Comparison of Changes in the EEG of Freely Moving Rats Induced by Diazepam by Invasive and Non-Invasive Methods

Introduction

Diazepam known to have sedative properties and increases beta-band activity and are high frequencies. The spectral content of EEG following administration of diazepam caused a decrease in the power of low frequency band (1-8Hz) and an increase in the power of high frequency bands (21-40Hz).¹An opposite drug-effect on arousal due to augmentation of the beta activity of the EEG, this EEG spectrum is known as pharmacological dissociation.²Diazepam known to enhance the ability of endogenous GABA, however diazepam induces sleep and increases the power of frequency of beta band.³ Allosteric modulator like benzodiazepines enhances the neuronal inhibition by GABA. This is one of the powerful therapeutic approaches for the treatment of central nervous system diseases such as sleep disturbances and anxiety disorders including seizures.⁴ During sensory processing and cognition, the EEG activity in the high frequency range (>20Hz) is prominent. Conscious and cognitive functions altered clinically by allosteric modulation of GABA_A receptors by benzodiazepines. Diazepam targets family of GABA_A receptor subtypes , α1,α2,α3, or α 5subunit.⁵ We investigated the effects of diazepam on rats by both methods, this involving recording of the EEG signals from the brain. The scalp electroencephalography is an effective technique to study neurological process of brain by judicious examination of brain electrical activity helps in studying neurological activities.⁶ Though there are various solutions for non-invasive EEG techniques during preclinical studies⁷ but dominated by implantation of intra-cortical electrodes.⁸ The complicated procedure of electrode grid placements in rodents requires invasive surgical procedures. The powerful tool, EEG finds its place in neurology and clinical neurophysiology. The electrical field generated by the nerve cells recorded as EEG signals.⁹ The relatively small signals, measured in microvolts (µv), the various frequency bands in EEG of scientific interest are delta (0.5-4Hz), theta (4-8Hz), alpha (8-13Hz) beta (13-30Hz) and gamma (>30Hz).

The electroencephalography is an useful tool to record the changes in brain electrical activity for investigational behavior and cognitive circumstances.¹⁰ Distinct pattern of EEG frequencies and its temporal dynamics altered in several situations and this helps in temporal analysis of neurophysiological process and due to this facilitate actions of drugs on central nervous system.¹¹ For the placement of electrodes by using stereotactic coordinates bregma is used as a reference point.¹² Animals have to undergo surgical method for the placement of electrodes under anesthesia and post-surgicalrecovery is indispensable to use animals for the research.¹³ Here a simple non-invasive procedure is employed to process the electrical signals of the brain using diazepam and an attempt is made to validate the signals generated by the diazepam invasive and conventional method involving stereotaxic surgery. In the past too non-invasive method was developed by Silva et al for bovine¹⁴ and modified for rat by Ferrari¹⁵ was fabricated to analyze EEG. The present study explains about the effect of diazepam induced changes analyzed by non-invasive procedure and validated using conventional method using stereotaxic surgery. This study addresses reliability of the non-invasive procedure to measure pharmacodynamic property of diazepam in brain.

Plastic foam electrode made from highly conductive commercially available thin wire and conductive gel is applied. The cyanoacrylate adhesive bonds EEG patch to the scalp strongly. This method is well suited for conscious and freely moving rats. This research addressed two inquiries: first, development of cost effective non-invasive method using diazepam and results substantiated by the technique of stereotaxic surgery using again same therapeutic dose of diazepam in normal Wistar rats of either sex.

Subjects and Methods

The experiments conducted in the institute after approval accorded from the Animal Ethical Committee (JCP/IAEC/2014). Twelve healthy Wistar rats of either sex , weighing 150±10 gm weight, 20-25 week of age used for the study and are purchased from Mahaveer Enterprise, Hyderabad. Diazepam was a gift sample from Vital Pharma, Hyderabad. The animals were caged individually in cages (12” x 12”x 11”) and provided pelleted feed (Lipton India Ltd. India) and water provided ad libitum. Pelleted food and water were withdrawn on the day of experiment, all twelve rats considered for recording EEG after administration of the diazepam.

Materials and Methods

To record the brain physiological changes disposable EEG patch developed using foam plastic electrical signals in the form of EEG was recorded (Figure 1). Cleanly shaved head or using depilatory agent the disposable EEG leads placed on scalp to record the brain physiology. The disposable EEG electrode patches are placed on the cortex of the left parietal region, the positive electrode and on right parietal region negative electrode and neutral electrode were placed on the nasal bone area. The size of the EEG leads measuring approximately 3 sq.mm containing conducting gel. In the present study brain signals are commonly sinusoidal when acquired from the animals.EEG activity classified into five distinct bands gamma (40Hz to 100Hz) delta <4Hz, theta activity 4-8Hz, beta activity 12 Hz-40Hz and alpha activity 8Hz-13Hz. Conducting gel used in this experiment contain potassium chloride for ion conductivity and are glycerin based gel containing (2-3%) water (80-90%), propylene glycol (10-20%), potassium chloride (2-5%) and hydroxyl propyl guar gum (1-3%). Approximately 25 mg of the conductivity gel used to place for each electrode and purchased from commercially available ECG leads (3M^TMIndia). The rat was held between two palms by retracting rat’s two forelimbs and the setup of foam plaster containing conducting gel placed on three placed on the rats head using plastic instant glue, a monomer called cyanoacrylate and is applied to the contour of the plastic foam and is fixed firmly onto the shaved head of the rat. The data acquisition system used to record EEG and signals are filtered through a high pass 0.5Hz and low pass 100Hz filters with a sampling rate of 200 samples/s. Data recorded using Biopac, USA. Post EEG recording artifact containing EEG was discarded before analysis.

EEG Recording

Animals made to adopt the experimental conditions for 15-20min,data acquisition system with the software AcqKnowledge 4.4 (Biopac system Inc., USA) used for EEG recording. Fast Fourier Transforms (FFT) used to assess the spectra between 0-100Hz Power Spectrum Density (PSD). Baseline EEG recording of 30 min of duration before administration of the drug diazepam examined for the data. Epochs of four seconds used for calculation of PSD and averaged for each segment of 30 min in baseline recording and 90 min recording considered after administration of the diazepam and are averaged.

Statistical Analysis

Statistical significance in EEG signals analyzed by paired t test and Power spectral analysis and for all analysesconsidered two-tailed significance. Test is considered to be significant if P< 0.05.

Surgical Procedures for EEG Recording

Animals are anaesthetized using ketamine (72 mg/kg, IP) and xylazime (8mg/kg, IP). To prevent any respiratory distress atropine (8mg/kg, IP) was administered. By making midline incision in the head, rat skull is exposed, stainless screws which are used as electrodes that are 1mm in diameter with a length of 2mm. the screws are fixed on parietal bone (AP 2mm; ML: 2mm and 2mm from bregma) to record electrophysiological properties of the brain, stainless screw fixed laterally to the midline of the nasal bone, which acts as ground electrode. Three wires connected to nine-pin connector was fixed on the skull with dental cement, post-operative care with an injection of dexamethasone (1.5mg/kg) to reduce brain inflammation and iodine-povidone solution to control infection was used for 5-6 days. Plastic chamber measuring (12”x 12” x11”) was used to record EEG after placing the rat, using Biopac data acquisition system electro encephalogram processed.

AcqKnowledge 4.4 (Biopac System Inc.USA) with EEG signals processed with high pass 5Hz and low pass 100Hz filters and run at 200 sampling rate/sec. Four seconds epochs employed for calculation and Fast Fourier Transform (FFT) used to asses spectra between 0-100 Hz. EEG data and Power Spectrum Density (PSD) used to record 30 min drug free baseline and 90 min recording was done post administration of the diazepam. Epochs of four seconds used to calculate PSD and average PSD of 30 min and 90 min recording considered for the estimation. Power of each of five frequency bands scrutinized for evaluation.

Results

The changes in various bands following administration of diazepam were analyzed before and after administration of diazepam. We investigated the effect of diazepam ascribed to its beta increasing effect and its effects on other bands. FFT is a modern signal processing technique, which converts signals to a series of different frequency sign wave. EEG waves divided according to its frequency, converting EEG by FFT to the sign wave and further EEG analysis.The research work depicting the effects of diazepam induced changes in EEG spectral content analyzed using a foam plastic electrode for non-invasive method and further validated using conventional method of stereotaxic surgery. Diazepam (5mg/kg body weight) in 2% acacia suspension administered in oral route for all experiments. Drug free baseline obtained in the presence of 2% acacia suspension (0.25ml), to the same animal diazepam 5mg/kg body weight orally administered and EEG recorded for 60 min.

The data acquisition system MP45 (Biopac Inc. USA) allowed us to record EEG activity in two different methods in rats, non-invasive and invasively. Diazepam induced physiological changes observed in the brain had characteristic EEG pattern and is observed in both methods. We calculated the power spectrum of the EEG from 0.5 to 100 Hz for both non-invasive and invasive methods of EEG. Diazepam in 5mg/kg body weight induced sedation of short duration and animals are never entered into the sleep in both methods. Vigilance state of the animals not affected. Diazepam induced changes persisted throughout study period. All the rats displayed major impairment of alertness after receiving 5mg/kg body weight. This impairment of alertness observed within 15-30 min after administration of the diazepam. The drug produced qualitatively identical changes in the spectra. Following electroencephalograph spectral analysis, diazepam induced changes in the five different bands mentioned in Tables 1 and 2. Every segment of 90 min after administration of diazepam EEG signals is analyzed in all bands. Analysis of EEG showed for both methods similar changes are observed. The changes observed frequency bands beta and gamma and in both methodsdelta, theta and alpha bands were not affected by the diazepam. The changes observed in the frequency bands in delta, theta and alpha were not statistically significant, though decreased in the power compared to the baseline. The changes were uniform among different bands in both methods. The statistically significant and characteristic increase in the beta and gamma bands are recorded.

Discussion

In both methods of EEG showed a pattern of increased frequencies in beta and gamma bands activity. The EEG activity was slightly lowered in delta, theta and alpha bands activity when comparison was made with baseline values. The effect of diazepam on EEG is an indicative of active behavior and was clearly observed in gamma and beta bands in both methods. The decreased alpha band activity specific for active behavior and is reflected in decrease in theta frequency (4-8Hz) synchronized with slow waves in delta frequency range (1-4Hz).

The dose of 5mg/kg diazepam chosen for both methods neurophysiological is measured. The parietal region of the EEG, the drug decreased delta (1-5Hz), theta and alpha frequencies. Especially higher frequency beta bands the increased in both cases. This impression of changes consistent with the conclusions of previous studies.¹⁶ Such changes in the beta frequency band steadily observed in many of the previous studies of benzodiazepines, a GABA receptor modulator. ^2,3,17,18Furthermore the beta band frequency increase was persistent over parietal regions of the brain. The increase in the gamma activity was observed similarly in the past investigation in rats, the increase in the beta and gamma EEG activity shown to be predominant for exhibiting the activeness but not for inactiveness of the rats. This increased beta and gamma activity is a prerequisite for the animal under sedation, this indicates that GABA a receptor modulators activate motor neurons. In the previous study too, the decrease in alpha activity specific for active behavior reflected in decrease in theta frequency.¹⁹ The drug diazepam decreased frequency in the range of 1-5 Hz(delta) and in the range of 9-10 Hz (alpha) activity and increased in higher frequencies in particularly beta and gamma.¹⁶

Conclusions

The small leads placed comfortably on the scalp of the rats and are well accepted without showing any uneasiness behavior. This inexpensive and easy to use EEG patch lead allowing the experiments to record the faithful EEG following administration of diazepam. To summarize, the effects of diazepam on neurophysiology of the rat, the drug acting as a modulator at GABA receptor complex resulting in increase in beta and gamma activity was most distinct and signature property of the diazepam and contributing for active behavior of the rats. This relation between large increase in beta and gamma activity by diazepam is a mechanism that shows active brain mechanism despite sedation.

Acknowledgement

The authors are thankful to All India Council of Technical Education (AICTE) New Delhi for providing financial assistance through Research Promotion Scheme (RPS) [F.No.8-125/RFID/RPS/Policy-3/2013-14] to work on the development and validation of non-invasive EEG in animal model.

Conflict of Interest

The authors declare no conflict of interest.