Rapid, reversible activation of AgRP neurons drives feeding behavior in mice

Abstract

Several different neuronal populations are involved in regulating energy homeostasis. Among these, agouti-related protein (AgRP) neurons are thought to promote feeding and weight gain; however, the evidence supporting this view is incomplete. Using designer receptors exclusively activated by designer drugs (DREADD) technology to provide specific and reversible regulation of neuronal activity in mice, we have demonstrated that acute activation of AgRP neurons rapidly and dramatically induces feeding, reduces energy expenditure, and ultimately increases fat stores. All these effects returned to baseline after stimulation was withdrawn. In contrast, inhibiting AgRP neuronal activity in hungry mice reduced food intake. Together, these findings demonstrate that AgRP neuron activity is both necessary and sufficient for feeding. Of interest, activating AgRP neurons potently increased motivation for feeding and also drove intense food-seeking behavior, demonstrating that AgRP neurons engage brain sites controlling multiple levels of feeding behavior. Due to its ease of use and suitability for both acute and chronic regulation, DREADD technology is ideally suited for investigating the neural circuits hypothesized to regulate energy balance.

Figure 1. Cre-dependent AAV-hM3Dq-mCherry is specifically expressed in the ARC of AgRP-Ires-cre mice and confers activation by CNO.

(A) Design of hM3Dq-mCherry AAV employing the FLEX Switch strategy, which uses 2 pairs of heterotypic, antiparallel loxP-type recombination sites to achieve Cre-mediated transgene inversion and expression (12). L-ITR, left-inverted terminal repeat; R-ITR, right-inverted terminal repeat; WPRE, woodchuck hepatitis posttranscriptional regulatory element. (B) Top: Schematic indicating the site of the imaged area in the ARC of the hypothalamus. Bottom: mCherry fluorescence exclusively in the ARC after bilateral injections of AAV-hM3Dq-mCherry into the hypothalamus of AgRP-Ires-cre mice crossed with Z/EG Cre-dependent reporter mice. Z/EG mice expressed GFP protein following Cre-mediated excision of an intervening sequence (Scale bar: 100 μm). (C) Colocalization of mCherry (anti-dsRed) and anti-GFP fluorescence in the ARC. Note that GFP is cytoplasmic and DREADD is expressed on the plasma membrane (scale bar: 10 μm). (D) Whole cell, current clamp recording from an AgRP neuron marked by mCherry fluorescence from an AgRP-Ires-cre mouse injected with AAV-hM3Dq-mCherry. CNO (5 μM) elicited rapid depolarization of the membrane potential and greatly increased the firing rate. This example trace is representative of 4 similar recordings. (E) Injection of CNO in vivo induces c-fos immunoreactivity in the ARC. Brains were obtained for c-fos analysis 90 minutes following injection of saline or CNO (0.3 mg/kg of body weight, i.p.) (scale bars: 120 μm).

Figure 2. Manipulation of AgRP neuron activity alters energy balance.

(A) Food intake. CNO (0.3 mg/kg of body weight, i.p.) or saline was injected 3 hours after the start of the 12-hour light cycle, and food intake was assessed between 30 minutes and 4 hours after injection (PI). Data are from male mice (mean ± SEM, n = 12; *P < 0.01). AgRP-i-Cre, AgRP-Ires-cre mice. (B) Oxygen consumption. Mice were acclimated in metabolic cages and injected with either saline (blunted arrow) or CNO (arrow) at 8:30 am. Black bars along the x axis indicate the 12-hour dark cycle. Data are from male mice (mean ± SEM, n = 6; *P < 0.01). (C–E) Chronic stimulation of AgRP neurons. (C) Body weight, (D) fat mass, and (E) food intake. AgRP-Ires-cre and wild-type control mice were injected twice daily (at 9:00 am and 5:00 pm) with saline from days 1–5, CNO (0.3 mg/kg of body weight, i.p.) from days 6–10 (arrow), and saline from days 11–15 (blunted arrow). Data are from female mice (mean ± SEM, n = 12; *P < 0.01). (F and G) Inhibitory DREADD (hM4Di). (F) Whole cell, current clamp recording from an AgRP neuron marked by mCherry fluorescence from a AgRP-Ires-cre mouse injected with AAV-hM4Di-mCherry. CNO (10 μM) hyperpolarized the membrane potential and decreased the firing rate. This example trace is representative of 5 similar recordings. (G) Inhibition of AgRP neurons decreases food intake. CNO (0.3 mg/kg of body weight, i.p.) or saline was injected at the start of the 12-hour dark cycle, and food intake was assessed between 30 minutes and 4 hours PI. Data are from male mice (mean ± SEM, n = 6; *P < 0.05).

Figure 3. Stimulating AgRP neurons drives a behavioral program to work for and search for food.

(A) Ad lib–fed animals injected with saline were first trained to associate a successful nose poke with a reward pellet using an FR1 schedule. Following the training period, the same cohort was injected with CNO (0.3 mg/kg of body weight, i.p.) or saline and then tested on a PR3. Acute stimulation of AgRP neurons in mice fed ad lib led to a significant increase in the break point, similar to the break point observed in fasted mice. Data shown are from male mice (mean ± SEM, n = 6, *P < 0.01). (B) Stimulation of AgRP neurons increases physical activity when food is absent but not when food is present. In the food-absent study, food was removed immediately following CNO or saline injection. The number of ambulatory episodes along the horizontal plane was assessed 0–1 hour, 1–2 hours, 2–3 hours, 3–4 hours, and 4–5 hours PI. The same cohort of mice was used in the food – and food + studies. Data shown are from male mice (mean ± SEM, n = 4, *P < 0.01).