Intravenous Reelin Treatment Rescues Atrophy of Spleen White Pulp and Correlates to Rescue of Forced Swim Test Immobility and Neurochemical Alterations Induced by Chronic Stress.

Reelin, an extracellular matrix protein with putative antidepressant-like properties, becomes dysregulated by chronic stress. Improvement in cognitive dysfunction and depression-like behavior induced by chronic stress has been reported with both intrahippocampal and intravenous Reelin treatment but the mechanisms responsible are not clear. To determine if treatment with Reelin modifies chronic stress-induced dysfunction in immune organs and whether this relates to behavioral and/or neurochemical outcomes, spleens were collected from both male (n = 62) and female (n = 53) rats treated with daily corticosterone injections for three weeks that received Reelin or vehicle. Reelin was intravenously administered once on the final day of chronic stress, or repeatedly, with weekly treatments throughout chronic stress. Behavior was assessed during the forced swim test and the object-in-place test. Chronic corticosterone caused significant atrophy of the spleen white pulp, but treatment with a single shot of Reelin restored white pulp in both males and females. Repeated Reelin injections also resolved atrophy in females. Correlations were observed between recovery of white pulp atrophy and recovery of behavioral deficits and expression of both Reelin and glutamate receptor 1 in the hippocampus, supporting a role of the peripheral immune system in the recovery of chronic stress-induced behaviors following treatment with Reelin. Our data adds to research indicating Reelin could be a valuable therapeutic target for chronic stress-related disorders including major depression.

Sex differences in basal reelin levels in the paraventricular hypothalamus and in response to chronic stress induced by repeated corticosterone in rats.

Repeated exposure to the stress hormone corticosterone results in depressive-like behaviours paralleled by the downregulation of hippocampal reelin expression. Reelin is expressed in key neural populations involved in the stress response, but whether its hypothalamic expression is sex-specific or involved in sex-specific vulnerability to stress is unknown. Female and male rats were treated with either daily vehicle or corticosterone injections (40 mg/kg) for 21 days. Thereafter, they were subjected to several behavioural tasks before being sacrificed to allow the analysis of reelin expression in hypothalamic nuclei. The basal density of reelin-positive cells in males was significantly higher in the paraventricular nucleus (19 %) and in the medial preoptic area (51 %) compared to females. Chronic corticosterone injections increased the immobility time in the forced swim test in males (107 %) and females (108 %) and decreased the exploration of the elevated plus maze in males (34 %). Corticosterone also caused a significant decrease in the density of reelin-positive cells in males, in both ventrodorsal (37 %) and ventrolateral (32 %) subdivisions of the paraventricular nucleus, while not affecting females. Moreover, in the paraventricular nucleus of males, 30 % of the basal reelin-positive cells co-expressed oxytocin while only 17.5 % did in females, showing a positive correlation between reelin and oxytocin levels. Chronic corticosterone did not significantly affect co-localization levels. For the first time, this study shows that there is a sexually dimorphic subpopulation of reelin-positive neurons in the paraventricular nucleus that can be differentially affected by chronic stress.

Reelin has antidepressant-like effects after repeated or singular peripheral injections.

Chronic stress is a significant risk factor for depression onset. The effects of chronic stress can be studied preclinically using a corticosterone (CORT)-administration paradigm that results in a phenotype of depressive-like behavior associated with neurochemical abnormalities in brain regions like the hippocampus. We have recently shown that intrahippocampal infusions of Reelin have a fast effect in normalizing CORT-induced behavioral and neurochemical alterations. Reelin is also expressed in multiple peripheral systems and is found in blood plasma which prompted us to investigate whether peripheral intravenous (i.v.) Reelin injections could also result in antidepressant (ATD)-like actions. Repeated i.v. injections of Reelin were effective in rescuing the CORT-induced increases in forced-swim-test immobility in male and female rats, decreases in Reelin-immunopositive cells in the dentate gyrus subgranular zone, the expression of hippocampal GABAAß2/3, GluA1, and GluN2B receptors, and serotonin transporter (SERT) membrane protein clustering (MPC) in blood lymphocytes. However, Reelin had only a partial effect on the number and maturation rate of dentate gyrus newborn cells. CORT and Reelin did not affect open field test behavior. After evaluating the effects of multiple Reelin injections, we demonstrated that a single Reelin injection administered at the end of CORT treatment could rescue in 24 h the behavioral (forced-swim-test and object-in-place test), as well as SERT MPC and neurochemical effects of CORT. These findings show that i.v. injections of Reelin have fast ATD-like effects associated with the restoration of hippocampal neurochemical deficits. Although additional mechanistic and pharmacokinetic studies are necessary, our data open the possibility to develop Reelin-based therapeutics with putative fast-ATD activity.

Prenatal Exposure to Bisphenols and Phthalates and Postpartum Depression: The Role of Neurosteroid Hormone Disruption.

CONTEXT: Postpartum depression (PPD) is a serious psychiatric disorder. While causes remain poorly understood, perinatal sex hormone fluctuations are an important factor, and allopregnanolone in particular has emerged as a key determinant. Although synthetic environmental chemicals such as bisphenols and phthalates are known to affect sex hormones, no studies have measured allopregnanolone and the consequences of these hormonal changes on PPD have not been interrogated. OBJECTIVE: To investigate associations of repeated measures of urinary bisphenols and phthalates in early and midpregnancy with serum pregnenolone, progesterone, allopregnanolone, and pregnanolone concentrations in midpregnancy and PPD symptoms at 4 months postpartum. METHODS: Prospective cohort study of 139 pregnant women recruited between 2016 and 2018. Bisphenols and phthalates were measured in early and midpregnancy urine samples. Serum sex steroid hormone concentrations were measured in midpregnancy. PPD was assessed at 4 months postpartum using the Edinburgh Postnatal Depression Scale (EPDS). Multiple informant models were fit using generalized estimating equations. Serum levels of allopregnanolone, progesterone, pregnanolone, and pregnenolone were examined as log-transformed continuous variables. PPD symptoms were examined as continuous EPDS scores and dichotomously with scores ≥10 defined as PPD. RESULTS: Di-n-octyl phthalate (DnOP) and diisononyl phthalate (DiNP) metabolites were associated with reduced progesterone concentrations. Log-unit increases in ∑DnOP and ∑DiNP predicted 8.1% (95% CI -15.2%, -0.4%) and 7.7% (95% CI -13.3%, -1.7%) lower progesterone, respectively. ∑DnOP was associated with increased odds of PPD (odds ratio 1.48; 95% CI 1.04, 2.11). CONCLUSION: Endocrine disrupting chemicals may influence hormonal shifts during pregnancy as well as contribute to PPD.

Ketamine Rescues Hippocampal Reelin Expression and Synaptic Markers in the Repeated-Corticosterone Chronic Stress Paradigm.

Depression is the leading cause of disability worldwide, which necessitates novel therapeutics and biomarkers to approach treatment of this neuropsychiatric disorder. To assess potential mechanisms underlying the fast-acting antidepressant actions of ketamine we used a repeated corticosterone paradigm in adult male rats to assess the effects of ketamine on reelin-positive cells, a protein largely implicated in the pathophysiology of depression. We also assessed the effects of reelin and ketamine on hippocampal and cerebellar synpatosomes, and on serotonin transporter clustering in peripheral lymphocytes to determine reelin and ketamine's impact at the synaptic and peripheral levels. Reelin and ketamine similarly rescue synaptic expression of mTOR and p-mTOR that were decreased by corticosterone. Reelin, but not ketamine, was able to rescue patterns of serotonin transporter clustering in the periphery. These findings display ketamine as a powerful modulator of reelin expression and lend strength to further evaluation of the putative fast antidepressant-like actions of reelin.

Fast-acting antidepressant-like effects of Reelin evaluated in the repeated-corticosterone chronic stress paradigm.

The present report examines the effects of repeated or single intrahippocampal Reelin infusions on measures of depressive-like behavior, cognition, and hippocampal neurogenesis in the repeated-corticosterone (CORT) paradigm. Rats received subcutaneous injections of CORT for 3 weeks and Reelin was infused through an inserted canula in the left hippocampus on days 7, 14, and 21, or only on day 21 of CORT injections. CORT increased immobility in the forced-swim test and impaired object-location memory. Notably, these effects were reversed by both repeated and single-Reelin infusions. CORT decreased both the number and complexity of doublecortin-labeled maturing newborn neurons in the dentate gyrus subgranular zone, and a single-Reelin infusion increased the number but not complexity of newborn neurons, while repeated Reelin infusions restored both. Injection of the AMPA antagonist CNQX blocked the rescue of the behavioral phenotype by Reelin but did completely block the effects of Reelin on hippocampal neurogenesis. Reelin is able to rescue the deficits in AMPA, NMDA, GABAA receptors, mTOR and p-mTOR induced by CORT. These novel results demonstrate that a single intrahippocampal Reelin infusion into the dorsal hippocampus has fast-acting antidepressant-like effects, and that some of these effects may be at least partially independent of Reelin actions on hippocampal neurogenesis.

Patterns of Membrane Protein Clustering in Peripheral Lymphocytes as Predictors of Therapeutic Outcomes in Major Depressive Disorder.

There is an utmost necessity of developing novel biomarkers of depression that result in a more efficacious use of current antidepressant drugs. The present report reviews and discusses a recent series of experiments that focused on analysis of membrane protein clustering in peripheral lymphocytes as putative biomarkers of therapeutic efficacy for major depressive disorder. This review recapitulates how the ideas were originated, and the main findings demonstrated that analysis of serotonin transporter and serotonin 2 A receptor clustering in peripheral lymphocytes of naïve depression patients resulted in a discrimination of two subpopulations of depressed patients that showed a differential response upon 8 weeks of antidepressant treatment. The paper also reviews the usefulness of animal models of depression for an initial evaluation of membrane protein clustering in lymphocytes, which provides a screening tool to determine additional proteins to be further evaluated in depression patients. Finally, the present review provides a brief discussion of the general field of biomarkers of depression in relation to therapeutic outcomes and suggests additional ideas to provide extra value to the reviewed studies.

Exploring the Potential Antidepressant Mechanisms of TNFα Antagonists.

Human and animal studies suggest an intriguing relationship between the immune system and the development of depression. Some peripherally produced cytokines, such as TNF-α, can cross the blood brain barrier and result in activation of brain microglia which produces additional TNF-α and fosters a cascade of events including decreases in markers of synaptic plasticity and increases in neurodegenerative events. This is exemplified by preclinical studies, which show that peripheral administration of pro-inflammatory cytokines can elicit depression-like behavior. Importantly, this depression-like behavior can be ameliorated by anti-cytokine therapies. Work in our laboratory suggests that TNF-α is particularly important for the development of a depressive phenotype and that TNF-α antagonists might have promise as novel antidepressant drugs. Future research should examine rates of inflammation at baseline in depressed patients and whether anti-inflammatory agents could be included as part of the treatment regimen for depressive disorders.

Changes in Membrane Protein Clustering in Peripheral Lymphocytes in an Animal Model of Depression Parallel Those Observed in Naïve Depression Patients: Implications for the Development of Novel Biomarkers of Depression.

Naïve depression patients show alterations in serotonin transporter (SERT) and serotonin 2A (5HT2A) receptor clustering in peripheral lymphocytes, and these alterations have been proposed as a biomarker of therapeutic efficacy in major depression. Repeated corticosterone (CORT) induces a consistent depression-like phenotype and has been widely used as an animal model to study neurobiological alterations underlying the depressive symptoms. In this experiment, we used the CORT paradigm to evaluate whether depression-like behavior is associated with similar changes in the pattern of SERT and 5HT2A membrane protein clustering as those observed in depression patients. We also analyzed the clustering of other proteins expressed in lipid rafts in lymphocytes. Rats received daily CORT or vehicle injections for 21 consecutive days. Afterward they underwent the forced swim test to evaluate depression-like behavior, and isolated lymphocytes were analyzed by immunocytochemistry coupled to image-analysis to study clustering parameters of the SERT, 5HT2A receptor, dopamine transporter (DAT), Beta2 adrenergic receptor (ß2AR), NMDA 2B receptor (NR2B), Pannexin 1 (Pnx1), and prion cellular protein (PrPc). Our results showed that CORT increases the size of protein clusters for all proteins with the exception of ß 2AR, which is decreased. CORT also increased the number of clusters for Pnx1 and PrPc only. Overall, these results indicate that alterations in SERT and 5HT2A protein clustering in naïve depression patients are paralleled by changes seen in an animal model of depression. The CORT paradigm may be a useful screen for examining additional proteins in lymphocytes as a preliminary step prior to their analysis as biomarkers of depression in human blood samples.

Mitochondria and Mood: Mitochondrial Dysfunction as a Key Player in the Manifestation of Depression.

Human and animal studies suggest an intriguing link between mitochondrial diseases and depression. Although depression has historically been linked to alterations in monoaminergic pharmacology and adult hippocampal neurogenesis, new data increasingly implicate broader forms of dampened plasticity, including plasticity within the cell. Mitochondria are the cellular powerhouse of eukaryotic cells, and they also regulate brain function through oxidative stress and apoptosis. In this paper, we make the case that mitochondrial dysfunction could play an important role in the pathophysiology of depression. Alterations in mitochondrial functions such as oxidative phosphorylation (OXPHOS) and membrane polarity, which increase oxidative stress and apoptosis, may precede the development of depressive symptoms. However, the data in relation to antidepressant drug effects are contradictory: some studies reveal they have no effect on mitochondrial function or even potentiate dysfunction, whereas other studies show more beneficial effects. Overall, the data suggest an intriguing link between mitochondrial function and depression that warrants further investigation. Mitochondria could be targeted in the development of novel antidepressant drugs, and specific forms of mitochondrial dysfunction could be identified as biomarkers to personalize treatment and aid in early diagnosis by differentiating between disorders with overlapping symptoms.

Comparative study of two protocols for quantitative image-analysis of serotonin transporter clustering in lymphocytes, a putative biomarker of therapeutic efficacy in major depression.

BACKGROUND: The pattern of serotonin transporter clustering on the plasma membrane of lymphocytes extracted from human whole blood samples has been identified as a putative biomarker of therapeutic efficacy in major depression. Here we evaluated the possibility of performing a similar analysis using blood smears obtained from rats, and from control human subjects and depression patients. We hypothesized that we could optimize a protocol to make the analysis of serotonin protein clustering in blood smears comparable to the analysis of serotonin protein clustering using isolated lymphocytes. RESULTS: Our data indicate that blood smears require a longer fixation time and longer times of incubation with primary and secondary antibodies. In addition, one needs to optimize the image analysis settings for the analysis of smears. When these steps are followed, the quantitative analysis of both the number and size of serotonin transporter clusters on the plasma membrane of lymphocytes is similar using both blood smears and isolated lymphocytes. CONCLUSIONS: The development of this novel protocol will greatly facilitate the collection of appropriate samples by eliminating the necessity and cost of specialized personnel for drawing blood samples, and by being a less invasive procedure. Therefore, this protocol will help us advance the validation of membrane protein clustering in lymphocytes as a biomarker of therapeutic efficacy in major depression, and bring it closer to its clinical application.

Reelin-Related Disturbances in Depression: Implications for Translational Studies.

The finding that reelin expression is significantly decreased in mood and psychotic disorders, together with evidence that reelin can regulate key aspects of hippocampal plasticity in the adult brain, brought our research group and others to study the possible role of reelin in the pathogenesis of depression. This review describes recent progress on this topic using an animal model of depression that makes use of repeated corticosterone (CORT) injections. This methodology produces depression-like symptoms in both rats and mice that are reversed by antidepressant treatment. We have reported that CORT causes a decrease in the number of reelin-immunopositive cells in the dentate gyrus subgranular zone (SGZ), where adult hippocampal neurogenesis takes place; that down-regulation of the number of reelin-positive cells closely parallels the development of a depression-like phenotype during repeated CORT treatment; that reelin downregulation alters the co-expression of reelin with neuronal nitric oxide synthase (nNOS); that deficits in reelin might also create imbalances in glutamatergic and GABAergic circuits within the hippocampus and other limbic structures; and that co-treatment with antidepressant drugs prevents both reelin deficits and the development of a depression-like phenotype. We also observed alterations in the pattern of membrane protein clustering in peripheral lymphocytes in animals with low levels of reelin. Importantly, we found parallel changes in membrane protein clustering in depression patients, which differentiated two subpopulations of naïve depression patients that showed a different therapeutic response to antidepressant treatment. Here, we review these findings and develop the hypothesis that restoring reelin-related function could represent a novel approach for antidepressant therapies.

Serotonin transporter clustering in blood lymphocytes predicts the outcome on anhedonia scores in naïve depressive patients treated with antidepressant medication.

BACKGROUND: We have shown that serotonin transporter (SERT) clustering in blood lymphocytes is altered in major depression and correlates with pharmacological therapeutic responses measured with the Hamilton scale. In the present report, we extend these results to the self-assessment anhedonia scale, as anhedonia is a cardinal symptom of major depression that is difficult to treat with first-line antidepressants. METHODS: We collected blood samples from 38 untreated depression patients at the time of enrolment and 8 weeks after pharmacological treatment. We used the self-assessment anhedonia scale to evaluate anhedonia symptoms before and after treatment. We also used quantitative immunocytochemistry to measure SERT clusters in blood lymphocytes. RESULTS: Evaluation of the distribution of SERT clusters size in the plasma membrane of lymphocytes identified two subpopulations of naive depression patients: Depression I (D-I) and Depression II (D-II). While naïve D-I and D-II patients initially showed similar anhedonia scores, D-II patients showed a good response in anhedonia symptoms after 8 weeks of psychopharmacological treatment, whereas D-I patients failed to show any improvement. Psychopharmacological treatment also induced an increase in the number of SERT clusters in lymphocytes in the D-II group, and this increase correlated with the improvement in anhedonia symptoms. CONCLUSIONS: SERT clustering in peripheral lymphocytes can be used to identify patient response to antidepressant therapy as ascertained by anhedonia scores.

Imipramine protects against the deleterious effects of chronic corticosterone on depression-like behavior, hippocampal reelin expression, and neuronal maturation.

We have hypothesized that a downregulation of reelin and deficient maturation of adult-born hippocampal neurons are important factors in the pathogenesis of depression. This hypothesis is based on previous work showing that depression-like behavior in rats treated with protracted corticosterone develops in concert with decreased dendritic complexity in newborn hippocampal granule neurons and decreased reelin expression in the proliferative subgranular zone of the dentate gyrus. In addition, heterozygous reeler mice with approximately 50% of normal brain levels of reelin are more vulnerable to the depressogenic effects of corticosterone than wild-type mice. The purpose of this experiment was to provide pharmacological validation for the link between reelin, neuronal maturation, and depression by examining whether the deleterious effects of corticosterone on these measures could be prevented by co-administration of the antidepressant imipramine. Rats received corticosterone injections, corticosterone injections plus either 10 or 15mg/kg imipramine injections, or vehicle injections for 21 consecutive days. They were then subjected to the forced swim test to assess depression-like behavior and sacrificed for immunohistochemical examination of immature neuron number and dendritic complexity and the presence of reelin+cells. We found that corticosterone increases depression-like behavior, decreases the number of reelin+cells in the subgranular zone, and decreases the number and complexity of immature neurons in the granule cell layer. All of these behavioral and cellular phenotypes were prevented by imipramine, providing further support for the idea that reelin is involved in the pathogenesis of depression.

Differential effects of corticosterone on the colocalization of reelin and neuronal nitric oxide synthase in the adult hippocampus in wild type and heterozygous reeler mice.

Repeated corticosterone (CORT) treatment induces a deficit in dentate gyrus subgranular zone reelin-positive cells, in maturation of newborn neurons, and results in a consistent depressive-like behavior. However, the molecular mechanisms underlying these processes are not known in detail. The purpose of the present study was to characterize the effect of three weeks of 20mg/kg CORT injections in the number of reelin and neuronal nitric oxide synthase (nNOS), as well as their colocalization, in hippocampal regions in wild type (WTM) and heterozygous reeler mice (HRM). ANOVA analysis shows a CORT×genotype interaction in the density of reelin+ cells co-localizing nNOS in the dentate subgranular zone and stratum-lacunosum moleculare, and in the density of nNOS+ cells in the hilus. There is a main effect of CORT in the density of both reelin+ and nNOS+ cells in the dentate subgranular zone and hilus, and in reelin+ cells in the molecular layer and CA3 stratum radiatum; and a main effect of genotype on the co-localization of both markers in the dentate subgranular zone, and in the density of reelin+ cell sin the stratum lacunosum moleculare. These alterations suggest a possible interconnection between reelin and nNOS expression that is altered by repeated CORT treatment.

Reelin as a putative vulnerability factor for depression: examining the depressogenic effects of repeated corticosterone in heterozygous reeler mice.

We examined a potential two-hit murine animal model of depression by assessing whether a genetic deficit in reelin increases vulnerability to the depressogenic effects of the stress hormone corticosterone. Stress is an identified risk factor for the onset of depressive symptoms, but depression also has a significant genetic component, suggesting that environmental factors and genetic background likely interact in the etiology of depression. Previous results have revealed that reelin levels are decreased in post-mortem hippocampal tissue from patients with schizophrenia, bipolar disorder and depression, and also in an animal model of depression. Therefore, we hypothesized that heterozygous reeler mice (HRM), with approximately 50% normal levels of reelin, would be more sensitive to the depressogenic effects of corticosterone than wild-type mice (WTM). Mice received subcutaneous injections of either vehicle or 5 mg/kg, 10 mg/kg, or 20 mg/kg of corticosterone for 21 consecutive days, and then they were assessed for changes in depression-like behavior, hippocampal reelin expression, and hippocampal neurogenesis. Corticosterone produced dose-dependent increases in depression-like behavior and decreases in reelin expression, neurogenesis, and cell maturation regardless of mouse genotype. There were no differences between the vehicle-injected HRM and WTM in these measures. However, the effects of CORT on behavior, the number of reelin-positive cells in the subgranular zone or hilus, and hippocampal neurogenesis were more pronounced in the HRM than in the WTM, providing support for the idea that mice with impaired reelin signaling may be more vulnerable to the deleterious effects of glucocorticoids. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Serotonin transporter clustering in blood lymphocytes of reeler mice.

Serotonin transporter clustering is an important feature for regulation of this transporter activity. We used immunocytochemistry to analyze alterations in serotonin transporter clustering in blood lymphocytes of reeler mice. Serotonin transporter immunolabelling is observed mostly as a patchy staining in lymphocytes membranes. Comparison of the number and size of serotonin transporter clusters in wild-type mice, heterozygous reeler mice, and homozygous reeler mice showed an increase in the number and size of clusters in heterozygous reeler mice, but only an increase in clusters size in homozygous reeler mice. Reelin is down-regulated in the brain of schizophrenia, autism, and mood disorders, and is also expressed in blood plasma. There is the possibility therefore that alterations in serotonin transporter clustering in blood lymphocytes associated with a decrease in reelin expression may be operative in some cardiovascular or immune system alterations showing comorbidity with these mental disorders.

The coexpression of reelin and neuronal nitric oxide synthase in a subpopulation of dentate gyrus neurons is downregulated in heterozygous reeler mice.

Reelin is an extracellular matrix protein expressed in several interneuron subtypes in the hippocampus and dentate gyrus. Neuronal nitric oxide synthase (nNOS) is also expressed by interneurons in these areas. We investigated whether reelin and nNOS are co-localized in the same population of hippocampal interneurons, and whether this colocalization is altered in the heterozygous reeler mouse. We found colocalization of nNOS in reelin-positive cells in the CA1 stratum radiatum and lacunosum moleculare, the CA3 stratum radiatum, and the dentate gyrus subgranular zone, molecular layer, and hilus. In heterozygous reeler mice, the colocalization of nNOS in reelin-positive cells was significantly decreased only in the subgranular zone and molecular layer. The coexpression of reelin and nNOS in several hippocampal regions suggests that reelin and nNOS may work synergistically to promote glutamatergic function, and the loss of this coexpression in heterozygous reeler mice may underlie some of the behavioral deficits observed in these animals.