Annika Lüttjohann

A revised Racine's scale for PTZ-induced seizures in rats

Behavioral scoring is commonly used to access seizure intensity in different seizure models. Racines scale, originally developed for the amygdala-kindling model, is also frequently used as an intensity measurement in other experimental seizure or epilepsy models. The aim of the present study is to assess the validity of Racines scale as an adequate seizure intensity measurement for the Pentylenetetrazole (PTZ) model. Male adult Wistar rats received systemic injections of PTZ starting with an initial dose of 20 mg/kg added up by 10 mg/kg every 15 min until the occurrence of a 5minute lasting convulsive seizure. Simultaneous EEG and video recordings were made. The PTZ-induced seizures gradually increased in intensity. Eleven behavioral categories were identified and statistically analyzed. Six different seizure intensity categories were found to differ from each other based on differences in onset latencies, the pattern of occurrence during high or low doses of PTZ and the EEG pattern. These categories were quite different from those of Racines scale. We suggest that Racines scale is not adequate for the assessment of the seizure intensity of PTZ-induced seizures and that an alternative scale with the six proposed behavioral seizure categories is a more adequate description of PTZ-induced seizures.

An algorithm for real-time detection of spike-wave discharges in rodents

The automatic real-time detection of spike-wave discharges (SWDs), the electroencephalographic hallmark of absence seizures, would provide a complementary tool for rapid interference with electrical deep brain stimulation in both patients and animal models. This paper describes a real-time detection algorithm for SWDs based on continuous wavelet analyses in rodents. It has been implemented in a commercially available data acquisition system and its performance experimentally verified. ECoG recordings lasting 5-8h from rats (n=8) of the WAG/Rij strain were analyzed using the real-time SWD detection system. The results indicate that the algorithm is able to detect SWDs within 1s with 100% sensitivity and with a precision of 96.6% for the number of SWDs. Similar results are achieved for 24-h ECoG recordings of two rats. The dependence of accuracy and speed of detection on program settings and attributes of ECoG are discussed. It is concluded that the wavelet based real-time detecting algorithm is well suited for automatic, real-time detection of SWDs in rodents.

On the Origin and Suddenness of Absences in Genetic Absence Models

The origin of spike-wave discharges (SWDs), typical for absences, has been debated for at least half a century. While most classical views adhere to a thalamic oscillatory machinery and an active role of the cortex in modifying normal oscillations into pathological SWDs, recent studies in genetic models such as WAG/Rij and GAERS rats have challenged this proposal. It seems now well established that SWDs originate from the deep layers of the somatosensory cortex, that the activity quickly spreads over the cortex and invades the thalamus. The reticular thalamic nucleus and other thalamic nuclei provide a resonance circuitry for the amplification, spreading and entrainment of the SWDs. Conclusive evidence has been found that the changed functionality of HCN1 channels is a causative factor for the changes in local excitability and age-dependent increase in SWD. Furthermore, upregulation of two subtypes of Na+ channels, reduction of GABAB and mGlu 2/3 receptors might also play a role in the local increased excitability in WAG/Rij rats. Signal analytical studies have also challenged the view that SWDs occur suddenly from a normal background EEG. SWDs are recruited cortical responses and they develop from increasing associations within and between cortical layers and subsequently subcortical regions, triggered by the simultaneous occurrence of theta and delta precursor activity in the cortex and thalamus in case both structures are in a favorable condition, and increased directional coupling between cortex and thalamus. It is hypothesized that the cortex is the driving force throughout the whole SWD and is also responsible for its end.

Electrical stimulation of the epileptic focus in absence epileptic WAG/Rij rats: assessment of local and network excitability

OBJECTIVE The study aims to investigate whether there is a higher excitability in the deep cortical layers of the peri-oral region of the somatosensory cortex as compared to other cortical regions in absence epileptic WAG/Rij rats and whether this is unique for this type of epileptic rats, as would be predicted by the cortical focus theory of absence epilepsy. METHODS Excitability of cortical structures was assessed in a double pulse paradigm (inter-pulse interval 400 ms, 400 μs pulse duration, varying stimulation intensities (20-100 μA)). Electrical stimulation was applied to the subgranular layers of the somatosensory and motor cortex of freely moving WAG/Rij and control Wistar rats. Electrical evoked potentials (EEPs) and afterdischarges (ADs) were recorded during wakefulness, drowsiness and non-REM sleep. RESULTS WAG/Rij rats, stimulated in the somatosensory cortex, showed higher amplitudes for the N1 and N3 components of the EEPs as compared to WAG/Rij rats stimulated in the motor cortex. This effect was present in all states of alertness and at all tested intensities. In addition, this effect was not (N1) or to much less extent (N3) present in nonepileptic control rats. Stimulation-induced 8 Hz ADs were predominantly found in WAG/Rij rats. ADs were longer after stimulation in the somatosensory than in the motor cortex and preferentially occurred during drowsiness. CONCLUSION There is a heightened excitability in the deep layer neurons of the perioral region of somatosensory cortex, which is unique for WAG/Rij rats. Moreover, the presence of 8 Hz ADs might point toward additional changes in the cortico-thalamo-cortical network. Drowsiness is an excellent state for 8 Hz ADs, mimicking spike and wave discharges (SWDs). The results are in good agreement with the cortical-focus theory of absence epilepsy.

The dynamics of cortico-thalamo-cortical interactions at the transition from pre-ictal to ictal LFPs in absence epilepsy

PURPOSE Generalized spike and wave discharges (SWD) are generated within the cortico-thalamo-cortical system. However the exact interactions between cortex and different thalamic nuclei needed for the generation and maintenance of SWD are still to be elucidated. This study aims to shed more light on these interactions via multisite cortical and thalamic local-field-potential recordings. METHODS WAG/Rij rats were equipped with multiple electrodes targeting layers 4 to 6 of the somatosensory-cortex, rostral and caudal RTN, VPM, anterior (ATN)- and posterior (Po) thalamic nucleus. The maximal-association-strength between signals was calculated for pre-ictal→ictal transition periods and in control periods using non-linear-association-analysis. Dynamics of changes in coupling-direction and time-delays between channels were analyzed. RESULTS Earliest and strongest increases in coupling-strength were seen between cortical layers 5/6 and Po. Other thalamic nuclei became later involved in SWD activity. During the first 500ms of SWDs the cortex guided most thalamic nuclei while cortex and Po kept a bidirectional crosstalk. Most thalamic nuclei started to guide the Po until the end of the SWD. While the rostral RTN showed increased coupling with Po, the caudal RTN decoupled. Instead, it directed its activity to the rostral RTN. CONCLUSIONS Next to the focal cortical instigator zone of SWDs, the Po seems crucial for their occurrence. This nucleus shows early increases in coupling and is the only nucleus which keeps a bidirectional crosstalk to the cortex within the first 500ms of SWDs. Other thalamic nuclei seem to have only a function in SWD maintenance. Rostral and caudal-RTN have opposite roles in SWD occurrence.

Dynamics of networks during absence seizure's on- and offset in rodents and man

Network mechanisms relevant for the generation, maintenance and termination of spike-wave discharges (SWD), the neurophysiological hallmark of absence epilepsy, are still enigmatic and widely discussed. Within the last years, however, improvements in signal analytical techniques, applied to both animal and human fMRI, EEG, MEG, and ECoG data, greatly increased our understanding and challenged several, dogmatic concepts of SWD. This review will summarize these recent data, demonstrating that SWD are not primary generalized, are not sudden and unpredictable events. It will disentangle different functional contributions of structures within the cortico-thalamo-cortical system, relevant for the generation, generalization, maintenance, and termination of SWD and will present a new “network based” scenario for these oscillations. Similarities and differences between rodent and human data are presented demonstrating that in both species a local cortical onset zone of SWD exists, although with different locations; that in both some forms of cortical and thalamic precursor activity can be found, and that SWD occur through repetitive cyclic activity between cortex and thalamus. The focal onset zone in human data could differ between patients with varying spatial and temporal dynamics; in rats the latter is still poorly investigated.

Peri-ictal network dynamics of spike-wave discharges: Phase and spectral characteristics

PURPOSE The brain is a highly interconnected neuronal assembly in which network analyses can greatly enlarge our knowledge on seizure generation. The cortico-thalamo-cortical network is the brain-network of interest in absence epilepsy. Here, network synchronization is assessed in a genetic absence model during 5 s long pre-ictal->ictal transition periods. METHOD 16 male WAG/Rij rats were equipped with multiple electrodes targeting layer 4 to 6 of the somatosensory-cortex, rostral and caudal RTN, VPM, anterior-(ATN) and posterior (Po) thalamic nucleus. Local field potentials measured during pre-ictal->ictal transition and during control periods were subjected to time-frequency and pairwise phase consistency analysis. RESULTS Pre-ictally, all channels showed spike-wave discharge (SWD) precursor activity (increases in spectral power), which were earliest and most pronounced in the somatosensory cortex. The caudal RTN decoupled from VPM, Po and cortical layer 4. Strong increases in synchrony were found between cortex and thalamus during SWD. Although increases between cortex and VPM were seen in SWD frequencies and its harmonics, boarder spectral increases (6-48Hz) were seen between cortex and Po. All thalamic nuclei showed increased phase synchronization with Po but not with VPM. CONCLUSION Absence seizures are not sudden and unpredictable phenomena: the somatosensory cortex shows highest and earliest precursor activity. The pre-ictal decoupling of the caudal RTN might be a prerequisite of SWD generation. Po nucleus might be the primary thalamic counterpart to the somatosensory-cortex in the generation of the cortico-thalamic-cortical oscillations referred to as SWD.

Thalamic stimulation in absence epilepsy

PURPOSE The site specific effects of two different types of electrical stimulation of the thalamus on electroencephalic epileptic activity as generated in the cortico-thalamo-cortical system were investigated in genetic epileptic WAG/Rij rats, a well characterized and validated absence model. METHODS First, 12 male rats received low frequency (double-pulse pairs of 2.5Hz, 150 μA intensity and 30s inter-pair-interval) open-loop stimulation to either the Ventral-Postero-Medial (VPM) or the Anterior Thalamic Nucleus (ATN) for 8h. Second, rats received high frequency (130Hz, pulse train of 1s) closed-loop stimulation applied to either VPM or ATN whenever a spike-wave discharge (SWD) was automatically detected. RESULTS Low frequency stimulation induced 8Hz SWD-like afterdischarges (AD). AD were frequently seen in VPM but rarely in ATN stimulated rats. AD, recorded in cortex and thalamus, showed a strong temporal coherence (visually assessed) and opposite spike polarities. Properties of AD and spontaneous SWD were equally affected by the stimulation. Closed-loop high frequency stimulation disrupted spontaneous SWD with no difference between ATN and VPM stimulated rats. 89% of SWD could be disrupted leading to a decrease in average SWD duration from 9 to 1.5s. CONCLUSION Low frequency stimulation induced AD, which strongly mimic SWD. Moreover, the effects were site-specific. High frequency thalamic stimulation disrupts ongoing SWD probable by interfering with the slow firing pattern of cortico-thalamo-cortical neurons seen during SWD cycle. The absence of stimulation site specificity for high frequency stimulation might be due to the fact that stimulation only started on average 1s after SWD onset when SWD are already fully expressed in the bidirectional cortico-thalamo-cortical resonance system.

Methods of automated absence seizure detection, interference by stimulation, and possibilities for prediction in genetic absence models

BACKGROUND Genetic rat models for childhood absence epilepsy have become instrumental in developing theories on the origin of absence epilepsy, the evaluation of new and experimental treatments, as well as in developing new methods for automatic seizure detection, prediction, and/or interference of seizures. METHOD Various methods for automated off and on-line analyses of ECoG in rodent models are reviewed, as well as data on how to interfere with the spike-wave discharges by different types of invasive and non-invasive electrical, magnetic, and optical brain stimulation. Also a new method for seizure prediction is proposed. RESULTS Many selective and specific methods for off- and on-line spike-wave discharge detection seem excellent, with possibilities to overcome the issue of individual differences. Moreover, electrical deep brain stimulation is rather effective in interrupting ongoing spike-wave discharges with low stimulation intensity. A network based method is proposed for absence seizures prediction with a high sensitivity but a low selectivity. Solutions that prevent false alarms, integrated in a closed loop brain stimulation system open the ways for experimental seizure control. CONCLUSIONS The presence of preictal cursor activity detected with state of the art time frequency and network analyses shows that spike-wave discharges are not caused by sudden and abrupt transitions but that there are detectable dynamic events. Their changes in time-space-frequency characteristics might yield new options for seizure prediction and seizure control.

Anti-epileptogenesis: Electrophysiology, diffusion tensor imaging and behavior in a genetic absence model

The beneficial effects of chronic and early pharmacological treatment with ethosuximide on epileptogenesis were studied in a genetic absence epilepsy model comorbid for depression. It was also investigated whether there is a critical treatment period and treatment length. Cortical excitability in the form of electrical evoked potentials, but also to cortico-thalamo-cortical network activity (spike-wave discharges, SWD and afterdischarges), white matter changes representing extra cortico-thalamic functions and depressive-like behavior were investigated. WAG/Rij rats received either ethosuximide for 2 months (post natal months 2-3 or 4-5), or ethosuximide for 4 months (2-5) in their drinking water, while control rats drank plain water. EEG measurements were made during treatment, and 6 days and 2 months post treatment. Behavioral test were also done 6 days post treatment. DTI was performed ex vivo post treatment. SWD were suppressed during treatment, and 6 days and 2 months post treatment in the 4 month treated group, as well as the duration of AD elicited by cortical electrical stimulation 6 days post treatment. Increased fractional anisotropy in corpus callosum and internal capsula on DTI was found, an increased P8 evoked potential amplitude and a decreased immobility in the forced swim test. Shorter treatments with ETX had no large effects on any parameter. Chronic ETX has widespread effects not only within but also outside the circuitry in which SWD are initiated and generated, including preventing epileptogenesis and reducing depressive-like symptoms. The treatment of patients before symptom onset might prevent many of the adverse consequences of chronic epilepsy.