Orexinergic projections to neocortex: potential role in arousal, stress and anxiety-related disorders.

Stress exacerbates many psychiatric conditions, and repeated stress contributes to the pathogenesis of disorders such as Post Traumatic Stress Disorder, Panic and Major Depressive Disorder. The mammalian cerebral cortex is responsible for our higher cognitive functions such as language, episodic memory, and complex perception. It interacts with various other structures, such as the thalamus, a large group of neurons that either relay sensory input to the cortex or mediate cortico-cortical interactions through these neurons. Our brain is not always paying attention to all details, and it does not always analyse detailed contexts. Its state is regulated by neuroendocrine factors. These are small molecules, such as orexin (hypocretin). Orexin is exclusively produced in neurons of the lateral hypothalamus. The orexin system is highly reactive to stress and regulates many physiological processes that are altered in stress-related mental illness, including sleep/wake patterns, appetite, and cognition. Changes in orexin levels have been reported in major depression and anxiety disorders, and genetic defects in the sensitivity to orexin (orexin 1 receptor polymorphism) have been associated with anxiety spectrum disorders, particularly in women who are twice as likely as men to suffer from stress-related mental illness.
Orexinergic neurons have wide projection targets across the entire central nervous system, including to other local (hypothalamic) neurons that are important for modulating arousal, appetite, and neuroendocrine functions. However, the role of projections to cortical circuits remains less well understood, although they may be involved in regulating cortical arousal and the cognitive responses to stress. Thus, they could represent promising targets for drug development that selectively target cortical, but not subcortical mechanisms involved in generating anxiety. Resolving the anatomical and functional connectivity between orexinergic neurons and cortical circuits, as well as the gender differences in this system, will be critical for starting to design orexinergic treatments for anxiety.
Within sensory cortex, layer 6b is the only orexin-sensitive layer. These neurons can be selectively labelled using Ctgf-/Drd1a-Cre transgenic mice, and we have very considerable expertise in studying this cell population. We demonstrated that there is a direct link between lateral hypothalamic orexin neurons and these cells. We also demonstrated that some of the orexin-sensitive layer 6b neurons selectively project to thalamic nuclei that are involved in higher cognitive functions. Sensory layer 6b neurons might therefore function as an orexin-gated circuit that amplifies feedback via cortico-thalamo-cortical loops and play an important role in regulating brain state and cognition.
In our preliminary experiments we genetically silenced a selected population of Drd1a-Cre neurons across the entire cerebral cortex and observed the behaviour of these Snap25 cKO mice. This manipulation did not influence circadian rhythms or locomotor activity when mice were exposed to a novel environment. However, it led to a strong reduction in anxiety-like behaviour, as measured in three different behavioural tasks. This suggests that some of the Drd1a-Cre neurons may act as a key component in the cortex for regulating emotional behaviours. We shall examine i) functional connectivity between orexin neurons and their cortical targets, ii) physiological responses of Ctgf-/Drd1a-Cre neurons to stress and arousal, and how this is modulated by orexin, and ii) involvement of these cells in anxiety and fear learning. The selective manipulation of these circuits presents unique therapeutic avenues for the intervention against anxiety, while not influencing autonomous functions. Our proposed experiments will dissect key components, cell type and gender differences of these circuits and shall test the molecular mechanisms that could be exploited in therapy.

Stress exacerbates many psychiatric conditions and repeated stress contributes to the pathogenesis of mental health disorders. Orexinergic neurons are highly reactive to stress and regulate many physiological processes altered in stress-related mental illness, including sleep/wake patterns, appetite and cognition. They project widely across the CNS, including hypothalamus, thalamus, brain stem, and cerebral cortex. The projections to other hypothalamic neurons and subcortical centres are important for modulating arousal, appetite and endocrine activity. The roles of projections to cortical circuits are poorly understood. They may be involved in regulating cortical arousal and cognitive responses.

We build on our expertise in cortical layer 6b (L6b) neurons, the only orexin sensitive cells in sensory cortex. We demonstrated that the Drd1a-Cre+ subgroup of L6b neurons selectively target higher order thalamic nuclei. 'Silencing' these neurons by Cre-mediated Snap25 cKO did not influence locomotor or circadian activity, but it reduced anxiety-like behaviour in the elevated plus maze and light-dark box (distinct from Rbp-4Cre neurons, Krone et al., 2021). This suggests Drd1a-Cre neurons regulate emotional behaviours as an orexin-gated cortico-thalamo-cortical circuit, most likely involving prefrontal cortex.

Here, we aim to resolve the functional connectivity between orexinergic neurons and cortical circuits including their inputs to Drd1a-Cre/Ctgf-Cre+ cortical cells, using optogenetics and calcium imaging ex vivo. Next, we will use high-density Neuropixel arrays to determine the involvement of orexin-sensitive cortical cells in sex specific responses to stress and arousal. Finally, we determine how silencing these neurons affects sex differences in anxiety-related behaviours. Our experiments will dissect key components, cell-type and gender differences of these circuits, and shall pave the way to identify molecular mechanisms that could be exploited in therapy.