Fig. 2: CRH-releasing afferents in the TRN originate from multiple central CRH sources.

a Schematic of the retrograde tracing approach: a Cre-dependent tdTomato-expressing virus was injected into the sensory TRN to label CRH-releasing neurons projecting to this region. Representative images showing retrogradely labeled CRH+ neurons (tdTomato, red) in several brain regions projecting to the TRN, including the basolateral amygdala (BLA, b), dorsal zona incerta (ZID, c), superior geniculate nucleus (SGN, d), and medial division of the medial geniculate nucleus (MGM, d). e Schematic of the anterograde tracing strategy to anatomically confirm projections from CRH-expressing neurons (GFP-labeled, green) in the identified regions. Examples of injection sites for anterograde tracing in BLA (f), ZID (g), SGN, and MGM (h). Anterogradely labeled CRH+ axons from BLA (i), ZID (j), and SGN/MGM (k) were observed contacting distinct regions of the TRN. l, m Retrograde tracing revealed CRH+ neurons in the ventrolateral parvocellular division of the geniculate nucleus (VLGPC) as a candidate TRN-projecting population; however, this projection was not confirmed with anterograde tracing. n No retrogradely labeled CRH+ neurons were found in the paraventricular nucleus of the hypothalamus (PVN), suggesting that TRN CRH inputs originate from non-neuroendocrine central CRH sources. o Summary diagram of confirmed CRH projections to the TRN, illustrating a complex and spatially organized network of CRH-releasing afferents. Schematic brain drawings are modified from ref. ⁸⁹ under a Creative Commons CC BY license. Independent observable for retrograde tracing: n = 14 (a, m, n); for anterograde tracing: BLA, n = 4 (f, i); ZID, n = 3 (g, j); MGM and SGN, n = 4 (h, k). All scale bars are 200 µm. Source data are provided as a Source data file.