Orexin neurons project to diverse sympathetic outflow systems

The viral transneuronal labeling method was used to demonstrate that orexin-containing neurons of the lateral hypothalamic area (LHA) are linked via multisynaptic connections to different sympathetic outflow systems. Two different types of transneuronal tracing experiments were performed: single- and double-virus studies. In the first series of experiments, Bartha pseudorabies virus (PRV), a retrograde transneuronal tracer, was injected into single sympathetic targets, viz., stellate ganglion, adrenal gland, celiac ganglion, and kidney. Six to 7 days post-injection, orexin (hypocretin) neurons were transneuronally labeled. In a second set of experiments, the double-virus tracing method was used to determine whether single orexin LHA neurons are linked to two different sympathetic outflow systems. Two isogenic forms of Bartha PRV were used that differed by a single gene. beta-Galactosidase Bartha PRV was injected into the stellate ganglion and green fluorescent protein Bartha PRV into the adrenal gland of the same rat. The reverse placement of viral injections was made in another set of rats. In both paradigms, some orexin LHA neurons were transneuronally labeled with both viruses, indicating that they are capable of modulating multiple sympathetic outflow systems. These findings raise the possibility that orexin LHA neurons regulate general sympathetic functions, such as those that occur during arousal or the fight-or-flight response.