Ana M. Genaro

Regulation of Hippocampal Gene Expression Is Conserved in Two Species Subjected to Different Stressors and Antidepressant Treatments

BACKGROUND Chronic stress has significant effects on hippocampal structure and function. We have previously identified nerve growth factor (NGF), membrane glycoprotein 6a (M6a), the guanine nucleotide binding protein (G protein) alpha q polypeptide (GNAQ), and CDC-like kinase 1 (CLK-1) as genes regulated by psychosocial stress and clomipramine treatment in the hippocampus of tree shrews. These genes encode proteins involved in neurite outgrowth. METHODS To analyze whether regulation of the above-mentioned genes is conserved between different species, stressors, and antidepressant drugs, we subjected mice to repeated restraint stress and tianeptine treatment and measured hippocampal messenger RNA (mRNA) levels by real time reverse transcription polymerase chain reaction (RT-PCR). RESULTS Chronically stressed mice displayed a reduction in transcript levels for NGF, M6a, GNAQ, and CLK-1. In addition, other genes implicated in neuronal plasticity, such as brain-derived neurotrophic factor (BDNF), cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), protein kinase C (PKC), neural cell adhesion molecule (NCAM), and synapsin I were downregulated in stressed mice. Tianeptine treatment reversed the stress effects for the genes analyzed. Alterations in gene expression were dependent on the duration of the stress treatment and, in some cases, were only observed in male mice. CONCLUSIONS These results suggest that genes involved in neurite remodeling are one of the main targets for regulation by chronic stress. The finding that this regulation is conserved in different stress models and antidepressant treatments highlights the biological relevance of the genes analyzed and suggests that they might be involved in stress-related disorders.

Effects of chronic mild stress on lymphocyte proliferative response. Participation of serum thyroid hormones and corticosterone

There is increasing evidence that stress produces changes in various immune processes. Some of these changes may be due to neurochemical and hormonal alterations including thyroid hormones levels. This work was carried out to study the impact of chronic mild stress (CMS) exposure on proliferative responses and its correlation with serum thyroid hormone levels. In addition, the influence of serum corticosterone levels on these responses was also studied. For this purpose, mice were submitted from1 to 6 weeks to a CMS model. After undergoing the stress schedule for 4 weeks, an alteration in the proliferative response was observed. Lymphocytes from exposed animals showed a decrease in T-cell response to concanavalin-A (Con A) and phytohemagglutinin (PHA) and an increase in B-cell proliferation to lipopolysaccharides (LPS). In parallel, a reduction in T3 and T4 serum levels was observed. On the contrary, serum corticosterone levels increased in animals exposed to CMS for 1 or 2 weeks and then return to normal values. Lowering serum thyroid hormone levels by propylthiouracil (PTU) treatment negatively modulates T-cell response without affecting B-cell response. On the other hand, the substitutive T4 treatment in stressed animals improved significantly the proliferative T-cell response. Non-significative changes in CD4/CD8 ratio were observed neither in stressed, PTU- or T4-treated animals. Taken together, our results suggest an impact of chronic stress on thyroid function that in turn alters T-cell response. These findings may help to elucidate the physiological mechanisms through which stress plays a roll in the etiology of many diseases.

Endogenous nitric oxide signalling system and the cardiac muscarinic acetylcholine receptor-inotropic response

1 In this paper we have determined the different signalling pathways involved in muscarinic acetylcholine receptor (AChR)‐dependent inhibition of contractility in rat isolated atria. 2 Carbachol stimulation of M2 muscarinic AChRs exerts a negative inotropic response, activation of phosphoinositide turnover, stimulation of nitric oxide synthase and increased production of cyclic GMP. 3 Inhibitors of phospholipase C, protein kinase C, calcium/calmodulin, nitric oxide synthase and guanylate cyclase, shifted the dose‐response curve of carbachol on contractility to the right. These inhibitors also attenuated the muscarinic receptor‐dependent increase in cyclic GMP and activation of nitric oxide synthase. In addition, sodium nitroprusside, isosorbide, or 8‐bromo cyclic GMP, induced a negative inotropic effect, increased cyclic GMP and activated nitric oxide synthase. 4 These results suggest that carbachol activation of M2 AChRs, exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositides turnover via phospholipase C activation. This in turn, triggers cascade reactions involving calcium/calmodulin and protein kinase C, leading to activation of nitric oxide synthase and soluble guanylate cyclase.

Altered Expression of Autonomic Neurotransmitter Receptors and Proliferative Responses in Lymphocytes from a Chronic Mild Stress Model of Depression: Effects of Fluoxetine

We studied beta-adrenergic and muscarinic cholinergic receptor (MR) expression and proliferative response in lymphocytes from animals under chronic mild stress (CMS) model of depression (CMS animals). Animals were subjected to CMS (periods of food or water deprivation, changes in lighting conditions, tilted cage, etc.) for 12 weeks. CMS lymphocytes showed an altered mitogen-induced proliferation. CMS-B and -T lymphocytes showed an increment on beta-adrenoceptor number and on intracellular responses to a beta-agonist. CMS-T cells showed higher MR expression and lower cGMP responses than normal lymphocytes. MR were not detectable in normal B cells while CMS-B cells showed both MR expression and cGMP response. Beta and muscarinic stimulation influenced lymphocyte proliferative responses, in accordance with cAMP and cGMP responses. After 12 weeks of the CMS procedure, animals were treated with fluoxetine while the CMS procedure continued. Fluoxetine treatment reverted the alterations induced by CMS. These findings suggest a possible mechanism for the immune alterations found in depressive disorders and for the effect of fluoxetine treatment on immune response.

Role of protein kinase C and cAMP in fluoxetine effects on human T-cell proliferation

In this work, we studied the effect of fluoxetine on human T-lymphocyte proliferation using optimal and suboptimal concanavalin A concentrations. In particular, we analyzed the influence of fluoxetine on the kinases that are involved in intracellular signalling after stimulation with mitogens. We found that fluoxetine promoted the Ca2+ -mediated proteolysis of protein kinase C (PKC) and increased cyclic-AMP (cAMP) levels, thereby impairing lymphocyte proliferation, when optimal concanavalin A concentrations were used. In contrast, when suboptimal concanavalin A concentrations were used, fluoxetine only increased PKC translocation, without modifying cAMP levels, leading to T-cell proliferation. According to our results, fluoxetine has a dual effect on T-cell proliferation by modulating the PKC and protein kinase A pathways. This mechanism is thought to be mediated through Ca2+ mobilization.

Altered lymphocyte catecholamine reactivity in mice subjected to chronic mild stress

There is considerable evidence that the sympathetic nervous system influences the immune response via activation and modulation of beta(2)-adrenergic receptors (beta(2)R). Furthermore, it has been suggested that stress has effects on the sympathetic nervous system. In the present study, we analyzed the influence of catecholamines on the reactivity of lymphocytes from mice exposed to a chronic mild stress (CMS) model of depression (CMS-animals). The effects of the CMS treatment on catecholamine and corticosterone levels and on beta(2)R lymphoid expression were also assessed. For this purpose, animals were subjected to CMS for 8 weeks. Results showed that catecholamines (epinephrine and norepinephrine) exert an inhibitory effect on mitogen-induced normal T-cell proliferation and a stimulatory effect on normal B-cell proliferation in response to selective B lymphocyte mitogens. Specific beta- and beta(2)-antagonists abolished these effects. Lymphocytes from mice subjected to CMS had an increased response to catecholamine-mediated inhibition or enhancement of proliferation in T and B cells, respectively. Moreover, a significant increase in beta(2)R density was observed in animals under CMS compared to normal animals. This was accompanied by an increment in cyclic AMP production after beta-adrenergic stimulation. On the other hand, neither catecholamine levels, determined in both urine and spleen samples, nor serum corticosterone levels showed significant variation between normal and CMS-animals. These findings demonstrate that chronic stress is associated with an increased sympathetic influence on the immune response and may suggest a mechanism through which chronic stress alters immunity.

Chronic stress influences the immune system through the thyroid axis

The aim of the present work was to analyze the effect of chronic stress on thyroid axis and its influence on the immune response. For this purpose a murine model of chronic stress was developed to evaluate and to correlate thyroid hormone levels with humoral alloimmune response. Results show a reduction in serum levels of thyroid hormones, specially a significant decrease in serum levels of triiodotyronine (T3) in stressed animals. On the other hand, alloimmunization was not able to induce an early increment in T3 and thyroxine (T4) levels as it was previously reported in normal animals. In addition, lower titers of alloantibodies were obtained in animals under stress conditions as compared to normal mice. The sustitutive T4 treatment in stressed animals increased significantly alloantibody production as well as the early increment in thyroid hormones after antigenic challenge. These findings suggest that chronic stress induces an alteration of the function of thyroid axis that alters the immune response.

Impaired T-cell dependent humoral response and its relationship with T lymphocyte sensitivity to stress hormones in a chronic mild stress model of depression

The humoral response and the role of catecholamines and corticosterone were analyzed in a chronic mild stress (CMS) model of depression. Mice subjected for more than 6 weeks to CMS showed a significant decrease in T-cell dependent antibody production. However, T-cell independent humoral response was not altered. Serum corticosterone levels and splenic norepinephrine (NE) contents showed an early increase but they were not altered after prolonged CMS exposure. Nevertheless, hormonal inhibitory effect on T lymphocyte reactivity was higher in 6-week CMS mice compared to non-exposed animals. Thus, our results suggest that the impaired T-cell dependent humoral response in a CMS model of depression is neither related to changes in glucocorticoids nor in NE levels but is correlated with an increment of T-cell sensitivity to stress hormones. These findings would underlie the involvement of catecholamines and glucocorticoid lymphocyte receptors in the immune alterations observed in stress and depression.

Stress induced cognitive deficit is differentially modulated in BALB/c and C57Bl/6 mice: Correlation with Th1/Th2 balance after stress exposure

This work shows a comparative study on the effects of chronic mild stress upon learning and memory and immunity, in BALB/c and C57BL/6 mice. Stressed BALB/c, but not C57Bl/6 mice, showed a poor learning performance, morphological alterations in the hippocampus with an increase in oxidative stress. A correlation between poor memory performance and the increase of the Th2/Th1 balance was found. Our results suggest that vulnerability to cognitive deficit associated with stress exposition could be related to a differential regulation of Th1/Th2 cytokine balance, suggesting a better learning performance for individuals that produce Th1 type cytokine after stress exposition.

Fluoxetine Action on Murine T-Lymphocyte Proliferation: Participation of PKC Activation and Calcium Mobilisation

The present study was undertaken to analyse the effect of fluoxetine upon murine T-lymphocyte proliferation. We found that fluoxetine exerted a dual effect, which depended on the degree of lymphocyte activation: at mitogenic concentration (2 microg/mL) of concavalin A (Con A), we observed an inhibitory effect on cellular proliferation, whereas, on submitogenic Con A concentration (1 microg/mL), fluoxetine stimulated the cellular response. Given these facts, we studied PKC activation and calcium mobilisation in both stimulatory and inhibitory effects of fluoxetine on T-cell proliferation. We observed that fluoxetine increased PKC translocation obtained with 1 microg/mL Con A concentration, whereas PKC was degraded when 2 microg/mL was used. This mechanism is thought to be mediated by calcium mobilisation. According to our results, fluoxetine seemed to modulate calcium influx, which, in turn, would influence PKC translocation, modulating the immune response.