Brain network reorganization differs in response to stress in rats genetically predisposed to depression and stress-resilient rats.

Treatment-resistant depression (TRD) remains a pressing clinical problem. Optimizing treatment requires better definition of the specificity of the involved brain circuits. The rat strain bred for negative cognitive state (NC) represents a genetic animal model of TRD with high face, construct and predictive validity. Vice versa, the positive cognitive state (PC) strain represents a stress-resilient phenotype. Although NC rats show depressive-like behavior, some symptoms such as anhedonia require an external trigger, i.e. a stressful event, which is similar to humans when stressful event induces a depressive episode in genetically predisposed individuals (gene-environment interaction). We aimed to distinguish neurobiological predisposition from the depressogenic pathology at the level of brain-network reorganization. For this purpose, resting-state functional magnetic resonance imaging time series were acquired at 9.4 Tesla scanner in NC (N=11) and PC (N=7) rats before and after stressful event. We used a graph theory analytical approach to calculate the brain-network global and local properties. There was no difference in the global characteristics between the strains. At the local level, the response in the risk strain was characterized with an increased internodal role and reduced local clustering and efficiency of the anterior cingulate cortex (ACC) and prelimbic cortex compared to the stress-resilient strain. We suggest that the increased internodal role of these prefrontal regions could be due to the enhancement of some of their long-range connections, given their connectivity with the amygdala and other default-mode-like network hubs, which could create a bias to attend to negative information characteristic for depression.

Reduced connectivity and inter-hemispheric symmetry of the sensory system in a rat model of vulnerability to developing depression.

Defining the markers corresponding to a high risk of developing depression in humans would have major clinical significance; however, few studies have been conducted since they are not only complex but also require homogeneous groups. This study compared congenital learned helpless (cLH) rats, selectively bred for high stress sensitivity and learned helplessness (LH) behavior, to congenital non-learned helpless (cNLH) rats that were bred for resistance to uncontrollable stress. Naïve cLH rats show some depression-like behavior but full LH behavior need additional stress, making this model ideal for studying vulnerability to depression. Resting-state functional connectivity obtained from seed correlation analysis was calculated for multiple regions that were selected by anatomy AND by a data-driven approach, independently. Significance was determined by t-statistic AND by permutation analysis, independently. A significant reduction in functional connectivity was observed by both analyses in the cLH rats in the sensory, motor, cingulate, infralimbic, accumbens and the raphe nucleus. These reductions corresponded primarily to reduced inter-hemispheric connectivity. The main reduction however was in the sensory system. It is argued that reduced connectivity and inter-hemispheric connectivity of the sensory system reflects an internal convergence state which may precede other depressive symptomatology and therefore could be used as markers for vulnerability to the development of depression.

Pharmacological inhibition of the lateral habenula improves depressive-like behavior in an animal model of treatment resistant depression.

Identifying new treatment approaches for treatment resistant depression (TRD) is an important topic for translational psychiatry. Functional inhibition of the lateral habenula (LHb) has recently been claimed to offer such an option for TRD. Rats which are bred for high susceptibility to develop learned helplessness provide a genetic model for TRD. We used the gamma-aminobutyric acid agonist muscimol to inhibit the LHb in Sprague-Dawley rats with congenital learned helplessness (cLH). Stereotactic pharmacological inhibition of the LHb exerted antidepressive effects in treatment resistant cLH rats.

Finasteride treatment inhibits adult hippocampal neurogenesis in male mice.

INTRODUCTION: The 5-alpha-reductase inhibitor finasteride is used for the treatment of androgenic alopecia, benign prostate hyperplasia and prostate cancer. Besides inhibiting the conversion of testosterone to the biologically more active 5alpha-dihydrotestosterone, it also inhibits the production of neurosteroids. Decreased neurosteroid levels are postulated to be involved in the pathophysiology of psychiatric disorders such as depression. As neurosteroids metabolized by 5-alpha-reductase influence neural plasticity, we investigated whether finasteride treatment alters adult hippocampal neurogenesis, implicated in the pathophysiology of depression. METHODS: Male C57BL/6N mice were treated subchronically (7 days) with finasteride or vehicle. Adult neurogenesis was assessed at two different time points after treatment (day 1; day 35) using immunohistochemistry. RESULTS: Finasteride treatment led to a significant decrease in brain 5alpha-dihydrotestosterone levels and induced a reversible reduction in the number of newborn cells and young neurons in the hippocampus. 35 days after the last finasteride injection, neurogenesis had returned to normal. DISCUSSION: These data indicate that inhibition of 5-alpha-reductase activity by finasteride treatment influences neuronal plasticity on a structural level. These changes might contribute to the pathophysiology of depressive episodes observed after finasteride treatment.

Reduced expression of glutamate transporters vGluT1, EAAT2 and EAAT4 in learned helpless rats, an animal model of depression.

BACKGROUND: It has been widely accepted that glial pathology and disturbed synaptic transmission contribute to the neurobiology of depression. Apart from monoaminergic alterations, an influence of glutamatergic signal transduction has been reported. Therefore, gene expression of glutamate transporters that strictly control synaptic glutamate concentrations have to be assessed in animal models of stress and depression. METHODS: We performed in situ-hybridizations in learned helplessness rats, a well established animal model of depression, to assess vGluT1 and EAAT1-4. Sprague-Dawley rats of two inbred lines were tested for helpless behavior and grouped into three cohorts according to the number of failures to stop foot shock currents by lever pressing. RESULTS: Helpless animals showed a significantly suppressed expression of the glial glutamate transporter EAAT2 (rodent nomenclature GLT1) in hippocampus and cerebral cortex compared to littermates with low failure rate and not helpless animals. This finding was validated on protein level using immunohistochemistry. Additionally, expression levels of EAAT4 and the vesicular transporter vGluT1 were reduced in helpless animals. In contrast, the transcript levels of EAAT1 (GLAST) and EAAT3 (EAAC1) were not significantly altered. CONCLUSIONS: These results strongly suggest reduced astroglial glutamate uptake and implicate increased glutamate levels in learned helplessness. The findings are in concert with antidepressant effects of NMDA-receptor antagonists and the hypotheses that impaired astroglial functions contribute to the pathogenesis of affective disorders.

Correlations and discrepancies between serum and brain tissue levels of neurotrophins after electroconvulsive treatment in rats.

INTRODUCTION: The neurotrophin brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are a central part of the molecular concepts on neuroplastic changes associated with stress, anxiety and depression. An increasing number of studies uses serum BDNF levels as a potential indicator for central nervous system alterations. METHODS: To analyze the relationship between brain tissue and serum BDNF and NGF levels, we used electroconvulsive shocks (ECS), an animal model of electroconvulsive therapy, and studied the temporal profile of neurotrophin expression in the hippocampus, prefrontal cortex and serum. 88 male Sprague-Dawley rats received single or serial ECS treatments and were killed between 3 hours and 14 days after the last treatment. RESULTS: We found a 2.8-fold rise for BDNF (1.3-fold for NGF) in the prefrontal cortex, and a 2.2-fold rise (1.2-fold for NGF) in the hippocampus after 5 ECS sessions. The temporal expression profile and correlation analyses between tissue and serum BDNF indicate that BDNF crosses the blood-brain barrier. No such correlation was found for NGF. DISCUSSION: The time course of central and peripheral BDNF changes may significantly differ. However, we demonstrate substantial evidence that it can be justified to measure serum BDNF levels with a time delay to monitor brain tissue neurotrophin alterations.

Reduced expression of GABA transporter GAT3 in helpless rats, an animal model of depression.

Mood disorders have been linked to glial and synaptic pathology such as disturbed neurotransmission of gamma-aminobutyric acid (GABA). We evaluated the expression of GABAergic marker genes in rats with helpless behaviour, an animal model of depression. Male Sprague-Dawley rats from inbred lines were tested for helpless behaviour and grouped according to failures in terminating foot shock currents. Expression levels of GABAergic marker genes were assessed using semiquantitative in situ-hybridization. Animals with congenital helpless behaviour (cH) were unable to escape current exposure in contrast to cH-animals derived from the same litters with low failure rates and to non-helpless animals (cNH). We found a significant downregulation of the GABA transporter GAT3 in cLH rats. GAT1 showed small changes, glutamic acid decarboxylase (GAD67) and the vesicular GABA transporter were not significantly altered. Reduced GABA transporter expression is well in concert with the behavioural phenotypes of knockout animals and strengthens the hypothesis of impaired glial functions in depression.

Learned helplessness: validity and reliability of depressive-like states in mice.

The learned helplessness paradigm is a depression model in which animals are exposed to unpredictable and uncontrollable stress, e.g. electroshocks, and subsequently develop coping deficits for aversive but escapable situations (J.B. Overmier, M.E. Seligman, Effects of inescapable shock upon subsequent escape and avoidance responding, J. Comp. Physiol. Psychol. 63 (1967) 28-33 ). It represents a model with good similarity to the symptoms of depression, construct, and predictive validity in rats. Despite an increased need to investigate emotional, in particular depression-like behaviors in transgenic mice, so far only a few studies have been published using the learned helplessness paradigm. One reason may be the fact that-in contrast to rats (B. Vollmayr, F.A. Henn, Learned helplessness in the rat: improvements in validity and reliability, Brain Res. Brain Res. Protoc. 8 (2001) 1-7)--there is no generally accepted learned helplessness protocol available for mice. This prompted us to develop a reliable helplessness procedure in C57BL/6N mice, to exclude possible artifacts, and to establish a protocol, which yields a consistent fraction of helpless mice following the shock exposure. Furthermore, we validated this protocol pharmacologically using the tricyclic antidepressant imipramine. Here, we present a mouse model with good face and predictive validity that can be used for transgenic, behavioral, and pharmacological studies.

Reduced sensitivity to sucrose in rats bred for helplessness: a study using the matching law.

Anhedonia is a core symptom of depression. As it cannot be directly assessed in rodents, anhedonia is usually inferred from a reduced consumption of, or preference for, a reinforcer. In the present study we tried to improve the measurement of anhedonia by performing a detailed preference analysis based on the generalized matching law and tested its sensitivity in rats congenitally prone (cLH) or resistant (cNLH) to learned helplessness. According to the current interpretation of learned helplessness as a model for depression, a reduction in the rewarding properties of sucrose in cLH rats was hypothesized. Our results revealed that the 'preference allocation' index provided by this test, but not the traditional measures of sucrose consumption or preference over water, was significantly lower in cLH rats, and was correlated with the helpless behaviour as measured in an escape procedure. Therefore, it is clear that more subtle preference measures provided by the analysis of choice using the matching law principles are more sensitive and discriminative than those based on consumption of, or preference for, a single concentration of sucrose over water. Moreover, our data are in agreement with the proposed relationship between helplessness and sucrose preference, and support the usefulness of the cLH and cNLH rats as a model of depression.

Basic pathophysiological mechanisms in depression: what are they and how might they affect the course of the illness?

Basic pathophysiological mechanisms in affective disorders are discussed. Studies carried out suggest that changes in neurogenesis do not underlie the behavioral changes which lead to helplessness. Since the behavioral changes leading to depressive or anxious behaviors are not correlated with changes in neurogenesis it appears unlikely that a decrease in the rate of neurogenesis is the basis for depression. A modified gene expression resulting in both functional and structural brain changes remains the most consistent hypothesis to explain how affective disorders may occur. An alternative candidate, synaptogenesis, appears as a likely candidate and requires further experimental testing.

Learned helplessness in the rat: improvements in validity and reliability.

Major depression has a high prevalence and a high mortality. Despite many years of research little is known about the pathophysiologic events leading to depression nor about the causative molecular mechanisms of antidepressant treatment leading to remission and prevention of relapse. Animal models of depression are urgently needed to investigate new hypotheses. The learned helplessness paradigm initially described by Overmier and Seligman [J. Comp. Physiol. Psychol. 63 (1967) 28] is the most widely studied animal model of depression. Animals are exposed to inescapable shock and subsequently tested for a deficit in acquiring an avoidance task. Despite its excellent validity concerning the construct of etiology, symptomatology and prediction of treatment response [Clin. Neurosci. 1 (1993) 152; Trends Pharmacol. Sci. 12 (1991) 131] there has been little use of the model for the investigation of recent theories on the pathogenesis of depression. This may be due to reported difficulties in reliability of the paradigm [Animal Learn. Behav. 4 (1976) 401; Pharmacol. Biochem. Behav. 36 (1990) 739]. The aim of the current study was therefore to improve parameters for inescapable shock and learned helplessness testing to minimize artifacts and random error and yield a reliable fraction of helpless animals after shock exposure. The protocol uses mild current which induces helplessness only in some of the animals thereby modeling the hypothesis of variable predisposition for depression in different subjects [Psychopharmacol. Bull. 21 (1985) 443; Neurosci. Res. 38 (200) 193]. This allows us to use animals which are not helpless after inescapable shock as a stressed control, but sensitivity, specificity and variability of test results have to be reassessed.

Brain-derived-neurotrophic-factor (BDNF) stress response in rats bred for learned helplessness.

Stress-induced elevation of glucocorticoids is accompanied by structural changes and neuronal damage in certain brain areas. This includes reduced expression of brain-derived neurotrophic factor (BDNF) in the hippocampus which can be prevented by chronic electroconvulsive seizures and antidepressant drug treatment. In the last years we have bred two strains of rats, one which reacts with congenital helplessness to stress (cLH), and one which congenitally does not acquire helplessness when stressed (cNLH). After being selectively bred for more than 40 generations these strains have lost their behavioural plasticity including their sensitivity to antidepressant treatment. We show here that in cLH rats, acute immobilization stress does not induce a reduction of BDNF expression in the hippocampus which is observed in Sprague--Dawley and cNLH rats. All animals tested exhibited elevated corticosterone levels when stressed, an indication, that in cLH rats regulation of BDNF expression in the hippocampal formation is uncoupled from corticosterone increase induced through stress. This may explain the lack of adaptive responses in this strain.

Acute stress decreases serotonin transporter mRNA in the raphe pontis but not in other raphe nuclei of the rat.

In addition to elevated corticosterone levels, stress produces structural changes and neuronal damage especially in the hippocampus. In this line it has been shown, that in rats single or repeated immobilisation markedly reduces brain-derived neurotrophic factor (BDNF) mRNA levels in the hippocampal formation. Since this neurotrophin also controls the efficacy of serotonergic neurotransmission, the aim of the current study was to investigate the effect of acute immobilization stress on the expression of serotonin transporter (SERT) mRNA in the raphe nuclei as a parameter of serotonergic innervation. We have examined the expression of SERT mRNA and of BDNF mRNA in rats upon acute immobilisation by quantitative in situ hybridisation with a (35)S-labelled oligonucleotide probe. Elevated corticosterone levels in stressed animals confirmed as internal controls the effect of stress under our conditions. Acute stress led to a significant decrease of BDNF mRNA in the hippocampus and of SERT mRNA in the raphe pontis, but not in other raphe nuclei investigated. These data provide evidence for fast interactions between neurotrophins, corticosterone and serotonergic neurotransmission under stress conditions.

Altered Ca(2+)-homeostasis in single T-lymphocytes of depressed patients.

After stimulation of T-lymphocytes from healthy volunteers with the mitogen phytohemagglutinin (PHA) 40% of the cells exhibit an oscillatory increase in intracellular calcium concentration ([Ca2+]i). During depression the number of cells responding to PHA is reduced to 20%. These cells show a marked decrease in [Ca2+]i-reaction to stimulation and flattened oscillations. This reduction of mitogen-induced Ca2+ signals in T-cells of depressed patients appears to be a reliable state marker in depressive illness and is reversed upon successful treatment with interpersonal psychotherapy (IPT).

K+ currents of human T-lymphocytes are unaffected by Alzheimer's disease and amyloid beta protein.

Alterations of Ca2+ homeostasis have been reported for both fibroblasts and T-lymphocytes of patients with Alzheimer's disease (AD). Considering the importance of K+ conductances for the cellular Ca2+ regulation and the recently reported absence of a K+ channel in Alzheimer fibroblasts, we investigated K+ currents in T-lymphocytes of patients with AD. In addition, the finding that amyloid beta protein affects the Ca2+ signal of T-lymphocytes and the function of K+ channels in fibroblasts prompted us to study a possible influence of amyloid beta protein fragments on K+ channels of T-lymphocytes. Our data, obtained by means of the whole-cell patch-clamp configuration on freshly isolated T-lymphocytes, indicate that K+ channels of these cells do not present any functional deficit in AD, and amyloid beta protein does not mediate an alteration of their currents.

Mitogen stimulated rise of intracellular calcium concentration in single T lymphocytes from patients with major depression is reduced.

1. The authors investigated the signal transduction in T-lymphocytes as a peripheral model for central neurons. 2. Intracellular free calcium concentration [Ca2+]i was measured using fura 2 in T-lymphocytes from 6 patients with major depression during and after depression and from 6 healthy controls. Patients were treated with interpersonal therapy (IPT) but not with psychotropic medication. 3. Phytohemagglutinin (PHA) triggers an oscillatory [Ca2+]i signal in human T-lymphocytes. This implies two mechanisms for [Ca2+]i regulation: inositol phophate (IP) mediated release from intracellular stores and [Ca2+]i influx from the extracellular medium. 4. PHA stimulates 49% of T cells from controls but only 17% of T cells from depressed patients. This finding explains previous results from cells in suspension indicating that [Ca2+]i signals after PHA-stimulation are reduced in cells from depressed patients. 5. Cells from depressed patients show less [Ca2+]i oscillations. Normal oscillation patterns are restored after clinical recovery from depression. 6. Thus altered [Ca2+]i oscillations in T-lymphocytes are a state phenomenon and may give us clues where to search for altered cellular mechanisms during depression.

Cytosolic free [Ca2+] in single T-lymphocytes from depressed patients and healthy controls.

Human lymphocytes are widely used as peripheral models for central neurones. Alterations in immune function have been reported in depressed patients, e.g. mitogen-induced proliferation is impaired during depression. One possible causative mechanism could be altered [Ca2+]i regulation. Phytohaemagglutinin (PHA)-induced rise of [Ca2+]i has been found to be diminished in lymphocyte suspensions from depressed patients (Ecker et al., this issue). We measured PHA-induced rise of [Ca2+]i in single Fura-2 AM-loaded T11+ lymphocytes of patients with major depression and controls to further analyse [Ca2+]i regulation in depression. The [Ca2+]i of resting lymphocytes was 57 +/- 2 nmol/l (mean +/- SEM). There was no difference in resting [Ca2+]i of resting lymphocytes of patients and controls. PHA evoked an increase of [Ca2+]i an 7 out of 14 cells from control subjects up to 400-500 nmol/l. In contrast, only 4 out of 13 cells from depressed patients showed an increase of [Ca2+]i up to 200 nmol/l. In a small fraction of cells from both groups the [Ca2+]i signal is oscillating. Our preliminary data confirm alteration of [Ca2+]i regulation in lymphocytes of depressed patients.