Prostaglandin EP3 receptor signalling as a contributor to sex differences in stress-related disorders mediated by the locus coeruleus

Stress and pain are deeply interconnected phenomena, with acute stress triggering adaptive responses, whereas chronic stress and chronic neuropathic pain lead to persistent alterations in neural circuits regulating affective and sensory processing. These processes are tightly interlinked at multiple levels, and prolonged nociceptive states can induce maladaptive plasticity that differs between males and females, reflecting welldocumented sex differences in stress responsivity. The noradrenergic locus coeruleus (LC), the brain’s main noradrenergic nucleus, together with its projection targets, including the dorsal reticular nucleus (DRt) and the basolateral amygdala (BLA), plays a central role in modulating nociception and anxiety-related behaviours and in coordinating acute and chronic stress responses. Notably, the LC exhibits sex-dependent anatomical and functional features that may shape vulnerability or resilience to stress-related disorders. Within this framework, prostaglandin signalling, particularly through the EP3 receptor expressed in LC neurons, has emerged as a key modulator of stress, nociception and affective behaviour, providing a mechanistic link between sex differences, stress regulation and chronic pain. The central hypothesis of this thesis is that sexual dimorphism within the LC, together with differential EP3 receptor signalling, contributes to sex-dependent vulnerability to stress-related disorders. Specifically, alterations in LC-EP3 signalling are proposed to underlie sex differences in behavioural and molecular responses to both acute stress and chronic neuropathic pain. To test this hypothesis, pharmacological and viral vector-based approaches were combined to selectively manipulate EP3 receptor signalling in LC noradrenergic neurons in male and female mice subjected to acute restraint stress or chronic constriction injury (CCI). Firstly, basal EP3 receptor expression within the LC was examined in naïve mice, revealing significantly higher levels in females than in males. In the acute restraint stress model, robust anxiety-like behaviour and analgesia were observed in both sexes. However, intra-LC administration of the EP3 agonist sulprostone during restraint selectively reversed anxiety-like behaviour and further potentiated analgesia in females, while no effects were observed in males. These evidences highlight a sex-specific role for LC-EP3 signalling in the regulation of stress responses. To further investigate the functional relevance of LC activity in females, chemogenetic activation of LC noradrenergic neurons was performed to mimic the effects of acute stress. LC activation produced a clear anxiogenic response, which was abolished by intra-LC sulprostone. In parallel, LC activation induced analgesia, which was further enhanced by sulprostone administration. Collectively, these findings demonstrate that EP3 receptor signalling within the LC exerts a female-specific modulatory and protective role during acute stress, providing a mechanistic basis for sex-dependent differences in behavioural stress responses. We next assessed the temporal contribution of LC noradrenergic neurons to nociceptive and anxiety-like behaviours in a chronic pain model using chemogenetic approaches. Characterisation of the LC during neuropathy progression revealed that LC function is temporally dynamic, with early activity exerting a protective effect in nociceptive processing, whereas late-stage dysregulation promotes a maladaptive, pronociceptive profile, with no detectable sex differences. In contrast, when considering affective behaviour, inhibition of noradrenergic LC neurons had a clear sex-specific effect at later stages of neuropathy, reducing anxiety-like behaviour in males but not in females. Similarly to the acute stress model, we examined the role of EP3 receptor signalling in LC activity during chronic neuropathic pain. In the CCI model, intra-LC sulprostone produced analgesic and anxiolytic effects in males but not in females. Western blot analysis revealed increased EP3 receptor expression in CCI males and decreased expression in CCI females relative to sham controls. In addition, prostaglandinsynthesising enzymes (mPGES1, PTGES3 and COX2) were upregulated in CCI males, whereas CCI females exhibited downregulation of mPGES1, PTGES3 and COX1, indicating a sex-dependent molecular environment regulating PGE₂ signalling. Proximity ligation assays demonstrated reduced EP3 receptor homodimerisation in CCI females, suggesting impaired receptor stability or availability. Functional assays further showed that intra-LC sulprostone decreased cAMP levels in CCI males but increased them in females, indicating sex-specific alterations in EP3 receptor coupling and signalling efficiency. Moreover, analysis of LC projection areas revealed that intra-LC sulprostone reduced pCREB levels in both the DRt and BLA of male CCI mice, whereas it increased pCREB expression in the same regions in females, indicating that EP3 receptor activation within the LC exerts sex-specific effects on downstream and upstream circuits. To investigate the causal role of EP3 signalling, EP3 receptor overexpression in the noradrenergic LC neurons was performed in a chronic pain model. EP3 receptor overexpression elicited analgesic effects in both sexes but anxiolytic effects exclusively in CCI males. Accordingly, the absence of anxiolytic effect in CCI females could not be attributed solely to receptor abundance and instead appeared to reflect downstream signalling alterations. Consistent with these findings, EP3 overexpression reduced pCREB expression in both regions in CCI males, while no significant changes were observed in CCI females. Overall, these findings demonstrate that sex-specific LC-EP3 signalling shapes differential behavioural and molecular responses in acute stress and chronic neuropathic pain. Elucidating these mechanisms provides a framework for the development of targeted and sex-specific therapeutic strategies focused on prostaglandin signalling pathways to improve stress and pain management.

Author dc:creator

• Martínez Cortés, Adrián

Advisors dc:contributor.advisor

• Berrocoso Domínguez, Esther María
• Llorca Torralba, Meritxell

Statement dc:rights

• Attribution-NonCommercial-NoDerivatives 4.0 Internacional

Licence dc:rights.uri

• http://creativecommons.org/licenses/by-nc-nd/4.0/

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/10498/39689

OAI identifier oai:identifier

oai:rodin.uca.es:10498/39689