The wake-promoting drug modafinil stimulates specific hypothalamic circuits to promote adaptive stress responses in an animal model of PTSD.

Pharmacotherapeutic intervention during traumatic memory consolidation has been suggested to alleviate or even prevent the development of posttraumatic stress disorder (PTSD). We recently reported that, in a controlled, prospective animal model, depriving rats of sleep following stress exposure prevents the development of a PTSD-like phenotype. Here, we report that administering the wake-promoting drug modafinil to rats in the aftermath of a stressogenic experience has a similar prophylactic effect, as it significantly reduces the prevalence of PTSD-like phenotype. Moreover, we show that the therapeutic value of modafinil appears to stem from its ability to stimulate a specific circuit within the hypothalamus, which ties together the neuropeptide Y, the orexin system and the HPA axis, to promote adaptive stress responses. The study not only confirms the value of sleep prevention and identifies the mechanism of action of a potential prophylactic treatment after traumatic exposure, but also contributes to understanding mechanisms underlying the shift towards adaptive behavioral response.

Elevation of Il6 is associated with disturbed let-7 biogenesis in a genetic model of depression.

Elevation of the proinflammatory cytokine IL-6 has been implicated in depression; however, the mechanisms remain elusive. MicroRNAs (miRNAs) are small non-coding RNAs that inhibit gene expression post-transcriptionally. The lethal-7 (let-7) miRNA family was suggested to be involved in the inflammation process and IL-6 was shown to be one of its targets. In the present study, we report elevation of Il6 in the prefrontal cortex (PFC) of a genetic rat model of depression, the Flinders Sensitive Line (FSL) compared to the control Flinders Resistant Line. This elevation was associated with an overexpression of LIN28B and downregulation of let-7 miRNAs, the former an RNA-binding protein that selectively represses let-7 synthesis. Also DROSHA, a key enzyme in miRNA biogenesis was downregulated in FSL. Running was previously shown to have an antidepressant-like effect in the FSL rat. We found that running reduced Il6 levels and selectively increased let-7i and miR-98 expression in the PFC of FSL, although there were no differences in LIN28B and DROSHA expression. Pri-let-7i was upregulated in the running FSL group, which associated with increased histone H4 acetylation. In conclusion, the disturbance of let-7 family biogenesis may underlie increased proinflammatory markers in the depressed FSL rats while physical activity could reduce their expression, possibly through regulating primary miRNA expression via epigenetic mechanisms.

Allele-specific programming of Npy and epigenetic effects of physical activity in a genetic model of depression.

Neuropeptide Y (NPY) has been implicated in depression, emotional processing and stress response. Part of this evidence originates from human single-nucleotide polymorphism (SNP) studies. In the present study, we report that a SNP in the rat Npy promoter (C/T; rs105431668) affects in vitro transcription and DNA-protein interactions. Genotyping studies showed that the C-allele of rs105431668 is present in a genetic rat model of depression (Flinders sensitive line; FSL), while the SNP's T-allele is present in its controls (Flinders resistant line; FRL). In vivo experiments revealed binding of a transcription factor (CREB2) and a histone acetyltransferase (Ep300) only at the SNP locus of the FRL. Accordingly, the FRL had increased hippocampal levels of Npy mRNA and H3K18 acetylation; a gene-activating histone modification maintained by Ep300. Next, based on previous studies showing antidepressant-like effects of physical activity in the FSL, we hypothesized that physical activity may affect Npy's epigenetic status. In line with this assumption, physical activity was associated with increased levels of Npy mRNA and H3K18 acetylation. Physical activity was also associated with reduced mRNA levels of a histone deacetylase (Hdac5). Conclusively, the rat rs105431668 appears to be a functional Npy SNP that may underlie depression-like characteristics. In addition, the achieved epigenetic reprogramming of Npy provides molecular support for the putative effectiveness of physical activity as a non-pharmacological antidepressant.

Long-term lithium treatment in bipolar disorder is associated with longer leukocyte telomeres.

Telomere shortening is a hallmark of aging and has been associated with oxidative stress, inflammation and chronic somatic, as well as psychiatric disorders, including schizophrenia and depression. Additionally, antidepressants have been found to protect against telomere shortening. However, pharmacological telomere studies are lacking in bipolar disorder (BD). Therefore, the objective of this study was to explore telomere length (TL) in patients with BD in the context of lithium treatment. We determined TL by quantitative real-time PCR using peripheral blood leukocytes. Participants were outpatients diagnosed with BD type 1 or 2 (n=256) and healthy controls (n=139). Retrospective case-control and case-case study designs were applied. Lithium response (LiR) was scored using the Alda-Scale. Lithium-treated BD patients overall, as well as those on lithium monotherapy, had 35% longer telomeres compared with controls (P<0.0005, partial η(2)=0.13). TL correlated positively with lithium treatment duration of >30 months (P=0.031, R(2)=0.13) and was negatively associated with increasing number of depressive episodes (P<0.007). BD patients responding well to lithium treatment had longer telomeres than those not responding well. This is the first study to report a positive effect of long-term lithium treatment on TL. Importantly, longer TL was also associated with a better LiR in BD patients. These data suggest that lithium exerts a protective effect against telomere shortening especially when therapeutically efficacious. We hypothesize that induction of telomerase activity may be involved in LiR in BD.

Emotional memory impairments in a genetic rat model of depression: involvement of 5-HT/MEK/Arc signaling in restoration.

Cognitive dysfunctions are common in major depressive disorder, but have been difficult to recapitulate in animal models. This study shows that Flinders sensitive line (FSL) rats, a genetic rat model of depression, display a pronounced impairment of emotional memory function in the passive avoidance (PA) task, accompanied by reduced transcription of Arc in prefrontal cortex and hippocampus. At the cellular level, FSL rats have selective reductions in levels of NMDA receptor subunits, serotonin 5-HT(1A) receptors and MEK activity. Treatment with chronic escitalopram, but not with an antidepressant regimen of nortriptyline, restored memory performance and increased Arc transcription in FSL rats. Multiple pharmacological manipulations demonstrated that procognitive effects could also be achieved by either disinhibition of 5-HT(1A)R/MEK/Arc or stimulation of 5-HT4R/MEK/Arc signaling cascades. Taken together, studies of FSL rats in the PA task revealed reversible deficits in emotional memory processing, providing a potential model with predictive and construct validity for assessments of procognitive actions of antidepressant drug therapies.

Impact of early life stress on alcohol consumption and on the short- and long-term responses to alcohol in adolescent female rats.

We examined the interaction between early life stress and vulnerability to alcohol in female rats exposed to prenatal restraint stress (PRS rats). First we studied the impact of PRS on ethanol preference during adolescence. PRS slightly increased ethanol preference per se, but abolished the effect of social isolation on ethanol preference. We then studied the impact of PRS on short- and long-term responses to ethanol focusing on behavioral and neurochemical parameters related to depression/anxiety. PRS or unstressed adolescent female rats received 10% ethanol in the drinking water for 4 weeks from PND30 to PND60. At PND60, the immobility time in the forced-swim test did not differ between PRS and unstressed rats receiving water alone. Ethanol consumption had no effect in unstressed rats, but significantly reduced the immobility time in PRS rats. In contrast, a marked increase in the immobility time was seen after 5 weeks of ethanol withdrawal only in unstressed rats. Hippocampal levels of neuropeptide Y (NPY) and mGlu1a metabotropic glutamate receptors were increased at the end of ethanol treatment only in unstressed rats. Ethanol treatment had no effect on levels of corticotropin-releasing hormone (CRH) in the hippocampus, striatum, and prefrontal cortex of both groups of rats. After ethanol withdrawal, hippocampal levels of mGlu1 receptors were higher in unstressed rats, but lower in PRS rats, whereas NPY and CRH levels were similar in the two groups of rats. These data indicate that early life stress has a strong impact on the vulnerability and responsiveness to ethanol consumption during adolescence.

Regulation of cytoskeleton machinery, neurogenesis and energy metabolism pathways in a rat gene-environment model of depression revealed by proteomic analysis.

The development of major depression requires both genetic and environmental factors. A brain proteomic investigation on the genetic model of Flinders sensitive and resistant line (FSL-FRL) rats was performed. Maternal separation (MS) was also applied to identify protein networks affected by stress exposure, since early-life trauma is considered an important antecedent of depression. Hippocampus (HIP) and prefrontal/frontal cortex proteins were extracted and separated by 2-Dimensional (2-D) gel electrophoresis. After image analysis, significantly modulated proteins in the different conditions analysed were identified by mass spectrometry. The expression of proteins involved in energy metabolism, cellular localization and transport, cytoskeleton organization and apoptosis differed in the two lines. Maternal separation differently affected the genetic backgrounds, by modulating cytoskeleton and neuron morphogenesis proteins in FSL; energy metabolism, cellular localization, neuron differentiation and intracellular transport in FRL. The present work shows that different mechanisms could be involved in the pathophysiology of depression and the vulnerability to stress, suggesting possible new cellular pathways and key markers for the study of affective disorders.

Animal models of depression and anxiety: What do they tell us about human condition?

While modern neurobiology methods are necessary they are not sufficient to elucidate etiology and pathophysiology of affective disorders and develop new treatments. Achievement of these goals is contingent on applying cutting edge methods on appropriate disease models. In this review, the authors present four rodent models with good face-, construct-, and predictive-validity: the Flinders Sensitive rat line (FSL); the genetically "anxious" High Anxiety-like Behavior (HAB) line; the serotonin transporter knockout 5-HTT(-/-) rat and mouse lines; and the post-traumatic stress disorder (PTSD) model induced by exposure to predator scent, that they have employed to investigate the nature of depression and anxiety.

Enhanced expression of the neuronal K+/Cl- cotransporter, KCC2, in spontaneously depressed Flinders Sensitive Line rats.

We used Flinder Sensitive Line (FSL) rats, a genetic model of unipolar depression, to examine whether changes in central GABAergic transmission are associated with a depressed phenotype. FSL rats showed an increased behavioral response to low doses of diazepam, as compared to either Sprague Dawley (SD) or Flinder Resistant Line (FRL) rats used as controls. Diazepam at a dose of 0.3 mg/kg, i.p., induced a robust impairment of motor coordination in FSL rats, but was virtually inactive in SD or FRL rats. The increased responsiveness of FSL rats was not due to changes in the brain levels of diazepam or its active metabolites, or to increases in the number or affinity of benzodiazepine recognition sites, as shown by the analysis of [(3)H]-flunitrazepam binding in the hippocampus, cerebral cortex or cerebellum. We therefore examined whether FSL rats differed from control rats for the expression levels of the K(+)/Cl(-) cotransporter, KCC2, which transports Cl(-) ions out of neurons, thus creating the concentration gradient that allows Cl(-) influx through the anion channel associated with GABA(A) receptors. Combined immunoblot and immunohistochemical data showed a widespread increase in KCC2 expression in FSL rats, as compared with control rats. The increase was more prominent in the cerebellum, where KCC2 was largely expressed in the granular layer. These data raise the interesting possibility that a spontaneous depressive state in animals is associated with an amplified GABAergic transmission in the CNS resulting from an enhanced expression of KCC2.

Defective group-II metaboropic glutamate receptors in the hippocampus of spontaneously depressed rats.

Spontaneously depressed flinders sensitive line (FSL) rats showed a reduced expression of mGlu2/3 metabotropic glutamate receptors in the hippocampus, as compared to "non-depressed" flinders resistant line (FRL) rats. No changes in mGlu2/3 receptor protein levels were found in other brain regions, including the amygdala, hypothalamus, and cerebral cortex. Biochemical analysis of receptor signalling supported the reduction of mGlu2/3 receptors in the hippocampus of FSL rats. Accordingly, the selective mGlu2/3 receptor agonist, LY379268 (1microM) reduced forskolin-stimulated cAMP formation by 56% and 32% in hippocampal slices from FRL and FSL rats, respectively. In addition, LY379268 enhanced 3,5-dihydroxyphenylglycine-stimulated inositol phospholipid hydrolysis from 65% to 215% in hippocampal slices from FRL rats, whereas it was inactive in slices from FRL rats. We also examined the behavioural response of FSL rats to systemic injection of LY379268 (0.5mg/kg, i.p., once a day for 1-21 days) by measuring the immobility time in the forced swim test, which is known to be increased in these rats. LY379268 was administered alone or combined with the classical antidepressant, chlorimipramine (10mg/kg, i.p.). LY379268 alone had no effect at any of the selected time-points, whereas chlorimipramine alone reduced the immobility time only after 21 days of treatment. In contrast, when combined with LY379268, chlorimipramine reduced the immobility time during the first 14 days of treatment. These data support the view that mGlu2/3 receptors might be involved in the pathophysiology of depressive disorders, and that pharmacological activation of these receptors may shorten the latency of antidepressant medication.

Impact of an intense stress on ethanol consumption in female rats characterized by their pre-stress preference: modulation by prenatal stress.

We examined the influence of prenatal stress on alcohol preference in adult female rats exposed to an intense stress. To take into account interindividual variability, the study was conducted in animals categorized as low or high alcohol preferring. After footshock, control high-preferring rats strongly reduced their alcohol consumption; in contrast, alcohol consumption was not changed in high-preferring rats that were prenatally stressed.

Exacerbated loss of cell survival, neuropeptide Y-immunoreactive (IR) cells, and serotonin-IR fiber lengths in the dorsal hippocampus of the aged flinders sensitive line "depressed" rat: Implications for the pathophysiology of depression?

Impairment of hippocampal neurogenesis has been proposed to provide a cellular basis for the development of major depression. Studies have shown that serotonin (5-HT) and neuropeptide Y (NPY) may be involved in stimulating cell proliferation in the dentate gyrus. The Flinders-sensitive line (FSL) rat represents a genetic model of depression with characterized 5-HT and NPY abnormalities in the hippocampus. Consequently, it could be hypothesized that hippocampal neurogenesis in the FSL rat would be impaired. The present study examined the relationship among 1) number of BrdU-immunoreactive (IR) cells, 2) NPY-IR cells in the dentate gyrus, and 3) length of 5-HT-IR fibers in the dorsal hippocampus, as well as volume and number of 5-HT-IR cells in the dorsal raphé nucleus, in adult and aged FSL rats and control Flinders-resistant line (FRL) rats. Surprisingly, adult FSL rats had significantly more BrdU-IR and NPY-IR cells compared with adult FRL rats. However, aging caused an exacerbated loss of these cell types in the FSL strain compared with FRL. The aged FSL rats also had shortened 5-HT-IR fibers in the dorsal hippocampus, indicative of an impaired 5-HT innervation of this area, compared with FRL. These results suggest that, for "depressed" FSL rats, compared with FRL rats, aging is associated with an excacerbated loss of newly formed cells in addition to NPY-IR cells and 5-HT-IR dendrites in the hippocampus. These observations may be of relevance to the depression-like behavior of the FSL rat and, by inference, to the pathophysiology of depression.

BDNF in schizophrenia, depression and corresponding animal models.

Understanding the etiology and pathogenesis schizophrenia and depression is a major challenge facing psychiatry. One hypothesis is that these disorders are secondary to a malfunction of neurotrophic factors. Inappropriate neurotrophic support during brain development could lead to structural disorganisation in which neuronal networks are established in a nonoptimal manner. Inadequate neurotrophic support in adult individuals could ultimately be an underlying mechanism leading to decreased capacity of brain to adaptive changes and increased vulnerability to neurotoxic damage. Brain-derived neurotrophic factor (BDNF) is a mediator involved in neuronal survival and plasticity of dopaminergic, cholinergic, and serotonergic neurons in the central nervous system (CNS). In this review, we summarize findings regarding altered BDNF in schizophrenia and depression and animal models, as well as the effects of antipsychotic and antidepressive treatments on the expression of BDNF.

Levetiracetam prevents changes in levels of brain-derived neurotrophic factor and neuropeptide Y mRNA and of Y1- and Y5-like receptors in the hippocampus of rats undergoing amygdala kindling: implications for antiepileptogenic and mood-stabilizing properties.

The amygdala-kindling model has been proposed as a model of sensitization processes with relevance to epilepsy as well as affective disorders. Levetiracetam is a novel anticonvulsant drug that delays the process of kindling, i.e., possesses antiepileptogenic properties. Preliminary reports also suggest a mood-stabilizing potential for levetiracetam. Brain-derived neurotrophic factor (BDNF) and neuropeptide Y (NPY) are central modulators of seizure activity, which undergo plastic changes during kindling epileptogenesis. Consequently, we investigated the regulation of BDNF and NPY mRNA and Y1-, Y2-, and Y5-like receptor binding in the hippocampus of vehicle-pretreated, partially and fully amygdala-kindled rats and corresponding levetiracetam-pretreated rats (40 mg/kg i.p.). The present data indicate that the process of kindling is associated with an upregulation of hippocampal BDNF and NPY mRNA levels and downregulation of Y1- and particularly Y5-like receptors. Pretreatment with levetiracetam markedly delays the progression of kindling and, in addition, exhibits a clear anticonvulsant effect. These effects are associated with abolition of the kindling-induced rise in BDNF and NPY mRNA and increasing levels of Y1- and particularly Y5-like receptors in all hippocampal subfields. Lastly, the present study reveals that an identical dose of levetiracetam reduced immobility in the rat forced swim test, the first experimental evidence indicative of an antidepressant and/or mood stabilizer-like profile of this drug. Considering that animal depression models display impairments in hippocampal NPY systems that become normalized following mood-stabilizing treatment, and that exogenous NPY exerts anticonvulsant as well as antidepressive-like activity in rodents, it is a heuristic possibility that increased hippocampal excitability and affective symptomatology may converge on an impaired hippocampal NPY function. Speculatively, the ability of levetiracetam to increase hippocampal Y1- and Y5-like receptor levels may have implications for the antiepileptic properties of levetiracetam, as well as its purported mood-stabilizing properties.

Identification of the bioactive peptide PEC-60 in brain.

PEC-60 is a 60-residue peptide originally isolated from pig intestine. It inhibits glucose-induced insulin secretion from perfused pancreas in a hormonal manner and also has biological activity in the immune system. PEC-60-like immunoreactive material has been reported in catecholamine neurons of the central and peripheral nervous systems, but the peptide has not been identified from that material. We have now isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is expressed in the gastrointestinal tract and the central nervous system. The specific regional brain distribution and interaction with classical neurotransmitters raise the possibility that PEC-60 may play a role in the central nervous system disorders involving dopamine dysregulation.

Early maternal deprivation alters hippocampal levels of neuropeptide Y and calcitonin-gene related peptide in adult rats.

Stressful events early in life are reported to be more prevalent among patients with an adult life psychiatric disorder. Early maternal deprivation is considered an animal model of early life stress. Maternally deprived adult rats display long-term alterations in the neuroendocrine system, brain and behavior that are in many ways analogous to depressive and schizophrenic symptomatology. Neuropeptide Y (NPY) and calcitonin-gene related peptide (CGRP) have been implicated in both disorders and also been suggested to play a role in the neuroadaptational response to stress. Consequently, male Wistar rat-pups were subjected to early maternal deprivation or control handling, on postnatal day (pnd) 9. On pnd 21, pups were weaned and split into two groups that were reared either on a saw-dust floor or on a grid-floor, considered to be a mild stressor. On pnd 67, all animals were subjected to the prepulse inhibition test. One week later, the animals were sacrificed, the brains removed and dissected on ice. Levels of NPY-like immunoreactivity (LI) and CGRP-LI were quantified by radioimmunoassay in brain regional extracts. Maternal deprivation led to a significant reduction in basal startle amplitude and disruption of prepulse inhibition. These findings were paralleled by significantly reduced levels of NPY and CGRP in the hippocampus and occipital cortex. It is hypothesised that these changes may be of relevance to aspects of schizophrenic and affective symptomatology. The present study further shows that brain NPY and, in particular, CGRP are sensitive to long-term mild stress and further implicate the involvement of these peptides in the neuroendocrine stress response.

Early maternal separation alters neuropeptide Y concentrations in selected brain regions in adult rats.

Human and animal studies support the involvement of neuropeptide Y (NPY) in the pathophysiology of depression. Thus, hippocampal NPY-LI is decreased in genetic models of depression, the Flinders Sensitive Line and Fawn Hooded rats. Maternal "deprivation" has been identified as one risk factor in the development of psychopathology, including depression in adulthood. In view of these findings we hypothesized that brain NPY may also be decreased in an animal model of early life maternal deprivation. To test this hypothesis, male and female Sprague-Dawley rats were maternally separated (MS) 6 h/day or briefly handled from postnatal day 2 (PN2) to PN6 and from PN9 to PN13. At 12 weeks of age the rats were sacrificed, the brains dissected and NPY-LI measured by radioimmunoassay. MS rats had lower NPY-LI in the hippocampus. NPY-LI was also lower in female compared to male rats in hippocampus. Lastly, NPY-LI was increased in the hypothalamus of both male and female MS rats. These findings support the hypothesis that altered NPY in the limbic region is a common denominator of several models of depression and might be a trait marker of vulnerability to affective disorders.

Dopamine receptor antagonists prevent the d-amphetamine-induced increase in calcitonin gene-related peptide levels in ventral striatum.

Microdialysis in conjunction with radioimmunoassay (RIA) were used to study the effects of acute d-amphetamine or dopamine (DA) receptor antagonists administration on extracellular concentrations of calcitonin gene-related peptide (CGRP) in the ventral striatum of the rat. One hour after the subcutaneous (s.c.) injection of saline, the DA-D(1) receptor antagonist SCH 23390 (0.3 mg/kg) or the DA-D(2/3) receptor antagonist raclopride (1.0 mg/kg), one additional s.c. injection of saline or d-amphetamine (1.5 mg/kg) was given. The dialysates were collected at 60-min intervals; CGRP-like immunoreactivities (-LI) were determined by RIA. d-Amphetamine significantly increased extracellular CGRP-LI concentrations compared to the control animals. Administration of either SCH 23390 or raclopride did not significantly affect CGRP-LI concentrations. Pretreatment with either SCH 23390 or raclopride abolished the stimulatory effect of d-amphetamine on CGRP-LI levels. The results show that d-amphetamine administration results in an increase in extracellular concentrations of CGRP in the ventral striatum through a mechanism that appears to involve stimulation of either DA-D(1) or DA-D(2/3) receptors. The results also indicate that changes in dopaminergic neurotransmission affect CGRP outflow in the ventral striatum in a phasic but not tonic manner.

Changed concentrations of tachykinins and neuropeptide Y in brain of a rat model of depression: lithium treatment normalizes tachykinins.

Lithium's therapeutic mechanism of action is unknown. In lithium-treated normal rats, increased striatal concentrations of neurokinin A (NKA)-like immunoreactivity (LI), substance P (SP-LI) and neuropeptide Y (NPY-LI) have been reported. To investigate whether these effects might be of therapeutic relevance, Flinders Sensitive Line rats (FSL), an animal model of depression, and control Flinders Resistant Line (FRL) rats were during a 6-week period fed chow to which either lithium or vehicle was admixed. Following sacrifice, the peptides were extracted from dissected brain regions and measured by radioimmunoassay. NKA-LI and SP-LI were markedly decreased in striatum and increased in frontal cortex in FSL compared to control FRL animals. Lithium treatment abolished these differences. Basal concentrations of NPY-LI were decreased in hippocampus of FSL rats, but unaffected by lithium. The present study suggests that changed tachykinins and NPY may underlie the characterized depressive-like phenotype of the FSL rats. It is hypothesized that altering tachykinin peptidergic neurotransmission in striatum and frontal cortex constitutes a mechanism of action of lithium and that such a mechanism might be of therapeutic relevance.