{"id":{"repo_id":"cadiz","oai_identifier":"oai:rodin.uca.es:10498/39689"},"canonical_url":"https://search.dev.ndltd.org/etd/cadiz/oai:rodin.uca.es:10498/39689","repository":{"repo_id":"cadiz","name":"Universidad de Cadiz","base_url":"https://rodin.uca.es/oai/request"},"display":{"title":"Prostaglandin EP3 receptor signalling as a contributor to sex differences in stress-related disorders mediated by the locus coeruleus","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Martínez Cortés, Adrián"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Berrocoso Domínguez, Esther María","Llorca Torralba, Meritxell"],"committee_chairs":[],"committee_members":[],"year":2026,"date_issued":"2026","date_published":"2026","updated_at":"2026-07-24T01:29:34Z","subjects":[],"languages":["eng"],"rights":["Attribution-NonCommercial-NoDerivatives 4.0 Internacional"],"rights_urls":["http://creativecommons.org/licenses/by-nc-nd/4.0/"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10498/39689","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Berrocoso Domínguez, Esther María","Llorca Torralba, Meritxell"]},{"key":"dc:contributor.other","label":"Dc Contributor Other","values":["Neurociencias"]},{"key":"dc:creator","label":"Author","values":["Martínez Cortés, Adrián"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2026-06-03T07:42:11Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2026-06-03T07:42:11Z"]},{"key":"dc:date.issued","label":"Date","values":["2026"]},{"key":"dc:type","label":"Dc Type","values":["doctoral thesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Attribution-NonCommercial-NoDerivatives 4.0 Internacional"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://creativecommons.org/licenses/by-nc-nd/4.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10498/39689"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.","El estrés y el dolor son fenómenos interconectados, ya que mientras que el estrés agudo desencadena respuestas adaptativas, el estrés crónico y el dolor neuropático crónico conducen a alteraciones persistentes en los circuitos neuronales que regulan el procesamiento afectivo y sensorial. Ambos procesos están estrechamente interrelacionados en múltiples niveles, y los estados nociceptivos prolongados pueden provocar una plasticidad maladaptativa que difiere entre machos y hembras, reflejando las conocidas diferencias sexuales en la respuesta al estrés. El locus coeruleus (LC), principal núcleo noradrenérgico del cerebro, junto con sus áreas de proyección, entre ellas el núcleo reticular dorsal (DRt) y la amígdala basolateral (BLA), desempeñan un papel importante en la modulación de la nocicepción y de las conductas relacionadas con la ansiedad, así como en la coordinación de las respuestas al estrés agudo y crónico. Cabe destacar que el LC presenta características anatómicas y funcionales dependientes del sexo que pueden determinar la vulnerabilidad o resiliencia frente a los trastornos relacionados con el estrés. En este contexto, la señalización mediante prostaglandinas, en particular a través del receptor EP3 localizado en neuronas del LC, ha surgido como un modulador clave del estrés, la nocicepción y la conducta afectiva, proporcionando un vínculo entre las diferencias sexuales, la regulación del estrés y el dolor crónico. La hipótesis central de esta tesis es que el dimorfismo sexual del LC, junto con la señalización diferencial del receptor EP3, contribuye a la vulnerabilidad dependiente del sexo a los trastornos relacionados con el estrés. En concreto, se propone que las alteraciones en la señalización LC-EP3 subyacen a las diferencias sexuales en las respuestas conductuales y moleculares tanto al estrés agudo como al dolor neuropático crónico. Para contrastar esta hipótesis, se combinaron aproximaciones farmacológicas y basadas en vectores virales para manipular selectivamente la señalización del receptor EP3 en neuronas noradrenérgicas del LC en ratones machos y hembras sometidos a estrés agudo por inmovilización o a un modelo de lesión nerviosa por constricción crónica (CCI). En primer lugar, se cuantificó la expresión basal del receptor EP3 del LC en animales naïve, observándose niveles significativamente más elevados en hembras comparados con los machos. En el modelo de estrés por inmovilización aguda se observó una marcada conducta tipo ansiosa y analgesia en ambos sexos. Sin embargo, la administración intra-LC del agonista EP3, sulprostone, durante la restricción revirtió selectivamente la conducta tipo ansiosa y potenció aún más el efecto analgésico en las hembras, sin observarse efectos en los machos. Estas evidencias subrayan un papel específico según el sexo de la señalización LC-EP3 en la regulación de las respuestas al estrés. Para profundizar en la importancia funcional de la actividad del LC en las hembras, se realizó la activación quimiogenética de neuronas noradrenérgicas del LC con el fin de imitar los efectos del estrés agudo. La activación del LC produjo una clara respuesta ansiogénica, que fue abolida por la administración intra-LC de sulprostone. De forma paralela, la activación del LC indujo analgesia, la cual fue intensificada por la administración de sulprostone. En conjunto, estos resultados demuestran que la señalización del receptor EP3 del LC ejerce una función moduladora y protectora específica de las hembras durante el estrés agudo, proporcionando un mecanismo para las diferencias sexuales observadas en las respuestas conductuales al estrés. A continuación, se evaluó la contribución temporal de las neuronas noradrenérgicas del LC en el dolor y la conducta tipo ansiosa en un modelo de dolor crónico mediante modulación quimiogenética. La caracterización del LC durante la progresión de la neuropatía reveló que su función es temporalmente dinámica en el procesamiento nociceptivo, con una actividad protectora temprana y una desregulación en fases tardías que promueve un perfil maladaptativo pronociceptivo, sin detectarse diferencias entre sexos. En contraste, con respecto a la conducta afectiva, la inhibición de las neuronas noradrenérgicas del LC mostró una clara dependencia del sexo en etapas avanzadas de la neuropatía, produciendo un efecto ansiolítico en los machos pero no en las hembras. De forma análoga al modelo de estrés agudo, se examinó el papel de la señalización del receptor EP3 en la actividad del LC durante el dolor neuropático crónico. En el modelo CCI, la administración intra-LC de sulprostone produjo efectos analgésicos y ansiolíticos en los machos, pero no en las hembras. El análisis por Western blot reveló un aumento en la expresión del receptor EP3 en los machos CCI y una disminución en las hembras en comparación con los controles sham. Asimismo, las enzimas implicadas en la síntesis de prostaglandinas (mPGES1, PTGES3 y COX2) se encontraron reguladas al alza en los machos, mientras que las hembras mostraron una disminución de mPGES1, PTGES3 y COX1, lo que indica la existencia de un entorno molecular dependiente del sexo que regula la señalización mediante PGE₂. Los ensayos de ligación de proximidad demostraron una menor homodimerización del receptor EP3 en las hembras CCI, lo que sugiere una estabilidad o disponibilidad reducida del receptor. Los análisis funcionales mostraron, además, que el sulprostone intra-LC disminuyó los niveles de AMPc en los machos CCI, mientras que los aumentó en las hembras, lo que indica alteraciones dependientes del sexo en el acoplamiento del receptor EP3 y en la eficiencia de su señalización. Adicionalmente, el estudio de las áreas de proyección del LC reveló que el sulprostone intra-LC redujo los niveles de pCREB en el DRt y la BLA de los machos CCI, mientras que los incrementó en esas mismas regiones en las hembras, lo que sugiere que la activación del receptor EP3 en el LC produce efectos dependientes del sexo sobre los circuitos descendentes y ascendentes. Para investigar el papel causal de la señalización EP3, se llevó a cabo la sobreexpresión del receptor EP3, específicamente en las neuronas noradrenérgicas del LC en el modelo de dolor crónico. La sobreexpresión del receptor produjo un efecto analgésico en ambos sexos, mientras que el efecto ansiolítico fue exclusivo de los machos CCI. En consecuencia, la ausencia de efecto ansiolítico en las hembras no puede atribuirse únicamente a la abundancia del receptor, sino que parece deberse a alteraciones en las vías de señalización. De forma consistente con estos resultados, la sobreexpresión de EP3 redujo la expresión de pCREB en ambas regiones en los machos CCI, mientras que no se observaron cambios significativos en las hembras. En conjunto, estos hallazgos demuestran que la señalización LC-EP3 dependiente del sexo determina las respuestas conductuales y moleculares diferenciadas frente al estrés agudo y el dolor neuropático crónico. La elucidación de estos mecanismos proporciona un marco conceptual para el desarrollo de estrategias terapéuticas dirigidas y específicas del sexo centradas en la señalización mediante prostaglandinas, con el fin de mejorar el abordaje del estrés y el dolor."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Prostaglandin EP3 receptor signalling as a contributor to sex differences in stress-related disorders mediated by the locus coeruleus"]}]}],"canonical_facts":{"dc:contributor.advisor":["Berrocoso Domínguez, Esther María","Llorca Torralba, Meritxell"],"dc:contributor.other":["Neurociencias"],"dc:creator":["Martínez Cortés, Adrián"],"dc:date.accessioned":["2026-06-03T07:42:11Z"],"dc:date.available":["2026-06-03T07:42:11Z"],"dc:date.issued":["2026"],"dc:description.abstract":["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.","El estrés y el dolor son fenómenos interconectados, ya que mientras que el estrés agudo desencadena respuestas adaptativas, el estrés crónico y el dolor neuropático crónico conducen a alteraciones persistentes en los circuitos neuronales que regulan el procesamiento afectivo y sensorial. Ambos procesos están estrechamente interrelacionados en múltiples niveles, y los estados nociceptivos prolongados pueden provocar una plasticidad maladaptativa que difiere entre machos y hembras, reflejando las conocidas diferencias sexuales en la respuesta al estrés. El locus coeruleus (LC), principal núcleo noradrenérgico del cerebro, junto con sus áreas de proyección, entre ellas el núcleo reticular dorsal (DRt) y la amígdala basolateral (BLA), desempeñan un papel importante en la modulación de la nocicepción y de las conductas relacionadas con la ansiedad, así como en la coordinación de las respuestas al estrés agudo y crónico. Cabe destacar que el LC presenta características anatómicas y funcionales dependientes del sexo que pueden determinar la vulnerabilidad o resiliencia frente a los trastornos relacionados con el estrés. En este contexto, la señalización mediante prostaglandinas, en particular a través del receptor EP3 localizado en neuronas del LC, ha surgido como un modulador clave del estrés, la nocicepción y la conducta afectiva, proporcionando un vínculo entre las diferencias sexuales, la regulación del estrés y el dolor crónico. La hipótesis central de esta tesis es que el dimorfismo sexual del LC, junto con la señalización diferencial del receptor EP3, contribuye a la vulnerabilidad dependiente del sexo a los trastornos relacionados con el estrés. En concreto, se propone que las alteraciones en la señalización LC-EP3 subyacen a las diferencias sexuales en las respuestas conductuales y moleculares tanto al estrés agudo como al dolor neuropático crónico. Para contrastar esta hipótesis, se combinaron aproximaciones farmacológicas y basadas en vectores virales para manipular selectivamente la señalización del receptor EP3 en neuronas noradrenérgicas del LC en ratones machos y hembras sometidos a estrés agudo por inmovilización o a un modelo de lesión nerviosa por constricción crónica (CCI). En primer lugar, se cuantificó la expresión basal del receptor EP3 del LC en animales naïve, observándose niveles significativamente más elevados en hembras comparados con los machos. En el modelo de estrés por inmovilización aguda se observó una marcada conducta tipo ansiosa y analgesia en ambos sexos. Sin embargo, la administración intra-LC del agonista EP3, sulprostone, durante la restricción revirtió selectivamente la conducta tipo ansiosa y potenció aún más el efecto analgésico en las hembras, sin observarse efectos en los machos. Estas evidencias subrayan un papel específico según el sexo de la señalización LC-EP3 en la regulación de las respuestas al estrés. Para profundizar en la importancia funcional de la actividad del LC en las hembras, se realizó la activación quimiogenética de neuronas noradrenérgicas del LC con el fin de imitar los efectos del estrés agudo. La activación del LC produjo una clara respuesta ansiogénica, que fue abolida por la administración intra-LC de sulprostone. De forma paralela, la activación del LC indujo analgesia, la cual fue intensificada por la administración de sulprostone. En conjunto, estos resultados demuestran que la señalización del receptor EP3 del LC ejerce una función moduladora y protectora específica de las hembras durante el estrés agudo, proporcionando un mecanismo para las diferencias sexuales observadas en las respuestas conductuales al estrés. A continuación, se evaluó la contribución temporal de las neuronas noradrenérgicas del LC en el dolor y la conducta tipo ansiosa en un modelo de dolor crónico mediante modulación quimiogenética. La caracterización del LC durante la progresión de la neuropatía reveló que su función es temporalmente dinámica en el procesamiento nociceptivo, con una actividad protectora temprana y una desregulación en fases tardías que promueve un perfil maladaptativo pronociceptivo, sin detectarse diferencias entre sexos. En contraste, con respecto a la conducta afectiva, la inhibición de las neuronas noradrenérgicas del LC mostró una clara dependencia del sexo en etapas avanzadas de la neuropatía, produciendo un efecto ansiolítico en los machos pero no en las hembras. De forma análoga al modelo de estrés agudo, se examinó el papel de la señalización del receptor EP3 en la actividad del LC durante el dolor neuropático crónico. En el modelo CCI, la administración intra-LC de sulprostone produjo efectos analgésicos y ansiolíticos en los machos, pero no en las hembras. El análisis por Western blot reveló un aumento en la expresión del receptor EP3 en los machos CCI y una disminución en las hembras en comparación con los controles sham. Asimismo, las enzimas implicadas en la síntesis de prostaglandinas (mPGES1, PTGES3 y COX2) se encontraron reguladas al alza en los machos, mientras que las hembras mostraron una disminución de mPGES1, PTGES3 y COX1, lo que indica la existencia de un entorno molecular dependiente del sexo que regula la señalización mediante PGE₂. Los ensayos de ligación de proximidad demostraron una menor homodimerización del receptor EP3 en las hembras CCI, lo que sugiere una estabilidad o disponibilidad reducida del receptor. Los análisis funcionales mostraron, además, que el sulprostone intra-LC disminuyó los niveles de AMPc en los machos CCI, mientras que los aumentó en las hembras, lo que indica alteraciones dependientes del sexo en el acoplamiento del receptor EP3 y en la eficiencia de su señalización. Adicionalmente, el estudio de las áreas de proyección del LC reveló que el sulprostone intra-LC redujo los niveles de pCREB en el DRt y la BLA de los machos CCI, mientras que los incrementó en esas mismas regiones en las hembras, lo que sugiere que la activación del receptor EP3 en el LC produce efectos dependientes del sexo sobre los circuitos descendentes y ascendentes. Para investigar el papel causal de la señalización EP3, se llevó a cabo la sobreexpresión del receptor EP3, específicamente en las neuronas noradrenérgicas del LC en el modelo de dolor crónico. La sobreexpresión del receptor produjo un efecto analgésico en ambos sexos, mientras que el efecto ansiolítico fue exclusivo de los machos CCI. En consecuencia, la ausencia de efecto ansiolítico en las hembras no puede atribuirse únicamente a la abundancia del receptor, sino que parece deberse a alteraciones en las vías de señalización. De forma consistente con estos resultados, la sobreexpresión de EP3 redujo la expresión de pCREB en ambas regiones en los machos CCI, mientras que no se observaron cambios significativos en las hembras. En conjunto, estos hallazgos demuestran que la señalización LC-EP3 dependiente del sexo determina las respuestas conductuales y moleculares diferenciadas frente al estrés agudo y el dolor neuropático crónico. La elucidación de estos mecanismos proporciona un marco conceptual para el desarrollo de estrategias terapéuticas dirigidas y específicas del sexo centradas en la señalización mediante prostaglandinas, con el fin de mejorar el abordaje del estrés y el dolor."],"dc:format":["application/pdf"],"dc:identifier.uri":["http://hdl.handle.net/10498/39689"],"dc:language.iso":["eng"],"dc:rights":["Attribution-NonCommercial-NoDerivatives 4.0 Internacional"],"dc:rights.uri":["http://creativecommons.org/licenses/by-nc-nd/4.0/"],"dc:title":["Prostaglandin EP3 receptor signalling as a contributor to sex differences in stress-related disorders mediated by the locus coeruleus"],"dc:type":["doctoral thesis"]},"updated_at":"2026-07-24T01:29:34Z"}