Ubicación y diseño experimental

The discovery that a focal zone can be found in the deep cortical layers of the somatosensory cortex in both WAG/Rij and GAERS rats, has shifted our attention towards more refined analyses of the local field potentials in the deep layers of the somatosensory area and the changes that occur between especially this local cortical focus and thalamic nuclei, both within the primary somatosensory loop and outside the somatosensory loop. In order to enable such a reinvestigation of network interactions at the start and end of SWDs, a first step was the development of a multichannel electrode system that allowed us to record in different cortical layers and in multiple specific thalamic nuclei. This system was physically implemented in the form of a prefabricated Teflon block, in which isolated electrode wires were inserted through thin holes, that were based on the projections from the desired positions in the brains on the Teflon block, glued and cut at the appropriate length. This block and a head stage are placed stereotactically on the animals head and the electrodes are aimed through the drilled holes in the skull (Figure 7).

Figure 8. Histological verification of electrode positions: electrodes aimed at layer 4, 5 and 6 of the

somatosensory cortex (A); rostral RTN and ATN (B); posterior thalamus and VPM (C); VPM (D); schematic illustration of the multi

Figure 7. New electrode system for multi

recording. It consists of a custom made Teflon block with holes in which electrodes are glued. The electrodes are cut at the desired length.

Histological verification of electrode positions: electrodes aimed at layer 4, 5 and 6 of the somatosensory cortex (A); rostral RTN and ATN (B); posterior thalamus and VPM (C); VPM (D);

the multi-electrode system (E).

New electrode system for multi-channel recording. It consists of a custom made Teflon block with holes in which electrodes are glued. The

cut at the desired length.

Histological verification of electrode positions: electrodes aimed at layer 4, 5 and 6 of the somatosensory cortex (A); rostral RTN and ATN (B); posterior thalamus and VPM (C); VPM (D);

In first recordings with this new system, electrodes were aimed at layer 5 and 6 of the somatosensory cortex, to obtain LFP recordings of the focal epileptic zone; the fourth layer of the somatosensory cortex as major input layer of the somatosensory loop; the VPM, with its reciprocal connections to the somatosensory cortex; both caudal and rostral RTN, receiving and sending collateral projections to TC and Cortico-Thalamic neurons; anterior nucleus, with its connections to the rostral RTN; and the posterior thalamic nucleus, which directly receives input from layer 5 of the somatosensory cortex without collaterals to the RTN (Figure 8) [107-109]. Recordings were bipolar, differential recordings with ground and reference electrodes on top of the cerebellum and a sample rate of 2048 Hz to ensure a high temporal resolution, needed for an investigation of possibly rapid temporal changes at the onset and offset of SWDs. In the resulting recordings, that now allowed the direct investigation and comparison between LFP activity in the local cortical zone and relevant thalamic nuclei, some phenomena that support both the idea that SWDs do not occur suddenly and the idea that SWDs have a local cortical origin in the deep layers of the somatosensory cortex, can be observed even in the raw EEG traces.

The first phenomenon is displayed in Figure 9a. It can be seen that prior to full-blown SWDs with rhythmic spike and wave activity in both cortex and thalamus there are already strong rhythmic oscillations in the dominant SWD frequency of 8 to 10 Hz. These oscillations are restricted to the deep cortical layers of the somatosensory cortex and start about one second prior to the emergence of the full-blown, generalized SWDs so that at least in recordings of the deep layers of the somatosensory cortex SWDs obviously do not emerge suddenly from a normal background EEG. The restriction of these oscillations to the deep cortical layers is also in good agreement with the concept that SWDs have a local cortical origin instead of a thalamic one.

It seems as if gradually more and more cortical cells become recruited until enough CT cells fire in synchrony, strong enough to entrain the thalamus into SWDs. This process might be reflected in a gradual increase in coupling strength of cortex and thalamus at the start of SWDs. Sitnikova et al. [105] found increases in Granger’s causality coefficient between frontal cortex and ventral basal thalamus, even more interesting would be to analyse this between the cortical focus and the thalamic VPM.

The concept of a local cortical focus in the deep layers of the somatosensory cortex is further supported by additional observations that can be done in the raw EEG traces: When zooming to the first “generalized” spike that can be seen in all cortical layers and all thalamic nuclei, it can be noticed that the spike in the deep somatosensory cortex precedes all thalamic spikes (Figure 9b), which is obviously in agreement with the guiding cortical influence during the first 500ms of a SWD as found by Meeren et al. [32]. Interestingly, this

temporal relationship with the cortical spikes preceding the thalamic one can not only be seen within the first 500 milliseconds but often throughout the whole SWD (Figure 9c). This might indicate that the cortex remains the

Figure 9. Multi-channel EEG recordings of the start of SWD of an adult WAG/Rij rat. A: Strong,

rhythmic oscillation in the deep layers of the somatosensory cortex precedes the generalized SWD with rhythmic spike and wave activity in cortex and thalamus. B: Temporal sequence of the first generalized spike. Note that the spike in layer 6 of the somatosensory cortex can be seen a few milliseconds earlier than all other cortical and thalamic ones. C: Temporal sequence

middle of the SWD. Note that again the spike in layer 6 of the somatosensory cortex can be seen a few milliseconds earlier than all other cortical and thalamic ones.

temporal relationship with the cortical spikes preceding the thalamic one can not only be seen within the first 500 milliseconds but often throughout the whole SWD (Figure 9c). This might indicate that the cortex remains the driving force throughout the whole SWD.

channel EEG recordings of the start of SWD of an adult WAG/Rij rat. A: Strong, rhythmic oscillation in the deep layers of the somatosensory cortex precedes the generalized SWD and wave activity in cortex and thalamus. B: Temporal sequence of the first generalized spike. Note that the spike in layer 6 of the somatosensory cortex can be seen a few milliseconds earlier than all other cortical and thalamic ones. C: Temporal sequence

middle of the SWD. Note that again the spike in layer 6 of the somatosensory cortex can be seen a few milliseconds earlier than all other cortical and thalamic ones.

temporal relationship with the cortical spikes preceding the thalamic one can not only be seen within the first 500 milliseconds but often throughout the whole SWD (Figure 9c). This

driving force throughout the whole SWD.

channel EEG recordings of the start of SWD of an adult WAG/Rij rat. A: Strong, rhythmic oscillation in the deep layers of the somatosensory cortex precedes the generalized SWD and wave activity in cortex and thalamus. B: Temporal sequence of the first generalized spike. Note that the spike in layer 6 of the somatosensory cortex can be seen a few milliseconds earlier than all other cortical and thalamic ones. C: Temporal sequence of a spike in the middle of the SWD. Note that again the spike in layer 6 of the somatosensory cortex can be seen a

Another observation in favor of the cortical focus theory is displayed

and wave-like oscillations, with most pronounced spiking in cortical layer 6, that remain localized within the layers of the somatosensory cortex and do not involve the thalamus. Incidentally, they even remain localized within a s

oscillations have a smaller spike amplitude compared to spikes of a full

even the oscillations preceding the full blown SWD, which might indicate that less CT cells might fire in synchrony, therefo

enough to also entrain the thalamus into the spike and wave oscillation.

Alternatively, the thalamus might not be in the optimal oscillatory state in terms of hyperpolarization of its cells, so that it does not start to reverberate to the cortical oscillatory input. It can be speculated whether these localized oscillations and probably also the oscillations that are seen in the somatosensory cortex preceding the SWDs are similar to the 4-12 Hz middle-voltage oscillations that precede SWDs in GAERS.

Another observation in favor of the cortical focus theory is displayed in Figure 10: Here spike like oscillations, with most pronounced spiking in cortical layer 6, that remain localized within the layers of the somatosensory cortex and do not involve the thalamus. Incidentally, they even remain localized within a single layer. These localized spike wave oscillations have a smaller spike amplitude compared to spikes of a full

even the oscillations preceding the full blown SWD, which might indicate that less CT cells might fire in synchrony, therefore the influence of the cortex on the thalamus is not yet strong enough to also entrain the thalamus into the spike and wave oscillation.

Alternatively, the thalamus might not be in the optimal oscillatory state in terms of so that it does not start to reverberate to the cortical oscillatory input. It can be speculated whether these localized oscillations and probably also the oscillations that are seen in the somatosensory cortex preceding the SWDs are similar to the

voltage oscillations that precede SWDs in GAERS.

in Figure 10: Here spike like oscillations, with most pronounced spiking in cortical layer 6, that remain localized within the layers of the somatosensory cortex and do not involve the thalamus. ingle layer. These localized spike wave-like oscillations have a smaller spike amplitude compared to spikes of a full-blown SWD and even the oscillations preceding the full blown SWD, which might indicate that less CT cells re the influence of the cortex on the thalamus is not yet strong

Alternatively, the thalamus might not be in the optimal oscillatory state in terms of so that it does not start to reverberate to the cortical oscillatory input. It can be speculated whether these localized oscillations and probably also the oscillations that are seen in the somatosensory cortex preceding the SWDs are similar to the

A last observation made in the raw EEG

in Figure 11, spike and wave oscillations do stop earlier in the thalamic recordings while the cortical layers continue to oscillate for a few seconds. Given the observation that the cortex seems to be the driving force throughout the seizure, this might mean that a seizure stops because of a decrease in directional coupling from cortex to thalamus. Alternatively, the thalamus either actively engages in another oscillatory pattern or is entrained to another rhythm by a third structure involved in seizure cessation. Signal analytical techniques such as non-linear association analysis, Granger causality or time

investigating the dynamics of corticothalamic network interactions at the end and s

seizure need to further elucidate this question as well as the generality for the above described observations done for the start of SWDs.

Figure 11. Multi-channel EEG recordings of the end of a SWD from a WAG/Rij rat. Note that the

spike and wave oscillations stop earlier in the thalamus than in the cortex. This might be due to a decrease in cortico-thalamic coupling.

A last observation made in the raw EEG traces concerns the end of SWDs. As can be seen in Figure 11, spike and wave oscillations do stop earlier in the thalamic recordings while the cortical layers continue to oscillate for a few seconds. Given the observation that the cortex iving force throughout the seizure, this might mean that a seizure stops because of a decrease in directional coupling from cortex to thalamus. Alternatively, the thalamus either actively engages in another oscillatory pattern or is entrained to another ythm by a third structure involved in seizure cessation. Signal analytical techniques such linear association analysis, Granger causality or time-frequency analyses, investigating the dynamics of corticothalamic network interactions at the end and s

seizure need to further elucidate this question as well as the generality for the above described observations done for the start of SWDs.

channel EEG recordings of the end of a SWD from a WAG/Rij rat. Note that the and wave oscillations stop earlier in the thalamus than in the cortex. This might be due to a

thalamic coupling.

traces concerns the end of SWDs. As can be seen in Figure 11, spike and wave oscillations do stop earlier in the thalamic recordings while the cortical layers continue to oscillate for a few seconds. Given the observation that the cortex iving force throughout the seizure, this might mean that a seizure stops because of a decrease in directional coupling from cortex to thalamus. Alternatively, the thalamus either actively engages in another oscillatory pattern or is entrained to another ythm by a third structure involved in seizure cessation. Signal analytical techniques such frequency analyses, investigating the dynamics of corticothalamic network interactions at the end and start of a seizure need to further elucidate this question as well as the generality for the above

channel EEG recordings of the end of a SWD from a WAG/Rij rat. Note that the and wave oscillations stop earlier in the thalamus than in the cortex. This might be due to a

CONCLUSIONS

It is well established that SWDs in WAG/Rij rats and in GAERS are initiated in the peri-oral region of the somatosensory cortex, more precisely, by neurons located in the deep cortical layers. Now the origin has been discovered, the early appearance of SWD-activity can be easily visualized by local field potentials from the deep layers. Also measurements of cortical excitability after local electrical stimulation demonstrate that the focal epileptic area is hyperexcitable.

In vitro or combined in vivo and in vitro studies provide clues to the mechanisms underlying increased cortical excitability and reduced presynaptic inhibition. Expression of two ion channels, the hyperpolarization-activated cyclic nucleotide gated (HCN) and Na+ channels, is directly linked to the age-dependent appearance of SWDs. Proepileptic changes in HCN1 subunit occurred prior to the age-related onset of SWDs. Moreover, an experimental treatment that increases the activity of HCN channels is known to prevent the occurrence of SWDs. It is hypothesized that the loss of cortical HCN channels and a diminished sensitivity of h current to its main modulator cAMP are crucial factors for epileptogenesis in these genetic models. Genetic factors are undoubtedly mainly responsible for the age dependent functional changes. The process of epileptogenesis responsible for the cortical focus is also accompanied by changes in the expression of Na+ channels, GABAB, iono- and metabotropic glutamate receptors and morphometric properties of pyramidal cells. It still remains to be established whether these changes are genetically programmed or they are the consequence of the hundreds SWDs per day.

Several types of EEG signal analysis demonstrate that SWDs in the genetic animal models do not arise suddenly. This has recently been indicated in GAERS by simultaneous electroencephalography-near-infrared spectroscopy (EEG-NIRS) study of the whole cortex [110]. Traditional spectral analysis of preictal EEG epochs in WAG/Rij rats displayed high variability of the immediate seizure-precursor activity. More detailed time-frequency analysis showed that SWDs are preceded by the simultaneous presence of delta and theta activity in cortex and thalamus. EEG coherence study revealed specific frequency associations between the epileptic cortical focus and the closest areas; this “local neuronal circuit” is primarily involved in the initiation of SWDs. The other cortico-cortical, cortico-thalamic and intrathalamic neuronal networks showed frequency-specific changes in coherence, suggesting that each part of thalamo-cortical circuitry operates in an own oscillatory mode during transition from preSWDs to SWDs. Granger causality and non-linear association analyses indicated that SWDs are accompanied by changes in directional coupling between cortex and thalamus. SWDs may terminate when the influence of cortex to thalamus fades away.

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