The lateral hypothalamic area (LHA) lies at the intersection of multiple neural and humoral systems and orchestrates fundamental aspects of behavior. which revealed unexpected neurochemical diversity. We found that single MCH and Hcrt/Ox neurons express transcripts for multiple neuropeptides and markers of both excitatory and inhibitory fast neurotransmission. Virtually all MCH and approximately half of the Hcrt/Ox neurons sampled express both the machinery for glutamate release and GABA synthesis in the absence of DAPT inhibition a vesicular GABA release pathway. Furthermore, we found that this profile is usually characteristic of a subpopulation of LHA glutamatergic neurons but contrasts with a broad population of LHA GABAergic neurons. Identifying the neurochemical diversity of Hcrt/Ox and MCH neurons will further our understanding of how these populations modulate postsynaptic excitability through multiple signaling mechanisms and coordinate diverse behavioral outputs. and kept on a 12/12 h light/dark cycle. Brain slice preparation for microdissection and single-cell dissociation Hypothalamic brain slices through the LHA were taken from five Ox-EGFP, 5 expression after removing cells absent for the transcript. Hierarchical clustering was performed using Wards DAPT inhibition method with complete linkage (Ward, 1963). For theory component analysis (PCA), gene expression was score normalized and processed using the princomp function in R. To examine potential subclusters and/or batch effects, we used both multiple hypothesis testing analysis using custom routines and the fisher.test function in R as well as PCA analysis using the princomp function in R. To quantitatively compare gene expression between Hcrt/Ox and MCH neurons, we performed multiple hypothesis testing around the 48 genes using Fishers exact test (Agresti, 1992) to report adjusted values, with the Benjamini-Hochberg procedure (Benjamini and Hochberg, 1995) to control the false discovery rate (FDR) at 5%. All statistical analyses were performed using R (The R Project for Statistical Computing; www.r-project.org, RRID: DAPT inhibition DAPT inhibition SCR_001905). Statistical power analysis We performed power analysis to assess whether the numbers of neurons used in this study are adequate to achieve sufficient statistical power in detecting differential gene expression. To this end, we used a simulation in which the sample sizes are fixed at the same values of the DAPT inhibition real data (Hcrt/Ox: 69; MCH: 89), and the true difference between the two probabilities of expression is set to various levels (0%, 15%, 25%, and 35%). With each simulation, presence/absence data are randomly generated, for which the Fishers exact test (Agresti, 1992) was performed at 5% significance level. The simulations were repeated 1000 COL1A1 times under each setting of true probabilities and effect size, and the proportion of times that this test is usually rejected is usually then an estimate of the corresponding power. Power analysis via simulation was performed using custom routines in R. Fluorescence hybridization (FISH) To prepare tissue sections for FISH, male juvenile (postnatal days P21-P24) wild type C57BL/6 mice were anesthetized with isoflurane, decapitated, and brains were dissected out into ice-cold sucrose. Brains were rapidly frozen on dry ice, embedded in OCT compound and cryosectioned at a thickness of 14 m onto SuperFrost Plus microscope slides. Sections were fixed with 4% paraformaldehyde (PFA) at 4C for 15 min, and then dehydrated in 50%, 70%, and 100% ethanol. RNAscope 2.5 Assay (Advanced Cell Diagnostics, ACD, RRID: SCR_012481) was used for all FISH experiments according to manufacturer’s protocols (Wang et al., 2012). All RNAscope FISH probes were designed and validated by ACD. Imaging and image quantification of FISH data Confocal images of FISH experiments were obtained using a Leica TSC Sp8 and confocal image files (lif) made up of image stacks were loaded into ImageJ (version 2.0.0, NIH, RRID: SCR_003070) and.