NR2B subunit of the NMDA glutamate receptor regulates appetite in the parabrachial nucleus

Diphtheria toxin-mediated, acute ablation of hypothalamic neurons expressing agouti-related protein (AgRP) in adult mice leads to anorexia and starvation within 7 d that is caused by hyperactivity of neurons within the parabrachial nucleus (PBN). Because NMDA glutamate receptors are involved in various synaptic plasticity-based behavioral modifications, we hypothesized that modulation of the NR2A and NR2B subunits of the NMDA receptor in PBN neurons could contribute to the anorexia phenotype. We observed by Western blot analyses that ablation of AgRP neurons results in enhanced expression of NR2B along with a modest suppression of NR2A. Interestingly, systemic administration of LiCl in a critical time window before AgRP neuron ablation abolished the anorectic response. LiCl treatment suppressed NR2B levels in the PBN and ameliorated the local Fos induction that is associated with anorexia. This protective role of LiCl on feeding was blunted in vagotomized mice. Chronic infusion of RO25-6981, a selective NR2B inhibitor, into the PBN recapitulated the role of LiCl in maintaining feeding after AgRP neuron ablation. We suggest that the accumulation of NR2B subunits in the PBN contributes to aphagia in response to AgRP neuron ablation and may be involved in other forms of anorexia.NMDA receptors form glutamate-gated ion channels that mediate many forms of synaptic plasticity under physiological conditions and neuronal death under excitotoxic pathological conditions (1, 2). NMDA receptors are tetrameric complexes composed of two obligatory NR1 subunits and two regulatory NR2 and/or NR3 subunits. Multiple subtypes of NMDA receptors are identified with distinct pharmacological and biophysical properties that are predominantly determined by the type of NR2 subunit (NR2A to NR2D). NR2A and NR2B subunits are the most abundant subunits in the adult brain and differ in channel properties, synaptic localization, and protein interaction elements, all of which can contribute to the induction of synaptic plasticity (3). Some recent studies suggest that NMDA receptors play a major role in regulating feeding behavior and energy homeostasis. For example, antagonism of hindbrain NMDA receptors increases food intake, whereas activation of NMDA receptors in the nucleus tractus solitarius (NTS) is necessary for cholecystokinin-induced reduction of food intake (4, 5). Injection of NMDA receptor agonists into the lateral hypothalamus elicits feeding in satiated animals, whereas NMDA blockade suppresses food intake and can reduce body weight (6). Genetic inactivation of NR1 subunits specifically in agouti-related protein (AgRP) neurons results in marked loss of food intake and body fat and impaired feeding after a fast (7). These findings implicate NMDA receptor signaling within several neuronal circuits in the control of feeding and energy balance.Emerging evidence suggests that a complex neural network including GABAergic AgRP neurons in the hypothalamic arcuate nucleus plays a pivotal role in the control of appetite and energy metabolism (8–10). Activation of AgRP neurons promotes food intake and body weight gain by inhibiting postsynaptic target neurons in the paraventricular hypothalamus (10). Ablation of AgRP neurons by administration of diphtheria toxin (DT) to mice that express the diphtheria toxin receptor (DTR) selectively in AgRP neurons (Agrp^DTR mice) results in aphagia and a fatal loss of body weight caused by the hyperactivity of postsynaptic neurons in the parabrachial nucleus (PBN) as a consequence of losing inhibitory GABAergic signals (11–13). This anorexic response can be prevented by benzodiazepine potentiation of GABA signaling, by genetic blockade of NMDA glutamate receptor signaling in the PBN, or by reducing glutamatergic input or output from the PBN, including knockdown of NMDA expression in the PBN (14). However, the physiological roles of NMDA receptors in the PBN in modulating appetitive behavior have not been identified.We explored the potential benefit of pretreatment with LiCl because this salt has been shown to affect the activity of the PBN and to modulate NMDA receptor functions. Lithium is commonly used to treat bipolar mood disorder, but it also can elicit robust neuroprotection against excitotoxicity in CNS neurons (15, 16). Long-term exposure to LiCl protects cultured cerebellar, cortical, and hippocampal neurons against glutamate-induced excitotoxicity; this protection can be attributed, in part, to the inhibition of NMDA receptor-mediated calcium influx (17). Significant weight gain is associated with prolonged lithium therapy in patients with bipolar disorder; these patients have enhanced preference for a high-calorie diet, but the mechanism(s) causing this metabolic effect are unknown (18). It is well documented that a single i.p. dose of LiCl produces gastrointestinal malaise that is coincident with the induction of Fos in certain brainstem structures, including the NTS and the PBN (19, 20). Exposure of rodents to a novel taste followed by LiCl treatment produces robust conditioned aversion to that taste, and electrolytic lesions of the PBN prevent LiCl-mediated conditioned taste aversion (21). These results are consistent with the idea that LiCl activates vagal afferents to the NTS, which then relays an excitatory, glutamatergic signal to the PBN where it induces Fos in a subpopulation of PBN neurons. We hypothesized that robust glutamatergic signaling induced in the PBN by LiCl treatment might lead to synaptic plasticity involving NMDA receptors and that these changes might influence the anorexia phenotype associated with the sudden loss of GABAergic input to the PBN from AgRP neurons.