Abstract
The hypothalamic-pituitary-adrenal (HPA) axis mediates the release of adrenal corticosteroids (CORT) under both basal conditions and in response to stress. The HPA axis is controlled by corticotropin-releasing hormone (CRH) neurons, which release CRH peptide to stimulate pituitary corticotrophs, leading to the secretion of adrenocorticotropic hormone (ACTH). Subsequently, ACTH is released into the bloodstream, where it triggers the adrenal cortex to synthesise and release CORT. In the absence of stress, CRH, ACTH, and CORT are secreted in a fluctuating pattern known as the ultradian rhythm (Henley et al., 2009; Windle et al., 1998; Ixart et al., 1993). In both humans (Henley et al., 2009) and rats (Windle et al., 1998), the ultradian rhythm in ACTH and CORT secretion is approximately one pulse per hour. Remarkably, the patterns of CRH neuron activity which control these ultradian rhythms remains unclear. In addition to controlling stress hormone release, CRH neurons have also been shown to control arousal and stress-associated behaviours (Fuzesi et al., 2016; Li et al., 2020). However, no studies to date have examined the relationship between CRH neuron activity and ultradian rhythms of arousal and stress hormone secretion. To investigate this, in vivo fibre photometry, automated blood sampling, and animal movement tracking were performed in a novel Crh-IRES-Cre rat.
Initially, RNAscope and immunohistochemistry (IHC) were used to characterise the Crh-IRES-Cre rat line. These experiments revealed a high level of co-localisation between Cre mRNA and Crh mRNA in the paraventricular nucleus (PVN) of Crh-IRES-Cre rats. IHC results also demonstrated Cre-recombinase protein labelling in the PVN.
I next performed GCaMP6s fibre photometry in order to record CRH neural activity in freely behaving Crh-IRES-Cre rats. These recording revealed a robust hourly rhythm (1.0 ± 0.09 pulses per hour) in CRH neuronal activity across the 24-hour day. Simultaneous animal movement tracking (as a measure of behavioural arousal) showed coordination between CRH neuronal activity and animal movement, suggesting a link between CRH neuron activity and arousal state. Causality analysis showed that there is a mathematical one-way influence of CRH neuronal activity on animal movement. Furthermore, CRH neuronal activity was consistently elevated when rats were exposed to a short white noise stressor.
In order to measure ultradian rhythms in CORT secretion, an automated blood sampling device was designed and optimised to collect consistent, small-volume, blood samples through a jugular vein catheter from freely moving rats. CORT levels showed robust ultradian rhythms of secretion, with 0.93 ± 0.12 pulses per hour. Simultaneous animal movement tracking showed a highly variable temporal coordination between CORT secretion and behaviour at an individual level.
Next, I performed simultaneous fibre photometry, blood sampling, and animal movement tracking to study the complex interplay between CRH neuronal activity, CORT secretion, and animal movement under both unstressed and stressed conditions. CRH neuronal activity exhibited an hourly rhythm of 1.14 ± 0.064 pulses per hour, whereas CORT secretion and animal movement showed ultradian rhythms of 0.90 ± 0.061 pulses per hour and 0.90 ± 0.041 pulses per hour, respectively. Cross-correlation analysis revealed a strong correlation between CRH neuronal activity, animal movement, and CORT levels under both unstressed and stressed conditions. This analysis showed that CRH neuronal activity preceded CORT secretion by approximately 15 minutes, which supports the hypothesis that pulsatile CRH neuronal activity drives ultradian CORT secretion. However, close examination of recordings revealed that not all pulses of CRH activity triggered pulses of CORT secretion. Likewise, not all pulses of CORT secretion occurred with a preceding pulse of CRH neuronal activity.
In summary, these data reveal the presences of ultradian rhythms in CRH neuronal activity, CORT secretion and animal movement in freely behaving rats. CRH neuronal activity was found to be highly correlated with animal movement and was shown to be a granger cause of animal movement using mathematical causality analysis. I have also shown CRH neuronal activity is correlated with CORT secretion with CRH neuronal activity preceding CORT by 15 mins. The mathematical modelling of the HPA axis also suggests that CRH neuronal activity determines the ultradian dynamics of CORT secretion. Overall, these results suggest that CRH neurons play an important role in coordinating ultradian rhythms of CORT secretion and behavioural arousal.