In Vitro Models Of Absence Epilepsy

The Ferret Dorsal Lateral Geniculate Nucleus Slice: A Model for the Transformation of

Spindle Waves into Spike-Wave Discharges

A number of thalamic in vitro models have been developed to investigate the mechanisms underlying the generation and spread of spike wave (SW)-like discharges in this brain structure. The ferret primary visual thalamic relay nucleus (dorsal part of the lateral geniculate nucleus, LGNd) and the associated section in the RT nucleus (termed the perigeniculate nucleus, PGN) have proven particularly useful. A sufficient part of the synaptic network is preserved in a slice preparation in vitro to generate spontaneous recur rent, spindle-like activity (Figure 3A), and this pattern of activity can be acutely transformed into SW-like discharges (Figure 3B) on pharmacologic manipulation (Bal et al., 1995a, b; von Krosigk et al., 1993).

Ferret thalamic slices can be obtained and studied using standard brain slicing and electrophysiologic recording techniques. Preparation includes decapitation of deeply anaesthetized (pentobarbital, 30-40mg/kg intraperitoneally) male or female animals 3 months to 3 years old. Sagittal slices (400-|mm thickness) from the forebrain of one hemisphere are prepared using a vibratome or vibroslicer. During preparation, tissue should be placed in a chilled solution in which NaCl is replaced with sucrose while maintaining an osmolarity of ~305 mOsm. After preparation, slices are placed in an interface style recording chamber and allowed at least 2 hours to recover. The bathing solution contains (in mM): NaCl, 126; KCl, 2.5; MgSO4, 1.2; NaH2PO4, 1.25; CaCl2, 2; NaHCOs, 26; dextrose, 10. ApH of 7.4 is achieved by gassing the solution with 95% O2 and 5% CO2. Bathing of the slices in an equal mixture of normal NaCl and the sucrose-substituted solutions for the first 20 minutes in the recording chamber may be beneficial. Intraspindle and particularly interspindle frequencies as well as the duration of each spindle wave are temperature sensitive; a bath temperature of 34 to 35° C is optimal for studying the cellular basis of spindle waves and SWDs in vitro. Typically each hemisphere of the ferret LGNd yields two or three slices that exhibit robust spontaneous spindling activity in more than 95% of experiments.

Extracellular multiunit recordings from ferret LGNd slices, using standard electrophysiologic techniques, revealed the occurrence of spontaneous spindle waves that were remarkably similar to those recorded in vivo in anaesthetized ferrets (interspindle period of 3-30 s, intraspindle frequency 6-10Hz, spindle duration 2-5 s). Intracellular recordings obtained with bevelled micropipettes pulled from medium-walled borosilicate glass and filled with 4M potassium acetate allowed the identification of the cellular mechanisms underlying spindling in TC neurons of the LGNd. It was shown that spindle waves are associated with barrages of IPSPs occurring at a frequency of 6 to 10 Hz, occasionally resulting in the generation of low-threshold Ca2+ spikes (Figure 3A). Most important, the bath application of 20 ||M (—)-bicuculline methiodide resulted in a prolongation and an increase in amplitude of these IPSPs, leading to a highly synchronized 2- to 4-Hz oscillation in which each TC cell generated action potential bursts on every cycle, thus forming a paroxysmal event resembling SWDs (Figure 3B). Bath application of the GABAB antagonist 2-OH-saclofen (250 ||M) abolished synchronized 2- to 4-Hz oscillations in ferret LGNd slices (Bal et al., 1995a); the effect of the classic antiabsence drug ethosuximide (ETX) has not been reported.

FIGURE 2 Correlated cellular activities in thalamus and neocortex during spike-wave discharges (SWDs). A: Simultaneous recording of multiunit activities in ventrobasal nucleus (VPM), rostral reticular thalamic nucleus (rRT), and somatosensory cortical sites. Epidural recordings of the bilateral electroencephalogram (EEG) (r, right; l, left hemisphere) from frontoparietal cortical areas. Calibration bars indicate 500 ||V for EEG recording, 100 ||V for unit activity. B: Inset demonstrates the temporal relationship of SWDs and burst-like activity at a faster time scale. C-E: Multisite unit recordings were simultaneously obtained from deep layers of the somatosensory cortex, rRT and caudal reticular thalamic (cRT) nucleus (C), cortex, rRT, ventroposterolateral thalamic (VPL) nucleus (D), and cortex, ventropostero-medial (VPM) and ventrolateral (VL) thalamic nucleus (E). Peristimulus-time histograms of unit activity (1-ms bins) triggered by the spike component on the EEG (upper traces in C—E show averaged SWDs) demonstrate phase locked unit activity. Stimulus-time histograms were averaged from 40 trials; n indicates number of animals. (Reprinted, with permission, from Seidenbecher et al., 1998.)

FIGURE 2 Correlated cellular activities in thalamus and neocortex during spike-wave discharges (SWDs). A: Simultaneous recording of multiunit activities in ventrobasal nucleus (VPM), rostral reticular thalamic nucleus (rRT), and somatosensory cortical sites. Epidural recordings of the bilateral electroencephalogram (EEG) (r, right; l, left hemisphere) from frontoparietal cortical areas. Calibration bars indicate 500 ||V for EEG recording, 100 ||V for unit activity. B: Inset demonstrates the temporal relationship of SWDs and burst-like activity at a faster time scale. C-E: Multisite unit recordings were simultaneously obtained from deep layers of the somatosensory cortex, rRT and caudal reticular thalamic (cRT) nucleus (C), cortex, rRT, ventroposterolateral thalamic (VPL) nucleus (D), and cortex, ventropostero-medial (VPM) and ventrolateral (VL) thalamic nucleus (E). Peristimulus-time histograms of unit activity (1-ms bins) triggered by the spike component on the EEG (upper traces in C—E show averaged SWDs) demonstrate phase locked unit activity. Stimulus-time histograms were averaged from 40 trials; n indicates number of animals. (Reprinted, with permission, from Seidenbecher et al., 1998.)

In Vitro Models of Absence Epilepsy ferret LGNd slice

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