The present study used male Wistar rats (n = 72 animals) aged 10–12 weeks and weighing 260 g (± 20 g). These animals were housed in standard cages in a controlled environment (22 ± 2°C; 12/12 h light/dark cycle, 55 ± 10% relative humidity) with ad libitum access to food and water. The experimental procedures were approved by the relevant Brazilian federal agencies and were in accordance with the Brazilian National Council for the Control of Animal Experimentation and the Ethics Committee on Use of Animals of the Biological Sciences Institute at the Federal University of Pará (CEUA/UFPA no. 9149220321). The data presented here were also collected in compliance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. All necessary precautions were taken to prevent animal suffering and distress.

The animals in this study were maintained in the research facility for at least 7 days prior to the experiment, for adaptation and acclimation (Figure 1), with the electrodes being implanted in the cortex 1 day prior to the application of the treatments. During the experiment, the rats were pretreated for 4 days with either saline, C. longa, DZP, or C. longa + DZP via the orogastric route (gavage) at 24-h intervals. On the fifth day (24 h after the last application), seizures were induced by a single dose of PTZ, intraperitoneally (Agarwal et al., 2013), with electroencephalographic and EMG records being collected over the subsequent 15 min. The rats were monitored for the subsequent 7 days (follow-up) prior to being euthanized. The brain was then extracted, sectioned, and stained with cresyl violet for cell counting. All these procedures were conducted strictly between 08:00 and 11:00 a.m.

The animals were divided into eight groups (each containing nine animals): (i) saline + saline (SAL); (ii) C. longa (CL) + saline; (iii) DZP + saline; (iv) CL/DZP + saline; (v) SAL + PTZ; (vi) CL + PTZ; (vii) DZP + PTZ; (viii) CL/DZP + PTZ. The seizure behavior was recorded after the application of PTZ.

In addition to the two drugs evaluated in the present study, three types of anesthetics were also used for handling the rats. C. longa was applied in the form of purified pharmaceutical grade curcumin, supplied as 250-mg capsules containing 50 mg of curcumin together with excipients (Aché Laboratórios Farmacêuticos S.A., Brazil). The curcumin was administered via oral gavage at a dose of 80 mg/kg (Akula and Kulkarni, 2014); while the DZP, 10 mg/2 ml (União Química, Embu-Guaçu, SP, Brazil), was administered at a dose of 5 mg/kg (V.O.). Three different types of anesthetic were used in the present study. Ketamine hydrochloride was obtained from the Köing Laboratory (Santana de Parnaíba, SP, Brazil) and xylazine hydrochloride was acquired from the Vallée Laboratory (Montes Claros, MG, Brazil), while the local anesthetic lidocaine, which was used to implant the electrodes, was obtained from the Hipolabor Laboratory (Sabará, MG, Brazil). The PTZ was obtained from Sigma Chemical Co. (St. Louis, MO, United States).

The EEGs were recorded as described by Estumano et al. (2019). For this, the animals were anesthetized and placed in a stereotaxic apparatus for the implantation of stainless-steel electrodes (exposed tip 1.0 mm in diameter) on the dura mater above the pre-frontal cortex at the bregma coordinates − 0.96 mm and ± 1.0 mm lateral, and were fixed with dental acrylic cement. The data were registered with the help of the electrodes using a digital data acquisition system composed of a high impedance amplifier (Grass Technologies, P511, United States), an oscilloscope (Protek, 6510, United States), and a data acquisition and digitalization board (National Instruments, Austin, TX, United States). Data were collected continuously at 1 kHz, at a low pass of 3 kHz, and a high pass of 0.3 Hz. During the recording sessions, the animals were confined to acrylic boxes (20 cm × 45 cm × 15 cm), and the EEG activity was recorded for 15 min immediately after the application of the PTZ or saline solution. The records collected using the digital data acquisition system were analyzed offline. The analyses were run at frequencies of up to 40 Hz, and then split into four bands, that is, the delta (1–4 Hz), theta (4–8 Hz), alpha (8–12 Hz), beta (12–28 Hz), and gamma (28–40 Hz) bands (Aminov et al., 2017).

The behavior of the animals was monitored during the seizures and compared with the latency patterns of the behaviors observed in the PTZ group. Latency was measured in relation to the onset of the following behaviors: (i) generalized tremor; (ii) spasms of the forelimbs; (iii) isolated clonic seizures with no loss of the posture reflex; (iv) generalized clonic seizures with transient loss of the posture reflex; and (v) tonic-clonic seizures with total loss of the posture reflex.

Electrodes were implanted in parallel to the masseter muscle, 5 mm above their point of insertion in the jaw to record the muscle activity during seizures in the PTZ groups. As for the EEG, the data were recorded for 15 min (Santos et al., 2021).

Once euthanized, the rats were perfused transcardially with phosphate-buffered saline (PBS, pH 7.4) at 4°C, followed by 4% formaldehyde (pH 7.4). The brain was extracted post-reperfusion, fixed in 4% formaldehyde for 72 h, cryoprotected in 30% sucrose for 24 h, and then cut into serial coronal sections (40 μm) and stained with Nissl (0.3% cresyl violet acetate). The number of cells of six coronal sections of the hippocampus (CA1 and CA3) of each rat were counted to provide a mean count for each group (n = 9 rats per group). The cell counts were based on the inspection of a field of 50 μm × 50 μm in each region (Wang et al., 2021). The counted (treatment-blind) cells were observed using the ImageJ digital imaging software (NIH, Bethesda, MD, United States).

The normality of the data variances was verified using the Kolmogorov–Smirnov test. All the data are presented as the mean and standard deviation (SD), and the F and p-values are included where pertinent. A p < 0.05 significance level was considered for all the analyses. The significance of differences between the pairs of groups was verified using Student’s t-test, while the variation among three or more groups was evaluated using an Analysis of Variance (ANOVA), either one-way or two-way, followed by Tukey’s test for pairwise multiple comparisons. The analyses were run in GraphPad Prism, version 9 (Graph-Pad Software Inc., San Diego, CA, United States).

The behavior of the rats was assessed to determine the evolution of the seizures (Table 1). The animals pretreated with saline that received PTZ progressed quickly to tonic-clonic seizures with the loss of the postural reflex after a mean interval of less than 5 min. Latency prior to the onset of seizure increased significantly in the group pretreated with C. longa, although the evolution to tonic-clonic seizure with loss of postural reflex was not interrupted.

The group pretreated with DZP that received PTZ presented a greater latency to the onset of seizures in comparison with the C. longa group, in addition to the stabilization of the symptoms, given that the rats presented only isolated clonic seizures with no loss of the postural reflex. Surprisingly, the combined pretreatment (C. longa + DZP) resulted in even better control of the seizure-related behavior, in comparison with the DZP + PTZ group, with the greatest latency to the onset of the spasms and only isolated clonic seizures. These results indicate that the association of C. longa and DZP may provide effective control and prevent the evolution of the seizure.

The EEGs were first obtained from the four saline groups (i–iv), that is, the animals that were pretreated with saline, C. longa, DZP, and C. longa + DZP, respectively, and then received saline on the fifth day (+ SAL). This provided a baseline for the verification of the possible effects of the pretreatment on brain activity. The animals pretreated with saline (group i) had amplitudes below 0.02 mV (Figure 2A), and the spectrogram reveals energy concentrations of below 10 Hz. None of the animals of the other groups (ii–iv) presented any significant difference in the brain activity (Figures 2B–D) in comparison with the control (i), which indicates that none of the pretreatments alter this activity.

In contrast, group v (SAL + PTZ) presented significant changes in the EEG trace, with peaks of amplitude of over 0.3 mV, and activity characterized by constant levels of spike-waves with a high frequency and amplitude (black arrow, Figure 2E). In group vi (CL + PTZ), amplitude varied up to 0.2 mV, while the frequency and amplitude of the spike-waves decreased (black arrow, Figure 2F). In group vii (DZP + PTZ), the changes in the EEG trace were less intense than in groups v and vi, that is, close to 0.1 mV (Figure 2G), which indicates control of the seizure. Finally, the combined pretreatment (CL/DZP + PTZ) obtained the best results in terms of seizure control, with an amplitude of 0.08 mV, and a reduction in the frequency and amplitude of the spike-waves (Figure 2H).

In addition, the total power did not vary significantly among the saline groups, i.e., groups i–iv [F_{(3, 32)} = 0.2671; p = 0.8486; Figure 3A]. The administration of PTZ to the saline group (v) resulted in a significant increase in the total power in comparison with group i (SAL + SAL: 0.1985 ± 0.0740 mV²/Hz × 10^–3 vs. SAL + PTZ: 5.509 ± 0.9856 mV²/Hz × 10^–3; p < 0.0001; Figure 3B). Significant variation [F_{(3, 32)} = 78.75; p < 0.0001; Figure 3C] was also found among the other PTZ groups (vi–viii), with all the different pretreatments reducing the total power of the PTZ-induced seizures. The mean total power of group vi (CL + PTZ) was 2.942 ± 0.5694 mV²/Hz × 10^–3, which was significantly lower (p < 0.0001) than the PTZ group (v). The mean total power of group vii (DZP + PTZ) was 2.066 ± 0.2846 mV²/Hz × 10^–3, significantly lower than that recorded for either group v (p < 0.0001: DZP + PTZ vs. SAL + PTZ) or vi (p = 0.0239: DZP + PTZ vs. CL + PTZ). However, the combined treatment (CL/DZP) resulted in the lowest total power of all (1.348 ± 0.3624 mV²/Hz × 10^–3), which was significantly lower than that recorded for the groups v–vii (p < 0.0001 in both cases), indicating that this treatment is the most effective one for the control of PTZ-triggered seizures.

The decomposition of the brainwaves was analyzed only for the PTZ groups (v–viii). In the case of the low-frequency waves, a significant increase (group v) was recorded in the bandpower of the delta waves [F_{(3, 32)} = 80.49; p < 0.0001; Figure 4A]. The animals that received PTZ presented brainwave patterns consistent with disorganized brain activity. However, pretreatment with C. longa (group vi) attenuated the effects of PTZ significantly (p < 0.0001 vs. SAL + PTZ), which indicates beneficial properties for the reduction of seizures. The attenuation of the delta waves in the two DZP groups (vii and viii) was also significantly greater in comparison with the animals pretreated only with C. longa (p < 0.0001 for DZP + PTZ and CL/DZP + PTZ vs. CL + PTZ).

A similar pattern was observed in the case of the theta waves (Figure 4B), with significant attenuation in the pretreatment groups (vi–viii) in comparison with the saline + PTZ group [F_{(3, 32)} = 275.0; p < 0.0001]. While C. longa alone significantly mitigated the effects of PTZ on the brain (p < 0.0001), the pretreatment with DZP (groups vii and viii) was significantly more effective (p < 0.0001).

The administration of PTZ also altered the alpha bandpower (Figure 4C) significantly [F(3, 32) = 244.8; p < 0.0001]. While all the different pretreatments had significant beneficial effects (p < 0.0001, for all comparisons with SAL + PTZ), CL/DZP was the most effective (CL + PTZ vs. CL/DZP + PTZ: p < 0.0001; DZP + PTZ vs. CL/DZP + PTZ: p = 0.0134), which indicates, once again, that the combination of C. longa and DZP provides better control than each compound on its own.

Similar results were also obtained for the higher-frequency waves, that is, the beta and gamma waves. As in the case of the alpha wave, all the different pretreatments had a positive effect on the beta wave (Figure 4D), thus reducing the oscillations significantly [F_{(3, 32)} = 388.0; p < 0.0001]. In this case, however, the C. longa treatments (CL or CL/DZP) reduced bandpower significantly more than the group that was pretreated only with DZP (CL + PTZ vs. DZP + PTZ: p = 0.0264; CL/DZP + PTZ vs. DZP + PTZ: p < 0.01). This indicates that C. longa may be especially beneficial for seizure control, in terms of the mechanisms that trigger the beta waves.

A similar pattern was recorded in the case of the gamma wave (Figure 4E), where all the pretreatments reduced the gamma wave bandpower significantly [F_{(3, 32)} = 69.62; p < 0.0001]. While the combined application of C. longa and DZP provided a better control for seizures than C. longa alone (p = 0.0008), it was no different from pure DZP (p = 0.1202).

As orofacial movement (chewing) is a diagnostic trait of PTZ-induced seizures, conjugated electrodes were implanted in the masseter muscle to evaluate its activity during the seizures. Following the application of PTZ, the seizures caused intense muscle contractions, with oscillations in amplitude of up to 0.5 mV in the electromyographic trace (Figure 5A). However, pretreatment with C. longa relieved the muscle contractions during the seizure, as revealed by a reduction and stabilization of this amplitude, and a significant reduction in the total power [F_{(3, 32)} = 93.35; p < 0.0001; SAL + PTZ vs. CL + PTZ: p = 0.0003; Figure 5B]. The use of DZP also resulted in intense myorelaxation, with a significant reduction in the total power in the EMG (Figure 5B; p < 0.0001). In the combined pretreatment (CL/DZP), the trace was less altered (Figure 5A), with significant improvement in the EMG in comparison with the SAL + PTZ group (Figure 5B, p < 0.0001), but not in comparison with the group treated with DZP alone (p = 0.8498).

The quantification of the Nissl-stained neuron-like cells in the CA1 and CA3 regions of the hippocampus (Figure 6A) indicated that the animals pretreated with saline that received PTZ on the fifth day suffered a significant reduction in the number of neuron-like cells in the CA1 region [F_{(7, 64)} = 226.9; p < 0.0001; SAL + (SAL and CL and DZP and CL + DZP) vs. SAL + PTZ: p < 0.0001; Figure 6B]. Although pretreatment with both C. longa (SAL + PTZ vs. CL + PTZ: p < 0.0001) and DZP (SAL + PTZ vs. CL + PTZ: p < 0.0001) resulted in the significant preservation of these cells, and C. longa performed significantly better than DZP (p < 0.0001), the combination of these two substances (CL/DZP) was the best pretreatment for the preservation of the neuron-like cells (SAL + PTZ vs. CL/DZP + PTZ: p < 0.0001; CL/DZP + PTZ vs. CL + PTZ: p < 0.0001; CL/DZP + PTZ vs. CL + PTZ: p < 0.0001). This indicates a protective effect on the cells in the CA1 region of the hippocampus 7 days after the seizures.

In the case of the CA3 region of the hippocampus (Figure 6C), a significant change was observed only in the saline group that received PTZ on the fifth day [F_{(7, 64)} = 62.69; p < 0.0001], which indicates that a lack of adequate preventive treatment for seizures may result in the damage of this layer. All the pretreatments tested in the present study provided significant prevention of the loss of the neuron-like cells (p < 0.001; for all comparisons).