Overexpression of KCNN4 channels in principal neurons produces an anti-seizure effect without reducing their coding ability.

Gene therapy offers a potential alternative to the surgical treatment of epilepsy, which affects millions of people and is pharmacoresistant in ~30% of cases. Aimed at reducing the excitability of principal neurons, the engineered expression of K+ channels has been proposed as a treatment due to the outstanding ability of K+ channels to hyperpolarize neurons. However, the effects of K+ channel overexpression on cell physiology remain to be investigated. Here we report an adeno-associated virus (AAV) vector designed to reduce epileptiform activity specifically in excitatory pyramidal neurons by expressing the human Ca2+-gated K+ channel KCNN4 (KCa3.1). Electrophysiological and pharmacological experiments in acute brain slices showed that KCNN4-transduced cells exhibited a Ca2+-dependent slow afterhyperpolarization that significantly decreased the ability of KCNN4-positive neurons to generate high-frequency spike trains without affecting their lower-frequency coding ability and action potential shapes. Antiepileptic activity tests showed potent suppression of pharmacologically induced seizures in vitro at both single cell and local field potential levels with decreased spiking during ictal discharges. Taken together, our findings strongly suggest that the AAV-based expression of the KCNN4 channel in excitatory neurons is a promising therapeutic intervention as gene therapy for epilepsy.

Morphological and Functional Alterations in the CA1 Pyramidal Neurons of the Rat Hippocampus in the Chronic Phase of the Lithium-Pilocarpine Model of Epilepsy.

Epilepsy is known to cause alterations in neural networks. However, many details of these changes remain poorly understood. The objective of this study was to investigate changes in the properties of hippocampal CA1 pyramidal neurons and their synaptic inputs in a rat lithium-pilocarpine model of epilepsy. In the chronic phase of the model, we found a marked loss of pyramidal neurons in the CA1 area. However, the membrane properties of the neurons remained essentially unaltered. The results of the electrophysiological and morphological studies indicate that the direct pathway from the entorhinal cortex to CA1 neurons is reinforced in epileptic animals, whereas the inputs to them from CA3 are either unaltered or even diminished. In particular, the dendritic spine density in the str. lacunosum moleculare, where the direct pathway from the entorhinal cortex terminates, was found to be 2.5 times higher in epileptic rats than in control rats. Furthermore, the summation of responses upon stimulation of the temporoammonic pathway was enhanced by approximately twofold in epileptic rats. This enhancement is believed to be a significant contributing factor to the heightened epileptic activity observed in the entorhinal cortex of epileptic rats using an ex vivo 4-aminopyridine model.

Ictal wavefront propagation in slices and simulations with conductance-based refractory density model.

The mechanisms determining ictal discharge (ID) propagation are still not clear. In the present study, we aimed to examine these mechanisms in animal and mathematical models of epileptiform activity. Using double-patch and extracellular potassium ion concentration recordings in rat hippocampal-cortical slices, we observed that IDs moved at a speed of about 1 mm/s or less. The mechanisms of such slow propagation have been studied with a mathematical, conductance-based refractory density (CBRD) model that describes the GABA- and glutamatergic neuronal populations' interactions and ion dynamics in brain tissue. The modeling study reveals two main factors triggerring IDs: (i) increased interneuronal activity leading to chloride ion accumulation and a consequent depolarizing GABAergic effect and (ii) the elevation of extracellular potassium ion concentration. The local synaptic transmission followed by local potassium ion extrusion and GABA receptor-mediated chloride ion accumulation underlies the ID wavefront's propagation. In contrast, potassium ion diffusion in the extracellular space is slower and does not affect ID's speed. The short discharges, constituting the ID, propagate much faster than the ID front. The accumulation of sodium ions inside neurons due to their hyperactivity and glutamatergic currents boosts the Na+/K+ pump, which terminates the ID. Knowledge of the mechanism of ID generation and propagation contributes to the development of new treatments against epilepsy.

Refinement of the Barnes and Morris water maze protocols improves characterization of spatial cognitive deficits in the lithium-pilocarpine rat model of epilepsy.

Temporal lobe epilepsy (TLE) often causes cognitive impairment, especially a decline in spatial memory. Reductions in spatial memory and learning are also common in rodent models of TLE. The Morris water maze and the Barnes maze are the standard methods for evaluating spatial learning and memory in rodents. However, animals with TLE may exhibit agitation, distress, and fail to follow the paradigmatic context of these tests, making the interpretation of experimental data difficult. This study optimized the procedure of the Morris water maze and the Barnes maze to evaluate spatial learning and memory in rats with the lithium-pilocarpine TLE model (LPM rats). It was demonstrated that LPM rats required a mandatory and prolonged habituation stage for both tests. Therefore, the experimental rats performed relatively well on these tests. Nevertheless, LPM rats exhibited a slower learning process compared to the control rats. LPM rats also showed a reduction in spatial memory formation. This was more pronounced in the Barnes maze. Also, LPM rats utilized a sequential strategy for searching in the Barnes maze and were incapable of developing a more efficient spatial search strategy that is common in control animals. The Barnes maze may be a better choice for assessing search strategies, learning deficits, and spatial memory in rats with TLE when choosing between the two tests. This is because of the risk of unexpected seizure occurrence during the Morris water maze tests, and the potential risks for animal welfare.

Alterations in the Properties of the Rat Hippocampus Glutamatergic System in the Lithium-Pilocarpine Model of Temporal Lobe Epilepsy.

Status epilepticus (SE) triggers many not yet fully understood pathological changes in the nervous system that can lead to the development of epilepsy. In this work, we studied the effects of SE on the properties of excitatory glutamatergic transmission in the hippocampus in the lithium-pilocarpine model of temporal lobe epilepsy in rats. The studies were performed 1 day (acute phase), 3 and 7 days (latent phase), and 30 to 80 days (chronic phase) after SE. According to RT-qPCR data, expression of the genes coding for the AMPA receptor subunits GluA1 and GluA2 was downregulated in the latent phase, which may lead to the increased proportion of calcium-permeable AMPA receptors that play an essential role in the pathogenesis of many CNS diseases. The efficiency of excitatory synaptic neurotransmission in acute brain slices was decreased in all phases of the model, as determined by recording field responses in the CA1 region of the hippocampus in response to the stimulation of Schaffer collaterals by electric current of different strengths. However, the frequency of spontaneous excitatory postsynaptic potentials increased in the chronic phase, indicating an increased background activity of the glutamatergic system in epilepsy. This was also evidenced by a decrease in the threshold current causing hindlimb extension in the maximal electroshock seizure threshold test in rats with temporal lobe epilepsy compared to the control animals. The results suggest a series of functional changes in the properties of glutamatergic system associated with the epilepsy development and can be used to develop the antiepileptogenic therapy.

Molecular and Cellular Mechanisms of Epilepsy 2.0.

Epilepsy is a prevalent neurological disorder [...].

Molecular and Cellular Mechanisms of Epilepsy.

Despite the availability of a large number of antiepileptic drugs, about 30% of patients with epilepsy, especially temporal lobe epilepsy (TLE), continue to experience seizures [...].

Alterations in Rat Hippocampal Glutamatergic System Properties after Prolonged Febrile Seizures.

Febrile seizures during early childhood may result in central nervous system developmental disorders. However, the specific mechanisms behind the impact of febrile seizures on the developing brain are not well understood. To address this gap in knowledge, we employed a hyperthermic model of febrile seizures in 10-day-old rats and tracked their development over two months. Our objective was to determine the degree to which the properties of the hippocampal glutamatergic system are modified. We analyzed whether pyramidal glutamatergic neurons in the hippocampus die after febrile seizures. Our findings indicate that there is a reduction in the number of neurons in various regions of the hippocampus in the first two days after seizures. The CA1 field showed the greatest susceptibility, and the reduction in the number of neurons in post-FS rats in this area appeared to be long-lasting. Electrophysiological studies indicate that febrile seizures cause a reduction in glutamatergic transmission, leading to decreased local field potential amplitude. This impairment could be attributable to diminished glutamate release probability as evidenced by decreases in the frequency of miniature excitatory postsynaptic currents and increases in the paired-pulse ratio of synaptic responses. We also found higher threshold current causing hind limb extension in the maximal electroshock seizure threshold test of rats 2 months after febrile seizures compared to the control animals. Our research suggests that febrile seizures can impair glutamatergic transmission, which may protect against future seizures.

Febrile Seizures Cause a Rapid Depletion of Calcium-Permeable AMPA Receptors at the Synapses of Principal Neurons in the Entorhinal Cortex and Hippocampus of the Rat.

Febrile seizures (FSs) are a relatively common early-life condition that can cause CNS developmental disorders, but the specific mechanisms of action of FS are poorly understood. In this work, we used hyperthermia-induced FS in 10-day-old rats. We demonstrated that the efficiency of glutamatergic synaptic transmission decreased rapidly after FS by recording local field potentials. This effect was transient, and after two days there were no differences between control and post-FS groups. During early ontogeny, the proportion of calcium-permeable (CP)-AMPA receptors in the synapses of the principal cortical and hippocampal neurons is high. Therefore, rapid internalization of CP-AMPA receptors may be one of the mechanisms underlying this phenomenon. Using the whole-cell patch-clamp method and the selective CP-AMPA receptor blocker IEM-1460, we tested whether the proportion of CP-AMPA receptors changed. We have demonstrated that FS rapidly reduces synaptic CP-AMPA receptors in both the hippocampus and the entorhinal cortex. This process was accompanied by a sharp decrease in the calcium permeability of the membrane of principal neurons, which we revealed in experiments with kainate-induced cobalt uptake. Our experiments show that FSs cause rapid changes in the function of the glutamatergic system, which may have compensatory effects that prevent excessive excitotoxicity and neuronal death.

Antiepileptogenic Effects of Anakinra, Lamotrigine and Their Combination in a Lithium-Pilocarpine Model of Temporal Lobe Epilepsy in Rats.

Temporal lobe epilepsy is a common, chronic disorder with spontaneous seizures that is often refractory to drug therapy. A potential cause of temporal lobe epilepsy is primary brain injury, making prevention of epileptogenesis after the initial event an optimal method of treatment. Despite this, no preventive therapy for epilepsy is currently available. The purpose of this study was to evaluate the effects of anakinra, lamotrigine, and their combination on epileptogenesis using the rat lithium-pilocarpine model of temporal lobe epilepsy. The study showed that there was no significant difference in the number and duration of seizures between treated and untreated animals. However, the severity of seizures was significantly reduced after treatment. Anakinra and lamotrigine, alone or in combination, significantly reduced neuronal loss in the CA1 hippocampus compared to the control group. However, the drugs administered alone were found to be more effective in preventing neuron loss in the hippocampal CA3 field compared to combination treatment. The treatment alleviated the impairments in activity level, exploratory behavior, and anxiety but had a relatively weak effect on TLE-induced impairments in social behavior and memory. The efficacy of the combination treatment did not differ from that of anakinra and lamotrigine monotherapy. These findings suggest that anakinra and lamotrigine, either alone or in combination, may be clinically useful in preventing the development of histopathological and behavioral abnormalities associated with epilepsy.

Beneficial Effects of Probiotic Bifidobacterium longum in a Lithium-Pilocarpine Model of Temporal Lobe Epilepsy in Rats.

Epilepsy is a challenging brain disorder that is often difficult to treat with conventional therapies. The gut microbiota has been shown to play an important role in the development of neuropsychiatric disorders, including epilepsy. In this study, the effects of Bifidobacterium longum, a probiotic, on inflammation, neuronal degeneration, and behavior are evaluated in a lithium-pilocarpine model of temporal lobe epilepsy (TLE) induced in young adult rats. B. longum was administered orally at a dose of 109 CFU/rat for 30 days after pilocarpine injection. The results show that B. longum treatment has beneficial effects on the TLE-induced changes in anxiety levels, neuronal death in the amygdala, and body weight recovery. In addition, B. longum increased the expression of anti-inflammatory and neuroprotective genes, such as Il1rn and Pparg. However, the probiotic had little effect on TLE-induced astrogliosis and microgliosis and did not reduce neuronal death in the hippocampus and temporal cortex. The study suggests that B. longum may have a beneficial effect on TLE and may provide valuable insights into the role of gut bacteria in epileptogenesis. In addition, the results show that B. longum may be a promising drug for the comprehensive treatment of epilepsy.

Modulation of seizure-like events by the small conductance and ATP-sensitive potassium ion channels.

Potassium ion channels are extensively involved in the regulation of epileptic seizures. The small conductance calcium-sensitive potassium channels (SK channels) and ATP-sensitive potassium (KATP) channels are activated by calcium ion entry and decrease ATP levels, respectively. These channels can underlie the post-burst afterhyperpolarization and be upregulated during seizures, providing negative feedback during epileptic activity. Using the whole-cell patch-clamp method in rat brain slices, we investigated the effect of SK- and KATP-affecting drugs on seizure-like events (SLEs) in the 4-aminopyridine model of epileptic seizures in vitro. We demonstrate that SK and KATP channels contribute to sustaining the high-frequency firing of the principal neurons in the deep layers of the entorhinal cortex during injections of depolarizing current and epileptiform discharges. Neither the pharmacological blockade nor the activation of these channels was able to prevent the epileptiform activity in brain slices. However, the blockade of KATP channels increases the SLE duration, suggesting that these channels may contribute to the termination of SLEs. Thus, KATP channels can be considered a promising target for pharmacological interventions for the treatment of epilepsy.

Optogenetic Low-Frequency Stimulation of Principal Neurons, but Not Parvalbumin-Positive Interneurons, Prevents Generation of Ictal Discharges in Rodent Entorhinal Cortex in an In Vitro 4-Aminopyridine Model.

Low-frequency electrical stimulation is used to treat some drug-resistant forms of epilepsy. Despite the effectiveness of the method in suppressing seizures, there is a considerable risk of side effects. An optogenetic approach allows the targeting of specific populations of neurons, which can increase the effectiveness and safety of low-frequency stimulation. In our study, we tested the efficacy of the suppression of ictal activity in entorhinal cortex slices in a 4-aminopyridine model with three variants of low-frequency light stimulation (LFLS): (1) activation of excitatory and inhibitory neurons (on Thy1-ChR2-YFP mice), (2) activation of inhibitory interneurons only (on PV-Cre mice after virus injection with channelrhodopsin2 gene), and (3) hyperpolarization of excitatory neurons (on Wistar rats after virus injection with archaerhodopsin gene). Only in the first variant did simultaneous LFLS of excitatory and inhibitory neurons replace ictal activity with interictal activity. We suggest that LFLS caused changes in the concentration gradients of K+ and Na+ cations across the neuron membrane, which activated Na-K pumping. According to the mathematical modeling, the increase in Na-K pump activity in neurons induced by LFLS led to an antiepileptic effect. Thus, a less specific and generalized optogenetic effect on entorhinal cortex neurons was more effective in suppressing ictal activity in the 4-aminopyridine model.

Prolonged Febrile Seizures Impair Synaptic Plasticity and Alter Developmental Pattern of Glial Fibrillary Acidic Protein (GFAP)-Immunoreactive Astrocytes in the Hippocampus of Young Rats.

Prolonged neonatal febrile seizures (FSs) often lead to cognitive decline and increased risk of psychopathology in adulthood. However, the neurobiological mechanisms underlying the long-term adverse effects of FSs remain unclear. In this study, we exposed rat pups to hyperthermia and induced FSs lasting at least 15 min. We investigated the short-term (one day) and delayed (11-13 and 41-45 days) effects of FSs on some parameters of morphological and functional maturation in the hippocampus. We noticed that FSs altered the developmental pattern of glial fibrillary acidic protein (GFAP) immunoreactivity. In rats aged 21-23 days, GFAP-positive astrocytes covered a smaller area, and their morphological characteristics resembled those of rats at 11 days of age. In post-FS rats, the magnitude of long-term synaptic potentiation was reduced compared to control animals of the same age. Applying the gliotransmitter D-serine, an agonist of the glycine site of NMDA receptors, restored LTP to control values. A decrease in LTP amplitude was correlated with impaired spatial learning and memory in the Barnes maze task in post-FS rats. Our data suggest that impaired neuron-glia interactions may be an essential mechanism of the adverse effects of FS on the developing brain.

Changes in Metabotropic Glutamate Receptor Gene Expression in Rat Brain in a Lithium-Pilocarpine Model of Temporal Lobe Epilepsy.

Preventing epileptogenesis in people at risk is an unmet medical need. Metabotropic glutamate receptors (mGluRs) are promising targets for such therapy. However, drugs acting on mGluRs are not used in the clinic due to limited knowledge of the involvement of mGluRs in epileptogenesis. This study aimed to analyze the changes in gene expression of mGluR subtypes (1-5, 7, 8) in various rat brain regions in the latent and chronic phases of a lithium-pilocarpine model of epilepsy. For this study, multiplex test systems were selected and optimized to analyze mGluR gene expression using RT-qPCR. Region- and phase-specific changes in expression were revealed. During the latent phase, mGluR5 mRNA levels were increased in the dorsal and ventral hippocampus, and expression of group III genes was decreased in the hippocampus and temporal cortex, which could contribute to epileptogenesis. Most of the changes in expression detected in the latent stage were absent in the chronic stage, but mGluR8 mRNA production remained reduced in the hippocampus. Moreover, we found that gene expression of group II mGluRs was altered only in the chronic phase. The study deepened our understanding of the mechanisms of epileptogenesis and suggested that agonists of group III mGluRs are the most promising targets for preventing epilepsy.

MTEP, a Selective mGluR5 Antagonist, Had a Neuroprotective Effect but Did Not Prevent the Development of Spontaneous Recurrent Seizures and Behavioral Comorbidities in the Rat Lithium-Pilocarpine Model of Epilepsy.

Metabotropic glutamate receptors (mGluRs) are expressed predominantly on neurons and glial cells and are involved in the modulation of a wide range of signal transduction cascades. Therefore, different subtypes of mGluRs are considered a promising target for the treatment of various brain diseases. Previous studies have demonstrated the seizure-induced upregulation of mGluR5; however, its functional significance is still unclear. In the present study, we aimed to clarify the effect of treatment with the selective mGluR5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP) on epileptogenesis and behavioral impairments in rats using the lithium-pilocarpine model. We found that the administration of MTEP during the latent phase of the model did not improve survival, prevent the development of epilepsy, or attenuate its manifestations in rats. However, MTEP treatment completely prevented neuronal loss and partially attenuated astrogliosis in the hippocampus. An increase in excitatory amino acid transporter 2 expression, which has been detected in treated rats, may prevent excitotoxicity and be a potential mechanism of neuroprotection. We also found that MTEP administration did not prevent the behavioral comorbidities such as depressive-like behavior, motor hyperactivity, reduction of exploratory behavior, and cognitive impairments typical in the lithium-pilocarpine model. Thus, despite the distinct neuroprotective effect, the MTEP treatment was ineffective in preventing epilepsy.

Delayed Impairment of Hippocampal Synaptic Plasticity after Pentylenetetrazole-Induced Seizures in Young Rats.

Data on the long-term consequences of a single episode of generalized seizures in infants are inconsistent. In this study, we examined the effects of pentylenetetrazole-induced generalized seizures in three-week-old rats. One month after the seizures, we detected a moderate neuronal loss in several hippocampal regions: CA1, CA3, and hilus, but not in the dentate gyrus. In addition, long-term synaptic potentiation (LTP) was impaired. We also found that the mechanism of plasticity induction was altered: additional activation of metabotropic glutamate receptors (mGluR1) is required for LTP induction in experimental rats. This disturbance of the plasticity induction mechanism is likely due to the greater involvement of perisynaptic NMDA receptors compared to receptors located in the core part of the postsynaptic density. This hypothesis is supported by experiments with selective blockades of core-located NMDA receptors by the use-dependent blocker MK-801. MK-801 had no effect on LTP induction in experimental rats and suppressed LTP in control animals. The weakening of the function of core-located NMDA receptors may be due to the disturbed clearance of glutamate from the synaptic cleft since the distribution of the astrocytic glutamate transporter EAAT2 in experimental animals was found to be altered.

Photostimulation activates fast-spiking interneurons and pyramidal cells in the entorhinal cortex of Thy1-ChR2-YFP line 18 mice.

The application of optogenetics in animals has provided new insights into both fundamental neuroscience and diseases of the nervous system. This is primarily due to the fact that optogenetics allows selectively activating or inhibiting particular types of neurons. One of the first transgenic mouse lines developed for the optogenetic experiment was Thy1-ChR2-YFP. Thy1 is an immunoglobulin superfamily member expressing in projection neurons, so it was assumed that channelrhodopsin-2 (ChR2) would be primarily expressed in projection neurons. However, the specificity of ChR2 expression under promoter Thy1 in different lines has to be clarified yet. Therefore, we aimed to determine the cell specificity of ChR2 expression in the entorhinal cortex of Thy1-ChR2-YFP line 18 mice. We have found that both pyramidal cells and fast-spiking interneurons in deep layers of the entorhinal cortex depolarized and fired in response to 470-nm photostimulation. To exclude the effect of synaptic activation of interneurons by pyramidal cells, we used a selective antagonist of AMPA receptors. Under these conditions, inhibitory postsynaptic currents decreased but did not disappear completely. Furthermore, gabazine inhibited these postsynaptic currents entirely, thus confirming the direct activation of interneurons by light. These data demonstrate that ChR2 is expressed in both pyramidal neurons and fast-spiking interneurons of the entorhinal cortex in Thy1-ChR2-YFP mice.

The application of the self-probing primer PCR for quantitative expression analysis of R607Q (un)edited GluA2 AMPA receptor mRNA.

Adenosine deaminase-dependent RNA editing is a widespread universal mechanism of posttranscriptional gene function modulation. Changes in RNA editing level may contribute to various physiological and pathological processes. In the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor GluA2 subunit, A-I editing in the Q607R site leads to dramatic changes in function, making the receptor channel calcium-impermeable. A standard approach for quantifying (un)edited RNAs is based on endpoint PCR (Sanger sequencing or restriction analysis), a time-consuming and semiquantitative method. We aimed to develop RT-qPCR assays to quantify rat Q607R (A-I) edited/unedited mRNA in samples in the present work. Based on self-probing PCR detection chemistry, described initially for detecting short DNA fragments, we designed and optimised RT-qPCR assays to quantify Q607R (un)edited mRNA. We used self-probing primer PCR technology for mRNA quantification for the first time. Using a novel assay, we confirmed that Q607R GluA2 mRNA editing was increased in 14-day- (P14) or 21-day-old (P21) postnatal brain tissue (hippocampus) compared to the embryonic brain (whole brains at E20) in Wistar rats. Q607R unedited GluA2 mRNA was detectable by our assay in the cDNA of mature brain tissue compared to that derived through classical methods. Thus, self-probing primer PCR detection chemistry is an easy-to-use approach for RT-qPCR analysis of RNA editing.

Insertion of Calcium-Permeable AMPA Receptors during Epileptiform Activity In Vitro Modulates Excitability of Principal Neurons in the Rat Entorhinal Cortex.

Epileptic activity leads to rapid insertion of calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) into the synapses of cortical and hippocampal glutamatergic neurons, which generally do not express them. The physiological significance of this process is not yet fully understood; however, it is usually assumed to be a pathological process that augments epileptic activity. Using whole-cell patch-clamp recordings in rat entorhinal cortex slices, we demonstrate that the timing of epileptiform discharges, induced by 4-aminopyridine and gabazine, is determined by the shunting effect of Ca2+-dependent slow conductance, mediated predominantly by K+-channels. The blockade of CP-AMPARs by IEM-1460 eliminates this extra conductance and consequently increases the rate of discharge generation. The blockade of NMDARs reduced the additional conductance to a lesser extent than the blockade of CP-AMPARs, indicating that CP-AMPARs are a more significant source of intracellular Ca2+. The study's main findings were implemented in a mathematical model, which reproduces the shunting effect of activity-dependent conductance on the generation of discharges. The obtained results suggest that the expression of CP-AMPARs in principal neurons reduces the discharge generation rate and may be considered as a protective mechanism.