The aim of this study was evaluation of the astrocytes number in different subfields of rat's Hippocampus after spatial learning with usage of Morris Water Maze technique and working memory method. In this study, between 2005-2006 years in Pasteur institute of Iran-Tehran and histological department of Gorgan University with usage of Morris Water Maze and working memory technique, we used 14 male albino wistar rats. Seventh rats were in control group and 7 rats in working memory group. After histological preparation, the slides were stained with PTAH staining for showing the Astrocytes. Present results showed significant difference in astrocytes number in CA1, CA2 and CA3 areas of hippocampus between control and reference memory group. The number of astrocytes is increased in working memory group. Then we divided the hippocampus to three parts: Anterior, middle and posterior and with compare of different area (CA1, CA2 and CA3) of hippocampus, we found that the differences between Anterior-middle and Middle-Posterior of CA1 and CA2 area of hippocampus were significant, whereas the difference between Anterior-Posterior parts was not significant in CA1 and CA2 areas. In CA3 area, the difference between Anterior-Middle and Anterior-Posterior parts was significant, whereas the difference between middle and posterior parts was not significant. We concluded that the number of astrocytes increased due to spatial learning and working memory technique. © 2008 Science Publications

Abbas, P.

Ahmad, H.

Mehrdad, J.

Naser, N.

Yousef, S.

American Journal of Animal and Veterinary Sciences

English

Golestan University of Medical Sciences Repository

                 American Journal of Animal and Veterinary Sciences 3 (1): 28-31, 2008ISSN 1557-4555© 2008 Science Publications                                                                                                                                                                                                                                                                  Working Memory Learning Method and Astrocytes Number in DifferentSubfields of Rat's Hippocampus1Jahanshahi Mehrdad, 2Sadeghi Yousef, 2Hosseini Ahmad, 3Naghdi Naser and 2Piriaie Abbas 1Department of Anatomy, Gorgan University of Medical Sciences, Gorgan, Iran 2Cellular and Molecular Research Center,  Shahid Beheshti University of Medical Sciences, Tehran, Iran 3Department of Physiology, Institute of Pasteur, Tehran, Iran  Abstract: The aim of this study was evaluation of the astrocytes number in different subfields of rat'sHippocampus after spatial learning with usage of Morris Water Maze technique and working memorymethod. In this study, between 2005-2006 years in Pasteur institute of Iran-Tehran and histologicaldepartment of Gorgan University with usage of Morris Water Maze and working memory technique,we used 14 male albino wistar rats. Seventh rats were in control group and 7 rats in working memorygroup. After histological preparation, the slides were stained with PTAH staining for showing theAstrocytes. Present results showed significant difference in astrocytes number in CA1, CA2 and CA3areas of hippocampus between control and reference memory group. The number of astrocytes isincreased in working memory group. Then we divided the hippocampus to three parts: Anterior,middle and posterior and with compare of different area (CA1, CA2 and CA3) of hippocampus, wefound that the differences between Anterior-middle and Middle-Posterior of CA1 and CA2 area ofhippocampus were significant, whereas the difference between Anterior-Posterior parts was notsignificant in CA1 and CA2 areas. In CA3 area, the difference between Anterior-Middle and Anterior-Posterior parts was significant, whereas the difference between middle and posterior parts was notsignificant. We concluded that the number of astrocytes increased due to spatial learning and workingmemory technique.Key words: Hippocampus, astrocytes, working memory, spatial learningINTRODUCTION   The hippocampal formation plays an important role in memory and learning. The Morris Water Maze (MWM) is a test of spatial learning for rodents that relies on distal cues to navigate from start locations around the perimeter of an open swimming arena to locate a submerged escape platform. Spatial learning is assessed across repeated trials and reference memory is determined by preference for the platform area when the platform is absent[1].  Learning needs some instrument for information storage and information maintenances mechanisms resemble to memory. In the other hand, the memory always accompany with learning[2].  The hippocampal formation consists of the subiculum, the hippocampus and the dentate gyrus[3]. The hippocampus can be subdivided into three subfields: The CA1, CA2 and CA3 areas[4].   The principal cells in hippocampus are pyramidal neurons and in the dentate gyrus are the granule cells. Apart from principal neurons, the hippocampal formation contains different types of glial cells[5].  Astrocytes, strategically positioned between the capillaries and neurons, are thought to play a role in neuronal energy metabolism [6,7]. Glycogen is localized in the brain almost exclusively in astrocytes[8,9].  Astrocytes and microglia play critical roles in CNS response to and recovery from injury[10,11,12]. Astrocytes have been shown to play important roles in nutrient supply, waste removal and axonal guidance. More recent work reveals that astrocytes play a more active role in neuronal activity, including regulating ion flux current, energy production, neurotransmitter release and synaptogenesis. The latter includes the activity of glial cell apposition to synapses and the regulation of synapse elimination by ensheatment[ known as glia swelling[12,13]  . Corresponding Author: Dr. Mehrdad Jahanshahi, Department of Anatomy, Gorgan University of Medical Sciences,  P.O. Box 49175-553, Gorgan, Iran Tel: 0098-171-4421651 28             29                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           American J. Animal & Vet. Sci., 3 (1): 28-31, 2008Recently, the researches showed that theastrocytes, not only receive the information fromenvironment, but also send the signals to neurons [14].According to our hypothesis, the number of astrocytesafter spatial learning must be increased, becauseastrocytes have a closely relationship to synapses. Theknowledge of changes in astrocytes number can help usthat know what amount these cells involve in memory,therefore the aim of this study was evaluation of theastrocytes number in different subfields of rat'sHippocampus after spatial learning with usage ofMorris Water Maze technique and working memorymethod.MATERIALS AND METHODDuring 2005-2006, 14 male albinos Wistar rats(200-250 g.) obtained from Pasteur institute of Iranwere used. Rats were housed in large plastic cage, foodand water were available. Animals were maintainedunder standard conditions and 12/12 h light/dark cyclewith lights on at 7.00 am. After accommodation withenvironment, we divided rats to Control and Workingmemory groups. We used of Morris Water Mazetechnique for spatial learning in working memorygroup.MWM Testing: The rats were placed in a circularplastic pool (diameter, 120 cm) with white inside walls,located in a normally equipped laboratory room,uniformly lighted by four neon lamps (40 Weach)suspended from the ceiling (3 m). No care was taken toenhance (or, vice versa, to impoverish) extra-mazecues, which were held in constant spatial relationsthroughout the experiments.The pool was filled with water (24°C), which was50 cm deep and made opaque by the addition of 2 L ofmilk. A white, steel escape platform (10 cm indiameter) was placed in the middle of one cardinalquadrant (NW, NE, SW, SE) 30 cm from the side walls,it was either submerged 2 cm below or elevated 2 cmabove the water level. Each rat was released gently intothe water always from the same cardinal wall point (S)facing the center of the pool. The animal was allowedto swim around to find the platform. Blocks of fourtrials were presented to each rat, two blocks of trials perday[15].Working memory testing in the water maze: On eachtrial, the rats were placed into the water at one of thefour cardinal points of the compass (N, E, S, W), whichvaried from trial to trial in a quasi-random order. Therats had to swim until they climbed onto the escapeplatform. If they failed to locate the platform within60 sec, they were guided there. The rats were allowedto stay on the platform for 20 sec.Two day after the reference memory pre-trainingphase, training on the working memory version of thenavigation task started. Only two trials per day weregiven until performance stabilized in the first trial(acquisition), the animal had to find the platform in anew position. The rats were allowed to stay there for20 sec before they were returned to the home cage. onthe second trial (retrieval ), which wasadministrated 75 min later, the platform was in itsprevious position but the animals was started from adifferent place to the preceding trial[16,17].After learning examinations, animals weredecapitated after ether anesthesia and the brains wereremoved for histological verification, at first the brainsfixed in formaldehyde 10% and two week laterimpregnated with paraffin wax. After histologicalprocessing, slices of 7 µm coronally (anterior toposterior of hippocampus) were produced with Leitzrotary microtome (One of 10 sections was selected forstaining and morphometeric measurements). Forastrocytes staining, we used PTAH (PhosphotanguesticAcid Haematoxylin) staining[18] because it is the specialstaining method for astrocyte cells and their processes.In this method the astrocytes appeared blue and theneurons appeared pink.Morphometric measurement was carried out usingon Olympus DP 12 digital camera and BX51microscope. We selected a field (75000 µm2) withinthe pyramidal layer of hippocampal subfield CA2.Randomly selected, non-overlapping photographs usinga ×40 objective lens were taken from the designatedareas. Images were saved by the Bioreporter programand further processed using the Adobe Photoshop 6.0program (Adobe System Inc., San Jose, CA, USA).For cell counts, photographs at a magnification of×40 (objective lens) were taken throughout thelongitudinal axis of the hippocampal subfields andfurther processed as described above. All of theastrocytes shown on this field counted and then themean and SD of astrocytes number were measured.Statistical analysis: Data was expressed as mean ±SDdifferences among areas were statistically evaluatedusing the one-way analysis of variance (ANOVA).Probabilities of p<0.05 were considered significant.RESULTS AND DISCUSSIONThere are significant differences in astrocytesnumber between control and working memory group inall subfields of hippocampus.             American J. Animal & Vet. Sci., 3 (1): 28-31, 2008   Table 1: The  mean  of  astrocytes  number  in  different  areas  of       hippocampus in control and working memory groups Compartment  Mean  Area (µm2)    Std. Error mean  CA1 c  49.00  75000  1.303  CA1 w  198.31  75000  2.980  CA2 c  48.82  75000  1.901  CA2 w  182.95  75000  3.769  CA3 c  41.95  75000  0.846  CA3 w  164.30  75000  2.300   c = Control      w = Working            Table 2: The mean of astrocytes number in different parts (Anterior, Middle   and Posterior)  of  control  and  working  memory groups Compartment  Mean  Area (µm2)    Std. Error mean  CA1 wa  195.67  75000  5.21 CA1 wm  208.92  75000  6.05 CA1 wp  190.53  75000  3.25  CA2 wa  192.15  75000  7.19 CA2 wm  165.18  75000  5.74 CA2 wp  190.00  75000  5.68  CA3 wa  155.28  75000  3.89 CA3 wm  169.60  75000  4.49 CA3 wp 167.00  75000  2.99       C = Control and w = Working                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          The mean and SD of the number of Astrocytes inshown area of hippocampus (per 75000 µm2) isdepicted in Table 1. In control group, the mean ofastrocytes number in CA1 and CA2 was similar andmore than CA3 subfield[19]. In working memory group,the number of astrocytes in CA1 and CA2 was similarand it was more than CA3 subfield.Then we divided the hippocampus to three parts:Anterior, Middle and Posterior one-thirds, according oftheir functional differences[20], the mean and SD of thenumber of Astrocytes in different parts (per 75000 µm2)is shown in Table 2.The differences of astrocytes number between allareas (CA1, CA2, CA3) of hippocampus in control andworking memory groups were significant. In all area,the number of astrocytes increased. Also, after thediviation of hippocampus to three parts: Anterior,Middle and Posterior one-thirds, because theirfunctional differences[20], we showed that in CA1 areaof working memory group, the most number ofastrocytes was in middle one-third, in CA2 area it wasin anterior one-third and in CA3 area the most numberof astrocytes was in middle one-third of hippocampus.These results indicated that the working memorymethod of spatial learning can cause increasing ofastrocytes number in posterior two-third ofhippocampus, especially in CA3 area.Physiologically, present results similar andresemble too many researches that worked on thespatial learning[16,17,21,22,23].Many studies provided the relationship betweenexercise and neurogenesis in hippocampus andespecially in dentate gyrus[24]. Physical exerciseincreases the neurogenesis in hippocampus as well asgenetic factors[24,25]. One of the exercise and learningmethod is the Morris Water Maze, that it can increaseneurogenesis in dentate gyrus[26].Keuker in 2003 with usage of water mazetechnique and Reference and working method (similarto present research), said that: The working memory inaged animals significantly differs from the younganimals, whereas the reference memory doesn't changeswith ages[27].Rusakov in 1997 said that: Memory formation isbelieved to alter neural circuitry at the synaptic level.Although the hippocampus is known to play animportant role in spatial learning, no experimental dataexist on the synaptic correlates of this process at theultrastructural level. Analysis of synaptic spatialdistribution showed a training-associated increase in thefrequency of shorter distances (i.e., clustering) betweensynaptic active zones in CA1, but not dentate, thusindicating alterations in local neural circuitry. Thisfinding indicates subtle changes in synapticorganization in area CA1 of the hippocampus followinga learning experience, suggesting that spatial memoryformation in mammalian hippocampus may involvetopographical changes in local circuitry withoutsynapse formation de novo[28].In conclusion these researches almost are resembleto our study and showed that spatial learning canincrease the synaptic location and indirectly we showedthat the increase of synaptic number, can increase thenumber of astrocytes.REFERENCES1.	 Vorhees, C.V. and M.T. Williams, 2006. Morriswater maze: Procedures for assessing spatial andrelated forms of learning and memory. Nat.Protocols, 1: 848-858.2.	 Markowitsch, H.J., 1995. Anatomical Basis ofMemory Disorders. In: Cognitive Neurosci,Gazzaniga, M.S. (Ed.). Cambridge, MA: Mit press,pp: 665-679.3.	 Knowles, W.D., 1992. Normal anatomy andneurophysiology of hippocampal formation. J.Clin. Neurophysiol., 9(2): 252-263.4.	 Amaral, D.G. and M.P. Witter, 1995. 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Working memory learning method and astrocytes number in different subfields of rat's Hippocampus

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Working memory learning method and astrocytes number in different subfields of rat's Hippocampus

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