) as a consequence of PTZ kindling. A correlation analysis revealed a significant correlation between seizure severity and PS deficit.
Results: Pir did not interfere with seizure severity, and the substance did not ameliorate the PS deficit. However, the substance disconnected the correlation between seizure severity and PS deficit. MK-801, which reduced the severity of kindled seizures, counteracted the PS deficit efficaciously.
Conclusions: The results suggest that seizure severity and alterations in sleep architecture are two factors in the comprehensive network underlying learning impairments associated with epilepsy. Considering the results obtained in the experiments with Pir, reduction of seizure severity does not guarantee the reduction of impairments in the domain of learning.
A body of evidence suggests that epilepsy can result in cognitive impairments leading to lowered educational and occupational levels of achievement. It was suggested that a plethora of multiple factors such as seizure type, seizure severity and frequency, seizure duration, and age at seizure onset underlie cognitive impairments associated with epilepsy (Lesser et al., 1986; Dodrill, 1992; Kalviainen et al., 1992; Devinsky, 1995; Aldenkamp et al., 1996; Vuilleumier et al., 1996; Aldenkamp et al., 2001; Samson 2002; Duncan and Thompson, 2003; Helmstaedter et al., 2003; Nolan et al., 2003; 2004; Dodrill, 2004; Sonmez et al., 2004).
The mechanism underlying these impairments is an object of debate and controversy. It was shown that sleep affects epileptic activity and vice versa. In epilepsy patients, altered sleep–wake cycles and an increased number of stage shifts were found. Moreover, quantitative aspects of different sleep stages were reported to be changed (Autret et al., 1997; Bazil and Walczak, 1997; Bazil, 2000; Bazil et al., 2000; Gigli and Valente, 2000; Janz, 2000; Bazil, 2003; 2005). Evidence indicates that sleep plays a role in the processes of learning and memory (Gais and Born, 2004; Paller and Voss, 2004; Maquet et al., 2005; Stickgold, 2005; Stickgold and Walker, 2005). However, alternative views exist (Vertes and Eastman, 2000; Vertes and Siegel, 2005). Discrete stages of sleep appear to be either permissive or obligatory for specific steps in memory formation (Drosopoulos et al., 2005; McNamara et al., 2005; Walker, 2005). Consequently, sleep disturbances accompanying epilepsy might be one factor contributing to cognitive deficits as found in epilepsy patients.
The kindling model is the most widely used model for studies on epileptogenic processes; epilepsy-related behavioral, neurophysiological, neurochemical, and neurohistopathological changes; and finally on drug targets by which epilepsy can be prevented or modified. Evidence suggests that different kindling protocols result in different behavioral outcomes (e.g., anxiety, learning impairments). Kindling refers to a process in which periodic application of initially subeffective chemical or electrical stimuli induces progressive intensification of evoked electroencephalographic and behavioral seizures. It was shown that electrical kindling (Lopes da Silva et al., 1986; Beldhuis et al., 1992; Becker et al., 1997a; Hannesson et al., 2001) and chemical kindling (Voigt and Morgenstern, 1990; Becker et al. 1992; 1995; Pohle et al., 1997; Rössler et al., 2000; Nagaraja et al., 2004; Mortazavi et al., 2005) worsened learning performance of animals that had acquired the kindling syndrome in a variety of learning models. Moreover, electrical kindling in rats (Stone and Gold, 1988; Cammisuli et al., 1997; Raol and Meti, 1998) and cats (Hiyoshi and Wada, 1990; Calvo and Fernandez-Mas, 1991; 1994; Gigli and Gotman, 1992) was found to modify sleep patterns. Previously, the noncompetitive glutamate antagonist MK-801 was shown to exert anticonvulsive effects and to counteract kindling-induced learning deficits (Grecksch et al., 1994), whereas the nootropic drug piracetam was ineffective in counteracting kindled seizures but effective in ameliorating kindling-induced learning deficits (Pohle et al., 1997). The substance showed protective effects (injection during kindling development) as well as restorative efficacy (injection after kindling completion before each shuttle-box session). Moreover, the substance was found to be effective in counteracting neuronal cell loss in distinct hippocampal structures (Pohle et al., 1997). In control animals, both substances did not change learning performance. To the best of our knowledge, the relation between sleep pattern and learning performance in pentylenetetrazole (PTZ)-kindled animals was not studied yet. The present study addresses the investigation of sleep pattern in rats before and after completion of kindling as well as effects of MK-801 and piracetam, which were administered in the process of kindling induction.
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Epilepsy is studied most commonly with kindling models because kindling reflects neurophysiologic, neurochemical, neurohistopathologic, and behavioral alterations associated with human epilepsy (McNamara et al., 1985; McNamara, 1986; Schmutz, 1987; Becker et al., 1992; Majkowski, 1999; Lagae et al., 2003). Several studies have shown the impact of epilepsy on higher cognitive function with special regard to memory impairment. Although the pathophysiologic mechanisms underlying these impairments are multifactorial, seizure frequency was reported to exert deleterious effects on cognition (Stafstrom, 2002; Tromp et al., 2003; Nolan et al., 2004; Hoie et al., 2005). Thus seizure suppression appears to be appropriate in the prevention of cognitive deficits after epilepsy. In a kindling experiment, it was shown that diazepam injected before each kindling stimulation prevented motor seizures and hippocampal cell loss (Becker et al., 1994, 1997b). However, the kindling-induced deficit in two-way active avoidance learning was evident regardless of the diazepam treatment, suggesting that motor seizures are only one component in the comprehensive network underlying cognitive impairments in epilepsy. Another factor in this network is the effect of various antiepileptic drugs that impair memory to quite different degrees (Devinsky, 1995; Drane and Meador, 1996; Bourgeois, 1998; Aldenkamp et al., 2003; Fritz et al., 2005). As a logical consequence, it was hypothesized that the use of memory-enhancing nootropic drugs might be a useful attempt at compensating for the cognitive deficits in epilepsy patients (Mondadori et al., 1984; Mondadori and Schmutz, 1986; Becker and Grecksch, 1995). The mode of action of this heterogeneous group of agents is not completely understood. Therefore the present study was designed to answer the following questions: (a) does chemical kindling result in changes in sleep pattern; and (b) do substances which are known to either counteract epilepsy-associated learning deficits or seizure attacks rebalance altered sleep patterns?
The principal findings of the present study are the following:
24 h after a single seizure attack, sleep patterns were found to be unchanged
pentylenetrazole kindling results in a reduction in paradoxical sleep
the nootropic drug piracetam did not normalize altered sleep pattern in kindled rats
the noncompetitive NMDA-receptor antagonist MK-801 ameliorated the deficit in paradoxical sleep in kindled rats.
Clinical observations in patients with epilepsy have shown altered sleep–waking cycles and an increased number of stage shifts. Conversely, it is well documented that sleep deprivation results in an impairment of memory retention (Fishbein and Gutwein, 1977; Smith, 1995; Forest and Godbout, 2000; Bjorness et al., 2005). Similar alterations were found in animal experiments that are considered to be useful tools in the study of correlative relations between sleep disturbances and cognitive deficits. After electrical kindling in different animal species and different brain structures, a reduced amount of sleep with special regard to paradoxical sleep was found (Stone and Gold, 1988; Hiyoshi and Wada, 1990; Calvo and Fernandez-Mas, 1991; 1994; Gigli and Gotman, 1992; Cammisuli et al., 1997; Raol and Meti, 1998). Interestingly, in chemically kindled rats, these changes in sleep patterns also are evident. Although the significant differences in paradoxical sleep parameters before and after completion of kindling appear to be marginal (e.g., Table 2), the size of the changes observed in sleep in the present experiment is comparable to the results obtained by other laboratories (Amici et al., 2001; Lena et al., 2004). A single seizure attack induced by PTZ, however, was without any obvious effect on sleep (Table 1). This is in line with other results showing that a single seizure attack did not modify glutamate binding or parameters of hippocampal long-term potentiation, as found in animals after kindling completion (Schröder et al., 1993; Ruethrich et al., 1996). In contrast,%PS was significantly reduced in the animals that acquired the kindling syndrome (Fig. 3), suggesting that altered sleep patterns are dependent on long-lasting plastic-adaptive alterations in central functioning. This well correlates with other reports. There it was concluded that an increase in PS sleep after a single seizure attack may represent an adaptive mechanism. Sustaining seizure activity breaks down this mechanism and results in loss of PS sleep (Raol and Meti, 1998).
PTZ kindling resulted in long-lasting learning impairment, which is still ascertainable 4 weeks after the last kindling stimulation (Becker et al., 1992). Detailed analysis revealed a significant relation between the learning impairment and seizure severity. A similar correlation was found between seizure severity and PS deficit (Fig. 4), which might suggest that the PS deficits contribute to kindling-induced learning impairment.
To elucidate the relation between sleep disturbances and learning impairments, we injected the nootropic drug Pir and the NMDA-receptor antagonist MK-801 in the course of kindling before each kindling injection. In previous studies, a dose of 100 mg/kg Pir was found efficaciously to counteract kindling-associated learning deficits without affecting seizure development (Becker and Grecksch, 1995). In contrast, MK-801 (0.3 mg/kg) significantly reduced seizure development, and it reduced the kindling-associated learning deficits (Grecksch et al., 1994). Surprisingly, Pir was without effect on the PS deficit in kindled rats, but this substance disconnected the significant correlation between seizure severity and PS deficit (Fig. 5). In previous experiments, Pir was found to enhance PS in rats. This is not contrasting because these results were obtained after brief Pir injection (Aldenkamp et al., 1996), whereas in the present experiment, sleep was analyzed after timely-spaced subchronic Pir treatment followed by washout (Wetzel, 1985). Conversely, MK-801–lowered seizure severity counteracted the kindling-induced PS and the kindling-associated learning deficit as well. This implies that beneficial effects on cognitive impairments in epilepsy may derive from both (i.e., reduction of seizure severity and rebalance in qualitative and quantitative parameters of paradoxical sleep). However, this does not exclude that factors other than seizure severity and changes in sleep structure are involved in learning impairments associated with epilepsy.
On the basis of animal experiments, certain AEDs may usefully be combined with nootropics (Mondadori et al., 1984). We are far from understanding impairments in the domain of cognition in epilepsy patients. Therefore more data are needed on the efficacy of AEDs and their combinations to control epilepsy-related cognitive dysfunctions.