Levetiracetam induction of theta frequency oscillations in rodent hippocampus in vitro

Background Levetiracetam (LEV), an antiepileptic drug, has been recently demonstrated to improve the cognitive function. Hippocampal theta rhythm (4–12 Hz) is associated with a variety of cognitively related behaviors, such as exploration, locomotion and spatial memory in both humans and animal models. We investigated the effects of LEV on the theta rhythm in the rat hippocampal CA3 area. Results We found that LEV increased the power of theta oscillation in a dose-dependent manner. The increase in theta power can be blocked by GABAA receptor or NMDA receptor antagonists but not by AMPA receptor antagonist, indicating the involvement of GABAA receptor and NMDA receptor in the induction of theta activity. Interestingly, LEV enhancement of theta power can be also blocked by taurine, indicating that LEV induction of theta may be related to the indirect boosting of GABA action via reduction of extrasynaptic GABAA receptor activation. Furthermore, the increased theta power can be partially reduced by mACh receptor antagonist atropine but not by nACh receptor antagonists, suggesting that mACh receptor activation provides excitatory input into local network responsible for LEV induction of theta. Conclusions Our study demonstrated that induction of a synchronized network oscillation, a novel role of LEV may especially benefit for the treatment of the neuronal disorders with impaired theta oscillation and cognitive function.

antagonist, indicating the involvement of GABAA receptor and NMDA receptor in the induction of theta activity. Interestingly, LEV enhancement of theta power can be also blocked by taurine, indicating that LEV induction of theta may be related to the indirect boosting of GABA action via reduction of extrasynaptic GABAA receptor activation.
Furthermore, the increased theta power can be partially reduced by mACh receptor antagonist atropine but not by nACh receptor antagonists, suggesting that mACh receptor activation provides excitatory input into local network responsible for LEV induction of theta. Conclusions Our study demonstrated that induction of a synchronized network oscillation, a novel role of LEV may especially benefit for the treatment of the neuronal disorders with impaired theta oscillation and cognitive function.

Background
Hippocampal theta rhythm (4-12 Hz) plays a key role in cognitive processes in both humans and animals [1,2] and is associated with a variety of cognitively related behaviors, including spatial coding, memory and sniffing exploratory locomotion [3]. The decreased theta activity is correlated with cognitive deficit seen in neurodegenerative disease such as epilepsy and Alzheimer's disease (AD) [4,5,6,7]. 3 The pharmacological induction of theta rhythm is determined by local neuronal circuits activated by specific receptor agonists including both metabotropic acetylcholine receptor (mAChR) and nicotinic acetylcholine receptor (nAChR) agonists in hippocampus [8,9], suggesting that cholinergic neurotransmission play a role in hippocampal theta generation.
Levetiracetam (LEV) is an anti-epileptic drug for the treatment of partial onset and generalized seizures [10,11]. Recently, LEV has been demonstrated to improve cognitive impairment of an Alzheimer's disease model [12] and to have a neuroprotective effects on brain injury [13,14,15] at its therapeutic plasma concentration (35-118µM) [16].
Accumulated evidence indicates that LEV improves the impairment in patients with amnestic mild cognitive impairment by reducing the hyperactivity of the hippocampus [17] and spatial memory impairment in a mouse kindling model and rat pilocarpine model of temporal lobe epilepsy(TLE) [18,19]. It has also been reported that LEV can improve the memory function defects of a ketamine-induced schizophrenia model [20]. GABA A receptor (GABA A R) appears to be critical for pharmacologically induced theta oscillations [8,9,21,22,23]. Taurine, rich in the brain, is an inhibitory amino acid, potently activates GABA A R in rat hippocampal CA1 area and consequently modulates theta via activating GABA A R [24, 25]. LEV has been reported to enhance GABA A R function [26], it is likely LEV modulates theta activity, which maybe affected by taurine, as there were negative effects of LEV on taurine production[27], which could indirectly boost GABA action via extrasynaptic GABA A R activation and increase network activity.
Whether LEV affects theta oscillation has not yet been reported. In this study, we found that LEV dramatically enhanced theta oscillations of the rat hippocampal CA3 area.

LEV-induced theta oscillations were mediated by NMDA receptors and GABA A receptors
In order to determine the mechanisms of LEV-induced theta oscillation, we examined the effects of the ionotropic glutamate receptor antagonist D-AP5 or NBQX or the ionotropic GABA A R antagonist bicuculline on LEV-induced theta oscillation. In a set of experiments (n 5 = 6), pretreatment of hippocampal slices with D-AP5 (50 μM) had no effect on baseline area power, further application of LEV (100 μM) caused a small increase without statistically significant difference (paired t-test, p>0.05, n = 6, figure 2 A1-3). In a different set of experiments (n = 8), we pretreated slices with NBQX (20 μM), further application of LEV (100 μM) caused a dramatic increase (383±111% vs NBQX 100%, paired t-test, p<0.01, n = 8, figure 2 B1-3) in area power. Compared with LEV alone, there was no significant difference (Student t-test, p>0.05). In another set of experiments (n = 6), pretreatment of hippocampal slices with bicuculline (2 μM) had no effect on baseline area power, further application of LEV (100 μM) failed to induce any oscillatory activity (paired t-test, p>0.05, n = 6, figure 2 C1-3). The results therefore indicated that LEV-induced theta oscillation is mediated by both NMDA receptor and GABA A receptor.

Taurine was involved in LEV-induced theta oscillations
Taurine is an inhibitory amino acid, potently acts on GABA A receptors located at both synaptic and non-synaptic sites, is functionally similar to the role of GABA [24]. LEV was reported to significantly reduce taurine level in the hippocampus [27]. To determine whether taurine is involved in LEV-induced theta oscillations, we studied the effects of taurine on LEV-induced oscillation. We pretreated hippocampal slices with taurine (100 μM) for 20min and further application of LEV (100 μM) caused little change on baseline area power (107±8.4% vs taurine 100%, paired t-test, p>0.05, n = 6, figure 3). The results showed that taurine pretreatment blocked LEV-induced theta oscillations. mACh receptors but not nACh receptors or L-type Ca 2 + channel mediate

LEV-induced theta oscillations
Previous study indicates that, we here determined whether LEV-induced theta oscillations are mediated by mACh receptors. Atropine pretreatment (50 μM) had no role on baseline, 6 further application of LEV (100 μM) caused a small but significant increase in area power (150±13.7% vs atropine 100%, paired t-test, p<0.01, n = 10, figure 4 A1-3). There was significant difference in area power between LEV and LEV+atropine (Student t-test, p<0.05), indicating that LEV-induced theta oscillation was partially blocked by atropine.
Previous study showed that nAChR agonist induced theta oscillation in hippocampus [9], we further determined whether nACh receptors is involved in LEV-induced theta oscillations by using α7 nAChR antagonist MLA (100 nM) and α4β2 nAChR antagonist DHβE (0.4 µM). Pretreatment of a combined MLA and DHβE, had no effect on baseline activity, further application of LEV (100 μM) caused a significant increase in area power (261±36.9% vs MLA and DHβE 100%, paired t-test, p<0.01, n = 6, figure 4 B1-3). Our results indicated that nACh receptors don't contribute to LEV-induced oscillatory activity.

Discussion
In this study, we demonstrate that LEV is able to elicit persistent oscillatory activity at theta frequency bands in hippocampal CA3 area, which are mediated by NMDA receptors and GABAA receptors as well as partially by mAChR.
Hippocampal theta rhythm is crucial for spatial memory and is thought to be generated by extrinsic synaptic transmissions. Ionotropic glutamatergic receptor agonist kainate or nAChR agonist nicotine can both induce theta oscillation in medial septal diagonal band (MSDB), which was blocked by GABAAR antagonist and was partially blocked by the AMPA/kainate or NMDA receptor (NMDAR) blocker [21,22]. Carbachol (a mAChR agonist) or nicotine induced theta oscillations in hippocampal CA3 area, which was also blocked by GABA A receptor antagonist and was partially blocked by the AMPA/kainate or NMDA receptor blocker [8,9,23]. Thus, it appears that these pharmacologically-induced oscillations were reduced by both GABA A R and ionotropic glutamate receptor antagonists, indicating the involvement of local GABAergic and glutamatergic neurons in the production of the rhythmic theta activity.
In this study, LEV-induced theta oscillation was also involved in the activation of local GABA A R and NMDAR. Surprisingly, we found that pretreatment with NBQX did not affect LEV-induced theta oscillations, suggesting that AMPA receptor (AMPAR) was not involved in LEV-induction of theta oscillation. This is in agreement with previous findings that GABA A receptors and inotropic glutamatergic receptors (NMDAR) are critical for the induction of theta oscillation [3]. AMPAR appears to be not involved in LEV induced oscillation, which is sort of similar to the theta oscillation induced by DHPG+ NBQX [28].
In our case, LEV+NBQX appears to induced a higher theta relative to LEV alone, suggesting that in DHPG+NBQX and LEV induced theta did not require AMPAR activation.
LEV had no effect on AMPA-induced glutamate release [29], which may explain that AMPAR was not involved in LEV-induced theta activity. Whereas others reported that LEV reduced the EPSC amplitude via affecting the presynaptic voltage-dependent calcium channel [30].

Conclusions
In summary, it is the first study to demonstrate that LEV is able to elicit persistent oscillatory activity at theta frequency bands in hippocampal CA3 area, which are mediated by NMDA receptors and GABA A receptors as well as partially by mAChR. LEV may especially benefit for the treatment of the neuronal disorders with impaired theta oscillation and cognitive function.

Experimental animals
Adult male Sprague-Dawley rats weighing 100-150 g were used in the current study. The

Statistical analysis
All data are expressed as mean ± standard error of mean (SEM) or medians ± min-max for non-normally distributed data. Statistical significance for comparison between two groups was performed using a student t-test if data appeared to be normally distributed or a Wilcoxon signed-rank test if the data were not normally distributed (nonparametric data).
Multiple comparisons among groups were analyzed using one-way repeated measures Analysis of Variance (ANOVA, post hoc Tukey test). If P < 0.05, the treatments were considered to have a statistically significant difference.

Availability of data and material
The datasets of this study are available from the corresponding author on request.

Competing interests
The authors declare that they have no competing interests.

Supplementary Files
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