Studies of Thalamocortical Epilepsy and Neurodevelopmental Disorders

John Huguenard, PhD

We are interested in the neuronal mechanisms that underlie synchronous oscillatory activity in the thalamus, cortex and the massively interconnected thalamocortical system. Such oscillations are related to cognitive processes, normal sleep activities and certain forms of epilepsy.  We are also interested in comorbidities in epilepsy, such as Autism  Spectrum Disorders, and whether the circuit abnormalities in ASD may overlap with those of epilepsy and explain in part why the incidence of epilepsy is so much higher in patients with ASD.

Our approach is an analysis of the discrete components that make up thalamic and cortical circuits, and reconstitution of components into both in vitro biological and in silico computational networks. Accordingly, we have been able to identify genes whose products, mainly ion channels, play key roles in the regulation of thalamocortical network responses.  Using this knowledge we have recently designed targeted optogenetic approaches to detect seizures at their onset, and then in real time disrupt them by instantly modifying the activity of key elements in the epileptic circuit.

Currently, projects focus on:

• Molecular pharmacology of inhibitory GABA_A receptors in the thalamus, and the role of endogenous benzodiazepine receptor ligands (endozepines) in regulating thalamic network activity and suppressing seizures.
• Analysis of progression and destabilization of widespread thalamic network activity using large microelectrode arrays
• Multiphoton imaging of neural activity in vitro and in vivo using Ca and voltage indicators.
• Real time interruption of seizures
• Regulation of activity in the thalamic reticular nucleus (TRN), a key site in the thalamocortical epileptic network, in which over-excitation appears to play an essential role in absence seizure generation.  The output fibers of TRN neurons are particularly susceptible to transmission failure, and we are studying the mechanisms underlying such failure, in the hope that we can prevent it.

The laboratory uses experimental techniques ranging from biophysical studies of single ion channels to in vivo recording to purely theoretical studies of network synchrony. Our toolbox includes:

• Use of mutant mouse models for analysis of gene function in circuit behavior. For example, knockout and knockin mice have been used to identify the specific GABA_A receptor isoforms that are critical for the therapeutic actions of benzodiazepines in thalamus.
• Patch clamp recording methods for single channels and whole cell currents, with both isolated neurons and those in situ in brain slices
• Multi-unit, multi-site extracellular recording techniques
• 2 photon imaging of neural activity in single neurons and in populations.
• Immunohistochemical techniques for cell identification and protein localization
• Molecular & genetic approaches for in situ hybridization of specific transcripts
• Microscopic techniques for computerized neuronal reconstruction (Neurolucida)
• Laser uncaging of photo-labile glutamate derivatives for circuit analysis
• Imaging of network activity via FRET glutamate nanosensors
• Paired intracellular recordings for analysis of single-axon synaptic connection
• Fluorometric detection of calcium indicator dyes in cells and circuits
• Local perfusion within slice micro-regions for pharmacological analysis
• Computer-based modeling of single cell and circuit behaviors