doi:10.1016/j.neuroscience.2006.09.059 
Copyright © 2006 IBRO Published by Elsevier Ltd.
Cellular neuroscience
Pharmacological and molecular characterization of ATP-sensitive K+ conductances in CART and NPY/AgRP expressing neurons of the hypothalamic arcuate nucleus
M. van den Topa, 1, D.J. Lyonsa, 1, K. Leeb, E. Coderrec, L.P. Renaudc and D. Spanswicka, 
, 
aDivision of Clinical Sciences, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK
bNeurology and GI CEDD, GSK, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
cNeurosciences, Ottawa Health Research Institute and University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9
Accepted 27 September 2006.
Available online 28 November 2006.
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Abstract
The role of hypothalamic ATP-sensitive potassium channels in the maintenance of energy homeostasis has been extensively explored. However, how these channels are incorporated into the neuronal networks of the arcuate nucleus remains unclear. Whole-cell patch-clamp recordings from rat arcuate nucleus neurons in hypothalamic slice preparations revealed widespread expression of functional ATP-sensitive potassium channels within the nucleus. ATP-sensitive potassium channels were expressed in orexigenic neuropeptide Y/agouti-related protein (NPY/AgRP) and ghrelin-sensitive neurons and in anorexigenic cocaine-and-amphetamine regulated transcript (CART) neurons. In 70% of the arcuate nucleus neurons recorded, exposure to glucose-free bathing medium induced inhibition of electrical excitability, the response being characterized by membrane hyperpolarization, a reduction in neuronal input resistance and a reversal potential consistent with opening of potassium channels. These effects were reversible upon re-introduction of glucose to the bathing medium or upon exposure to the ATP-sensitive potassium channel blockers tolbutamide or glibenclamide. The potassium channel opener diazoxide, but not pinacidil, also induced a tolbutamide and glibenclamide-sensitive inhibition of electrical excitability. Single-cell reverse transcription–polymerase chain reaction revealed expression of mRNA for sulfonylurea receptor 1 but not sulfonylurea receptor 2 subunits of ATP-sensitive potassium channels. Thus, rat arcuate nucleus neurons, including those involved in functionally antagonistic orexigenic and anorexigenic pathways express functional ATP-sensitive potassium channels which include sulfonylurea receptor 1 subunits. These data indicate a crucial role for these ion channels in central sensing of metabolic and energy status. However, further studies are needed to clarify the differential roles of these channels, the organization of signaling pathways that regulate them and how they operate in functionally opposing cell types.
Key words: hypothalamic slice; whole-cell patch-clamp; energy balance; glucose
Abbreviations: ACSF, artificial cerebrospinal fluid; AgRP, agouti-related protein; ARC, hypothalamic arcuate nucleus; CART, cocaine-and-amphetamine-regulated transcript; DMSO, dimethyl sulfoxide; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; Hepes, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid); KATP channels, ATP-sensitive potassium channels; KCO, potassium channel opener; KIR, inward rectifying potassium channel; Na-ATP, ATP disodium salt; NGS, normal goat serum; NPY, neuropeptide Y; POMC, pro-opiomelanocortin; RT-PCR, reverse transcription–polymerase chain reaction; SUR1, sulfonylurea receptor 1; SUR2, sulfonylurea receptor 2; TBS-T, Tris-buffered saline containing 1% Triton X-100; TTX, tetrodotoxin; VMH, ventromedial nucleus of the hypothalamus
Fig. 1. NPY/AgRP neurons express functional KATP channels. (A) Whole-cell current-clamp recording from an ARC NPY/AgRP showing glucose-free-induced hyperpolarization and reduction in neuronal input resistance indicated by the decrease in amplitude of electrotonic potentials (downward deflections indicate electrotonic potentials evoked in response to current pulses: 0.2 Hz, 0.5 s, −30 pA). Effects of glucose-free were partly reversed by orexin and subsequently by tolbutamide. (B) Current voltage (IV) relationships of an NPY/AgRP neuron in the presence (top) and absence (bottom) of extracellular glucose. (C) Plot of data shown in B indicating a reversal potential close to that for potassium ions (point of intersection).
Fig. 2. Ghrelin sensitive neurons express functional KATP channels. (A) Samples of a continuous record showing glucose-free-induced inhibition and subsequent depolarization and associated increased membrane resistance induced by ghrelin. This neuron was also sensitive to tolbutamide. (Bi) Same neuron as A showing effects of glucose-free, ghrelin and tolbutamide on I–V relations. (Bii) Plots of data shown in Bi. Note, reversal potentials for glucose-free, ghrelin and tolbutamide close to the reversal potential for potassium.
Fig. 3. ARC CART neurons express functional KATP channels. (A) Continuous whole-cell recording showing glucose-free-induced inhibition and subsequent reversal by tolbutamide in a CART-expressing neuron revealed retrospectively by labeling with Alexa 595 (red) from recording pipette and immunocytochemically with a CY2-labeled antibody for CART (green) and shown in B. (Ci) Same neuron showing effects of glucose-free on I–V relations in this CART neuron. (Cii) Plots of data shown in Ci. Note the reversal potential for glucose-free close to potassium.
Fig. 4. Glucose, ATP and tolbutamide sensitivity of ARC neurons. (A) Glucose-free bathing medium induced inhibition with 2 mM Na-ATP in the recording pipette, an effect reversed by tolbutamide (200 μM). (B) With 2 mM ATP in pipette solution, tolbutamide or reintroduction of glucose (10 mM) reversed the effects of glucose-free. (C) With no ATP in the pipette solution glucose-free induced hyperpolarization, an effect reversed by tolbutamide. (D) Plot of time taken to reach peak steady-state inhibition following recording with no intracellular ATP, 0 mM ATP and glucose-free and 2 mM intracellular ATP and glucose glucose-free. The * indicates the significant reduction in the rundown time when ATP is omitted from the recording solution (P<0.01). (E) Current–voltage (IV) plot of the glucose-free-induced currents as observed in the presence (red) and absence (black) of intracellular ATP.
Fig. 5. Concentration-dependent sensitivity of ARC neurons to tolbutamide. (A) Continuous current clamp recording in glucose-free and TTX showing the concentration-dependence of tolbutamide-induced depolarizations in this neuron. (B) Concentration-response curve for tolbutamide. Data are from three neurons maintained between −60 and −65 mV in glucose-free. (Ci) Effects of tolbutamide on IV relations in TTX and glucose-free. (Cii) Plot of data shown in Ci measured at point indicated by solid circle in Ci. (D) Averaged current responses (n=3) obtained in response to voltage-clamp ramps −126 to −46 mV, 10 mV/s in the presence (red trace) and absence (black trace) of tolbutamide under glucose-free conditions. Point of intersection indicates the reversal potential of the tolbutamide-induced current.
Fig. 6. Glibenclamide sensitivity of ARC KATP conductances. (A) Continuous current clamp recording showing glucose-free-induced hyperpolarization was reversed by glibenclamide. (Bi) IV relationships of a neuron hyperpolarized in the absence of extracellular glucose obtained under control and glucose-free conditions in the presence and absence of glibenclamide. (Bii) Plot of data shown in Bi. (C) Averaged current responses (n=3) obtained in voltage-clamp in the presence (red trace) and absence (black trace) of glibenclamide in glucose free. Point of intersection indicates reversal potential, approaching potassium under our recording conditions.
Fig. 7. Arc neurons express SUR1. (A) Continuous current clamp record showing an ARC neuron insensitive to pinacidil (600 μM) was subsequently inhibited by diazoxide (500 μM), an effect reversed by tolbutamide (200 μM). (B) Pooled data showing effects of pinacidil (600 μM; left) and diazoxide (500 μM; right). * Indicates a significant difference at P<0.001. (Ci) Samples of a continuous record showing effects of diazoxide and tolbutamide on IV relations. (Cii) Plot of data shown in Ci diazoxide-induced response and reversal by tolbutamide. Note the reversal potential around −80 mV. (D) Averaged current responses (n=4) obtained in voltage-clamp under control (black trace) and in the presence of diazoxide and diazoxide/tolbutamide (red and green traces, respectively). (E) Single-cell RT-PCR revealed expression of SUR1 but not SUR2.
Table 1.
Properties of ARC neurons recorded in the presence or absence of 2 mM [ATP]i
The group denoted “all neurons” reflects the pooled data from all neurons exposed to glucose-free environment included in The Effects of Intracellular ATP on KATP = Mediated Inhibitions section. The number of neurons included per group is indicated above the relevant columns in parentheses. Statistical significance was determined using the Student’s two-tailed t-tests for independent populations.
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