Modulation of stress consequences by hippocampal monoaminergic, glutamatergic and nitrergic neurotransmitter systems

Abstract

Several findings relate the hippocampal formation to the behavioural consequences of stress. It contains a high concentration of corticoid receptors and undergoes plastic modifications, including decreased neurogenesis and cellular remodelling, following stress exposure. Various major neurotransmitter systems in the hippocampus are involved in these effects. Serotonin (5-HT) seems to exert a protective role in the hippocampus and attenuates the behavioural consequences of stress by activating 5-HT_1A receptors in this structure. These effects may mediate the therapeutic actions of several antidepressants. The role of noradrenaline is less clear and possibly depends on the specific hippocampal region (dorsal vs. ventral). The deleterious modifications induced in the hippocampus by stress might involve a decrease in neurotrophic factors such as brain derived neurotrophic factor (BDNF) following glutamate N-methyl-d-aspartate (NMDA) receptor activation. In addition to glutamate, nitric oxide (NO) could also be related to these effects. Systemic and intra-hippocampal administration of nitric oxide synthase (NOS) inhibitors attenuates stress-induced behavioural consequences. The challenge for the future will be to integrate results related to these different neurotransmitter systems in a unifying theory about the role of the hippocampus in mood regulation, depressive disorder and antidepressant effects.

• Allostasis
• antidepressant
• BDNF
• depression
• inescapable stress
• dopamine

Introduction

Several hypotheses about the pathophysiology of depression and other stress-related disorders have been proposed. There is an extensive scientific literature describing the involvement of the hippocampal formation in the neurobiology of such disorders, mainly based on observations that similar neurochemical and morphological changes can be found in the hippocampus of stressed animals and depressed humans, which can be prevented or reversed by repeated antidepressant treatment. Much attention has been focused on hippocampal serotonergic and noradrenergic systems. However, recent pieces of evidence has linked other hippocampal neurotransmitters, such as glutamate and nitric oxide (NO), to the development of stress-induced behavioural consequences. In the present work, we review recent data that reinforces the involvement of the hippocampus in behavioural changes induced by stress and how local transmitter systems may interact to mediate stress adaptation and antidepressant effects.

Stress and coping

Selye (1936) first described that exposure to different noxious stimuli could lead to physiologic responses aimed at allowing adaptation of the organism to the new demands. In order to characterize this phenomenon, Selye coined the term stress to describe a potential or real threat to homeostasis imposed by different noxious stimuli that could lead to several physiological alterations and eventually, death. Selye postulated a three stage stereotyped physiologic response in reaction to a stressor, called the general adaptation syndrome. The first stage is the alarm reaction, in which the adrenal medulla releases epinephrine and the adrenal cortex produces glucocorticoids (GCs) to help to restore homeostasis. Restoration of homeostasis leads to the resistance stage, in which defence and adaptation are sustained. If the stressor persists, the adaptive response ceases and the stage of exhaustion follows, with illness and death being possible consequences. Nowadays, it is well recognized that not only physical and chemical agents can act as stressors but also psychological factors, such as novelty and social problems, can cause significant physiological and behavioural changes.

Bruce McEwen and his research team have reinterpreted Selye's alarm response and proposed a new terminology for linking the protective and damaging effects of the biological responses to stressors (McEwen 1998, 2005a,b; Goldstein and McEwen 2002). They proposed the concept of allostasis to refer to a system by which stability is achieved through change—i.e. adaptation. When one's set points vary beyond the limits of homeostatic mechanisms, these variables are referred as being in allostatic states (altered and sustained activity aimed at integrating energetic and the associated behavioural modifications in response to changing environment and challenges). The cumulative results of an allostatic state would then lead to allostatic load, which also provides, within limits, adaptation. However, if the additional load of unpredictable events in the environment is superimposed, then allostatic load can increase and become allostatic overload. Allostatic overload has no useful purpose and predisposes the individual to disease. Thus, according to McEwen, Selye's alarm response could be reinterpreted as the process leading to adaptation, or allostasis, in which GCs, as well as other mediators, promote adaptation to the stressor. But if the alarm response is sustained, with prolonged hormonal secretion, an allostatic state may ensue leading to allostatic overload, which replaces Seley's stage of exhaustion. In other words, Selye's diseases of adaptation would be the result of the allostatic state leading to allostatic overload and resulting in the exacerbation of pathophysiological changes.

A prominent mechanism by which the brain reacts to stress is by activating the hypothalamic–pituitary–adrenal (HPA) axis (Herman and Cullinan 1997; Sapolsky et al. 2000; Tsigos and Chrousos 2002; Carrasco and Van de Kar 2003). Neurons in the paraventricular nucleus of the hypothalamus (PVN) secrete corticotrophin-releasing factor (CRF), which stimulates the synthesis and release of adrenocorticotropin (ACTH) from the anterior pituitary. ACTH then stimulates the synthesis and release of GCs (cortisol in humans and corticosterone in rodents). GCs, in turn, exert effects on metabolism and affect behaviour by acting on two specific receptors (glucocorticoid receptors—GRs and mineralocorticoid receptors—MRs) in different brain regions (Fuxe et al. 1996; Carrasco and Van de Kar 2003). GCs can also exert inhibitory feedback on the HPA axis activity by acting on its receptors in the PVN, hippocampus and cortex (Herman and Cullinan 1997; de Kloet 2000; Carrasco and Van de Kar 2003). Thus, the stress response, with resultant activation of the HPA axis, is meant to be acute or at least of a limited duration. The time-limited duration of this process renders its accompanying antireproductive, antigrowth, catabolic and immunosuppressive effects, temporarily beneficial rather than damaging, allowing adaptation to occur. In contrast, chronicity of the stress system activation would lead to damage and induce pathological states.

In accordance with this proposal, chronic exposure to uncontrollable stresses and consequently, to high GCs levels, has been related to the aetiology of several diseases, including depression and post-traumatic stress disorder (PTSD) (Baungartner et al. 1985; Peeters and Broekkamp 1994; Heim et al. 2000; Sheline 2000; Bonne et al. 2004; Charney 2004; McEwen 2005a). For example, an increased prevalence of life stress episodes before the onset of major depression has been documented, suggesting that they could have a main role in its development (Post 1992). In laboratory animals, the exposure to uncontrollable and severe stressful events can induce physiological and behavioural alterations that resemble human depression, such as motor deficits, weight and sleep changes, anhedonia, memory impairments and excessive GC secretion (Willner 1986, 1990; Willner and Mitchell 2002; Anisman and Matheson 2005). Therefore, most animal models aimed at studying the neurobiology of stress-related disorders, such as depression, are based on behavioural modification induced by exposure to different stressors that can be prevented or reversed by antidepressant drugs (Willner 1986, 1990; Willner and Mitchell 2002).

HPA axis hyperactivity has been implicated in the initiation and maintenance of some stress-induced alterations that could contribute to depression. This hypothesis is supported by several clinical and experimental findings (Checkley 1992; Heim et al. 2000; Tsigos and Chrousos 2002; Keller et al. 2006). Abnormal, excessive activation of HPA axis is observed in depressed individuals (Whiteford et al. 1987; Gold et al. 1995; Keller et al. 2006), who often show increased cortisol production that is unresponsive to the administration of the synthetic GC dexamethasone (Baungartner et al. 1985). Moreover, there is a high co-morbidity between depression and Cushing's syndrome, which is a disease characterized by high levels of circulating cortisol (Starkman et al. 1981, 1986, 1992). Consistent with these human data are the observations that rodents reared in isolation or submitted to inescapable shocks show abnormalities in HPA axis function (Heidbreder et al. 2000) and that chronic GC treatment can lead to depressive-like behaviour in rats (Johnson et al. 2006).

However, the mechanisms that mediate the stress effects on behaviour and mood are not fully understood. It has been suggested that the levels of GCs, particularly if sustained over long periods of time, might be high enough to induce toxic effects in some neuronal circuits, especially those related to mood regulation, such as the hippocampus, cortex and amygdala (Sapolsky 2000; Sapolsky et al. 2000; Lee et al. 2002b,a). Impaired functions in these structures might be expected to contribute to some of the cognitive abnormalities observed in depressed patients.

Stress and the hippocampus

The hippocampus is a brain structure that has been traditionally reported to be involved in spatial learning and in episodic, declarative and contextual memory (Fanselow 2000; Riedel and Micheau 2001; Knierim 2003; Shu et al. 2003; Buckley 2005; Frankland and Bontempi 2005; Moscovitch et al. 2005, 2006). However, the hippocampus is the brain region showing the highest density of receptors for corticosteroids (McEwen et al. 1968; Gerlach and McEwen 1975; de Kloet 2000) and has been consistently linked to the brain's response to stress and to the regulation of the HPA axis activity (de Kloet 2003; Herman et al. 2005). It has been proposed to play an important role in coping with threatening stimuli. According to Gray and McNaughton (2000), during threatening or novel situations the hippocampus is involved with the detection and resolution of conflicts between incompatible goals or response tendencies. Specifically, when this type of conflict is detected, the hippocampus outputs a signal that increase the weight or valence of affectively negative information, thereby reducing or inhibiting the tendency to approach the goal. During extinction, the hippocampal output would augment the negative affect produced by nonreward (e.g. frustration). In accordance with this proposal, neuroimaging studies have shown hippocampal/parahippocampal activation during perception of several negatively valenced stimuli and/or experiencing of negatively valenced affective states (Blood et al. 1999; Isenberg et al. 1999; Mirz et al. 2000). In addition, the hippocampus could have an important protective role after repeated stress. An impairment of this adaptive effect induced by severe stress could facilitate the development of behavioural deficits and clinical symptoms (Deakin and Graeff 1991; Graeff et al. 1996).

Stress effects on the hippocampus seem to be mediated largely by the lower affinity GRs which become heavily occupied with corticosteroids in response to stress (Joels 2001; de Kloet 2003; de Kloet and Derijk 2004). GC initial action on these receptors is involved in terminating the endocrine response to stress by attenuating HPA axis activation (Herman and Cullinan 1997; Feldman and Weidenfeld 1999; Herman et al. 2005) and promoting memory storage in preparation for future events (Oitzl and de Kloet 1992; Sandi et al. 1997; de Kloet et al. 1999; Oitzl et al. 2001; Avital et al. 2006; Diamond et al. 2006). Since corticosteroid receptors function as transcriptional regulators, the first step that leads to their ultimate effect on adaptive behaviour involves the altered expression of responsive genes. In the hippocampus, the activation of MRs or GRs leads to the altered expression of several genes that underlie aspects of cell metabolism, structure and synaptic transmission (Datson et al. 2001). Tables I and II summarize the main findings relating hippocampal gene expression changes, stress and antidepressant treatments.

Table I.  Gene expression changes in the hippocampal formation induced by stress.

Hippocampal regions	Molecular approach	Stress procedure	Effect	References
All	ISH	Shaking stress	↑ GPDH	Nichols et al. (1990)
CA1	ISH	Restraint plus tail electrical shocks	↑ c-fos, ↑ zif/268	Schreiber et al. (1991)
DG	ISH	Acute restraint	↓ c-fos	Titze-de-Almeida et al. (1994a,b)
All	ISH	Acute and repeated restraint	↓ c-fos	Melia et al. (1994)
CA1, DG	ISH	Acute restraint	↓ MR hmRNA	Herman and Watson (1995)
CA1	ISH	Maternal deprivation	↓ MR, ↓ GR	Vazquez et al. (1996)
DG, CA1, CA3	ICC	Chronic social stress	↑ c-fos	Matsuda et al. (1996)
DG, CA1, CA3	ISH	Maternal deprivation	↑ GR	Liu et al. (1997)
DG	ISH, ICC	Acute restraint	↑ c-fos mRNA, ↔ Fos protein	Del Bel et al. (1998)
All	ISH;	Acute prolonged stress (restraint, 2 h; forced swim, 20 min and ether anaesthesia); CVUS	Acute: ↓ GR and ↓ MR; CVUS: ↔ GR and MR	Liberzon et al. (1999)
DG, CA1	ISH	Acute prolonged stress (restraint, 2 h; forced swim, 20 min and ether anaesthesia); CVUS	Acute: ↓ 5-HT1A, CVUS: ↔ 5-HT1A	Lopez et al. (1999)
All	ISH, Northern blot	Chronic stress	↓ GR	Kitraki et al. (1999)
CA1-3, DG	ISH	Acute restraint	↑ CRH	Givalois et al. (2000)
All	Quantitative RT-PCR	Repeated restraint	↑ GluR1, ↔ NMDA R1	Schwendt and Jezova (2000)
CA1-3, DG	ISH	Acute or repeated restraint	↔ nNOS	de Oliveira et al. (2000)
DG, CA1, CA3	ISH	Acute or repeated restraint	↓ Synaptophysin ↑ synaptotagmin	Thome et al. (2001)
DG, CA1, CA3	ISH	Repeated social stress	↓ GR, ↑ MR	Meyer et al. (2001)
All	ISH, RNAse protection assay	Maternal deprivation	↓ BDNF; ↓ NMDA (NR-2A and NR-2B)	Roceri et al. (2002)
All	RNAse protection assay	Acute restraint	↑ BDNF	Marmigere et al. (2003)
All	ICC	Acute restraint	↑ PPA2	Morinobu et al. (2003)
All	SAGE	Forced swimming test	53 Genes differently expressed	Drigues et al. (2003)
All	ICC	Repeated social stress	↓ NGF, ↓ M6a, ↓ CLK-1, ↓ GNAQ,	Alfonso et al. (2004)
CA1, CA2, CA3, DG	ICC	Acute restraint	↑ c-fos;	de Medeiros et al. (2005)
All	RT-PCR, microarray	Learned helplessness	Distinct gene expression profile compared to controls	Kohen et al. (2005)
All	real-time RT-PCR	Repeated restraint	↓ NGF, ↓ M6a, ↓ GNAQ, ↓ CLK-1, ↓ BDNF, ↓ CREB, ↓ PKC, ↓ NCAM, ↓ synapsin I	Alfonso et al. (2006)
All	Real time RT-PCR	Social defeat	↑ CRF, ↑ GR	Marini et al. (2006)
CA1, CA3	Microarray, Real time RT-PCR, ISH	Repeated restraint	444 Genes differentially expressed, ↓ Grb2, ↓ Pip5k1b, ↓ Gstp2	Ejchel-Cohen et al. (2006)
All	RT-PCR	Repeated Morris water maze stress	↓ GR, ↑ pro-CRH and CRH-R1	Aguilar-Valles et al. (2005)
CA1	ISH	Acute restraint and adrenalectomy	↔ Arc	Mikkelsen and Larsen (2006)

ISH, in situ hybridization; ICC, immunocytochemistry; CVUS, chronic variable unpredictable stress; DG, dentate gyrus; SAGE, serial analysis of gene expression; GPDH, glycerol phosphate dehydrogenase; MR, mineralocorticoid receptor; GR, glucocorticoid receptor; PPA2, phosphatase 2A; NGF, nerve growth factor; M6a, membrane glycoprotein 6a; CLK-1, CDC-like kinase 1; GNAQ, G-protein alpha q; NCAN, neural cell adhesion molecule; Grb2, growth factor receptor-bound protein 2; Pip5k1b, phosphatidylinositol-4-phosphate 5-kinase, type 1 beta; and Gstp2, glutathione S-transferase, pi2

Table II.  Gene expression changes in the hippocampal formation induced by antidepressant treatments.

Hippocampal regions	Molecular approach	Antidepressant treatment	Effect	References
All	ISH, RT-PCR, Southern blot	SSRI	↓ SERT	Lesch et al. (1993)
DG	ISH	Imipramine	- ↑ 5-HT2C	Tohda and Watanabe (1996)
All	ISH;	Desipramine or fluoxetine	Fluoxetine: ↑ CREB and- ↓ SP1; desipramine: ↑ GRE	Frechilla et al. (1998)
CA1, CA2, CA3, CA4,	ISH;	Imipramine or citalopram	↔ Zeta, ↓ epsilon 1, ↓ epsilon 2-subunits of NMDA receptor	Boyer et al. (1998)
All	Quantitative RT-PCR	Fluoxetine	↑ AA-NAT	Uz and Manev (1999)
Hippocampal cultured cells	RT-PCR, ICC	Desipramine, amitriptyline, mianserin or paroxetine	↑ GR	Okugawa et al. (1999)
CA3, DG	ICC in CRE–LacZ transgenic mice.	Fluoxetine	↑ CRE-mediated gene expression, ↑ phosphorylation of CREB	Thome et al. (2000)
CA1-3, DG	ISH	Buspirone, fluoxetine, 8-OH-DPAT or moclobemide	↑ GR (8-OH and moclobemide), ↓ GR (buspirone and fluoxetine), ↑ NGFI-A (moclobemide, 8-OH-DPAT, fluoxetine)	Bjartmar et al. (2000)
DG, CA1-4	ISH	Fluoxetine or venlafaxine	↓ MR	Yau et al. (2001)
DG	ISH	Fluoxetine, paroxetine, sertraline or tranylcypromine	Time-dependent ↓ (4 h) or ↑ BDNF (24 after last injection)	Coppell et al. (2003)
CA!	ISH	Venlafaxine, paroxetine or desipramine	↑ Arc mRNA;	Pei et al. (2003)
All	SAGE	Forced swimming test with moclobemide, clorgyline or amitriptyline	53 Genes differently expresses, 89 genes changed by drugs	Drigues et al. (2003)
All	Two-dimensional protein gel electrophoresis	Venlafaxine or fluoxetine	23 Protein spots modulated by drug treatment	Khawaja et al. (2004)
All	Real-time RT-PCR	Psychosocial stress with clomipramine	Stress ↓ NGF, M6a, CLK-1, ↑ GNAQ clomipramine prevented stress-induced effects	Alfonso et al. (2004)
DG	ICC	7-Nitro-indazole	↑ PSA-NCAM, pCREB, 5-HT and TPH	Park et al. (2004)
DG, CA1, CA3,	ISH	Fluoxetine	↑ GC	Yau et al. (2004)
CA1, CA2, CA3, DG	ICC	Imipramine with restraint stress	↓ Stress induced ↑ c-fos	de Medeiros et al. (2005)
All	RT-PCR	Fluoxetine	↑ Serotonin N-acetyltransferase, per2, clock, Bmal1, cry1, NPAS2 and ↓ per1	Uz et al. (2005)
DG, CA1	ISH	Fluoxetine	↑ BDNF	Molteni et al. (2006)
All	Real time RT-PCR	Imipramine and metyrapone	↑ BDNF	Rogoz and Legutko (2005)
All	Real time RT-PCR	Mirtazepine	↑ BDNF	Rogoz et al. (2005)
All	RT-PCR	Fluoxetine, desipramine or phenelzine with corticosterone	Fluoxetine and phenelzine prevented ↓ BDNF mRNA induced by corticosterone	Dwivedi et al. (2006)
All	Northern blot	MPEP (selective mGlu5 receptor antagonist)	↑ BDNF	Legutko et al. (2006)

SERT, serotonin transporter; GRE, glucocorticoid response element; AA-NAT, N-acetyltransferase; Bmal1, brain and muscle ARNT-like protein; Cry1, cryptochrome 1; CRE, cAMP response element; CREB, CRE binding protein; NGFI-A, nerve growth factor-induced clone A; Npas2, neuronal PAS2; NGF, nerve growth factor; M6a, membrane glycoprotein 6a; CLK-1, CDC-like kinase 1; GNAQ, G-protein alpha q; per1-2, period homolog 1-2; PSA-NCAM, polysialylated-neural cell adhesion molecule; and TPH, tryptophan hydroxylase.

Although GC-mediated processes are adaptive in nature, repeated exposure to high levels of GCs can be deleterious to the structure and function of the hippocampus. This is supported, for example, by numerous studies that consistently report that stress decreases the proliferation of new neurons in the subgranular zone of the hippocampus. Adult neurogenesis is decreased by many different types of stressors, including predator odour (Tanapat et al. 2001), social stress (Gould et al. 1997; Czeh et al. 2001), acute and chronic restraint stress (Pham et al. 2003; Rosenbrock et al. 2005), inescapable shocks (Malberg and Duman 2003; Vollmayr et al. 2003; Bland et al. 2006) and chronic mild stress (Alonso et al. 2004). These stress-induced effects are thought to be corticosteroid-dependent since administration of corticosterone significantly decreases neurogenesis in the hippocampus (Gould et al. 1992; Cameron et al. 1998) and the blockade of corticosteroid actions prevents stress-induced decrease in neurogenesis (Cameron and Gould 1996; Mayer et al. 2006). Moreover, it seems that the suppressive action of GCs on cell proliferation is not direct but occurs through an N-methyl-d-aspartate (NMDA) receptor-dependent excitatory pathway (Gould et al. 1997; Cameron et al. 1998; Gould and Tanapat 1999; Nacher and McEwen 2006). For a complete and recent review of the literature on the regulation of neurogenesis by stress, see Dranovsky and Hen (2006), Mirescu and Gould (2006) and Schmidt and Duman (2006).

In addition to a decrease in neurogenesis, stress also influences the structure of mature neurons in the adult hippocampus. Repeated stress exerts effects similar to GCs on dendritic remodelling in CA3. Chronic restraint stress results in reduction in apical dendritic length and branch number in rat CA3 pyramidal neurons (Watanabe et al. 1992; Vyas et al. 2002). Other repeated stress paradigms, such as repeated variable stress and psychosocial stress (Magariños and McEwen 1995a; Magariños et al. 1996; Fuchs et al. 2001), result in similar dendritic remodelling in the CA3 region. Repeated stress-induced dendritic remodelling may also be mediated by sustained high levels of GCs (Sapolsky et al. 1990; Magariños and McEwen 1995b; Fuchs and Flugge 1998; Fuchs et al. 2001) and glutamate (Magariños and McEwen 1995b; McEwen 1996).

Stress also decreases the expression of the brain derived neurotrophic factor (BDNF, Vaidya et al. 1997; Nibuya et al. 1999; Rasmussen et al. 2002; Murakami et al. 2005; Smith 2005; see Duman and Monteggia (2006) for a complete review). BDNF is required for and may induce neurogenesis (Lee et al. 2002a; Scharfman et al. 2005), neuronal survival (Sairanen et al. 2005) and neuronal branching (Danzer et al. 2002; Govindarajan et al. 2006). Moreover, BDNF exerts direct effects on membrane excitability (Schinder and Poo 2000). Therefore, it is possible that a stress-induced decrease of BDNF levels is involved in neurogenesis, synaptic morphology and membrane excitability changes, thus leading to modification of hippocampal synaptic transmission, connectivity and function. Stress and adrenal steroids may impair long-term potentiation (LTP) in the rodent hippocampus (Foy et al. 1987; Shors et al. 1990; Pavlides et al. 1993; Diamond and Rose 1994; Pavlides et al. 2002; Kim et al. 2005; Aleisa et al. 2006; Kim et al. 2006), a synaptic model postulated to be fundamental to memory storage (Lynch 2004). On the other hand, stress seems to facilitate hippocampal theta-rhythm (Shors et al. 1997; Yamamoto 1998), a rhythmic waveform that occurs in alert, immobile rats presented with threatening stimuli, possibly facilitating fear memory acquisition and retrieval (Seager et al. 2002; Seindenbecher et al. 2003; Vertes 2005). In agreement with these electrophysiological data, it is reported from studies employing behavioural models that stress exerts complex effects on memory processing, ranging from impairment of hippocampal-dependent spatial memory to facilitation of fear-related learning and memory (see Kim and Diamond (2002) for review).

These findings have been related to several neuropsychiatric diseases, especially depression and PTSD. In agreement with this hypothesis, several studies have found a significant reduction in hippocampal volume in patients suffering from these disorders (Sheline et al. 1996, 1999, 2003; Stein et al. 1997; Bremner et al. 2000; Mervaala et al. 2000; Steffens et al. 2000; Frodl et al. 2002; MacQueen et al. 2003; Smith 2005; Karl et al. 2006). Interestingly, Fuchs and co-workers (Ohl et al. 1999, 2000; Fuchs et al. 2001) have shown that chronic psychological stress in primates reduces the proliferation of new cells in the hippocampus, causing a decrease of neuronal function and viability. This was associated with a trend towards a decrease in total hippocampal volume. Again, this seems to be mediated by high corticosteroid levels, since it can also be induced by long-term cortisol exposure (Lupien et al. 1998; Ohl et al. 1999) and reduced hippocampal volume can be observed in Cushing's patients (Starkman et al. 1992). Recent evidence, on the other hand, suggests that in PTSD a small hippocampus probably precedes the symptoms and predicts the susceptibility to develop the disorder after severe stress exposure (Gilbertson et al. 2002; Gross and Hen 2004). Together, however, these results support the proposal that in depression and PTSD there is an impairment of a stress protective role played by the hippocampus.

The fact that chronic antidepressant treatment is able to attenuate these stress-induced effects strengthens the involvement of hippocampal atrophy in pathophysiology of stress-related disorders (Sheline et al. 2003; Bremner 2006). Depression, therefore, could be a consequence of stress-induced neuronal death and decreased cellular resilience within the hippocampal circuitry with consequent alteration in information processing. Antidepressants would work by restoring normal connectivity and function in this region (Castrén 2005). According to this hypothesis, increased BDNF release would select those neurons and connections that mediate useful neuronal activity to the target neurons and helps to eliminate connections that produce random noise. Therefore, “although antidepressant-induced neurogenesis increases overall activity, only those neurons that best mediate useful neuronal activity to the target cells would be selected and prevail in the competition. Increased granule neuronal turnover produced by antidepressant treatments indicates that there are more competing neurons available for selection, which may improve the ability of the hippocampus to rapidly adapt to emerging environmental challenges” (Sairanen et al. 2005). Corroborating this hypothesis, it was shown that behavioural effects of antidepressants depend on intact BDNF signalling (Saarelainen et al. 2003) and hippocampal neurogenesis (Santarelli et al. 2003). Moreover, hippocampal BDNF administration prevents cognitive impairment induced by stress (Radechi et al. 2005) and induces antidepressant-like effects in animal models (Sciuciak et al. 1997; Shirayama et al. 2002).

In summary, a wealth of evidence indicates that impairment of normal hippocampal functions could lead to stress-related disorders, maybe by impairing protective mechanisms and enhancing the processing of aversive memories, predisposing the individual towards negatively valenced affective states. The increase in cell turnover and connectivity induced by antidepressants would restore hippocampal function.

Stress, serotonin and the hippocampus

Since the discovery that the selective inhibition of serotonin reuptake induces antidepressant effects, several studies have investigated the role of the serotonergic neurotransmission in the neurobiology of depression. Serotonin was shown to be essential to the effects of antidepressant drugs by Delgado et al. (1990). They reported that remitted depressed patients receiving antidepressants experience an acute symptom relapse after a procedure (tryptophan depletion) that decreases serotonin brain levels (Delgado et al. 1990; Heninger et al. 1996). More recently, it was shown that reducing serotonin synthesis induced depressive-like symptoms in normal subjects submitted to uncontrollable stress (Richell et al. 2005). Therefore, it seems that serotonin is not only necessary for the therapeutic effects of antidepressants but it is also required to facilitate resilience to uncontrollable stress. Also, the acute relapse of depressive symptoms induced by serotonin depletion suggests that other mechanisms in addition to increased neurogenesis and cellular resilience are involved in the therapeutic effects of antidepressants.

Deakin and Graeff (1991) proposed that the serotonergic pathways originated in the dorsal raphe nuclei (DRN) regulate adaptive responses aimed at terminating acute stress, such as escape, fight and flight reactions. However, in case of chronic exposure to inescapable aversive stimuli, where coping with stress is needed the pathway connecting the median raphe nucleus (MRN) to the hippocampus would help adaptation to occur by inhibiting the consolidation of stressful memories and thus disconnecting the aversive events from the behavioural outcomes (Deakin and Graeff 1991). A failure in this mechanism would favour the development of stress-induced behavioural deficits in animals and depression in humans. Several experimental findings have supported this hypothesis (see Graeff et al. (1996) for review). For example, electrolytic lesions of the MRN significantly enhanced plasma corticosterone levels and incidence of gastric ulcers (Andrade et al. 1999) and lesions of serotonin neurons located in the MRN impaired the behavioural adaptation to repeated restraint stress (Netto et al. 2002). Moreover, adaptation to chronic mild stress was followed by increased levels of serotonin in the dorsal hippocampus (Storey et al. 2006) and post-stress facilitation of hippocampal serotonergic neurotransmission prevented the development of stress-induced behavioural deficits (Guimarães et al. 1993; Padovan and Guimarães 1993; Joca et al. 2003, Figure 1), findings which are consistent with the hypothesis that increased serotonin release in the hippocampus may be implicated in the mechanisms underlying coping with inescapable stress.

Figure 1 Post-stress facilitation of serotonin-mediated neurotransmission in the dorsal hippocampus prevents the behavioural consequences of stress. Male Wistar rats (n = 7–23) were submitted to inescapable footshocks (40 shocks, 1 mA, 10 s) or habituation (30 min) in a shuttle box. Immediately afterwards, they received bilateral intra-hippocampal injections of saline (Sal) or zimelidine (Zim, 20 or 100 nmols/0.5 μl), a selective serotonin reuptake inhibitor, and were tested 24 h later wit escapable footshocks (30 sound-signalled shocks, 0.8 mA, 10 s). Data are expressed as the mean ± SEM number of escape and/or avoidance failures in each block (summation of five individual trials). * Indicates significant difference from respective Sal-treated group (p < 0.05, ANOVA followed by Duncan test, modified from Joca et al. (2003), with kind permission from Elsevier).

In agreement with these data, it was shown in rodents that behavioural adaptation to repeated restraint stress is followed by serotonergic supersensitivity (Kennett et al. 1985a,b) and that post-stress facilitation of serotonergic neurotransmission by 5-HT_1A agonist administration is able to prevent the development of stress-induced behavioural consequences (Kennett et al. 1987). These results, therefore, suggest that adaptation to stress involves augmentation of 5-HT_1A receptor function.

Hippocampal 5-HT_1A receptors are likely to be the main target of the MRN-serotonergic pathway to promote stress adaptation. Post-stress microinjections of a selective serotonin reuptake inhibitor (SSRI) or 5-HT_1A agonists into the dorsal hippocampus reversed the deficits induced by restraint stress of open arm exploration in an elevated plus-maze (Guimarães et al. 1993, Netto and Guimarães 1996). More recently, we showed similar effects in the learned helplessness model (Joca and Guimarães 2006, Figure 1).

The mechanisms by which 5-HT_1A receptors mediate adaptation to stress are still unknown. One possible mechanism could be interference with consolidation of stressful memories (Guimarães et al. 1993). Activation of 5-HT_1A receptors negatively modulates LTP in the hippocampus (Corradetti et al. 1992; Sakai and Tanaka 1993; Kojima et al. 2003; Tachibana et al. 2004), a phenomenon traditionally linked to learning and memory (Vertes 2005). Consistent with these physiological data, systemic or intra-hippocampal administration of 5-HT_1A agonists impaired acquisition and consolidation of spatial and fear-related memories (Carli and Samanin 1992; Carli et al. 1992a,b; Stiedl et al. 2000). Moreover, mice with knockout of 5-HT_1A receptors show impaired hippocampal-dependent learning and memory (Sarnyai et al. 2000), and their fear response is biased toward threatening cues (Gross et al. 2000; Klemenhagen et al. 2006). Therefore, activation of hippocampal 5-HT_1A receptors could help adaptation to stress by attenuating the emotional impact of aversive stimuli and consequently inhibiting the consolidation of stressful memories. Another possibility is that the activation of 5-HT_1A receptors could attenuate hippocampal hyperactivity, which can be observed in stressed rats (Petty and Sherman 1981; Shumake et al. 2002) and depressed humans (Mayberg et al. 2000; Goldapple et al. 2004), by attenuating the release of glutamate (Matsuyama et al. 1996; Strosznajder et al. 1996). Finally, 5-HT_1A mediates some trophic actions attributed to serotonin, such as the increase in neurogenesis (Brezun and Daszuta 1999; Brezun and Daszuta 2000; Radley and Jacobs 2002; Banasr et al. 2004) and the release of neurotrophic factors (Galter and Unsicker 1996). It is possible that these effects could protect hippocampal circuitry from the deleterious effects induced by repeated exposure to stress and GCs.

Under conditions of repeated exposure to severe inescapable stress and high corticosteroid levels 5-HT_1A-mediated functions could be impaired, thus predisposing to the development of behavioural deficits. In fact, different kinds of severe stressors can induce down-regulation and desensitization of 5-HT_1A receptors within the hippocampus (Flugge 1995; Karten et al. 1999; van Riedel et al. 2003). This seems to be corticoid-dependent, since the administration of corticosterone to rats induces the same effect (Mendelson and McEwen 1992; Meijer and de Kloet 1994; Beck et al. 1996; Neumaier et al. 2000; Bijak et al. 2001) and removal of the adrenal glands enhances 5-HT_1A expression in the hippocampus (Chalmers et al. 1993). Besides, MR, GR and 5-HT_1A receptors are highly colocalized within the hippocampus (Lopez et al. 1998, Lopez et al. 1999), which strengthens the possibility that 5-HT_1A expression is under the control of GCs. However, altered 5-HT_1A function and hypocortisolemia are also observed after repeated exposure to severe stress (Harvey et al. 2003, 2004a). Harvey and co-workers suggested that this could be the result of a hypersensitive feedback regulation on HPA-axis regulation that would follow the acute hypercortisolemia induced by the first stress exposure. The hypocortisolemia over sustained periods could also contribute to altered 5-HT_1A function and abnormal behaviour. This can be of special importance in the neurobiology of PTSD, where hypocortisolemia is a hallmark characteristic of the disorder. The finding that stressed rats show cognitive impairment along with 5-HT_1A receptor changes (Harvey et al. 2004a) and that chronic antidepressant treatment reverses the stress effects upon 5-HT_1A receptors (Bijak et al. 2001) further suggest that 5-HT_1A-mediated functions are important for normal behaviour and mood.

Several clinical studies suggest an impairment of hippocampal 5-HT_1A-mediated neurotransmission in depression. Post-mortem brains of depressed suicides show reduced number of hippocampal post-synaptic 5-HT1 receptors (Cheetham et al. 1990) and an increased number of 5-HT inhibitory autoreceptors (Stockmeier et al. 1998). In addition, depressed patients have a widespread reduction in 5-HT_1A receptor binding measured by positron emission tomography scans with [C]WAY-100635 (Drevets et al. 2000; Sargent et al. 2000) and show a marked attenuation of prolactin release in response to intravenous tryptophan (Deakin et al. 1990). Finally, most effective antidepressants progressively enhance serotonin function in the hippocampus (Blier and De Montigny 1994, Haddjeri et al. 1998; Blier 2003; Castro et al. 2003).

Therefore, it can be suggested that severe inescapable stress impairs the hippocampal serotonergic system, particularly 5-HT_1A receptors, which would limit adaptation to subsequent aversive stimuli, leading to helpless-like behaviour and depressive symptoms in humans. Chronic antidepressant treatment, by facilitating 5-HT_1A-mediated transmission within the hippocampus, would ameliorate these behavioural changes.

Beyond serotonin

In addition to serotonin, several neurotransmitter systems in the hippocampal formation may also participate in stress responses. Ninety per cent of pyramidal and granule cells within the hippocampus are glutamatergic and 10% are GABA-producing interneurons (Vizi and Kiss 1998). Moreover, the hippocampal neuropil is enriched by noradrenergic, serotonergic, dopaminergic and cholinergic axon terminals. These systems are afferent pathways originating from the locus coeruleus, raphe nuclei, ventral tegmental area (VTA) and septal nuclei, respectively (reviewed in Vizi and Kiss 1998).

Noradrenergic system

The noradrenergic system has long been related to behavioural responses to stress (Leonard 2001; Morilak et al. 2005) and to the mechanism of action of antidepressant drugs (Heninger et al. 1996; Brunello et al. 2002; Nutt 2006). Noradrenergic neurons are excited in response to stress, leading to enhanced noradrenaline release in several brain regions, including limbic regions thought to be involved in mediating a variety of behavioural, cognitive, affective, autonomic and neuroendocrine responses to stress (Morilak et al. 2005).

However, noradrenaline and serotonin may play different roles in the hippocampus. In several behavioural paradigms (open-field, passive avoidance and Geller-Seifter), intra dorsal-hippocampus administration of serotonin or noradrenaline induced opposite behavioural effects (Plaznick et al. 1983). Serotonin and noradrenaline also show differences in the regulation of hippocampal functional activity. While serotonin inhibits theta rhythm (Hajos-Korcsok et al. 2000) and LTP (Corradetti et al. 1992), noradrenaline seems to facilitate both processes (Katsuki et al. 1997; Hajos et al. 2003; Almaguer-Melian et al. 2005). In this regard, noradrenaline release into the hippocampus in response to aversive stimuli would signal relevant biological outcomes (“alarm-system”), promoting arousal and storage of fear-related memories (Gray and McNaughton 2000). Thus, the persistence of this system in the active mode could lead to the development of behavioural disorders, such as anxiety and depression. In accordance with this hypothesis, the anxiogenic drug FG-7142 facilitates noradrenergic transmission in the dorsal hippocampus (Ida et al. 1991). Similar effects were reported for animals that showed potentiated behavioural response to stress (Rosario and Abercrombie 1999). Moreover, facilitation of noradrenergic neurotransmission within the dorsal hippocampus failed to show antidepressant-like effects and seems to facilitate helpless-like behaviour in non-stressed rats (Figures 2 and 3, Joca et al. 2006), suggesting that the blockade of the noradrenergic system in this region could prevent the development of stress-induced behavioural deficits. In agreement with this proposal, systemic administration of a β-adrenergic receptor antagonist attenuated the development of PTSD in humans that had recently been exposed to severe trauma (Vaiva et al. 2003).

Figure 2 Post-stress facilitation of noradrenaline-mediated neurotransmission in the dorsal hippocampus does not prevent the behavioural consequences of stress. Male Wistar rats (n = 11–14) were submitted to inescapable footshocks (40 shocks, 1 mA, 10 s) or habituation (30 min) in a shuttle box. Immediately afterwards, they received bilateral intra-hippocampal injections of Sal or desipramine (DIM, 3 or 30 nmols/0.5 μl), a selective noradrenaline reuptake inhibitor and were tested 24 h later with escapable footshocks (30 sound-signalled shocks, 0.8 mA, 10 s). Data are expressed as the mean ± SEM number of escape and/or avoidance failures in each block (summation of five individual trials). There was no significant difference between treatments (modified from Joca et al. (2006), with kind permission from Elsevier).

Figure 3 Pre-stress facilitation of noradrenaline-mediated neurotransmission in the dorsal hippocampus also does not prevent the behavioural consequences of stress. However, it facilitates helpless behaviour in control (non-stressed) animals. Male Wistar rats (n = 7–19) received bilateral intra-hippocampal injections of Sal or desipramine (DIM, 30 nmols/0.5 μl) and immediately afterwards were submitted to inescapable footshocks (40 shocks, 1 mA, 10 s) or habituation (30 min) in a shuttle box. All animals were tested 24 h later with escapable footshocks (30 sound-signalled shocks, 0.8 mA, 10 s). Data are expressed as the mean ± SEM number of escape and/or avoidance failures in each block (summation of five individual trials). * Indicates significant difference from respective Sal-treated group (t-test, p < 0.05, (modified from Joca et al. (2006), with kind permission from Elsevier).

On the other hand, facilitation of noradrenergic neurotransmission within the ventral hippocampus seems to protect against stress effects (Petty and Sherman 1980; Sherman and Petty 1980) and prevents learned helpless development. Rats with enhanced noradrenaline levels within this region do not develop learned helplessness in response to inescapable footshocks (Petty and Sherman 1980; Sherman and Petty 1980). Moreover, noradrenaline release is increased in the ventral hippocampus during habituation to repeated stress (Hajos-Korcsok et al. 2000).

These data are in agreement with recent evidence indicating the existence of important functional differences between dorsal and ventral regions of the hippocampus, the first being closely related to learning/memory processes while the latter would be more involved in anxiety (Kjelstrup et al. 2002; Bannerman et al. 2004; Degroot and Treit 2004; Bertoglio et al. 2006). Thus, it could be speculated that the facilitation of noradrenergic neurotransmission in the ventral hippocampus, but not in the dorsal hippocampus, could attenuate the stress-induced aversive state and, as a consequence, its emotional impact, leading to antidepressant-like effects.

Another possibility to explain the involvement of noradrenergic drugs in stress adaptation would be the facilitation of serotonergic neurotransmission in the hippocampal formation. Mongeau et al. (1997) proposed that the main mechanism of antidepressant treatments in the hippocampus would be to enhance 5-HT_1A and/or to reduce β-adrenergic-mediated neurotransmission. Both effects could be produced by antidepressant treatments through parallel or serial mechanisms involving interactions between the serotonergic and noradrenergic systems.

Noradrenaline, therefore, could play a different role in modulating stress responses depending on the hippocampal region. Antidepressant effects induced by noradrenergic drugs could involve either an interference with noradrenergic neurotransmission in the ventral hippocampus or a facilitation of serotonergic neurotransmission in the dorsal hippocampus. Moreover, the behavioural effects of antidepressants may also involve interference with noradrenergic neurotransmission in other brain structures apart from the hippocampal formation such as the amygdala (Strange et al. 2003).

Dopaminergic system

Dopamine is another monoamine proposed to play an important role in the regulation of mood and behaviour, particularly in motivated and reward-related behaviour (Koob 1996; Berridge and Robinson 1998; Hyman et al. 2006). It is also implicated in the neurobiology of depression as well as in the mechanism of action of antidepressants (Gambarana et al. 1995; D'Aquila et al. 2000; Basso et al. 2005; Gershon et al. 2006; Nestler and Carlezon 2006). However, much of the attention aimed at understanding the role of dopamine in the pathophysiology of depression has been directed to the mesolimbic dopamine pathways, where a dopaminergic hypofunction has been related to the decreased motivation and/or anhedonia observed in human depressive states (Nelson and Charney 1981; Cabib and Puglisi-Allegra 1996; Willner 1997; D'Aquila et al. 2000; Nestler and Carlezon 2006).

Not much is known about the role of hippocampal dopaminergic neurotransmission in the neurobiology of stress-related disorders. Dopamine levels are altered in the hippocampus in response to stress (Vijayakumar and Meti 1999; Zangen et al. 1999; Harvey et al. 2005; Dronjak and Gavrilovic 2006). These levels are increased in congenitally “depressive” rats (Zangen et al. 1999; Yadid et al. 2000) or in rats submitted to an acute exposure to different inescapable stressors (Harvey et al. 2005). On the other hand, chronic exposure to stress induces significant reduction of dopamine content in the hippocampus (Sunanda et al. 2000; Dronjak and Gavrilovic 2006). Chronic antidepressant treatment can counteract the acute neurochemical imbalances induced by stress (Zangen et al. 1999; Yadid et al. 2000), increasing and decreasing the responsiveness of hippocampal dopamine D₂ and D₁-like receptors, respectively (Bijak and Smialowski 1988; Bijak 1993). In accordance with these data, the mRNA for D₅ receptors (D₁ family) is increased in the hippocampus of patients suffering from unipolar depression (Knable et al. 2004)

Several pieces of evidence indicate that the facilitation of dopaminergic neurotransmission induces neuroprotective effects in the hippocampus (Takata et al. 2000; Chlan-Fourney et al. 2002; Bai et al. 2003). Subchronic treatment with D₂ and D₃ receptor agonists, for example, induces a significant increase in expression of the anti-apoptotic protein Bcl-2 in the hippocampus (Takata et al. 2000). Corroborating these results, several groups have shown that chronic treatment with D₂ antagonists induces significant decreases in the hippocampal expression of BDNF mRNA and protein (Angelucci et al. 2000; Lipska et al. 2001; Chlan-Fourney et al. 2002; Bai et al. 2003) and TrkB (Angelucci et al. 2000). These results suggest that the facilitation of dopaminergic neurotransmission within the hippocampus may enhance BDNF expression, which is believed to mediate the behavioural effects of antidepressants (Saarelainen et al. 2003). Studies using chronic treatment with preferential inhibitors of dopamine uptake that possess antidepressant properties could test this hypothesis. These data support the involvement of the hippocampal dopaminergic system in the neurobiology of stress-related disorders and in the behavioural effects of antidepressant drugs. However, additional evidence is needed to clarify the role of hippocampal dopaminergic neurotransmission in adaptation to stress and in the mechanism of action of antidepressants. For example, infusion of drugs that modulate dopaminergic neurotransmission into the hippocampus of rats submitted to appropriate models predictive of antidepressant effects could further clarify the role of hippocampal dopamine in stress neurobiology and in the mechanism of antidepressant drugs.

Glutamatergic and nitrergic systems

Several preclinical studies have indicated that repeated treatment with NMDA receptor antagonists possesses antidepressant-like properties in different animal models (Sofia and Harakal 1975, Trullas and Skolnick 1990; Petrie et al. 1999; Berman et al. 2000). These data are supported by clinical evidence showing that drugs that reduce glutamatergic tone may play a role in the treatment of depression (Sanacora et al. 2004, 2006; Zarate et al. 2004, 2006). For example, chronic treatment with riluzole, a drug that decreases glutamate release (Wang et al 2004) and increases glutamate uptake (Azbill et al. 2000; Frizzo et al. 2004), induces antidepressant and anxiolytic effects (Zarate et al. 2004; Mathew et al. 2005). It also improves the mood of depressed patients resistant to antidepressants (Sanacora et al. 2006).

Although the mechanisms responsible for these effects are not still understood, several lines of evidence have pointed to the hippocampus as an important target for these drugs. The hippocampal glutamatergic system is activated by stress leading to local increase of glutamate release (Moghaddam 1993; Sunanda et al. 2000) through a mechanism that seems to be corticoid-dependent (Abraham et al. 1998). This increased glutamate level has been proposed to mediate stress-induced morphological damage to the hippocampus (Magariños and McEwen 1995b; McEwen 1996; Gould et al. 1997; Cameron et al. 1998; Gould and Tanapat 1999; Nacher and McEwen 2006). Moreover, glutamate is crucial for LTP (Lynch 2004) and positively modulates hippocampal theta rhythm (Puma and Bizot 1999; Bonansco et al. 2002; Leung and Shen 2004), thus facilitating learning/memory processes. Systemic administration of a NMDA receptor antagonist prevents the activation of the hippocampus in response to inescapable stress (Titze-de-Almeida et al. 1994a,b). Also, blockade of hippocampal glutamatergic neurotransmission disrupts the acquisition of fear conditioning (Cammarota et al. 2004; Quinn et al. 2005; Roesler et al. 2006). Therefore, stress-induced glutamate release within the hippocampus may facilitate the formation of aversive memories and at high levels, promote hippocampal damage. These processes would then contribute to the development of stress-induced behavioural changes.

The aforementioned hypothesis is further supported by the observation that intra-hippocampal administration of NMDA antagonists prevents the anxiogenic effect induced by stress (Padovan et al. 2000) and induces antidepressant-like effects in rats (Padovan and Guimarães 2004). In accordance with these behavioural data, chronic treatment with antidepressants reduces glutamate release in hippocampal synaptosomes (Bonanno et al. 2005), downregulates NMDA receptors (Boyer et al. 1998) and decreases glutamate responsiveness (Zahorodna and Bijak 1999). Besides, antidepressant drugs that reduce glutamate levels, such as tianeptine, prevent stress-induced alterations in hippocampal neuronal morphology (McEwen and Chattarji 2004; Reagan et al. 2004) whereas drugs that block glutamate receptors enhance neurogenesis (Cameron et al. 1995, 1998; Yoshimizu and Chaki 2004; Nacher and McEwen 2006) and prevent stress-induced atrophy (Magariños and McEwen 1995b; McEwen 1996). Therefore, modulation of the glutamatergic system seems to play a key role in the regulation of synaptic plasticity in the hippocampus. Normalization of stress-induced alterations in glutamate function, on the other hand, would produce antidepressant effects. This normalization could be achieved via mechanisms targeting the glutamatergic synapse directly or indirectly, for example, involving other neurotransmitters such as serotonin, noradrenaline, or GABA. All these systems closely interact in the hippocampal circuitry (Vizi and Kiss 1998).

The observation that glutamate, by acting on NMDA receptors, can evoke the release of NO (Garthwaite et al. 1988) lead to the suggestion that the antidepressant-like effects produced by NMDA antagonists would result from reduced NO production in the brain. Jefferys and Funder (1996) first reported that systemic inhibition of the enzyme nitric oxide synthase (NOS) produces an antidepressant-like effect in the forced swimming test (FST) in rats, an effect reversed by pretreatment with l-arginine, the substrate for NOS. Besides this observation, it was reported that chronic treatment with nitro-l-arginine, a NOS inhibitor, induces down-regulation of cortical β-adrenoceptors, an effect observed in rodents following chronic treatment with many clinically effective antidepressants (Karolewicz et al. 1999). These effects seem to be due to the inhibition of neuronal NOS (nNOS) because the administration of preferential inhibitors of this isoform also induced dose-dependent antidepressant-like effects in rodents (Yildiz et al. 2000).

Experimental and clinical studies suggest the existence of a dysregulation of the nitrergic system in stress-related disorders. Depressed patients show elevated plasma nitrate levels (Suzuki et al. 2001) and significant mood improvement in response to the systemic administration of methylene blue, a drug that inhibits NOS/ guanylate cyclase (Naylor et al. 1987). Moreover, enhanced hippocampal expression of the nNOS enzyme is reported in post-mortem brain of depressed patients (de Oliveira et al. 2000a,b). Again, stress seems to contribute to this neurochemical dysregulation, since repeated exposure to inescapable stress leads to enhanced expression of NOS and enhanced levels of nitrogen oxides in the hippocampus (Harvey et al. 2004b, 2005).

Systemic administration of clinically effective antidepressants reduces NOS activity in the hippocampus (Luo and Tan 2001; Wegener et al. 2003) and local administration of a nNOS inhibitor immediately before or after an inescapable stress induces antidepressant-like effects in rats (Joca and Guimarães 2006). Increased NO levels in the hippocampus, therefore, could contribute to the development of stress-induced behavioural consequences. The mechanisms mediating these effects, however, are not completely understood. NO is proposed to modulate synaptic transmission in several ways, the most common being through activation of soluble guanylate cyclase (sGC) and nitrosilation of proteins and enzymes (Snyder and Ferris 2000; Prast and Philippu 2001). As a result, NO can modulate neuronal excitability and neurotransmitter release (Snyder and Ferris 2000; Prast and Philippu 2001). It can decrease serotonin levels in several brain regions (Kiss 2000; Prast and Philippu 2001), including the hippocampus (Weneger et al. 2000). The antidepressant-like profile of NOS inhibitors in the FST was similar to that of serotonin selective reuptake inhibitors and depended on intact serotonin neurotransmission (Harkin et al. 2003). Taken together, these data suggest that the increase in NO production that follows stressful situations can impair serotonergic transmission in the brain, interfering with the adaptation to repeated stress exposure. On the other hand, activation of 5-HT_1A receptors negatively regulates glutamate release (Dijk et al. 1995; Matsuyama et al. 1996) and NO synthesis (Strosznajder et al. 1996), suggesting that these systems mutually interact in the hippocampal formation to modulate behaviour under stressful conditions.

NO could also change neuronal function by direct neurotoxic effects. In primary cortical culture administration of NOS inhibitors prevents cell death elicited by NMDA and related excitatory amino acids, thus suggesting that NO mediates glutamate-induced neurotoxicity (Dawson et al. 1991). Besides, in cultured hippocampal neurons, NO can decrease BDNF release and inhibition of nNOS enhances hippocampal BDNF expression (Canossa et al. 2002). We observed similar effects in vivo after treatment with a nNOS inhibitor that produced antidepressant-like effects in the FST (Figure 4). Moreover, systemic inhibition of NO is reported to increase hippocampal neurogenesis (Pacher et al. 2003; Cardenas et al. 2005; Zhu et al. 2006). These data suggest that stress-induced release of NO could mediate pathological processes within the hippocampus that account for the plastic changes and behavioural alterations seen in stressed animals. Therefore, inhibition of NO synthesis could protect the hippocampus from stress-induced effects, thus allowing behavioural adaptation to occur in situations of chronic exposure to stress.

Figure 4 Subchronic treatment with a NOS inhibitor increases BDNF expression in the hippocampus. The figure shows stained cells for BDNF in the hippocampal formation after (a) vehicle, (b) imipramine (15 mg/kg) and (c) 7-nitro-indazole (60 mg/kg), a preferential nNOS inhibitor, treated rats. They received three i.p. injections (0, 5 and 24 h) and were killed 24 h after the last injection, the hippocampus was removed and processed for BDNF immunohistochemistry (primary anti-BDNF antibody: rabbit polyclonal antibody raised against a peptide mapping at the N-terminus of BDNF of human origin, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:800). Note BDNF signal (dark stained cells) only in (b) and (c). DG, dentate gyrus; CA, ammons horn. Bar = 150 μm.

Based on these data, it is possible to speculate that an enhanced NMDA/NO-pathway facilitates the development in animals of behavioural deficits induced by stress and thus may contribute to the pathophysiology of stress-related disorders, such as depression and PTSD. Drugs that attenuate NMDA and/or NO-mediated neurotransmission would offer an alternative treatment for these disorders, either alone or in combination with antidepressant drugs (Zarate et al. 2006).

Conclusion

Stress-induced behavioural changes are a complex phenomenon that certainly involves more than one structure. However, numerous pieces of evidence have pointed to the hippocampal formation as an important target for stress and antidepressant-induced effects. Several main neurotransmitter systems in the hippocampus are related to stress effects. Serotonin, by acting on 5-HT_1A receptors in the dorsal hippocampus, facilitates adaptation to severe inescapable stress. A failure in this system induced by severe stress and/or high corticoid levels would predispose to the development of stress-induced behavioural deficits. This process would be facilitated by glutamate and NO. Drugs that facilitate 5-HT_1A- or attenuate glutamatergic/nitrergic-mediated neurotransmission in the hippocampal formation, on the other hand, would promote adaptation to stress and induce antidepressant-like effects (Figure 5). The role of noradrenaline in these processes is less clear and may differ depending on the specific hippocampal region (dorsal vs. ventral).

Figure 5 Severe stress, activating the NMDA/NO pathway, can impair normal hippocampal functioning, predisposing to helplessness and depressive symptoms. Conversely, serotonin can counteract the stress-induced damage to the hippocampus, restoring normal functioning and allowing coping and stress adaptation. These effects are probably mediated by 5-HT_1A receptors. GCs have a permissive role in NMDA damaging effects and at high levels, can impair 5-HT_1A-mediated neurotransmission. Clinical studies using tryptophan depletion and results obtained with direct intra-hippocampal drug injections suggest that serotonin, glutamate and NO can also interfere with hippocampal function independently of cellular remodelling and neurogenesis.

Acknowledgements

The authors acknowledge J. C. de Aguiar and E. L. T. Gomes for their helpful technical assistance. This research was supported by grants from CAPES, CNPq and FAPESP (02/13197-2 and 04/01738-4).

Behavioral procedures described in this manuscript were conducted in conformity with the Brazilian Society of Neuroscience and Behavior guidelines for the care and use of laboratory animals, which are in compliance with international laws and policies. The experimental protocols were approved by the local Ethical Committee and all efforts were made to minimize animal suffering.