350µm Coronal Sections of mouse Suprachiasmatic & Paraventricular Nuclei using the 7000smz-2.

Abstract (from: https://doi.org/10.1186/s12915-020-00871-8)

Background: Daily variations in mammalian physiology are under control of a central clock in the suprachiasmatic nucleus (SCN). SCN timing signals are essential for coordinating cellular clocks and associated circadian variations in cell and tissue function across the body; however, direct SCN projections primarily target a restricted set of hypothalamic and thalamic nuclei involved in physiological and behavioural control. The role of the SCN in driving rhythmic activity in these targets remains largely unclear. Here, we address this issue via multielectrode recording and manipulations of SCN output in adult mouse brain slices.

Results: Electrical stimulation identifies cells across the midline hypothalamus and ventral thalamus that receive inhibitory input from the SCN and/or excitatory input from the retina. Optogenetic manipulations confirm that SCN outputs arise from both VIP and, more frequently, non-VIP expressing cells and that both SCN and retinal projections almost exclusively target GABAergic downstream neurons. The majority of midline hypothalamic and ventral thalamic neurons exhibit circadian variation in firing and those receiving inhibitory SCN projections consistently exhibit peak activity during epochs when SCN output is low. Physical removal of the SCN confirms that neuronal rhythms in ~ 20% of the recorded neurons rely on central clock input but also reveals many neurons that can express circadian variation in firing independent of any SCN input.

Conclusions: We identify cell populations across the midline hypothalamus and ventral thalamus exhibiting SCN dependent and independent rhythms in neural activity, providing new insight into the mechanisms by which the circadian system generates daily physiological rhythms.

Slice Preparation

Coronal slices (350 µm thickness) containing the mid-rostrocaudal extent of the SCN and the PVN were prepared using a 7000 smz-2 vibrating microtome (Campden Instrument, UK). For a subset of experiments (n = 9), brain slices were prepared such that a portion (~ 500 µm) of optic nerve was also retained. Slicing was performed in an ice cold cutting solution (4 °C, bubbled with 95% O2/5% CO2) of composition: 189 mM sucrose, 10 mM D-glucose, 26 mM NaHCO3, 3 mM KCl, 5 mM MgSO4, 0.1 mM CaCl2, 1.25 mM NaH2PO4. Slices were subsequently transferred to aCSF (oxygenated as above) for maintenance and recording of composition: 124 mM NaCl, 3 mM KCl, 24 mM NaHCO3, 1.25 mM NaH2PO4, 1 mM MgSO4, 10 mM glucose, 2 mM CaCl2 (2 mM) and supplemented with 0.0001% gentamicin (Sigma-Aldrich, UK). Slice were rested at room temperature for ~ 20 min prior to transfer to recording chamber where they were further equilibrated for 1–2 h prior to the start of electrophysiological data collection.

Keywords: Electrophysiology, Circadian, Paraventricular nucleus, Subparaventricular zone, Channelrhodopsin