Introduction: Astrocytes play an important role in the hippocampus, probably in memory and learning. The aim of this study was to evaluate the number of astrocytes in the CA3 subfield of the rat hippocampus after spatial learning using the Morris water maze with reference and working memory methods. Methods: 45 male albino wistar rats were divided into three groups, with 15 rats in the control group and 15 rats in each of the other two groups. The two study groups of rats underwent spatial learning using the Morris water maze, with one group trained using the reference memory and the other, the working memory technique, respectively. After histological processing, the slides of the brains were stained with the phosphotanguestic acid haematoxylin staining method for detection of the astrocytes. Results: There was a significant difference in the number of astrocytes in the CA3 area between the control and reference memory groups. The difference between control and working memory groups was significant as well. Additionally, when comparing the two learning groups, we also found significant differences between them. Conclusion: The number of astrocytes increased due to spatial learning

Hosseini, A.

Jahanshahi, M.

Marjani, A.

Naghdi, N.

Sadeghi, Y.

English

Golestan University of Medical Sciences Repository

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   O r i g i n a l A r t i c l e Singapore Med J 2008; 49(5) : 388 The effect of spatial learning on the number of astrocytes in the CA3 subfield of the rat hippocampus Jahanshahi M, Sadeghi Y, Hosseini A, Naghdi N, Marjani A Department of Anatomy, Gorgan University of Medical Sciences, PO Box 49175-553, Gorgan, Iran Jahanshahi M, PhD Assistant Professor Department of Biochemistry Marjani A, PhD Assistant Professor Cellular and Molecular Research Center, Department of Anatomy, Shahid Beheshti University of Medical Sciences, Daneshjoo Avenue, Velenjak, Tehran, Iran Sadeghi Y, MD, PhD Professor Hosseini A, PhD Professor Department of Physiology, Pasteur Institute, Pasteur Street, Pasteur Square, Tehran, Iran Naghdi N, PhD, Associate Professor Correspondence to: Dr Mehrdad Jahanshahi Tel: (98) 171 442 1651Fax: (98) 171 442 Email: mejahanshahi @yahoo.com ABSTRACT Introduction: Astrocytes play an important role in the hippocampus, probably in memory and learning. The aim of this study was to evaluate the number of astrocytes in the CA3 subfield of the rat hippocampus after spatial learning using the Morris water maze with reference and working memory methods. Methods: 45 male albino wistar rats were divided into three groups, with 15 rats in the control group and 15 rats in each of the other two groups. The two study groups of rats underwent spatial learning using the Morris water maze, with one group trained using the reference memory and the other, the working memory technique, respectively. After histological processing, the slides of the brains were stained with the phosphotanguestic acid haematoxylin staining method for detection of the astrocytes. Results: There was a significant difference in the number of astrocytes in the CA3 area between the control and reference memory groups. The difference between control and working memory groups was significant as well. Additionally, when comparing the two learning groups, we also found significant differences between them. Conclusion: The number of astrocytes increased due to spatial learning. Keywords: astrocytes, CA3 subfield, hippocampus, Morris water maze technique, spatial learning Singapore Med J 2008;49(5):388-391 INTRoduCTIoN The Morris water maze (MWM) is a test of spatial learning for rodents, which 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) The theory of neuron­astrocyte interaction may be useful in provoking a reformulation of concepts of some disorders affecting the nervous system. The presence of glial signaling in the brain and interactions between neurons and glia, were previously demonstrated.(2) Recent studies have shown that astrocytes are necessary for the formation, function, and stability of central nervous system (CNS) synapses in vitro and have provided increasingly provocative evidence that astrocytes also actively participate in synaptic plasticity within the developing brain.(3) Astrocytes, strategically positioned between the capillaries and neurons, are thought to play a role in neuronal energy metabolism.(4,5) Glycogen is localised in the brain almost exclusively in astrocytes.(6,7) Astrocytes and microglia play critical roles in CNS response to, and recovery from, injury.(8-10) 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 ensheathment (known as glia swelling).(10,11) The number of astrocytes in spatial learning has rarely been documented. The aim of this study was to evaluate the number of astrocytes in the CA3 subfield of the rat hippocampus after spatial learning by using the MWM with the reference and working memory learning methods. MeTHodS Between 2005 and 2006, 45 male albino Wistar rats (weighing 200–250 g) were obtained from Pasteur Institute of Iran, Tehran, Iran. Rats were housed in large plastic cages, and food and water were made available. Animals were maintained under standard conditions, with 12-hour light/dark cycles with lights switched on at 7.00 a.m. After adaptation to the environment, we divided the rats into control, reference and working memory groups. We used the MWM for spatial learning in the reference and working memory groups. For 1289                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Singapore Med J 2008; 49(5) : 389 MWM testing, the rats were placed in a circular plastic pool (diameter 120 cm) with white inside walls, located in a normally-equipped laboratory room, uniformly lighted by four neon lamps (40 W each) suspended from the ceiling (3 m). No modification was made to enhance or impoverish extra-maze cues, which were held in constant spatial relations throughout the experiments. The pool was filled with water (24°C), which was 50 cm deep and made opaque by the addition of 2 L of milk. Awhite steel escape platform (10 cm in diameter) was placed in the middle of one cardinal quadrant (NW, NE, SW, SE) 30 cm from the side walls; it was either submerged 2 cm below or elevated 2 cm above the water level. The animal was allowed to swim around to find the platform. Blocks of four trials were presented to each rat, two blocks of trials per day.(12) In each trial of the reference memory test group, the rats were placed into the water at one of the four cardinal points of the compass (N, E, S, W), which varied from trial to trial in a quasi-random order. The rats had to swim until they climbed onto the escape platform. If they failed to locate the platform within 60 seconds, they were guided there. The rats were allowed to stay on the platform for 20 seconds. After the final trial, the rats were towel dried and placed in a holding cage under a heating lamp before they were returned to the home cage. The route of the rats was recorded by an infra-red digital camera, and the route and time of each trial were recorded with computer aid. In the working memory test group, training on the working memory version of the navigation task started two days after the reference memory pre-training phase. Only two trials per day were given until performance stabilised in the first trial (acquisition), where the animal had to find the platform in a new position. The rats were allowed to stay there for 20 seconds before they were returned to the home cage. On the second trial (retrieval), which was administrated 75 minutes later, the platform was in its previous position but the animals was started from a different place to the preceding trial.(13,14) After the learning examinations, animals were decapitated after ether anaesthesia and the brains were removed for histological assessment. At first, the brains were fixed in 10% formaldehyde and two weeks later, they were impregnated with paraffin wax. After histological processing, 7-µm thick coronal slices (serial section of anterior to posterior hippocampus) were produced with a Leitz rotary microtome (HM 325, Microm International GmbH, Walldorf, Germany). One of ten sections were selected for staining; therefore, we had about 40 slides for morphometeric measurement). For astrocyte staining, we used phosphotanguestic acid haematoxylin (PTAH) staining(15) because it is the special staining method for Fig. 1 Photomicrograph shows the astrocytes (PTAH stain, ×100). astrocyte cells and their processes. In this method the astrocytes appeared blue and the neurons appeared pink (Fig. 1). Morphometric measurement were carried out using on Olympus DP 12 digital camera and BX 51 microscope (Olympus Optical, Tokyo, Japan). We selected a field (75,000 µm2) within the pyramidal layer of the hippocampus subfield CA3. Randomly-selected, non-overlapping photographs using a 40× objective lens were taken from the designated areas. Images were saved by the Bioreporter programme and further processed using the Adobe Photoshop 6.0 programme (Adobe System Inc, San Jose, CA, USA). For cell counts, photographs at a magnification of × 40 (objective lens) were taken throughout the longitudinal axis of the hippocampus CA3 subfield and further processed as described above. All of the astrocytes shown on this field were counted, and then the mean and standard deviation (SD) of astrocytes number were measured. Data was expressed as mean ± SD. Differences between areas were statistically evaluated using the one-way analysis of variance (ANOVA). p-value < 0.05 was considered significant. ReSulTS The mean, SD and standard error of the mean (SEM) of the number of astrocytes in the CA3 area of the hippocampus (per 75,000 µm2) in the control, reference and working memory study groups are depicted in Table I. There were significant differences in the number of astrocytes between                                                                                                                                                                                                                                                                                                                                          Singapore Med J 2008; 49(5) : 390     Table I. The mean, Sd and SeM of the number of astrocytes in the CA3 area of the hippocampus in the three groups.  Group  Mean astrocyte no.  Area (µm2)  SD SEM  Control  41.95   75,000  11.22 0.846  Reference memory  116.6  75,000  31.14 2.34  Working memory  164.3  75,000  30.51 2.3 2a Fig. 2 Photomicrographs show CA3 subfield of the hippo-campus in the (a) control group; (b) reference group; and (c) working group. Yellow asterisks indicate the astrocytes (PTAH stain, × 40). 2b the control and reference memory groups, between the control and working memory groups and also between the reference and working memory groups. The number of astrocytes in the working memory group was more than the reference memory group, and those in the reference memory group were more than those in the control group (Fig. 2). dISCuSSIoN The differences in the astrocyte numbers among all groups were significant. These results indicated that the reference and working memory methods of spatial learning can cause an increase of astrocyte number in the CA3 subfield of the hippocampus. Physiologically, our results are similar to and resemble those of many researchers who have worked on spatial learning.(13-18) Many studies have provided the relationship between exercise and neurogenesis in the hippocampus, and especially in the dentate gyrus.(19) Physical exercise increases the neurogenesis in the hippocampus, apart from genetic factors.(19,20) One of the exercise and learning methods, which can increase neurogenesis in dentate gyrus is the MWM.(21) Keuker et alused the MWM technique and the reference and working methods (similar to our research), and reported that the working memory in aged animals differed significantly from that in young animals, while the reference memory did not change with age.(22) 2c Rusakov et al found that memory formation is believed to alter neural circuitry at the synaptic level. Although the hippocampus is known to play an important role in spatial learning, no experimental data exist on the synaptic correlations of this process at the ultrastructural level. Analysis of synaptic spatial distribution showed a training-associated increase in the frequency of shorter distances (i.e. clustering) between synaptic active zones in the CA1, but not the dentate, thus indicating alterations in local neural circuitry. This finding indicates subtle changes in the synaptic organisation in area CA1 of the hippocampus following a learning experience, suggesting that spatial memory formation in mammalian hippocampus may involve topographical changes in local circuitry without synapse formation de novo.(23) In conclusion, according to our hypothesis, the number of astrocytes is increased in the CA3 subfield after learning, because they are the third structure of synapses.(24) The knowledge of changes in astrocyte                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   Singapore Med J 2008; 49(5) : 391 numbers can help us study the extent of how these cells are involved in memory. Our study corroborated with previous research, which showed that spatial learning can increase the synaptic location and indirectly showed that the increase of synaptic numbers can also increase the number of astrocytes. ReFeReNCeS 1.	 Vorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 2006; 1:848-58. 2.	 Antanitus DS. A theory of cortical neuron-astrocyte interaction. Neuroscientist 1998; 4:154-9. 3.	 Ullian EM, Christopherson KS, Barres BA. Role for glia in synaptogenesis. Glia 2004; 47:209-16. 4.	 Pellerin L, Magistretti PJ. Food for thought: challenging the dogmas. J Cereb Blood Flow Metab 2003; 23:1282-6. 5.	 Forsyth R, Fray A, Boutelle M, et al. A role for astrocytes in glucose delivery to neurons? Dev Neurosci 1996; 18:360-70. 6.	 Gruetter R. Glycogen: The forgotten cerebral energy store. J Neurosci Res 2003; 74:179-83. 7.	 Tsacopoulos M, Magistretti PJ. Metabolic coupling between glia and neurons. J Neurosci 1996; 16:877-85. 8.	 Rabcheusky AG. Influences of activated microglia/brain macrophages on spinal cord injury and regeneration. In: Streit WJ, ed. Microglia in the Regeneration and Degenerating Cerebral Nervous System. New York: Springer-Verlag 2002: 209-26. 9.	 Bechmann I, Nitsch R. Astrocytes and microglial cells incorporate degenerating fibers following entorhinal lesion: a light, confocal, and electron microscopical study using a phagocytosis-dependent labeling technique. Glia 1997; 20:145-54. 10. Teter B, Ashford JW. Neuroplasticity in Alzheimer’s disease. J Neurosci Res 2002; 70:402-37. 11. Laming PR, Kimelberg H, Robinson S, et al. Neuronal-glial interactions and behaviour. Neurosci Biobehav Rev 2000; 24:295-340. 12. Leggio MG, Molinari M, Neri P, et al. Representation of actions in rats: The role of cerebellum in learning spatial performances by observation. Proc Natl Acad Sci U S A 2000; 97: 2320-5. 13. Naghdi N, Asadollahi A. Genomic and nongenomic effects of intrahippocampal microinjection of testosterone on long-term memory in male adult rats. Behav Brain Res. 2004; 153:1-6. 14. Sarihi A, Motamedi F, Naghdi N, Rashidy-Pour A. Lidocaine reversible inactivation of the median raphe nucleus has no effect on reference memory but enhances working memory versions of the Morris water maze task. Behav Brain Res 2000; 11:1-9. 15. Bancroft JB, Stevens A. Theory and practice of histological techniques. Edinburgh: Churchill Livinsgstone, 1990: 360-1. 16. Redish AD, Touretzky D. The role of the hippocampus in solving the morris water maze. Neural Comput 1998; 10:73-111. 17. Isgor C, Sengelaub DR. Prenatal gonadal steroids affect adult spatial behavior, CA1 and CA3 pyramidal cell morphology in rats. Horm Behav 1998; 34:183-98. 18. Bronders R, Brandy SY, Yehuda S. The use of the MWM in the study of memory and learning. J Neurosci 1989; 48:29-62. 19. Van Praag H. Christic BR. Sejnowski TJ, Gage FH. Runing enhances neurogenesis, learning, and long term potentiation in mice. Proc Natl Acad Sci U S A 1999; 96:13427-31. 20. MadsenTM,Yeh DD,Valentine GW, Duman RS. Electroconvulsive seizure treatment increases cell proliferation in rat frontal cortex. Neuropsychopharmacology 2005; 30:27-34. 21. Rosenzweig ES, Redish AD, McNaughton BL, Barnes CA. Hippocampal map realignment and spatial learning. Nat Neurosci 2003; 6:609-15. 22. Keuker JI, de Biurrun G, Luiten PG, Fuchs E. Preservation of hippocampal neuron numbers and hippocampal subfield volumes in behaviorally characterized aged tree shrews. J Comp Neurol2004; 468:509-17. 23. Rusakov DA, Davies HA, Harrison E, et al. Ultrastractural synaptic correlates of spatial learning in rat hippocampus. Neuroscience 1997; 80:69-77. 24. Ventura	 R, Harris KM. Three-dimensional relationships between hippocampal synapses and astrocytes. J Neurosci 1999; 19:6897-906. 

The effect of spatial learning on the number of astrocytes in the CA3 subfield of the rat hippocampus

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The effect of spatial learning on the number of astrocytes in the CA3 subfield of the rat hippocampus

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