In animal models, psychosocial stress-induced (neuro)inammation, apoptosis andreduced neurogenesis are associated to the onset of depression

anf Scowicivers

iland

a r t i c l e i n f o

Article history:Received 14 January 2010Received in revised form 28 August 2010Accepted 29 August 2010Available online 7 September 2010

2010 Elsevier Inc. All rights reserved.

Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 744759

Contents lists available at ScienceDirect

Progress in Neuro-Psychopharmacology & BiologicalPsychiatry

j ourna l homepage: www.e lsev ie r.com/ locate /pnp1. Introduction

External stress is widely acknowledged as a predisposing andprecipitating factor of depression especially in genetically predis-

posed persons. An enhanced responsivity to external stress isassociated with the early phases of depression. Early life experiencesmodulate the development of appropriate/inappropriate responses toexternal stressors and thus the vulnerability for depressive episodes.

Abbreviations: IO&NS, inammatory, oxidative and nitrosative stress; I&ND, inammatory and neurodegenerative; CMS, chronic mild stress; LH, learned helplessness; LPS,lipopylysaccharide; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; sIL-2R, soluble IL-2 receptor; sTNFR, soluble TNF receptor; TRYCATs, tryptophan catabolites; IDO,

indoleamine 2,3-dioxygenase; COX-2, cyclooxygenase 2;barrier; PG, prostaglandin; BDNF, brain-derived nuclear fa1ra, interleukin-1 receptor antagonist; CRF, corticotropin-molecular patterns;DAMPs, damage-associatedmolecularVHF, ventral hippocampal formation; IL-1R, IL-1 receptinhibitor; SNRI, serotoninnorepinephrine reuptake inhityrosine kinase; MAOI, monoamine oxidase inhibitor; RIM Corresponding author. Maes Clinics @ TRIA, Piyavat

E-mail addresses: kubera@if-pan.krakow.pl (M. Kubdr.michaelmaes@hotmail.com (M. Maes).

0278-5846/$ see front matter 2010 Elsevier Inc. Aldoi:10.1016/j.pnpbp.2010.08.026It is concluded that external stressors may provoke depression-like behaviors through activation ofinammatory, oxidative, apoptotic and antineurogenic mechanisms. The clinical efcacity of antidepressantsmay be ascribed to their ability to reverse these different pathways.Keywords:CytokinesDepressionInammationNeurodegenerationNeurogenesisOxidative stressa b s t r a c t

Recently, the inammatory and neurodegenerative (I&ND) hypothesis of depression was formulated (Maeset al., 2009), i.e. the neurodegeneration and reduced neurogenesis that characterize depression are caused byinammation, cell-mediated immune activation and their long-term sequels. The aim of this paper is toreview the body of evidence that external stressors may induce (neuro)inammation, neurodegeneration andreduced neurogenesis; and that antidepressive treatments may impact on these pathways.The chronic mild stress (CMS) and learned helplessness (LH) models show that depression-like behaviors areaccompanied by peripheral and central inammation, neuronal cell damage, decreased neurogenesis andapoptosis in the hippocampus. External stress-induced depression-like behaviors are associated with a)increased interleukin-(IL)1, tumor necrosis factor-, IL-6, nuclear factor B, cyclooxygenase-2, expression ofToll-like receptors and lipid peroxidation; b) antineurogenic effects and reduced brain-derived neurotrophicfactor (BDNF) levels; and c) apoptosis with reduced levels of Bcl-2 and BAG1 (Bcl-2 associated athanogene 1),and increased levels of caspase-3. Stress-induced inammation, e.g. increased IL-1, but not reducedneurogenesis, is sufcient to cause depression. Antidepressants a) reduce peripheral and centralinammatory pathways by decreasing IL-1, TNF and IL-6 levels; b) stimulate neuronal differentiation,synaptic plasticity, axonal growth and regeneration through stimulatory effects on the expression of differentneurotrophic factors, e.g. trkB, the receptor for brain-derived neurotrophic factor; and c) attenuate apoptoticpathways by activating Bcl-2 and Bcl-xl proteins, and suppressing caspase-3.ROS, reactive oxygen species; RNS, reactive nitrogen specictor; GC, glucocorticoids; OB, olfactory bulbectomized; aporeleasing factor; TACE, TNF converting enzyme; NFB, nupatterns;MAPK,mitogenactivatedproteinkinases; GSK-3or; NGF, nerve growth factor; NMDA, N-methyl-D-aspartabitor; NRI, noradrenergic reuptake inhibitor; rrTNF, recomA, reversible inhibitor of monoamine oxidase A.e Hospital, 998 Rimklongsamsen Road, Bangkok 10310,era), eobuchowicz@slam.katowice.pl (E. Obuchowicz), ga

l rights reserved.Oil and Gas Institute, Lubicz 25, 31-503 Krakw, Polande Maes Clinics @ TRIA, Piyavate Hospital, 998 Rimklongsamsen Road, Bangkok 10310, ThaMarta Kubera a, Ewa Obuchowicz b, Lisa Goehler c, Joa Department of Experimental Endocrinology, Institute of Pharmacology, Polish Academy ob Department of Pharmacology, Medical University of Silesia, Medykow 18, PL 40-752 Katc Center for the Study of Complementary and Alternative Therapies, School of Nursing, Undna Brzeszcz d, Michael Maes e,iences, Smetna 12, PL 31-343 Krakw, Polande, Polandity of Virginia, Charlottesville, VA, 22904, USAes; TBARS, thiobarbituric acid reactive substances; BBB, blood-brainE/, apolipoprotein E knockout; I.C.V., intracerebroventricular; IL-clear factor B; TLR, Toll-like receptor; PAMPs, pathogen associated, glycogen synthase kinase-3; BAG-1, Bcl-2 associated athanogene1;te; TCA, tricyclic antidepressant; SSRI, selective serotonin reuptakebinant rat TNF; ERK, extracellular signal-related kinase; trtB, TrkB

Thailand.ykema@virginia.edu, goehler@virginia.edu (L. Goehler),

745M. Kubera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 744759The latter, in turn, may be triggered by external stressors, such asnegative life events (Maes, 1999, 2001).

There is now evidence that depression is accompanied byactivation of immune, inammatory, oxidative and nitrosative stress(IO&NS) pathways (Maes, 1993, 2010; Maes et al., 1990). Reviewpapers in this special issue summarize that different IO&NS pathwaysare key features of depression (Maes, 2010; Maes et al., 2010;Szewczyk et al., 2010; Zunszain et al., 2010; Song and Wang, 2010;Gardner and Boles, 2010). External stressors, like stressful life events,and/or internal stressors, e.g. inammatory conditions, may inducethe previously mentioned pathways and consequently are involved inthe etiology of depression (Maes, 1995, 2008; Maes et al., 1995, 2009;Anisman, 2009; Miller et al., 2009).

There is also evidence that depression is accompanied bystructural changes in the hippocampus, prefrontal cortex, amygdala,anterior cingulate and basal ganglia (Campbell and MacQueen, 2006).The selective loss of hippocampal volume is caused by a) hippocampalneuronal death, neuronal and glial cell modications, and othercellular changes as well (Stockmeier et al., 2004); and b) decreasedneurogenesis (Sapolsky, 2004). The inammatory and (neuro)degenerative (I&ND) hypothesis of depression states that the above-mentioned changes in depression are caused by IO&NS pathways(Maes et al., 2009). The reviews included in this special issue focus onthe different IO&NS pathways that play a role in the I&ND hypothesisof depression (Maes et al., 2010; Szewczyk et al., 2010; Zunszain et al.,2010; Song and Wang, 2010; Gardner and Boles, 2010).

Translational research, including animal models of depression, isneeded to decipher the exact pathways and molecular mechanisms bywhich external and internal stressors activate peripheral and centralIO&NS pathways and cause neurodegeneration, which ultimately leadto depression-like behaviors. Findings from animal models can serve astemplates for identifying targeted brain regions for further assessmentsin humans. Adequate animal models of depression should a) closelysimulate the etiology, the symptomatology, and the course ofdepression and should respond to antidepressive treatments estab-lished in human depression; and b) have sufcient face and predictivevalidity and be reproducible between investigators.

The aim of this paper is to review the body of evidence that a)external stressors may cause systemic inammation, neuroinamma-tion, neurodegeneration and reduced neurogenesis; and b) thesestress-induced changes may be blocked by antidepressants. Towardthis end, we will review well-validated, external stress-inducedanimal models of depression, such as the chronic mild stress model(CMS) and the learned helplessness (LH) paradigm. In this specialissue, another review on animal models summarizes the pathwaysthat link internal stressors, for example depressive-like behaviorsinduced by lipopolysaccharide (LPS), to microglial activation andneuroregression (Song and Wang, 2010). We will start with a briefreview on the I&ND hypothesis of depression in humans and thepathways by which peripheral inammatory pathways may drivecentral biochemical and inammatory changes thereby causingdepressive-like behaviors.

2. The inammatory and neurodegenerative (I&ND) hypotheses ofdepression

2.1. IO&NS pathways in depression

There is now evidence that pro-inammatory cytokines, producedby monocytes, macrophages and brain microglia, and by activated Tlymphocytes, play an important role as mediators of external andinternal stress responses (Maes, 1995, 2010). Based on various ndingsin clinical depression and animal models, the cytokine hypothesis wasformulated, i.e. depressionmay be caused by an increased production ofpro-inammatory cytokines that are caused by external or internal

stressors (Maes, 1993, 1995, 2008; Maes et al., 2009; Anisman, 2009;Miller et al., 2009). The ndings include a) inammatory abnormalitiesin clinical depression, e.g. increased production of pro-inammatorycytokines, such as interleukin-1 (IL-1), IL-6, and tumor necrosisfactor- (TNF); and an acute phase response (Maes, 1995). b) Cell-mediated immune activation as indicated by increased production ofinterferon- (IFN), neopterin, and IL-12; increased levels of the solubleinterleukin-2 receptor (sIL-2R) and sTNFRs; and increased synthesis oftryptophan catabolites (TRYCATs) through cytokine induction ofindoleamine 2,3-dioxygenase (IDO) (Maes, 2010). c) Cytokine-basedimmunotherapy in hepatitis C and cancer patients often causesdepression (Maes, 1995). d) Cytokine administration or systemicadministration of LPS induces depression-like symptoms in animalmodels of depression (Dunn and Swiergiel, 2005; Goshen and Yirmiya,2009). e) Antidepressants have anti-inammatory effects in depressedpatients and in animal models of depression as well (Maes et al., 1999;Kubera et al., 2000a,b,c). f) In experimental and clinical studies, anti-inammatory drugs which block the production or activity of pro-inammatory cytokines, e.g. cyclooxygenase (COX)-2 inhibitors, efer-nazept or 3-fatty acids, have antidepressant activities (Mller et al.,2009). g) Clinical depression and animal models of depression areaccompanied by O&NS, including increased levels of radical oxygenspecies and radical nitrogen species (ROS/RNS) and thiobarbituric acidreactive substances (TBARS), an index for lipid peroxidation, whileantidepressants have antioxidative effects (review: Maes et al., 2010).

2.2. Peripheral inammation may provoke neuroinammation

Pro-inammatory cytokines, and in particular IL-1, TNF and to alesser extent IL-6, generated in the periphery, are able to initiatecytokine synthesis in the CNS. Although cytokines produced by immunecells in theperiphery cannot diffuse across the intact blood-brain barrier(BBB) they cana)diffuse into thebrain throughBBBdecientbrain areasor be actively transported into the CNS by endothelial cell transporters;b) activate BBB endothelial cells to produce various soluble factors e.g.prostaglandin (PG)E2, which can activate neurons via activation ofmicroglia and astrocytes; and 3) activate afferent neuronal signals,particularly afferent vagus nerve, which signals the CNS of the presenceof systemic inammation (Goehler et al., 2000; Banks, 2005; Turrin andRivest, 2004; Pavlov and Tracey, 2005). This explains why peripherallyborn cytokines through their effects on brain neuroinammation areable to induce physiological and behavioral responses in the CNS typicalfor depression. Thus, in animals and humans, systemic inammatorypathways provoke sickness behavior, including behaviors such asanhedonia; reduction of locomotor activity, exploration and grooming;lethargy; anxiety; sleepiness; anorexia; weight loss; hyperalgesia; andfailure to concentrate or memory disturbances (Holmes and Miller,1963; Hart, 1988; Maier et al., 1993; Kelley et al., 2003; Yirmiya, 1996;Exton, 1997; Qin et al., 2007). Pro-inammatory cytokines, such as IL-1and TNF, may cause sickness behavior by bottom-up signaling fromthe peripheral blood to the brain (Maier et al., 1993; Kelley et al., 2003;Qin et al., 2007). Systemic LPS administration induces a peripheralinammation and central neuroinammation or microglial activation,resulting in chronically elevated IO&NS pathways, e.g. increased TNFlevels that may even be elevated for months (Qin et al., 2007).

2.3. The I&ND hypothesis of depression

Neurodegeneration and decreased hippocampal neurogenesis areother pathways that are involved in depression (Sapolsky, 2004; Hennand Vollmayr, 2004). In this respect, only a few ndings are mentionedhere. Using MRI in depressed patients, Campbell and MacQueen(2006) observed volumetric changes in the hippocampus, amygdala,prefrontal cortex, anterior cingulate and basal ganglia. Stockmeier et al.(2004) found signicant neuronal and glial cell modications in thepostmortem hippocampus. Signs of lowered hippocampal volume are

related to neurocognitive disorders in depression (Brown et al., 2004).

746 M. Kubera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 744759Reduced hippocampal neurogenesis is now suggested as a nalcommon pathway in many brain disorders associated with mood(Duman, 2004) and cognitive dysfunction, e.g. geriatric depression; andthe depression mild cognitive impairment dementia complex(Duman, 2004; Hayley et al., 2005; Henn and Vollmayr, 2004; Henn etal., 2004). In healthy brains, neurogenesis occursmainly in two regions:the subventricular zone, which generates new neurons in the olfactorybulb; and the subgranular zone, which generates new neurons in thedentate gyrus of the hippocampus. Studies on adult neurogenesis aremostly restricted to the hippocampus. Decreased neurogenesis indepression is associated to lowered levels of neurotrophins, such asbrain-derived neurotrophic factor (BDNF) (Angelucci et al., 2005; Smithet al., 1995).

Initially, most research on neurodegeneration and decreasedneurogenesis examined the effects of glucocorticoid hypersecretionin depression since glucocorticoids (GCs) provoke adverse effects inthe hippocampus and therefore contribute to neurodegeneration(Sapolsky, 2004). However, the I&ND hypothesis (Maes et al., 2009)considers that sequels of cell-mediated immune activation andinammatory pathways in depression are the causes for neurode-generation in depression (Maes et al., 2009). First, increased levels ofglucocorticoids in depression are at least partly caused by increasedproduction of pro-inammatory cytokines, like IL-1 and IL-6 (Maes,1995). Secondly, pro-inammatory cytokines, such as TNF and IL-1,and to a lesser extent IL-6, and O&NS result in neurodegeneration anddecreased neurogenesis (Maes et al., 2009). The mechanisms wherebythese pro-inammatory cytokines and O&NS pathways provokeneurodegenerative disorders and antineurogenic effects are reviewedelsewhere (Maes et al., 2009, 2010).

3. External stressors activate IO&NS pathways and cause reducedneurogenesis and neurodegeneration

3.1. External stress models

In this paper we will review the data obtained in two externalstress-induced models of depression, i.e. CMS and LH. The CMSparadigm involves the exposure of animals to a series of mild andunpredictable stressors such as isolation, crowded housing, altera-tions of darklight cycle, restricted food access etc. for up to 3 months.After CMS, the animals show a variety of symptoms resemblinghuman depression, such as long-lasting changes of locomotor activity,weight loss, altered diurnal rhythms, sleep disturbances, andanhedonia. The model is pharmacologically sensitive and theanhedonia following the exposure to CMS is reversed by antidepressanttreatments.

The LH model was initially developed by Overmier and Seligman(1967) in dogs, and subsequently demonstrated in rats, mice andhuman beings. LH is induced through application of uncontrollableand unpredictable aversive stimuli, in the form of an acute stressor.The latter can be an electric foot shock or tail shock or loud acousticsounds. Rodents subjected to severe inescapable tail shock stressusing the LH paradigm displayed marked impairments in learning ofan active task, i.e. they learned to be helpless (Maier, 1990). Inaddition to escape behavior decits, helpless animals show weightloss, agitated locomotor behavior, sleep changes, decreased libido,decits in cognitive and rewarded behavior and elevated corticoste-rone concentrations. The animals appear to maintain helplessbehavior for about 10 days to 2 weeks. Only a part of the animals,around 20%, develop signs of helplessness, a nding that is explainedby differences in animal strains; animal supplier sources; andpotential geneenvironment interactions (Henn and Vollmayr,2003). Different classes of antidepressants reverse helplessness(Henn and Vollmayr, 2003).

Other external stress models that we will review comprise

repeated unpredictable stress, which entails exposure of rats to twoof eight different stressors twice daily (forced swim, restraint stress,overnight lights, etc.) for 10 days; and maternal separation is ananimal model of early life stress in which rat pups are deprived ofmaternal contact once or repeatedly during the rst postnatal weeks(Hofer, 1996). The effectiveness of antidepressant drugs can also betested in the forced swimming test (Porsolt test) (Kubera et al.,2006a), the tail suspension test, motherinfant separation test,prenatally stressed rodents, and the olfactory bulbectomized (OB)model of depression.

3.2. External stressors induce IO&NS pathways and provoke depression-likebehavior

In this section we will review stress-related animal models ofdepression, e.g. the CMS and the LH model, and the associationsbetween stress-induced depression-like behaviors and the increasedproduction of IL-1, IL6 and TNF, and neurodegeneration as well.There is nowevidence that external stress in humans is accompanied byan increasedproductionof pro-inammatory cytokines, including IL-1,IL-6, and TNF (review: Maes, 1999, 2001). Therefore, it was positedthat increased production of pro-inammatory cytokines followingexternal stressors play a role in the pathophysiology of stress-induceddepression (Maes, 2001). In experimental animals, psychologicalstressors induce the production of pro-inammatory cytokines, suchas IL-1 and IL-6 (Maes, 1999, 2001). Immobilization stress increases IL-1mRNAexpression in thehypothalamus andbiologically active IL-1 in thehypothalamus (Minami et al., 1991; Shintani et al., 1995a,b). Inescap-able shock increases brain IL-1 in the adrenalectomized rats 2 h afterstress (Nguyen et al., 1998). Mild inescapable footshock signicantlyincreases the stimulated production of IL-1 and TNF by isolatedalveolar macrophages (Persoons et al., 1995). Immobilization stress,exposure to electric footshock, physical restraint, open eld stress orconditioned, aversive stimuli elevate plasma IL-6 and IL-6 mRNAexpression in midbrain (Nguyen et al., 1998; Shizuya et al., 1997;Takaki et al., 1994; LeMay et al., 1990).

Kubera et al. (1996) found that the depression-like state induced bychronic (3-week) exposure ofWistar rats to CMS increased the ability ofsplenocytes to produce IL-1. After undergoing stress for three-weeks,C57BL/6mice show increased IL-1production (Kuberaet al., 1998).Also,Grippo et al. (2005) found that IL-1was signicantly increased in thebrain and blood in CMS animals. Moreover, brain cytokine concentra-tions are negatively correlated with sucrose intake, suggesting that thehigher levels of these cytokines are responsible for the greater degree ofanhedonia. This suggests that increased levels of pro-inammatorycytokines in the CNS are related to the severity of anhedonia. In a recentstudy that examines the effects of CMS on atherosclerosis, apolipopro-tein E knockout (apoE/) mice subjected to daily CMS for someweeks showed increased IL-1production (Gu et al., 2009). The key roleof IL-1 in the onset of depression-like behavior was demonstrated byGoshenet al. (2008). These authors observed that CMS caused increasedhippocampal IL-1 levels.Moreover,mice chronically administratedwithIL-1 via osmotic minipumps for 4 weeks, without any exposure tostressors, expressed similar behaviors as mice that were exposed toCMS. These ndings suggest that IL-1 is sufcient for the manifestationof depressive-like behaviors (Goshen and Yirmiya, 2009).

LH is modulated by inammatory mediators, such as IL-1 and LPStreatment. Thus, acute and chronic administration of IL-1 signi-cantly increases the latency of escape to a foot shock (Bonaccorsoet al., 2003). Similarly, intracerebroventricular (i.c.v.) administrationof the IL-1 receptor antagonist (IL-1Ra) 1 h before inescapable shockblocked the subsequent interference with escape learning andenhancement of fear conditioning normally produced by thistreatment (Maier and Watkins, 1995). These ndings are supportedby recent studies showing that pre-treatment with IL-1Ra did notcause a footshock-induced reduction of social interaction, supporting

the view that brain IL-1 plays a role as a mediator of stress-induced

747M. Kubera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 744759responses (Arakawa et al., 2009). In rats, repeated open eld or forcedswim exposition increased IL-1 in the amygdala and in thehippocampus after acute and chronic stress (Badowska-Szalewskaet al., 2009). Other studies, however, were unable to detect thatexposure to forced swim causes higher central IL-1 production, whichsuggests that the central IL-1 system is not always important as astress mediator following this stressor (Deak et al., 2003).

Not only IL-1, but also TNF is signicantly increased in the brainand blood of CMS animals (Grippo et al., 2005). Thus, 4 weeks ofexposure of male, SpragueDawley rats to CMS provoked anhedoniathat was associated with increased plasma levels of TNF andcorticosterone (Grippo et al., 2005). Pan et al. (2006) observed thatCMS-treated rats showed increased levels of TNF and IL-1 andserum corticotropin-releasing factor (CRF) and corticosterone levels.In the abovementioned study by Gu et al. (2009), CMS provokedincreased TNF production in apoE/ mice. Xiu et al. (2010)detected that CMS reduces the average sucrose consumption andincreases serum TNF in both tumor-bearing and non-tumor-bearingrats. Importantly, stressed tumor-bearing rats had less sucroseconsumption and increased TNF levels, while an inverse associationwas detected between serum TNF and sucrose consumption (Xiuet al., 2010). In the brain, the active forms of TNF, TNF convertingenzyme (TACE), and the receptors TNF-R1 (p55) and TNF-R2 (p75)are detected. Immobilization stress increases TACE and TNF levels inthe brain cortex (Madrigal et al., 2002) and increases TNF-R1 anddecreases TNF-R2 expression (Caso et al., 2006). In other stressexperiments, decreased brain TNF levels were found (O'Connoret al., 2003), that may cause a decreased neuroprotective activity ofthis cytokine via TNF-R2.

In the abovementioned study by Xiu et al. (2010), CMS increasedserum IL-6. Grippo et al. (2005) found no signicant alterations in IL-6in the brain and blood in CMS animals. Chourbaji et al. (2006), on theother hand, found that IL-6(/) mice showed resistance to stress-induced helplessness. These data suggest that IL-6 plays a role in themolecular mechanisms in depressive-like behaviors because externalstressors increase IL-6 in wild-type hippocampi and a lack of IL-6confers resistance to depression-like behavior (Chourbaji et al., 2006).Rats subjected to IL-6 administration in the amygdala and hippocam-pus show an increased immobility time in the forced swimming test(Wu and Lin, 2008). Importantly, inhibiting IL-6 in the amygdala andthe hippocampus signicantly reduced the immobility time in theforced swimming test (Wu and Lin, 2008).

There are now many data that external stressors activate O&NSpathways in brain cells (astrocytes and microglia) and brain macro-phages. For example, CMS signicantly reduced sweet food intake,increased the production of superoxide production in many brainstructures and increased TBARS in cortex (Lucca et al., 2009). In anotherstudy, CMS provoked a reduction in weight gain and an increase inimmobility time in the forced swimming test and signicantly increasedTBARS (Tagliari et al., 2010). CMS has been shown to increase O&NS inthe amygdala, hippocampus and anterior thalamus in male but notfemale rats (Mllo et al., 2009). Inmale streptozotocin-induced diabetesWistar rats, the forced swimming test resulted in signicantly increasedTBARS in the serum and hippocampus (da Silva Haeser et al., 2007).Pedreanez et al. (2006) reported increased numbers of superoxideanions in the cerebrum and cerebellum in rats with depressive-likebehaviors. Madrigal et al. (2001) andOlivenza et al. (2000) showed thatacute or chronic immobilization stress increases iNOS and ONOOexpression in the brain. Inhibition of iNOS prevents the development ofanxiety and depressive-like behavior in response to acute and chronicstressors (Sevgi et al., 2006; Mutlu et al., 2009; Garca-Bueno et al.,2008a). For more references and details on the O&NS pathways thereader is referred to another review article in this special issue (Maeset al., 2010). All in all, external stressors may induce IO&NS pathwaysthat may be related to the onset of depression-like behaviors (Maes

et al., 2010).3.3. Other mechanisms involved in external stress-induced depression-likebehaviors

Besides IL-1, TNF and IL-6, there are a number of otherinammatory molecules that are responsive to external stress andthus may play a role in stress-induced (neuro)inammation, e.g.nuclear factor B (NFB), COX-2 and LPS-related mechanisms.Therefore, we will briey discuss the effects of external stressors onthese molecules. Fig. 1 shows that activation of NFB is one of theearliest events in the stress-inammatory response. NFB recognizesspecic DNA sequences in the promoter of target genes, such as iNOSand COX-2, andmany other genes encoding pro-inammatory factors.CMS mice not only show signicantly increased IL-1 and TNFlevels, but also increased NFB (Gu et al., 2009). This is in line withother results showing that restraint stress activates, as soon as 4 hafter stress termination, signicant increases in brain NFB expression(Madrigal et al., 2001). Munhoz et al. (2006) showed that chronicunpredictable stress exacerbates LPS-induced activation of NFB inthe frontal cortex and hippocampus. All in all, we may speculate thatchronic external stress via activation of NFB may play a role in theincreased production of pro-inammatory cytokines following stress.

External stressors activate COX enzymes that enable the produc-tion of lipophilic molecules including prostaglandins, e.g. PGE2,leukotrienes, and thromboxanes. There are three forms of COXenzymes: COX-1, COX-2 and COX-3. COX-1 is constitutively expressedin the brain tissue, whereas COX-2 is rapidly induced by cytokines andbacterial endotoxines. COX-2 is able to produce high levels ofperoxides and prostanoids, like PGE2, sometimes 1020 times greaterthan the physiological levels produced by COX-1 (Seibert et al., 1995;Garca-Bueno et al., 2008a,b). External stressors, such as immobiliza-tion stress (Madrigal et al., 2003) and swimming stress (Yamagataet al., 1993), and also exposure to elevated temperatures (Katafuchiet al., 2003) increase COX-2 expression and/or activity in the brain.There is now evidence that COX-1 and COX-2 pathways are involvedin depressive-like behavioral induced by systemic inammation.COX-1 is responsible for the early onset of behavioral effects, whileCOX-2 is involved in late behavioral changes. COX-2 inhibitors andantibodies against IL-1, TNF and IL-6 attenuated IL-1- but not LPS-induced sickness behavior. The behavioral effects induced by LPS arereversed by indometacin, a nonspecic COX1/2 inhibitor, whichpoints towards the role of COX-1 in systemic inammation (Teelinget al., 2007; Teeling and Perry, 2009). It is thought that low gradesystemic inammation, can stimulate COX-1 dependent production ofCRH, which viamicroglial CRHR1 stimulates the production of pro-inammatory cytokines and this response may be exaggerated ifprimed with LPS (Teeling and Perry, 2009).

Another receptor that responds to LPS, i.e. the Toll-Like receptor(TLR), is sensitive to external stressors. TLRs recognize pathogenassociated molecular patterns (PAMPs), i.e. conserved microbialstructural motifs; and damage-associated molecular patterns(DAMPs), i.e. endogenous molecules released from disrupted cellsand degraded cellular matrix. The interaction between the TLR andPAMPs plays a key role in innate immune responses. Ligandrecognition by TLR activates twomajor intracellular pathways leadingto activation of NFB or mitogen activated protein kinases (MAPK)(p38 and jun amino-terminal kinase). Although TLRs are mainlyexpressed on cells of the immune system, they are also found onmicroglia, astrocytes and neurons. In the CNS, TLRs are mainlyactivated by DAMPs, and probably this process is increased duringstress responses (Lewthwaite et al., 2002; Garca-Bueno et al., 2008a).Thus, Gu et al. (2009) showed that CMS provoked signicant increasesin the expression of TLR4. Social stressors signicantly increase theexpression of TLRs on the surface of splenicmacrophages (Bailey et al.,2007). TLR4-decient mice show better behavioral performance anddecreased inammatory responses and lipid peroxidation in brain

tissue in response to immobilization stress in comparison to mice

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748 M. Kubera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 744759EXTERNAL STRESSO

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Chronic mildChronic mildstress

O&NSLipid

peroxida

Depression-like TNFaCaspaseDepression-likebehavior

TNFaIL-1b

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Fig. 1. External stressors in animals, such as chronic mild stress and learned helplessnebehavior is accompanied by peripheral and central inammation, with increased levelwith normal TLR4 expression (Caso et al., 2008). Thus, externalstressors upregulate TLR4 expression which suggests that thepresence of these stressors may aggravate (neuro)inammation dueto internal stressors, e.g. PAMPs and DAMPs, and thus may cause asuperinduction of (neuro)inammatory responses.

3.4. External stressors, depression-like behaviors and antineurogeniceffects

In this section we will review the possible associations betweenthe behavioral and antineurogenic effects of external stressors inanimals. CMS reduces hippocampal neurogenesis and cytogenesis inwild mice but not in IL-1 receptor knockout mice (Goshen andYirmiya, 2009). Jayatissa et al. (2006) found that CMS signicantlydecreased sucrose consumption and cytogenesis in the hippocampusas measured by means of bromodeoxyuridine (BrdU), a marker ofproliferating cells. Moreover, these authors found an associationbetween recovery from depressive-like behaviors due to CMS andincreased cytogenesis in the hippocampus. In Swiss albino mice, CMScaused severe neuronal cell damage, as measured by BrdU positivecells (Li et al., 2008). In male SpragueDawley rats administered BrdUbefore or after CMS, K.J. Lee et al. (2006) found that CMS signicantlydecreased the survival of new-born cells in granule cell layer of thehippocampus, and had no effect on the proliferation or differentiationof new-born cells. Rodents subjected to CMS show signicantdecreases in hippocampal neurogenesis (Alonso et al., 2004). Huaet al. (2008) also reported that CMS induces depressive-like behaviorsand impairs hippocampal neurogenesis. In addition to increasedneurodegeneration and reduced neurogenesis, external stressors may

(TNFa) and IL-6. The external triggers and cytokines may induce nuclear factor B (NFB)nitrosative stress (O&NS) pathways, and cyclooxygenase (COX)-2. Through these pathway(RNS) species, including O2 and NO that results in the generation of peroxynitrite. COXincrease the expression of Toll-like receptors (TLR4), which increase the sensitivity to internmicrobial structural motifs, such as lipopolysaccharide (LPS), and damage-associated molecuand consequently provoke activation of neuronal N-methyl-D-aspartate (NMDA) receptors. Esuch as brain-derived neurotrophic factor (BDNF) and VGF. Finally, external stressors causeincreased levels of caspase-3. Cytokines, O&NS, NFB, COX-2, prostaglandins, e.g. PGE2; excicontribute to the neurodegenerative processes and reduced neurogenesis which are observ

More sensitive to internal stressors: PAMPs (LPS) and DAMPs

TLR4

PAMPs (LPS) and DAMPs

GlucocorticoidsNFB

activation

COX-2

PGE2PGJ2

S Excitotoxic glutaminergic effectsNMDA receptors

SNS

rogenesis, apoptosis and cell damage

re accompanied by depressive-like behaviors. External stress-induced depression-likepro-inammatory cytokines, such as interleukin-1 (IL-1), tumor necrosis factor-also provoke impairments in brain plasticity, e.g. in long-termsynaptic plasticity. Thus, CMS facilitates long-term synaptic depres-sion but has no effect on long-term potentiation as examined in thehippocampus bywhole-cell patch clampmeasurements in brain slices(Holderbach et al., 2007). In the CMS model, immune activation isrelated to neuroendocrine and behavioral changes and alterations inneuroplasticity (Koo and Duman, 2008; Grippo et al., 2005).

CMS not only induces depressive-like behaviors but also neurocog-nitive decits. Therefore, it is important to examine the associationsbetween reduced neurogenesis and neurocognitive functioning follow-ing CMS. Thus, CMS increases IL-1, TNF and IL-6 and provokesneurocognitive defects, as estimated by the water maze task, the objectrecognition and object location test (Li et al., 2008). In another study,CMS signicantly reduced the surviving of new brain cells in thehippocampus and the subventricular zone and an impairment inhippocampus-dependent learning, while hippocampus-independentlearning was not affected (Mineur et al., 2007). While CMS impairedhippocampus-dependent learning and reduced neurogenesis no directcausative relationships could be established between the behaviors andreduced neurogenesis (Mineur et al., 2007).

In the LH model, the stressor decreased the formation of newdentate gyrus cells and these changes correlated with the behavioraldespair several days after exposure to the stressor in the LH model ofdepression (Malberg and Duman, 2003). In another study, helplessbehavior developed before the decrease of dentate gyrus cellproliferation was maximal, suggesting that there is no signicantassociation between cell proliferation and the induction or mainte-nance of LH (Vollmayr et al., 2003). In any case, the stressor acutelydownregulated dentate gyrus cell proliferation in animals that

, which in turn induces the expression of pro-inammatory cytokines, oxidative ands, external stress may cause increased amounts of reactive oxygen (ROS) and nitrogen-2 may generate prostaglandins (PG), such as PGE2 and PGJ2. External stressors alsoal stressors, including pathogen associated molecular patterns (PAMPs), i.e. conservedlar patterns (DAMPs). External stressors increase glucocorticoids and glutamate releasexternal stressors cause antineurogenic effects by downregulating neuroptrophic factors,apoptosis with lowered levels of Bcl-2 and BAG1 (Bcl-2 associated athanogene 1), andtotoxic glutaminergic effects, apoptotic pathways and reduced neurotrophic substancesed in depression-like behaviors.

749M. Kubera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 744759developed helplessness and in those that did not develop helplessness(Vollmayr et al., 2003). Moreover, the latter authors found thatimmobilization stress did not cause learned helplessness although itreduced cell proliferation. The rate of neurogenesis is not simplyrelated to the behavioral changes seen in depression, since only asmall proportion (20%) of the animals exposed to a foot shockdeveloped LH, whereas all animals examined had exactly the samedecrease in neurogenesis rate (Henn and Vollmayr, 2004b).

3.5. Are stress-induced antineurogenic effects sufcient to explaindepression-like behaviors?

The abovementioned data shows that not all results suggest thatreduced neurogenesis determines depression-like behavioral andneurocognitive defects following external stressors (Vollmayr et al.,2007). This thesis is substantiated by recent research that scrutinizedwhether there are associations between behaviors and antineurogeniceffects following external stressors. First, Jayatissa et al. (2010)examined CMS rats with stereological estimations of the number ofproliferatingprogenitors, number of granule cells, and the volumeof theventral hippocampal formation (VHF). These authors found that in CMS,the depression-like state occurs prior to reduced cell proliferation and isnot directly associatedwith reduced numbers of granule cells in the VHF(Jayatissa et al., 2010). These ndings suggest that CMS reduces cellproliferation and the total number of granule cells in the VHF but thatthose changes are not associated with the onset of depressive-likebehaviors. Second, in another study hippocampal neurogenesis waseliminated by means of focal hippocampal irradiation to disrupt cellproliferation (Surget et al., 2008). It was found that this procedure hasno effect on the sensitivity to CMS, which shows that reducedneurogenesis is not associated to external stress-induced depression-like behaviors (Surget et al., 2008). Also, CMS provokes reductions inshort-term neurogenesis that however may be fully recovered in thelong term,while CMS provokes lasting anhedonia (Elizalde et al., 2010).This shows that decreases of neurogenesismaynot be sufcient to causebehavioral defects following CMS. Indeed, the behaviors may occurwithout reducing neurogenesis, while reduced neurogenesis is notnecessarily the cause of the disturbed behaviors. In this respect, it ishypothesized that stress-induced antineurogenic effects may uncoupleemotions from their external context which may contribute to anegative mood state (Perera et al., 2008).

3.6. Mechanistic explanations of the neuro-changes induced by externalstress

3.6.1. Inammatory mechanismsIn this section we will review that the neurodegeneration that

accompanies stress-induced depressive-like behavior is not onlycaused by the neurogenic effects thatwe have discussed in Section 2.2,but also by the effects of activated IO&NS, and increased corticoste-rone and glutamate levels (Maes et al., 2009).

Administration of IL-1 suppresses hippocampal cell proliferation,whereas blockade of the IL-1 receptor (IL-1R) by using either aninhibitor or IL-1R nullmiceblocks the antineurogenic effect of stress andblocks the anhedonic behavior caused by chronic stress exposure. Invivo and in vitro studies demonstrate that hippocampal neuralprogenitor cells express IL-1R and that activation of this receptordecreases cell proliferation via the NFB signaling pathway. Thesendings conrm that IL-1 is a critical mediator of the antineurogenicand depressive-like behavior caused by acute and chronic stress (KooandDuman, 2008). IL-1 is not only a pivotal pro-inammatory cytokine,but also an important mediator of CNS injuries (Pinteaux et al., 2009;GoshenandYirmiya, 2009).As discussed, external stressors can increasethe expression of IL-1 in the central nervous system, e.g. in thehypothalamus (O'Connor et al., 2003),whereas administration of the IL-

1ra prevents the stress-induced depressive-like behaviors and rescuesneurons from apoptosis via reduction of ROS production (Y.J. Lee et al.,2006). IL-1-induced neurotoxicity is mediated via actions on glia cells,mainly astrocytes and possibly other cells, e.g. endothelial cells, and isdose dependent. Such activation causes the production of glialneurotoxic factors such as free radicals and metaloproteinases whichmay cause neuronal death, e.g. MMP9 produced by astrocytes andactivated by neuronal urokinase plasminogen activator (Pinteaux et al.,2009; Thornton et al., 2006, 2008). IL-1 impairs BDNF signaltransduction (Tong et al., 2008).

TNF may via TNF-R1 activate MAPK and increase NFB causingneuronal apoptosis (Hallenbeck, 2002), whereas activation of theTNF-R2 probably results in neuroprotective effects (Williams et al.,2005). Thus, the two TNF receptors may exhibit antagonistic functionson neurodegenerative processes whereby TNF-R1 aggravates andTNFR2 reduces neuronal cell loss, respectively (Hallenbeck, 2002;Williams et al., 2005). Both TNF-R1 and TNF-R2 may induce the NFBpathway, be it with distinguishable kinetics and upstream acti-vating components. For example, TNF-R1 induces a transient NFBactivation, whereas TNFR2 facilitates long-term phosphatidylinositol3-kinase-dependent NFB activation (Marchetti et al., 2004). Munhozet al. (2004) reported that TNF plays a key role on the persistence ofbrain cellular damage two weeks after repeated stress.

The role of IL-6 in response to external stressors is very complex. IL-6may protect neurons but in other conditions may have detrimentaleffects. For example, intracerebral administration of IL-6 beforeexposition of rodents to ischemic or excitotoxic insults inhibits neurondeath (Lucas et al., 2006). Mice with chronic IL-6 overexpression, showmarked neurodegeneration and psychological distress that are relatedto increased IL-6 plasma and/or brain concentrations (Allan andRothwell, 2003). The involvement of O&NS is exemplied in a studyshowing that CMS-induced behavioral depression and impairedhippocampal neurogenesis are prevented in mice that are treatedwith a neuronal NOS (nNOS) inhibitor and in null mutant mice lackingthe nNOS gene (nNOS/) (Zhou et al., 2007). There is now evidencethat NFB plays a role in neurodegenerative processes. For example,inhibiting NFB results in neuroprotective effects through inhibition ofNFB-mediated gene transcription of pro-inammatory cytokines(Hwang et al., 2010). In Alzheimers' Disease, for example, microglialNFB signaling is a key component in neuronal death caused byamyloid-beta (Chen et al., 2005).

COX-2 is not only a key component in neuroinammation, but playsalso a role inneurodegenerative disorders (Yang andChen, 2008). Theseeffects are caused by the ability of COX-2 to modify synaptic activities(Yang and Chen, 2008). Elevation of COX-2 enhances the excitatoryglutamatergic neurotransmission and long-term potentiation, effectsthat are mediated by PGE2, and the PGE2 receptor (Chen et al., 2002).PGE2 by acting on EP2 receptors may aggravate cAMP-dependentexcitotoxic neurodegeneration (Takadera and Ohyashiki, 2006). PGE2and other prostanoidsmay be neurotoxic and generate cellular death byapoptosis via inducing glutamate release from astrocytes and ROSproduction (Takadera et al., 2002; Phillis et al., 2006). PGE2 may causemotor neuron death by its effects on glutamate release from astrocytesand formation of reactive oxygen species (Izecka, 2003). Li et al. (2004)observed that COX-2 induction and the concomitant PGJ2 productioncauses an autotoxic loop between both compounds that furtheraggravates neurodegeneration. There are, however, some data showingthat COX-2 and PGE2 may have neuroprotective effects. Thus, Uchidaet al. (2002) reported that PGE2 plays an important role in cellproliferation in the subgranular zone. Injections of sulprostone, a PGE2analogue, increased the number of 5-bromo-2-deoxyuridine-positivecells (Uchida et al., 2002). Different prostaglandins, including PGE2,increase the secretion and synthesis of BDNF and nerve growth factor(NGF), indicating that these compounds have neurotrophic properties(Toyomoto et al., 2004).

Moreover, stress may induce the release of glutamate and

aspartate into the synaptic cleft, which can induce excitotoxic necrotic

750 M. Kubera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 744759brain injury (Joca et al., 2007). CMS provokes reductions in short-termneurogenesis and lasting changes in c-Fos immunoreactivity andglutamate and GABA levels (Elizalde et al., 2010). Extracellularglutamate binds to the N-methyl-D-aspartate (NMDA) receptorswhich may induce further glutamate release and a dramatic increasein intracellular Ca2+ levels and neuronal death as a result ofoveractivation of Ca2+-dependent enzymes (e.g. proteases, lipases,peroxidases) and generation of free radicals. Injured neurons and glialcells, in turn, release pro-inammatory cytokines, which may causeneurodegeneration. An important role in stress-induced neuroin-ammation is played by stress-induced inhibition of expression of theexcitatory amino acid transporters, which enhance the uptake ofglutamate from the synaptic cleft (Garca-Bueno et al., 2007, 2008a).

Glucocorticoids (GC) together with catecholamines (adrenaline,noradrenaline, dopamine) are among the main mediators releasedduring stress. The opinion that glucocorticoids have only anti-inammatory properties has been changed because high levels of GCmay induce immune cell extravasation, migration, production ofinammatory messengers and activation of pro-inammatory tran-scription factors (Sorrells and Sapolsky, 2007, Garca-Bueno et al.,2008a,b). For example, unpredictable stress increases TNF, IL-1 andiNOS protein levels in the hippocampus, cortex and prefrontal cortexwhen LPS is administered peripherally (Munhoz et al., 2006; De Pabloset al., 2006). This effect appears to be GC receptor mediated by priorexposure to GC and can result in a priming of the immune responseto a subsequent pro-inammatory response (Garca-Bueno et al.,2008a). GCs exacerbate glutamate-induced neuronal loss in inam-matory context (Sapolsky, 2004) and induce expression of enzymesrelated to prostaglandin (lipocalin type-prostaglandin synthesis) andleukotriene synthesis (5-lipooxygenase) in CNS (Garcia-Fernndez etal., 2000). GC secreted by stress increase the sensitivity to LPS-inducedinammation in different brain areas as indicated by increasedimmunostaining of molecular markers of activated microglia andastrogliosis, e.g. glial brillary acid protein (Nair and Bonneau, 2006).

3.6.2. Antineurogenic mechanismsExternal stressors may also exert direct antineurogenic effects since

they reduce the production of neurotrophic compounds. Thus,signicant reductions in the secretion of hippocampal BDNF wereobserved 5 weeks after CMS exposure. Chronic restraint stress signif-icantly reducedhippocampal cell proliferation andBDNFexpression (Xuet al., 2006). Hu et al. (2010) were able to conrm that CMS provokes areduction in sensitivity to reward and decreased mRNA and proteinBDNF levels. In Swiss albino mice, CMS caused a signicant reducedexpressionof BDNF indentate gyrus of thehippocampus (Li et al., 2008).These ndings suggest that CMS impairs cognitive behaviors throughantineurogenic effects. In another study, however, CMS did not changeBDNF mRNA levels in the granule cell layer of the hippocampus,suggesting that the survival of new-born cells may be reduced in thepresence of normal BDNF levels (K.J. Lee et al., 2006). BDNFadministration is able to reverse learned helplessness suggesting thatthe LH-induced decreases in BDNF synthesis are involved in depressive-like behavior. Thus, Siuciak et al. (1997) showed that midbrain BDNFinfusions blocked LH and decreased the immobility time by 70% in theforced swimming test. Greenwood et al. (2007) showed that bilateralinjections of BDNF into the dentate gyrus prior to stress preventedstress-induced reductionsof hippocampalBDNF, but did not prevent LH,whereas physical activity together with uoxetine conferred resistanceto the development of LH. Female BDNF conditional knockout miceshow normal locomotor activity but an increase in depressive-likebehavior estimated in the sucrose preference test and in the forcedswimming test (Monteggia et al., 2007).

The trkB receptor (TrkB tyrosine kinase), the high afnity receptorfor BDNF, responds to stressors. This receptor is activated by BDNF andmediates synaptic plasticity, survival and neuronal differentiation of

cells.Mice lacking TrkB in the neural progenitor cells in thehippocampusshow an impaired neurogenesis (Li et al., 2008). Nibuya et al. (1999)found that repeated immobilization stress increased catalytic trkBmRNA, but not truncated TrkB transcripts in rat hippocampus. Thus,while repeated immobilization stress decreases BDNF mRNA theincreased trkB receptor levels may be regarded as a compensatorymechanism to counteract the effects of the external stressors.

Both the learned helplessness and forced swim test paradigmsdownregulate VGF nerve growth factor in the hippocampus (Thakker-Varia et al., 2007). This neuropeptide plays a role in regulatingsynaptic plasticity and may act downstream of BDNF to reversedepressive-like behaviors due to external stressors (Thakker-Variaet al., 2007). CMS also increases glycogen synthase kinase-3 (GSK-3)but decreases synapsin-I expression in the hippocampus. Specicinhibition of GSK-3 blocks the effects of CMS in depressive-likebehavior (Silva et al., 2008). GSK-3 is a signal transducing moleculethat plays a role in neuronal loss in neurodegenerative diseases, suchas Parkinson's disorder (Petit-Paitel, 2010). Synapsins are a family ofproteins that regulate synaptic functions, including neurotransmitterrelease, by modulating the number of synaptic vesicles that will bereleased (Evergren et al., 2007).

3.6.3. ApoptosisApoptosis has been proposed to be another mechanism contrib-

uting to stress-related depression-like behavior. Chronic stress in treeshrew provoked mild decreases in the entire hippocampal volumeafter 28 days of chronic stress while apoptosis was signicantlyreduced in the stratum radiatum but increased in the hilus (Lucassenet al., 2001). Enhanced apoptosis was detected in the brain structuresof rats tested in animal models of depression, such as repeatedunpredictable stress (Kosten et al., 2008) and maternal separation(Lee et al., 2001). Tobe et al. (2005) reported that Fos expression doesnot adapt to repeated maternal stress in the fetal paraventricularnucleus and that the latter shows a strong vulnerability to cell death,including apoptosis. Social stress administered prior to mild ischemicinsult (stroke) strongly compromises the expression of Bcl-2 (DeVrieset al., 2001). Also, unpredictable stress reduced the expression of Bcl-2mRNA in the rat limbic structures (Jiao et al., 2005; Kosten et al.,2008). Bcl-2 is an anti-apoptotic endogenous membrane protein thatprotects against apoptosis and cell death, including that caused byO&NS (Kowaltowski et al., 2004). Bcl-2 promotes neuritogenesis andaxon regeneration (Chen et al., 1997). The gene that binds to Bcl-2,called Bcl-2 associated athanogene (BAG-1), enhances the anti-apoptotic effects of Bcl-2. BAG-1 regulates proteosomal proteinelimination pathways (Takayama et al., 1995). CMS has been shownto decrease the expression of BAG-1 in the hippocampus (Silva et al.,2008). These ndings suggest that the induction of apoptotic path-ways in CMS is at least partially related to Bcl-2/BAG1-relatedmechanisms. In another study, CMS (5 weeks treatment) provokedanhedonia that is associated by increases in caspase-3 positiveneurons in the cerebral cortex (Bachis et al., 2008). Caspase-3 is anapoptosis executioner that is activated in apoptotic cells by deathligands and mitochondrial pathways (Salvesen, 2002). Increasedcaspase-3 following CMS therefore indicates increased apoptosis.Also, Liu et al. (2010) found that CMS caused anhedonia andassociated increases in TUNEL-positive neurons, caspase-3 and Baxin the hippocampus. Bax is another pro-apoptotic molecule thatinduces apoptosis with an early release of cytochrome c precedingcaspase activation and subsequent proteolysis (Finucane et al., 1999).

4. The effects of antidepressants on IO&NS pathways andneurogenesis

4.1. Effect of antidepressants on IO&NS pathways

In this section we will discuss the effects of antidepressants on the

IO&NS and neurogenic pathways that play a role in the external

751M. Kubera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 744759stress-related animal models of depression. Since peripheral cyto-kines directly or indirectly affect brain function (Kronfol and Remick,2000), the effects of antidepressants on plasma cytokine concentra-tions or their production by isolated splenocytes, lymphocytes or bywhole blood cultures were examined. In the CMS model, theantidepressant effect of imipramine was associated with a decreasein concanavalin A-stimulated IL-1 production by splenocytes (Kuberaet al., 1996). Prolonged treatment with desipramine increased theproduction of IL-10 by lymphocytes isolated from chronic mild stressexposed and unexposed mice (Kubera et al., 2001b). IL-10 is an anti-inammatory cytokine that counteracts inammation and cell-mediated immune responses. Contrary to CMS-exposed rats, innaive rats chronic administration of imipramine had no effect onthis splenocytic activity. The results of in vivo and ex vivo studies aredepicted in Table 1. In OB rats, chronic treatment with desipramine, atricyclic antidepressant (TCA), remarkably diminished LPS-stimulatedplasma concentration of TNF and IL-1 (Connor et al., 2000). Innaive rats, chronic desipramine administration reduced LPS-inducedTNF levels and markedly augmented LPS-induced IL-10 plasmalevels (Shen et al., 1999). Moreover, desipramine normalized thehyperactivity in bulbectomized rats (Connor et al., 2000) andprevented LPS-induced anorexia, loss of body weight, antidipsogeniceffect and hypoactivity in naive rats (Shen et al., 1999). These resultssuggest that desipramine may exert its antidepressant effect at leastpartly through its anti-inammatory activity.

In contrast, the LPS-induced increase in TNF and IL-1 mRNAexpression in the rat spleen was not altered by a chronic treatmentwith imipramine, another TCA, or uoxetine, a selective serotoninreuptake inhibitor (SSRI), although these drugs are able to affect theLPS-induced behavioral and endocrine changes. Hence, it has beensuggested that facilitation of the recovery from the LPS-inducedsuppression of food consumption, body weight and plasma cortico-sterone levels by uoxetine or imipramine are not mediated by theirinuence on peripheral cytokines (Yirmiya, 1996; Yirmiya et al.,2001). Chronic treatment with paroxetine, an SSRI, and venlafaxine, aserotoninnorepinephrine reuptake inhibitor (SNRI), had no inu-ence on the LPS-induced levels of TNF and IL-10 in naive rats andthey also failed to alter any of the LPS-elicited behavioral effects (Shenet al., 1999). However, chronic treatment with paroxetine preventedthe IFN-induced alterations in cytokine production estimated insupernatants of the mitogens (Con A and LPS)-stimulated wholeblood culture. The levels of TNF and IL-1were lower, whereas IL-10was higher in cell cultures obtained from paroxetine-pretreated ratsin comparison to rats treated with IFN alone. Notably, it was alsoobserved that paroxetine prevented the anxiety behavior displayed byIFN-treated rats in the open eld test (Myint et al., 2007).

Atypical antidepressants can also inhibit behavioral decits associ-ated with immune challenges. Tianeptine, an antidepressant with anti-stress activity,which increases serotonin reuptake andhas aweak effecton noradrenergic system, when given repeatedly attenuated the effectsof peripheral LPS administration, anorexia, body, weight loss, hypoac-tivity, decreased social exploration and increased corticosterone levelsand also alleviated sickness behavior induced by peripheral IL-1(Castanon et al., 2001) but had a slight inuence on the plasma cytokinelevels. Tianeptine attenuated the LPS-stimulated TNF plasma concen-tration but did not affect signicantly IL-1 and IL-10 (Castanon et al.,2004). Acute and repeated administration of mirtazapine, an atypicalantidepressant that enhances noradrenergic and serotonergic neuro-transmission by blocking 2-adrenergic receptors, decreased IL-6release and increased IL-4 release by splenocytes of noradrenalinetransporter knockout (NET-KO) mice. NET-KO mice display a highersusceptibility to the immunosuppressive effect of mirtazapine thanwild-type mice, suggesting that the noradrenergic system plays animportant role in the immunological effects of mirtazapine. A decreasein immobility time in the forced swimming test was observed in C57BL/

6Jmice after acute but not repeatedmirtazapine administration. InNET-KOmice, mirtazapine did not induce this typical behavioral effect in theforced swimming test (Kubera et al., 2006b).

Table 2 summarizes the studies, which examined the effects ofantidepressants on brain cytokines. In naive rats, single injection ofdesipramine reduced LPS-stimulated cortical gene expression of IL-1and TNF (O'Sullivan et al., 2009). Paroxetine pre-treatmentprevented the increase in hypothalamic IL-1 and IL-10 induced byIFN (Myint et al., 2007). Among all brain structures studied, thesignicant effects of antidepressants were only observed in thehypothalamus. Long-term treatment with tianeptine (Castanon et al.,2004) decreased LPS-stimulated IL-1, IL-6 and TNF mRNAs levelsand increased marginally IL-10 mRNA in the hypothalamus.

More pronounced effects of antidepressants than those reported invivo were observed in vitro. For example, amitriptyline, a TCA, and itsmetabolite nortryptyline signicantly decreased LPS-stimulated pro-duction of IL-1 and TNF in primary mixed glial cell cultures andmicroglial cultures (Obuchowicz et al., 2006). Imipramine, uvox-amine, an SSRI, and reboxitine, a selective noradrenergic reuptakeinhibitor (NRI), inhibited IL-6 secretion by stimulated microglial cellculture (Hashioka et al., 2007). Desipramine suppressed LPS-inducedrelease of IL-1, IL-6 and TNF by neuronal stem cells (Huang et al.,2007), but failed to alter mRNA expression of IL-1 and TNF inprimary cortical glial cells (O'Sullivan et al., 2009). Hwang et al.(2008) showed that two TCAs, i.e. clomipramine and imipramine,signicantly reduced TNF and NO production in microglia andastrocyte cultures; and iNOS, IL-1 and TNF mRNA levels;translocation of NFB (p65 subunit) to the nucleus; and phosphor-ylation of MAPK in LPS-stimulated microglia cells. These results showthat both TCAs attenuate the IO&NS pathways during microglialactivation.

Reynolds et al. (2004) provided evidence that TNF plays a keyrole in themechanism of antidepressant action of desipramine. In rats,chronic i.c.v. microinfusion of polyclonal anti-TNF antibodies anddesipramine decreased the immobility time in the forced swimmingtest and increased norepinephrine release from hippocampal slicesisolated from these rats in comparison to the control group. Theopposite effect was induced by cerebral infusion of recombinant ratTNF (rrTNF). Moreover, chronic co-administration of rrTNF withdesipramine prevented an enhanced norepinephrine release inducedby desipramine. These results suggest that the efcacy of desipramineas an antidepressant drug is due to a decreased level of TNF becausecontinual decrease in this cytokine level stimulated norepinephrinerelease directly and indirectly by switching the function of 2-adrenergic receptors from inhibition to facilitation. This indicates thatantidepressants are effective because they decrease TNF.

All in all, pronounced effects of antidepressants on cytokines weredetected in studies conducted in glial cell cultures and studies thatexamined peripheral cytokines in animal models of depression. Thesestudies provided evidence that antidepressants of different classessuppress the production or expression of pro-inammatory cytokines,while increasing the levels of anti-inammatory cytokines, such as IL-10 and IL-4 (Shen et al., 1999; Kubera et al., 2001a, 2006a).

4.2. Effects of antidepressants on neurogenesis and apoptotis

As described previously, CMS provokes anhedonia and decreasedcytogenesis in the hippocampal formation (Jayatissa et al., 2006). Thelatter authors reported that chronic treatment with escitalopram, anSSRI, blocked the CMS-induced reduction in hippocampal cytogenesis inanimals that recovered and not in those that did not recover fromanhedonia. Rats subjected to chronic restraint stress were characterizedby decreased hippocampal cell proliferation, while repeated adminis-tration of quetiapine and venlafaxine prevented the stress-inducedreduction in cell proliferation (Xu et al., 2006). WAY-100635, a 5-HT1Areceptor antagonist, augmented the efcacy of citalopram in reversing

the anhedonia and post-stroke neurogenesis in the dentate gyrus of rats

uegue

s

s

s

s

s

BL/r rattar

752 M. Kubera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 744759Table 1Effects of antidepressants on peripheral cytokines.

Antidepressant and dose Treatment duration Animals

In vivo studiesDesipraminea

7.5 mg/kg ip.17 days Bulbectomized Sprag

Sham operated Spra

Desipramineb

7.5 mg/kg ip.21 days SpragueDawley rat

Imipraminec

10 mg/kg ip.5 weeks SpragueDawley rat

Fluoxetinec

10 mg/kg ip.5 weeks SpragueDawley rat

Paroxetineb

7.5 mg/kg ip.21 days SpragueDawley rat

Venlafaxineb

10 mg/kg ip.21 days SpragueDawley rat

Tianeptined

10 mg/kg ip.21 days twice daily Wistar rats

Ex vivo studiesDesipraminee

10 mg/kg ip.5 weeks CMS-exposed and

CMS-unexposed C57Imipraminef

10 mg/kg ip.5 weeks CMS-exposed Wista

CMS-unexposed WisParoxetineg

10 mg/kg po.14 days then paroxetine+IFN- sc. 50000 IU/kg 3 days/weekfor 5 weeks

Wistar rats

Mirtazapineh

20 mg/kg ip.Single injection7 daysSingle injection

NET-KO miceC57BL/6J miceexposed to CMS after ischemic surgery (Wang et al., 2010). Chronicadministration of antidepressants attenuates the external stress-induced antineurogenic effects (Hitoshi et al., 2007; Czeh et al., 2001).Antidepressant treatments increase cell proliferation andblock theeffectof stress onhippocampalneurogenesis (Czeh et al., 2001;Dranovsky andHen, 2006). The long-lasting decrease in neurogenesis, a consequence ofexposure to inescapable stress, is reversed by antidepressant treatmentand correlates to the reversal of behavioral decits in LH (Malberg andDuman, 2003). Treatments with different antidepressants, such asuoxetine, tranylcypromine, a monoamine oxidase inhibitor (MAOI),and reboxetine, increases BrdU-labeled hippocampal cells (Malberget al., 2000). Not only antidepressants, but also electro-shock treatment,results in neurogenic effects and in increased hippocampal synapsenumbers (Chen et al., 2009; Madsen et al., 2000).

Other results suggest that imipramine and uoxetine may notretain their therapeutic efcacy in CMS mice, when neurogenesis isblocked by irradiation (Surget et al., 2008). This suggests thathippocampal neurogenesis is needed by antidepressants to counteractthe depressogenic effects of stress (Surget et al., 2008). Bessa et al.(2009), on the other hand, showed that the clinical efcacy ofuoxetine and imipramine on learned helplessness and anhedoniaremained even when neurogenesis was blocked by methylazoxy-methanol, a cytostatic agent. These authors found that the clinicalefcacy of antidepressants depends on the re-establishment ofsynaptic contacts and dendritic remodeling in the frontal cortex andhippocampus, rather than neurogenesis. Therefore, it is safe to positthat the mood-improving effects of antidepressants in part depend onincreased neurogenesis and neuronal plasticity as well.

7 days

LPS, lipopolysaccharide; CMS, chronic mild stress; Con A, concanavalin A;, no effect; , ia Connor et al. (2000).b Shen et al. (1999).c Yirmiya et al. (2001).d Castanon et al. (2004).e Kubera et al. (2001b).f Kubera et al. (1996).g Myint et al. (2007).h Kubera et al. (2006a,b).Effects Region

Dawley ratsDawley rats

LPS-induced TNF-, IL-1 levels LPS-induced IL-1 levels LPS-induced TNF-

Plasma

LPS-induced TNF- levels LPS-induced IL-10 levels

Plasma

LPS-induced TNF- and IL- mRNA Spleen

LPS-induced TNF- and IL- mRNA Spleen

LPS-induced TNF-, IL-10 levels Plasma

LPS-induced TNF-, IL-10 levels Plasma

LPS-induced TNF- levels LPS-induced IL-1, IL-10 levels

Plasma

LPS-induced TNF-, IL- mRNA m Spleen

6 mice Con A-induced IL-10 production Con A-induced IL-2, IL-4, IFN- production

Splenocytes

srats

Con A-stimulated IL-1, IL-2 production Con A-stimulated IL-1, IL-2 production

Splenocytes

IFN--induced IL-, TNF- and IL-10 production stimulated by Con A+LPS

Whole blood culture

Production stimulated by Con A: IL-6, IFN-, IL-4 IL-6, IFN-, IL-4

SplenocytesThere is now also information on the molecular pathways thatunderpin the neurogenic mechanisms of antidepressants. Antide-pressants target the MAPK/extracellular signal-related kinase (ERK)and GSK-3 signaling pathways and this ultimately promotes neuro-genesis and neuronal process growth (review: Hunsberger et al.,2009). For example, acute blockade of MAPK signaling producesdepressive-like behaviors and counteracts the behavioral effects ofantidepressants (Duman et al., 2007). Besides upregulating GSK-3 andMAPK/ERK, antidepressants may exhibit neurogenic effects byupregulating BAG1 and endogenous neurotrophic and neuroprotec-tive molecules, e.g. BDNF and NGF (Hunsberger et al., 2009).

Table 3 shows the outcome of studies that focused on the effects ofantidepressants on BDNF, a molecule involved in promoting synapticplasticity and neurogenesis, and the trkB receptor. Chronic treatmentwith antidepressants increases, while acute treatment either reducesor did not have any effects on BDNF mRNA (De Foubert et al., 2004;Martnez-Turrillas et al., 2005). Venlafaxine given chronically coun-teracted the down-regulation of BDNF expression in rats subjected toCMS (Xu et al., 2006). Loss of BDNF attenuated the action ofdesipramine in the forced swimming test in both male and femaleknockout mice (Monteggia et al., 2007). Chronic treatment withimipramine counteracted the down-regulation of BDNF expression inimmobilization-stressed rats (Duric and McCarson, 2006). In naiverats, chronic treatment with desipramine decreased (Torregrossaet al., 2005) or moderately increased BDNF mRNA expression in thedentate gyrus (Jacobsen and Mork, 2004) or increased BDNF mRNAexpression in rat hippocampus (Nibuya et al., 1995; Dwivedi et al.,2006). Fluoxetine given chronically had no effect on the expression of

IL-6, IFN-, IL-4 IL-6, IFN-, IL-4

ncrease; , decrease.

y ra

em c

753M. Kubera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 744759BDNF mRNA in the dentate gyrus (Torregrossa et al., 2005) and BDNFin different brain regions (Conti et al., 2007). Fluoxetine increasedBDNF mRNA expression in the hippocampus (Nibuya et al., 1996;Dwivedi et al., 2006). Escitalopram, an SSRI, did not affect BDNFmRNAexpression but decreased BDNF protein levels in the frontal cortex andhippocampus (Jacobsen and Mork, 2004) whereas sertraline, another

Table 2Effects of antidepressants on brain cytokines.

In vivo studies

Antidepressant and dose Treatment duration Animals

Desipraminea

15 mg/kg ip.Single injection SpragueDawle

Paroxetineb

10 mg/kg po.14 days followed by paroxetine+IFN- sc. 50000 IU/kg 3 days/weekfor 5 weeks

Wistar rats

Tianeptinec

10 mg/kg ip.21 days twice daily Wistar rats

In vitro studies

Antidepressant Cell culture

Amitryptylined

NortryptylinePrimary mixed glial cellsMicroglia cells

Imipraminee

FluvoxamineReboxetine

Microglia 6-3 cells

Desipraminea Cortical mixed glia cellsDesipraminef Hippocampus-derived adult neuronal stClomipramineg

ImipramineBV-2 microglia cells

LPS, lipopolysaccharide;, no effect; , increase; , decrease.a O'Sullivan et al. (2009).b Myint et al. (2007).c Castanon et al. (2004).d Obuchowicz et al. (2006).e Hashioka et al. (2007).f Huang et al. (2007).g Hwang et al. (2008).SSRI, increased BDNF mRNA expression (Nibuya et al., 1995). Studiesinvestigating the antidepressant effect on BDNF protein or BDNFmRNA levels have focused mainly on the hippocampus but it wasobserved that uoxetine upregulated BDNF mRNA levels in themesocortico-limbic system (Molteni et al., 2006), while tianeptineincreased BDNF expression in the rat amygdala (Reagan et al., 2007).In a study performed on transgenic and knockout mice, BDNFsignaling was required for an increased neurogenesis after imipra-mine or uoxetine, and that antidepressants when given chronicallyincreased the turnover (neurogenesis and elimination) of hippocam-pal neurons (Sairanen et al., 2005).

There is also evidence that acute as well as chronic antidepressanttreatments induced trkB activation in the cerebral cortex andhippocampus (Saarelainen et al., 2003). Various antidepressantsinduce a rapid activation of TrkB signaling (Rantamki et al., 2007).In the forced swim test, the effects of citalopram, but not reboxetine,depend on trkB signaling (Rantamki et al., 2007). Chronic tranylcy-promine, sertraline, and desipramine administration signicantlyincreased trkB receptor mRNA in the rat hippocampus (Nibuya et al.,1995, 1996). Koponen et al. (2005) found that trkB.TK+ mice, whichshow increased activity of trkB had antidepressant-like responses inthe forced swimming test. The same authors found that activation oftrkB mediated the effects of antidepressants. The trkB.T1-overexpres-sing transgenic mice, which show reduced trkB signaling in brain, andBDNF null mice were resistant to the effects of antidepressants in theforced swimming test. Data reported by Li et al. (2008) suggest thattrkB is not only essential in hippocampal neurogenesis, but also in theclinical efcacity of antidepressants. In mice lacking trkB in hippo-campal neural progenitor cells, repeated antidepressant administra-tion did not result in behavioral modications, whereas ablation oftrkB in dentate gyrus neurons responded normally to antidepressants(Li et al., 2008). These data suggest that lowered neurogenesis may bean important factor in treatment resistance to antidepressants. All inall, there is some evidence that BDNF/trkB receptorsmay contribute tothe clinical effects of antidepressants and that BDNF/trkB signalingmay be required for the behavioral effects of antidepressants.

Effects

ts LPS-stimulated IL-1, TNF- mRNAs in the cortex

Normalized IFN--stimulated IL-1, IL-10 levels in the hypothalamus

LPS-stimulated IL-1, TNF-, IL-6 mRNAs and a tendency for IL-10 mRNAin the hypothalamus without changes in the hippocampus and pituitary

Effects

LPS-stimulated IL-1, TNF- release

IFN--stimulated IL-6 release

LPS-stimulated IL-1, TNF- mRNAsells LPS-stimulated IL-1, IL-6, TNF- release

LPS-stimulated TNF- levels LPS-stimulated IL-1, TNF- mRNAs without anychanges in the unstimulated gene expressionIn this paragraph we review the anti-apoptotic effects of antide-pressants rst in the external stress studies and then in in vivo and invitro studies. Bachis et al. (2008) reported that desipramine reversed thepro-apoptotic effect of CMS. In adult rats subjected to unpredictablestress, the effects of chronic treatment with tranylcypromine, rebox-etine and uoxetine on Bcl-2, Bcl-xl and Bax mRNA expression wereexamined. The Bcl-2 and Bcl-xl proteins have a well-known anti-apoptotic activity. Bcl-xl supports neuronal survival but does notpromote axonal regeneration (Jiao et al., 2005). The antidepressantsupregulated Bcl-2 mRNA in the rat limbic structures and frontal cortex.Fluoxetine administered during one week of social isolation, whichstarted on postnatal day l4, decreased the number of TUNEL-positiveand caspase-3-positive cells (apoptotic cells) in the dentate gyrus. Inuoxetine-treated pups, cell proliferation and volume of the dentategyrus were increased compared to animals that had experiencedmaternal separation (Lee et al., 2001). Table 4 shows the effects ofantidepressants on anti- and pro-apoptotic proteins in vivo and in vitrostudies. Tranylcypromine and reboxetine increased Bcl-xl mRNA, whileuoxetine decreased Bax expression in hippocampal subregions(Kosten et al., 2008). Repeated administration of citalopram, an SSRI,imipramine and amitriptyline upregulated Bcl-2 expression in the rathippocampus (Murray and Huston, 2007). The anti-apoptotic effects ofantidepressants were also conrmed by Xu et al. (2003), who found anenhanced intensity of Bcl-2 immunostaining in the hippocampal mossybers following chronic administration of amitriptyline and venlafaxinein low (5 mg/kg) but not in higher dosage (10 mg/kg). This effect wasassociatedwith anelevated intensity of BDNF immunostainingpotentialin the CA1CA4 but not in the dentate granular cell layer.

The effect of antidepressants on the expression of cell survivalgenes has also been examined in in vitro studies. The study by Huang

s.

ley

awle

ecr

754 M. Kubera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 744759Table 3Effects of antidepressants on brain-derived neurotrophic factor (BDNF) in in vivo studie

Antidepressant and dose Treatment duration Animals

Desipraminea

15 mg/kg ip.21 days SpragueDawley rats

Desipramineb

10 mg/kg ip.21 days Wistar rats

Desipraminec

15 mg/kg ip.21 daysSingle injection

SpragueDawley rats

Imipramined

15 mg/kg ip.21 days Immobilization-stressed

SpragueDawley ratsFluoxetinee

10 mg/kg po.(once daily)

Single administration SpragueDawley rats

4 days14 days21 days

Fluoxetinea

15 mg/kg ip.21 days SpragueDawley rats

Fluoxetinef

10 mg/kg ip.21 days SpragueDawley rats

Escitalopramb

10 mg/kg/day(by osmotic pomp)

21 days Wistar rats

Sertralinec

10 mg/kg ip.21 daysSingle injection

SpragueDawley rats

Venlafaxineg

5 mg/kg ip.21 days CRS-exposed SpragueDaw

Tianeptineh

10 mg/kg ip.21 days CRS-subjected SpragueD

Non-stressed rats

VTA, ventral tegmental area; CRS, chronic restraint stress;, no effect; , increase; , da Torregrossa et al. (2005).b Jacobsen and Mork (2004).c Nibuya et al. (1995).d Duric and McCarson (2006).e De Foubert et al. (2004).f Molteni et al. (2006).g Xu et al. (2006).et al. (2007) deserves special attention. They found that desipramineexerted neuroprotective effects because this drug has anti-inamma-tory and anti-apoptotic properties. Desipramine increased theproliferation and survival rate of cultured neural stem cells derivedfrom hippocampal tissue; inhibited LPS-induced release of IL-1,TNF, and IL-6; facilitated 5-HT and noradrenaline (NE) releasethrough activation of the MAPK/ERK pathway; reduced LPS-stimu-lated caspase-3 and -8 activities; and increased Bcl-2 mRNA levels.The latter effect was essential because the blockade of its biosynthesisby Bcl-2 gene silencing with the use of small interfering RNA (siRNABcl-2) diminished all effects of desipramine. Thus, Bcl-2 may mediatethe abovementioned effects of desipramine. These results indicatethat IO&NS pathways and apoptosis are closely related in the brainand that the Bcl-2 is one link between both factors.

In the same in vitromodel, imipramine induced neuroprotection viaan upregulation of BDNF and Bcl-2 mRNA and protein expression; anddecreased LPS-induced apoptosis. Desipramine also exerted anti-inammatory effects, including reduced LPS-stimulated IL-6, IL-1,and TNF release; and promoted serotonergic ber differentiation viathe modulation of the BDNF/MAPK/ERK pathway/Bcl-2 cascades (Penget al., 2008). Moreover, in two other studies, the same authors observedan enhanced expression of mRNA for the anti-apoptotic proteins Bcl-2,and Bcl-xl inuoxetine- ormoclobemide-treated hippocampus-derivedneural stemcells (Chiou et al., 2006;Chen et al., 2007).Moclobemide is areversible inhibitor of monoamine oxidase A (RIMA). In moclobemide-treated cells, the expression of the pro-apoptotic proteins Bax and Fasremained unaltered. Moclobemide prevented FasL-induced apoptosis;stimulated neurite outgrowth; and promoted the differentiation ofneural stem cells into functional serotonergic neurons via extracellularsignal-regulated kinases 1 and 2 activation (Chiou et al., 2006). All in all,the abovementioned in vivo studies and experiments on culturedneuralstem cells suggest that antidepressants have signicant anti-apoptotic

h Reagan et al. (2007).Effects

BDNF mRNA in the dentate gyrus without changes in thehippocampal CA1, CA3 and the frontal cortex BDNF mRNA in the dentate gyrus without changes in CA3and the frontal cortex BDNF, trkB mRNAs in the hippocampus BDNF, trkB mRNAs in the hippocampusNormalized BDNF mRNA in the hippocampus

BDNF mRNA, BDNF levels in the hippocampus, medialhabenular and paraventricular thalamic nuclei BDNF mRNA, BDNF levels in the mentioned structures BDNF mRNA, BDNF levels in the mentioned structures BDNF levels in the hippocampus BDNF mRNA in the hippocampal regions(CA3, CA1, dentate gyrus) and the frontal cortex BDNF mRNA in the VTA, prefrontal cortex nucleus accumbens (shell),hippocampus; no changes in the striatum and substantia nigra BDNF mRNA in the dentate gyrus, CA3, frontal cortex; BDNF levelsin the frontal cortex and hippocampus

BDNF, trkB mRNAs in the hippocampus BDNF, trkB mRNAs in the hippocampus

rats Normalized decreased by stress BDNF immunoreactivity in thehippocampal regions (CA3, CA1, dentate gyrus)

y rats BDNF mRNA and BDNF levels in the amygdala BDNF levels in the amygdala

ease; trkB receptor mediates survival-promoting BDNF effects.effects that contribute to their cell survival effects (Drzyzga et al., 2009).By their effects on Bcl-2 proteins, antidepressants may promote axonalgrowth and regeneration, contribute to synaptic stability and/orstimulate neuronal differentiation.

5. Conclusions

External stress, which is widely acknowledged as a predisposingand precipitating factor in depression, causes an increased expressionof pro-inammatory cytokines and activation of various inamma-tion-related pathways in the CNS. External stress models ofdepression show that depression-like behaviors are accompanied byperipheral and central activation of IO&NS pathways, includingincreased IL-1, IL-6 and TNF levels. External stress-inducedincreases in IL-1 are sufcient to explain the depressive-likebehaviors induced by the stressors. External stressors also induceincreases in NFB, COX-2, TLR4 expression and oxidative stress,including lipid peroxidation. There is also evidence that externalstress causes neuronal cell damage, a marked decrease in neurogen-esis and apoptosis in the hippocampus. External stress-inducedapoptosis is associated with increased caspase-3 and loweredexpression of Bcl-2. The external stress-induced pathways thatinitiate the cascade of events leading to the reduction in synapticconnection and decit in neuronal function are: a) the detrimentaleffects of pro-inammatory cytokines, like IL-1, TNF and IL-6;NFB, COX-2 and its products, PGE2; ROS; apoptotic pathways,including caspase-3; and the antineurogenic GSK-3; and b)suppression of neurotrophic factors, such as BDNF and VGF; andanti-apoptotic pathways, e.g. Bcl-2 and BAG-1.

Chronic administration of antidepressants attenuates the externalstress-induced depressive-like behaviors, (neuro)inammatory reac-tions, and antineurogenic effects. The mechanisms that explain the

ls

eD

ice

Amitriptyline 5 mg/kg ip.c 21 days SpragueD

cell

cell

cellcell

mphrote

755M. Kubera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 744759Imipraminee Hippocampus-derived neural stem

Fluoxetinef Hippocampus-derived neural stemMoclobemideg Hippocampus-derived neural stem

LPS, lipopolysaccharide;, no effect; , increase; , decrease; Bcl-2, B-cell leukemia/lyBcl-2 and Bcl-xl anti-apoptotic proteins; Bax, Bad pro-apoptotic proteins; FasL p

a Kosten et al. (2008).b Murray and Huston (2007).c Xu et al. (2003).d Huang et al. (2007).e Peng et al. (2008).f Chen et al. (2007).gVenlafaxine 5 mg/kg ip.Amitryptiline 10 mg/kg ip

In vitro studies

Antidepressant Cell culture

Desipramined Hippocampus-derived neural stemTable 4Effects of antidepressants on pro- and anti-apoptotic proteins.

In vivo studies

Antidepressant and dose Treatment duration Anima

Fluoxetine 5 mg/kg ip.a

Reboxetine 20 mg/kg ip (twice daily)Tranylcypromine 7.5 (10 days) then 10 mg/kg ip.

21 days Spragu

Citalopram 20 mg/kg po.b

Imipramine 10 mg/kg ip.Amitriptyline 20 mg/kg ip

14 daysSingle administration

CD1 mclinical efcacy of antidepressants in external stress-induced depres-sive-like behaviors are very complex. Thus, chronic administration ofantidepressants reduces peripheral and central inammatory pathwaysand stimulates neuronal differentiation, synaptic plasticity, axonalgrowth and regeneration, and has anti-apoptotic effects. The anti-inammatory effects are attributed to direct suppressive effects ofantidepressants on pro-inammatory cytokines, like IL-1, TNF andIL-6, and stimulatory effects on anti-inammatory cytokines, e.g. IL-10.The neurogenic/regenerative effects are attributed to stimulatory effectson the expression of different neurotrophic factors, in particular trkB,the receptor for BDNF; and down-regulation of apoptotic pathways byactivating Bcl-2 and Bcl-xl proteins, and suppressing caspase-3 and -8and Bax.

Acknowledgment

This study was partly supported by research grants POIG.01.01.02-12-004/09-00 and N 401c130 31/2871 from the Ministry of Sciencesand High Education Warszawa, Poland.

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