Supplementary Figure 1: CRH neurons in the aBNST and CeA directly project to the VTA and SNc. | Nature Neuroscience

Supplementary Figure 1: CRH neurons in the aBNST and CeA directly project to the VTA and SNc.

From: Chronic CRH depletion from GABAergic, long-range projection neurons in the extended amygdala reduces dopamine release and increases anxiety

Supplementary Figure 1

Extension of Fig. 1a,b. AAVs expressing a Cre-dependent Synaptophysin (Syp)-GFP fusion protein (AAV-Ef1a::DIO-Syp-GFP) were unilaterally injected into the aBNST or CeA of Crh-ires-Cre mice. Presynaptic Syp-GFP puncta are shown in green, immunostaining for tyrosine hydroxylase (TH), a dopaminergic marker, in red. The anterogradely labeled presynaptic terminals (Syp-GFP puncta) represent direct projection sites of aBNST and CeA CRH neurons. Prominent Syp-GFP labeling is observed throughout the VTA and SNc following AAV-Ef1a::DIO-Syp-GFP injections into the aBNST or CeA of Crh-ires-Cre mice. The density of Syp-GFP puncta in the VTA was higher in aBNST-injected compared to CeA-injected mice. The opposite was observed for the SNc, which received stronger input from CeA CRH neurons (a,b, bottom panels). Additional projection sites of aBNST and CeA CRH neurons, as well as of other CRH-expressing brain regions, are listed in Supplementary Table 1. Monosynaptic innervation of the VTA by CRH-expressing neurons of the CeA and aBNST has also been demonstrated by others13,14,50. (c) Accuracy of the tracing approach was verified by co-injecting AAV-Ef1a::DIO-Syp-GFP and AAV-Ef1a::DIO-RFP into the PVN. AAV-Ef1a::DIO-RFP helped to identify correctly targeted neurons at the injection site, since most Syp-GFP is transported to presynaptic terminals and thus often not visible in the cell soma. The highest Syp-GFP puncta density originating from the PVN was observed in the zona externa of the median eminence, the most prominent projection site of parvocellular CRH-positive PVN neurons. From there CRH is released into the hypophysial portal vasculature to initiate activation of the hypothalamic-pituitary-adrenal axis. (d) Projections from CeA and BNST to the VTA were further verified by retrograde tracing using Fluorogold (FG). FG injected to the VTA of Crh-ires-Cre;Ai9 mice revealed a significant number of CRH/tdTomato positive neurons in the BNST and CeA co-labeled with FG. (e) Similarly, injection of retrobeads into the VTA of Crh-ires-Cre;Ai9 mice resulted in CRH/tdTomato neurons co-labeled with retrobeads. All tracing experiments were independently replicated three times. Abbreviations: Anterior BNST (aBNST), anterior dorsal BNST (adBNST), anterior ventral BNST (avBNST), basolateral amygdala (BLA), central amygdala (CeA), median eminence (ME), paraventricular nucleus of the hypothalamus (PVN), substantia nigra pars compacta (SNc), substantia nigra pars reticularis (SNr), synaptophysin (Syp), third ventricle (3V), Ventral tegmental area (VTA). Scale bars represent 100 µm.

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