Electrophysiology in 300µm mouse DMH Slices in Study Investigating Regulation of AGRP Neurons

Abstract

(from Food cue regulation of AGRP hunger neurons guides learning, Berrios et al, 2021)

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Agouti-related peptide (AGRP)-expressing neurons are activated by fasting—this causes hunger, an aversive state that motivates the seeking and consumption of food5,6. Eating returns AGRP neuron activity towards baseline on three distinct timescales: rapidly and transiently following sensory detection of food cues, slowly and longer-lasting in response to nutrients in the gut, and even more slowly and permanently with restoration of energy balance. The rapid regulation by food cues is of particular interest as its neurobiological basis and purpose are unknown. Given that AGRP neuron activity is aversive, the sensory cue-linked reductions in activity could function to guide behaviour. To evaluate this, we first identified the circuit mediating sensory cue inhibition and then selectively perturbed it to determine function. Here, we show that a lateral hypothalamic glutamatergic → dorsomedial hypothalamic GABAergic (γ-aminobutyric acid-producing) → AGRP neuron circuit mediates this regulation. Interference with this circuit impairs food cue inhibition of AGRP neurons and, notably, greatly impairs learning of a sensory cue-initiated food-acquisition task. This is specific for food, as learning of an identical water-acquisition task is unaffected. We propose that decreases in aversive AGRP neuron activity mediated by this food-specific circuit increases the incentive salience of food cues, and thus facilitates the learning of food-acquisition tasks.

Method

Electrophysiology

Mice were deeply anaesthetized, and intracardially perfused with ice-cold dissection buffer (in mM: 2.5 KCl, 1.25 NaH2PO4, 20 HEPES, 10 MgSO4•7H2O, 0.5 CaCl2•2H2O, 92 choline chloride, 25 glucose, 2 thiourea, 5 sodium ascorbate, 3 sodium pyruvate, and 20 NaHCO3) bubbled with 95% O2, 5% CO2. Brains were then rapidly removed and immersed in ice-cold dissection buffer. DMH sections were dissected and 300 μm thick coronal slices were prepared using a vibrating microtome (Campden 7000smz 2). Slices recovered for 10 min in a 35 °C submersion chamber filled with oxygenated dissection buffer. Slices were then transferred to a secondary 35 °C submersion chamber filled with oxygenated artificial cerebrospinal fluid (ACSF; in mM: 125 NaCl, 2.5 KCl, 25 NaHCO3, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 glucose) and allowed to recover for an additional 15 min. Slices were then kept at room temperature in oxygenated ACSF for ≥ 30 min until use.