Our results reveal that low-intensity, pulsed FUS sonication suppressed the number of epileptic signal bursts observed in EEG recordings after the induction of acute epilepsy via intraperitoneal injection of PTZ. The presence of the suppressive effect was found in terms of the number of epileptic EEG spikes from the analysis of the unfiltered (Figure 4A) and theta-band (Figure 4B) EEG activity. FUS-mediated reduction of epileptic EEG activity was most notably observed in the theta band. EEG theta activity has also been consistently reported to have a positive correlation with the level of epilepsy [33–35]. Thus, our findings may offer more information with regard to the possible mechanisms involved in the reduction of epileptic activity (for example, the region-specific efficacy of FUS and its manifestation in theta band activity). The assessment of EEG patterns associated with sonication, in the absence of induced epilepsy, will offer rich information on the excitatory or inhibitory influence of FUS on neural circuitry, and it will provide more information on the applicability of FUS to non-pathological conditions. We also found that the second sonication session further enhanced the suppressive effect beyond that of the first sonication session. Based on the analysis of the Racine scores, it consistently appears that the FUS-treated group recovered from an epileptic state more quickly than the unsonicated group.
Taken together, these findings suggest that transcranial FUS sonication provided a significant suppressive effect on PTZ-induced epileptic activity in rats. These observations are in good agreement with previous studies on the temporary suppression of spontaneous activity in the excised crayfish ventral nerve cord  and on the suppression of visual activity in cats mediated through insonication of non-focused ultrasound . Although the inferior colliculus of rats is responsive to ultrasound  and can even induce audiogenic seizures [39, 40], our observations are unlikely to be associated with the auditory responsiveness of the inferior colliculus of rats to ultrasound frequencies. This is because the ultrasound frequency of the present study (e.g., 690 KHz) was far greater than the audible range (applicable to rodents, approximately 30 to 70 KHz) of ultrasound frequencies in which the maximal responsiveness of the inferior colliculus of rats was observed . Generally, the rodent species used in this study can process ultrasound up to approximately 80 KHz [41–43].
Since epileptic activity is caused by abnormally excessive or synchronous neural activity in the brain , and synaptic contacts could potentially be disrupted by ultrasound waves , FUS sonication might reduce the propagation of epileptic discharges across the brain. Alternatively, a different hypothesis can be put forth to explain our findings: FUS sonication may have caused a reduction in epileptic EEG activity by regulating thalamic GABAergic inhibitory neurons implicated in epilepsy . Evaluation of the extracellular neurotransmitter levels (such as GABA) may offer useful information to clarify some of these hypotheses through the use of microdialysis techniques that assay various types of neurotransmitters directly from the brain .
Although little is known about the detailed mechanism underlying FUS-mediated neuro-modulation, it has long been reported that ultrasound can significantly affect the neurophysiology of in vitro local neural circuitry [48, 49]. Gavrilov et al.  reported that the main effect of FUS in stimulating neural structures is due to mechanical force that could produce alterations in membrane potential, thus resulting in the stimulation of neural structures. It has also been proposed that ultrasound sonication may influence membrane fluidity, turbidity and permeability [50, 51]. Accordingly, the activity of ion-channels or receptors on the membrane can be influenced by ultrasound sonication , and the trans-membrane concentrations or passage of ions or neurotransmitters can be subsequently altered. FUS-mediated structural alterations in soma/axonal/dendritic connections may also have attributed to our findings and thus requires further investigation.
It has been consistently reported that ultrasound sonication activates voltage-gated Na+ and Ca2+ channels  and that a FUS-mediated mechanical force can activate several mechano-sensitive ion channels, allowing cation entry [52–55] and resulting in alterations in membrane potential . Therefore, FUS-mediated dysfunction of functional molecules, such as cell membrane transporters that are sensitive to trans-membrane ion-concentrations, may lead to biochemically altered states in the sonicated area. For example, activation of the serotonin transporter (SERT) is modulated by the trans-membrane gradient of Na+ and K+ , and trans-membrane ion concentrations are potentially altered by FUS sonication. Consequently, abnormal SERT activity, possibly by FUS sonication, may actuate a change in the extracellular level of serotonin (5-HT).
In terms of biological safety, it is noteworthy that ultrasound sonication can potentially generate free radicals [57, 58]. For example, ultrasound sonication can decompose water into hydrogen and hydroxyl radicals . These free radicals, although short-lived, are extremely unstable and can react easily with other surrounding biological molecules, possibly resulting in tissue damage and inflammatory response . However, these free radicals are typically produced at high acoustic intensities that are associated with cavitation . Since the current study uses an acoustic intensity much lower than those that produce cavitation and free radicals, sonication in the present study is unlikely to adversely affect the brain tissue.
As shown in the histological results (cf. Figure 5), the sonication employed in the present study did not cause any inadvertent biological damage to the target region. The intensity of sonication used in the present study was 130 mW/cm2 (Ispta), which is far less than the upper regulatory limit for non-obstetric ultrasound imaging (720 mW/cm2; ). It has been reported that when a short duration of sonication (5 sec) is used, ultrasound intensity up to 430 W/cm2 (at 936 KHz) can be applied without inducing mechanical damages to the brain tissue . The MI of the present study was 0.33, which is sufficiently within the range of safety guidelines (i.e., 1.9; ). Collectively, our sonication parameters are all within the range of safety guidelines for clinical ultrasound imaging and demonstrate a significant reduction of epileptic activity characterized in EEG and behavioral monitoring.
The present study has several technical limitations to overcome. First of all, since the intraperitoneal injection of PTZ elicits hyper-excitability over the distributed regions of the brain, region-specific anti-epileptogenic effects of FUS were not demonstrated. In order to examine the utility of FUS in suppressing region-specific epileptogenic activity, a regional chemical kindling model such as an intracortical injection of kainic acid (KA) can be adopted to induce focal epileptic lesions in an animal model. KA induces nonconvulsive status epilepticus, followed by the chronic occurrence of spontaneous recurrent seizures and massive hippocampal damage [64–66]. The regional application of FUS to a KA-kindled epileptogenic focus for probing its potential utility in the treatment of chronic focal epilepsy constitutes one of our future subjects of investigation.
Another technical limitation of the study is the spatial error introduced while positioning the sonication focus. There are several sources that can contribute to potential spatial error while targeting the sonication focus to the thalamic area. These sources include the inherent mechanical repositioning error of the mechanical 3-axis stage that mounted the transducer as well as spatial error associated with acoustic field distortion during transcranial FUS application. Since these sources typically introduce errors that are significantly smaller than the acoustic focus, the major source of spatial error during positioning of the focus can be traced to the use of an external anatomical landmark, i.e., the ear canal and associated inter-aural lines, during stereotactic positioning of the animal with respect to the sonication apparatus. Based on the work by Rubins et al. , the potential spatial error associated with the procedure can be estimated to be on the order of 0.5 mm, which is approximately 15% of the short-axis diameter of the FUS focus (3.5 mm in diameter). The characterization of the exact location and size of the sonication focus in the brain would clearly improve the spatial accuracy of sonication delivery. The use of magnetic resonance imaging (MRI) enables an elaborate spatial guidance system for the application of focused acoustic energy to a defined anatomical location [11, 68, 69]. For example, an MRI-compatible stereotactic positioning system [70, 71] would allow users to track the coordinates of the sonication focus. Localization of the sonication focus can also be accomplished by the guidance of acoustic radiation force impulse (ARFI) imaging which can visualize the degree of acoustic force imposed on tissues without the generation of heat [72–74].
It is also noteworthy that FUS can elicit neuronal stimulation with different sets of FUS-parameters (i.e., TBD = 50 msec, PRF = 10 Hz in rabbits or TBD = 0.4 msec, PRF = 1500 Hz in rats, and both achieved at a higher acoustic intensity of Ispta 4~6 W/cm2; unpublished data). Therefore, it is reasonable to predict that FUS could further exacerbate neuronal hyperactivity in epilepsy. Accordingly, further studies on parameter-dependent efficacy of the method and careful selection of the sonication parameters are needed to develop appropriate treatment guidelines.
FUS-mediated region-specific functional neuro-modulation promises new, powerful ways to study brain function and brain-behavior relations. As a result, we anticipate that this technique may influence the development of new modalities for neurotherapeutic treatments across a wide clinical range. For instance, neurological conditions that are associated with subcortical structures (i.e., pain and movement disorders related to abnormalities in the thalamus and elements of the limbic system) may potentially benefit from FUS due to its ability to reach deep brain regions in a non-invasive way. Similarly, the modulatory effects of FUS can be utilized to modify aberrant brain activity and neurotransmission associated with various psychiatric conditions, such as depression or post-traumatic stress disorder.