Neurophotonics’ methods in approach to in vivo animal epileptic models: advantages and limitations

Abstract

Neurophotonic technology is a rapidly growing group of techniques that are based on the interactions of light with natural or genetically modified cells of the neural system. New optical technologies make it possible to considerably extend the tools of neurophysiological research: from the visualization of functional activity changes to control of brain tissue excitability. This opens new perspectives for studying the mechanisms underlying the development of human neurological diseases. Epilepsy is one of the most common brain disorders, it is characterized by recurrent seizures which affects over ~1% of the world's population. However, how seizures occur, spread, and terminate in a healthy brain is still unclear. Therefore, it is extremely important to develop appropriate models to accurately explore the causal relationship of epileptic activity.

The use of neurophotonic technologies in epilepsy research falls into two broad categories - the visualization of neural epileptic activity, and the direct optical influence on neurons in order to induce or suppress epileptic activity. An optogenetic variant of the classical kindling model of epileptic seizures in which activatable cells are genetically defined, is called optokindling. Research is also underway concerning the application of neurophotonics techniques for suppressing epileptic activity, aiming to bring these methods into clinical practice. This review aims to systematize and describe new approaches that use combinations of different neurophotonics methods to work with in vivo models of epilepsy. These approaches overcome many of the shortcomings associated with classical animal models of epilepsy and thus increasing the effectiveness of developing new diagnostic methods and anti-epileptic therapy.

INTRODUCTION

Epilepsy is a severe neurological disorder that has a significant impact on a patient’s quality of life. Epilepsy manifests in the form of paroxysmal periodic activation of brain neurons that synchronize excitation in a common network. Antiepileptic pharmacological drugs are the first-line therapy for epilepsy. As a rule, they can provide excellent control over seizures [1]. However, their action is directed at reducing epileptic symptoms and not at eliminating the root causes of epilepsy. In addition, the drugs are not effective for all patients and not for all forms of epilepsy. A separate serious problem is drug-resistant epilepsy [2]. Studies using animal models can help advance our understanding of the mechanisms behind the development and termination of epileptic seizures. Furthermore, they are useful in the context of testing the effectiveness of new antiepileptic drugs.

Over the course of the past, various genetic and non-genetic models have been successfully used in the study of seizures and in the search for new antiepileptic drugs [2]. The gold standard for epilepsy research has always been electroencephalography (EEG), including its branch, electrocorticography (ECoG). Despite its advantages, this method has significant drawbacks - its ability to localize the epileptic focus is extremely limited [3][4]. The widespread use of functional magnetic resonance tomography (fMRI) and positron emission tomography (PET) played a significant role in the development of epileptology, but could not radically improve the situation [5][6][7][8]. The spatial resolution of these methods remains limited, and in addition, it is often possible to localize the epileptic focus only while the seizure is occurring, further complicating the evaluation. When working with epileptic animal models, the use of these technologies is even more difficult due to the small size of the animal brain and the high cost of these methods [9][10].

Epilepsy models can be divided into three large groups in accordance with their mechanism of action - physical, chemical, and genetic [11]. In the first one, the role of an epileptogenic is played by a physical effect, such as an electric current or elevated temperature. In the second, chemical agents with epileptogenic properties are used. And in the third one, the models are genetically modified animals that demonstrate certain features of the epileptic phenotype [11][12].

Such models include the 6-Hz mouse model of partial seizures, the intrahippocampal kainate model in mice, and animals in which spontaneous recurrent seizures develop following the induction of status epilepticus by chemical or electrical stimulation [13][14]. A model based on intracortical administration of 4-Aminopyridine (4-AP) has proved to be very useful for the study of epileptic seizures, the main clinical component of epilepsy [15]. All of these models have serious limitations since the forms of epilepsy are very diverse. However, although the mechanism of epileptic seizures has been studied in many animal models using chemical or electrical induction, or spontaneous manifestation of epileptic activity in genetically modified animals, interpretation of the results of such studies is difficult.

Optical techniques have revolutionized many directions of neurobiological research [2][7]. Their introduction has radically changed the development and investigation of epileptic models [16]. After the development of optical imaging of intrinsic signals, the process of creating new optical imaging methods has developed exponentially [16][17][18][19]. Currently, a large number of them are widely used when working with in vivo epileptic models, and the number of neurophotonic technological solutions used in this case is growing [20]. The development of neurophotonics had a significant impact not only on the methods of using epileptic models but also on the models themselves [21].

The development of neurophotonics has made it possible to use combinations of optical and genetic methods for the study of epileptogenesis. Such a strategy circumvents the shortcomings associated with classical animal models and provides new opportunities in the development of antiepileptic strategies.

1. THE OPTICAL IMAGING OF THE EPILEPTIC SEIZURES

1.1. Optical Intrinsic Signal Imaging

If, with a certain degree of conventionality, we talk about several generations in the method of functional brain optical imaging, then optical intrinsic signal imaging (OISI) certainly belongs to the first generation [22][23]. This method is based on the difference in the optical properties of oxy- and deoxyhemoglobin, and numerous epileptic studies have been carried out with its help. The technique is constrained by its low temporal resolution; however, it offers several advantages, notably simplicity and the elimination of the necessity to employ dye indicators that may possess toxic properties (Figure 1A). Despite the low temporal resolution, OISI remains a valuable method for monitoring epileptic seizures in many studies. Thus, using the well-known 4-AP mouse model of epileptiform activity, heterogeneity of hemodynamic changes in different areas of the cortex during seizures was demonstrated [24]. Using OISI, a persistent drop in the light reflectance around the center of epileptic activity was found, which corresponds to the dynamics of hemoglobin oxygenation. This phenomenon is called "epileptic dip". Under similar conditions, a similar phenomenon is also observed using functional magnetic resonance imaging [25].

Figure 1.

In vivo optical imaging of the epileptic activity. When using IOSI (A), a region of the cortex is illuminated with near-infrared monochromatic light. The intensity of light reflection at each point is mainly determined by the ratio of oxy- and deoxyhemoglobin at the current time. This makes it possible to carry out functional mapping without the use of chemical indicators introduced from outside. Voltage-sensitive dye imaging (B) is based on the administration in the brain of voltage-sensitive dyes. Being embedded into the neural membrane, molecules of these dyes change their fluorescent properties depending on the transmembrane electric field. This makes it possible to perform functional imaging with very high temporal resolution but requires more sophisticated technology. The excitation and emitted light must be separated, usually, a dichroic mirror is used for this purpose.

From a physical point of view, functional near-infrared spectroscopy is a kind of OISI, but transcranial. Because of this, fNIRS has an extremely low spatial resolution, but this method is harmless and can be easily applied to awake patients. fNIRS has been successfully applied to the detection and prediction of epileptic seizures [26].

Being safe for the patient, the OISI method has found application for the surgical treatment of epilepsy [27]. Both spontaneous and stimulation-evoked epileptiform activity has been successfully mapped using OISI [27]. In more recent OISI epileptic studies, functional cortical maps have demonstrated evidence of a hierarchy for sensory response patterns in the human primary somatosensory cortex [28]. Further development of the OISI method as applied to the human brain during neurosurgical operations led to the development of a method for differentiating epileptic cortical zones using the analysis of low-frequency oscillations of the optical signal. This method detects epileptogenic cortices by exploiting the biophysical features contributed by the altered vessel networks within the epileptogenic cortex thereby facilitate clinical decision-making [29].

Finally, OISI was successfully used in the clinic to acquire high-resolution maps of epileptiform activity in patients undergoing surgery for medically intractable epilepsy. This technology made it possible to observe both spontaneous and stimulation-evoked epileptiform activity on the open brain. Potentially OISI has a perspective to become a tool for a new approach to the surgical treatment of epilepsy [30].

Photoacoustic imaging is also a hybrid technology based on a combination of laser scanning and ultrasonic imaging of the object under study [31][32]. Photoacoustic tomography (PAT) uniquely combines the high contrast advantage of optical imaging and the high-resolution advantage of ultrasound imaging in a single modality. PAT is also able to provide functional information, including blood volume and local oxygenation but unfortunately, the low temporal resolution is a disadvantage of this method [33]. However, PAT has been successfully applied to the imaging of epileptic seizures in vivo. Moreover, epileptic activity in the thalamo-cortical loop was successfully visualized using PAT [31][34][35].

1.2. Voltage-sensitive dyes imaging

Subsequently, following the implementation of OISI, other imaging technologies emerged and progressed [36]. Voltage-sensitive dyes imaging (VSDi) appeared, which made it possible to directly monitor neuronal activity with a temporal resolution commensurate with the resolution of electrophysiological methods [22][37][38]. In contrast with OISI, optical imaging, based on the VSD, reflects neural activity directly, and therefore demonstrates very high spatial temporal resolution. Being fluorophores, sensitive to the electric field, molecules of VSD are embedded into the neural membrane and make it possible to monitor membrane potentials by recording the fluorescent optic signal (Figure 1B). Thus, using VSDi, the synchronization of neuronal activity during focal epileptic seizures caused by 4-AP was studied in vivo. It demonstrated a statistically significant increase in synchronization during seizure activity [39]. Using VSDi in combination with OISI made it possible to simultaneously register changes in the membrane potential and local oxygenation in vivo. As it turned out, epileptic seizures consist of several dynamic multidirectional waves of changes in the membrane potential. Local blood flow in the focus of epileptic activity changes much more slowly [40].

Voltage-sensitive dyes for the study of epileptic seizures are used not only for the direct fluorescent imaging of membrane potentials. Interesting results have been obtained by combining VSD with photoacoustic imaging of optical absorbent [41]. This photoacoustic technology is based on differences in the photoacoustic effect when a photon is absorbed by various biomolecules [42][43]. The absorption of photons by voltage-sensitive dye molecules is significantly higher than the absorption by surrounding tissues. In addition, this absorption varies depending on the state of the cell membrane to which these molecules are bound. This allows the use of photoacoustic for functional imaging. A significant disadvantage of this method is strong photobleaching since photoacoustic requires the use of fairly powerful lasers [41][44].

Thus, next-generation models were developed, and optical techniques have revolutionized this process, as have many other directions of neurobiological research [2][7]. The introduction of optical methods into practice has radically changed the work with epileptic models [16]. After the development of optical imaging of intrinsic signals, the process of creating new optical imaging methods has developed exponentially [16]. Currently, a large number of them are widely used when working with in vivo epileptic models, and the number of technological solutions used in this case is growing [16][20]. The development of neurophotonics has had an impact not only on the methods of using epileptic models but also on the models themselves [21].

1.3. GENETICALLY ENCODED INDICATORS OF NEURAL ACTIVITY

It should be noted that VSDs are highly toxic and subject to photobleaching, so they are only suitable for use in acute experiments. The genetically encoded indicators lack this disadvantage [45]. Genetically expressed calcium indicators (GECI) are suitable for the optical recording of epileptiform activity but have a significantly lower temporal resolution [46]. This disadvantage is largely compensated by the high level of fluorescence, which makes calcium imaging particularly suitable for recording the activity of individual neurons.

Genetically encoded fluorescent indicators (GEVI) provide good cellular resolution, and temporal resolution can be achieved by combining GEVI with voltage-sensitive fluorescent indicators in appropriate imaging setup, [47][48]. A common disadvantage of all optical methods is their limited depth, which is determined by the optical properties of the brain parenchyma.

The performance of existing GEVIs is limited by the brightness and photostability of fluorescent proteins. These shortcomings are gradually being overcome, and recently GEVIs called Voltron and Positron have been developed that use photostable synthetic dyes instead of protein-based fluorophores [49][50][51]. This increased the effectiveness of the indicator by an order of magnitude [49]. Visualization of the membrane potential of genetically strictly defined cells provides a unique opportunity to obtain information about the spatial and temporal dynamics of electrical signals at the cellular level in many situations. There is no doubt that this technique will soon be applied to the study of epileptic activity [49][51][52].

The use of modern GEVIs in combination with modern microscopic technologies makes it possible to combine subcellular spatial resolution with millisecond resolution from identified neuronal populations in living brain slices [53][54]. For transient expression, indicators can be injected directly into the animal's brain using lentiviral or adenovirus vectors. An alternative way is to obtain transgenic animals expressing these indicators under the control of tissue-specific or constitutive promoters [47][55][56][57].

In addition to GEVI, numerous fluorescent indicators with extremely high molecular selectivity have been developed in recent years. Using these indicators, we can optically monitor the release of neurotransmitters, changes in acidity, and ion concentrations. Thus, there is a fluorescent initiator of GABA, the main inhibitory mediator of the central nervous system [58]. Based on the GABA peptide receptor iGABASnFR, this indicator detects the release of GABA, causing an easily detectable increase in fluorescence in vivo in mice and zebrafish [58]. Methods for fluorescent monitoring of glutamate have also been developed. The glutamate indicator iGluSnFR allows visualization of neurotransmission with high cell specificity. The novel fluorescence probe iGluSnFR3 exhibits fast, nonsaturating activation kinetics and reports synaptic glutamate release with increased specificity in mouse visual cortex [58]. Recently it was reported about genetically-encoded sensors GPCR-Activation-Based-DA (GRABDA) that enable measure dopamine (DA) changes reliably and specifically with high spatiotemporal precision [59]. In response to DA releasing, GRABDA sensors exhibit large fluorescence increases with subcellular resolution [59]. This sensor can resolve a-single-electrical-stimulus evoked DA release and detect DA release in flies, fish, and mice. Thus, GRABDA sensors enable DA monitoring with high spatial and temporal resolution in a variety of organisms [59]. Among the neurotransmitters that can be studied using genetically encoded fluorescent indicators, serotonin is no exception [60]. Such Indicators allow researchers to achieve remarkable spatiotemporal resolution when studying serotonergic circuits in a variety of preclinical models of nervous system diseases [60]. Single-neuron resolution dynamic fluorescence imaging of activity with fluorescent tracers holds great promise for epilepsy research in in vivo experimental model systems [61]. In recent years, Genetically encoded fluorescence pH sensors have also been developed, and sensors sensitive to zinc, chlorine, magnesium and potassium ions have appeared [62]. An extremely interesting method for pre-clinical studies of epilepsy may be imaging of chloride ions in cortical neurons. Chloride currents are associated with ionotropic GABA A receptors [63]. The regulation of chlorine transmembrane currents is impaired in some brain pathologies, including epilepsy. There are both chemical and genetically encoded probes that allow monitoring of chlorine ions by optical methods [63]. These methods have significantly revolutionized the strategy of neurobiological research, but their use directly in working with epileptic models is still quite limited.

In conclusion, it should be noted that optical intrinsic signal imaging provides a general picture of epileptic seizures in open-brain animal models, but does not provide information about cellular interaction in the epileptic focus. However, this method is suitable for chronic experiments, and to some extent applicable in clinical practice, since it does not require additional chemical or genetic manipulations with the brain. Voltage-sensitive dye imaging provides high temporal resolution, and in some cases, high (down to the cellular level) spatial resolution, but is applicable only in acute animal experiments due to the dye toxicity. Genetically expressed calcium indicators are also suitable for the optical recording of epileptiform activity but have a significantly lower temporal resolution. This disadvantage is largely compensated by the high level of fluorescence, which makes calcium imaging particularly suitable for recording the activity of individual neurons.

2. THE OPTICAL STIMULATION OF NEURONS IN MODELS OF EPILEPSY

2.1. Methods for stimulating neuronal activity using light

One of the common methods of provocation of epileptiform activity is a model of the development of epilepsy by repeated electrical stimulation of brain tissue and the consequent development of spontaneous seizures. While electrical stimulation is widely used to study neural circuits, optogenetics offers the opportunity to stimulate or suppress the activity of neurons in certain subpopulations. One of the main goals of modeling epileptic activity is to find the cellular basis for the formation of seizures. Experiments that combine artificially induced seizures with imaging methods reveal areas of the brain involved in seizures [64]. In most cases, however, such methods fail to identify the cell population involved in seizures. Optogenetic methods make it possible to stimulate or inhibit strictly defined populations of neurons, or selectively recording their activity [65].

Optogenetic stimulation can induce responses in neurons that express a light-sensitive transmembrane protein, usually channelrhodopsin. Like electrical stimulation, optical stimulation can be used in models of epilepsy (Figure 2A). Numerous mouse models of epilepsy are based on overheating since an increase in body temperature is known to cause a decrease in seizure readiness and the development of a generalized epileptic seizure [66][67]. Impressive results have been obtained using both opto- and chemogenetic effects on hippocampal neurons. Such exposure, shortly before the application of overheating, increased the sensitivity of the brain to elevated temperatures and contributed to the development of a generalized seizure [66].

Figure 2.

Simplified diagram of feedback disruption caused by optogenetic stimulation (A) and optokindling (B). Neurons expressing channelrhodopsin-2 (ChR2+) located in the anterior piriform cortex (aPCx) are activated by light. The piriform cortex (pPCx) neurons do not have photosensitive peptides and do not respond to photostimulation, but receive excitatory inputs (red arrows) from the aPCx neurons. However, light-activated aPCx neurons also affect inhibitory interneurons, which in turn send inhibitory signals (black arrow) to pPCx neurons. (A). After optokindling the GABA synthesis inhibitory interneurons is reduced (B). As a result, a decrease in GABA synthesis in inhibitory neurons (yellow) reduces the inhibitory effect on pyramidal neurons, which leads to an increase in convulsive readiness.

2.2. EPILEPSY MODELS BASED ON OPTOKINDLING

Prolonged repeated stimulation of particular brain structures, so-called kindling, leads to the gradual development of a focus of persistent excitation and the spread of local epileptic discharges to neighboring zones [68][69]. The state of increased convulsive activity formed as a result of kindling in the brain persists for a long time, possibly a lifetime of the experimental animal. In the conditions of kindling, one can clearly monitor the onset of seizures and the staging of their development. Behavioral and electrographic convulsive manifestations observed during kindling conditions exhibit a notable level of reproducibility, making it easy to quantify and analyze their properties. In numerous epileptic studies, it is crucial for the kindling animals to develop spontaneous seizures without the requirement of intentional kindling stimulation during testing [68].

In electrical kindling models, the animal's brain receives repeated electrical pulses at subconvulsive levels, but this stimulation will eventually induce convulsions [70]. Thus, seizures are not always spontaneous, they are often triggered by a slowly developing local process, but may become spontaneous over time. However, along with the advantages, kindling also has a number of disadvantages. Electrostimulation can cause tissue damage and inflammation. In addition, the population of neurons involved in kindling is not controlled, since electrostimulation does not have cell selectivity.

Methods of optogenetics help to circumvent these shortcomings. An alternative to the classic kindling - optokindling - was proposed in 2019, when a technique was developed to induce seizures in the neocortex using optogenetics instead of electrical stimulation [71].

It should be noted that in the recent study, evidence was obtained for the effectiveness of optokindling not only by monitoring seizures but also at the cellular level [72][73]. This was done using Fos-expression, a histological method to identify neuronal activity. In animals euthanized on day I of kindling, increased Fos-expression was limited to the directly stimulated area of the anterior piriform cortex [74]. In contrast, in animals euthanized on day four of optokindling, Fos expression was widespread in unstimulated cells of the anterior and posterior piriform cortex [72]. Experiments on surviving brain slices have shown that in optokindled animals, compared with controls, there was a significantly higher ratio of excitatory to inhibitory (E/I) postsynaptic currents in the area of application of kindling, primarily due to a decrease in the amplitude of the inhibitory current. It was concluded that the synaptic contacts of kindled neurons had an increased E/I ratio [73][75]. A protocol for optokindling has been developed in detail and published. It has been shown that optokindling is broadly similar to electrophysiological kindling [73].

Optokindling allows control of the activity of genetically defined neurons without serious damage or inflammation [64]. Epileptic seizures can be induced in initially healthy animals by repeated light stimulation of neurons expressing Channelrhodopsin-2 (ChR2), which is a light-switched cation-selective ion channel [64]. Simultaneously, it was possible to conduct chronic EEG recordings, while video monitoring can be used to assess behavioral activity.

Optogenetic methods have been adopted in epileptic studies to study both stopping and initiating seizures [64][71]. Optokindling protocol eventually produces seizures that increased in number and severity with additional stimulation sessions. It was shown that seizures are associated with long-term plasticity, but not with damage to neurons or astrocytes. The effect obtained by optokindling was kept for several weeks [71]. It can be considered proven that optokindling has many similarities with classical kindling but has the added benefit of being able to study the role of specific populations of neurons. This allows the study of the relationship between long-term plasticity and epileptogenesis.

The mechanisms of optokindling are not entirely clear but are being actively studied. As with electrical kindling, regular optogenetic stimulation leads to epileptogenesis. However, selective activation of key neurons involved in this process may enhance recurrent excitation. (Figure 2A). Conversely, optokindling attenuates feedback inhibition by disrupting GABA synthesis and release [72]. In a recent study, optogenetic stimulation was used to activate populations of mouse piriform cortex (PCx) principal neurons in vivo. Already after 3–4 days of stimulation, massive generalized seizures could be induced earlier by subconvulsive stimuli (Figure 2B) [72].

Optokindling can increase the strength of recurrent excitation through one or more of three mechanisms: (1) by increasing the likelihood of presynaptic vesicle release, (2) by increasing the postsynaptic response to glutamate release (3) by increasing the number of synaptic contacts between neighboring neurons [72]. But as is well known, in epileptic models’ seizures can be induced by exposing neurons of different ergodicity. Not only hyperactivation and lowering of excitation thresholds of glutamatergic neurons but also inactivation of GABAergic inhibitory interneurons provokes epileptiform activity. A comprehensive study based on optokindling has been undertaken to identify the molecular processes by which patterns of brain activity can gradually and pathologically disrupt the balance of cortical excitation and inhibition [72]. The obtained data led the authors to the conclusion that during optokindling inhibition is weakened due to a decrease in the expression, synthesis, or availability of GABA, in other words, optokindling depletes GABA and weakens feedback inhibition.

It seems important that the authors attempted to determine whether the expansion of the spread of optically induced neuronal activity is the result of a shift in the ratio of excitatory and inhibitory (E/I) synaptic inputs to pyramidal neurons. For this purpose, in vitro experiments were carried out using brain living slices of control animals and animals after optokindling [71]. Cells affected by optokindling were positively identified by the expression of yellow fluorescent protein (YFP), associated with ChR2. It was shown that upon optical stimulation of ChR2+ neurons, optokindled animals demonstrated a significantly higher E/I ratio of postsynaptic currents due to a decrease in the amplitude of the inhibitory current. Wherein electrical stimulation did not reveal any differences between the slices of control and optokindled animals. Histological analysis showed that there were no changes in the number of interneurons or density of GABA terminals, but there was a marked decrease in GABA levels after optical kindling [75]. It can be said with a high degree of certainty that optokindling of pyramidal neurons leads to a deficiency in the synthesis, transport, and/or packaging of GABA in adjacent interneurons (Figure 2). This decrease in GABA levels, in turn, leads to impaired disynaptic inhibition, leading to the pathological spread of excitation [75]. However, since there is no excitatory synaptic disturbance, there is no excitotoxicity. This is certainly an advantage of the optokindling-based model of epilepsy, but it should be noted that the role of excitotoxicity in animal models of epilepsy remains somewhat unclear [75].

Probably, during the formation of epileptiform activity, the mechanism described above may not be the only one. The hypothesis that changes in synapses similar to those caused by long term potentiation (LTP) contribute to the formation of epileptiform activity seems quite plausible [71]. Mechanisms of memory formation like LTP are similar to the synaptic changes underlying kindling-induced epileptogenesis. Kindling as well as LTP are most effectively induced by high-frequency stimuli, which trigger N-methyl-D-aspartate (NMDA) receptor-inducible calcium cascade and synthesis of peptides that regulate synaptic transmission [76]. LTP is a long-term enhancement of synaptic transmission, which is cellular correlate of learning and memory. LTP-induced changes result in a long-lasting postsynaptic increase in the sensitivity of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors and this increase may play a role in lowering excitability thresholds, leading to increased epileptiform activity [77].

In another recent study, the authors explored optokindling regulation of the excitability of neural circuits [78]. The level of excitability is affected by synaptic efficiency and internal excitability of neurons. The complex relationship between these factors largely determines the functioning of neural circuits and their susceptibility to seizures. Optokindling stably induced epileptiform activity, and systematic modulation of light intensity made it possible to accurately assess the threshold between normal and abnormal neuronal activity [78]. This approach certainly provides a new platform for testing the effects of therapeutic interventions on neural circuits.

A huge advantage of optogenetic methods lies in the ability to selectively and specifically express photosensitive peptides in local populations of neurons. For these purposes, adeno-associated virus (AAV) was used to express ChR2 in excitatory neurons of the ventral hippocampus. The serotype of the AAV itself can confer a degree of selectivity through specific tropism [79]. Selectivity of expression was achieved by using a promoter of calcium/calmodulin-dependent protein kinase II (CaMKIIa) [80]. In research using AAV, evidence was obtained for the spread of epileptic activity during kindling from the ipsilateral to the contralateral hemisphere [81].

Although successful, these approaches have a number of limitations. Target in the brain can be very small or very large or difficult to access or inject with the adeno-associated vector. As a result, brain areas may obtain different levels of opsin expression in cells from the injection site after the introduction of the vector [82].

The transgenic mouse approach can overcome many of these limitations. In such situations, a frequent strategy is to use the Cre recombinase-loxP targeting strategy. In this situation, one group of animals expresses Cre under a specific promoter (eg, in neurons expressing parvalbumin [PV]). They are crossed with another group of animals that express the opsin in a Cre-dependent manner. Double genetically-modified animals will only express particular genes under the control of a specific promoter (Figure 3A, B). Using a chimeric CreERT2 (encoding a Cre recombinase fused to a mutant estrogen ligand-binding domain of ERT2), this method allows the regulation of Cre recombinase activity at the target locus. In the presence of tamoxifen, the Cre recombinase translocated to the nucleus and inverts the effector orientation, which initiates effector synthesis (Figure 3C).

Figure 3.

The diagram of using the Cre recombinase-loxP targeting strategy in transgenic mouse approach to optokindling. A – the neuron expresses both Cre recombinase and the effector opsin in a Cre-dependent manner; B – Cre recombinase is synthesized, but localized in the neuron’s cytoplasm; transcription is disabled; C – after tamoxifen treatment Cre recombinase moves to nucleus and inverts the effector orientation, that initiates the effector synthesis.

This allows initiating the expression of the target light-sensitive protein in neurons of a certain brain region and at a certain time. This methodological approach avoids possible abnormalities in CNS development in model animals expressing effector opsin since it is read-only after tamoxifen treatment. Some experimental targets require high levels of ChR2 expression due to the relatively small optical current mediated by each opsin molecule. For this reason, the Cre-dependent ChR2 gene has been most often introduced into the Rosa26 locus by incorporating a CAG promoter and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) [82].

Chimeric CreERT2, driven by enhanced synaptic activity-responsive element (E-SARE-CreERT2) was recently used to label the neuronal ensemble activated during focal seizures, and separately - the neural ensemble, activated during interictal periods in mice’s anterior piriform cortex [83].

The optokindling as well as electrokindling processes potentiated synaptic transmission and promoted neural circuit rearrangement which increases the intensity and number of seizures. Using a sophisticated combination of opto- and chemogenetic, electrophysiological, histological, and behavioral methods, it was shown that a strictly determined population of neurons is responsible for the formation of epileptic seizures. The neurons associated with this population are likely to be used to control epileptic seizures [83]. Such a study clearly demonstrates the possibility of identifying neural ensembles responsible for the formation of an ictal event. On the other hand, the neuronal ensemble activated in the period between seizures can also serve as a target for preventing epileptic seizures by various therapeutic methods [83].

3. OPTOGENETIC INHIBITION FOR CONTROL OF EPILEPTIFORM ACTIVITY

Since optogenetics allows both stimulation and inhibition of neuronal activity with a high degree of selectivity, it was logical to apply optogenetic methods to control epileptiform activity directly. Optogenetic inhibition of neural activity has been successfully employed to suppress epileptic seizures in different animal models [84][85][86][87][88][16]. At least many types of focal epileptic activity arise as a result of a disturbance in the balance of excitation and inhibition, which is often associated with a deficient efficiency of the GABAergic system. In epileptic models, experimental reduction of inhibition by blocking GABAergic neurotransmission induces epileptiform activity.

Optogenetic inhibition of neuronal activity has been successfully used to suppress epileptic activity in various models [84][88]. In all these studies, optogenetic stimulation likely shifted the balance of excitation/inhibition in favor of the latter, and inhibition was carried out by GABAergic neurons. Standing apart is the method of optogenetic suppression of epileptic activity using a closed-loop system to stop spontaneous seizures in mice with chronic epilepsy. According to the authors, this technique is applicable not only to epilepsy but can be used to relieve other pathological conditions of the brain with the help of optogenetic interventions [85].

The ability of interneurons of different subtypes to suppress epileptic activity has been the focus of attention of epileptologists for many years [74]. There is evidence that, paradoxically, some subtypes of interneurons play a role in the initiation and maintenance of epileptiform activity. This phenomenon has also been investigated using optogenetic stimulation of epileptiform activity. The optogenetic results confirm that synchronous interneuron activity is sufficient to trigger burst activity [74].

Optogenetic suppression of epileptic seizures was successfully realized by global optogenetic activation of mixed interneuron populations in cortical-hippocampal living slices [89]. In this way, it was demonstrated that optogenetic activation of mixed interneuron populations inhibits epileptiform activity in the hippocampus due to the inhibition of principal neurons by GABA released from these interneurons [89].

Thus, optogenetic approaches have been successfully used to suppress epileptic seizures. To do this, an inhibitory opsin - usually halorhodopsin - can be used in excitatory neurons to suppress their activity during seizures. Halorhodopsin is the main opsin that inhibits neuronal activity. It acts as a chloride pump driven by yellow light with a wavelength of 580 nm. At the same time, for each received yellow photon, one chlorine ion enters through the membrane, which hyperpolarizes the neuron [86]. Its expression has been successfully used to control electrically and chemically induced epileptiform activity in vitro as well as in vivo [90][91][92][93][94].

The idea of suppressing epileptic seizures with the help of optogenetic influence in practice can be implemented in a variety of ways. Thus, cortical epileptiform neurons, in which halorhodopsin was expressed by lentiviral delivery, are a convenient cellular target for optogenetic influence [95]. Epileptic activity under such exposure is suppressed extremely effectively, which has been demonstrated in chronic experiments in vivo [95]. Thus, it was shown that hyperexcitability in the hippocampus can be optogenetically suppressed by hyperpolarization of principal cells [96].

The experimental paradigm applied in most studies using optogenetic inhibition of epileptiform activity was quite simple. There are light-sensitive channels, the activation of which causes hyperpolarization of the neuronal membrane, and therefore with the help of these channels, we can suppress or reduce spike activity. But other possibilities were soon discovered to control epileptic seizures using optogenetics [97][98]. As already mentioned, repeated stimulation of neuronal circuits (kindling) increases convulsive readiness. Optogenetic stimulation can shift the neuronal network to a hyperexcitable state, but continued stimulation (optokindling) can, on the contrary, increase resistance to the development of epileptic seizures [97].

Indeed, it was quite logical to expect that frequent brain stimulation, causing seizures, would intensify epileptogenesis. The surprise was that continuous low frequency repeated stimulation led to a sharp decrease in the convulsive response to the stimulus. Probably, a well-chosen stimulation paradigm causes the release of adenosine, an endogenous inhibitory mediator, by glial cells. This makes the neural circuits more resistant to seizures. We fully share the conclusion of the authors that the appearance of such resistance is associated with brain plasticity and, most likely, can be used in antiepileptic therapy [97]. It is known that adenosine affects both the generation of seizures (ictogenesis) and the development of epilepsy (epileptogenesis), moreover, the synthesis of adenosine and its release by glial cells are regulated by the mechanisms of plasticity [99].

Light stimulation of halorhodopsin suppressed the spike activity of glutamatergic neurons in the hippocampus in vitro [100]. In this mouse model, lentiviral delivery of genetic material to pyramidal neurons CA1 and CA3 was used and the use of light at 573–613 nm caused their hyperpolarization, suppressing epileptiform activity [100]. Even then it was possible to show that input impedance, the amplitude, threshold and other parameters of the action potential were the same in halorhodopsin neurons compared to controls. Thus, the extremely important conclusion was made that neurons with artificially expressed light-sensitive channels have no other functional differences from neurons without such channels [100]. This experimental paradigm has also been applied in vivo, and also successfully [95].

It should be noted that photosensitive channels-inhibitors have serious limitations when using them to suppress epileptic activity. Their chronic use in vivo is limited due to short-term photocurrents and tissue heating during optogenetic stimulation. To a large extent, these shortcomings are devoid of a new optogenetic potassium channel silencer called PACK [101]. The PACK tool consists of two components: Beggiatoa's photoactivated adenylate cyclase (bPAC) and the cAMP-dependent potassium channel SthK. This channel provides a continuous potassium current. Activation of the PACK silencer by short light pulses causes a decrease in neuronal excitation both in vitro and in vivo [101].

A study of the consequences of ictal events, namely, Sudden Unexpected Death in Epilepsy (SUDEP) has also been attempted using optogenetic methods [102]. Seizure-induced respiratory arrest (S-IRA) has been described both in patients and in animal models. Serotonin (5-HT), which is an important modulator of many vital functions, including respiration, is thought to play an important role [102]. Optogenetic stimulation of dorsal raphe neurons reduced the likelihood of respiratory arrest caused by seizures. In the same study, it was shown that the anti-seizure effects of optogenetic stimulation were inhibited by a 5-HT receptor antagonist. The optogenetic suppression of the effect of seizures on respiration was independent of model type, suggesting that a deficiency in 5-HT neurotransmission in the dorsal raphe nucleus (DRN) is associated with the effect of seizures on respiration in DBA/1 mice. Therefore, selective targeting of neurons in the DR could be used to suppress epileptic seizures and prevent SUDEP [102].

Optogenetic stimulation allows selective action on certain populations of neurons, which can increase the effectiveness and safety of low-frequency stimulation for the suppression of epileptic activity [103]. The effectiveness of this approach has been successfully demonstrated in living slices of the entorhinal cortex, in which epileptic activity was induced by 4-AP. Due to the selectivity of optogenetic stimulation, it was possible to distinguish between the activation of both excitatory and inhibitory neurons, or to activate only inhibitory interneurons, or to hyperpolarize excitatory neurons [103]. It turned out that the simultaneous stimulation of excitatory and inhibitory neurons suppresses ictal activity. Probably, in this case, low-frequency optogenetic stimulation caused changes in the K⁺ and Na⁺ concentration gradients which intensifies the Na⁺- K⁺ -pump and led to an antiseizure effect [103].

4. Nonspecific photobiomodulation to suppress the epileptiform activity

Anti-epileptic strategies include not only the search for new antiepileptic drugs but also the exploration of non-pharmacologic approaches. Such a subset in the field of neurophotonics is the use of photobiomodulation (PBM) [104]. From a physical point of view, PBM, including transcranial photobiomodulation therapy (tPBT) is the use of non-ionizing electromagnetic radiation to trigger a series of photochemical reactions in living cells [104]. It is believed that mitochondria play an important role in this process [105]. The most commonly used energy is visible red and near-infrared radiation (NIR). It is likely that this energy is absorbed by the mitochondria and affects ATP synthesis, and cytochrome oxidase C plays a key role in this process [105].

Using in vivo epilepsy model, it was demonstrated that tPBM (808 nm) attenuates seizures and status epilepticus (SE) induced by pentylenetetrazole (PTZ) in rats [106]. Probably, the biological basis for this action of PBM is the reduction of neuronal damage in the cortex and cortical structures. In addition, it turned out that PBM reduces apoptosis of parvalbumin-positive hippocampal interneurons. These neurons are GABAergic interneurons, and thus being functionally eliminated from neural circuits increases the likelihood of developing seizures and SE. When using shorter wavelength light (780 nm) tPBM reduces epileptiform discharges in a rat model of induced stroke-induced epilepsy [107]. Longer wavelength photons likely penetrate biological tissue more easily since they carry less energy and their overall absorption is less [108]. Epileptiform activity increases neuroinflammation, and tPBM reduces the level of neuroinflammatory cytokines [108]. This effect has been shown in the hippocampus after SE [106][109].

When using PBM, the key is to use the correct wavelength, usually in the range of 633-810 nm. The physical basis of PBM is the Grotthuss–Draper law, which in a simplified form is: for light to produce an effect upon matter it must be absorbed [110]. It is technically necessary to take into account the photon intensity (power density (Watt / cm²)). It must be adequate, otherwise, the absorption of photons will be insufficient to achieve the desired effect. However, if the intensity is too high, the photon energy will be converted into excess heat, which can damage cells.

5. Perspectives on optogenetic modulation in clinical practice

The use of loop-back and feed-back paradigms in combination with optogenetics opens up new possibilities for therapeutic intervention not only in various forms of epilepsy but also, for example, in Parkinson's disease [111][112][113]. So, for in vivo experiments, an intracranial implant containing a light-emitting diode has recently been used, which makes it possible to stimulate neurons directly in the brain. Of course, target neurons must be genetically modified to make them sensitive to light.

In carrying out such studies, several scientific groups were guided by the following principles. In the brain, nerve cells generate rhythmic activity, which is the source of the EEG. In pathological conditions, EEG rhythms are often disrupted, causing abnormal patterns of activity. In epilepsy, the abnormal activity is often localized to a small focus but may spread to adjacent areas [85][114].

At the time of the appearance of such activity, it can be identified automatically on the EEG, and the implant provides an optogenetic effect on certain neurons. When the EEG normalizes, the optogenetic influence stops. Such a system allows you to amplify or suppress brain waves in accordance with the protocol used. With the help of this method, it became possible to modulate the intensity of the abnormal EEG pattern, that is, for example, to reduce the intensity of an epileptic seizure [85][114]. Such studies have so far been conducted only on animal models of epilepsy, but we do not see any fundamental problems that would make the use of this technology in clinical medicine impossible [85].

It should be noted that optogenetic stimulation is increasingly being considered as an alternative to the well-known deep brain stimulation (DBS) method, which has been demonstrating its effectiveness in the treatment of Parkinson's disease for many years [111][115]. In some cases, DBS is also used to treat epilepsy. A technique similar to DBS is electrical stimulation of the vagus nerve. This method provides a reduction in the frequency of seizures, but its mechanism of action remains unknown. With good reason, it is believed that in many cases electrical stimulation can be replaced by optogenetic one, and research in this area is ongoing. Moreover, hybrid technologies based on the interaction of nanoscintillators and X-rays have appeared, which in the future can further increase the prospects for the use of optogenetic stimulation in clinical practice [115][116].

In principle, the availability of both inhibitory and excitatory photosensitive peptides from the optogenetic arsenal, along with the ability to target the corresponding excitatory or inhibitory neurons, allows the use of neurophotonic technologies to control abnormal neuron activity in clinical practice [117]. Animal studies show realism in such scenarios and have provided encouraging results. However, until now, sufficiently reliable clinical protocols have not been developed, and, likely, some number of problems will have to be solved for their implementation. The combination of neural feedback with optogenetic methods may open up significant prospects in this area. Nevertheless, optogenetics has not yet found its application in the clinic, although work in this area will continue for more than a year.

CONCLUSION

There are several applications of neurophotonic methods when working with animal models of epilepsy. The first and oldest of these is the use of functional brain optical imaging to localize an epileptic focus and study the onset, spread, and termination of epileptic seizures. Some of these techniques apply not only to animal models but also to humans – during neurosurgical interventions, as well as transcranially. At the next stage, when working with genetic models of epilepsy, optogenetic technologies found widespread use. The "read-in" format made it possible to apply optogenetic technologies for kindling models. Optokindling, in contrast to classical kindling, has shown several advantages, primarily due to its selective effect on target cells. The "read-out" format, specifically the use of fluorescent cellular metabolism indicators, has found wide application for monitoring and functional mapping of epileptic activity. Indicators of intracellular calcium and membrane potential are especially widespread among genetically expressed fluorescent indicators, but the number of indicators of neurotransmitters is also growing. Selective effect on the cells involved in the generation of ictal events makes it possible not only to provoke epileptic seizures, but also to suppress epileptiform activity. This opened new perspectives not only in the study of epileptic activity but also in the development of new approaches for epilepsy treatment.

Highlights:

• Ongoing advancements are continuously enhancing animal models of epilepsy – one of such enhancements is the extensive use of neurophotonics technologies

• Neurophotonics methods employed in epileptic models include optical imaging technologies and methods of stimulating or silencing neurons

• The development of neurophotonic technologies facilitates their application not only in experimental epileptology, but also in a clinical setting