Effect of low frequency stimulation of olfactory bulb and olfactory epithelium on epileptiform activity and synaptic plasticity following pentylenetetrazol administration in rats

doi:10.21203/rs.3.rs-6214600/v1

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Effect of low frequency stimulation of olfactory bulb and olfactory epithelium on epileptiform activity and synaptic plasticity following pentylenetetrazol administration in rats

https://doi.org/10.21203/rs.3.rs-6214600/v1

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Background: The anticonvulsant effect of olfactory bulb (OB) and olfactory epithelium (OE) electrical stimulation was investigated in anesthetized and freely moving animals.

Methods: Male Wistar rats were anesthetized with urethane (1.5 g/kg). Stimulating electrodes were bilaterally placed either in OB or OE. Another electrode was placed in the CA1 area for recording epileptiform discharges (EDs) following pentylenetetrazol (PTZ, i.v.) injection and evoked field potentials following Schaffer collateral stimulation. Rats were divided into PTZ and control groups. Each group received stimulation (1 Hz) either in OB (OBS) or OE (OES). ED threshold and duration, and the ability to generate long-term potentiation (LTP) were assessed. Finally, the effect of OBS on acute PTZ-induced seizure and working memory was investigated in freely moving animals. OBS significantly increased the ED threshold when applied at 250 µA and decreased ED duration when applied at 125 and 250 µA.

Results: Applying OES had a small effect on the ED threshold but significantly decreased ED duration when applied at 125 and 250 µA. Both OBS and OES mitigated the PTZ-induced increase in basal synaptic transmission. Meanwhile, OBS and OES significantly restored the LTP generation following PTZ injection in anesthetized rats. In addition, applying OBS in freely moving animals reduced the seizure severity and restore working memory impairment.

Conclusions Obtained data showed that the OB and OE may be considered as effective targets for electrical brain stimulation to attenuate epileptiform activity and seizure severity. In addition, both OBS and OES decreased the seizure-induced impairment in LTP generation.

Deep brain stimulation

Seizure

Olfactory bulb

Olfactory epithelium

Synaptic plasticity

Epilepsy is a common neurological disease that is characterized by recurrent seizures, mainly caused by an imbalance of excitatory and inhibitory activity in the brain. Although antiepileptic drugs are the main treatment of epilepsy, about 20–30% of patients treated with more than two anticonvulsant drugs are not seizure free and have drug-resistant epilepsy (1). Furthermore, some patients are not candidates for seizure-focus resection due to the difficulty in determining the precise seizure focus and the potential side effects of surgery (2). Therefore, finding treatment alternatives is necessary.

Deep brain stimulation (DBS) is a neuromodulation method that has been widely used for treatment of neurological disorders, including Parkinson’s disease, and recently is being used as an alternative treatment for pharmacoresistant epilepsy. Animal and human studies suggest that DBS may be an effective therapy for seizure prevention (3). Various patterns of antiepileptic stimulation have been employed, and one of the parameters which has a profound impact on the anticonvulsant effects of DBS is frequency (4). Both high frequency stimulation (HFS) and low frequency stimulation (LFS) can be effective in reducing epileptiform activity and seizures. However, HFS may cause tissue damage (5).

There are several studies showing the antiepileptic effects of LFS. Applying LFS on amygdala during amygdala kindling acquisition significantly decreases afterdischarge (AD) duration, behavioral seizure score and increased the number of stimulations required to become full-kindled (6). Moreover, hippocampal LFS decreased the amygdala stimulus-induced epileptic seizures (the seizure duration and afterdischarge frequency) and increased GABA_A receptor expression in pharmacoresistant epileptic rats (7). In the temporal lobe epilepsy model of pilocarpine, LFS of the subiculum completely inhibited spontaneous seizures (8).

In addition to DBS parameters, the DBS target is also an important factor in determining the DBS anticonvulsant action. Several brain regions have been considered as DBS target in both clinical and experimental research. These targets may be either the seizure focus, or other structures related to the region of seizure generation or propagation.

The olfactory system has functional and anatomical connectivity with the structures involved in seizure generation and propagation in temporal lobe epilepsy (9). In addition, nasal respiration entrains an oscillatory pattern in neural activity of olfactory system including piriform cortex and other regions related to olfaction, such as the hippocampus and amygdala. In some types of epilepsy, such as medial temporal lobe epilepsy, an olfactory aura may precede or accompany the seizure occurrence as an unpleasant, perceived odor (10). In addition, it is demonstrated that epilepsy in mice that received bulbectomy surgery becomes progressively more severe over time (11).

The olfactory bulb, as the primary area for receiving olfactory inputs, sends projections to the piriform cortex through olfactory tract fibers. The piriform cortex, as the primary olfactory cortex, is a part of limbic system and has outputs to various subcortical areas, including the entorhinal cortex and amygdala, which are related to temporal lobe epilepsy. A noteworthy point is that the piriform cortex is considered as a region of seizure generation and propagation. In a clinical study, resection of at least half the piriform cortex in patients with temporal lobe epilepsy increased the probability of seizure freedom (12). Piriform cortex tissue damage has been observed in status epilepticus animal models of chemoconvulsants kainic acid and pilocarpine (13). Furthermore, reduction in the volume of the temporal piriform cortex was found in temporal lobe epilepsy, suggesting the engagement of this area with temporal lobe epilepsy networks (14).

Recently, the olfactory system has been introduced as a potential target for DBS. It has been reported that applying LFS to the anterior piriform cortex decreased the frequency and severity of spontaneous seizures in the kainic acid model of epilepsy (15). In addition, applying LFS to the olfactory bulb had anticonvulsant effect in the hippocampal electrical kindling model of seizure (16).

Considering the relationship between olfactory systems and the structures involved in epilepsy, we aimed to study whether olfactory epithelium stimulation, as a non-invasive alternative method for DBS, would be as effective as olfactory bulb stimulation in seizure suppression. For this purpose, we used an in vivo model of epileptiform activity in anesthetized rat to be able to insert the stimulating electrode into the rat’s nasal cavity. Then, the threshold and duration of ED, as well as the ability of long-term potentiation (LTP) generation was assessed in the dorsal hippocampus.

Animals

138 male adult Wistar rats, aged 8–10 weeks and weighing 260–290 g, were used in this study. Animals were obtained from Pasteur Institute (Tehran, Iran) and kept in the animal facility under controlled conditions, including regular 12 h light/dark cycle, ambient temperature of 24 ± 2°C, and free access to food and water. Subjects passed a seven-day adaptation period before surgery. Ethics approval for this study was granted by the ethical committee of Faculty of Medical Sciences in Tarbiat Modares University, Iran (IR.MODARES.REC.1398.108). The experiments were run according to the guidelines of the ethics committee that were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals guidelines.

Electrode implantation in anesthetized animal

The animals were anesthetized with urethane (1.5 g/ kg, i.p.) and fixed in a stereotaxic frame. Stainless steel, teflon-coated electrodes (127 µm bare diameter, 212 µm coated diameter, A-M Systems, USA) were used. A monopolar recording electrode was implanted in the CA1 region of the right hippocampus (2.8 mm posterior to the bregma, 1.8 mm lateral and 2.3–2.5 mm below dura) according to the rat brain atlas (Paxinos and Watson, 2006). This electrode was used for ED recording.

For applying olfactory bulb stimulation (OBS), two bipolar electrodes were implanted in the right and left olfactory bulbs (8.5 mm anterior to the bregma, ± 1.1 mm lateral and 1.6 mm below dura). To record evoked field post-synaptic potentials (fEPSPs) in CA3-CA1 synapses in the right dorsal hippocampal CA1, a bipolar stimulating electrode was inserted in the Schaffer collaterals (3.1 mm posterior to the bregma, 3.1 mm lateral and 2.6-3.0 mm below dura), and fEPSPs were recorded from the CA1 by the monopolar electrode. Finally, a monopolar reference electrode was connected to a stainless-steel screw positioned at the left parietal bone.

For applying olfactory epithelium stimulation (OES), two bipolar stimulating electrodes were inserted into both nostrils of animals. Because the electrode insertion in the nostrils causes irritation and discomfort to the animal, we placed the stimulating electrodes in the nasal cavity of anesthetized animal. The electrode-depth in the nostrils was adjusted such that the bare tip of the electrode touched the olfactory epithelium. All experiments and recordings were run at least 15 min after surgery.

Another group of animals were anesthetized by ketamine and xylazine (100 mg/kg and 20 mg/kg, respectively). A monopolar recording electrode was implanted into the CA1 region of the dorsal hippocampus, and a bipolar stimulating electrode was inserted into the olfactory bulb at the same stereotaxic coordination and the same manner that explained above.

In vivo induction of epileptiform discharges in anesthetized animal

We used pentylenetetrazol (PTZ) for epileptiform discharges (ED) induction in anesthetized rats. For intravenous (i.v.) injection of PTZ, a 26 G plastic IV cannula was inserted into the lateral tail vein. The cannula was connected to a 5 ml syringe prefilled with heparinized PTZ solution via a polyethylene tube. The syringe was mounted in the infusion pump. The correct positioning of the cannula in the vein was verified by the presence of blood in the cannula, and once confirmed, the cannula was secured to the tail using adhesive tape.

PTZ was injected at a constant flow rate of 2.5 ml/min by an infusion pump (WPI, UK). PTZ injection was stopped immediately after observing the EDs in LFP recording. LFPs were recorded for 30 min after PTZ injections. EDs were detected as oscillations with the amplitude of more than twice the baseline, the frequency of more than 0.25 Hz. The ED duration was also calculated as the summation of primary and secondary EDs.

Evoked field potential recording in anesthetized animal

Evoked field potentials were recorded from dorsal hippocampal CA1 region when the Schaffer collaterals were stimulated in anesthetized rats. Animals were fixed in a stereotaxic apparatus that was placed in a Faraday cage. The depth of recording electrode in stratum radiatum of the CA1 region and the stimulating electrodes in Schaffer collaterals were changed smoothly to record a suitable field excitatory post-synaptic potential (fEPSPs). For fEPSP recording, Schaffer collaterals were stimulated while the fEPSPs were recorded from stratum radiatum of the CA1 area in the sink location. The fEPSP slopes were calculated as an index of excitatory synaptic transmission in the hippocampus.

The basal synaptic transmission was recorded for 20 min (to confirm the signal stability) by stimulation of the Schaffer collaterals at test-pulse intensity. The test-pulse intensity was calculated based on an input-output (I/O) curve and was determined as the intensity that elicits 40–60% of the maximum fEPSP slope. Stimulations were applied at test pulse at the frequency of 0.1 Hz. The fEPSP slope of 12 consecutive responses were calculated and averaged to be considered as the response at each time point in the time-line curves. The evoked filed potentials were filtered with a low pass at 3 kHz, digitized at a sampling rate of 10 kHz, and saved on disk using BIODAC ES1721 (Trita Health Technology Co., Tehran, Iran).

Following DBS application, we examined the impact of DBS on LTP generation. Therefore, a new test pulse was measured at about 1 h after applying DBS (there was no significant difference between this new test pulse and the previous measured test pulse). The baseline recording of evoked field potential was run for 20 min. LTP was induced by applying a prime burst stimulation (PBS) as a single pulse followed by a burst of 10 pulses at 100 Hz at 170 ms later, and the entire train was repeated 8 times at 10 s intervals. Subsequently, evoked field potentials were recorded for 60 min to evaluate the induction and maintenance of LTP.

The short-term plasticity was evaluated by measuring the paired-pulse index (PPI) using paired-pulse stimulation at the inter-pulse intervals (IPIs) of 50 ms and at the test pulse intensity. PPI was calculated by dividing the slope of the second fEPSP by the slope of the first fEPSP. The body temperature and blood glucose were monitored during the experiment.

Acute PTZ-induced seizure

To evaluate whether OBS had anticonvulsant action in freely moving animals, a PTZ acute model of seizure was used. PTZ was injected intraperitoneally at the dose of 60 mg/kg. Then, the animals’ behavior was video monitored, and the local field potentials were recorded for 30 min.

Animal stimulation

In anesthetized rats, stimulations were applied bilaterally as either OBS or OES before PTZ administration. The stimulation protocol was according to our previous experiments (17, 18). We applied OBS or OES in the pattern of low frequency stimulation (900 monophasic square pulses at 1 Hz and 0.1 ms pulse duration at the intensity of 125 µA or 250 µA) using BIODAC ES1721 (Trita Health Technology Co., Tehran, Iran).

Bilateral OBS was also applied in freely moving animals at the same pattern before PTZ injection.

Open filed test in freely moving animals

The locomotor activity of subjects was assessed by open field test. Rats were put into the open field arena, that was a cubed box (60×60×60 cm), and their activities were video monitored by a camera mounted on the top of the box for 5 min. The box was carefully cleaned with 70% ethanol before putting the animal inside it. The recorded videos were transferred to a PC containing Ethovision software 11 (Noldus Information Technology, Wageningen, The Netherlands) to measure the traveled distance, velocity, center time and border time for each animal.

Y-maze test in freely moving animals

To evaluate the working memory, we performed the Y-maze test before and about 24 h after PTZ injection. The Y-maze apparatus consisted of a black plastic maze with three arms (50 cm long, 32 cm high and 16 cm wide) that were intersected at 120◦. A rat was placed at the end of one arm and allowed to move freely in the maze for 8 min. Entries into all arms were noted (4 paws had to be inside the arm for a valid entry) and a spontaneous alternation was counted if an animal entered three different arms consecutively. The percentage of spontaneous alternation was calculated according to below Eq. (19).

All behavioral tests were done from 10 a.m. to 12 p.m.

Experimental procedures

In the first experiment, the effect of applying OBS was evaluated on ED threshold and duration and LTP generation. The animals were divided into PTZ and control groups. In the PTZ group, animals either received OBS at the intensities of 125 µA (PTZ + OBS125 group) or 250 µA (PTZ + OBS250 group) or did not receive OBS (PTZ group). Similarly, animals in the control group also received OBS at the intensities of 125 µA (control + OBS125 group) or 250 µA (control + OBS250 group) or did not receive OBS (control group). The time-line protocol of experiments is shown in Fig. 1. The test pulse intensity was determined in the PTZ + OBS group after a 15 min recovery period post electrode implantation, and the basal evoked potentials were recorded in the dorsal CA1 for 20 min. Then, OBS was applied for 15 min and PTZ injection was started immediately after the last pulse of OBS. PTZ injection continued until the onset of EDs. The LFP recording continued up to 30 min. Then, PBS was applied and LTP generation was evaluated for 1 h after. In the PTZ group the same protocol was run, but OBS was not applied. In the control and control + groups, the experimental procedure was the same, but the animals received saline instead of PTZ.

In the second experiment, the effect of applying electrical stimulation in olfactory epithelium (OES) was evaluated on ED threshold and duration and LTP generation. The animals were divided into PTZ and control groups and each group either received OES (as PTZ + OES and control + OES) or did not receive OES (as PTZ and control). The whole experimental procedures were completely the same as experiment 1 and only the stimulation target was olfactory epithelium instead of olfactory bulb (Fig. 1).

In the third experiment, the same pattern of OBS (250 µA) was applied in freely moving animals before acute PTZ injection. Animals were divided into control (received saline) and PTZ (received acute PTZ). Each group either received OBS or did not stimulate. The behavioral and electrophysiological seizure parameters were evaluated in PTZ and PTZ + OBS groups. Then, the open field and Y-maze test were performed in all experimental groups before and 24 h after PTZ or saline injection.

Statistical analysis

Data were expressed as means ± SEM. Statistical analysis was performed using GraphPad Prism version 9 (GraphPad Software). The statistical difference between groups data was assessed using paired t- test, one-way and two-way ANOVA analysis followed by Tukey’s post hoc test. Significance levels were set at p < 0.05.

The effect of applying OBS and OES on epileptiform discharges

OBS or OES were bilaterally applied at two intensities (125 and 250 µA) and ED threshold was determined based on the dose of PTZ (mg/kg). A one-way ANOVA test indicated that applying OBS prior to PTZ administration significantly (F_{(2, 41)} = 5.363, p < 0.01) raised the ED threshold at the intensity of 250 µA (55.31 ± 2.21 mg/kg in PTZ + OBS250 group (n = 13) vs 46.36 ± 1.57 mg/kg in PTZ group (n = 12), p < 0.01, Cohen’s f = 0.48). While the lower intensity did not yield significant impact, it still had a medium effect in reducing the ED threshold (52.22 ± 1.60 mg/kg in PTZ + OBS125 group (n = 19), Cohen’s f = 0.34) (Fig. 2B).

On the other hand, no significant change was observed in ED threshold following the bilateral application of OES (F_(2,33) = 1.443, p = 0.25) (Fig. 2C). Calculating the size effect also showed a small effect of OES on the ED threshold at both intensities of 250 µA (45.12 ± 1.35 mg/kg in PTZ + OES250 (n = 14), Cohen’s f = 0.08), and 125 µA (49.24 ± 2.34 mg/kg in PTZ + OES125 (n = 10), Cohen’s f = 0.18) (Fig. 2C).

The total ED duration was measured by summing the duration of primary and secondary EDs throughout the entire LFP recording period. A one-way ANOVA showed that applying OBS before PTZ application significantly (F_(2,38) = 14.97, p < 0.001) reduced the ED duration at both intensities of 125 µA (869.0 ± 55.82 s in PTZ + OBS125 group (n = 16), p < 0.01) and 250 µA (706.6 ± 53.06 s in PTZ + OBS250 group (n = 12), p < 0.001) compared to PTZ group (1092 ± 21.63 s, n = 13) (Fig. 2E). Similar effects were also observed following applying OES at 125 µA (772.2 ± 83.82 s (n = 9), p < 0.01) and 250 µA (860 ± 62.40 s (n = 13), p < 0.05) (F_{(2, 32)} = 8.446, p < 0.01) (Fig. 2F).

Then, we compared the percentage of changes in the ED threshold (relative to PTZ group) in PTZ + OBS250 and PTZ + OES250 groups. A two-way ANOVA showed no significant difference between the PTZ + OBS250 and PTZ + OES250 groups (119.3 ± 4.78% in PTZ + OBS250 group compared to 97.33 ± 2.91% in PTZ + OES250) (F_{(1, 52)} = 3.627, p = 0.06) (Fig. 2D). There was also no significant difference in the percentage of changes in ED duration (relative to the PTZ group) among different experimental groups (F_{(1, 46)} = 3.863, p = 0.06) (Fig. 2G). These data showed a similar anticonvulsant effect of OBS and OES.

Effect of applying OBS and OES on synaptic transmission

The effect of OBS and OES on synaptic transmission was evaluated by comparing the slope of fEPSPs before (as baseline) and 30 min after stimuli application. In control group (n = 11), the fEPSP slope was 104.9 ± 3.13 µV/ms. Applying OBS or OES had a moderate effect on reducing the fEPSP slope at both intensities (76.28 ± 11.03 µV/ms in control + OBS125 group (n = 8), Cohen’s f = 0.41; 77.91 ± 11.91 µV/ms in control + OBS250 group (n = 9), Cohen’s f = 0.40; 63.69 ± 10.54 µV/ms in control + OES125 group (n = 4), Cohen’s f = 0.58; 80.68 ± 21.00 µV/ms in control + OES250 group (n = 4), Cohen’s f = 0.34). PTZ administration potentiated basic synaptic transmission (122.50 ± 10.60 µV/ms, n = 13) with a larger effect compared to the control group (Cohen’s f = 0.62). Applying OBS at both intensities, significantly prevented the potentiation effect of PTZ on field potential slopes (82.33 ± 6.19 µV/ms in PTZ + OBS125 group (n = 11), p < 0.05; and 62.10 ± 6.94 µV/ms in PTZ + OBS250 group (n = 8), p < 0.001) compared to PTZ group (Fig. 3A). Similar effects were observed following applying OES at 125 µA with medium effect (80.72 ± 12.73 µV/ms (n = 7), Cohen’s f = 0.51) and at intensity of 250 µA (56.41 ± 8.53 µV/ms (n = 9), p < 0.01) (Fig. 3B).

Effect of applying OBS and OES on long term synaptic plasticity

Our previous experiments showed the restoring effect of DBS on synaptic plasticity and LTP in kindled animals (20, 21). Therefore, we tried to find out whether OBS or OES had similar effect on LTP generation in the present in vivo model of epileptiform activity. Anesthetized animals were stimulated at test pulse and evoked fEPSPs were recorded from stratum radiatum of dorsal hippocampus in response to stimulation of Schaffer collaterals. No significant difference was observed in test pulse intensity among different experimental groups.

LTP was induced by applying PBS in anesthetized rats and fEPSPs recording was continued for 60 min post PBS. Following application of OBS, a one-way ANOVA showed significant difference in LTP magnitude among different experimental groups (F_(3,41) = 6.516, p < 0.001) (Fig. 4B). A post-hoc Tukey test revealed that PTZ administration significantly decreased LTP magnitude compared to the control group (111.8 ± 4.14 (n = 13) vs 185.5 ± 16.90 (n = 13) respectively; p < 0.001). Applying bilateral OBS at both 125 and 250 µA prior to PTZ administration prevented the decrease in LTP magnitude, so that there was no significant difference between PTZ + OBS125 and PTZ + OBS250 groups and control animals. There was a significant difference in LTP magnitude only between PTZ + OBS250 (169.1 ± 20.98, n = 8), but not PTZ + OBS125 (156.6 ± 8.41, n = 11), and PTZ group (p < 0.05). However, applying OBS 125 µA had medium effect on LTP magnitude compared to PTZ group (Cohen’s f = 0.35) (Fig. 4).

Although a one-way ANOVA showed significant difference among experimental groups following bilateral OES administration (F_(3,37) = 6.853, p < 0.001), however, according to the post-hoc Tukey test, applying OES had not significant effect on LTP magnitude compared to PTZ group (Fig. 4). Of course, there was no significant difference between PTZ + OES and control group, which indicated the effectiveness of OES on LTP generation in PTZ + OES groups. In addition, OES had medium effect on LTP magnitude when applied at the intensities of 250 µA (146.4 ± 10.12 (n = 8), Cohen’s f = 0.30) and 125 µA (139.7 ± 16.49 (n = 7), Cohen’s f = 0.22) compared to PTZ group. Therefore, applying OES prior to PTZ administration may impact LTP generation in a manner similar to, but weaker than, that of OBS application.

Application of OBS or OES at both intensities (125 and 250 µA) in control groups had no significant effect on LTP magnitude and no significant difference was observed between control + OBS, control + OES and control groups (Fig. 4C).

Effect of applying OBS and OES on short term synaptic plasticity

To evaluate the effect of OBS and OES on short-term synaptic plasticity, paired-pulse index (PPI) was calculated using an inter-pulse interval of 50 ms before and after applying PBS in all experimental groups. There was no significant difference in PPI among different experimental groups before PBS. Similar to previous reports (20), PPI was significantly (p < 0.001) reduced in the control group (n = 13) after PBS application, as determined by a paired t-test. However, the PPI did not show a significant change in the PTZ group after PBS, indicating a decrement in short-term plasticity following PTZ administration and epileptiform activity. Applying OBS at both intensities restored the ability of short-term plasticity. There was a significant reduction in PPI post PBS in PTZ + OBS125 group (n = 11, p < 0.05) and in PTZ + OBS250 group (n = 8, p < 0.05) compared to before PBS values. Applying OES at both intensities had no significant effect on the ability of short-term plasticity following PBS application (Fig. 5).

Effect of applying OBS on seizure severity and working memory in freely moving animals

To evaluate the effect of OBS on seizure severity in freely moving rats, the effective pattern of OBS (i.e., 250 µA) was applied before the PTZ injection. An unpaired t-test showed a significant increase in latency to forelimb clonus (p < 0.05) and a significant reduction in epileptiform discharge duration compared to the PTZ group (p < 0.01). Moreover, the mortality rate was decreased in the PTZ + OBS group (1 out of 8 rats) compared to the PTZ group (4 out of 11 rats) (Fig. 6B).

Working memory was evaluated by Y-maze test about 24 hours after PTZ injection. There was an impairment in working memory following PTZ injection and a two-way ANOVA showed a significant decrease in spontaneous alternation percentage in the PTZ group (n = 6) compared to the control group (n = 5) (p < 0.05) (Fig. 6C). Applying OBS significantly increased the percentage of spontaneous alternation in the PTZ + OBS group compared to PTZ group (p < 0.01). Applying OBS in control + OBS group (n = 7) had no significant effect on the percentage of spontaneous alternation. In addition, there was not any significant difference in the total number of entrances among experimental groups (Fig. 6C).

Analyzing open field test data showed that there was no significant difference in locomotor activity among different experimental groups.

Data obtained in the present study showed that olfactory system may be considered as a target for anticonvulsant action of brain stimulations. We introduced both the olfactory bulb and olfactory epithelium as appropriate targets for electrical stimulation in seizure treatment. The results demonstrated that applying OBS and OES increased the ED threshold and reduced the ED duration, indicated that both treatments attenuated the epileptiform activity following acute PTZ administration. These data are in line with previous observations that applying LFS to the olfactory bulbs (16) or piriform cortex (15, 22), had anticonvulsant action.

Inserting the stimulating electrodes into the nasal cavity on the OE is very difficult in freely moving animals, because OE is a very sensitive organ in rats (23). This is why we planned the experiments on anesthetized animals. Of course, even the anesthetized animals showed a withdrawal reflex as soon as the electrode touched the OE. Considering this technical limitation, we could not evaluate the seizure behaviors in this experiment, and only ED parameters were analyzed for determining the seizure severity. In addition, in the last experiment, we could only investigate the anticonvulsant effect of OBS in freely moving rats. OBS had anticonvulsant action in freely moving animals and reduced the mortality due to decreasing the seizure severity. The similarity between data obtained in anesthetized and freely moving rats indicates that the in vivo model of epileptiform activity may be considered as a suitable model for investigating the anticonvulsant action of OBS and OES.

To be considered as a suitable target for anticonvulsant action of DBS, a brain area needs to have anatomical and functional connections with the regions involved in seizure generation or propagation. There are many studies that document a heavy connection between the activity of olfactory system and the hippocampus, which are among the most important brain areas in seizure generation and propagation (24). OB, and therefore OE, have both anatomical (25) and functional (26) connections with the hippocampus. The respiration-entrained oscillations, depending on breathing rate, have been revealed in rats and mice dorsal hippocampus (26, 27).

Human studies have also shown that respiration-locked activity occurs in several regions of the human brain and natural breathing synchronizes electrical activity in limbic-related brain areas, including the hippocampus (28, 29). In addition, a fMRI study demonstrated that in the entorhinal cortex (as the main source of hippocampal inputs), the neural activity and β oscillations increases following OB electrical stimulation, suggesting the involvement of these structures in olfactory information processing (30). Similarly, in a human study, Zhou et al. 2021 found that the hippocampus oscillates with respiratory rhythms even at rest, showing a coherence in olfactory-hippocampal networks (31). These data show the strong functional connection between olfactory system and the hippocampus as an important area involved in epilepsy and seizures.

On the other hand, many researchers showed the role of OB in convulsive seizures. For example, it has been demonstrated that increased neuronal excitability after bulbectomy in mice resulted in progressively more severe epilepsy over time (11). In another study, it was reported that pilocarpine-induced status epilepticus resulted in neuronal degeneration in the external plexiform layer and glomerular layer of the olfactory bulb (32). Moreover, significant impairment in olfactory function and a decrease in olfactory bulb volumes have been shown in epilepsy patients (33). All these documents confirm the hypothesis that olfactory bulb and olfactory epithelium may be suitable targets for DBS and brain stimulation in epilepsy treatment.

Similar to our previous study in freely moving animals (20, 34), in hippocampal slices of kindled animals (21), and in vitro epileptiform activity induction (18), a single dose injection of PTZ in anesthetized rats led to LTP impairment. Applying both OBS and OES had a restoring effect on LTP induction, although again OBS had stronger effect compared to the OES. Furthermore, considering the role of LTP induction in memory formation, evaluating the working memory by Y-maze test in freely moving animals also showed that applying OBS significantly restored working memory impairment induced by PTZ injection. These data also confirm that our in vivo epileptiform induction model is similar to both freely moving and in vitro epileptiform models for seizure study.

In the present study we also investigated the effects of OBS and OES on short-term plasticity by measuring the paired-pulse index. Like previous reports, paired-pulse facilitation was lower in the PTZ group compared to control before PBS application. This difference may be related to the increase in releasing probability following PTZ injection (35). In addition, while paired-pulse index reduced significantly following PBS in control group (because of synaptic potentiation and increment of releasing probability), there was no significant change in paired-pulse index following PBS in PTZ group (because of the inability of synapses to be potentiated following PBS). Interestingly, applying OBS significantly restored the changes in short-term plasticity and the paired-pulse index in PTZ + OBS groups. Applying OES had very weak effect on short-term plasticity impairment.

The exact mechanism of OBS and OES remains unclear and needs further study to shed light on it. However, the restoring effect of OBS and OES on synaptic plasticity may confirm the synaptic modulation hypotheses of DBS (36). In addition, our data showed that applying OBS and OES decreased basal synaptic transmission and prevented the PTZ-induced increase in excitability. Administration of OBS and OES prior to PTZ injection may suppress the olfactory bulb-hippocampal network and prevent the destructive effects of PTZ.

Interestingly, our results showed similar anticonvulsant effects following both OBS and OES application. Considering the direct projections from olfactory epithelium to olfactory bulb, the clinical translation of these data is that OES may be considered as a potential non-invasive brain stimulation method for epilepsy treatment.

Consistent with chemical olfactory system stimulation, it appears that electrical stimulation of the olfactory epithelium could yield positive results. Some studies indicated that sensory stimulation of the olfactory system by specific odorants could interfere with the synchronization typical of seizure activity and have an anticonvulsant effect (37). However, chemical stimulation demonstrated in experimental studies might not be useful for humans, as chemical inhalation may have carcinogenic effects in the nasal cavity and lower respiratory tract. Taking this into account, Li et al 2009 suggested using electrical stimulation of the olfactory mucosa for epilepsy treatment (38).

The other important result of the present study was that both OBS and OES exerted anticonvulsant action at low frequency. Our previous studies also showed the effectiveness of low-frequency DBS in suppressing seizure behaviors (16). However, in the case of OES as a probable, non-invasive method for brain stimulation, it is difficult to apply high frequency stimulation, as it may be intolerable for patients. More studies are needed to understand the exact anticonvulsant mechanism of OES, to find the optimal stimulation pattern and design the suitable instrument for applying OES in epilepsy patients.

DBS, as a new FDA approved therapeutic manner for drug-resistive epileptic syndromes, has recently attracted a lot of attention, especially for finding its best target. Obtained data showed that the OB and OE may be considered as effective targets for electrical brain stimulation to attenuate epileptiform activity and seizure severity. The similarity between the anticonvulsant actions of OES and OBS indicates that OES as a noninvasive brain stimulation method may be as effective as invasive DBS methods and is a potential alternative for epilepsy treatment. In addition, both OBS and OES decreased the seizure-induced impairment in LTP generation and memory.

Afterdischarges

DBS

Deep brain stimulation

Epileptiform discharges

fEPSPs

Field excitatory post-synaptic potential

HFS

High frequency stimulation

IPI

Inter-pulse interval

LFS

Low frequency stimulation

LTP

Long-term potentiation

Olfactory bulb

OBS

Olfactory bulb stimulation

Olfactory epithelium

OES

Olfactory epithelium stimulation

PBS

Prime burst stimulation

PPI

Paired-pulse index

PTZ

Pentylenetetrazol

Ethics approval consent to participate

All experiments and procedures were performed in accordance with the Tarbiat Modares University guidelines for animal care and approved by the Ethics Committee of the Faculty of Medical Sciences, Tarbiat Modares University (IR.MODARES.REC.1398.108).

Availability of data and materials

The data that support the findings of this study are available on request from the corresponding author.

Competing Interest

The authors declare that there is no conflict of interest.

Funding

This study was supported by a grant from Vice-Chancellor for Research of Tarbiat Modares University and a grant #4031507 from Iranian National Science Foundation (INSF).

Author contributions

SC carried out the experiment and analyzed the data and wrote the first draft of the manuscript; MZ and MR participated in the methodology; YF, MR and AS participated in the design of the study and in the results interpretation; VB participated in reviewing and editing; JM‑Z contributed in the design of the experiments, data analysis, writing the manuscript and preparing the funds.

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No competing interests reported.

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Effect of low frequency stimulation of olfactory bulb and olfactory epithelium on epileptiform activity and synaptic plasticity following pentylenetetrazol administration in rats

Status:

Version 1

Abstract

Figures

Background

Methods

Animals

Electrode implantation in anesthetized animal

Evoked field potential recording in anesthetized animal

Acute PTZ-induced seizure

Animal stimulation

Open filed test in freely moving animals

Y-maze test in freely moving animals

Experimental procedures

Statistical analysis

Results

The effect of applying OBS and OES on epileptiform discharges

Effect of applying OBS and OES on synaptic transmission

Effect of applying OBS and OES on long term synaptic plasticity

Effect of applying OBS and OES on short term synaptic plasticity

Effect of applying OBS on seizure severity and working memory in freely moving animals

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1