The sodium glucose co-transporter 2 inhibitor dapagliflozin ameliorates the fluid-retaining effect of the endothelin A receptor antagonist zibotentan.

BACKGROUND: Endothelin A receptor antagonists (ETARA) slow chronic kidney disease (CKD) progression but their use is limited due to fluid retention and associated clinical risks. Sodium-glucose co-transporter 2 inhibitors (SGLT2i) cause osmotic diuresis and improve clinical outcomes in CKD and heart failure. We hypothesized that co-administration of the SGLT2i dapagliflozin with the ETARA zibotentan would mitigate the fluid retention risk using hematocrit (Hct) and bodyweight as proxies for fluid retention. METHODS: Experiments were performed in 4% salt fed WKY rats. First, we determined the effect of zibotentan (30, 100 or 300 mg/kg/day) on Hct and bodyweight. Second, we assessed the effect of zibotentan (30 or 100 mg/kg/day) alone or in combination with dapagliflozin (3 mg/kg/day) on Hct and bodyweight. RESULTS: Hct at Day 7 was lower in zibotentan versus vehicle groups [zibotentan 30 mg/kg/day, 43% (standard error 1); 100 mg/kg/day, 42% (1); and 300 mg/kg/day, 42% (1); vs vehicle, 46% (1); P < .05], while bodyweight was numerically higher in all zibotentan groups compared with vehicle. Combining zibotentan with dapagliflozin for 7 days prevented the change in Hct [zibotentan 100 mg/kg/day and dapagliflozin, 45% (1); vs vehicle 46% (1); P = .44] and prevented the zibotentan-driven increase in bodyweight (zibotentan 100 mg/kg/day + dapagliflozin 3 mg/kg/day = -3.65 g baseline corrected bodyweight change; P = .15). CONCLUSIONS: Combining ETARA with SGLT2i prevents ETARA-induced fluid retention, supporting clinical studies to assess the efficacy and safety of combining zibotentan and dapagliflozin in individuals with CKD.

Impaired Aversive Memory Formation in GPR37L1KO Mice.

GPR37L1 is an orphan G-protein-coupled receptor, which is implicated in neurological disorders, but its normal physiological role is poorly understood. Its close homologue, GPR37, is implicated in Parkinson's disease and affective disorders. In this study, we set out to characterize adult and middle-aged global GPR37L1 knock-out (KO) mice regarding emotional behaviors. Our results showed that GPR37L1KO animals, except adult GPR37L1KO males, exhibited impaired retention of aversive memory formation as assessed by the shorter retention latency in a passive avoidance task. Interestingly, the viral-mediated deletion of GPR37L1 in conditional knockout mice in the hippocampus of middle-aged mice also showed impaired retention in passive avoidance tasks, similar to what was observed in global GPR37L1KO mice, suggesting that hippocampal GPR37L1 is involved in aversive learning processes. We also observed that middle-aged GPR37L1KO male and female mice exhibited a higher body weight than their wild-type counterparts. Adult and middle-aged GPR37L1KO female mice exhibited a reduced level of serum corticosterone and middle-aged GPR37L1KO females showed a reduced level of epinephrine in the dorsal hippocampus in the aftermath of passive avoidance task, with no such effects observed in GPR37L1KO male mice, suggesting that lack of GPR37L1 influences behavior and biochemical readouts in age- and sex-specific manners.

The Effect of Early Life Stress on Emotional Behaviors in GPR37KO Mice.

GPR37 is an orphan G-protein-coupled receptor, a substrate of parkin which is linked to Parkinson's disease (PD) and affective disorders. In this study, we sought to address the effects of early life stress (ELS) by employing the paradigm of limited nesting material on emotional behaviors in adult GPR37 knockout (KO) mice. Our results showed that, while there was an adverse effect of ELS on various domains of emotional behaviors in wild type (WT) mice in a sex specific manner (anxiety in females, depression and context-dependent fear memory in males), GPR37KO mice subjected to ELS exhibited less deteriorated emotional behaviors. GPR37KO female mice under ELS conditions displayed reduced anxiety compared to WT mice. This was paralleled by lower plasma corticosterone in GPR37KO females and a lower increase in P-T286-CaMKII by ELS in the amygdala. GPR37KO male mice, under ELS conditions, showed better retention of hippocampal-dependent emotional processing in the passive avoidance behavioral task. GPR37KO male mice showed increased immobility in the forced swim task and increased P-T286-CaMKII in the ventral hippocampus under baseline conditions. Taken together, our data showed overall long-term effects of ELS-deleterious or beneficial depending on the genotype, sex of the mice and the emotional context.

Peripubertal stress-induced heightened aggression: modulation of the glucocorticoid receptor in the central amygdala and normalization by mifepristone treatment.

Despite the enormous negative impact of excessive aggression for individuals and societies, there is a paucity of treatments. Here, using a peripubertal stress model of heightened aggression in rats, we investigated the involvement of the glucocorticoid system and tested the effectiveness of antiglucocorticoid treatment to normalize behavior. We assessed peripubertal stress-induced changes in glucocorticoid (GR) and mineralocorticoid (MR) gene expression in different amygdala nuclei and hippocampus, and report a specific increase in GR mRNA expression in the central amygdala (CeA). Administration of mifepristone (10 mg/kg), a GR antagonist, before stressor exposure at peripuberty prevented the habituation of plasma corticosterone responses observed throughout the stress protocol. This treatment also prevented the increase in aggression and GR expression in the CeA observed in peripubertally stressed rats at adulthood. Viral downregulation of CeA GR expression at adulthood led to reduced aggression. Subsequently, we showed that a brief, 3-day, treatment with mifepristone at adulthood was effective to normalize the abnormal aggression phenotype in peripubertally stressed rats. Our results support a key role for GR actions during peripubertal stress for the long-term programming of heightened aggression. Strikingly, they also support the translational interest of testing the effectiveness of mifepristone treatment to diminish reactive aggression in early adversity-related human psychopathologies.

CRHR1 links peripuberty stress with deficits in social and stress-coping behaviors.

Stressful life events during childhood and adolescence are important risk factors for the development of psychopathologies later in life. The corticotropin releasing hormone (CRH) and the CRH receptor 1 (CRHR1) have been implicated in the link between early life adversity and adult anxiety and depression, with rodent studies identifying the very early postnatal period as highly susceptible to this programming. Here, we investigated whether stress exposure during the peripubertal period - comprising juvenility and puberty - is effective in inducing long-lasting changes in the expression of CRHR1 and CRHR2 in the hippocampus and amygdala, and whether treating animals with a CRHR1 antagonist following stress exposure could reverse behavioral alterations induced by peripuberty stress. We show that peripuberty stress leads to enhanced expression of the Crhr1, but not Crhr2, gene in the hippocampal CA1 and the central nucleus of the amygdala, in association with social deficits in the social exploration test and increased stress-coping behaviors in the forced swim test. Treatment with the CRHR1 antagonist NBI30775 (10 mg/kg) daily for 1 week (from P43 to P49), immediately following peripuberty stress exposure, prevented the occurrence of those psychopathological behaviors at adulthood. These findings highlight peripuberty as a period of plasticity for the enduring modulation of the CRHR1 system and support a growing body of data implicating the CRHR1 system in the programming effects of early life stress on eventual psychopathology. They also support recent evidence indicating that temporarily tackling CRHR1 during development might represent a therapeutic opportunity to correct behavioral trajectories linking early stress to adult psychopathology.

Long-term behavioral programming induced by peripuberty stress in rats is accompanied by GABAergic-related alterations in the Amygdala.

Stress during childhood and adolescence is a risk factor for psychopathology. Alterations in γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain, have been found following stress exposure and fear experiences and are often implicated in anxiety and mood disorders. Abnormal amygdala functioning has also been detected following stress exposure and is also implicated in anxiety and social disorders. However, the amygdala is not a unitary structure; it includes several nuclei with different functions and little is known on the potential differences the impact of early life stress may have on this system within different amygdaloid nuclei. We aimed here to evaluate potential regional differences in the expression of GABAergic-related markers across several amygdaloid nuclei in adult rats subjected to a peripuberty stress protocol that leads to enhanced basal amygdala activity and psychopathological behaviors. More specifically, we investigated the protein expression levels of glutamic acid decarboxylase (GAD; the principal synthesizing enzyme of GABA) and of GABA-A receptor subunits α2 and α3. We found reduced GAD and GABA-A α3, but not α2, subunit protein levels throughout all the amygdala nuclei examined (lateral, basolateral, basomedial, medial and central) and increased anxiety-like behaviors and reduced sociability in peripubertally stressed animals. Our results identify an enduring inhibition of the GABAergic system across the amygdala following exposure to early adversity. They also highlight the suitability of the peripuberty stress model to investigate the link between treatments targeting the dysfunctional GABAergic system in specific amygdala nuclei and recovery of specific stress-induced behavioral dysfunctions.

Increased corticosterone in peripubertal rats leads to long-lasting alterations in social exploration and aggression.

Stress during childhood and adolescence enhances the risk of psychopathology later in life. We have previously shown that subjecting male rats to stress during the peripubertal period induces long-lasting effects on emotion and social behaviors. As corticosterone is increased by stress and known to exert important programming effects, we reasoned that increasing corticosterone might mimic the effects of peripubertal stress. To this end, we injected corticosterone (5 mg/kg) on 7 scattered days during the peripuberty period (P28-P30, P34, P36, P40, and P42), following the same experimental schedule as for stress administration in our peripubertal paradigm. We measured play behavior in the homecage and, at adulthood, the corticosterone response to novelty and behavioral responses in tests for anxiety- and depression-like behaviors, aggression, and social exploration. As compared to vehicle, corticosterone-treated animals exhibit more aggressive play behavior during adolescence, increased aggressive behavior in a resident-intruder (RI) test while reduced juvenile exploration and corticosterone reactivity at adulthood. Whereas the corticosterone treatment mimicked alterations induced by the peripuberty stress protocol in the social domain, it did not reproduce previously observed effects of peripuberty stress on increasing anxiety-like and depression-like behaviors, respectively evaluated in the elevated plus maze (EPM) and the forced swim tests. Our findings indicate that increasing corticosterone levels during peripuberty might be instrumental to program alterations in the social domain observed following stress, whereas other factors might need to be recruited for the programming of long-term changes in emotionality. Our study opens the possibility that individual differences on the degree of glucocorticoid activation during peripuberty might be central to defining differences in vulnerability to develop psychopathological disorders coursing with alterations in the social realm.

Early stress evokes age-dependent biphasic changes in hippocampal neurogenesis, BDNF expression, and cognition.

BACKGROUND: Adult-onset stressors exert opposing effects on hippocampal neurogenesis and cognition, with enhancement observed following mild stress and dysfunction following severe chronic stress. While early life stress evokes persistent changes in anxiety, it is unknown whether early stress differentially regulates hippocampal neurogenesis, trophic factor expression, and cognition across the life span. METHODS: Hippocampal-dependent cognitive behavior, neurogenesis, and epigenetic regulation of brain-derived neurotrophic factor (Bdnf) expression was examined at distinct time points across the life span in rats subjected to the early stress of maternal separation (ES) and control groups. We also examined the influence of chronic antidepressant treatment on the neurogenic, neurotrophic, and cognitive changes in middle-aged ES animals. RESULTS: Animals subjected to early stress of maternal separation examined during postnatal life and young adulthood exhibited enhanced hippocampal neurogenesis, decreased repressive histone methylation at the Bdnf IV promoter along with enhanced BDNF levels, and improved performance on the stress-associated Morris water maze. Strikingly, opposing changes in hippocampal neurogenesis and epigenetic regulation of Bdnf IV expression, concomitant with impairments on hippocampal-dependent cognitive tasks, were observed in middle-aged ES animals. Chronic antidepressant treatment with amitriptyline attenuated the maladaptive neurogenic, epigenetic, transcriptional, and cognitive effects in middle-aged ES animals. CONCLUSIONS: Our study provides novel insights into the short- and long-term consequences of ES, demonstrating both biphasic and unique, age-dependent changes at the molecular, epigenetic, neurogenic, and behavioral levels. These results indicate that early stress may transiently endow animals with a potential adaptive advantage in stressful environments but across a life span is associated with long-term deleterious effects.