Abstract
Disruptions to the brain circuits for escape behaviours are associated with anxiety disorders. The Periaqueductal Grey (PAG) is a critical brain region in controlling fear and anxiety behaviours. However, the neural circuits that innervate the PAG output remain poorly characterised. Fear and anxiety behaviours are also modulated by corticotrophin-releasing hormone (CRH) signalling. However, it is unknown how CRH controls these behaviours. CRH is expressed by several discrete neural populations in the brain. Therefore, this study aimed to determine whether CRH neurons project to the PAG and, if so, identify the specific population(s) of CRH neuron involved.
A retrograde adeno-associated viral vector encoding td-Tomato (a red fluorescent protein) was administered into the PAG of adult male CRH-cre transgenic mice to transduce td-Tomato expression exclusively in cre-expressing neurons. The vector is engineered to infect synaptic terminals and travel retrogradely to the cell body. Hence, all CRH neurons projecting to the PAG will be identified by td-Tomato expression at the cell body.
PAG-projecting CRH neurons were identified in the anterodorsal and anteromedial (AD: 18 ± 5.68 SEM cells, AM: 21 ± 9.28 SEM cells, n=6) thalamus. No Td-Tomato expressing cells were observed in wildtype control mice (?² = 3.11, df= 2, p < 0.5, n=2). Additionally, td-Tomato labelled fibres were identified in the periventricular thalamus and reuniens nucleus (PVT: 7 ± 0.75 SEM fluorescence per pixel; RE: 10 ± 0.73 SEM fluorescence per pixel, n = 6) with significantly higher fluorescence than the background fluorescence in wild-type controls (n=2), confirming the difference (p<0.0001; F (1,35), two-way ANOVA with total n=8, Sidak). Therefore, the PAG appears to be innervated by thalamic CRH neurons that project via the PVT and RE, highlighting a bidirectional Thalmus to PAG CRH circuitry. While this study has identified a novel neural circuit connecting CRH-expressing neurons and the PAG that might underpin fear and anxiety behaviours, future research is required to determine whether this circuit becomes dysfunctional in fear and anxiety disorder models.