Possible appositions was checked visually to verify the accuracy of this quantitative procedure. The number of VGAT or VGLUT2 appositions was normalized to a set distance of biocytin-filled proximal process (1 m). To determine the overall density of VGAT and VGLUT2 boutons in the ARH, we analyzed only the 633 nm HeNe laser images. Each VGATor VGLUT2-labeled synaptic bouton was defined as three-dimensional object (voxel to voxel). The ratio of labeled synaptic boutons for VGAT or VGLUT2 was obtained by dividing the average density of each age to the average density of P13 15. DiI implants in postnatal mice:. Both male and female pups from NPYhr-GFP mice at P15 and P21 were anesthetized and perfused transcardially with saline followed by AMG9810 site ice-cold 4 PFA, pH 7.4. Perfused brains were removed immediately and stored in fixative at 4 until they could be blocked and embedded in 3 agarose. Embedded brains were sectioned coronally rostral to caudal to expose the dorsal medial hypothalamus (DMH). The surface of each block was stained with methylene blue to visualize neuroanatomical features. A small crystal of DiI (1,1dioctadecyl-3,3,3,3-tetramethylindo carbocyanine perchlorate; Invitrogen) was placed unilaterally into the DMH of each brain under a dissection scope. Implanted brains were stored in 4 PFA at 37 for 3 weeks. After the diffusion period, 50 M coronal slices were cut through the hypothalamus using a Vibratome (Leica VT1000S). Hypothalamic sections were mounted onto gel-subbed glass slides and coverslipped with slowfade (Invitrogen). Before mounting sections were incubated with DAPI (1:4000) for 1 min. Hypothalamic slices containing DMH or ARH were imaged on a Leica laser scanning confocal microscope using a 488 nm AR laser for NPY-GFP, a 561 nm DPSS laser for DiI, and a 405 nm laser for DAPI. qPCR. Micropunches from the ARH were collected from 10 Mequitazine chemical information animals at ages P12 13. We combined two animals per tube and isolated RNA using Trizol and the RNeasy micro kit with on-column deoxyribonu-8560 ?J. Neurosci., June 3, 2015 ?35(22):8558 ?Baquero et al. ?Synaptic Distribution in Arcuate Nucleus NeuronsFigure 1. Comparison of circularity and area of synaptic boutons in NAG neurons. A, Quantification of circularity and area of VGAT synaptic boutons (n 6 ?8 optical sections per animal from 9 animals). B, Quantification of circularity and area of VGLUT2 synaptic boutons (n 6 ?8 optical sections per animal from 9 animals). Error bars indicate mean SEM. clease I treatment (Qiagen). Quality and integrity of RNA was determined using the Agilent 2100 Bioanalyer (Agilent Technologies). Reverse transcriptase reactions were prepared using 300 ng of RNA and iScript cDNA Synthesis Kit (Bio-Rad). Quantitative real-time PCR was completed using TaqMan probes (Applied Biosystems) for NKCC1 (Mm01265951_m1), KCC2 (Mm00803929_m1), and housekeeping gene -actin (Mm00607939_s1) was used as an endogenous control to normalize each sample and gene. PCRs were in a 10 l volume using 0.5 l TaqMan probe, 6 ng cDNA template, 5 l TaqMan Gene Expression Master Mix II with UNG (Applied Biosystems), and 2.5 l DNase/RNase molecular grade water (Qiagen). Real-time PCR was run using an Applied Biosystems 7900HT Fast Real-Time PCR system with an initial denaturing at 50 for 2 min, 95 for 10 min, followed by 40 cycles at 95 for 15 s, and annealing at 60 for 1 min. Results were calculated by the relative standard curve method. Data analysis. All data are expressed as means SEM.Possible appositions was checked visually to verify the accuracy of this quantitative procedure. The number of VGAT or VGLUT2 appositions was normalized to a set distance of biocytin-filled proximal process (1 m). To determine the overall density of VGAT and VGLUT2 boutons in the ARH, we analyzed only the 633 nm HeNe laser images. Each VGATor VGLUT2-labeled synaptic bouton was defined as three-dimensional object (voxel to voxel). The ratio of labeled synaptic boutons for VGAT or VGLUT2 was obtained by dividing the average density of each age to the average density of P13 15. DiI implants in postnatal mice:. Both male and female pups from NPYhr-GFP mice at P15 and P21 were anesthetized and perfused transcardially with saline followed by ice-cold 4 PFA, pH 7.4. Perfused brains were removed immediately and stored in fixative at 4 until they could be blocked and embedded in 3 agarose. Embedded brains were sectioned coronally rostral to caudal to expose the dorsal medial hypothalamus (DMH). The surface of each block was stained with methylene blue to visualize neuroanatomical features. A small crystal of DiI (1,1dioctadecyl-3,3,3,3-tetramethylindo carbocyanine perchlorate; Invitrogen) was placed unilaterally into the DMH of each brain under a dissection scope. Implanted brains were stored in 4 PFA at 37 for 3 weeks. After the diffusion period, 50 M coronal slices were cut through the hypothalamus using a Vibratome (Leica VT1000S). Hypothalamic sections were mounted onto gel-subbed glass slides and coverslipped with slowfade (Invitrogen). Before mounting sections were incubated with DAPI (1:4000) for 1 min. Hypothalamic slices containing DMH or ARH were imaged on a Leica laser scanning confocal microscope using a 488 nm AR laser for NPY-GFP, a 561 nm DPSS laser for DiI, and a 405 nm laser for DAPI. qPCR. Micropunches from the ARH were collected from 10 animals at ages P12 13. We combined two animals per tube and isolated RNA using Trizol and the RNeasy micro kit with on-column deoxyribonu-8560 ?J. Neurosci., June 3, 2015 ?35(22):8558 ?Baquero et al. ?Synaptic Distribution in Arcuate Nucleus NeuronsFigure 1. Comparison of circularity and area of synaptic boutons in NAG neurons. A, Quantification of circularity and area of VGAT synaptic boutons (n 6 ?8 optical sections per animal from 9 animals). B, Quantification of circularity and area of VGLUT2 synaptic boutons (n 6 ?8 optical sections per animal from 9 animals). Error bars indicate mean SEM. clease I treatment (Qiagen). Quality and integrity of RNA was determined using the Agilent 2100 Bioanalyer (Agilent Technologies). Reverse transcriptase reactions were prepared using 300 ng of RNA and iScript cDNA Synthesis Kit (Bio-Rad). Quantitative real-time PCR was completed using TaqMan probes (Applied Biosystems) for NKCC1 (Mm01265951_m1), KCC2 (Mm00803929_m1), and housekeeping gene -actin (Mm00607939_s1) was used as an endogenous control to normalize each sample and gene. PCRs were in a 10 l volume using 0.5 l TaqMan probe, 6 ng cDNA template, 5 l TaqMan Gene Expression Master Mix II with UNG (Applied Biosystems), and 2.5 l DNase/RNase molecular grade water (Qiagen). Real-time PCR was run using an Applied Biosystems 7900HT Fast Real-Time PCR system with an initial denaturing at 50 for 2 min, 95 for 10 min, followed by 40 cycles at 95 for 15 s, and annealing at 60 for 1 min. Results were calculated by the relative standard curve method. Data analysis. All data are expressed as means SEM.