Sake yeast compounds modulates thalamocortical rhythmic burst activity in C57BL/6J mice via adenosine A2A receptors

Introduction: Slow rhythmic oscillatory activity in the thalamocortical network which is seen as delta waves in the EEG during periods of deep sleep has a number of physiological functions. Oscillatory activity in the thalamus is based on the cyclic interaction between different thalamic cell types, including thalamocortical relay neurons and reticular thalamic neurons, leading to rhythmic activation of T-type Ca2+ channels and burst firing in both cell types. Modern lifestyle is frequently associated with insufficient sleep which is correlated with increased risks for obesity, hypertension and cerebral infarction. On the search for non-pharmacological compounds increasing the quality and duration of sleep, it was recently found that Japanese sake yeast supplementation selectively increased delta sleep which was accompanied by the subjective improvement of sleep quality. However the neuronal mechanisms underlying this effect are unknown. Therefore in this study we investigated the effects of different types of sake yeast suspensions on dampened rhythmic bursting in horizontal thalamic slices of C57BL/6J mice. Methods: In this study we used 8 to 10 weeks old C57BL/6J mice. Animals were sacrificed under isoflurane anesthesia and brains were rapidly removed from the skull and placed in an ice-cold slicing solution. Horizontal thalamic slices (400 μm thickness) were obtained using a microtome (Leica VT 1200s, Leica, Wentzler, Germany) and incubated in an oxygenated physiological solution (32°C) for at least 1 hour before recording. Brain slices were transferred to an interface chamber and recordings were performed at 32±1°C. Intrathalamic rhythmic burst activity was induced through stimulation of the internal capsule (IC) using a pair of tungsten electrodes (with 50–100 MΩ resistance) and network activity (in form of local field potentials, LFPs) were measured and evaluated in the VB complex using a glass electrode (GC150T-10; Clark Electromedical Instruments, Pangbourne, UK) with a resistance of 0.5–2 MΩ. Burst firing was defined by at least 3 high-frequency spikes with an intra-burst frequency interval of > 100 Hz and an inter-burst interval of not less than 500 ms when multiple bursts were triggered. The activity was measured 50-100 ms after stimulation of the IC and analyzed up to 2-3 s following stimulus onset. In this study we perfused brain slices with suspensions of sake yeast powder (GSP6 powder, Lion Corporation, Japan; WLP705 liquid, White Labs, Belgium) or supernatant of yeast cultures to test their effects on rhythmic burst activity. The IC was stimulated by a single pulse once per minute. Baseline was recorded for 20 minutes followed by drug perfusion. Response to the drug was noticed within 20-30 min after starting perfusion. Total duration of recording per slice was 1hr. Results: Preliminary results showed a significant and dose-dependent (suspensions of sake yeast powder with 40 and 80 µg mL–1) increase in the number of bursts, the number of spikes per burst and the duration of bursting in comparison to baseline in response to a single stimulus. Very similar effects were induced with liquid sake yeast (100 μL mL–1) and the supernatant of sake yeast cultures (100 μL mL–1). Notably the latter exerted the strongest effect on all analyzed parameters. Our findings indicated that sake yeast increased and prolonged slow rhythmic bursting in thalamic slices via adenosine A2A receptors thereby pointing to a possible neuronal network correlate of increased delta sleep following food supplementation in clinical trials. In addition the active compounds seemed to be released from cultured sake yeast cells and were effective following separation of the cell culture supernatant.Conclusion: The present study for the first time points to the neuronal mechanisms involved in increased delta activity following sake yeast supplementation in that components of the thalamic burst firing machinery are positively modulated resulting in increased bursting. Future studies have to identify the active compounds and their signaling pathways.

Keywords:

Sake yeast , thalamocortical activity , C57BL/6J mice , local field potential recordings , brain slices , burst activity