RT doctoral thesis
T1 Prostaglandin EP3 receptor signalling as a contributor to sex differences in stress-related disorders mediated by the locus coeruleus
A1 Martínez Cortés, Adrián
A2 Neurociencias
AB Stress and pain are deeply interconnected phenomena, with acute stress triggeringadaptive responses, whereas chronic stress and chronic neuropathic pain lead to persistentalterations in neural circuits regulating affective and sensory processing. These processesare tightly interlinked at multiple levels, and prolonged nociceptive states can inducemaladaptive 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 acentral role in modulating nociception and anxiety-related behaviours and in coordinatingacute and chronic stress responses. Notably, the LC exhibits sex-dependent anatomicaland functional features that may shape vulnerability or resilience to stress-relateddisorders. Within this framework, prostaglandin signalling, particularly through the EP3receptor expressed in LC neurons, has emerged as a key modulator of stress, nociceptionand affective behaviour, providing a mechanistic link between sex differences, stressregulation and chronic pain.The central hypothesis of this thesis is that sexual dimorphism within the LC, togetherwith differential EP3 receptor signalling, contributes to sex-dependent vulnerability tostress-related disorders. Specifically, alterations in LC-EP3 signalling are proposed tounderlie sex differences in behavioural and molecular responses to both acute stress andchronic neuropathic pain. To test this hypothesis, pharmacological and viral vector-basedapproaches were combined to selectively manipulate EP3 receptor signalling in LCnoradrenergic neurons in male and female mice subjected to acute restraint stress orchronic 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 stressmodel, robust anxiety-like behaviour and analgesia were observed in both sexes.However, intra-LC administration of the EP3 agonist sulprostone during restraintselectively reversed anxiety-like behaviour and further potentiated analgesia in females,while no effects were observed in males. These evidences highlight a sex-specific role forLC-EP3 signalling in the regulation of stress responses. To further investigate thefunctional relevance of LC activity in females, chemogenetic activation of LC noradrenergic neurons was performed to mimic the effects of acute stress. LC activationproduced a clear anxiogenic response, which was abolished by intra-LC sulprostone. Inparallel, LC activation induced analgesia, which was further enhanced by sulprostoneadministration. Collectively, these findings demonstrate that EP3 receptor signallingwithin the LC exerts a female-specific modulatory and protective role during acute stress,providing a mechanistic basis for sex-dependent differences in behavioural stressresponses. We next assessed the temporal contribution of LC noradrenergic neurons tonociceptive and anxiety-like behaviours in a chronic pain model using chemogeneticapproaches. Characterisation of the LC during neuropathy progression revealed that LCfunction is temporally dynamic, with early activity exerting a protective effect innociceptive processing, whereas late-stage dysregulation promotes a maladaptive, pronociceptive profile, with no detectable sex differences. In contrast, when consideringaffective behaviour, inhibition of noradrenergic LC neurons had a clear sex-specific effectat 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 inLC activity during chronic neuropathic pain. In the CCI model, intra-LC sulprostoneproduced analgesic and anxiolytic effects in males but not in females. Western blotanalysis revealed increased EP3 receptor expression in CCI males and decreasedexpression 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. Proximityligation assays demonstrated reduced EP3 receptor homodimerisation in CCI females,suggesting impaired receptor stability or availability. Functional assays further showedthat intra-LC sulprostone decreased cAMP levels in CCI males but increased them infemales, indicating sex-specific alterations in EP3 receptor coupling and signallingefficiency. Moreover, analysis of LC projection areas revealed that intra-LC sulprostonereduced pCREB levels in both the DRt and BLA of male CCI mice, whereas it increasedpCREB expression in the same regions in females, indicating that EP3 receptor activationwithin the LC exerts sex-specific effects on downstream and upstream circuits.To investigate the causal role of EP3 signalling, EP3 receptor overexpression in thenoradrenergic LC neurons was performed in a chronic pain model. EP3 receptoroverexpression elicited analgesic effects in both sexes but anxiolytic effects exclusively in CCI males. Accordingly, the absence of anxiolytic effect in CCI females could not beattributed solely to receptor abundance and instead appeared to reflect downstreamsignalling alterations. Consistent with these findings, EP3 overexpression reducedpCREB expression in both regions in CCI males, while no significant changes wereobserved in CCI females.Overall, these findings demonstrate that sex-specific LC-EP3 signalling shapesdifferential behavioural and molecular responses in acute stress and chronic neuropathicpain. Elucidating these mechanisms provides a framework for the development oftargeted and sex-specific therapeutic strategies focused on prostaglandin signallingpathways to improve stress and pain management.
YR 2026
FD 2026
LK http://hdl.handle.net/10498/39689
UL http://hdl.handle.net/10498/39689
LA eng
DS Repositorio Institucional de la Universidad de Cádiz
RD 21-sep-2026