Referee 1's review (Vincenzo Di Marzo):

Review of the original manuscript:
In this article, Kola et al address the very important issue of whether or not the actions of ghrelin on food intake, AMPK activation and PVN anorexic neuron activity are directly mediated by CB1 receptors and endocannabinoids. The data presented strongly argue in favour of such a possibility. The study has been mostly well performed and the article is well written. The data are very important since they fill a crucial gap in our knowledge of the mechanism of action of both ghrelin and endocannabinoids. I have, however, some major points that need to be addressed by the authors and should strengthen this important study:
1) the hypothalamic levels of 2-AG seem to be considerably (almost 10-fold) higher than those previously reported by others in the field. Furthermore, it is not clear why, whilst rimonabant reduces 2-AG levels, these levels are higher in CB1 KO mice. In a previous study, surprisingly not quoted by the authors, Di Marzo et al (J Neurochem, 2000) showed no changes in either 2-AG or anandamide levels in the hypothalamus of both male and female CB1 KO mice seemingly of the same strain, background and age as those used in this study. Furthermore, the authors refer to a preliminary work (ref. 20) by Bequet et al., which has never been published in full. These authors, however, did find that rimonabant causes changes in hypothalamic endocannabinoid levels, but the changes found with anandamide were opposite to those with 2-AG, whereas the present authors report here similar changes for 2-AG and anandamide. All these discrepancies should be discussed rather than just being ignored, and, importantly, the results with anandamide should be also shown.
2) The authors have reported effects of rimonabant on ghrelin-induced AMPK and endocannabinoid levels. Yet, they seem to have used a per se non-inactive dose of rimonabant (Fig. 1b,c). It is possible that what they see is not antagonism, but rather the sum of two opposite effects. Control experiments with an inactive per se dose of rimonabant should be carried out;
3) Given for granted that the authors deal with the above two points, they should discuss further why the effect of ghrelin on endocannabinoid levels is not observed in the presence of inactive CB1 receptors. They mention a positive feed-back loop on endocannabinoid biosynthesis. This possibility might be supported by the previous finding that also NPY is inactive in CB1 KO mice (Poncelet et al., Neurosci Lett. 2003) and yet that CB1 stimulation enhances NPY levels (Gamber et al., Neuropharmacology, 2005), two other very relevant papers that should have been quoted by the authors. It is possible that ghrelin stimulates EC levels both directly and indirectly via NPY, whose levels are then further amplified by CB1 stimulation, thus leading to further EC biosynthesis. This positive feed-back mechanism would require both ghrelin and CB1 receptors. By converse, tonic CB1 receptor stimulation alone (without concomitant ghrelin receptor stimulation, necessary to trigger both NPY and endocannabinoid levels) might not be sufficient to elevate EC levels, and this might explain why CB1 KO mice do not exhibit lower EC levels in the hypothalamus. The authors might wish to discuss this possibility.
4) The electrophysiology experiments are very important but perhaps should be accompanied by a little bit more pharmacology. For example, in order to establish a true link between the effects of ghrelin on food-intake and EC levels and its effect on PVN neuron activity, it would be important to see if a DAGLalpha inhibitor also blocks the effect of ghrelin in these experiments. In fact, rimonabant might behave as an inverse agonist in vitro, and BAPTA-AM is not specific for endocannabinoid biosynthesis. DAGL inhibitors such as THC have been widely used recently in similar studies on the role of endocannabinoids as retrograde messengers.

Minor point: 1) since the effect of ghrelin on endocannabinoid levels is a crucial part of this work, recent reviews and experimental papers showing the effects, or lack thereof, of other hypothalamic hormones on endocannabinoid levels should be quoted in the references. 2) The name of the last author in reference 9 is mispelt.

Review of the first revised manuscript:
The authors have answered satisfactorily to my comments

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.