Video telemetric EEG system in physiological and pathological conditions

The radio-telemetry is a technology of radio-signals recording from chronically implanted radio-transmitters to investigate less invasively physiological parameters from freely moving animals. The video image is an innovative methodology which allows capturing the behavioural patterns and dedicated software-hardware systems (es. EthoVision XT), using video tracking technologies, allow the quantitative and qualitative measure of the behavioural activities. The aim of present research activities is the application of a new Video-Telemetry EEG (Video-tEEG) Model in the Rat. Thirty-four male Sprague-Dawley rats were intraperitoneally implanted with a telemetric transmitter (type TL10M3-F50-EEE); EEG leads were placed on the dura mather for cortical EEG (EEGc) recording and deep to the dentate gyrus for the hippocampal EEG (EEGhipp) recording; the third couple of leads was secured to the musculus cervicoauricolaris (neck muscle) for the EMG recording. This research had three principal objectives: 1- Analyze the relation between environmental condition (enrichment or not, olfactory stimuli or not), behavioural/sleep pattern and theta activity; Following differential experiences provided by enriched environmental conditions, several studies have shown significant changes in laboratory animals at morphological and behavioural levels, but only few and not recent works evaluated effects of these conditions on sleep pattern. Besides, some data suggested the correlations between cortex, hippocampal electrical activities and olfaction. 8 rats were placed into the experimental PhenoTyper® cages for habituation. 4 of 8 animals received novel environmental enrichment. From these rats EMG, EEGc and EEGhipp signals by telemetry and the general activity information by video were recorded continuously for 24h immediately after this enrichment change (Phase 1) then continuously for 24h after 3 days of permanence of the animal into the enriched cage (Phase 2). Three days after this last registration, all 8 animals received olfactory stimuli (2 drops of bergamot and 2 of lemon essential oil) every morning for 4 days. From these rats 24h telemetric signals (EEGc, EEGhipp and EMG) and videos were recorded immediately after the first olfactory stimuli (Phase 3) and on the 4th day of olfactory stimulation (Phase 4). From the whole 24h period videos, spontaneous motor activity was analysed by automated video-tracking with Ethovision XT software. Sleep analysis by EEGc and EMG traces and theta activity by EEGhipp were performed in all recorded 24h telemetric traces. The video-tracking analysis showed that the novel environmental enrichment induces in rats an increase of motor activity which is not observed after 2 days by novel objects introduction. The novel enrichment takes animals to enhance cage’s exploration and so to increase their motor activity, but this effect is not observed after some days by novelty, likely due to animals to become accustomed to the enrichment presence. Instead, after introduction of novel olfactory stimuli (Phase 3) and after 4 daily reinforced olfactory stimuli (Phase 4), no differences were found between enriched and not enriched environmental groups of the same phase in any considered motor parameters. The theta activity analysis showed that there is a light difference between time of theta activity during the first day by introduction of enrichment or olfactory stimuli in all animals with or without environmental enrichment. Otherwise the interesting result is the value of delta considering the differences between olfactory phase (Phase 4) and enrichment phase (Phase 2) and so focus on the stimuli after “habituation”. In details, the animals which didn’t receive enrichment showed an increase of theta activity almost three times than animals with environmental enrichment. This result should indicate that a single stimulus (olfactory stimuli) induces an increase of theta activity bigger than more stimuli associated together. All these results suggest how it is important to check any possible influence of test conditions and also the routine laboratory procedures like cleaning, in order to ensure the reliability and daily repeatability of experimental data. 2- Analyze the relation between seizures and convulsions (EEGs traces versus behavioural observations); One of the most frequently used model for assessing pro-convulsant/anticonvulsant drugs activity and to identify pharmaceuticals that may alter seizure susceptibility is the pentylentetrazol (PTZ)-induced convulsion model. Six telemetric animals were injected with vehicle (saline) and, the following day, with PTZ freshly dissolved in the vehicle and administered intraperitoneally at 40 mg/kg dose. Two weeks before PTZ treatment and the day of treatment, telemetric signals (EMG, EEGc and EEGhipp traces) and videos were recorded simultaneously and continuously, starting 1 hour before treatment until 23 hours after treatment. On each experimental condition (baseline, vehicle- and PTZ-injection) from 23h period videos, spontaneous motor activity was analysed by automated video-tracking with Ethovision XT software. Telemetry traces were then analysed with Neuroscore Software starting 1 hour before treatment until 4 hours after treatment. Paroxysmal events were categorized as seizures when at least 4 peaks were detectable in an EEGc recording (≥ 3 sec; 1 sec minimal interval), characterized by a 1.5 fold amplitude increment than baseline. EEGc alterations, shorter than 3 sec, were categorized as spike trains. PTZ-injection group showed a significant increase in motor parameters compared with baseline and vehicle-injection groups. Since EEGc and EEGhipp traces presented similar features, we focus our attention and quantitative analysis on cortical EEG trace. We should note that, independently to convulsion-latency, four animals up to five showed electrical EEG seizures alterations. The animal showing no seizure before tonic-clonic convulsive state was however characterized by a much higher number of spike trains, confirming the extreme importance of this first window, which is absolutely neglected in a mere behavioural observation. Considering the two subpopulations based on the latency of convulsions, we can observe that animals characterized by shorter latency will develop more severe post-convulsive seizure activity, with an augmented seizure frequency. We can thus postulate that the singular susceptibility to PTZ is predictive of the severity of the pathology. This research work demonstrates the importance of performing EEG radio-telemetry combined with synchronised video monitoring, in the acute PTZ-induced convulsion model. In particular the video-tEEG model distinguishes abnormalities in EEG patterns, in the absence of behavioural signs, also taking into account the inter-individual variability in seizure activity and behavioural manifestation. 3- Assess the technical feasibility for the integration of a Video-telemetry Electroencephalography (video-tEEG) system with a set up for sequential blood bleeding. The chronic femoral vein cannulation is a method widely used in pre-clinical studies to allow collection of blood samples at established time-points and to obtain a Pharmacokinetic-Pharmacodinamic (PK-PD) profile. Six telemetric animals were submitted to a second surgical procedure for the chronic cannulation of the femoral vein. The catheter was exteriorized in the interscapular region and rat was equipped with a jacket to allow for protection of the catheter and its connection with the tether. After a post-surgery recovery of 5-6 days all animals were placed in PhenoTyper® cages. The catheter was inserted into a 45 cm long metallic tether connected with a balanced harm which was directly fixed to the PhenoTyper® cage’s wall. In order to maintain patency of femoral catheter during whole experimental procedure, it was continuously infused saline with heparin 25UI/mL at 0.2 mL/h by infusion pump. In order to obtain time-right plasma concentrations exactly when a motor behavioural convulsion event was observed, all rats were treated with a test compound known to induce behavioural convulsions; videos and telemetric signals (EMG, EEGc and EEGhipp traces) were recorded simultaneously and continuously, starting 1 hour before treatment until 4 hours post-dose. Blood samples (300 µL blood/time point) from the catheter were collected manually at pre-dose, at three established post-dose time points, and at each occasion when a motor behavioral convulsive episode was observable. All blood samples required were collected successfully including on the occasion of motor behavioral convulsion events. The videos were analysed by the Ethovision XT software and the software was able to track animals also in presence of the additional equipment required for the bleeding (i.e., jacket, tether and balance harm), confirming that this set up did not compromise in any way the spontaneous motor activity analysis made by this software. In addition no interferences with the telemetric recordings due to the bleeding equipment were noted. The combined video-tEEG Model and the blood bleeding systems can be used in pre-clinical investigations to better define the PK/PD profile of drug-induced convulsions or seizures, allowing for safer investigations in the clinical phase.