S.04.02 Neurobiological and behavioral effects of chronic mild stress in glucocorticoid receptor-impaired transgenic mice

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5.04. Hot topics in preelin~cal neuropsyef~opBarmaeology

could there%re contribute to psychotogenesis. DARFP-32 is a key regulator of kinase/phosphatase signaling cascades modulated by dopaminergic, serotonergic and glutamatergic neurotransmission. I-fern we implicate a common signaling pathway in mediating the behavioral effects of dopaminergic agonists (such as D-amphetamine), serotonet'gic agonists (such as LSD) and glutamatergic antagonists (such as PCP). In this pathway DARPP32 is phosphorylated at Thr34 and Set130 or dephosphorylated at Thr75, in a pattern predicted to cause a synergistic inhibition of protein phosphatase-1. The psychotomimetics also increased the phosphorylation state of Ser1`33-CREB and Serg-GSK-3fO. To clarify the role of Thr34-, Thr75-, and Ser130-DARPP-`32 in the actions of psychotomimetics, we utilized gene knock-in methodology to generate three lines of mutant mice in which Thr34-, Thr75-, or Serl.30-DARFP-.32 was mutated to alanine to prevent phosphorylation. Using these phoaphomutant mice, it was found that the psychotomimetic-induced increases of P-Ser133CREB and P-Serg-OSK-3f(? were significantly reduced in frontal cortex and striatum from T34A and 8130A mutants, but unaltered in T75A mutants. These studies demonstrate that DARPP-.32 signaling pathway~ play an integral role in the effects of all three psychotomimetics on phosphorylation of OSK-3f(3 and CREB. To further characterize the involvement of DARPP-32 in mediating the actions of the psychotomimetics, c-fos mRNA was studied in w i l d t ~ e and phosphomutant mice. C-fos, an immediate early gene stimulated by Ser9-OSK-3f(3 [via an indirect disinhibitory mechanism], and Serl3.3-CRE]; [via direct positive regulation], is a measure of post-~naptic biochemical activation. The ability of all the psychotomimetics to increase c-fos mRNA in cingulate cortex and in the paraventricular region of striatum was reduced in T34A- and Sl`30A-mntants. To further test the functional importance of this pathway, the effects of D-amphetamine, LSD, and PeP on sensorimotor gating and repetitive movements were compared in wildtype, in DARPP-`32 knockout (KO), DARPP32 phosphomutant mice. Prepulse inhibition (PPI) of the startle reflex is, both in humans and in rodents, the most appropriate test for sensorimotor gating currently available. Similarly, repetitive behaviors are often seen in schizophrenics, and, drugs that elicit psychosis in humans also increase repetitive movements in rodents. D-amphetamine, LSD, and PCP disrupted PPI and caused an increase in repetitive movements in w i l d t ~ e mice, but not in DARFP-32 KO mice or in T34A- or S130A-DARFP-32 mice. A general pattern cruet'yes from this studhz Three pathways, involving regulation of the state of phosphorylation of Thr34-, Thr75and Ser130-DARPP-32, inhibit PP-1, leading to an increased state of phosphorylation of various PP-1 substrates. Future studies may identify the precise PP-1 substrates involved in the behavioral effects of psychotomimetic drugs.

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Neurobiological and behavioral effects of chronic mild stress in glucocorticoid receptor-impaired transgenic mice

N. Froger 1, E. Palazzo 1, C. Boni 1, N. Hanoun 1, C. Joubert 2, I. Dutriez-Casteloot 3, N. ];arden 4, C. CohenSalmon 2, M. Hamon 1, L. Lanfumey 1. I~SERM U 28~, Neurop~yctcop[zarmacology, Facu#g de Mgdecine ~tigSalpStri~re, 91 Bd de t'H@ital, 75535 Pari~ cedex 13, France; 2 CNi~hi-U3/IR 7593, personnalit~s et conduites adaptations, Facu#d de Mddecine Pitid-galpgtri~re, 91 Bd de l 'H@ital, 75635 Paris oedex 13, France," 3Neuroendoorinologie du d~oeloppemer~, UPRES-EA 2701, Un~oersit~ de Lille l, 59555 Villeneuve d'Ascq, France; 4Laval University Hospital i~esearcf¢ Center and Dpt. of Anat. and Physiol., University Laoal, Ste Foy, Quebec GIV 4G2, Canada Vulnerability to major affective disorders, particularly depression that is associated with hyperactivity of the hypothalamic-pituitaryadrenal (HPA) axis and hypoactivity of the serotoninergic (5-HT) system, has been shown to involve both genetic and environmental factors. HPA axis activity is controlled by a feedback mechanism triggered mainly through the stimulation of glucocortiooid receptors (OR), and a reduced OR function has been proposed to contribute to ttPA axis alterations in depression. In addition, depression-associated deficit in ttPA axis regulation might be related to the decreased serotoninergic neurotransmission evidenced in depressed patients because OR expression is actually dependent on the central 5-I-IT tone. Transgenic mice (GR-i) bearing a transgene encoding a glucocorticoid receptor (OR) antisense RNA that down regulates OR expression specifically in the central nervous system, have been proposed as a genetic model of vulnerability to affective disorders [1]. We further assessed the relevance of this model by investigating the functional status of both the hypothalamic-pituitary-adrenal (HPA) axis and the serotoninevgic system in OR-i mice which had been subjected to chronic mild stress (CMS) in an attempt to trigger behavioral deficits responsive to antidepressant therapies [2]. Corticosterone levels were measured by radioimmunoassay, OR protein and mRNA expression were quantified using [31-I]oorticosterone binding and in situ hydridization, 5-HT1A autoreceptor functioning was estimated by in vitro electrophysiology and [35S]OTP-~'-S binding. Operant behaviour was analyzed in a previously described decision-making task [.3]. Statistical analyses were performed using a two-way ANOVA with stress and genotype as main factors, and followed, in case of significance, by unpaired two-tailed Student's t-test. OR-i mice showed no suppression of corticosterone secretion in response to dexamethasone administration, and exhibited a decrease in both OR specific binding (-50% in the hippocampus) and OR mRNA levels (-25% in the hippocampus and the dorsal raphe nucleus (DR.N) compared to wild-type (WT) mice. ARer exposure to CMS (for 4 weeks), hippocampal OR mRNA levels were significantly reduced (-32%) in WT but increased in GR-i mice (+83%). Under control non stress%l conditions, neither in v#ro electrophheiologioal recordings in brainstem slices nor [35S]GTP-,(-S binding measurements showed alterations in 5-t-IT1A autoreoeptor functioning in the DRN of GR-i mutants compared to WT mice. In contrast, clear-cut differences were revealed by stress since exposure to CMS produced a functional desensitization of DRN 5-NT1A autoreceptors in WT but not in OR-i mice. In addition, CMS was found to facilitate choice behaviour of WT but not OR-i mice in the decision-making task. These data emphasize the existence of profound deficits in the

S.04 Hot topics in predin~ca[ neuropsyel~op~a~acology adaptive responses to chronic stress in GR-i mutants compared to paired wild-type mice and confirm that the GR-i mouse is a relevant genetic model to assess how stress can contribute to trigger neurobiological and behavioral alterations associated with depression.

References [1] Holsboer, R and Barden, iN. Antidepressants and hypothalamiopituitary-adrenoeortical regulation. Endoor Rev, 1996. 17: p. 187-205. [2] Wilin% i~, Validity, rdiability and utility of the &tonic mild strms model of depression: a 10-year review and evaluation. Psychopharmaoolo~ (B~I), 1997. 134: p. 319-329. [3] Pardon, M.C., et al., Influence of a &ronioultramild stressprocedure on decision-making in mice. J Psychiatry Neurosci, 2000.25: p. 167-177.

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Leptin treatment in activity-based anorexia

I. Hillebrand 1, M. Kas 2, A. Scheurink 2, G, van Dijk 2, A. Adan 2,

izRudolf Magnus I~sgitute of Neuro~cience, Depart~nent of P#armacology and Anatomy, Utrecht, The Net#erlands; 2The Netherlands Activity-based anorexia (At~A) is an animal model for anorexia nervosa, which models hypophagia and hyperactivity. In ABA, rats are food restricted (one hour food access per day) and have access to a running wheel. The combination of these two factors leads to a fast and severebody weight loss; rats eat even less than is possible in one hour and running wheel activity is increased (Routtenberg and Kuznesof, 1967, Kas et al, 2003). The reason for this paradoxical behavior is not clear. Here we hypothesize that ABA is caused by decreased leptin signaling as a consequence of starvation. Indeed, it was shown before that leptin treatment of starved rats decreased hyperactivity in a semi-starvation induced hyperactivity model (SIN) (Exner et al, 2000). From this study it was proposed that leptin treatment in acute anorexia could possibly reduce hyperactivity in anorexia nervosa patients, which could be beneficial for further treatment. The SIH model used by Exner differs at several points from our ABA model, the most important being amount and time of food intake, making the At)A model more severe than SIH. In the stu@ described here, we further investigated the role of leptin in the ABA model. Female rats were allowed free wheel running under ad libitum feeding conditions. After 10 days icy cannulae were placed into the lateral ventricle,,which were connected to osmotic minipumps infusing leptin (4 fYg/day) or saline for 5 days. Immediately after surgery, food was restricted and was only provided during the first hour of the dark phase. After 5 days of exposure to the model, rats were decapitated, trunk blood was collected and brains were isolated. Gent expression of (an)orexigenic arcuate neuropeptides was analyzed using in situ hybridization. Data were analyzed using t-test, p values<0.05 were considered significantly different. Rats exposed to the ABA model displayed a fast decrease of adipose tissue mass (p=0.000) and plasma leptin (p=0.001) levels compared to palrfed rats without running wheels. ICV inNsion of leptin in the ABA model resulted in practically absence of running wheel activity in the dark phase (p=0.001) and light phase (p=0.001). As well leptin treatment further decreased food intake (p=0.000) in ABA rats. POMC expression levels in the arcuate nucleus were less decreased (p=0.001), while NPY and AgRP expression levels tended to be less increased (p=0.328, p=0.169) compared to saline treated ABA rats after 5 days of exposure to the model. We conclude that leptin treatment did not improve physical condition of rats exposed to the A1;A model,

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but in fact, decreased survival in the At)A model. The beneficial effect of decreased energy expenditure as in running activity is counterbalanced by hypophagia. We hypothesize that leptin also influences other aspects of energy expenditure, e.g. metabolic rate, which might contribute to a decreased survival in the ABA model. From this experiment it is therefore concluded that leptin trea~nent might be considered as pharmacotherapy in anorexia, nervosa patients to suppress hyperactivity, but only if sufficient food intake is secured. References

P,_outtenbetg, A~ Kuznesof, A.W., 1967. Self-starvation of rats living in activity wheels on a restricted feeding soheclule. J. Comp. Physiol. Psy&ol. 64, 3, 414-21. Kas, M.J., van Dijlg G, S&eurink, A.J, Adan, P,.A., 2003. Agouti-related protein prevents self-starvation. Mol. Psychiatry. 8, 2, 235-40. gxne~ C, Hebebrand J, Rems&midt. H, Wewetzer C, Ziegler A, He~pettz S~ Sohweiget U, Blum W• Preibis& G, Hddmaier G~ Klingenspor M., 2000. Leptin suppresses semi-starvation induced hyperactivity in rats: implications for anorexia nervosa. Mol. Psy&iatr> 5, & 476-81.

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Temporal and anatomical characterization of stress-related changes on BDNF expression in t~e rat central nervous system

R, Molteni I , F, Fumagalli I , M, Roceri I, E Bedogni I , E Cirulli2,

G, R a c ~ n i 1, M,A, Riva I , 1 Center of NeuropBarmacolo~,

U~ioer~t¢7 of Milan, Department of P~armacological Sciencex and Center of Excellence for Neuroc&generatioe Disorders, Milan, Italy; 2Section of Bd~aoioral Neurosoieno< Department of Cell Biology and Neurosciences, Istituto Euper~ore di 5fanim ', P~ale Regina Elena 299, 001~i Rome, Italy Several evidence indicates that the origin of psychiatric disease,s, included schizophrenia and depression, is probably related to a complex interaction between genetic and environmental factors. Among the environmental factors, stress plays a crucial role by influencing the progression of the disease as well as the outcome of the pharmacological therapy [1]. Although it is well established that stressful experiences impact neuronal function throughout the interaction of multiple components, the neurobiological mechanisrm underlying this effect are still unclear. One of the molecular mediator of the central effect of stress is Brain-derived neurotrophic factor (BDNF), a neurotrophin strongly involved in the regulation of synaptic function and plasticity [2]. Although it is well accepted that BDNF expression is altered following stress in experimental animal models, it should be considered that not only the type,, but also the timing and the duration of the stressful experience, seem to be crucial for the effect on brain function. Accordingly, the purpose of our study was to evaluate the effect of different stress paradigms on B D N F expression in rat hippocampus and prefrontal cortex, two brain regions related to cognitive impairment observed in neuropsychiatric disorders. Sprague-Dawley adult male rats were exposed to acute and repeated stress paradigms; for each experiment rats were randornly assigned to two groups: Control (animals left undisturbed in their home cages) and Stress (animals exposed to the stressful stimuli). The rats nndergoing acute stress were immobilized for 2 hours in restraint containers and killed by decapitation at different time points after the end of the stress session (0, 3, 20 h). The rats exposed to repeated stress were placed 5 rain daily for 2 weeks in a bucket filled with cold water deep enough so that the animal's paws did not touch the bottom and killed 1 hour after