Seizure-like thalamocortical rhythms initiate in the deep layers of the cortex in a co-culture model

Abstract

The oscillatory rhythms underlying many physiological and pathological states, including absence seizures, require both the thalamus and cortices for full expression. A co-culture preparation combining cortical and thalamic explants provides a unique model for investigating how such oscillations initiate and spread. Here we investigated the dynamics of synchronized thalamocortical activity by simultaneous measurement of field-potential recordings and rapid imaging of Ca²⁺ transients by fluorescence methods. Spontaneous sustained hypersynchronized “seizure-like” oscillations required reciprocal cortico-thalamocortical connections. Isolated cortical explants can independently develop brief discharges, while thalamic explants alone were unable to do so. Rapid imaging of Ca²⁺ transients demonstrated deep-layer cortical initiation of oscillatory network activity in both connected and isolated explants. Further, cortical explants derived from a rat model of genetic absence epilepsy showed increased bursting duration consistent with an excitable cortex. We propose that thalamocortical oscillatory network activity initiates in deep layers of the cortex with reciprocal thalamic interconnections enabling sustained hyper-synchronization.

Introduction

Strong reciprocal connections between the thalamus and cortex are responsible for generating rhythmic oscillatory neuronal network activity. These thalamocortical oscillations are known to have important neurophysiological functions that include sensor–motor information integration and modulation of sleep (Andolina et al., 2007, Iyengar et al., 2007, Pinault and Deschenes, 1992, Razak et al., 2009). Aberrant thalamocortical oscillations in this network are also responsible for pathological conditions, of which the best characterised are absence seizures (Avoli and Gloor, 1982, Blumenfeld and McCormick, 2000, Kostopoulos et al., 1981, Pinault, 2003, Steriade and Contreras, 1995). The thalamus and the cortex are both essential for initiation and propagation of these epileptic thalamocortical oscillations (Vergnes and Marescaux, 1992). Despite the well-recognised interactions between these brain structures the specific roles that each play in initiation, evolution and maintenance of pathophysiological oscillatory network activity are still unclear.

In vivo electrophysiological analysis in animal models of absence epilepsy suggests the spike-and-wave discharges (SWD), that are a hallmark of such seizures, are initiated in the cortex and secondarily engage the thalamus (Meeren et al., 2002, Meeren et al., 2001, Pinault, 2003, Polack et al., 2007). However, a major limitation of in vivo electrophysiology studies is relatively poor spatial sampling due to the limited number of electrodes that can be used at once. While studies using functional magnetic resonance imaging have attempted to explore this issue, this approach does not have high temporal resolution and is an indirect measure of neuronal activity (McKeeff et al., 2007). Rapid imaging technologies now enable higher spatial and temporal resolution of neuronal activity (Berger et al., 2007, Derdikman et al., 2003). These techniques are limited in vivo to cortical surface recordings or to in vitro studies that can encompass deeper brain structures.

Here we use an in vitro thalamocortical co-culture preparation (Caeser et al., 1989, Lotto and Price, 1994, Molnar and Blakemore, 1991, Rennie et al., 1994, Yamamoto et al., 1989) that is amenable to fast imaging, to investigate temporal and spatial aspects of oscillatory thalamocortical network activity. This has two major advantages in the context of this study. First, the neuronal circuitry that is being measured is contained within the sampled field enabling us to rule out initiation or influences of other neuronal populations. Second, organotypic cultures lose the opaque dead cell layer often seen in acute slices thereby increasing the signal-to-noise. The results demonstrate that sustained network events were initiated in the deep layers of the cortex and secondarily spread to involve the thalamus and more widespread cortical areas. This activity was dependent on the reciprocal interconnectivity of the thalamus and cortex. Further, cortex-only explants from a genetic model of absence epilepsy (GAERS) exhibited significantly more hyperexcitable activity compared to non-epileptic control explants. This demonstration of how oscillatory thalamocortical network activity develops and evolves in the co-culture model may give insight into how this occurs in vivo in physiological and pathophysiological settings, and in particular the importance of the inter-relationship between the cortex and the thalamus.