Vagal Nerve Stimulation: The Effect on the Brain Oscillatory Field Potential.

More than thirty years of medical treatment with the use of vagal nerve stimulation (VNS) has shown that this therapeutic procedure works in a number of homeostatic disturbances. Although the clinical usage of VNS has a long history, our knowledge about the central mechanisms underlying this treatment is still limited. In the present paper we review the effects of VNS on brain oscillations as a possible electrophysiological bio-marker of VNS efficacy. The review was prepared mainly on the basis of data delivered from clinical observations and the outcomes of electrophysiological experiments conducted on laboratory animals that are available in PubMed. We consciously did not focus on epileptiform activity understood as a pathologic oscillatory activity, which was widely discussed in the numerous previously published reviews. The main conclusion of the present paper is that further, well-designed experiments on laboratory animals are absolutely necessary to address the electrophysiological issues. These will fill a number of gaps in our present knowledge of the central mechanisms underlying VNS therapy.

Hippocampal theta rhythm induced by vagal nerve stimulation: The effect of modulation of electrical coupling.

Previously we have demonstrated that vagal nerve stimulation (VNS) is capable of inducing hippocampal formation (HPC) theta rhythm (Broncel et al., 2017). The neuronal substrate underlying this novel phenomenon is poorly known, though the cholinergic and GABAergic profile of VNS-induced theta rhythm in anesthetized rats has just recently been addressed (Broncel et al., 2018a, 2019). In this study we extended our earlier observation concerning the pharmacological profile of VNS-induced theta oscillations. Specifically, the purpose of the present study was to test the hypothesis that VNS-induced hippocampal theta rhythm could be modulated by local HPC gap junctions (GJs) transmission. Two GJs agents were used: carbenoxolone, nonspecific GJs blocker and trimethylamine, a nonspecific opener of GJs. Two basic parameters of theta rhythm were evaluated: frequency and power. It was demonstrated that carbenoxolone inhibits VNS-induced theta while trimethylamine facilities it. These observations indicate that HPC electrical coupling mediates the theta rhythm induced by vagal nerve stimulation.

GABAergic mediation of hippocampal theta rhythm induced by stimulation of the vagal nerve.

The key question to be answered in the present study was whether the medial septal GABAergic receptors are engaged in the pharmacological profile of the hippocampal formation (HPC) theta rhythm induced by vagal nerve stimulation (VNS). It was demonstrated that the medial septal microinfusion of GABAA and GABAB agonists (muscimol and baclofen) resulted in a progressive reduction of the power of VNS-induced theta. The medial septal microinfusion of GABAA and GABAB antagonists (bicuculline and 2-OH saclofen) resulted in the generation of VNS-induced theta with increased power. The effect of the combined medial septal microinfusion of GABAA agonist and antagonist and GABAB agonist and antagonist on VNS-induced theta rhythm was also evaluated: in the presence of GABAA,B antagonists the effect of agonists predominated. In separate experiments, GABAA and GABAB antagonists were administrated in anesthetized rats pretreated with i.v. administration of atropine sulfate. Atropine was found to abolish spontaneous theta and VNS-induced theta examined in the presence of bicuculline or 2-OH saclofen. The present data provide evidence that the medial septal GABAA and GABAB receptors are involved in the central mechanisms responsible for modulation of VNS-induced HPC theta oscillations. Furthermore, the results of the present study also demonstrate that, in fact, both GABAergic and cholinergic involvement is necessary for the appearance of VNS-induced theta.

GSK3ß activity alleviates epileptogenesis and limits GluA1 phosphorylation.

BACKGROUND: Glycogen synthase kinase-3ß (GSK3ß) is a key regulator of cellular homeostasis. In neurons, GSK3ß contributes to the control of neuronal transmission and plasticity, but its role in epilepsy remains to be defined. METHODS: Biochemical and electrophysiological methods were used to assess the role of GSK3ß in regulating neuronal transmission and epileptogenesis. GSK3ß activity was increased genetically in GSK3ß[S9A] mice. Its effects on neuronal transmission and epileptogenesis induced by kainic acid were assessed by field potential recordings in mice brain slices and video electroencephalography in vivo. The ion channel expression was measured in brain samples from mice and followed by analysis in samples from patients with temporal lobe epilepsy or focal cortical dysplasia in correlation to GSK3ß phosphorylation. FINDINGS: Higher GSK3ß activity decreased the progression of kainic acid induced epileptogenesis. At the biochemical level, higher GSK3ß activity increased the expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel 4 under basal conditions and in the epileptic mouse brain and decreased phosphorylation of the glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 at Serine 831 under basal conditions. Moreover, we found a significant correlation between higher inhibitory GSK3ß phosphorylation at Serine 9 and higher activating GluA1 phosphorylation at Serine 845 in brain samples from epileptic patients. INTERPRETATION: Our data imply GSK3ß activity in the protection of neuronal networks from hyper-activation in response to epileptogenic stimuli and indicate that the anti-epileptogenic function of GSK3ß involves modulation of HCN4 level and the synaptic AMPA receptors pool.

Medial septal cholinergic mediation of hippocampal theta rhythm induced by vagal nerve stimulation.

BACKGROUND: Electrical vagal nerve stimulation (VNS) has been used for years to treat patients with drug-resistant epilepsy. This technique also remains under investigation as a specific treatment of patients with Alzheimer's disease. Recently we discovered that VNS induced hippocampal formation (HPC) type II theta rhythm, which is involved in memory consolidation. In the present study, we have extended our previous observation and addressed the neuronal substrate and pharmacological profile of HPC type II theta rhythm induced by VNS in anesthetized rats. METHODS: Male Wistar rats were implanted with a VNS cuff electrode around the left vagus nerve, a tungsten microelectrode for recording the HPC field activity, and a medial septal (MS) cannula for the injection of a local anesthetic, procaine, and muscarinic agents. A direct, brief effect of VNS on the HPC field potential was evaluated before and after medial-septal drug injection. RESULTS: Medial septal injection of local anesthetic, procaine, reversibly abolished VNS-induced HPC theta rhythm. With the use of cholinergic muscarinic agonist and antagonists, we demonstrated that medial septal M1 receptors are involved in the mediation of the VNS effect on HPC theta field potential. CONCLUSION: The MS cholinergic M1 receptor mechanism integrates not only central inputs from the brainstem synchronizing pathway, which underlies the production of HPC type II theta rhythm, but also the input from the vagal afferents in the brain stem.

Postnatal Development of the Posterior Hypothalamic Theta Rhythm and Local Cell Discharges in Rat Brain Slices.

Theta rhythms have been recorded from rat brain slices of the posterior hypothalamic area (PHa), including the supramammillary and posterior hypothalamic nuclei. Additionally, in numerous studies theta-related neurons were identified in the PHa according to the classification of Bland and Colom (Progress in Neurobiology, 41, 157-208, 1993). It is currently widely accepted that the PHa contributes to the process of HPC theta frequency programming at least in certain behavioral states. The postnatal development of the HPC and its ability to generate theta has also been a subject of studies. Specifically, it was found that theta oscillations are present in the HPC of 8-10 days old rat pups and turn into a well-synchronized and high-amplitude activity in the following few days. In our current study, we therefore focused on the postnatal development of cholinergically-induced theta rhythm and theta-related neuronal activity in PHa slices obtained from 8 to 24 days old rat pups. Theta activity was observed in the PHa preparations at the age of 8-10 days and then progressively increased its probability of occurrence, amplitude and synchrony up to the age of 22-24 days when it reached a plateau phase. A steady increase in the number of recorded neurons correlated with local theta oscillations was also observed.

Some technical issues of vagal nerve stimulation. An approach using a hippocampal formation theta rhythm.

Previously, we have demonstrated that hippocampal (HPC) theta rhythm can be produced, depending on current intensity, directly during vagal nerve stimulation (VNS) or with a time delay following stimulation. This suggests that theta EEG pattern can also be used as a bio-indicator of the efficiency of VNS. In the present study, we focused on three specific, technical issues related to the stimulation procedure of the vagal nerve: i/does the type of the electrode used for VNS and the technique of its implantation affect the parameters of the HPC theta rhythm? ii/does the type of electrode used determine the current intensity threshold of VNS-induced HPC theta? iii/is the repeatability of the VNS effect determined by the type of electrode used? We demonstrated that a platinum-iridium cuff electrode offers some important advantages over a tungsten electrode. Firstly, despite some possible mechanical and compression nerve damage related to permanent contact with the vagal nerve, it offers a lower current intensity threshold for inducing theta oscillations. Secondly, and most importantly, the cuff electrode offers repeatability of the VNS effect on the HPC theta rhythm. However, one disadvantage of using this type of an electrode is that the permanent pressure on the vagal nerve by the cuff itself may decrease the amplitude of the investigated field potential.

Vagus nerve stimulation produces a hippocampal formation theta rhythm in anesthetized rats.

Vagus nerve stimulation (VNS) has been used for years to treat patients with drug-resistant epilepsy. In the present study, the effect of different stimulation protocols of VNS on the hippocampal formation (HPC) type II theta field potentials were evaluated in anesthetized rats. The following theta parameters were analysed: power, frequency and duration of theta epochs. We documented for the first time the presence of HPC type II theta in response to the application of VNS. A VNS-induced theta rhythm appeared in different experimental protocols and, depending on the current intensity, could occur directly during VNS (brief effect) or after vagal stimulation (delayed effect), using lower intensity stimuli.

Is electrical coupling involved in the generation of posterior hypothalamic theta rhythm?

Data obtained in in vitro experiments and urethane anaesthetized animals have revealed that the mechanisms responsible for the generation of hippocampal cholinergic theta rhythm are specifically affected by the administration of broad spectrum gap junctions (GJs) blocker - carbenoxolone (CBX). The aim of this study was to examine the effect of GJs modulation on the production of posterior hypothalamic theta. Specifically, we were interested in evaluating whether CBX could attenuate the theta rhythm recorded from the supramammillary nucleus and posterior hypothalamic nuclei, in both in vitro and in vivo preparations. The data we obtained from in vitro and in vivo preparations demonstrated that the administration of CBX did not suppress cholinergically induced theta in posterior hypothalamic area (PHa) slices nor the theta rhythm observed in the PHa of urethane anaesthetized rats. Moreover, the application of trimethylamine, while very effective in the enhancement of hippocampal theta rhythm, did not produce any changes in theta oscillations observed in either in vitro or in vivo posterior hypothalamic area preparations. These data show that electrical coupling via GJs is not involved in theta rhythm generation in the PHa. Surprisingly, we observed a significant enhancement of theta activity in response to the carbenoxolone administration in both in vitro and in vivo PHa preparations. The theta rhythm enhancement detected in those experiments was attenuated by the application of spironolactone (mineralocorticoid receptors antagonist). We suggest that the observed excitatory effects of CBX on posterior hypothalamic oscillatory activity in the theta band could be mediated by mineralocorticoid receptors.

Cell discharge correlates of posterior hypothalamic theta rhythm. Recipe for success in recording stable field potential.

The theta rhythm discovered in the posterior hypothalamus area (PHa) differs from theta observed in the hippocampal formation. In comparison to hippocampal spontaneous theta, the theta recorded in the PHa is rarely registered, has lower amplitude, often disappears, and sometimes returns after a few minutes. These features indicate that spontaneous theta recorded in the PHa is not an appropriate experimental model to search for the correlation between PHa cell discharges and local field potential. In this paper we present standard experimental conditions necessary to record theta-related cells in the PHa in anesthetized rats. Three pharmacological agents were used in the experiments to induce PHa theta rhythm in urethanized rats: carbachol (CCH), carbenoxolone and kainic acid, which are potent enough to induce well-synchronized PHa theta. However, CCH was found to be the best pharmacological tool to induce PHa theta oscillations, due to its longest duration of action and lack of preliminary epileptogenic effects. It seems that CCH-induced theta can be the most suitable pharmacological model for experiments with the use of protocol of long-lasting recordings of PHa theta-related cell discharges.

Cell discharge correlates of posterior hypothalamic theta rhythm recorded in anesthetized rats and brain slices.

Kowalczyk et al. (Hippocampus 2014; 24:7-20) were probably the first to conduct a systemic study of posterior hypothalamic area (PHa) theta rhythm in anesthetized rats. They demonstrated that local PHa theta field potentials were tail-pinch resistant and could be generated in urethane-anesthetized rats independently of ongoing hippocampal formation theta rhythm. These in vivo data were also confirmed in PHa slice preparations perfused with cholinergic agonist, carbachol. In the current experiments we extend our earlier observations concerning PHa theta rhythm. Specifically, PHa field potentials were analyzed in relation to the ongoing local cell firing repertoire. Single-unit discharge patterns of cells localized in the posterior hypothalamic and supramammillary nuclei were characterized according to the criteria that was developed previously to classify theta-related cells in the hippocampal formation. The present study demonstrated that in addition to the earlier described theta-related cells (theta-on, theta-off and gating cells) the PHa also contains cells discharging in a very regular manner, which were labelled "timing cells". This type of neuron has not been previously documented. We suggest that "timing cells" form a part of the ascending brainstem synchronizing pathway, provideing a regular rhythmic signal which facilitates the transduction of tonic discharges of cells localized in the brain stem into theta-frequency rhythmic discharges. © 2016 Wiley Periodicals, Inc.

Development of theta rhythm in hippocampal formation slices perfused with 5-HT1A antagonist, (S)WAY 100135.

Numerous studies have revealed that median raphe nuclei stimulation induces desynchronization of hippocampal field activity in vivo. Some findings provide evidence for tonic regulation of the theta oscillation of the septo-hippocampal system via the serotonergic system. To date the involvement of serotonergic transmission in theta rhythm generation in hippocampal slices has never been investigated. Thus the aim of the present study was to test whether HPC in vitro preparation is capable of producing theta in the presence of compounds modulating the activity of 5-HT1A receptors. To achieve this a series of experiments designed to determine the effect of different concentrations of the 5-HT1A receptor antagonist (S)WAY 100135 on HPC field activity was carried out. The dominant field potential pattern recorded within HPC slices was epileptiform activity, with a maximum frequency ranging from 0.19 ± 0.06 Hz to 0.69 ± 0.10 Hz. In addition, after the bath application of (S)WAY 100135 in concentrations 3 and 10 µM rhythmic epochs in the theta frequency range were also noted. The highest probability of theta rhythm production was observed after the bath perfusion with a solution of (S) WAY 100135 at a concentration of 10 µM. These theta rhythm epochs were characterized by a higher-than-average amplitude compared to carbachol-induced theta epochs and shorter time duration, with no apparent differences in the average frequency and duration of intervals between theta epochs. These results obtained herein of in vitro studies provide direct evidence for the involvement of serotonergic receptors in the depression of oscillatory activity in the HPC theta band.

Orexinergic theta rhythm in the rat hippocampal formation: In vitro and in vivo findings.

Previous in vivo data suggested that orexin neuropeptides (ORX(A) and ORX(B) ) synthetized in hypothalamic neurons were involved in the mechanism of generation of the hippocampal formation theta rhythm. Surprisingly, this suggestion has never been directly proved by experiments using intraseptal or intrahippocampal administration of orexins. In this study, involving the use of in vitro hippocampal formation slices and in vivo model of anesthetized rat, we provide the first convergent electropharmacological evidence that in the presence of both ORX(A) and ORX(B) the hippocampal formation neuronal network is capable of producing oscillations in the theta band. This effect of orexin peptides was antagonized by selective blockers of orexin receptors (OX1 R and OX2 R), SB 334867 and TCS OX2 29, respectively. These results provide evidence for a novel, orexinergic mechanism responsible for the production of theta rhythm in the hippocampal formation neuronal network.

The electrical coupling and the hippocampal formation theta rhythm in rats.

Gap junctions (GJs) were discovered more than five decades ago, and since that time enormous strides have been made in understanding their structure and function. Despite the voluminous literature concerning the function of GJs, the involvement of these membrane structures in the central mechanisms underlying oscillations and synchrony in the neuronal network is still a matter of intensive debate. This review summarizes what is known concerning the involvement of GJs as electrical synapses in mechanisms underlying the generation of theta band oscillations. The first part of the chapter discusses the role of GJs in mechanisms of oscillations and synchrony. Following this, in vitro, ex vivo, and in vivo experiments concerning the involvement of GJs in the generation of hippocampal formation theta in rats are reviewed.

Atropine-sensitive theta rhythm in the posterior hypothalamic area: in vivo and in vitro studies.

Theta rhythm is the largest, most prominent, and well-documented electroencephalography activity present in a number of mammals, including humans. Spontaneous theta activity recorded locally in the posterior hypothalamic area (PHa) has never been the subject of detailed studies. The authors have shown that local theta field potentials could be generated in urethane-anesthetized rats in the supramammillary (SuM) nuclei and posterior hypothalamic (PH) nuclei. Theta recorded in the PHa was produced independently of simultaneously occurring hippocampal theta. These data were confirmed in the PHa maintained in vitro. Local theta field activity was recorded in the SuM and PH nuclei of PHa slice preparations perfused with cholinergic agonist carbachol. Both in vivo and in vitro recorded PHa theta rhythmicity had a cholinergic-muscarinic profile, that is, it was antagonized by muscarinic antagonist atropine sulfate.

Development of NMDA-induced theta rhythm in hippocampal formation slices.

During the past 20 years experimental evidence has accumulated demonstrating that the appearance of theta rhythm requires a certain level of excitation of local neuronal networks. In this study we extended our earlier in vitro observations concerning the involvement of cholinergic and GABAergic neurotransmission in hippocampal theta production. Specifically, we investigated whether the hippocampal neuronal network is capable of generating theta oscillations in the presence of N-methyl-d-aspartic acid (NMDA) in a brain slice preparation. To answer this question, the effect of different concentrations of NMDA (Experiment I) and the effect of interaction between NMDA and GABAA/B agonists and antagonists on field potentials recorded in the CA3c region of hippocampal formation (HPC) slice preparations (Experiment II) was examined. We demonstrated for the first time that apart from the epileptiform activity recorded in almost all series of Experiments I and II, only the perfusion of HPC slices with NMDA in doses of 30 and 50 µM, as well as the perfusion of HPC slices with NMDA and GABAB agonist baclofen (50 µM NMDA+50 µM BACL), resulted in the appearance of individual theta epochs. The best synchronized theta oscillations obtained after administration of 50 µM NMDA+50 µM BACL resembled theta activity induced by a bath perfusion of 50 µM carbachol. In light of the obtained results we conclude that besides the cholinergic and GABAergic input, NMDA glutamatergic drive is also important for the appearance of theta oscillations in HPC in vitro.

Spatiotemporal characterization of mTOR kinase activity following kainic acid induced status epilepticus and analysis of rat brain response to chronic rapamycin treatment.

Mammalian target of rapamycin (mTOR) is a protein kinase that senses nutrient availability, trophic factors support, cellular energy level, cellular stress, and neurotransmitters and adjusts cellular metabolism accordingly. Adequate mTOR activity is needed for development as well as proper physiology of mature neurons. Consequently, changes in mTOR activity are often observed in neuropathology. Recently, several groups reported that seizures increase mammalian target of rapamycin (mTOR) kinase activity, and such increased activity in genetic models can contribute to spontaneous seizures. However, the current knowledge about the spatiotemporal pattern of mTOR activation induced by proconvulsive agents is rather rudimentary. Also consequences of insufficient mTOR activity on a status epilepticus are poorly understood. Here, we systematically investigated these two issues. We showed that mTOR signaling was activated by kainic acid (KA)-induced status epilepticus through several brain areas, including the hippocampus and cortex as well as revealed two waves of mTOR activation: an early wave (2 h) that occurs in neurons and a late wave that predominantly occurs in astrocytes. Unexpectedly, we found that pretreatment with rapamycin, a potent mTOR inhibitor, gradually (i) sensitized animals to KA treatment and (ii) induced gross anatomical changes in the brain.

The generation of theta rhythm in hippocampal formation maintained in vitro.

The most spectacular example of oscillations and synchrony which appear in the brain is the rhythmic slow activity (theta) of the limbic cortex. Theta rhythm is the best synchronized electroencephalographic activity that can be recorded from the mammalian brain. Hippocampal formation is considered to be the main structure involved in the generation of this activity. Although detailed studies of the physiology and pharmacology of theta-band oscillations have been carried out since the early 1950s, the first demonstration of atropine-sensitive theta rhythm, recorded in completely deafferented hippocampal slices of a rat, was performed in the second half of the 1980s. Since the discovery of cholinergically induced in vitro theta rhythm recorded from hippocampal formation slices, the central mechanisms underlying theta generation have been successfully studied in in vitro conditions. Most of these experiments were focused on the basic question regarding the similarities between the cholinergically induced theta activity and theta rhythm examined in vivo. The results of numerous in vitro experiments strongly suggest that cholinergically induced theta rhythm recorded in hippocampal slices is a useful analogue of theta observed in intact animals, and could be helpful in searching for the mechanisms of oscillations and synchrony in the central nervous system neuronal networks. The objective of the present review is to discuss the main results of experiments concerning theta oscillations recorded in in vitro conditions. It is our intent to provide, on the basis of these results, the characteristics of essential mechanisms underlying the generation of atropine-sensitive in vitro theta.

Theta-related gating cells in hippocampal formation: in vivo and in vitro study.

In this study we extended our earlier in vitro findings concerning the discovery of a novel type of theta-related cells, which we have termed gating cells. There were two main objectives of our present investigations. The first was to determine the distribution of theta gating cells in the separated CA1 and CA3 generators in three different pharmacological conditions: (i) the presence of a cholinergic agonist-carbachol, (ii) the presence of carbachol and GABA(A) ergic antagonist-bicuculline, (iii) the presence of carbachol and GABA(B) ergic antagonist-2-hydroxysaclofen. The second objective of our studies was to verify our earlier in vitro findings and to demonstrate, for the first time, gating cells in intact hippocampus during the generation of Type II theta in urethane anaesthetized rats. Two hundred ninety-nine theta-related cells were isolated and recorded from in vivo and in vitro hippocampal formation. Twenty out of all 299 neurons (6.6%) were classified as gating cells. The neuron was classified as a gating cell if it met one of the following criteria: (i) the cell discharges occurred precisely in the beginning and at the end of each theta epoch (gating cell A); (ii) the cell began to discharge just before the transition from non-theta interval/LIA into the theta epoch (gating cell B); (iii) the cell began to discharge just after the transition from the theta epoch into non-theta interval/LIA (gating cell C). Our data demonstrates that the appearance of theta epochs and their length, as well as the appearance of non-theta states (in vivo recorded LIA or in vitro recorded intervals between theta epochs) and their length, may require the existence of a specific population of hippocampal neurons which we termed gating cells.

Does an anti-oxidant ascorbic acid improve the condition of hippocampal formation slice preparations? A micro-EEG approach.

The objective of this study was to assess whether ascorbic acid (AA), an intracellular anti-oxidant critical for neuronal protection, when added to artificial cerebrospinal fluid (ACSF), is able to protect hippocampal (HPC) formation slice preparations from ageing. In this research, the micro-electroencephalographic (EEG) technique was applied. Experiments were performed on 72 HPC formation slices obtained from 12 male Wistar rats. Two series of experiments were conducted: the control experiment, in which ACSF was used as an incubation medium, and the research one, in which ACSF was supplemented with 200 µM AA. The experimental model of carbachol-induced EEG theta rhythm was applied. The following parameters of theta rhythm after 15, 30 and 45 min of recording were analysed: frequency, power, time duration of theta epochs and time duration of intervals between theta epochs. The results show that AA causes a statistically significant decrease in the power of theta rhythm after 15, 30 and 45 min of recording. The time duration of intervals between theta epochs was almost twice as long in slices incubated in ACSF + AA than in ACSF after 45 min of recording. The data obtained indicate that AA does not improve the condition of HPC slices. On the contrary, it worsens the ability of slice preparations to generate theta oscillations. We hypothesize that our data may result from the Fenton reaction or changes in the conformation of connexins.