Christopher R Pryce Daniela Riiedi Bettschen Andrea C Dettling and Joram Feldon

Destroy Depression

Treatment for Depression

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Behavioural Neurobiology Laboratory, Swiss Federal Institute of Technology Zurich, Schorenstrasse 16,

CH-8603 Sehwerzenbach, Switzerland

Abstract: Depression is now one of the most common human illnesses and is of immense clinical and economic importance. Considerable preclinical research efforts have been made to establish animal models of depression, and more recently the human evidence derived from brain imaging studies has provided important insights into the functional neuroanatomical correlates of depression. Despite this, knowledge of the neurobiology of both depression and its pharmacological treatment is limited and so, consequently, is the efficacy of antidepressant pharmacology. In terms of etiology, whilst evidence for specific factors and mechanisms is sparse, it is well established from human epidemiological and clinical studies that genetic and environmental factors, and of course their interaction, are involved. With regard to the environment, acute stressors induce adaptive behavioral and physiological changes in adult mammals that resemble the symptoms of depression but are transient, whereas chronic stressors lead to chronic changes in these behavioral and physiological states, such that they constitute the symptoms, e.g. low mood, helplessness, anhedonia, hyposomnia, and associated abnormalities of depression, e.g. elevated catecholamine output, hyper-cortisolemia. The environment in which human infants and children develop is fundamental to how they develop, and it is clear that parental loss but also infant or child exposure to emotional or physical neglect or abuse, impact on development including increased vulnerability to depression and associated physiological abnormalities, across the life span. It would be important to establish the mechanisms mediating specific forms of abnormal offspring-parent relationship and development, including whether this early experience induces depressed traits per se and/or traits of increased vulnerability to depression that are triggered by events in later life. Studies of early-life environmental manipulations in rodents and primates can potentially yield evidence that abnormal developmental experience leading to dysfunction of the neurobiology, physiology and behavior of emotion is a general mammalian characteristic, and therefore that this approach can be used to develop animal models for depression research.

Introduction

The major aim of this chapter is to review the current evidence that specific manipulations of the postnatal environment in rat and monkey infants can result in changes in their behavioral and physiological status, such that they provide important models of specific symptoms and associated abnormalities of

"•Corresponding author. Tel.: +41 1 655 7386; Fax.: +41 1 655 7203; E-mail: pryce(abehav.biol.ethz.ch the major mood disorder, depression. We begin with a brief overview of depression in terms of its major symptomatology and associated features, and also its etiology. With regard to the latter, we emphasize the quite considerable and growing body of human evidence, clinical and epidemiological, that the environment experienced by human infants is extremely important in shaping the likelihood of developmental psychopathology, including posttraumatic stress disorder due to parental loss, neglect or abuse, as well as the vulnerability to depression in adulthood. We then review some of the most prominent literature describing the study of the long-term neurobehavioral impacts of experimental postnatal manipulations in rats and monkeys. This is followed by a review of some of our own recent findings in this area, obtained using the unusual but important approach of performing very similar studies in parallel in rats and monkeys. Finally, we assess the progress made with and future directions of development of validated rodent and primate models of depression based on postnatal manipulations.

Depression: symptoms, associated features and aetiology

Symptoms and associated features

Clinical disorders of the representation and regulation of mood and emotion, most notably depression and anxiety disorders, can occur during childhood, adolescence, adulthood and senescence. Depression is the most common psychiatric disorder, with the lifetime risk for major depressive disorder being 10-25% for women and 5-12% for men. The diagnostic symptoms of depression are either chronically depressed mood (sadness, helplessness, irritability) and/or chronic loss of interest/pleasure in nearly all activities (anhedonia), together with additional symptoms, including changes in sleep patterns, appetite and psychomotor activity, feelings of worthlessness, and cognitive impairment (APA, 1994). Although there is currently no biological marker that is diagnostic for depression, the above reported or observable diagnostic symptoms are often associated with a constellation of physiological and anatomical changes. Thus, atypical nocturnal electroencephalography (EEG) is common, including increased wakefulness (Jindal et al., 2002). Sampling of cerebrospinal fluid (CSF), blood or urine has revealed dysregulation in the stress hormone systems of depressed patients. Often there is hyperactivity in the hypothalamic-pituitary-adrenal (HPA) system, related to altered limbic regulation thereof, including elevated basal and stress-related titres of corticotropin releasing factor (CRF), corticotropin (ACTH) and Cortisol, and resistance of Cortisol to suppression by dexamethasone challenge (Holsboer, 2000; Wong et al., 2000). Depression-related hyperactivity in the sympatho-adrenomedullary (SAM) system is indicated by elevated adrenaline (ADR) and noradrenaline (NA) titres (Koslow et al., 1983). Concomitant with HPA and SAM hyperactivity, structural imaging studies have demonstrated adrenal hypertrophy in depressives (Heim et al., 1997a, b). Functional neuro-imaging studies have identified over-activity in brain regions proposed to modulate emotional expression (e.g. orbital prefrontal cortex) and mediate emotional and stress responses (e.g. amygdala), and underactivity in brain areas implicated in integrating emotional, attentional and cognitive sensory processing (e.g. anterior cingulate cortex) (Rolls, 2000; Drevets, 2001; Davidson et al., 2002). Morphometric neuro-imaging has identified reduced volume of the hippocampus in patients with major depression, and hippocampal dysfunction could underlie the context-inappropriate emotional responding that characterises the disorder (Sheline, 2000). Post-mortem studies have identified cellular and synaptic pathologies in some of these same brain regions (Eastwood and Harrison, 2001; Harrison, 2002).

Neurotransmitters implicated in the pathophysiology (and of course therapeutic pharmacology) of depression include the NA (Wong et al., 2000), serotonin (5-HT) (Maes and Meltzer, 1995) and dopamine (DA) (Naranjo et al., 2001) pathways and the glutamate and gamma-aminobutyric acid (GABA) systems (Duman et al., 1997; Manji et al., 2001). Cellular and molecular theories of depression propose a complex pathway based on inter-relationships between: (1) elevated CRF and corticosteroid activity related to environmental stress (see above and below); (2) altered activity of and interactions between NA, 5-HT, DA, glutamate and GABA; (3) reduced activity in messenger pathways and gene transcription underlying synthesis of neurotrophic factors (e.g. brain derived neurotrophic factor, BDNF) in limbic and cortical neurons; (4) reduced resilience and plasticity of these neurons; and (5) disrupted mood and emotion processing (Duman et al., 1997; McEwen, 1998; Sapolsky, 2000; Manji et al., 2001). There follow some examples of lines of evidence that provide support for such inter-relationships. The CRF synthesised in the central amygdala is released from efferents synapsing with the locus coeruleus (LC) in the brainstem, the principal site of cell bodies for NA neurons. Descending LC NA efferents innervate the sympathetic autonomic nervous system and ascending LC NA efferents project to the limbic and pre/frontal neocortical regions implicated in depression (see above). Stress activates amygdala CRF release and NA neurotransmission (Valentino et al„ 1998; Sanchez et al„ 2001), and chronic NA activation can lead to receptor down-regulation and reduced BDNF levels (Manji et al, 2001). Stress also activates glutamate release that in turn reduces energy capacity in post-synaptic neurons. Cortisol acts by facilitating glutamate signalling at NMDA receptors and by inhibiting glucose transport (Manji et al, 2001). Cortisol can also exert direct neuronal actions, in terms of regulation of synthesis of neurotrophic factors and modulation of synaptic transmission (de Kloet et al, 1998; Joels 2001). This is primarily achieved via its transcription factor receptors, the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), that can either activate or deactivate expression of target genes. Central MR distribution is primarily localised in the hippocampus and amygdala, whereas GRs are widespread, including hypothalamic, limbic and neocortical expression (de Kloet et al, 1998; Sanchez et al, 2001).

Genetic and environmental aetiology

Determinants of the risk for developing depression are complex as so is the disease aetiology. Basic, clinical and epidemiological studies indicate a complex interplay between genetic susceptibility, environmental experience, and maturation/aging. Families with a high genetic load for depression can be identified (Lauer et al, 1998). Furthermore, healthy probands in these families demonstrate, as traits, atypical activity in physiological systems, e.g. HPA, that function abnormally in depression, possibly reflecting system-specific vulnerability (Holsboer, 2000). Various physiological states that are characteristic of depression are also characteristic of the adaptive stress response to acute environmental challenge, e.g. reduced appetite and sexual interest, wakefulness, HPA and SAM hyperactivity (de Kloet et al, 1998; McEwen, 2000). The adaptive stress response is followed by a rapid return to homeostasis mediated by negative feedback systems, but chronic stress or allostatic load can induce altered plasticity and cellular resilience in the brain, including loss of negative feedback function (McEwen, 2000; Manji et al, 2001). Whilst the aetiological relationships between the systemic, neurochemical and cellular consequences of chronic stress, on the one hand, and the development of depression, on the other, remain to be elucidated, it is clear that depression often presents in an environment of chronic psychogenic stress (e.g. related to interpersonal relationships) or following a profound environmental event with chronic psychogenic consequences. With regard to the latter it is very important that increased risk of major depressive disorder exists in individuals with posttraumatic stress disorder (PTSD), and that the co-morbidity of these two disorders is high (APA, 1994). PTSD develops following exposure to an extreme traumatic stressor that elicits fear or helplessness and can occur at any age, including childhood. Further diagnostic criteria are persistent re-experience of the traumatic event, persistent avoidance of stimuli associated with the trauma, reduced responsiveness to and interest in the daily environment, and persistent behavioral and physiological symptoms of hyperarousal. Several physiological and anatomical commonalities with depression exist, including disturbed sleep, SAM hyperactivity and reduced hippocampal size (Charney et al, 1993; Baker et al, 1999; Davidson et al, 2002). Low Cortisol titres and supersuppression of Cortisol by dexamethasone challenge are also characteristics of PTSD, thereby providing an intriguing contrast with glucocorticoid status in depression (Heim et al, 1997a,b).

There is overwhelming evidence for a greater role for psychogenic stressors and traumas in association with the first episode of depression than with subsequent episodes, strongly suggesting that chronic sensitization of specific physiological systems and neurobiological circuits, presumably encoded at the level of gene expression, increases vulnerability to develop subsequent episodes (Post, 1992). Physiological and neuroanatomical abnormalities associated with depression are often state-dependent but they can also persist after remission, and the likelihood of this is positively correlated with depression episode number (APA, 1994). Finally here, what is particularly striking and of marked relevance to this chapter, is that this developmental-experiential process can begin in infancy (Heim and Nemerolf 1999, 2001).

Early adverse experience as a developmental risk factor

As stated above, depression aetiology is intimately related to traumatic events and chronic stress, and both of the latter can occur in the context of social relationships. In humans, indeed in mammals generally, there is no more important or intimate relationship than that between infant and caregiver, developing to that between child and caregiver, and leading eventually to offspring independence (Rutter and Rutter, 1993). The offspring-caregiver relationship normally constitutes a dynamic process of bi-directional species-typical behaviors. However, inappropriate infant- and child-directed behavior is a serious public health problem in terms of its incidence rate and its impact on mental health development (De Bellis et al., 1999a, b). Maltreatment of minors includes physical neglect, emotional neglect, physical abuse and sexual abuse. There is now a considerable body of epidemiological evidence demonstrating that early adverse experience in the form of maltreatment is a pre-eminent factor in the development of affective disorders: maltreatment can be the direct cause of PTSD, can markedly increase the risk to develop PTSD in response to trauma in adulthood, and can markedly increase the risk to develop depression in adulthood. Furthermore, PTSD and depression are often co-morbid in individuals who experienced such early-life adversity (McCauley et al., 1997).

As with adulthood-onset affective disorder, physiological and neuroanatomical abnormalities are frequent in children who have suffered maltreatment and are presenting with affective disorder. Furthermore, there is the possibility that even in the absence of clinical symptoms, abnormal traits are present that mediate the high long-term vulnerability that maltreated individuals exhibit (Heim and Nemeroff, 2001). Children with PTSD and co-morbid depressive symptoms secondary to past maltreatment experiences demonstrate elevated urinary NA, ADR and DA titres (De Bellis et al., 1999b). Maltreated children with PTSD exhibit increased systolic blood pressure and heart rate (Perry, 1994). Studies investigating HPA function in maltreated children have not yielded consistent findings, but it is important to note that between-study comparison is confounded by differences in type of and age at maltreatment, and psychopathology at the time of study. There is evidence that severely neglected children with psychopathology exhibit low peak basal Cortisol titres and that children with PTSD symptoms exhibit enhanced dexamethasone suppression of Cortisol, in common with PTSD adults (Heim and Nemeroff, 2001). Neuroimaging studies in maltreated children with PTSD have demonstrated smaller intracranial, cerebral and hippocampal volumes compared with matched controls (Bremner et al., 1997; De Bellis et al., 1999b).

Two recent key studies have investigated the impact of childhood abuse on HPA and autonomic function in adulthood (Heim et al., 2000, 2001). Using 2x2 experimental designs, both studies compared women with a clinical history of childhood physical and/or sexual abuse either with or without a current diagnosis of major depression with their respective matched controls (non-abused depressed or non-abused non-depressed). PTSD prevalence was higher in the group of abused women with current depression than in the abused women without current depression. In one study (Heim et al., 2000) responsiveness to a standardized and validated psychosocial stress protocol was investigated: The women with a history of childhood abuse exhibited greater plasma ACTH responsiveness than did the respective controls; the increase was more pronounced in the abused women with current depression, and these women also exhibited greater Cortisol responsiveness than did each of other the three groups (which were similar in their Cortisol profiles) and greater heart rate responsiveness than controls. In a second study (Heim et al., 2001), complementary to the first, subjects were examined in terms of their responses to CRF and ACTH provocative neuroendocrine challenge tests, with blood sampling being conducted pre- and post-challenge via indwelling catheters. Firstly, basal plasma Cortisol titres were lower in the abused women with and without current depression than in the control women. In terms of challenge responsiveness, CRF challenge stimulated a greater ACTH response in abused women without current depression than in their control group, whereas abused depressives and control depressives both exhibited blunted ACTH responsiveness. In the ACTH stimulation test, abused women who were not depressed exhibited blunted Cortisol responsiveness relative to each of the other three groups, who responded similarly. The authors interpret their findings in terms of sensitisation of the pituitary (CRF-ACTH) and counter-regulative adaptation of the adrenal gland (ACTH-cortisol) in abused women without current depression. On exposure to stress, they propose, such women may hyper-secrete CRF (see Heim et al. (2000)) resulting in depression but pituitary CRF receptor down-regulation (Heim et al., 2001).

Clearly, therefore, human early life adversity exerts marked and consistent effects on stress systems, the brain, and behavior, and dramatically increases vulnerability to development of affective psycho-pathology. Given the robustness of this relationship, the high prevalence of infant -child maltreatment, and the continuum between developmental and adulthood affective psychopathology, then robust animal models of affective disorders based on early life stress would be of immense value in terms of increased neurobiological understanding of the aetiology, symptomatology and pharmacology of these most prevalent psychiatric disorders.

Rat studies of the effects of early adverse experience

Although the evidence for a relationship between human early life adversity and chronically increased vulnerability to affective disorders is compelling, it is of course clear that such early life adversity can take many different forms and, as such, can have very varied consequences. The clinical studies that have been achieved to-date report primarily on cases of sexual abuse in mid- and late childhood as opposed to adversity experienced in the infancy phase of early life. A prospective experimental design, whilst fraught with ethical and practical considerations, is realistically the only approach that would allow for adequate definition of the form of neglect or abuse experienced and, therefore, for the study of the acute and chronic consequences of specific forms of early life stress. In laboratory animals, prospective, controlled studies are of course the norm, and for the laboratory rat (Rattus norvegicus) there is a huge body of literature describing the acute and chronic effects of specific manipulations of the rat pup's social environment on its neurobiological, physiological and behavioral phenotype. A great deal of the rat evidence on the acute effects of such postnatal manipulations is relevant to the present chapter, and a large number of the studies describing chronic effects are relevant here also. There follows below only a very brief overview of this extensive evidence.

Short-term effects of pup-dam manipulation

The rat pup is born in an immature or altricial state of development in a litter of 8-16. Rat maternal behavior occurs in regular bouts separated by periods when the dam is away from the intact litter (Stern, 1996). Maternal behavior is stimulated by multi-sensory pup cues. The consequences, indeed functions, of this behavior are to promote infant growth and maintain infant homeostasis via the regulation of physiology and behavior. The mother-infant regulators have been divided into three general categories, namely (1) thermal-metabolic, (2) nutritional-intero-ceptive and (3) tactile/sensorimotor, and the infant systems that are regulated include metabolic systems, the autonomic nervous system, neuroendocrine axes, neurochemical systems, and the central nervous system (CNS) and behavior (Hofer, 1994a, b). For example, the thermal input provided by the dam to the pup maintains oxygen consumption and heart rate at constant levels; provision of milk maintains consistent sleep-wake cycles, corticosterone titres, and cardiophysiology (via interoceptors); and tactile stimulation maintains consistent growth hormone titres and brain catecholamine titres (Hofer 1994a, b). That maintenance of homeostasis in infancy is dependent on another conspecific is of course a major difference to older life stages. In addition, some specific physiological systems also have a different level (or set point) of basal activity in infancy relative to older life stages. Particularly noteworthy in the present context is that postnatal days (PNDs) 3-14 of rat life are characterized by the so called stress hyporesponsive period (SHRP), comprising low basal blood titres of ACTH and corticosterone (CORT), and attenuated responsiveness of the pituitary-adrenal system to physical events (e.g. ether exposure, saline injection) that elicit marked ACTH and CORT stress responses in older conspecifics (Walker et al., 1986). Maintenance of the SHRP is dependent on maternal care: Isolation of the litter from the dam for a single period of at least 8h leads, per se, to an increase in ACTH and CORT titres and also leads to increased pituitary-adrenal responsiveness to discrete stressors (Levine et al., 1992; van Oers et al., 1998a, b). Repeated pup separation from the dam and the litter for 1 h per day on PNDs 2-8 leads to a marked stress response (i.e. amelioration of the SHRP), at least when the stressor constitutes 1 h pup isolation, on PND 9 (McCormick et al., 1998). Furthermore, it has been demonstrated that: exposure of rat pups to pharmacological hypercorticoidism during the SHRP leads to inhibition of neurogenesis, gliogenesis and synaptogenesis, and disrupted conditioned learning in adulthood (Bohn, 1984); and that exposure to hypercorticoidism in the adult physiological range during the SHRP actually leads to adult offspring that exhibit reduced CORT and behavioral stress responsiveness and improved conditioned learning (Catalani et al., 2000). Whilst these findings are extremely important, it is at the same time prudent to bear in mind that the demonstration of the SHRP may have resulted in exaggeration of the importance of the HPA system and in particular of CORT in terms of responding to and mediating the effects of postnatal manipulations. As described above, the rat dam is a regulator of many physiological and neurobiological infant systems, and the disruption of pup homeostasis via manipulation of the dam-pup relationship such that the pup is released from maternal regulators could involve acute changes in a large number of systems that then lead to chronic developmental effects. For example, brief, daily separation of the litter from the dam ("early handling", see below) has been demonstrated to yield transient increases in: thyroid hormone titres, hippocampal serotonin (5-HT) activity, hippocampal cAMP formation, protein kinase A titres, and mRNA and protein levels of cAMP-responsive transcription factors. In turn, it has been proposed that it is the increased activity in this pathway that underlies the increased density of glucocorticoid receptors in the hippocampus that is exhibited by adult rats that experienced such early handling (Meaney et al., 1996, 2000).

Long-term effects of pup-dam manipulations: general issues

Table 1 provides an overview of the major infant-mother manipulations that have been studied in rats and monkeys and that are covered in this review. In rats, the first postnatal manipulation model to be investigated in terms of its chronic consequences for long-term development was the comparison between early handling (EH) and early non-handling (NH). Early handling constitutes the experimenter picking up the pup, removing it from the breeding cage and isolating it in a small compartment for several minutes, repeated across at least PNDs 1-7, whereas NH constitutes the complete absence of handling, both experimental and maintenance related (Levine, 1960). As described above, EH elicits acute endocrine and neurochemical responses from pups (Meaney et al., 2000). Furthermore, chronically, a remarkable constellation of neurobiological, physiological and behavioral effects has been demonstrated with the EH-NH model. From the earliest studies of the EH-NH model (Levine et al., 1955; Levine, 1957; Denenberg, 1964), it has been demonstrated that, relative to NH, EH leads to adult offspring that are less anxious and fearful when exposed to environmental challenge. For example, behaviorally, EH adults are more active and spend more time in the centre of an open field, they exhibit less reflexive startle behavior to an acoustic stimulus, and exhibit increased active avoidance in a two-way foot-shock shuttle box (Levine et al., 1955; Weiner et al., 1985; Caldji et al., 2000; Pryce et al., 2001a, 2003). Physiologically, EH adults exhibit an attenuated pituitary-adrenal endocrine stress response to environmental challenge, as evidenced by a combination of lower peak stress responses and more rapid poststress return to basal levels in terms of circulating ACTH and CORT (Plotsky and Meaney 1993; Meaney et al., 1996; Liu et al., 2000). Inputs to which EH and NH rats exhibit such differential responsiveness include both unconditioned stressors such as restraint (Plotsky and Meaney, 1993) and conditioned stressors such as acoustic stimuli that

Table 1. Key procedural characteristics of commonly studied postnatal environments in rats and monkeys

Species

Procedure

Characteristics

Major comparison group

Rat, Monkey

Non-handling (NH)

Animal facility rearing (AFR)

Early handling (EH, H)

Maternal separation (MS)

Rat, Monkey

Rat, Monkey

Early deprivation (ED)

Maternal Privation (MP)

Rat, Monkey Control (CON)

Mother and infants not exposed to human physical contact, and exposed to only minimal distal human disturbance, e.g. room entry restricted to one person only.

Mother and infant(s) exposed to human physical contact during cage cleaning and to distal human disturbance but not to additional procedures.

Mother and infants exposed daily to human physical contact and 15 min exposure to a different physical environment. Mother and infants always separated. Infants separated from dam either as an intact litter or as individual pups.

Mother and infants exposed daily to human physical contact and 3-6 hours of separation of the intact litter from the dam. In some cases the litter is exposed to a different physical environment and the mother remains in the home cage, in other cases the mother is removed and it is the litter that remains in the home cage.

Mother and infants exposed daily to human physical contact and 0.5-6 hours of separation of individual infants from littermates and from the mother, in a different physical environment. The mother remains in the home cage.

Infants removed from mother at 1-2 days after birth and reared using human hand-rearing (monkey) or artificial feeding device (rat). In the case of MP monkeys, at 3-6 months of age they are sometimes placed with MP peers.

The physical human contact aspects of the manipulation are controlled but mother and infant are not exposed to additional experimental procedures, e.g. Picking up and placing in a transport cage for rats, and catching and briefly restraining the parent carrying the infant for monkeys. It is important to note that effects of these control procedures on subsequent maternal care cannot be controlled for.

Early handling Maternal separation Early deprivation Animal facility rearing Maternal separation (Rat), Early deprivation (Rat, Monkey)

Non-handling Maternal separation

Non-handling Early handling Animal facility rearing

Non-handling (Rat) Early handling (Rat) Animal facility rearing (Rat) Control (Rat, Monkey)

Animal facility rearing (Rat, Monkey)

Early handling (Rat)

Early deprivation (Rat, Monkey)

have peen paired previously with mild electro foot-shock (Pryce et al., 2003). As such, the EH-NH model has yielded considerable evidence relating directly to anxiety disorders but not to depression. Of course, it is also the case that anxiety disorders and depression exhibit high co-morbidity (APA, 1994) and furthermore, certain aspects of the model are directly relevant to depression. For example, depression is associated with either increased levels of CRF in the CSF, or at least with an absence of the expected decrease in CRF levels in the presence of the hyper-cortisolism (vis. negative feedback) that is often associated with depression (Wong et al, 2000). Adult rats that were non-handled in infancy exhibit both increased basal CRF mRNA in the hypothalamus and increased CRF in the median eminence, relative to EH adults (Plotsky and Meaney, 1993). Also, depressed patients exhibit a pronounced increase in basal CSF NA levels, and although basal levels are unaffected, relative to EH, NH adults exhibit increased NA levels in the paraventricular nucleus of the hypothalamus (PVNh) following restraint stress (a difference that could contribute to the ACTH and CORT hyper-reactivity of NH rats) (Liu et al, 2000).

Without doubt a very interesting and biomedically relevant characteristic of the EH-NH model is that EH leads to a phenotype of reduced reactivity to aversive environmental challenge. From the point of view of animal modeling of psychiatric disorders, however, it is intuitively problematic that it is the non-manipulated group, i.e. NH, that yields the symptom-like phenotype. The separation time of 15min that is typically used for EH is actually less than what pups are exposed to in the natural situation where dams have to leave the nest to forage. Furthermore, it has been demonstrated that EH leads to increased levels of the maternal behaviors of licking and nursing in comparison with NH (Lee and Williams 1974; Liu et al, 1997). Relative to NH, EH constitutes a constellation of human handling of the dam and the pups, exposure of the pups to a different environment, and increased maternal care (licking and nursing), and which of these factors is/are responsible for the hypo-anxious phenotype remains to be elucidated (Liu et al, 1997; Denenberg, 1999; Pryce and Feldon, 2003). One obvious approach to investigating postnatal environmental manipulations that, relative to the appropriate control, do lead to long-term effects of relevance to preclinical depression research is to increase the duration and the severity of the manipulation. A large number of laboratories have used such an approach of exposing pups to marked deviation from the typical postnatal environment. Obviously, there are an extremely large number of factors that can be varied in any such manipulation including, among others: the number of days that the manipulation is performed across pup development, the duration of each episode of manipulation, whether pups are separated from the dam as an intact litter or are isolated completely, whether the manipulation is performed during the dark or the light phase, and the ambient temperature of the manipulation environment. A review by Lehmann and Feldon (2000) details these factors as well as a large number of experimental design factors that are important in this research area.

Recently, we proposed a nomenclature that facilitates recognition of at least some of the important variables and can provide a framework for postnatal manipulation research (Pryce et al, 2002; Pryce and Feldon, 2003; Table 1). The term maternal separation is used to describe separation of the intact litter from the dam for one or more hours per day across several postnatal days, and single maternal separation to describe separation of the intact litter from the dam for a single 24 h period. Infant or early combined with either isolation or deprivation is used to describe separation of the pup from the dam and the litter for one or more hours per day across several postnatal days (Fig. 1A). Our preference is for early deprivation (ED), because this indicates similarity to EH and thereby emphasises the important and reciprocal relationship between these two manipulations: the patent form of EH constitutes separation of the pup from the litter and the dam (Levine, 1960; Denenberg et al, 1967), as does ED; EH does not constitute deprivation in that the isolation period is shorter than species-typical periods between successive bouts of maternal care, whereas ED clearly does.

Another important and complex issue in the study of potentially severe postnatal manipulations and their long-term effects is that of the control group. That NH leads to the development of an atypical phenotype on a number of neurobehavioral parameters relative to EH has been summarized above (see also Pryce and Feldon, 2003). Furthermore, recent studies have demonstrated consistent adulthood differences between NH rats and those exposed as infants to the human interventions (e.g. handling during cage cleaning) inherent to rodent maintenance or, to use the term given to this comparison group, animal facility rearing (AFR) (Huot et al, 2001). Therefore, studies in which EH-NH-AFR have been compared directly reveal that NH adults exhibit increased CORT responsiveness to restraint, reduced locomotor activity in an open field, and increased acoustic startle reactivity, relative to both EH and AFR, with the latter two groups exhibiting similar

Dettling Daniela

Fig. 1. Photographs of the early deprivation procedure in (A) rat pups and (B) a common marmoset infant. In the case of rat pups they are placed in open plastic compartments on a thin layer of saw dust. The apparatus is either placed directly on a table so that the manipulation is conducted at ambient room temperature (cold-ED) or on top of heating pads (warm-ED). In our laboratory the manipulation is typically conducted in the dark and during the dark phase of a reversed light-dark cycle. In the case of the marmoset infant it is placed in a standard mouse cage fitted with a lid to which it can cling. Absorbent paper is placed on the bottom of the cage for urine and faeces. The cage is placed in an isolation chamber that is illuminated with a 4 W bulb; no other source of heat is provided. A small video camera placed above the cage allows for the marmoset's behaviour to be observed and recorded.

Fig. 1. Photographs of the early deprivation procedure in (A) rat pups and (B) a common marmoset infant. In the case of rat pups they are placed in open plastic compartments on a thin layer of saw dust. The apparatus is either placed directly on a table so that the manipulation is conducted at ambient room temperature (cold-ED) or on top of heating pads (warm-ED). In our laboratory the manipulation is typically conducted in the dark and during the dark phase of a reversed light-dark cycle. In the case of the marmoset infant it is placed in a standard mouse cage fitted with a lid to which it can cling. Absorbent paper is placed on the bottom of the cage for urine and faeces. The cage is placed in an isolation chamber that is illuminated with a 4 W bulb; no other source of heat is provided. A small video camera placed above the cage allows for the marmoset's behaviour to be observed and recorded.

phenotypes in each of these environments (Pryce et al, 2001a; Pryce and Feldon. 2003).

Given that the EH-NH model has been most studied in terms of effects on anxiety-like behavior and stress reactivity (see above), then it is logical that it is these parameters that have been most investigated in studies of long-term effects of MS and ED. When MS constitutes 3-h litter-dam separation on PNDs 2-14 it does exert some specific long-term neuroendocrine effects relative to NH, namely increased basal CRF mRNA levels in the hypothalamus, higher CRF titres in the median eminence (Plotsky and Meaney, 1993) and higher restraint stress-induced NA release into the paraventricular nucleus of the hypothalamus (Liu et al, 2000). The MS adults tend to exhibit a more prolonged ACTH stress response but not a higher peak ACTH or CORT stress response relative to NH adults (Plotsky and Meaney, 1993; Liu et al, 2000). Behaviorally, MS is without anxiogenic effect relative to NH, including no effect on: locomotion or exploration in an open field, novelty-induced suppression of feeding, or reflex acoustic-startle reactivity (Caldji et al, 2000). Relative to AFR, MS does yield chronic anxiogenic effects; thus, MS rats demonstrate higher ACTH and CORT peak responses and more prolonged responses to an air-puff stressor, and increased anxiety-like behavior in the elevated plus maze (Huot et al, 2001). Turning to ED, we have studied a form of this postnatal manipulation that has several points in common with MS, the most important of which is that it was performed daily from a very early stage of development (PNDs 1-21) and involved an extended period of separation each day (4 h) (Pryce et al, 2001a, b). Because of the high hypothermic stress associated with isolating the young pup not only from the dam but also the litter (Denenberg et al, 1967), ED was conducted not at room temperature, the typical conditions for MS, but at 30°C and therefore under moderate rather than severe thermal stress (Blumberg and Sokoloff 1998). As adults, ED rats did not differ from NH or AFR in terms of basal CORT levels, but they did demonstrate a significantly reduced CORT response to restraint compared with NH rats, as did AFR rats (Pryce et al, 2001a). Behaviorally, ED adults were more active in a novel open field compared with NH (with AFR intermediate), exhibited, as did AFR, a reduced acoustic startle reflex compared with NH, and enhanced two-way active avoidance compared with NH (with AFR intermediate) (Pryce et al, 2001a, 2003; Pryce and Feldon, 2003). What is clear, therefore, is that although ED does constitute a severe postnatal manipulation in terms of the complete absence of stimuli derived from dam and littermates for an extended period per day, its long-term effects on anxiety-like and stress-related pheno-types, rather than being in the opposite direction to those of EH relative to NH are actually in the same direction as, and in absolute terms very similar to those of, EH relative to NH.

Against this background, it will be clear that the MS and ED manipulations described above would not be expected to yield symptom-like effects in tests related to depression research, certainly not in experiments that deploy NH as the comparison group. With regards to MS, one approach has been to investigate its effects relative to AFR rather than NH; with regards to our own ED research, we have maintained NH as the comparison group but have increased the severity of the manipulation with regards to temperature conditions. These MS-AFR and ED-NH studies are reviewed below. Prior to this, we provide details of the major tests used in rat depression research.

Behavioral tests used in rat depression research

As described in the Introduction, the two major diagnostic symptoms of depression are depressed mood and anhedonia, with at least one of these present continuously for a minimum of two weeks (APA, 1994). With regards to depressed mood, articulated by the patient as feelings of sadness, emptiness or helplessness, and observable in behavior as, for example, tearfulness (and in children and adolescents as irritable behavior), it is probably true that there is no rat behavioral test for which it is claimed that the state induced by the test is sadness-like or irritable-like. There are tests for which the terms "helplessness" or "despair" are used to infer the psychological state underlying the behavioral responses exhibited by the rat to the test conditions. These tests might possess some face validity relative to the symptoms of feelings of helplessness and worthlessness, and of psychomotor retardation (APA, 1994), with the descriptive term of impaired coping ability perhaps providing a parsimonious description of a psychological state that is applicable to rat experimental subject and human patient. Anhedonia, i.e. diminished interest or pleasure in all or most activities most of the day, is the second major diagnostic symptom of depression and there are several behavioral tests that are described as tests of rat anhedonia. There follow brief summaries of the learned helplessness test, the forced swim test of behavioral despair, the sucrose preference test of anhedonia, and the progressive ratio schedule reinforcement test of anhedonia, in terms of the principle and methodology of each test and its predictive validity with respect to anti-depressant action. The rationale for focussing on these tests is that they are the tests that have been applied in the investigation of the potential for specific postnatal manipulations to induce long-term depression-like effects in the rat.

Learned helplessness (LH) is an inferred psychological state used to account for the behavioral phenomenon in which animals exposed to uncontrollable aversive events, i.e. exposure to an aversive unconditioned stimulus (US) that is not predictable, escapable or avoidable, exhibit deficits in their instrumental responding in terms of escaping from subsequent aversive events. That is, even though the US is now predictable (because it is preceded by a conditioned stimulus (CS) such as a tone or light) and escapable or avoidable, the animal exhibits a temporary deficit in establishing the now adaptive instrumental contingency, namely US-escape response (Overmier and Seligman, 1967; Seligman and Maier, 1967; Gray, 1987). The LH test constitutes two stages: typically, rats are placed in a shock chamber for 40-60 min and exposed to inescapable foot shocks of around 1-2 mA and 5-15 sec duration on a variable interval schedule, such that the total shock duration is approximately one-half of the session duration (Overmier and Seligman, 1967; Vollmayr and Henn, 2001). This is the US-preexposed (US-PE) group and the control comprises rats placed in the shock chambers without US exposure (US-NPE). (The original LH studies included a group that could escape the US and a yoked group that could not, so that it was thereby possible to demonstrate the importance of the uncontrollability of US exposure to development of the LH state.) Twenty-four hours later, US-PE and US-NPE rats are placed in a conditioning chamber, either a lever-containing operant chamber or a two-way shuttle chamber divided in two by a barrier, in which the same form of aversive US and a novel CS (e.g. tone, light) are presented.

Taking the example of the two-way shuttle chamber, each trial constitutes exposure to a CS (e.g. 12 sec tone) that predicts delivery of the US (e.g. 2 sec foot shock contiguous with final 2 sec of the CS). The session can comprise 100 trials delivered on a VI-schedule, analyzed in blocks of 10 trials each, and three types of trial outcome are possible: escape failure, i.e. subject does not perform the response of crossing the barrier that is instrumental in terminating CS and US during CS+US exposure; escape response, i.e. subject crosses the barrier during CS+US exposure and thereby terminates these stimuli; avoidance response, i.e. subject crosses the barrier during CS exposure and thereby terminates this stimulus and avoids the US. Given that three possible outcomes are possible at each trial, then LH in the US-PE subjects can in principle be observed and defined, relative to US-NPE controls, as increased escape failures per se, increased escape failures and decreased avoidance responses, or decreased avoidance responses per se. There are several alternative psychological explanations for rat LH behavior and the extent to which a transient intervening state is involved that has commonalities with any of the depression symptoms, vis. face validity, is debatable (Gray, 1987). Nonetheless, the escape deficit induced by US-PE is attenuated ("prevented") by daily pre-treatment with antidepressant for at least 1 week prior to US-preexposure (a broad spectrum of antidepressants act in this manner; Gray, 1987; Weiss and Kilts, 1998; Chen et al„ 2001). The robust escape deficit does not persist beyond 24 h, an interval too short for the study of antidepressant-mediated reversal of ("recovery from") the escape deficit, with even the most rapid antidepressant reversal effects in rats requiring 1 week of administration (Sherman et al., 1982). A modification of the test, aimed at providing a model for the study of antidepressant reversal (rather than prevention) of escape deficit, involves initial US-preexposure followed by exposure to a small number of foot shocks every 48 h across a 3 week period followed by testing of escape behavior. This chronic stress normally acts to sustain the escape deficit induced by the acute US-PE session, and antidepressant treatment beginning after the US-preexposure and maintained up to the escape test is reported to reverse this stress-sustained escape deficit (Gambarana et al., 2001).

The forced swim test (FST) in the rat is, like the LH test, a two-stage paradigm. Unlike the LH test, however, exposure to the first stage (pre-test) induces a behavioral change in the second stage (test) that is reversible by acute antidepressant treatment between pre-test and test. The FST has therefore gained the status of a rat model of depression to the extent that it is sensitive to and therefore of screening potential for antidepressant compounds, vis. predictive validity (Porsolt et al., 1977; Willner, 1997; Cryan et al., 2002). During the pre-test phase, rats are placed in a cylinder of water from which they cannot escape by swimming or climbing, and in which they cannot make foot contact with the cylinder bottom whilst maintaining the head above the water surface. The pre-test usually lasts 15min and initially the rat's behavior is oriented to escape in the form of swimming or climbing that are both commensurate with a high amount of locomotion. As the pre-test progresses, then the rat gradually increases its immobility, with amount of immobility (manual measure) or total distance moved (automated measure) gradually increasing and decreasing, respectively, across time (Hedou et al., 2001; Cryan et al., 2002). The test phase of the FST is typically conducted 24 hours later and is of 5-min duration. The untreated or vehicle-treated control rat rapidly acquires the high immobility that characterised its performance at the end of the pre-test; however, this is prevented by antidepressant treatment immediately following the pretest and prior to the test. In fact, selective serotonin reuptake inhibitors (SSRIs) and selective noradrenaline reuptake inhibitors (SNRIs) both exert mobility-enhancing but different effects: SSRIs increase swimming at test and SNRIs increase climbing (reviewed in Cryan et al., 2002). With regard to transient intervening states that can account for the observed behavior and psychopharmacology of the FST, it has been proposed that the rapid development of high immobility at the test phase reflects either behavioral despair in terms of learning that escape-directed behavior is not reinforced in this environment, or disengagement from actively coping with a stressful situation and switching to passive behavior (Lucki, 1997). These intervening variables correspond to some extent to the diagnostic symptoms of depression (APA, 1994). However, it is arguable that in the existing battery of rat behavioral tests for depression, those aimed at the symptom of anhedonia possess the highest face validity; some of these tests have been used to investigate the long-term effects of postnatal manipulations, and these are described next.

At the level of stimulus representation and processing, depression constitutes increased emphasis of negative or aversive stimuli and decreased emphasis of positive or appetitive stimuli. In the rat, appetitive stimuli that have been studied in terms of developing tests of anhedonia include sweet-tasting nutrients (typically sucrose), estrous females for males and pups for parous females, with the major focus on nutrients. In the sucrose consumption test, rats are presented with a drinking bottle containing the typically highly palatable sucrose solution (1-2%) and their consumption of this is measured across one or more days (Willner, 1997). Reduced sucrose consumption is taken as a measure of anhedonia, an interpretation typically validated with the independent demonstration that the same rats do not exhibit reduced consumption when given water (if the decreased consumption of fluid exhibited with respect to sucrose was non-specific then a variable such as decreased thirst rather than anhedonia, would constitute the more parsimonious explanation). In the sucrose preference test, rats are presented in the home cage with two drinking bottles, one containing a sucrose solution and the other water, with both bottles containing a volume in excess of what the rats will consume and with relative positioning of the bottles reversed between tests to control for side preferences and to prevent development thereof. Sucrose consumption is calculated as a proportion of total liquid consumption across days to yield a sucrose preference index (value of 0.5 represents absence of a sucrose-versus-water preference) (Willner, 1997). Further to a comparison of sucrose consumption relative to water consumption, it is possible to analyze the effects on consumption of changes in the concentration, and therefore presumably of changes in the expected versus actual reward value, of the sucrose solution. The contrast effect test measures the behavioral response to replacement of a familiar reward with a novel one of a greater or lesser intrinsic reward value (Flaherty 1982). Relative to anhedonia, both a blunted response to a positive contrast (concentration of sucrose solution 2 > solution 1, with both solutions on the upward slope of the sucrose concentration-consumption curve) and a blunted response to a negative contrast could be analogous to the blunted responsiveness to appetitive stimuli in human depression (Matthews et al., 1996).

In each of the above sucrose consumption tasks, there is very little distinction between the rat's appetitive and consummatory behavior relative to the reinforcer: the rat approaches the spout of the drinking bottle and licking it results in drinking the sucrose solution. Using operant schedules of conditioned reinforcement it is possible to divorce appetitive behavior regulated by incentive motivation for the sucrose from sucrose consumption, i.e. to divorce consumption of reward from the motivation to obtain reward, and perhaps thereby to increase the relevance of the rat's behavior to those aspects of depressive-anhedonic human behavior that incorporate disturbed appetitive behavior, e.g. social withdrawal. The progressive ratio (PR) schedule is the reinforcement schedule that has been most used in this context, and constitutes a systematic increase in the response requirement for each successive reinforcement (Hodos, 1961; Richardson and Roberts, 1996). With the PR schedule, it is possible to identify the maximum response requirement that an individual will perform to support reinforcement delivery under the specific conditions of the test. Often a maximum response latency is built into the test parameters, and the response ratio achieved before the subject first exceeds this latency is used to define its "break point". In depression research, the PR schedule has typically been used with sucrose as the reinforcer and with the rats under food and water deprivation (e.g. (Barr and Phillips, 1999)), such that treatment effects on PR schedule performance could be mediated by differences in hunger/thirst as well as motivation to obtain reward.

Long-term effects of pup-dam manipulations: depression-model research

The major research focus to date in terms of long-term depression-like effects of rat postnatal manipulations has been on the sucrose-based tests for anhedonia. In one of the earliest studies in this field, Crnic and colleagues (Crnic et al., 1981) investigated the effects of 12 h MS at 30°C on PNDs 1-21 in terms of sucrose consumption and sucrose preference in adulthood. The comparison groups were AFR and a group in which dams but not pups were handled daily. The initial sucrose concentration was 1 % and this was increased daily, with subjects tested each day. There was no effect of MS on either sucrose consumption, expressed relative to metabolically active body tissue (body weight0'75), or on sucrose preference (Crnic et al., 1981). Using the same 3 h MS as that studied intensively in terms of its anxiogenic effects (see above), Shalev and Kafkafi (2002) also tested for MS effects in adulthood in the sucrose preference test, using 0.5, 1.0 and 3.0% sucrose in a repeated measures design, relative to NH and EH. There was no effect of MS relative to NH or EH, and also no effect of NH relative to EH. In the same study, it was also demonstrated that MS did not affect PR schedule performance for sucrose solution in non-deprived rats, across a concentration range of 0.3-10%, relative to NH and EH, with these two groups also again exhibiting similar performance (Shalev and Kafkafi, 2002). One further study worthy of note here compared 3h MS rats with AFR and EH rats in a preference test of sucrose (2.5%) versus ethanol (8%) + sucrose (2.5%) (Huot et al., 2001). The MS subjects consumed markedly more etha-nol+sucrose solution and slightly less sucrose solution than both AFR and EH rats, which were very comparable in their respective consumptions of both solutions. Unfortunately, it is impossible to interpret these results in terms of sucrose preference because of the absence of a sucrose-versus-water test. Nonetheless, the findings are worthy of inclusion here because in the same study it was found that MS sucrose consumption increased and ethanol+sucrose consumption decreased in response to chronic treatment with the SSRI paroxetine (21 days, 7 mg/kg per day, via osmotic minipump; Huot et al., 2001).

Using a 6-h MS on 10 days spaced randomly between PNDs 5-20, Matthews et al. (1996) investigated the effects of this MS on adulthood sucrose preference and sucrose contrast effects relative to EH adults. The sucrose preference test was conducted with solutions of 1, 15 and 34%, and two separate cohorts, namely with or without 23 h food and water deprivation. There was no postnatal treatment effect on sucrose preference at any of the sucrose concentrations, in either condition.

Overall, the fasted MS and EH rats exhibited a higher sucrose preference than did the sated rats (Matthews et al., 1996). To investigate contrast effects, the positive contrast test comprised sessions of 5, 5 and 15% on three successive days, and the negative contrast test comprised sessions of 15, 15 and 2.1% on three successive days. MS subjects, relative to EH, exhibited an attenuated increase in sucrose consumption in response to the 5%-to-15% positive contrast and also an attenuated decrease in sucrose consumption in response to the 15%-to-2.1% negative contrast. This latter treatment elfect appeared to be primarily due to the average decreased consumption of the 15% sucrose by the MS rats (Matthews et al., 1996). Therefore, studies in several laboratories have identified that MS does not lead to altered sucrose consumption or an altered sucrose preference, and furthermore it has been demonstrated by Shalev and Kafkafi (2002) that MS does not impact on motivation to obtain reward, as measured using the PR schedule of reinforcement. However, reactivity to changes in sucrose concentration, in both directions, was reduced in MS relative to EH adults, suggesting that perception of differences in and therefore perhaps the overall hedonic value of reward, is chronically blunted by MS relative to EH (Matthews et al., 1996).

In a previous section we described our findings with an ED postnatal manipulation in terms of its long-term effects on anxiety-like behavior and stress reactivity, and specifically that this ED was without effect relative to AFR and EH but, and to a very similar extent to AFR and EH, yielded adult offspring that were less anxious and stress reactive than NH adults (Pryce et al., 2001a, 2003; Pryce and Feldon, 2003). This ED involved providing pups with some exogenous warmth above the typical rodent room ambient temperature of 21-22°C, specifically 30° C. As is typical for altricial mammals, during the first two weeks of life rat pups exhibit limited homeo-thermic capacity such that their body temperature is dependent on that of their surroundings. Below 25° C, body and skin temperatures decrease as does respiration; sleep is reduced and ultrasonic vocalization is increased (Blumberg and Sokoloff, 1998). Several sources of evidence highlight the importance of ambient temperature in determining the acute and chronic effects of postnatal manipulations. In one of the earliest studies, Denenberg et al, (1967) demonstrated that 3 min EH in the form of placement of 2-day-old pups singly in a novel environment leads to an acute increase in CORT levels if performed at colony-room temperature but not at nest temperature. In a study based on a 6h ED, ED at 20°C resulted in pups losing a similar amount of weight during such cold-ED to that exhibited by warm-ED (nest-temperature) pups; nonetheless, growth and development of cold-ED pups was substantially retarded relative to the warm-ED pups and the EH comparison group (Zimmerberg and Shartrand, 1992). In our exploratory studies aimed at attempting to identify an ED that induces long-term depression-like effects, we decided to perform ED at colony room temperature and therefore within the extreme thermal stress zone (Blumberg and Sokoloff, 1998).

We perform this cold-ED (hereafter referred to as ED) from lOOOh to 1400 h and therefore during the dark active phase, in rats maintained on a reversed light-dark cycle (lights off 0700-1900) (Fig. 1). Pups are placed on sawdust in individual compartments (Pryce et al, 2001b; Pryce et al, 2002). The comparison groups we have used in studies to-date are NH and EH. The NH procedure involves the complete absence of human contact with dam or pups across PNDs 1-14; furthermore, only the experimenter has access to the NH colony room, for food and water provision and, in some studies, for observation of maternal behavior. In the case of EH, the same apparatus is used as for ED but with the isolation lasting 15min only (Pryce et al, 2001b). ED pups are of lower body weight than NH pups with EH pups intermediate in body weight. ED pups also receive more licking and nursing throughout the day than do NH pups (Riiedi-Bettschen et al, in press a). At PND 21, subjects are weaned and caged in isosexual groups for study in adulthood. We have conducted the ED manipulation with three different strains of laboratory rat, primarily with the outbred Wistar strain but also the inbred and histocompatible Fischer and Lewis strains that are derived from the Wistar strain. Given that all of our (warm) ED studies to-date have been conducted with Wistar rats, this was the obvious strain in which to study ED effects. We selected the Fischer rat because of the general evidence that this strain is stress hyper-responsive relative to outbred strains, at least in adulthood; to the best of our knowledge there are no existing comparative studies of the sensitivity of Fischer rats to postnatal manipulations. The Fischer rat has been studied in parallel with the Lewis rat in terms of postnatal manipulation effects: the two strains react very differently to ED, as they have already been demonstrated to do in other environmental situations (e.g. restraint stress reactivity (Stohr et al, 2000), aversive conditioning (Pryce et al, 1999)). However, the Fischer-Lewis comparison is beyond the scope of the present chapter, where we describe some of our major findings with Wistar and Fischer male rats in terms of ED effects in depression-related tests.

In the FST, we have demonstrated that ED leads to adult male Wistar rats that exhibit reduced mobility at the test stage specifically, relative to NH. The FST was performed as described above, with a water depth of 35 cm and a 15-min pre-test and 5-min test. At the onset of the pre-test the NH and ED subjects demonstrated a high level of activity, measured automatically as total locomotion per min (see Hedou et al, 2001) followed by a significant and monotonic decrease as the session progressed. There p>0.40

Pre-Test

Test

Fig. 2. Effects of early deprivation conducted at ambient room temperature (cold-ED) on swimming activity in the forced swimming test, using non-handling (NH) as the comparison group. Subjects were adult male Wistar rats, JV=8 per treatment, and the data presented are the meanis.e.m. distance swum per minute measured using a validated automated procedure (Hedou et al, 2001). The pre-test conducted on day 1 had a duration of 15 min and there was no treatment effect on swimming distance; the test conducted on day 2 had a duration of 5 min and there was a significant main effect of treatment that reflected the overall reduction in distance swum by the cold-ED rats (Riiedi-Bettschen et al, in press b).

Pre-Test

Test

Fig. 2. Effects of early deprivation conducted at ambient room temperature (cold-ED) on swimming activity in the forced swimming test, using non-handling (NH) as the comparison group. Subjects were adult male Wistar rats, JV=8 per treatment, and the data presented are the meanis.e.m. distance swum per minute measured using a validated automated procedure (Hedou et al, 2001). The pre-test conducted on day 1 had a duration of 15 min and there was no treatment effect on swimming distance; the test conducted on day 2 had a duration of 5 min and there was a significant main effect of treatment that reflected the overall reduction in distance swum by the cold-ED rats (Riiedi-Bettschen et al, in press b).

was no significant main effect of postnatal treatment and no time x treatment interaction (Fig. 2). At test, 24 h later, there was a significant main effect of postnatal treatment, reflecting the decreased locomotion of ED subjects across the 5-min test relative to NH (Fig. 2). Therefore, in the FST, designed to screen antidepressants in terms of attenuation of the decrease in activity (increase in immobility) at the test phase, ED resulted in decreased activity and specifically at the test phase. Invoking the interpretations given for reduced activity during the test phase relative to the pre-test phase of the FST, this increased sensitivity in ED adults could reflect a trait of relatively high susceptibility to develop a state of behavioral despair and/or motor retardation in a stressful environment.

The same Wistar male subjects were investigated in terms of the effects of ED on PR schedule operant behavior for 7% sucrose solution under conditions of minimal water deprivation. Under conditions of 23 h water deprivation, rats were shaped and then trained on a fixed ratio-1 schedule (FR-1) to bar press in order to obtain 0.1 ml sucrose solution. Subjects were then shifted to a

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