A

20 DM bicuculline

FIGURE 3 Block of g-amino-butyric acid (GABA)a receptors leads to spike-wave discharge (SWD)-like synchronized oscillations in ferret dorsal part of the lateral geniculate nucleus (LGNd) slices. Intracellular recording from a thalamocortical (TC) neuron during the generation of a normal spindle wave. B: Bath application of (-)- bicuculline methiodide (20 ||M) results in a slowing of the frequency of oscillations from 5 to 2.4 Hz and to a marked enhancement of low-threshold spike (LTS)-associated burst firing. (Adapted from Bal et al., 1995.)

FIGURE 3 Block of g-amino-butyric acid (GABA)a receptors leads to spike-wave discharge (SWD)-like synchronized oscillations in ferret dorsal part of the lateral geniculate nucleus (LGNd) slices. Intracellular recording from a thalamocortical (TC) neuron during the generation of a normal spindle wave. B: Bath application of (-)- bicuculline methiodide (20 ||M) results in a slowing of the frequency of oscillations from 5 to 2.4 Hz and to a marked enhancement of low-threshold spike (LTS)-associated burst firing. (Adapted from Bal et al., 1995.)

The Horizontal Thalamic Slice: A Model for Intrathalamic Rhythmicity

A rodent in vitro thalamic slice preparation has been developed that contains interconnected VB and RT neurons and that is capable of generating sustained low-frequency (2-4 Hz) oscillations (Huguenard and Prince, 1994b). The horizontal thalamic slice provides a preparation that can be obtained easily and that allows straightforward analyses of genetic mutations or pharmacologic interventions on intrathalamic rhythmicity.

Horizontal thalamic slices are taken through the middle portion of the RT nucleus (stereotaxic levels 2.1-4.1 mm posterior from bregma), which contains the adjacent ventral posterior lateral nucleus (see Paxinos and Watson, 1997). Rat pups P8-P26 are anesthetized (50mg/kg pentobarbital) and decapitated. Brains are rapidly removed and placed in chilled (4°C) low-Ca2+/low-Na+ slicing solution consisting of (in mM): sucrose, 234; glucose, 11; NaHCOs, 24; KCl, 2.5; NaH2PO4, 1.25; MgSO„; CaCl2, 0.5; equilibrated with a mixture of 95% O2, 5% CO2. Thereafter a block of brain containing the thalamus is transferred to a vibratome, and 400-mm slices are obtained in the horizontal plane, hemi-sected and submerged in preheated (33° C), oxygenated physiological saline containing (in mM): NaCl, 126; glucose, 11; NaHCO3, 26; KCl, 2.5; NaH2PO„, 1.25; MgSO„, 0.63; CaCl2, 2; equilibrated with a mixture of 95% O2, 5% CO2. After 1 hour the heat is turned off and slices are allowed to cool to room temperature.

For electrophysiologic recordings, slices are placed in an interface-type recording chamber with continuous perfusion (2ml/min) of physiologic saline at 33° C. Multiunit extracellular recordings can be obtained from thalamic relay neurons in VB and from GABAergic neurons from the RT

nucleus using sharpened tungsten electrodes (0.1MW resistance). Recordings are amplified and filtered above 100 Hz. Intrathalamic network oscillations driven by synaptic interactions between RT and VB neurons can be evoked by stimulation of the internal capsule. Bicuculline methiodide (1-10 |mM) added to the bathing medium results in a prolongation and increase in synchronization of oscillatory activity. On the other hand, application of the classic antiabsence drug ETX has a robust anti-oscillatory effect. Typical responses evoked by stimulating the internal capsule (40 |ms shocks of 40-V intensity) consist of one to five repetitive bursts, at a frequency of 2 to 4 Hz, that last for 2 to 8 seconds. Recordings are typically stable for more than an hour.

The horizontal thalamic slice preparation represents a simple experimental setup that is ideal for screening of antiepileptic drugs and the effects of gene knockout (Huguenard and Prince, 1994a, b; Huntsman et al., 1999; Yue and Huguenard, 2001). Besides the extracellular recordings described previously, the horizontal thalamic slice can be used in conjunction with conventional sharp intracellular (Sohal et al., 2003) or patch-clamp recording techniques (use of a submerged rather than an interface-type chamber is highly recommended for the latter). Whereas blind patch-recordings were described originally (Huguenard and Prince, 1994b), the use of a standard upright microscope equipped with infrared differential interference contrast optics (Dodt and Zieglgansberger, 1990) allows for the visual identification of single neurons.

The Thalamocortical Slice: A Model for Synchronous Thalamocortical Activity

To account for the joint contribution of both thalamus and cortex to the generation of SWDs, in vitro preparations including both regions of the brain have been developed. Originally the mouse thalamocortical slice was introduced as a suitable system for studying the physiology and pharmacology of the thalamocortical synapse and for exploring the synaptic circuitry of the somatosensory cortex (Agmon and Connors, 1991). Later a similar preparation was introduced for rats (Tancredi et al., 2000; Zhang et al., 1996). This thalamocortical slice preparation is a reproducible model that enables investigators to analyze thalamocortical synchronization and to understand the pathogenesis of absence epilepsy (D'Arcangelo et al., 2002; Tancredi et al. 2000).

To achieve thalamocortical slices Wistar rats (P15-P28) are decapitated under halothane anesthesia, and their brains are quickly removed and placed in cold, oxygenated artificial cerebrospinal fluid (ACSF) containing (mM): NaCl, 124; KCl, 2; KH2PO4, 1.25; MgSO4, 0.5; CaCl2, 2; NaHCO3, 26; glucose, 10; a pH of 7.4 is achieved by bubbling control

71 mV

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