Our results establish the importance of GABA signaling by AgRP neurons onto post-synaptic GABAA
receptors for maintenance of feeding behavior. Two lines of evidence support this conclusion: starvation resulting from AgRP neuron ablation can be prevented by chronic delivery of a GABAA
receptor agonist, and viral-mediated inactivation of GABA biosynthesis in the ARC inhibits feeding. Furthermore, ablation of AgRP neurons in mice lacking AgRP and NPY, the other known neuromodulators made by these neurons, results in starvation; hence, loss of these neuropeptides is not responsible for the starvation phenotype (Phillips and Palmiter, 2008
We suggest that loss of GABA signaling from AgRP neurons onto post-synaptic GABAA
receptors results in unopposed excitation of post-synaptic cells as revealed by Fos
and astrocyte activation. Our observation that inactivation of GABA synthesis in the ARC leads to anorexia suggests that direct GABA signaling by AgRP neurons to critical brain regions is important to maintain feeding after AgRP neuron ablation; however, we cannot rule out the contribution by other neurons in the ARC that produce GABA such as the POMC neurons (Hentges et al., 2004
). The anorexia observed with viral inactivation of GABA synthesis was more severe than that observed by Tong et al (2008)
but still not as great as that observed with AgRP neuron ablation, presumably because only a fraction of the AgRP neurons was transduced by the virus.
We assume that chronic delivery of bretazenil during AgRP neuron ablation provides sufficient inhibitory tone to suppress the excitability of post-synaptic cells in critical feeding circuits, but suppression of cytokine production by astrocytes may also be involved. We predicted that those brain regions where Fos mRNA was lowered the most would reveal the most critical brain regions. We have identified two brain targets of AgRP neurons (PBN and LS) that appear to be particularly important. The most dramatic reduction of Fos expression was in PBN and that is the only brain region where direct delivery of bretazenil was able to prevent starvation, although delivery of bretazenil to the LS was partially effective. Furthermore, injection of DT into the PBN (but not the LS) produced anorexia indicating that signaling by AgRP neurons to this brain region is particularly important. Nevertheless, experiments involving chronic delivery of bretazenil to the 3rd or 4th ventricle suggest that excessive neuronal activity in both forebrain and hindbrain may contribute to the eating disorder. The pharmacology of the agonists and antagonists used in this study indicate that GABA signaling from AgRP neurons onto GABAA receptors is sufficient to prevent starvation, but activation of GABAB receptors may also be involved. The close apposition of axonal fibers from AgRP neurons with Fos-positive neurons in the lateral PBN suggests that there may be direct GABA signaling onto the critical PBN neurons.
The PBN is a major relay for gustatory and visceral information and mediates anorexia associated with malaise induced by intraperitoneal injection of LiCl or lipopolysaccharide – treatments that are used to mimic the effects of toxic or rancid foods and bacterial infections, respectively (Rinaman and Dzmura, 2007
). Lesions of PBN prevent the acquisition of conditioned taste aversion (Trifunovic and Reilly, 2002
) and conditioned taste preferences (Reilly and Trifunovic, 2000
). The PBN is the most responsive brain region where benzodiazepine injection promotes taste reactivity (Berridge and Pecina, 1995
). Direct injections of cannabinoid or mu-opioid receptor agonists into the PBN stimulate feeding (Chaijale et al., 2008
;DiPatrizio and Simansky, 2008
;Wilson et al., 2003
). Thus, the PBN can process both aversive and appetitive ascending visceral information to modulate feeding behavior. LiCl-induced anorexia induces robust Fos expression in the PBN and other brain regions (Andre et al., 2007
;Lamprecht and Dudai, 1995
;Swank and Bernstein, 1994
). The Fos-positive cells in the PBN after LiCl induction are in the same region of the PBN as those induced after AgRP neuron ablation. Thus, it is likely that loss of GABAergic input to the PBN after AgRP neuron ablation activates circuits that normally promote nausea-induced anorexia. Our findings that bretazenil infusion into the PBN prevents anorexia induced by AgRP neuron ablation, while infusion of bicuculline into the PBN of normal mice promotes anorexia are consistent with this hypothesis. The role played by the LS in feeding is more obscure. Because the LS relays signals related to sensory input and reward and projects to the lateral hypothalamus with a well-established role in feeding behavior (Bernardis and Bellinger, 1996
), aberrant activity within the LS may produce anorexia via its projections to the lateral hypothalamus. Pharmacological, electrophysiological, and lesion experiments suggest a role of the LS in modulation of feeding (Scopinho et al., 2008
) but the precise role of the GABAergic inputs from AgRP neurons to this brain region require further investigation.
In addition to the consensus view that AgRP neurons oppose melanocortin signaling by POMC neurons in pathways that regulate appetite and metabolism (Cone, 2005
;Morton et al., 2006
;Saper et al., 2002
), we propose that there are some brain regions such as the PBN where AgRP neurons have largely melanocortin-independent effects. In our view (), ablation (or inhibition) of AgRP neurons leads to activation of POMC neurons and the melanocortin-signaling pathway, as well as a pathway involving the PBN, both of which inhibit feeding. Activation of AgRP neurons has the opposite effect. The profound anorexia that ensues from activation of the PBN – perhaps mimicking severe gastrointestinal malaise or aversive gustatory input - could mask the role of AgRP neurons in regulating appetite and metabolism by counteracting melanocortin signaling. The transient ~10% loss of body weight that still occurs when bretazenil is infused may reflect the melancortin-dependent role of AgRP neurons on feeding and metabolism. If so, this effect is gradually compensated because the mice eventually regain their body weight after ~ 10 days, which may account for the relatively mild effect of chronic loss of GABA signaling by AgRP neurons (Tong et al., 2008
Diagram illustrating how loss of GABAergic signaling from AgRP neurons leads to starvation
It is noteworthy that abrupt withdrawal of bretazenil during 4th
ventricle delivery results in a more precipitous decline of body weight than that achieved by ablation of AgRP neurons alone. This result mimics the loss of body weight and appetite, a common symptom of the “benzodiazepine withdrawal syndrome” seen in patients who discontinue chronic use of benzodiazepines (Pecknold, 1993
). In parallel with a clinical study showing that flumazenil treatment alleviated the withdrawal syndrome (Gerra et al., 2002
), our results demonstrate that bretazenil-mediated restoration of food intake was completely abolished by the 4th
ventricle administration of flumazenil. On the other hand, after cessation of chronic infusion of bicuculline into the PBN of wide-type mice, their anorexia switched to prolonged hyperphagia that was inversely proportional to the dosage of bicuculline. These complementary results illustrate robust dynamics and sensitivity of a hindbrain GABA signaling network that governs appetite and food palatability.
An unexpected finding is that feeding persists in AgRP neuron-ablated mice after the bretazenil-eluting minipumps are depleted or removed. This result is reminiscent of the observation that ablation of AgRP neurons in neonatal mice does not result in starvation (Luquet et al., 2005
;Phillips and Palmiter, 2008
). We suggested previously (Luquet et al., 2005
) that ablation of AgRP neurons in neonates has a minimal effect on feeding because adaptations can take place during the 2 to 3 weeks between ablation and weaning - when AgRP neurons mature and independent feeding becomes important (Nilsson et al., 2005
). We originally thought that the adaptations might involve establishing new neuronal circuits, but the present results suggest a different explanation. We now suggest that the hyperactive post-synaptic neurons adapt by reducing the effects of excitatory inputs and/or enhancing alternative inhibitory inputs (Horvath, 2006
). The increase in GAD67 staining in the PBN after AgRP neuron ablation may reflect such compensatory changes. Synaptic plasticity could also involve changes in the number of excitatory and/or inhibitory synapses as well as changes in abundance and activity of various receptors, including ion channels and G-protein-coupled receptors. Examination of the mechanisms involved will depend on more precise identification of the post-synaptic neurons with elevated Fos signal. Our results suggest that these hypothetical adaptations are incomplete and mostly ineffective 6 days after DT treatment, but can be established within 11 days with the aid of chronic GABAA
-receptor activation. In this view, bretazenil suppresses the excitability of post-synaptic neurons and reduces local gliosis, which in turn renders sufficient time and a favorable physiological setting for the adaptations to occur.