Visually guided whole cell patch clamp of mouse supraoptic nucleus neurons in cultured and acute conditions

Abstract

Recent advances in neuronal culturing techniques have supplied a new set of tools for studying neural tissue, providing effective means to study molecular aspects of regulatory elements in the supraoptic nucleus of the hypothalamus (SON). To combine molecular biology techniques with electrophysiological recording, we modified an organotypic culture protocol to permit transfection and whole cell patch-clamp recordings from SON cells. Neonatal mouse brain coronal sections containing the SON were dissected out, placed on a filter insert in culture medium, and incubated for at least 4 days to allow attachment to the insert. The SON was identifiable using gross anatomical landmarks, which remained intact throughout the culturing period. Immunohistochemical staining identified both vasopressinergic and oxytocinergic cells present in the cultures, typically appearing in well-defined clusters. Whole cell recordings from these cultures demonstrated that certain properties of the neonatal mouse SON were comparable to adult mouse magnocellular neurons. SON neurons in both neonatal cultures and acute adult slices showed similar sustained outward rectification above -60 mV and action potential broadening during evoked activity. Membrane potential, input resistance, and rapidly inactivating potassium current density (IA) were reduced in the cultures, whereas whole cell capacitance and spontaneous synaptic excitation were increased, perhaps reflecting developmental changes in cell physiology that warrant further study. The use of the outlined organotypic culturing procedures will allow the study of such electrophysiological properties of mouse SON using whole cell patch-clamp, in addition to various molecular, techniques that require longer incubation times.

Characterization of Cortical Neuronal and Glial Alterations during Culture of Organotypic Whole Brain Slices from Neonatal and Mature Mice

“…It is proposed that as neonatal organotypic hippocampal cultures display increased synaptogenesis over time, then increased periods of culture would result in physiological conditions similar to that seen in vivo , and different to that observed in acute brain slice cultures [11]. However, no significant difference was noted in the electrophysiological properties of hippocampal organotypic cultures and acute brain slices [9], [36].…”

TRPV1 Gene Deficiency Attenuates Miniature EPSC Potentiation Induced by Mannitol and Angiotensin II in Supraoptic Magnocellular Neurons

“…The pH was adjusted to 7.3, and the osmolality of all solutions ranged between 298 and 303 mOsmol/kg. The perfusion medium for the mice slice preparation was modified by increasing osmolality to 312 mOsmol/kg, according to the natural basal serum osmolarity observed in this strain of mice (Stachniak and Bourque, 2006). For hyperosmotic stimulation, mannitol was added to the perfusion medium.…”

Performance, Properties and Plasticity of Identified Oxytocin and Vasopressin Neurones In Vitro

“…As the title of our review suggests, these studies have been aimed at the VP and OT neuronal firing patterns (performance), their underlying properties (focusing on spike afterpotentials) and, for OT neurones, the plasticity associated with reproductive state. Although this review will focus on studies largely from rat, in vitro studies of magnocellular neurosecretory neurones studies have been performed in guinea pigs (19–21), mice (22, 23) and cats (24).…”