Osmoregulatory Circuits in Slices and En Bloc Preparations of Rodent Hypothalamus

Synaptic control of rat magnocellular neurosecretory cells by warm‐sensing neurons in the organum vasculosum lamina terminalis

“…If thermosensitive SON‐projecting OVLT neurons make synaptic contacts with MNCs in the SON, then increases in temperature should cause a rise in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) or spontaneous postsynaptic inhibitory currents (sIPSCs) recorded in MNCs. To test this hypothesis, we performed whole cell patch clamp recordings from SON MNCs in angled horizontal hypothalamic slices that preserve OVLT‐SON circuitry 34,36 . MNCs voltage clamped at −60 mV displayed readily detectable inward and outward synaptic events respectively identified as glutamatergic sEPSCs and GABAergic sIPSCs (data not shown).…”

“…Hypothalamic explants prepared as described previously 34 were superfused at 1.2 ml/min with carbogenated (95% oxygen, 5% CO 2 ) artificial cerebrospinal fluid (ACSF; pH 7.2–7.3, adjusted to 295 ± 2 mosmol/kg with mannitol) comprising (in mM) NaCl (104), NaHCO 3 , 26 D‐glucose, 10 KCl, 3 NaH 2 PO 4 (1.25), MgCl 2 (1.25), and CaCl 2 2 . For extracellular recording glass microelectrodes (10–20 MΩ) filled with 1 M NaCl were connected to an Axoclamp‐2A amplifier and voltage signals (0.8–2 KHz) were digitized at 10 KHz through a Digidata 1440 (both from Molecular Devices LLC) with a PC running Clampex 10.2 software (Molecular Devices).…”

“…A single angled horizontal slice (400 μm thick) was prepared from each animal as described previously 34 . Briefly, a trimmed block of brain was glued cortex down with the rostral pole facing upwards to a mounting block angled ~40° relative to the horizontal plane.…”

“…Hypothalamic explants prepared as described previously 34 the optic tract at the level of the middle cerebral artery, and recordings were obtained within 200 μm from the surface, which is an area highly enriched in VP neurons. For OVLT recordings glass microelectrodes were positioned within the OVLT inside a region extending no more than 100 μm laterally from midline and $400 μm rostrally from the tip of the preoptic recess, which corresponds to the part of the OVLT that lacks a blood-brain barrier.…”

“…A single angled horizontal slice (400 μm thick) was prepared from each animal as described previously. 34 Briefly, a trimmed block of brain was glued cortex down with the rostral pole facing upwards to a mounting block angled $40 relative to the horizontal plane. The assembly was then placed in a vibratome and a first cut was made to discard the tissue lying anterior and ventral to the optic tracts and most of the optic chiasma.…”