Abstracts

Rationale: Intra-thalamic oscillatory activity between the reticular nucleus (nRT) and relay cells (e.g. somatosensory VB) is involved in the generation of sleep spindles. This network can be transformed to generate slower oscillations similar to those seen in absence seizures. Inhibitory output from nRt is known to mediate these slow oscillations by hyperpolarizing relay neurons and allowing them to fire T-type calcium channel-dependent rebound bursts. This in turn causes glutamate release in nRT, which excites relay neurons and results in the release of GABA back onto relay neurons. This rhythmic pattern of activity generates oscillatory activity lasting for several seconds in vitro. We investigated glutamate fluctuations using fluorescence resonance energy transfer (FRET)-based glutamate nanosensor imaging with concurrent thalamic field potential recordings. This approach allowed visualization of the glutamate release from relay neurons into nRT and analysis of the temporal and spatial pattern of activation. Methods: Thalamic brain slices (400 μM) were prepared from rats (P12-P16). Animals were anesthetized and the brains removed and placed in chilled slicing solution. The slices were then incubated in 32° C oxygenated aCSF for 1 hour, and allowed to cool to room temperature. Brain slices were then placed on a plate insert in a tissue culture dish with aCSF, placed in an interface incubation chamber and incubated with 50 μL of concentrated glutamate nanosensor protein for at least 15 minutes. During recording, slices were completely submerged in aCSF and superfused continuously with aCSF + 50 uM picrotoxin and 300 pM apamin. Extracellular field potentials were recorded using glass micropipettes (1 MΩ). A stimulating electrode was placed outside nRT to stimulate descending corticothalamic axons every 30-60 sec. Imaging used single excitation (433 nm) of both glutamate sensor fluorophores (CFP & YFP) and FRET ratiometric analysis was performed.Results: Glutamate nanosensor imaging demonstrated that glutamate is released in nRt with each active phase of the oscillation. The glutamate signal tended to have a large initial peak followed by smaller peaks which were synchronized with the electrically recorded oscillation. Glutamate nanosensor signal was limited to the nRt and spread within the nRt with each active phase of the oscillation and lasted until the electrical oscillation was complete. In some experiments the glutamate showed gradual accumulation over the course or the oscillation while in other experiments the signal returned to baseline in between each active phase of the oscillation.Conclusions: Glutamate fluctuations in nRt are detectable using nanosensor imaging. Glutamate is released first focally near the site of stimulation and then spreads within nRt from this site during the oscillation. This imaging technology will be useful for mechanistic studies of the initiation and termination of epileptiform activity.