LEV-induced theta oscillations in hippocampal slices
The basal activity of field potentials were recorded at CA3 area of rat hippocampal slices, perfusion of aCSF for 80 min had no effect on basal activity of field potentials (n=4, Fig. 1A-B). The application of LEV (3-100 µM) induced persistent theta oscillation (4-12Hz) in CA3 area of rat hippocampal slices. LEV-induced theta rhythm had a small but insignificant decrease within one hour after removal of LEV (data not show). Examples of LEV-induced theta oscillation were shown in figure 1C-F. The peak frequency of LEV-induced theta oscillation was 8.1±1.6 Hz (vs control 5.0±1.3 Hz, paired t-test, p<0.05, n=6), 9.7±0.9 Hz (vs control 5.4±1.1 Hz, paired t-test, p<0.01, n=6), 8.8±0.6 Hz (vs control 5.4±1.0 Hz, paired t-test, p<0.01, n=6) and 9.5±1.6 Hz (vs control 5.8±1.0 Hz, paired t-test, p<0.01, n=11) for 3µM, 10µM, 30µM and 100µM, respectively(figure 1F). Compared with the control, there was a significant difference in the mean peak frequency after applying LEV and there was no significant difference in the mean peak frequency among the various concentrations of LEV treatments (ANOVA, p>0.05).The area power of LEV-induced theta oscillation was 9.6±1.6 µV2 (vs control 7.7±1.2 µV2), 6.9±1.0 µV2 (vs control 5.0±0.8 µV2), 23.9±9.1 µV2 (vs control 15.5±6.0 µV2) and 24.9±6.3 µV2 (vs control 8.0±1.2 µV2) for 3µM, 10µM, 30µM and 100µM, respectively. Compared with the control, LEV dose-dependently increased the area power by 22.7% (p>0.05, n=6), 43.9% (p>0.05, n=6), 70.2% (p<0.05, n=6) and 174.3% (p<0.01, n=11) for 3µM, 10µM, 30µM and 100µM, respectively (ANOVA, post hoc Tukey test, p<0.01, figure 1G).
LEV-induced theta oscillations were mediated by NMDA receptors and GABAA 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 GABAAR antagonist bicuculline on LEV-induced theta oscillation. In a set of experiments (n=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 (383.6±111.8% 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 GABAA receptor.
Taurine was involved in LEV-induced theta oscillations
Taurine is an inhibitory amino acid, potently acts on GABAA receptors located at both synaptic and non-synaptic sites, is functionally similar to the role of GABA[22]. LEV was reported to significantly reduce taurine level in the hippocampus [24]. 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 (106.8±8.4% vs taurine 100%, paired t-test, p>0.05, n=6, figure 3A). The results showed that taurine pretreatment blocked LEV-induced theta oscillations. To further verify that extrasynaptic GABAA receptors are involved in LEV-induced theta oscillations, we studied the effects of THIP on LEV-induced oscillation. We pretreated hippocampal slices with THIP (30 μM) for 40min and further application of LEV (100 μM) caused little change on baseline area power (112.6±5.7% vs THIP 100%, paired t-test, p>0.05, n=4, figure 3B). The results showed that THIP pretreatment blocked LEV-induced theta oscillations.
mACh receptors but not nACh receptors or L-type Ca2+ channel mediate LEV-induced theta oscillations
Previous study indicates that, mACH receptors mediate oscillations activity [27]. We here determined whether LEV-induced theta oscillations are mediated by mACh receptors. Atropine pretreatment (50 μM) had no role on baseline, further application of LEV (100 μM) caused a small but significant increase in area power (150.4±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 [8], 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 (261.3±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.
In another set of experiments (n=6), L-type Ca2+ channel antagonist nifedipine (10µM) was applied to pretreat hippocampal slices, further application of LEV (100 μM) caused a significant increase in area power (221.3±38.0% vs nifedipine 100%, paired t-test, p<0.01, n=6, figure 4 C 1-3). Thus, these results indicate that L-type Ca2+ channel did not involve in the regulation of LEV-induced theta oscillations.