Terminology. Terminology. Terminology. Terminology. Terminology. Bromodeoxyuridine

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1 Kateřina Náměstková, Zuzana Šimonová, Eva Syková Behavioural Brain Research Bromodeoxyuridine : Doublecortin : DCX Glial Fibrillary Acidic Protein : GFAP Trace eye blink conditioning 1 Volume 163 : pp , , a compound that is structurally similar to thymidine, one of the building blocks of DNA. gets incorporated into DNA instead of thymidine. Deoxyribose sugar Phosphate A : Adenine T : Thymine G : Guanine 3 C : Cytosine Bromodeoxyuridine 4 Thymidine Daughter DNA Strand DCX, a component of the microtubule cytoskeleton, is one such molecule which are involved specifically in neuronal path-finding and movement. Parental DNA Strand DCX is associated with migration of neuronal precursors during development of the nervous system. Therefore, measuring the cells with indicates how many cells are replicating. Therefore, measuring the cells with DCX indicates newborn neurons. 5 6

2 Neurons Astrocyte Glial fibrillary acidic protein (GFAP) is an intermediate filament protein. In the central nervous system, GFAP is expressed in astrocytes and but not in other glial cells. Corneal Air Puff Elicits Eyeblink Response Corneal Air Puff Given with Tone Tone Given Alone Elicits Eyeblink Response Microglial cell Therefore, measuring the cells with GFAP indicates astrocyte. 7 (tone) CS (air puff) US Time Trace 8 on off on off Hippocampus is an important structure for spatial learning and memory. Inner blade hilus Outer blade New neurons are continuously born in the granular cell layer of the hippocampus throughout life in a number mammalian species, including humans. (Altman and Das, 1965; van Praag H et al., 1999; Brown JP et al., 2003) Newborn cells can be detected by labeling with [ 3 H] thymidine or. Neuronal phenotype can be determined by using various neuronal markers such as doublecortin (DCX). 9 Copyright 2001 by Allyn & Bacon 10 Several conditions influence the rate of proliferation and the survival and function of the newborn cells During aging Exposure to stress Antidepressant Tx Physical activity Enrich environment Some task of hippocampusdependent learning Suppress granule cell proliferation (Gould E and Tanapat P, 1999) Promote proliferation and survival (Malberg JE and Duman RS, 2003; Stressful experiences Duman RS et al., 2001; Malberg JE et al., 2000) Corticosterone levels Suppress proliferation (Gould E and Tanapat P, 1999; Duman RS et al., 2001) Basal levels of corticosterone may influence proliferation throughout the entire lifespan.

3 In the study of Gould E and Tanapat P (1999) In the first 2-postnatal weeks old rats Chronic application of several classes of antidepressants (including selective serotonin re-uptake inhibitor : fluoxetine) Low levels of corticosterone that are accompanied by a stress hyporesponsive period a high rate of granular cell proliferation Age Basal levels of corticosterone 13 Rate of proliferation Expression of the trophic factor BDNF Rate of granular cell proliferation (Malberg JE et al., 2000) Physical activity also up-regulates the transcription of BDNF gene and to be additive to antidepressant treatment. (Neeper SA et al., ; Russo-Neustadt AA et al., 2000) Hippocampus-dependent learning as short as 4 days in the Morris water maze (MWM) Acquisition of 800 trials of a trace eye blink conditioning Animal exposed to an enriched environment for 8 weeks Voluntary wheel running for 30 days Number of surviving cells (Gould E et al., 1999) Performance in the hippocampus-dependent spatial learning in the Morris water maze (Nilsson M et al., 1999; van Praag H et al., 1999) Presumably due to the contribution of newborn cells to hippocampus formation Objectives of this study Physical activity Morris water maze task = Stressful component To investigate whether spatial learning in the Morris water maze (MWM) will affect the proliferation of granule cells in the hippocampus of adult rats. Affect the proliferation of granular cells in the hippocampus of adult rats. To investigate the effects of antidepressant treatment with fluoxetine on the change in proliferative response to the MWM task

Animals 3-month-old male Wistar rats (320-340 g) Housed under standard conditions, 2-3 per cage Maintained on a 12 h light and dark cycle Study were approved by the ethical committee (Institute of

4 Animals 3-month-old male Wistar rats ( g) Housed under standard conditions, 2-3 per cage Maintained on a 12 h light and dark cycle Study were approved by the ethical committee (Institute of Experiment Medicine, Academy of Sciences of the Czech Republic) Rats were randomly assigned to 4 groups and then underwent 15 days of 4 different treatments. Control group (C) 1.5 ml saline (i.p.) Water maze group (WM) Saline MWM Fluoxetine group (F) 5 mg/kg Fluoxetine (i.p.) Fluoxetine treated and Water maze group (FWM) Fluoxetine MWM Experimental schedule Sacrificed Morris Water Maze 3-month-old rats i.p. injection - C group - WM group - F group Treatment 15 days - FWM group 8 hr 16 hr Day 1 Day 15 Day Tissue processing and immunohistochemistry Rats swim in a pool of water to find a hidden platform. Rats were exposed to 4 trial / day with an intertrial interval of 60 sec. For each trial, rat was placed in a random quadrant of the tank. Provided 60 sec to locate and mount the platform. Latency to reach the platform, was recorded. 22 camera Morris Water Maze Light Day Invisible platform Invisible platform platform Invisible platform 23 Cold water (22 ± 2 C) 24

Tissue processing and immunohistochemistry 4% paraformaldehyde Add Primary Antibody Mouse monoclonal directed against 5-bromo-2-deoxyuridine Doublecortin Goat anti-doublecortin C-18 GFAP

visualized by DAB Doublecortin 25 Vectastain Elite ABC kit 26 Add Primary Antibody Mouse monoclonal directed against 5-bromo-2-deoxyuridine Doublecortin Goat anti-doublecortin C-18 GFAP

sections Count cells every 6 th section Separately count in GCL and in hilus DCX cells count in 3 sections from each animal Doublecortin Alexa Flour 488-conjugated donkey anti-goat IgG 27 28 Number

5 Tissue processing and immunohistochemistry 4% paraformaldehyde Add Primary Antibody Mouse monoclonal directed against 5-bromo-2-deoxyuridine Doublecortin Goat anti-doublecortin C-18 GFAP Cy3-conjugated rabbit anti-rat GFAP 40 μm thick coronal sections Peroxidase immunohistochemistry Add Secondary Antibody After saturation sucrose Peroxidase-conjugated rabbit anti-mouse (goat) IgG, visualized by DAB Doublecortin 25 Vectastain Elite ABC kit 26 Add Primary Antibody Mouse monoclonal directed against 5-bromo-2-deoxyuridine Doublecortin Goat anti-doublecortin C-18 GFAP Cy3-conjugated rabbit anti-rat GFAP Images and cell counting Microscope (63x) Indirect immunofluorescence Alexa Flour 594 (488) -conjugated goat anti-mouse IgG Add Secondary Antibody Coronal brain sections Count cells every 6 th section Separately count in GCL and in hilus DCX cells count in 3 sections from each animal Doublecortin Alexa Flour 488-conjugated donkey anti-goat IgG Number of cells and DCX cells Statistical analyses Photomicrographs of a rat hippocampus stained with anti- (newborn cell) Number of cells and DCX cells expressed as mean ± S.E.M. / hippocampus in a slice Comparisons between groups using Student s t-test Statistical significance P

6 Photomicrographs of a rat hippocampus stained with anti-dcx (newborn neuron) From Photomicrographs Show GFAP processes forming a basket around only some of the cells in the cluster of cells. Survival of newborn cells is likely to be successful when I. Stimulating substances diffusing from the blood stream II. Impulses from the functional neuronal network Both are delivered through the astrocytic syncytium Sub-granular zone astrocytes are the primary precursors of the D cells that differentiate into neuron. Syková E et al. study (2002) Learning deficits in aged rats are related to the disorganization of astrocytic processes and the loss of extracellular matrix in the hippocampus. SGL astrocytes Dark cells (Doetsch F. Curr 35 Opin Genet Dev 2003; 13: ) 36

7 Effects of the treatments on the number of cells per hippocampus in one slice (mean ± S.E.M.) Immunohistochemical staining for DCX to determine the number of cells differentiating into new neurons born during approximately the last 14 days preceding sacrifice. The time period when newborn cells show this early neuronal marker. * p < ** p < 0.01 Effects of the treatments on the number of cell and DCX cells per hippocampus in one slice (mean ± S.E.M.) * p > 0.05 Learning curves of the 2 groups of animals that underwent the MWM learning task (WM and FWM) Suggest that fluoxetine treatment increases proliferation but not the differentiation and survival of newborn neurons. Supporting for this results In the work of Shors et al. (2002) Blocking neurogenesis with cytostatic Spatial learning performance in the water maze Trace eye blink and fear conditioning intact impaired Present study Shows that spatial learning acquisition trials in the MWM and fluoxetine treatment affect the rate of cell proliferation in the neurogenic region. Complex stimulation and learning in the MWM for 15 days Suggested that the acquisition of spatial memory in the water maze may be a task that requires only a small percentage of new neurons. 41 Down-regulation of granular cell proliferation 42

8 has been considered as a factor that positively influences the generation of new neurons in adulthood. Hippocampus-dependent learning of temporal or spatial events Present results contradict previous study In an experiment done by Lemaire et al. (2000) 5 days of spatial task learning in the MWM Rate of proliferation Survival of newborn cells There are important differences between the studies I. Length of exposure to the MWM task this study 15 days Lemaire et al. study 5 days Morris water maze task = Spatial learning Physical activity Stressful component II. injection after the training before the training The motivation for finding the location of the hidden platform is to escape from a life-threatening situation in a unpleasant environment of cold water The different results of the two studies suggest that Prolonged exposure to a stressful experience Benefit from proliferation factors (such as physical activity) Results in this study can be compared to van Praag et al. study van Praag et al. study (1999) Mice were divided into 2 groups and underwent 12 days of training (inject everyday) only to swimming water maze acquisition 47 48

9 Result in the van Praag et al. study (1999) Number of labeled cells was not different between two groups. Down-regulating effect of stress was probably masked by a normal rate of neurogenesis prior to the decline in the proliferative rate during the water maze training.? First question Further study In this study have not examined the survival of newborn cells for an extended period of time. It would be of interest to see if the combination of fluoxetine and learning in MWM would change the number of surviving cells 4 weeks after labeling Second question? Mechanism of action of stressful learning combined with fluoxetine on the rate of neurogenesis. This study Show the negative effect of long-term exposure to the MWM on the proliferation of new neurons. To be a potential substrate for new learning capacity 51 Speaker : Mr. Nattapon Sookprasert SIPS/M

Are Both Embryonic Migratory Pathways Preserved in the Adult Brain Cerebral Cortex?

Prague Medical Report / Vol. 107 (2006) No. 1, p. 71 80 71) Are Both Embryonic Migratory Pathways Preserved in the Adult Brain Cerebral Cortex? Šimonová Z., Dutt J. Department of Neuroscience of the Institute