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Assessing antidepressant activity in rodents: recent developments and future needs John F. Cryan, Athina Markou and Irwin Lucki Animal models are indispensable tools in the search to identify new antidepressant drugs and to provide insights into the neuropathology that underlies the idiopathic disease state of depression. As new targets are developed, both serendipitously and through hypothesis-driven research, existing animal paradigms are being modified and new tests are being developed to detect antidepressant actions of compounds acting on a broad range of neural and genetic targets. This review focuses on recent findings regarding some of the most widely employed animal models used currently to predict antidepressant potential. Emphasis is placed on recent modifications to such paradigms that have increased their utility and reliability. Furthermore, some key issues that need to be addressed for future discovery of novel antidepressant agents are examined, and the available data on genetically altered mice that might lead to the discovery of novel targets for antidepressant action are collated.

John F. Cryan* Neuromodulation Unit, Nervous System Research, Novartis Pharma AG, WSJ 386.344, Basel, CH-4002, Switzerland. *e-mail: john_f.cryan@ pharma.novartis.com Athina Markou Dept of Neuropharmacology, CVN-7, The Scripps Research Institute, 10550 North Torrey Pines Rd, La Jolla, CA 92037, USA. Irwin Lucki Depts of Psychiatry and Pharmacology, University of Pennsylvania, 538A Clinical Research Building, Philadelphia, PA 19104, USA.

Depression is a serious disorder in today’s society, with estimates of lifetime prevalence as high as 21% of the general population in some developed countries [1]. As defined by the American Psychiatric Association [2], depression is a heterogeneous disorder often manifested with symptoms at the psychological, behavioral and physiological levels (Box 1). As with all diseases, approximations of both the disorder and the actions of corrective medications in laboratory animals are essential for the development of effective therapies. The wide spectrum of disruptions that characterize depression highlight the difficulty posing researchers to mimic the disorder in the laboratory. Indeed, two human symptoms, recurring thoughts of death or suicide, or excessive thoughts of guilt, are impossible to model in laboratory animals. The question remains impenetrable as to whether we can ever know whether a laboratory animal is ‘depressed’ (Fig. 1). Nonetheless, numerous attempts have been made to create animal models of depression, or at least of the symptoms of depression, and criteria for their evaluation have been established. Some of the most widely cited criteria were developed by McKinney and Bunney >30 years ago [3]. They proposed that the minimum requirements for an animal model of depression are: (1) it is ‘reasonably analogous’ to the human disorder in its manifestations or symptomatology; (2) there is a behavioral change that can be monitored objectively; (3) the behavioral changes observed should be reversed by the same treatment modalities that are effective in humans; and http://tips.trends.com

(4) it should be reproducible between investigators. Most models of depression in use at that time were based on primate separation experiments that attempted to model the entire syndrome of depression. However, subsequent efforts to delineate validity criteria for animal models often do not take into account the reliability and usability of the paradigm in the everyday rodent laboratory setting and are often based on esoteric, theoretical principals rooted in comparing the etiological basis between the human condition and the syndrome in the animal model ([4], but also see [5]). Unlike other medical disorders where the pathology is well defined, such as diabetes or Parkinson’s disease, the underlying pathophysiology of depression is still unresolved, thus making it virtually impossible to fulfill criteria solely based on etiology. Most recently, it has become clear that a more useful strategy might be to model single endophenotypic differences (i.e. one clear-cut behavioral output) relevant to the disease state as opposed to a syndrome [4]. Geyer and Markou [4] have proposed that the only criteria that are necessary and sufficient for initial use are that the paradigm has strong predictive validity and that the behavioral readout be reliable and robust in the same laboratory and between laboratories. The satisfaction of other criteria such as construct or discriminant validity might have heuristic value and are desirable but not essential for the model to provide important initial uses in both basic neurobiological research and drug discovery. Various paradigms have been developed and are instrumental in detecting the antidepressant-like potential of novel compounds in preclinical settings. The models commonly used are diverse and were developed originally based on the behavioral consequences of stress, drug, lesion or genetic manipulations (Table 1). Many of these models have undergone iterative improvements to keep pace with continuing advances in the development of drugs with an increasingly wide array of pharmacological actions. Moreover, such improvements to models continue to be necessary to detect antidepressant effects more precisely in genetically engineered animals and after modifications of cellular and molecular targets. In this review, some of the recent improvements to models that enhance their utility to detect antidepressant effects are highlighted.

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Box 1. Symptoms of major depression [a] • Depressed mood most of the day (in children and adolescents, irritability might signify a depressed mood) • Markedly diminished interest or pleasure in all or most activities most of the day • Large increase or decrease in appetite • Insomnia or excessive sleeping • Psychomotor agitation (evident by, for example, hand wringing) or slowness of movement • Fatigue or loss of energy • Indecisiveness or diminished ability to think or concentrate • Feelings of worthlessness or excessive or inappropriate guilt • Recurrent thoughts of death or suicide Reference a American Psychiatric Association (1994) Diagnostic and Statistical Manual of Mental Disorders (4th edn), American Psychiatric Press

Animal models of antidepressant-like activity The forced swim test

The forced swim test (FST) was developed by Porsolt and colleagues [6] in the rat and, subsequently, in the mouse [7]. This test is the most widely used tool for assessing antidepressant activity preclinically. The widespread use of this model is largely a result of its ease of use, reliability across laboratories and ability to detect a broad spectrum of antidepressant agents [8]. The test is based on the observation that rats, following initial escapeoriented movements, develop an immobile posture when placed in an inescapable cylinder of water. If they are replaced in the testing apparatus 24 h later, they resume this posture quickly. The immobility is thought to reflect either a failure of persistence in escape-directed behavior (i.e. behavioral despair) or the development of passive behavior that disengages the animal from active forms of coping with stressful stimuli [9]. If antidepressant

Fig. 1. A ‘depressed’ rat? It is an impossible quest to mimic major depressive disorders completely in rodents. Instead of anthropomorphizing the human condition, as in the cartoon, investigators have developed paradigms that detect specific behavioral endophenotypic differences (clear-cut behavioral outputs) that are sensitive to the effects of antidepressant treatments (both pharmacological and non-pharmacological).

treatments are given between the two exposures, the subjects will actively persist engaging in escapedirected behaviors for longer periods of time than after vehicle treatment. For reasons not yet elucidated, in mice, one exposure is sufficient to generate a stable immobility readout that can be countered by acute pretreatment with antidepressant agents. However, the major drawback of the traditional FST is that it is unreliable in the detection of the effects of selective 5-HT reuptake inhibitors (SSRIs) [9], which are the most widely prescribed antidepressant drugs today.

Table 1. Widely used rodent models sensitive to the effects of antidepressant agentsa Animal model

Ease of use Reliability Specificity Applicable Comments to mice

Forced swim test

High

High

Highb

Yes

Modified forced swim test High

High

Highb

?

Tail suspension test

High

High

Highb

Yes

Olfactory bulbectomy

Medium

High

High

Yes

Learned helplessness

Medium

Medium

High

Yes

DRL-72 Neonatal clomipraminec Prenatal stress Chronic mild stress

Medium Medium Medium Low

Medium Medium ? Low

Medium ? ? High

? Yes Yes Yes

Resident intruder

Low

?

Medium

?

High

Medium

Yes

Drug-withdrawal-induced Low changes in ICSS aAbbreviations:

Sensitive to acute antidepressant treatments; does not reliably detect SSRIs Sensitive to acute antidepressant treatments; differentiates antidepressants from different classes including SSRIs Sensitive to acute antidepressant treatments; certain strains climb their tail Behavioral effects evident only following chronic treatment; mechanism of action poorly understood Sensitive to short-term antidepressant treatments; ethical restrictions in some countries Sensitive to short-term antidepressant treatments Only limited testing of antidepressants have been conducted Only limited testing of antidepressants have been conducted Reliability has been questioned repeatedly; behavioral effects evident only following chronic treatment Distinguishable behavioral effects only following chronic treatment; requires further validation in other laboratories Requires further validation; cannot assess baseline strain differences easily

DRL-72, differential reinforcement of low-rate 72 second schedule; ICSS, intracranial self-stimulation; SSRI, selective 5-HT reuptake inhibitor. can be screened out by complementary locomotor activity studies. cClomipramine is a nonselective 5-HT reuptake inhibitor. bStimulants

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Refs [6,7] [9] [7,58] [17,18] [7,29,30] [7] [29] [59,60] [31,39,61] [62] [36,37,40,41]

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Fig. 2. Rats undergoing forced swim test (FST) behaviors. The modified FST is used to assess the role of monoamines in antidepressant action. (a) Rats can engage in at least three different forms of behavior: immobility, swimming and climbing. (b) The differential effects of the selective 5-HT reuptake inhibitor (SSRI) fluoxetine and the noradrenaline reuptake inhibitor reboxetine on active behaviors in the FST are shown. Thus, antidepressants that primarily potentiate 5-HT-mediated neurotransmission increase swimming behavior whereas those with primary actions through catecholamines increase climbing behavior. Fluoxetine data are reproduced, with permission, from [10]; reboxetine data are reproduced, with permission, from [11].

The modified forced swim test

In an effort to enhance the sensitivity of the traditional FST in the rat so that it can be SSRI responsive, several simple procedural modifications have been made [9]. These developments include increasing the water depth to 30 cm from traditional depths of 15–18 cm, and using a time sampling technique to rate the predominant behavior over a 5-s interval. These alterations enabled investigators to distinguish specific behavioral components of active behaviors, namely: (1) climbing behavior (also known as thrashing), which is defined as upward-directed movements of the forepaws along the side of the swim chamber; (2) swimming behavior, the movement (usually horizontal) throughout the swim chamber that also includes crossing into another quadrant; and (3) immobility, which is defined, as in the traditional Porsolt test, as when no additional activity is observed other than that required to keep the rat’s head above the water (Fig. 2). As a result of the increase in water depth, there is considerably less immobility than in the traditional test because the animals cannot have contact with the cylinder bottom. The major advance of http://tips.trends.com

the modified FST over its traditional counterpart is that it reveals that catecholaminergic agents decrease immobility with a corresponding increase in climbing behavior, whereas 5-HT-related compounds such as SSRIs also decrease immobility but increase swimming behavior [9,10]. Recent studies have shown that 5-HT2C receptors play an instrumental role in mediating the effects of the SSRI fluoxetine in the test [10]. Furthermore, it has been shown that the antidepressant-like behavioral effects of the noradrenaline reuptake inhibitor reboxetine in the FST are dependent on an intact ventral tegmental noradrenaline-mediated system but not the locus coeruleus system [11]. One major drawback of the FST (as with many antidepressant-sensitive paradigms) is the fact that short-term antidepressant treatments reverse the immobility whereas in the clinic it can take weeks for the same antidepressants to elevate mood. However, it has been demonstrated that doses of antidepressant drugs that are inactive acutely elicit antidepressant-like effects when administered chronically, which further validates the modified paradigm [12]. Many other research groups [13–16] have used this strategy with much success, indicating the reliability of the modified paradigm. Olfactory bulbectomy

The bilateral removal of the olfactory bulbs of a rat (hamsters and mice have also been used) results in a complex constellation of behavioral, neurochemical, neuroendocrine and neuroimmune alterations, many of which are correlated with changes observed in major depression [17]. To many investigators the bulbectomy model seems to be an obscure test because the rationale for its use as an animal model has been often questioned based on construct and etiological validity arguments [5]. However, this model has one of the best portfolios for the prediction of known antidepressant compounds following repeated administration and is reliable between laboratories [17]. The most consistent behavioral change caused by bulbectomy is a hyperactive response in a novel, brightly lit open field apparatus, which is reversed almost exclusively by chronic, but not acute, antidepressant treatment [17,18]. Reasons for this time-dependent reversal are not fully understood but have generated much interest of late. Recent studies have shown that the hyperactive response might be related to increases in defensive behaviors [19] or alterations in aversively motivated behavior [20]. Furthermore, it has been shown that antidepressant compounds preferentially enhance habituation to novelty in the bulbectomized rat, and that these effects are not secondary to anosmia (loss of the sense of smell) [21]. Concurrent with these studies, other groups have focused on neurochemical and physiological alterations that might account for the antidepressant-sensitive behavioral alterations. Much interest has been placed on the serotonergic system with a 5-HT hyperinnervation of the frontal

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*

* *

*

*

90

90 12 36 42 60 84 108 132 156 T Hours following withdrawal

12 3642 60 84 108 132 156 T Hours following withdrawal

Fig. 3. Withdrawal from chronic amphetamine and nicotine results in a marked elevation in brain reward thresholds. The combination of a 5-HT1A receptor antagonist, p-MPPI ((4-iodo-N-[2-[4methoxyphenyl]-1-piperazinyl]ethyl)-N-2-pyridinyl-benzamide hydrochloride), with the selective 5-HT reuptake inhibitor (SSRI) fluoxetine (as opposed to either drug alone) results in a return to baseline threshold values within hours after the acute drug treatment (T, arrow). The animals maintain this baseline threshold for the remainder of the testing period. This strategy of combination therapy has been used clinically to accelerate the onset of antidepressant activity and the current data suggest that this model be one of the few that is sensitive to such actions. Reproduced, with permission, from [40].

cortex [22] and stressor-induced alterations in 5-HT-mediated activity [23] observed subsequent to bulbectomy. Furthermore, increased striatal glutamate release during novelty exposure-induced hyperactivity has been demonstrated that might have a modulatory role on the antidepressant-sensitive response [24]. Increases in the concentrations of the neuropeptides (or their encoding genes) corticotropin-releasing factor, thyrotrophin-releasing factor, somatostatin [25] and neuropeptide Y [26], which might play a role in mediating the antidepressant-sensitive behaviors, have also been demonstrated. Imaging studies demonstrated alterations in signal intensities in cortical, hippocampal, caudate and amygdaloid regions in olfactory bulbectomized animals, compared with sham-operated controls [27]. In addition, ventricular enlargement was evident in bulbectomized animals. It has been suggested that these structural changes correlate somewhat with those seen in depressed patients [27]. Comparing the behavioral and biochemical effects of bulbectomy in young versus aged rats, Slotkin and colleagues [28] suggest that this test might provide a useful animal model with which to test therapeutic interventions for geriatric depression. Learned helplessness

The learned helplessness paradigm is based on the fact that following repeated uncontrollable shocks, animals demonstrate escape deficits that are reversible by antidepressant agents [29]. The behavioral deficits are sensitive to a broad spectrum of antidepressant compounds usually following shortterm treatment. The major drawback of the model is that most of the depression-like symptomatology does not persist beyond 2–3 days following cessation of the uncontrollable shock [29]. A recent modification of the rat learned helplessness procedure incorporates aspects of the chronic mild stress paradigm [30]. http://tips.trends.com

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By chronic exposure to mild stressors the effects of the uncontrollable shock can be maintained for a prolonged period, and chronic treatment with the SSRI fluoxetine and the nonselective monoamine uptake inhibitor imipramine reversed these changes. The chronic stress procedure involves restraint and novel housing, and avoids the problems associated with food deprivation used in the traditional chronic mild stress procedure [31]. Vollmayer and Henn [32] have recently proposed key factors that can be manipulated to enhance both the usability and the reliability of the rat learned helplessness paradigm. These include using a larger testing apparatus, a mild shock presentation and a relatively difficult shock avoidance task [7]. Furthermore, they point out that animals can artefactually avoid shock as a result of their position in the apparatus, which should also be taken into account [7]. Models based on drug-withdrawal-induced anhedonia

Reward deficits associated with withdrawal from drugs of abuse can represent an animal model of the symptom of ‘diminished interest or pleasure (anhedonia)’ with construct, convergent and predictive validities [4,33]. Recent studies showed that amphetamine withdrawal is characterized by decreased breaking-points under a progressive ratio schedule for a sucrose solution reinforcer [34]. Under the progressive ratio schedule animals are required to increase their operant responding for a fixed reward until they reach a ‘break-point’, which determines the maximal amount of effort the animals will expend to procure the desired rewarding stimulus. Thus, the break point provides an objective measure of the subject’s motivation [34]. Amphetamine withdrawal is also associated with decrements in anticipatory and motivational measures for sexual reinforcement [35] and elevations in brain reward thresholds in rats [4]. The use of the intracranial self-stimulation (ICSS) paradigm has provided investigators with a reliable and quantitative behavioral readout that enables the assessment of reduced brain reward function following withdrawal from a variety of drugs of abuse [36–38]. ICSS has also been used successfully to examine behavioral deficits in the chronic mild stress model of depression [39]. In certain situations, antidepressant compounds have been shown to attenuate the withdrawal-induced reward deficits [36,37,40,41]. Recent data indicated that drugwithdrawal-induced changes in ICSS in rats is one of the few models sensitive to the proposed fast-acting antidepressant effects of a 5-HT1A receptor antagonist in combination with an SSRI (Fig. 3) [40]. Further validation is required before this model can be used routinely to detect antidepressant-like activity. Strain differences in animal models of depression

The burgeoning use of genetically altered mice in behavioral pharmacology has resulted in much

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Box 2. Down and (knocked)-out: the use of genetically altered mice to assess antidepressant-related phenotypes There has been an upsurge in the development of mice with genetically altered expression of a specific protein, be it a receptor, transporter, enzyme or signal transduction protein. These new tools have the potential to examine novel targets for antidepressant activity for which there are few established pharmacological tools. Table I lists some of the genetically modified mice that have been shown to have depressive or antidepressant-related behaviors. The majority of studies use simple tests such as the forced swim test (FST) or tail suspension test (TST) to elucidate their behavioral changes. Some examples of knockout mice, such as those with targeted deletion of the 5-HT1A receptor and the noradrenaline transporter, are expected to show antidepressant-related phenotypes given the large body of evidence implicating these proteins in antidepressant action. In other examples, where selective pharmacological tools have been unable to penetrate, such as α2-adrenoceptor subtypes and signaling molecules such as the G protein Gzα, the behavioral evidence in mutant mice implicates these targets in antidepressant action and provides new directions for drug discovery. However, the much-discussed caveats associated with interpretation of the behavioral effects in genetically altered animals should not be understated [42], the major two being background strain differences and compensatory adaptive changes. The ability to see the same phenotype across different strains, as in the case of 5-HT1A receptor knockout mice, gives further credence to the reliability of the phenotype [a–c]. The full potential of regionally selective and inducible knockout and transgenic mice has yet to be realized, but such strategies offer many advantages over currently used techniques. Such mice certainly will be welcome tools to dissect regionally specific circuits that might influence the actions of antidepressants. The ability to restore, albeit transiently, the phenotype in noradrenaline-deficient mice by administering the synthetic precursor L-deoxyphenylserine is a novel way to confirm that the phenotype is related to noradrenaline function as opposed to adaptive changes resulting from being reared without this monoamine [d]. Specific behavioral changes can be conformed by conducting multiple types of behavioral tests such as FST, TST and learned helplessness. Other physiological analyses such as tests for locomotor activity, pain sensitivity or cognition might be necessary to implicate behavioral changes to stress-induced depression. Such caveats cannot be underestimated and overinterpretation of antidepressant-like phenotypes must be avoided. For example, muscarinic acetylcholine M1 receptor knockout animals are hyperactive and correspondingly have an artefactual antidepressant-like phenotype in the FST [e]. Brain-derived neurotrophic factor (BDNF) heterozygote knockout mice show altered behavior in the learned helplessness paradigm but this has been ascribed to their reduced sensitivity to pain as opposed to a depressionrelated phenomenon [f]. Therefore, appropriate caution using other convergent tests that draw on different antidepressant-related endophenotypes, and complementary physiological analyses provide a program of information concerning whether a given phenotype is functionally relevant to depression-related pathology.

emphasis being placed on studying individual strain differences in both baseline behavior and in the response to psychotropic medications in mice [42]. In almost all of the behavioral models outlined above, substantial strain differences have been observed. Further analyses of both inbred and outbred strains might help reveal phenotypic behavioral differences that might have an underlying genetic basis relevant to antidepressant action. In a recent survey of 11 different http://tips.trends.com

References a Ramboz, S. et al. (1998) Serotonin receptor 1A knockout: an animal model of anxiety-related disorder. Proc. Natl. Acad. Sci. U. S. A. 95, 14476–14481 b Parks, C.L. et al. (1998) Increased anxiety of mice lacking the serotonin1A receptor. Proc. Natl. Acad. Sci. U. S. A. 95, 10734–10739 c Heisler, L.K. et al. (1998) Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice. Proc. Natl. Acad. Sci. U. S. A. 95, 15049–15054 d Cryan, J.F. et al. (2001) Use of dopamine-β-hydroxylasedeficient mice to determine the role of norepinephrine in the mechanism of action of antidepressant drugs. J. Pharmacol. Exp. Ther. 298, 651–657 e Miyakawa, T. et al. (2001) Hyperactivity and intact hippocampus-dependent learning in mice lacking the M1 muscarinic acetylcholine receptor. J. Neurosci. 21, 5239–5250 f MacQueen, G.M. et al. (2001) Performance of heterozygous brain-derived neurotrophic factor knockout mice on behavioral analogues of anxiety, nociception, and depression. Behav. Neurosci. 115, 1145–1153 g Mayorga, A.J. et al. (2001) Antidepressant-like behavioral effects in 5-hydroxytryptamine(1A) and 5-hydroxytryptamine(1B) receptor mutant mice. J. Pharmacol. Exp. Ther. 298, 1101–1107 h Trillat, A.C. et al. (1998) C. R. Seances Soc. Biol. Fil. 192, 1139–1147 i Schramm, N.L. et al. (2001) The α2a-adrenergic receptor plays a protective role in mouse behavioral models of depression and anxiety. J. Neurosci. 21, 4875–4882 j Sallinen, J. et al. (1999) Genetic alteration of the α2-adrenoceptor subtype c in mice affects the development of behavioral despair and stress-induced increases in plasma corticosterone levels. Mol. Psychiatry 4, 443–452 k Xu, F. et al. (2000) Mice lacking the norepinephrine transporter are supersensitive to psychostimulants. Nat. Neurosci. 3, 465–471 l Cases, O. et al. (1995) Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice lacking MAOA. Science 268, 1763–1766 m Grimsby, J. et al. (1997) Increased stress response and β-phenylethylamine in MAOB-deficient mice. Nat. Genet. 17, 206–210 n Filliol, D. et al. (2000) Mice deficient for delta- and mu-opioid receptors exhibit opposing alterations of emotional responses. Nat. Genet. 25, 195–200 o Yang, J. et al. (2000) Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs. Proc. Natl. Acad. Sci. U. S. A. 97, 9984–9989 p Montkowski, A. et al. (1995) Long-term antidepressant treatment reduces behavioural deficits in transgenic mice with impaired glucocorticoid receptor function. J. Neuroendocrinol. 7, 841–845

strains of mice in the FST [43], there was a tenfold difference in baseline immobility scores in the 4-min test between strains. Furthermore, the baseline level did not correlate with sensitivity to antidepressants. The noradrenaline reuptake inhibitor desipramine reduced immobility in seven of the strains with DBA/2J and C57/B6 being the most responsive strains. By contrast, only three strains (DBA/2J, BALB/cJ and NIH-Swiss) were sensitive to the effects of fluoxetine.

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Table I. Genetically altered mice that exhibit depression or antidepressant-related behaviora Genetically altered mouse

Depression or antidepressant-related phenotype

Test used

Refs

5-HT1A receptor knockout 5-HT1B receptor knockout

Antidepressant-like effects Increased sensitivity to the effects of SSRIs Blockade of antidepressant-like effects Blockade of antidepressant-like effects of antidepressants from variety of classes Depressive-like effects and blockade of the antidepressant-like effect of imipramineb Antidepressant-like effects Depressive-like effects Antidepressant-like effects Antidepressant-like effects Antidepressant-like effects Antidepressant-like effects Depressive-like effects Blockade of antidepressant-like effects of desipramine and reboxetinec Antidepressant-like effects Antidepressant-like effects

FST, TST TST FST FST

[a–c] [g] [h] [d]

FST

[i]

FST FST FST, TST FST FST FST FST FST

[j] [j] [k] [l] [m] [n] [n] [o]

FST FST

[p] [q]

Antidepressant-like effects Antidepressant-like effects Antidepressant-like effects Antidepressant-like effects Antidepressant-like effects Blockade of the antidepressant-like effects of Hypericum perforatumd Antidepressant-like effects Reduced sensitivity to fluoxetinee Antidepressant-like effects

FST FST FST FST, TST FST, TST FST FST FST FST, TST

[r] [s] [t] [u] [v] [w] [x] [y] [z]

Dopamine-β-hydroxylase knockout α2A-Adrenoceptor knockout α2C-Adrenoceptor knockout α2C-Adrenoceptor overexpressing Noradrenaline transporter knockout Monoamine oxidase A knockout Monoamine oxidase B knockout Mu opioid receptor knockout Delta opioid receptor knockout Gzα G-protein knockout Glucocortocoid-receptor-impaired transgenic Glutamic acid decarboxylase (65-kDa isoform) knockout Neural cell adhesion molecule knockout Tumor necrosis factor α knockout Angiotensinogen knockout Adenosine A2A receptor knockout Tachykinin NK1 receptor knockout Interleukin-6 knockout Dopamine D5 receptor knockout DARPP-32 knockout CREB mutant (α and ∆ isoforms)

aAbbreviations: CREB, cAMP response element-binding protein; DARPP-32, dopamine- and cAMP-regulated phosphoprotein of molecular weight 32 000; FST, forced swim test; SSRI, selective 5-HT reuptake inhibitor; TST, tail suspension test. bImipramine is a nonselective monamine reuptake inhibitor. cDesipramine and reboxetine are noradrenaline reuptake inhibitors. dActive constituent of St John’s wort. eFluoxetine is an SSRI.

q Stork, O. et al. (2000) Postnatal development of a GABA deficit and disturbance of neural functions in mice lacking GAD65. Brain Res. 865, 45–58 r Stork, O. et al. (2000) Recovery of emotional behaviour in neural cell adhesion molecule (NCAM) null mutant mice through transgenic expression of NCAM180. Eur. J. Neurosci. 12, 3291–3306 s Yamada, K. et al. (2000) Neurobehavioral alterations in mice with a targeted deletion of the tumor necrosis factor-α gene: implications for emotional behavior. J. Neuroimmunol. 111, 131–138 t Okuyama, S. et al. (1999) Reduction of depressive-like behavior in mice lacking

angiotensinogen. Neurosci. Lett. 261, 167–170 u El Yacoubi, M. et al. (2001) Adenosine A2A receptor antagonists are potential antidepressants: evidence based on pharmacology and A2A receptor knockout mice. Br. J. Pharmacol. 134, 68–77 v Rupniak, N.M. et al. (2001) Comparison of the phenotype of NK1R–/– mice with pharmacological blockade of the substance P (NK1) receptor in assays for antidepressant and anxiolytic drugs. Behav. Pharmacol. 12, 497–508 w Calapai, G. et al. (2001) Interleukin-6 involvement in antidepressant action of Hypericum perforatum. Pharmacopsychiatry 34, S8–S10

Similarly, Liu and Gershenfeld [44] examined the baseline and imipramine-induced behaviors of 11 strains of mice in the tail suspension test. These data also revealed a broad range in baseline scores, and only three strains (DBA/2J, NMRI and FVB/NJ) responded to the antidepressant. These data suggest that strain comparisons might be needed to prevent false negative screening of compounds in various tasks. Strain differences were also demonstrated in rat http://tips.trends.com

x Holmes, A. et al. (2001) Behavioral characterization of dopamine D5 receptor null mutant mice. Behav. Neurosci. 115, 1129–1144 y Svenningsson, P. et al. (2002) Involvement of striatal and extrastriatal DARPP-32 in biochemical and behavioral effects of fluoxetine (Prozac). Proc. Natl. Acad. Sci. U. S. A. 99, 3182–3187 z Conti, A.C. et al. cAMP response element-binding protein is essential for the upregulation of brain-derived neurotrophic factor transcription, but not the behavioral or endocrine responses to antidepressant drugs. J. Neurosci. (in press)

paradigms with the Wistar-Kyoto rat being less responsive than many other strains to antidepressant treatments in various paradigms [45,46]. To discriminate genetic influences on ‘depressivelike’ behavior, several investigators have undertaken selective breeding programs of animals based on the individual responsiveness in animal models of depression. These breeding efforts include animals susceptible to learned helplessness [47], high and low

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FST responders [29] and animals bred for spontaneous high or low immobility scores in the tail suspension test [48]. Other genetic models have been developed based on an underlying alteration in the function of a selective neurotransmitter system, be it acetylcholine-receptormediated [49] or 5-HT1A-receptor-mediated [50] responses. Moreover, forward genetic approaches are under way whereby a mutant line of animals is induced randomly by mutagens such as ethylnitrosonurea, and behavior is analyzed subsequently [51]. Positional cloning would enable the identification of gene targets relevant for the behavioral response. Many genetic models inevitably use simple screening paradigms such as the FST procedures to determine that the mice indeed have relevant phenotypes (Box 2). The use of single endophenotypic differences such as reaction to stress or pronounced elevation in ICSS thresholds in response to manipulations, as opposed to a syndrome, might be a useful strategy to identify genetic factors. Therefore, both genetic and behavioral strategies should be viewed as complementary and serving overlapping purposes, and together might yield further information about the idiopathic disease state. The demonstration of strain differences in the response to antidepressant drugs could provide new models for the detection of genes that influence the clinical effects of antidepressants. Such models might eventually match individual patients to the most effective types of therapy for their genetic constitution or type of depression. Species differences – new targets

Acknowledgements A.M. was supported by a Novartis Pharma AG Research grant and I.L. was supported by grants USPHS R01 MH36262 and P05 MH48125 from the National Institute of Mental Health. We would like to thank Paul J. Kenny and Alasdair M. Barr for their helpful comments on the manuscript. We would also like to thank Peggy Myer from the Dept of Biomedical Graphics of The Scripps Research Institute for her assistance with graphics, and Mike Arends for editorial assistance. This is publication 14527-NP from The Scripps Research Institute.

It is becoming apparent that there are distinct species differences in the primary targets of certain psychotropic agents, which makes translation or prediction of effects from rat or mouse to human difficult. Tachykinin NK1 and 5-HT1B receptors are but two examples of receptors relevant to antidepressant action whereby important structural differences between the human and the rat and/or mouse interfere with the ability to predict pharmacological effects across species [52,53]. Although neurochemical studies have been carried out in species with receptor pharmacology more similar to that in humans such as gerbils, hamsters, ferrets and guinea-pigs, the use of these species for assessing antidepressant-like activity behaviorally has not been firmly established. Just as most tests developed for rats were refined for use in mice with the advent of genetically altered mice [42], it will be crucial to devise new tests, and modify current tests for other species, to encapsulate the real pharmacological actions of potential antidepressant compounds with novel mechanisms of action. The recent report of the humanization of the mouse 5-HT1B receptor using a ‘knock-in’ approach of the human receptor gene demonstrates one novel strategy that

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can be used to overcome species differences in antidepressant targets [52]. Future directions

Traditionally, the use of animal models has been instrumental in detecting antidepressant compounds based on a known pharmacology. Models such as the FST, olfactory bulbectomy and learned helplessness, which were validated originally for detection of tricyclic antidepressants and monoamine oxidase inhibitors, should detect related compounds with ease. It remains unclear as to whether currently used paradigms can systematically detect antidepressant agents with non-monoamine mechanisms of action. That said, robust antidepressant-like activity has been demonstrated by such paradigms for compounds whose mechanisms of action are primarily through corticotropin-releasing factor [54,55], neuropeptide Y [16], glucocortocoid [56], glutamate [57] and substance P [53]. Furthermore, neurochemical and behavioral analysis of the effects of novel compounds will be most relevant in ‘depressed’ animals in addition to ‘normal’ healthy animals. The further validation of models such as those involving prenatal stress, maternal deprivation, neonatal clomipramine (a nonselective 5-HT reuptake inhibitor) administration or amphetamine withdrawal might provide the appropriate substrates for such analyses. Knockout strategies have opened up an entire new avenue for selection of drug targets in depression. The future will also bring the more systematic use of genechip microarray analysis in animal models of depression. This tool will enable the powerful comparison of changes in gene expression in various animal models before, during and after antidepressant treatment. Such genetic approaches in rodent paradigms might provide novel targets for antidepressants that might translate therapeutically to the human situation. Concluding remarks

It is clear from our experience that current models need to be refined continuously or new models developed to reveal the therapeutic potential of a broad range of compounds, as was the case with the introduction of SSRIs. Indeed, battery-style testing of various paradigms modeled on different endophenotypes of the depression syndrome, be it anhedonia or stress-induced coping, is encouraged. The refinement of animal models over past years has demonstrated that many traditional paradigms are receptive to further modification. Such improvements will enhance the models’ utility for both the detection of novel targets for antidepressant activity and contributing to a better understanding of the underlying pathophysiology of depression.

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