One of the major limiting factors in the successful management of type 1 diabetes is Recurrent Hypoglycaemia (RH) resulting from supraphysiological insulin supplementation, which augments the already impaired counterregulatory response to hypoglycaemia. The hypothalamus is essential for the maintenance of whole body glucose homeostasis, including glucose counterregulation (3). Glucosensing (GS) neurons of the hypothalamus use glucose not only as a fuel source but also as a signalling molecule, in order to modulate their electrical activity according to central glucose availability. The molecular mechanisms underlying the function of GS neurons still remain unclear but they are thought to contain the same glucose sensing cellular machinery as pancreatic β-cells, such as ATP-sensitive K+ channels (KATP), glucokinase, glucose transporter proteins and AMPK (5). It is well established that KATP channels regulate the release of insulin from pancreatic β-cells in response to blood glucose levels (1), however they also modulate the electrical activity of GS neurons and are vital for initiation of the counterregulatory response to hypoglycaemia (2,3). The aim of the current study was to determine whether the response to hypoglycaemia of GS neurons is altered following RH, and whether this defect is driven by prior KATP activation. We have recently established that GT1-7 cells, a population of mouse hypothalamic GS neurons, display the characteristic features of a GS system as electrical activity of these cells is directly regulated by glucose metabolism. Patch-clamp electrophysiology was used to examine the electrical activity of GT1-7 cells after exposure to RH (3 hours of 0.1mM glucose for 3 days) or antecedent KATP opener NN414 (3 hours during the previous day) versus appropriate controls. Radiolabeled metabolic assays were used to investigate if any change occurred in glucose oxidation capacity or uptake kinetics. After exposing GT1-7 cells to RH, the magnitude of the ΔVm hyperpolarising response to 0.5mM glucose was reduced by 66.7% compared to control (ΔVRH=4.9 ± 1.0mV vs. ΔVCont=14.7 ± 0.9mV, p<0.0001; n = 7-10). Antecedent NN414 exposure (5μM) also produced a defect in the ability of the cells to hyperpolarise to 0.5mM glucose by 73.1% compared to control (ΔVNN414=3.8 ± 1.7mV vs. ΔVCont=14.2 ± 1.4mV, p<0.01; n=6). Maximum KATP conductance and glucose uptake and oxidation were unaffected by RH and antecedent NN414 treatments. Values are means ± S.E.M., compared by ANOVA. Thus exposure of GT1-7 cells to RH induces defective GS, which was mimicked by antecedent pharmacological activation of KATP channels, independently of any change in glucose uptake or oxidation. This suggests that defective GS, following RH, may be dependent on prior KATP activation.