A GABAergic projection from the central nucleus of the amygdala to the ventrolateral periaqueductal gray: functional implications and modulation by orexin

Conditioned fear to a tone is associated with disinhibition of brainstem-projecting neurons in the central nucleus of the amygdala (CeA). This is thought to trigger freezing via projections to the ventrolateral periaqueductal gray (VLPAG). However, the proportion of these projections containing GABA and glutamate is unknown. Using in situ hybridisation for glutamic acid decarboxylase (GAD67) and vesicular glutamate transporters (VGLUT1, VGLUT2) combined with retrograde labelling from the VLPAG, we showed that 90% of VLPAG-projecting CeA neurons contained GAD67, and none contained VGLUT1 or VGLUT2. Next, by combining Fos immunohistochemistry with retrograde labelling from the VLPAG, we found that sustained re-exposure to a feared context induced Fos expression in only 0.7% of VLPAG-projecting CeA neurons. We then attempted to selectively activate neurons in the medial CeA with orexin-A in freely moving rats, whilst recording cardiovascular and behavioural response. Orexin was associated with enhanced heart rate (HR), mean arterial pressure (MAP), activity, tail temperature and arousal. Next, we administered the same treatment to rats before sustained re-exposure to a fear-conditioned context. We showed that orexin prevented normal within-trial reductions in freezing, but had no effect on HR or MAP responses, activity or total time spent freezing. In a follow up re-exposure, rats previously injected with orexin were treated with saline. These rats did not show any sign of previous extinction learning, and spent more time freezing than rats receiving saline in the first re-exposure. Conversely, rats injected with orexin which were previously injected with saline displayed low levels of freezing. Finally, we sought to determine the behavioural and cardiovascular effects of microinjection of orexin-A in the VLPAG, an important structure in passive coping responses. We showed that orexin enhanced arousal, activity, HR, MAP and tail temperature. These findings support the