Table 1.

The time-line of the study design/experimental procedure

	Immunohistochemistry for ghrelin and GOAT
Day 1	Placement of P5 rat pups under 18-h fasting
Day 2	Group 1: Preparation of hippocampal slice culture from P6 pups of control and 18-h fasted rats, GHSR1a KO mice, and wild-type mice
	Group 2: Cardiac perfusion of age-matched pups from control and 18-h fasted rats
Day 3–7	Group 1: Incubation and culturing of slices prepared in Day 2
Day 8	Group 1: Overnight fixation of cultured slices (check condition/viability of slices prior to fixation) In some experiments, antagonists/inhibitor will be applied before fixation
	Group 2: Cryo-sectioning of cardially-fixed brains
Day 9–10	Application of primary antibody to fixed slices and cryo-sectioned fixed brain specimens
Day 11	Application of secondary antibody and coverslip
Day 12	Imaging and data acquisition with a confocal microscope
	Fluorescent ghrelin binding in fixed slices
Day 1	Preparation of hippocampal slice culture from P6 pups of control rats, GHSR1a KO mice, and wild-type mice
Day 2–6	Incubation and culturing of slices prepared in Day 1
Day 7	Overnight fixation of cultured slices (check condition/viability of slices prior to fixation) In some experiments, antagonists/inhibitor will be applied before fixation.
Day 8	Two-hour application of F302 to one-half of the fixed slices and F203 to the other half of fixed slices
Day 9	Imaging and data acquisition with a confocal microscope
	Fluorescent ghrelin binding in live slices
Day 1	Prepare hippocampal slice culture from P6 pups of control rats, GHSR1a KO mice, and wild-type mice
Day 2–6	Incubation and culturing of slices prepared in Day 1
Day 7	Check condition/viability of slices In some experiments, antagonists/inhibitor will be applied.
Day 8	Overnight application of F302 to one-half of the live slices and F203 to the other half of live slices
Day 9	Imaging and data acquisition from live slices with a confocal microscope