In Vitro Hippocampal Electrophysiology and in Vivo Quantitative EEG Revealed Robust Neurophysiological Effects of the Antivertigo-Agent Vertigoheel® in a Rat Study

Vertigo is a common symptom with impact on daily life. Vertigoheel (VH-04) has demonstrated to be effective for Vertigo in former studies. This paper aims to investigate the mode of action of the medicinal product VH-04 in the rat brain. In an in vitro study neurophysiological recording from hippocampal slices from adult male Sprague Dawley rats was performed in order to substantiate a possible direct effect on the brain of VH-04 in different concentrations. In an in vivo cross-over study with 11 Fischer 344 rats, a neurophysiological method was applied to systemically analyse VH-04’s activity in the rat brain. This method combines quantitative assessments of telemetrically transmitted field potentials after drug treatment with subsequent discriminant analysis to classify the compound. The database used for the analysis of classification contained numerous chemicals and medicinal products of different dosages, all tested in the same paradigm, which is continuous wireless monitoring of the EEG of freely moving rats before and after drug intake. Following single stimuli on the Schaffer collaterals in the presence of VH-04 in different concentrations, in vitro responses of pyramidal cells increased depending on the VH-04 concentration (0.25 4 ml/L). Results were statistically significant for concentrations above 2.5 ml/L. Long-term potentiation was only marginally affected. Out of several specific glutamate receptor antagonists the effect of VH-04 was only antagonized by AMPA and kainic acid receptor-mediated signalling. Their enhancement indicates better information processing in the hippocampus, a brain structure primarily involved in memory processes. The in vivo characterisation of VH-04-induced changes in EEGHow to cite this paper: Dimpfel, W., Seilheimer, B. and Schombert, L. (2019) In Vitro Hippocampal Electrophysiology and in Vivo Quantitative EEG Revealed Robust Neurophysiological Effects of the Antivertigo-Agent Vertigoheel® in a Rat Study. Neuroscience & Medicine, 10, 407-425. https://doi.org/10.4236/nm.2019.104030 Received: November 29, 2019 Accepted: December 24, 2019 Published: December 27, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
The terms vertigo, dizziness, and unsteadiness are used inconsistently by the public as well as professionals and there is a general demand for a more precise definition of vertigo in clinical practice as shown by Blakley et al. [1]. Anyhow, the lifetime prevalence for vertigo of 7.8% in the general population was estimated with a significant rise with age as published by Neuhauser et al. [2]. Vertigo is a symptom, and depending on its origin, vertigo is distinguished between vestibular and non-vestibular vertigo. Vestibular vertigo is distinguished between peripheral and central disorders. To the peripheral vestibular disorders belong vestibular neuritis, benign paroxysmal positional vertigo, Ménière's disease and bilateral vestibulopathy. Non-vestibular vertigo has different etiologies such as metabolic disorders (diabetes mellitus), drug-induced dizziness, orthostatic dysregulation, stroke affecting other cerebral areas and psychiatric disorders (anxiety, depression). Although a number of specific conditions are defined that can cause vertigo, the condition is often multifactorial mainly in, but not restricted to the elderly [2] complicating the diagnosis and treatment of the symptom. Therefore, the diagnostic and therapeutic approach must be multi-systemic and oriented to the visual, proprioceptive, and vestibular systems according to Fernández et al. [3].
Several studies have investigated the medicinal product VH-04. In a randomized double-blind controlled clinical study Weiser et al. [4] compare the treatment of vertigo to the treatment of betahistine. It could be demonstrated on 105 patients in 15 study centers, receiving either VH-04 or betahistine hydrochloride, that both treatments reduced the frequency, duration, and intensity of vertigo attacks during a six-week treatment period. Klopp et al. [5]  sesses a complex repertoire of chemicals that belong to a variety of substance classes, which is in contrast to defined single drug combinations. Therefore, activity monitoring requires a reconsideration of the conventional methods. According to Wink [6], the pharmacological activity of multicomponent mixtures cannot be assigned to a single substance, and the contained phytochemicals typically act in an unspecific and widespread manner. It is the summation of activities that possibly leads to a potentiation of effects and promotes a prominent outcome. Conversely, the fractionation or isolation of principal constituents from extracts has been shown to end up in a loss of previously detected activities according to Keith et al. [7]. Numerous theories propose that the interplay and interference of single components in a mixture is the rationale for the advanta- An overview of glutamatergic receptor-specific chemicals is given in Table 2.

Animals and Trial Setting
In vivo studies were performed on twenty-two eight-month-old Fisher 344 ® rats (Charles River Laboratories, Sulzfeld, Germany) which were kept in sterile filter-top cages on a reversed day/night cycle and provided with food and water Ad libidum. The implantation of bipolar concentric steel electrodes into the FC, the HC, the ST and the RF were achieved by screws drilled into the scull and fixated by red dental cement. Signals were wirelessly transmitted by a radio-telemetric system (TSE-Systems GmbH, Bad Homburg, Germany), using 40 megahertz (MHz) as a carrier frequency, and were amplified and processed to give the desired power spectra of a 0.25 Hertz (Hz) resolution according to Dimpfel et al. [13]. The animals were granted a two-week recovery period before drug treatment. The reversed day and night cycle was continued for the whole study. The study was performed during the four weeks after surgery. A time course is documented in Figure 1. After a forty-five-minute pre-drug baseline recording, single doses of VH-04 or vehicle control were administered as indicated ( Figure   2). After a five-minute recovery interval, EEG-signals were then recorded for one hour. Different doses of VH-04 were tested in a crossover design. A total of eleven animals were exposed to one dose of the medicinal product per experiment followed by a one-week drug-free interval. One animal from the highest dose group was excluded due to technical issues. Principles of laboratory (NeuroCode

Hippocampal Slices in Vitro
HC slices were obtained from forty-two adult male Sprague Dawley ® rats (Charles River Laboratories, Sulzfeld, Germany). The rats were kept under a reversed day/night cycle for two weeks prior to the experiments, in order to record in vitro activity in slices during the active phase of their circadian rhythm according to Dimpfel et al. [14] and Dimpfel [15]. Animals were sacrificed, brains removed, and hippocampi isolated under micro-stereoscopic view. The midsection of the HC is fixed to the table of a vibrating microtome using a cyanoacrylate adhesive, submerged in chilled bicarbonate-buffered saline, and cut into slices of 400 μ thickness. All slices are pre-incubated for at least one hour in Carbogen saturated artificial cerebrospinal fluid (ACSF; 124 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 2 mM MgSO 4 , 26 mM NaHCO 3 , 10 mM glucose; pH = 7.4) in a pre-chamber before use, according to Dimpfel [15]. The methodology has been described earlier by Dimpfel [16] in more detail. During the experiment, slices were held and treated in a special super-fusion chamber (List Electronics, Darmstadt, Germany) according to Haas et al. [17] and kept at 35˚C as reported in the literature [18].
Four slices were used from one rat per day under one of the test conditions, either control or different concentrations of the test preparation. After obtaining stable responses to singular stimulus (SS), long-term potentiation was induced by applying a theta burst stimulus (TBS) type pattern [19].

EEG Recording in Vivo
Animals were day-night converted to allow recording during the active phase.
EEG-recordings were performed at the same time of day to exclude circadian effects. During recording, rats were able to move freely but had no access to food in order to avoid artefacts caused by chewing. Signals were transmitted and Neuroscience & Medicine processed to give power spectra of a 0.25 Hz resolution as described earlier in Dimpfel [20]. Briefly, artefacts were automatically rejected and signals of 4 s units were collected and Fast Fourier transformed using a Hanning window, 512 Hz sampling frequency. Four values were averaged to give a final sampling frequency of 128 Hz. The resulting electrical power spectra were divided into six defined frequency ranges (Table 3). Spectra were averaged in three-minute intervals during recording in order to supervise the data acquisition and in sixty-minute intervals during offline analysis for final evaluation.

Discriminant Analysis
The effects of VH-04 were further compared with those of reference drugs using twenty-four variables, six frequencies times four brain areas, by linear discriminant analysis according to Fischer as described previously in Dimpfel [21]. The  (Table 4). This positioning is useful because it quantitatively describes the relational similarities and differences to all plotted medicinal products.

Statistical Analysis
Averages of EEG-signals in µV 2 were expressed as percent of pre-drug baseline values, and data of all animals per group were summarised as mean ± standard    after SS. In the presence of TBS, however, a slight, statistically insignificant increase of the population spike amplitude was seen ( Figure 5). Nearly identical results were obtained in the presence of SYM 2206 according to Pelletier et al. [27], another non-competitive AMPA receptor antagonist ( Figure 5). In order to test a possible interference of VH-04 with kainate receptors-glutamatergic neurotransmission was modulated by UBP 30, a very potent and selective competitive kainate receptor antagonist according to More et al. [28]. In the presence of 50 nM UBP 301 alone, no change in the signal amplitude could be measured

EEG Recordings in Vivo
The changes of electrical activity at several locations of the brain recorded in the presence of VH-04 in different dosages (the so-called electropharmacogram, EPG) were recorded continuously for one hour after drug administration following the forty-five minutes lasting baseline. Changes in spectral power within the four brain regions following physiological saline administration could hardly be detected, which is documented for the first hour after administration in Figure 6. Spectral  Concomitantly with the telemetric recording, motion of the freely moving rats was monitored. There were no obvious changes in motion in the presence of VH-04 compared to control of 0.9% NaCl.

Discussion
In this report, we demonstrate that VH-04 is able to alter the EEG-signature of rats, so it could be characterised as a CNS-active medication. We have chosen to go for a holistic method in order to analyse its mode of action systematically in vivo and in vitro. The i.p. injection of VH-04 dose-dependently attenuated delta, theta, alpha 2 and beta 1 waves in the HC, as well as in the FC. In the ST and RF, smaller changes in EEG-signals were observed, but they only reached statistical significance in the ST at 1 ml/kg and in the RF at the highest dosage of 2 ml/kg. The attenuation of alpha 2 waves in the HC and FC, could be interpreted as an activation of the dopaminergic system, because dopamine receptor agonists attenuate alpha 2 waves and dopaminergic receptor antagonists increase alpha 2 waves in this test-system [20]. The prefrontal cortex and the HC formation are densely innervated by dopaminergic afferents, and learning and memory are strongly modulated by acetylcholine and dopamine activity. There is some evidence that the norepinephrine-system affects the incorporation of memories, involving the modulation of synaptic plasticity [32]. Interestingly, both visual and vestibular stimulations have been shown to influence power and frequency of EEG delta, theta and alpha waves in several brain regions of humans [33]. As an outcome of the analysis, the discriminant analysis showed certain similarities in comparison to cognition enhancing cholinergic Metanicotine and Tacrine as evidenced by its positioning in Figure 8, particularly indicated by the close proximity of VH-04 to the reference medicinal product [21]. Many allopathic drugs for vertigo include anticholinergic compounds impairing cognitive functions. However, this is highly speculative and needs to be examined in further studies. Hypothetical effects of VH-04 on cognition are justifiable concerning its effectiveness in the treatment of vestibular vertigo [34], suggesting that information provided by the vestibular system directly affects the HC. Thus, if vestibular input is impeded by bilateral vestibular dysfunction [35] or chronic bilateral vestibular loss [36], spatial memory is impaired in humans. Moreover, vestibular loss results in HC atrophy [36]. If, on the contrary, the vestibular system is stimulated by caloric irrigation or electrical stimulation of the inner ear, cognitive processes are expedited in humans and memory may be enhanced [37] [38]. Another factor is that VH-04 exerts vasorelaxant effects, presumably by regulating smooth muscle cell contraction or relaxation, and there is preliminary evidence for a close relationship between resistance vessel function and neuropsychological performance [39]. Hemispheric malperfusion and altered cerebral blood flow regulation are potentially related to cognitive impairment [40]. For VH-04 subcutaneous microcirculatory improvements caused by VH-04 were demonstrated [5]. Picrotoxin, a GABA antagonist and a secondary metabolite of Anamirta cocculus prevented cognitive impairments in a mouse model of Alzheimer's disease, according to Yoshiike et al. [41]. Direct exposure of the HC in vitro to several concentrations of VH-04 led to significant increases of the population spike amplitude after SS. Several specific glutamate receptor antagonists were tested to attenuate VH-04 induced increases of the population spike amplitudes. Only compounds interacting with the AMPA and the kainic acid receptor attenuated the effects of VH-04. Dingledine [42] postulates that here is ample evidence that the HC slice preparation in vitro provides an effective method for investigating the communication structure of the brain regarding neurophysiological parameters. Of special interest are surrogate parameters, which reflect cognitive features. Two parameters dominate the analysis of HC function in vitro. The first one consists in the response to the stimulation of the Schaffer collaterals by SS, resulting in the excitation of pyramidal cells. The second one is the initiation of long-term potentiation after theta burst stimulation. Both parameters increase in the presence of memantine, an antidementive medicinal product for the treatment of Alzheimer's disease, see Dimpfel [43]. VH-04 at rather low concentrations, dilution of 1:4000 of the original preparation, was able Neuroscience & Medicine to increase these responses. These data corroborate electrophysiological data obtained during the course of in vivo experiments in the second part of the present investigation, where the obtained EPG in rats provides evidence for a stimulatory action of VH-04 on several brain regions. Kainate receptors play a crucial role in the control of synaptic integration and spike transmission efficacy at the HC mossy fiber synapses and are highly implicated in spatial information processing [44]. According to Johnson et al. [45], an AMPA potentiating mechanism might be related also to neuroprotection. In regards to the field potentials and neurotransmission, Dimpfel [15] used more or less specific drugs with known influence on neurotransmission to examine a relationship between field potentials and neurotransmitter activity. Numerous antagonists and agonists of dopamine, norepinephrine, serotonin, glutamate and acetylcholine receptors led to reproducible changes of the frequency content. This indicates that changes in these frequency ranges have a physiological meaning. However, interpretation of these frequency changes is limited, since the activity of several neurotransmitters can change simultaneously in response to a drug. Thus, it is often only possible to assign a dominant change within a particular frequency range to one specific neurotransmitter.

Conclusion
We conclude that VH-04 has a significant activity on the CNS and that it significantly affects alpha 2 waves in all brain areas. Preliminary observations revealed certain similarities of activity with cognition enhancing drugs, but further experiments are required to support this concept. Preclinical studies are to further investigate the potential effects of VH-04 on memory and cognition, and if these effects can be verified, to further elucidate its mechanism of action.