Glucose sensing by neurons isolated from the hypothalamus

Neurons within the hypothalamus sense nutrient and peptide levels to modulate feeding behavior. Neurons were isolated from the hypothalamus of newborn (day 4) rat brains and were loaded with the Ca²⁺ fluoroprobe Fluo-3 AM. At zero glucose, many neurons demonstrate Ca²⁺ oscillations, but these oscillations are blocked by addition of 0.5mM glucose. Immunocytochemical analysis demonstrated the presence of glucokinase and the glut-2 glucose transporter, required for glucose sensing by pancreatic beta cells, in a small number (∼1%) of isolated neurons. Consistent with this observation, one to five percent of neurons in a culture exhibit either elevations, depressions or oscillations in cellular Ca²⁺ concentration in response to increasing glucose from 0.5 to 20mM. Neurons that elevated Ca²⁺ in response to glucose show repetitive firing of action potentials with super-threshold depolarizing current injection from a -65 mV baseline potential. Hyperpolarizing currents evoked negative voltage responses with a time-dependent voltage sag attributed to an inward cationic conductance. Elevation of glucose elicited a slow developing membrane depolarization, and loss of the rectifying current in these neurons. Current research is assessing the effect of glucose on excitable membranes and its dose response characteristics. These results indicate the feasibility of isolating and identifying nutrient sensitive neurons from the hypothalamus providing a valuable model for studying the mechanisms underlying hypothalamic regulation of feeding behavior.