Organotypic slice culture of the hypothalamic paraventricular nucleus of rat

Abstract

Organotypic slice cultures have been developed as an alternative to acute brain slices because the neuronal viability and synaptic connectivity in these cultures can be preserved well for a prolonged period of time. This study evaluated a stationary organotypic slice culture developed for the hypothalamic paraventricular nucleus (PVN) of rat. The results showed that the slice cultures maintain the typical shape of the nucleus, the immunocytochemical signals for oxytocin, vasopressin, and corticotropin-releasing hormone, and the electrophysiological properties of PVN neurons for up to 3 weeks in vitro. The PVN neurons in the culture expressed the green fluorescent protein gene that had been delivered by the adenoviral vectors. The results indicate that the cultured slices preserve the properties of the PVN neurons, and can be used in longterm studies on these neurons in vitro.

Fig. 1

A schematic diagram of a stationary organotypic slice culture. After decapitating young rats at postnatal 7-10 days, the isolated brain was dissected to 200 µm using a Vibratome and placed on a filter insert under sterile conditions. Filter inserts were placed in 6-well plates containing the culture medium, and the cultures were grown in a CO₂ incubator.

Fig. 2

Low magnification (× 40) views of a rat hypothalamus slice-explant at 0 (A), 4 (B), 10 (C), and 14 DIV (D). Note that the slice became more translucent on later days, indicating that slice thickness decreases with the length of incubation. DIV, day in vitro.

Fig. 3

The paraventricular nucleus and the electrophysiological recording from PVN neurons in the slice culture. (A) and (B), Cresyl violet staining of the cultured hypothalamic slices containing the PVN after 11 DIV (× 40). The area of the nucleus marked by a square in (A) is presented at higher magnification in (B) (× 100). (C), PVN neurons in a slice culture at 17 DIV. Scale bar = 10 µm. (D), Voltage responses to a depolarizing current pulse (60 pA, 250 ms) with a hyperpolarizing prepulse (-90 pA, 250 ms) from the neuron marked by the arrow in (C).

Fig. 4

Immunocytochemistry of oxytocin, vasopressin, and CRH in the PVN of the cultured slices. The PVN neurons were stained with antibodies against oxytocin (A), vasopressin (B), and CRH (C and D). D shows the CRH-immunoreactive cells in the area marked in C. Note that oxytocin and vasopressin neurons are distributed in the magnocellular regions, but the CRH neurons are distributed in both the magnocellular and parvocellular regions. 3V; third ventricle. bar = 100 µm.

Fig. 5

Expression of EGFP in the neurons in the cultured slices. The EGFP gene was delivered by the adenoviral vectors. Low magnification (× 40) views at the bright-field (A) and under the fluorescent filter for EGFP (B). High magnification (× 400) views of the neurons in the left (C) and right PVN (D) showing the detailed anatomy of the neurons, including the cell bodies, processes, and possible contacts between neurons. The 2 horizontal lines are parts of the stabilizing net in the chamber.