Figure 1

Growth hormone is expressed in excitatory and inhibitory cells in the amygdala. Double in situ hybridization against growth hormone (GH; red) and GAD67 (green), a marker of inhibitory interneurons, was conducted in the rat amygdala to quantify nuclear expression of these transcripts. (a) Top: the bar graphs display the overall percentage of GH+ cells (left), the overall percentage of GAD67+ cells (middle) and the percentage of GH+ cells that are GAD67+ (right) in the lateral nucleus of amygdala (LA). Bottom: representative double-positive cells from the LA are shown. Blue signal represents nuclear DAPI staining (n=1340). Red signal indicates GH mRNA (n=617), green signal indicates GAD67 mRNA (n=535) and yellow signal corresponds to overlap of GH and GAD67 mRNA. (b) Top: bar graphs show the percentages of different cell populations in the basolateral nucleus (BL), as shown in a (Top). Bottom: representative double-positive cells from the BL. Blue signal represents nuclear DAPI staining (n=1341). Red arrows indicate representative nuclei containing only GH mRNA (n=624), green arrows indicate representative nuclei containing only GAD67 mRNA (n=493) and yellow arrows signify representative nuclei containing both GH and GAD67 mRNA. The bar graphs reveal that GH mRNA is broadly expressed across LA and BL (35–40% of nuclei). Scale bars represent 35 μm. The 'n' reported represent the total number of cells counted across rats (three rats). (c) For some brain sections containing LA and BL, in situ hybridization was performed without adding the probe to target GH (negative control); all other steps were identical to those used for other sections. This enabled visualization of background staining. We observed no red puncta for the GH signal with this method. Some diffuse red haze was present, indicating nonspecific binding. Thus, the discrete red puncta observed in a and b represent true, albeit sparse, signal. (d) Coronal sections of the rat basolateral complex of the amygdala (BLA) were stained for GH protein (green), the neuronal marker NeuN (red), and the nuclear marker DAPI (blue). A colorized merged image of a single slice in the z axis is shown (left; × 63). The individual channels for GH and NeuN are also depicted (middle, right). GH is clearly expressed by excitatory pyramidal neurons in the BLA. Scale bar indicates 20 μm. (e) Primary cell cultures of amygdala were generated and stained for GH (green), mitogen-activated protein kinase (MAPK; red) and DAPI (blue). GH is strongly expressed in the dendrites and cell bodies of excitatory pyramidal amygdala neurons ( × 63). Scale bars represent 20 μm. (f) GH and GFP (GH group) or GFP only (GFP group) was overexpressed in the BLA before auditory fear conditioning. Conditional freezing during fear conditioning, context extinction and tone extinction are depicted. All data shown are means±s.e.m. GFP, green florescent protein.