Whole-cell Current Clamp on Mouse ARC POMC Neurons; Transverse 200µm Slices prepared with 7000smz-2

Abstract

(from: https://doi.org/10.3390/plants10050837)

Background: Obesity due to an excessive intake of nutrient disturbs the hypothalamus mediated energy metabolism subsequently develops metabolic disorders. In this study, we investigated the effect of pine needle extract (PNE) on the hypothalamic proopiomelanocortin (POMC) neurons involved in the regulation of energy balance via melanocortin system and fat tissue metabolism.

Methods: We performed electrophysiological and immunohistochemical analyses to determine the effect of PNE on POMC neurons. Mice were fed a normal or high-fat diet for 12 weeks, then received PNE for the last 2 weeks to measure the following physiological indices: Body weight, food intake, fat/lean mass, glucose metabolism, and plasma leptin levels. In addition, changes of thermogenic, lipolytic, and lipogenetic markers were evaluated in brown adipose tissue (BAT) and white adipose tissue (WAT) by western blotting, respectively. Results: PNE increased hypothalamic POMC neuronal activity, and the effect was abolished by blockade of melanocortin 3/4 receptors (MC3/4Rs). PNE decreased body weight, fat mass, plasma leptin levels, and improved glucose metabolism after high-fat-induced obesity. However, PNE did not change the expression of thermogenic markers of the BAT in HFD fed groups, but decreased only the lipogenetic markers of WAT. This study suggests that PNE has a potent anti-obesity effect, inhibiting lipogenesis in WAT, even though HFD-induced leptin resistance-mediated disruption of POMC neuronal activity.

Method

Transverse sectioned brain slices (200 μm thickness) were prepared by vibratome (7000smz-2; Campden Instruments, Loughborough, UK). The pipette solution contained 130 mM of K-gluconate, 5 mM of CaCl2, 10 mM of EGTA, 10 mM of HEPES, 2 mM of MgATP, 0.5 mM of Na2GTP, and 10 mM of phosphocreatine. To record membrane potentials, brain slices were placed in a recording chamber and superfused with artificial cerebrospinal fluid (containing: 113 mM of NaCl, 3 mM of KCl, 1 mM of NaH2PO4, 26 mM of NaHCO3, 2.5 mM of CaCl2, 1 mM of MgCl2, and 5 mM of glucose in 95% O2/5% CO2) at 1.5–2 mL/min. The recording chambers were placed on the stage of an upright and infrared differential interference contrast microscope (Olympus BX51WI; Olympus, San Jose, CA, USA), which was mounted on a Gibraltar X-Y table. The prepared brain slices were visualized by infrared microscopy with a 40X water immersion objective. Whole-cell current-clamp recordings were obtained from visualized ARC POMC neurons of the POMC-eGFP mice brain slices at a holding potential of −70 mV. Electrophysiological signals were low-pass filtered at 2–5 kHz, saved on a desktop PC and analyzed offline with pClamp 11 software (Molecular Devices, San Jose, CA, USA). For each recording, membrane potentials measured every 30 s were considered as single data points. We compared a total of 10 data points before and after the application of PNE were compared using paired t-tests. Membrane potentials were recorded using a multi-clamp 700 B (Molecular Devices, San Jose, CA, USA) in the whole-cell configuration. All recordings were conducted at 30 ± 2 °C.