Cognitive Function Test. NOR & spatial NOR task. Introduction. Novel Object Recognition (NOR) Estrogen Receptor (ER) in Brain
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1 Cognitive Function Test Human Rodent Sutcliffe JS, Marshall KM, Neill JC. Behavioral Brain Research 177(2007) Radial Arm maze Morris Water maze Siriporn Vongsaiyat 16 th Feb 2007 Novel Object Recognition (NOR) Test Novel Object Recognition (NOR) NOR & spatial NOR task NOR task Acquisition trial inter-trial interval retention trial spatial NOR task Acquisition trial inter-trial interval retention trial Longer Inter-trial interval = harder task Introduction Hypothalamic-pituitary-gonadal axis Estrogen Receptor (ER) in Brain HPA axis HPG axis Hypothalamic-pituitary-adrenal axis can influence learning & memory strategies and capacities Shughrue et al., 1997 Hippocampus play a significant role in working & spatial memory Hippocampus contains both ERα & ERβ Gonadal steroids have the ability to influence the structural properties of the brain regions that sub-serve learning memory Cerebral cortex Hippocampus Amygdala 1
2 Action of Gonadal Hormones Alter hippocampal electrophysiology Estrogen (E) excitatory Progesterone (P) & its metabolites inhibitory Androgen (A) less clear Hippocampal plasticity alters across the estrous cycle of the rat in response to cyclic changes in estrogen and progesterone Pro-estrous phase CA1 pyramidal cell spine synapses number & density Estrous phase synaptic number & density Estrogen Progestrone LH FSH Diestrous 2 Proestrous Estrous Diestrous 1 These cyclical changes could induce significant behavioral changes Estrogen Female foraging hypothesis Verbal fluency & motor skills (Kimura,1999) Clinical research to facilitate the storage of new memory to provide protection for neuronal circuits improve cognitive function in Alzheimer s disease (Amantea, 2005) Menopausal women (Birzniece, 2006) Schizophrenia (Cutter, 2003) Verbal skill & muscular coordination (Kimura,1994) Females should be better than males at remembering the identity of objects Frick et al.(1999) > in remembering the identity & location of objects Confounding factor = estrous cycle Walf et al.(2006) Positive effect of estrogen administration on object recognition in ovariectomised rats object recognition in pro-estrous stage > other stages The aim of this study To investigate the influence of gender and estrous cycle phase on working and spatial memory in the NOR paradigm Hypothesis Female will demonstrate variable performance in spatial and working memory task, which may be related to changes in the hippocampal structure in response to natural cyclic hormonal fluctuation 2
3 Subjects Methods Mature hooded Lister (hl) rats 373 ± 17 g 234 ± 17 g 5 rats/cage 12 h light:12 h dark cycle 21 ± 2 C 40 50% free access to food & water Experiments were approved by the University of Bradford Ethical Review Process Apparatus Experimental Procedure VDO Camera 40 cm 7 days Daily Handled 3 days 30 min Habituated Testing day 52 cm Re-habituated Acquisition 1h 2h 3h 30 min 4h 5h 24h 48h Inter-trial Interval (ITI) Retention 52 cm NOR Task Spatial NOR Task VDO Camera VDO Camera Acquisition Inter-trial trial Interval Retention Acquisition Inter-trial trial Interval Retention 3
4 Exp1: Effect of phase of estrous cycle 2 Week before starting experiment Vaginal smears 09:00-11:00 am. insertion of 0.2 ml of 0.9% (w/v) saline solution Exp2: Gender effects in the NOR task Re-habituated Acquisition 1h 2h 3h 30 min 4h 5h 24h 48h Inter-trial Interval (each n=6) Retention VDO Camera Proestrous (N=6) Estrous (N=6) Di-estrous (early) (N=6) Di-estrous (late) (N=6) NOR and Spatial NOR Task (ITI = 1h) 54 rats 54 rats Exp3: Gender effects in the spatial NOR task Re-habituated Acquisition 1h 2h 3h 30 min 4h 5h 24h 48h Inter-trial Interval (each n=6) Retention Discrimination Index (DI) DI = time spent exploring the novel/moved object - time spent exploring the Familiar/stationary object total time spent on all object exploration in the retention trial VDO Camera 54 rats 54 rats Value < 0; animals exploring the familiar/stationary object more than the novel/moved object Value > 0; animals exploring the novel/moved object more than the familiar/stationary object Statistical analysis Differences in object exploration for both trials for all group means were tested for significance by two-way ANOVA using objects and ITI as set variables. A paired t-test was used to evaluate any statistical difference between exploration for each object, in each trial, for each experimental condition. Result One-way ANOVA followed by post hoc Dunnett's t-test was used to determine discrimination index (DI) and LMA differences for each ITI, gender and stage of the oestrous cycle in each experiment. Statistical evaluations were made using SPSS for Windows v13.0 (significant when p <0.05). 4
5 Working memory Experiment 1: effect of phase of estrous cycle in the NOR and spatial NOR task * p < 0.05, ** p < 0.01 Spatial memory Working & Spatial memory * p < 0.05 In female rats Experiment 2: gender effect in NOR task * p < 0.05, ** p <
6 In male rats Table 2 ** p < 0.01, *** p < In female rats Experiment 3: gender effect in the spatial NOR task ** p < 0.01 In male rats Table 3 ** p <
7 Discussion Experiment 1: effect of phase of estrous cycle in the NOR and spatial NOR task Discuss: Exp.1 Discuss: Exp.1 Walf et al.(2006) significant effect of the estrous cycle during the NOR no estrous cycle effect were found following a 1 h ITI During pro-estrous compared to other stages of the cycle significant object discrimination beneficial effects of the observed E peak at pro-estrous In Exp.2 : can identify a novel object following an ITI of 3 h E during pro-estrous enables object recognition beyond 3 h Discuss: Exp.1 A significant discrimination for the moved object was only observed during estrous phase E & P object location memory E and/or P may play a role in object location memory Frye et al. (1995) Discuss: Exp.1 sex differences in water maze tasks were limited to differences between females in pro-estrous and males E has an inhibitory effect on spatial memory 7
8 Discuss: Exp.1 Estrous cycle differences may be difficult to measure Behavioral response are subject to hormonal influence via classical genomically mediated steroid receptor pathway significant lag time between Experiment 2: gender effect in NOR task serum level changes & target response Discuss: Exp2 Discuss: Exp2 Ghi et al.(1999) could not identify the novel object after 1 h could remember up to 90 min Frick et al.(1999) > in remembering the identity & location of objects Confounding factor = estrous cycle Levy (2005) & Neave (2005) > in object array tasks Men encode less information about specific visual features Experiment 3: gender effect in the spatial NOR task Sexual dimorphism in spatial working memory is seen in both human and other laboratory animal Duff SJ el al. (2001), Eals MS et al. (1997), Frye CA et al. (1995) poor spatial ability E during pro-estrous /prior to ovulation/when pharmacologically manipulated. Birzniece V el al. (2006), Cutter WJ (2003), Danial JM et al.(1997), Frye CA et al. (1995) 8
9 Morris Water Maze cue platform Sex difference may be attributed to effects of sex hormones during growth & development of animals and in adulthood (William et al, 1990) Circulating androgen, estrogen, progestagens will exert activational effects in steroid responsive neural circuits Estradiol Testosterone 1.3 meter perform better than Warren et al. (1997), Frye et al. (1995) During growth & development Steroids are responsible for alteration in regional cell (cell number, size, and packing density) (Fitch et al, 1998) Life pre-defined neuronal circuitry should have a significant impact on subsequent cognitive abilities due to fundamental gender differences or hormonal fluctuations during estrous cycle Locomotor activity (LMA) sexually dimorphic behaviours may affect performance in the NOR task LMA differences were ruled out by the lack of group differences both sexes & both trials Mechanism for gender differences Hippocampus spatial learning Structural changes throughout the estrous cycle show more dentritic spines at pro-estrous > estrous phase HPA fluctuations across the estrous cycle may be involved in spatial performance E & P influence the concentration of enzymes, receptors, transporter mechanisms associated with neurotransmission Adequate storage & processing of memories The structural features of the brains may rely on the gonadal hormone levels 9
10 Conclusion Recent work has shown clear sexual dimorphisms in a multitude of neurotransmitter systems NOR is a suitable, well-validated behavioural paradigm to assess changes in cognitive function The value of hormones as an adjunct therapy for the treatment of cognitive impairments associated with age, disease and physical injury remains an under-explored and exciting field Thank you 10
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