Beta‐klotho (KLB) is a coreceptor required for endocrine fibroblast growth factor (FGF) 15/19 and FGF21 signaling in the brain. Klb is prominent within the hypothalamus, which is consistent with its metabolic functions, but diverse roles for Klb are now emerging. Central Klb expression is low but discrete and may govern FGF‐targeted sites. However, given its low expression, it is unclear if Klb mRNA is more widespread. We performed in situ hybridization to label Klb mRNA to generate spatial maps capturing the distribution and levels of Klb within the mouse hypothalamus, hippocampal region, subiculum, and amygdala. Semiquantitative analysis revealed that Klb‐labeled cells may express low, medium, or high levels of Klb mRNA. Hypothalamic Klb hybridization was heterogeneous and varied rostrocaudally within the same region. Most Klb‐labeled cells were found in the lateral hypothalamic zone, but the periventricular hypothalamic region, including the suprachiasmatic nucleus, contained the greatest proportion of cells expressing medium or high Klb levels. We also found heterogeneous Klb hybridization in the amygdala and subiculum, where Klb was especially distinct within the central amygdalar nucleus and ventral subiculum, respectively. By contrast, Klb‐labeled cells in the hippocampal region only expressed low levels of Klb and were typically found in the pyramidal layer of Ammon's horn or dentate gyrus. The Klb‐labeled regions identified in this study are consistent with reported roles of Klb in metabolism, taste preference, and neuroprotection. However, additional identified sites, including within the hypothalamus and amygdala, may suggest novel roles for FGF15/19 or FGF21 signaling.

Fibroblast growth factor 21 (FGF21) has emerged as a critical endocrine factor for understanding the neurobiology of obesity, such as by the regulation thermogenesis, food preference, and metabolism, as well as for neuroprotection in Alzheimer’s disease and traumatic brain injury. FGF21 is synthesized primarily by the liver and pancreas then crosses the blood brain barrier to exert its effects in the brain. However, the sites of FGF21 action in the brain is not well-defined. FGF21 action requires the activation of FGF receptor 1c as well as its obligate co-receptor beta klotho (KLB). In order to determine the sites of FGF21 action, we mapped the distribution of Klb mRNA by in situ hybridization using RNAscope technology. We labeled Klb distribution throughout the rostrocaudal axis of male wildtype mice by amplifying Klb hybridization using tyramine signal amplification and visualizing Klb hybridization using Cyanine 3 fluorescence. The resulting Klb signal appears as punctate red “dots,” and each Klb neuron may express low (1–4 dots), medium (5–9 dots), or high levels (10+ dots) of Klb hybridization. We then mapped individual Klb expressing neuron to the atlas plates provided by the Allen Brain Atlas in order to determine Klb distribution within the substructures of each brain region, which are defined by Nissl-based parcellations of cytoarchitectural boundaries. The distribution of Klb mRNA is widespread throughout the brain, and the brain regions analyzed thus far point to notable expression in the hypothalamus, amygdala, hippocampus, and the cerebral cortex. The highest expression of Klb was localized to the suprachiasmatic nucleus in the hypothalamus, which contained low and medium Klb-expressing neurons in the lateral hypothalamic area and ventromedial hypothalamic nucleus while low expressing Klb neurons were seen in the paraventricular and dorsmedial hypothalamic nucleus. Hippocampal Klb expression was limited to the dorsal region and largely restricted to the pyramidal cell layer of the dentate gyrus, CA3, CA2, and CA1 but at low levels only. In the amygdala, low and medium Klb expressing cells were seen in lateral amygdala nucleus while low levels were observed in the basolateral amygdala nucleus. Cortical Klb expression analyzed thus far included low Klb-expressing neurons in the olfactory areas, including layers 2 and 3 of piriform cortex and nucleus of the lateral olfactory tract. These findings are consistent with the known roles of FGF21 in the central regulation of energy balance, but also implicates potentially wide-ranging effects of FGF21 such as in executive functions.