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Master internship at the Netherlands Institute for Neuroscience The influence of environment on neuronal development in mouse CA1 M. Rijnsburger Supervisor: G.J.A. Ramakers The influence of environment on neuronal development in mouse CA1 M. Rijnsburger Supervisor: G.J.A. Ramakers Abstract. The aim of this study was to investigate the normal development of dendrites in the mouse hippocampal area CA1 and the influence of an enriched environment on this development. A large increase in dendrite length and spine density with subsequent pruning was expected in both conditions. The enriched environment was hypothesized to either cause more synaptogenesis in the early stages of development or less pruning in the later stages of development. To investigate this, wild-type mice were assigned randomly to either a standard or an enriched environment starting at starting at 1 week and 3 weeks respectively. Mice were sacrificed at different ages and were distributed over 7 age groups (1, 2, 3, 4 ,6, 8 & 12 weeks postnatal). The brain were stained using a Golgi-Cox procedure. Pyramidal cells in the CA1 hippocampal area were randomly selected and digitally reconstructed manually. The expected increase in dendrite length during the first weeks of development occurred and there were indications for pruning between 4 and 8 weeks in both conditions. Surprisingly, a second significant increase occurred in dendrite length after pruning. Our observations suggest that enrichment led to longer dendrite lengths and that maximum length was reached later than in the standard condition. However, no significant differences between conditions were found. Although not conclusive, our observations suggest that the window of development is lengthened in the enriched condition. Keywords: Enrichment, enriched environment, synaptogenesis, pruning, hippocampus, CA1, window of development 1. Introduction In recent years, researchers have become increasingly interested in the influence of environment on brain development, especially synaptogenesis. An explosion of synapse formation in the human cortex occurs between the third trimester of pregnancy and the first three years of age and is characterized by an overshoot of synapses (Huttenlocher and Dabholkar, 1997). As shown in figure 1, different regions in the cortex show the same pattern of synaptogenesis, although with a different timing. For instance, the density of synapses in the visual cortex was found to be greatest at 8-12 months (Huttenlocher, 1982), while the prefrontal cortex appears to acquire synaptic junctions more slowly (Huttenlocher and Dabholkar, 1997). Fig. 1 Mean human synaptic density development in synapses/100 μm3. Open circles: visual cortex, filled circles: auditory cortex, x: prefrontal cortex. All regions show the same pattern of development, but with different timing. Adapted from Huttenlocher and Dabholkar, 1997. After an initial overshoot of synapses during synaptogenesis, a period of synapse elimination (pruning) follows, during which synaptic density decreases to about 60% of the maximum (figure 1, Huttenlocher and Dabholkar, 1997). In mice, there seems to be an initial overshoot of synaptogenesis at 8 weeks with subsequent pruning (10-15 %) between 8 and 12 weeks (Keuken and Ramakers, 2008). Pruning seems to be activity dependent; repeated neuronal activity in certain brain circuits results in stabilization of those circuits, circuits that are not activated will be eliminated during the pruning process. This had been shown by Huizen et al. (1985, 1987, see figure 2), where the bioelectrical activity of rat cerebral cortex cultures was blocked by tetrodotoxin (TTX). This prevented the large scale elimination of synapses. Furthermore, when the cultures grown in TTX-supplemented medium were switched to a normal medium, the synapse density remained at a high level, suggesting the presence of a critical period of pruning where after elimination no longer occurs. On the other hand, they showed that picrotoxin (PTX), a GABA receptor blocker, increased bioelectrical activity and thereby accelerated the synaptic network formation. Probably due to this acceleration, the pruning period occurred 1 week earlier than normal. It is suggested that the decrease in synapse density is necessary to optimize cognitive performance (Ramakers, 2005). It has been shown that several forms of mental retardation (MR) are associated with abnormalities in the morphology and density of synapses as well as a reduction in dendritic branching (Reviewed in Huttenlocher, 1991). In conclusion, pruning is thought to be necessary for optimal cognitive performance and activity appears to be very important in this „critical period‟ of synapse elimination. Figure 2. Number of synapses per 1000 µm3 tissue for control and TTX (left) and PTX (right) treated cultures for synapses on spines. Adapted from Huttenlocher et al., 1985 and 1987. There are many indications that the environment could influence various processes in the brain (e.g., Rosenzweig et al., 1962, Walsh, 1980, Van Praag et al., 2000). One example is given by the enriched environment paradigm, in which the environment is „enriched‟ in relation to standard housing conditions. Hebb was the first to propose the „enriched environment‟ as an experimental concept (1947, referred in van Praag et al., 2000). He reported that rats that he took home as pets showed behavioural improvements over their litter mates kept at the laboratory. Rosenzweig and colleagues (1962) introduced enriched environments as a testable scientific concept. The standard definition of an enriched environment is “a combination of complex inanimate and social stimulation”, because it has been shown that social grouping alone cannot account for cerebral effects of the enriched environment alone (Rosenzweig et al., 1987). In general, the „enriched‟ animals are kept in larger cages and in larger groups with the opportunity for social interaction. The environment is complex and is varied over the period of the experiments: tunnels, nesting material, toys and (often) food locations are changed frequently. In addition, animals are often given the opportunity for voluntary physical activity on running wheels, although it has been shown that physical activity does not influence neuronal changes (Faherty et al., 2003). An enriched environment affects different aspects of brain development. It has been shown that EE enhances the number of neurons (cell survival) in the dentate gyrus, increases brain size, enhances gliogenesis, neurite branching and synapse formation in the cortex, and increases the synapse-to-neuron ratio (Reviewed in van Praag et al., 2000). A larger number of dendritic spines (Rosenzweig, 1973) and density of non-perforated synapses have been found in the hippocampal CA1 after enrichment (Rampon et al., 2000). The effects of an enriched environment may be mediated by an increase in electric activity due to increased sensory stimulation. Studies in cultured neuronal networks have shown that depolarization of neurons induces a strong increase in total axon and dendrite length (Ramakers et al., 1998, Ramakers, 2005), mainly by stimulating branch formation. Although it is evident that enrichment enhances synaptic density, it is not clear how this occurs during development. One possibility is that EE influences the production of synapses in the CA1 in the early stages of development. Another hypothesis is that it influences the pruning process in the later phase of development. The last hypothesis reveals a paradox; as experiences cause more pruning and at the same time a better network how it is possible then, that EE results in more synapses, which also has a positive effect on the organism‟s behavior? At this moment it is not yet clear whether pruning actually occurs in CA1. The variety of animal species and their ages, brain region and cell type studied are all factors that contribute to the great diversity of reported results on spine plasticity (Segal et al., 2000). Schwartzkroin et al. (1982) showed some pruning in rabbits in the dendritic neuronal network between the first postnatal month and adulthood in CA1, but it is not yet known if this pruning process also occurs in mice. Ramakers and Keuken (2008, unpublished) found 10 to 15 % pruning in mice hippocampus between 8-12 weeks of development under normal environmental conditions in the basal dendritic tree but not in the apical dendritic tree. This was far less than the pruning of 40% in the human visual, auditory and prefrontal cortex (Huttenlocher and Dabholkar, 1997). On the other hand, Jose Overbeek, who investigated pruning in mice as part of her bachelor project in 2009, found no pruning in the last weeks (8-12) of development. The aim of the present study is to investigate the effect of EE on dendrites and spines in CA1 pyramidal neurons and how it is caused; by an increased production in the early stages of development, or by a reduction in pruning in the later phase. These questions were investigated by comparing the development of the morphology of hippocampal CA1 pyramidal cells of wilde-type (WT) mice in an enriched environment starting at the age of weaning with that of WT mice housed in standard conditions. Mouse brains were studied at 1, 2, 3, 4, 6, 8 and 12 weeks. The reason for the CA1 as area of interest is that pyramidal neurons in the CA1 area of the hippocampus show a reasonably uniform morphology. Moreover, this brain area has been functionally associated with learning and memory, and may therefore be a primary area of modification by the enriched environment because the objects in the EE were rearranged within the cage during the experiment (Faherty et al., 2003). Brains were stained using a Golgi-cox method followed by quantitative analysis of pyramidal neurons in the CA1. Apical and basal dendrites were studied separately because it has been shown that they differ considerable in their dendritic morphology (Alpar et al., 2006). The enriched environment should result in more branching and longer dendrites as expected from previous studies (Van Praag et al., 2000). A large increase in spine density was expected within the first weeks in both conditions, reaching a maximum at 8 weeks. After that, pruning was expected to occur, leading to an outcome of about 15-20% lower spine density than the maximum at 8 weeks. The enriched environment is expected to end up with a higher spine density compared to the standard condition. 2. Materials & Methods 2.1. Animals This study used 68 male wild-type (WT) mice. At 21 days the animals were weaned and thereafter housed individually in a standard or enriched environment. The animals were sacrificed at 1, 2, 3, 4, 6, 8 and 12 weeks (see table 1). Three mice were lost due to mistakes with the Golgi embedding procedure. One mouse died prematurely for unknown reasons. This resulted in a total of 64 mice. Five mice were moved to the EE from 42 until 84 days (called the „late enrichment‟ group). Table 1. Number of mice in each condition Age 1 2 3 Condition Standard 2 8 8 Enriched (EE) 4 6 5 8 2 5 12 4 5 12, late 5 7 1 8 Late enrichment 2.2. Environment At the age of weaning (21 days), the male pups were randomly assigned to either a standard or an enriched environment. The standard condition as well as the enriched environment cages consisted of nesting material and water and food continuously available. The enriched environment consisted of PVC pipes, sticks, marbles, pots, stones, steps, play houses, rubber gloves and ping pong balls. A change in position of materials was made every day. Once a week, the objects were replaced for other objects, with ascending complexity. 2.2. Tissue preparation The mice were anesthetized with carbon dioxide (CO2). After that, they were decapitated and the brains were immediately removed and placed in a Golgi-Cox solution for 21 days. The brains were then rinsed, dehydrated and embedded in 12% celloidine. Coronal sections of 200 µm were cut using a sledge microtome (Reichert Jung Polycut). The 4 sections then went through a staining procedure and were mounted on microscope slides with histomount. See Appendix A for the protocols used. 2.3. Data acquisition The CA1 area of the hippocampus was located in the sections using a Zeiss Axioplan 2 microscope. Pyramidal neurons were traced in sections posterior to the location where the corpus callosum is not anymore present in the sections. Pyramidal neurons were recognized by the presence of a basilar and an apical dendritic tree, with a single apical shaft. The neurons were recorded in a Z-stack of 99 planes with a distance of 1 µm using the 40x objective lens (Plan-apochromat, 0.95 N.A), an Evolution QEi FAST (monochrome, 12 bit) camera and Image-Pro Plus 5.0 software (Media Cybernetics, Silver Spring, USA). The condition that must be met were that the pyramidal cell had to be fully stained, not damaged or broken, that the cell was clearly distinguishable from the cells in the vicinity and that the cell was horizontally orientated within the section. To avoid cut-ends, only neurons in the middle of the sections were selected. Five or six neurons were recorded per mouse. A total of 325 neurons were recorded. Table 2. Number of recorded neurons in each condition Age condition standard enriched 1 13 2 51 3 45 4 28 29 6 6 12 8 26 41 12 12, late 30 44 The recorded neurons were drawn in 3D by manually tracing the dendrites through the Zstack and labeling different structures (cell body, apical/basal dendrites, spines) using Neurodraw, an ImagePro Plus 5.0 application (K. de Vos, J. van Heerikhuize and C. W. Pool). Neurodraw stores the coordinates of each drawing point and a label indicating the type of structure. The variables analyzed in this study were: cell body surface area, number of dendrites and total dendrite length per cell. The variables separated for the apical and basal side are: number of branch points, total dendrite length, intermediate segment length, terminal segment length and spine density. Statistics In this study there are two independent variables: environment and age. Therefore the data were statistically analyzed using two-way analysis of variance (ANOVA) using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Due to the non-normal distribution of the data, Mann-whitney U tests were used to test for differences between the conditions. The variables mentioned in the previous paragraph are the dependent variables. 3. Results Standard Enriched Soma area 3.1 Cell characteristics 210 The cell body (soma) surface area of mice 200 in the standard condition (ST mice) 190 showed a significant decrease of ± 33% 180 μm2 between 1 and 12 weeks (F(6, 192) = 170 8.838, P < 0.001. Fig. 2). The cell body of 160 EE mice also decreased significantly from 150 4 to 6 weeks (F(3, 122) = 5.273, P < 0.01) 140 but then increased again from 6 to 8 weeks * 130 120 (n.s.) to decrease significantly from 8 to 12 weeks there after (F(3, 122) = 5.273, P < 0.05). The cell body surface at 8 weeks 0 2 4 6 8 10 12 14 Age (weeks) Figure 2. Cell body surface per condition (Yellow = standard, pink = enriched), x-axis: Age in weeks. was significantly larger in EE mice than in Total Dendrite Length ST mice (U = 381, P = 0.05). The surface Standard Enriched 2100 of 12 weeks EE mice was also ±10% 1900 larger compared to ST mice (n.s.). 1700 The total dendrite length per cell showed a 0.001) in the ST mice between 1 and 4 μm 1500 significant increase (F(6, 192) = 7.080, P < 1300 1100 weeks (Fig. 3). After that a decrease 900 occurred between 4 and 8 weeks (F(6, 700 192) = 7.080, P < 0.05), whereafter the 500 0 2 4 8 to 12 weeks (F(6, 192) = 7.080, P < 6 8 10 Age (weeks) total dendrite length increased again from Figure 3. Total dendrite length per neuron per condition 0.05). Total dendrite length was longer in EE mice compared to ST mice at 6, 8 and 12 weeks and the maximum was reached at 6 weeks (n.s.). As in the ST mice, an increase was observed from 8 to 12 weeks in the EE mice (± 17% (n.s.)). Both conditions 12 14 thus showed a developmental effect and the dendrite length in the EE condition was larger compared to the standard Standard Enriched # basal dendrites 6 5,5 condition. The ST mice showed an increase of ± 30% (n.s.) from 1 to 6 weeks in the 5 4,5 number of basal dendrites per neuron (Fig. 4). The number of dendrites was larger in the standard condition at all age weeks compared with the EE mice (n.s). Furthermore the ST mice showed a 4 3,5 0 2 4 6 8 10 12 14 Age(weeks) Figure 4. Number of basal dendrites per neuron per condition decline of ± 17% (n.s.) between 6 and 8 weeks. The total dendrite length increased in both conditions between 8 and 12 weeks (± 4% in both conditions, n.s.), although not as large as the increase in the total dendrite length. In the next few paragraphs a closer look will be taken on the components that are responsible for changes in the total dendrite length. 3.2. Apical dendrites In apical dendrite length showed a large increase in the ST mice between 1 and 4 weeks (F(6, 192) = 6.617, P < 0.001, Fig. 4). After that a decrease of ±14% occurred between 4 and 8 weeks (n.s.), where after the total dendrite length increased again from 8 to 12 weeks (± 28%, n.s.). The apical dendrite length was longer in EE mice compared to ST mice at 4, 6, 8 and 12 weeks and the maximum length was reached later in the EE condition (n.s.). A significant increase was observed from 8 to 12 weeks in the EE mice (F(3, 122) = 3.396, P < 0.01). Both conditions thus showed a developmental effect and the dendrite length in the EE condition was larger compared with to standard condition. Standard Enriched Apical Dendrite Length Mean Apical TermSegmLenght Standard Enriched 65 1.200 60 55 1.000 50 µm µm 800 600 * 45 40 35 30 400 25 200 0 2 4 6 8 10 12 14 Age(weeks) Figure 5. Total apical dendrite length per neuron per condition 20 0 2 4 6 8 10 14 Figure 6. Length of apical terminal segments per neuron per condition The age and condition differences are most likely the result of differences in mean terminal segment length. In figure 6 can be observed that the mean terminal segment length showed a sort like increase as the dendrite length in ST mice between 1 and 6 weeks (F(6, 192) = 10.287, P < 0.01) and a significant decrease between 6 and 8 weeks (F(6, 192) = 10.287, P < 0.05). There after, the terminal segment length increased slightly (± 14%, n.s.). The mean apical terminal segment length was larger for the EE mice at 6, 8 and 12 weeks compared to the standard condition (only significant at 12 weeks: U = 462, P < 0.05). 12 Age(weeks) The number of apical branch points and inter-segment length did not contribute to the larger total apical dendrite length in both conditions and no significant differences between the two conditions were observed there (See fig. 7 and 8). # Apical Branch Points Standard Standard Mean Apical Intersegment Length Enriched Enriched 20 35 18 33 31 16 29 14 µm 27 12 25 23 10 21 19 8 17 15 6 0 2 4 6 8 10 12 Age(weeks) Figure 7. Number of apical branch points per neuron per condition 14 0 2 4 6 8 Age(weeks) 10 12 Figure 8. Length of apical inter-segments per neuron per condition 14 3.3 Basal dendrites The ST mice showed an increase from 1 to 4 weeks in the total basal dendrite length (F(6, 907) = 3.407, P < 0.001. See fig. 9). After that a decrease of ±18% occurred between 4 and 8 weeks (n.s.), where after the total dendrite length increased again slightly from 8 to 12 weeks (± 10%, n.s.). Just as with the apical dendrite length, the total basal dendrite length of the EE mice was longer compared to the ST mice for all ages (except week 4, all differences were n.s.). The maximum of basal dendrite length of the EE mice was reached about 2 weeks later then the ST mice, at 6 weeks. Basal Dendrite Length Standard Mean Basal TermSegmRootLength Enriched Enriched 80 210 75 190 70 170 65 60 150 µm µm 230 Standard 130 55 50 110 45 90 40 70 35 50 30 0 2 4 6 8 10 12 14 0 2 4 Age(weeks) Figure 9. Total basal dendrite length per neuron per condition 6 8 10 12 14 Age(weeks) Figure 10. Mean basal terminal segment root length The condition differences for the basal Mean TermSegmRootDistance dendrite length are most likely the result of Standard Enriched 65 length and mean terminal segment root distance (Fig. 10 and 11). Both showed differences between conditions, significant at 60 55 50 µm differences in mean terminal segment root 45 8 weeks (U = 8047, P < 0.05 and U = 7947, P 40 < 0.01 respectively). In figure 12, 13 and 14 35 can be observed that the number of basal 30 0 branch points, mean inter-segment length and mean terminal segment length did not 2 4 6 8 10 12 Age(weeks) Figure 11. Mean basal terminal segment root distance 14 contribute to the larger total basal dendrite length in EE mice and no significant differences between the two conditions were observed there. Nevertheless, a clear developmental trend is visible in the ST mice in the graphs. Standard # Basal Branch Points Mean Basal Intersegment Length Enriched 3 18 2,8 16 2,6 14 2,4 12 µm 2,2 2 Standard Enriched 10 8 1,8 6 1,6 4 1,4 2 1,2 0 1 0 2 4 6 8 Age(weeks) 10 12 0 14 Standard Enriched 50 µm 40 30 20 10 0 2 4 6 8 Age(weeks) 10 Figure 14. Mean basal terminal segment length per neuron per condition 12 6 8 10 12 14 Figure 13. Mean basal inter-segment length per neuron per condition 60 0 4 Age(weeks) Figure 12. Number of basal branch points per neuron per condition Mean Basal TermSegmLength 2 14 4. Discussion The aim of this study was to investigate the effect of environmental enrichment on dendrites and spines in CA1 pyramidal neurons. In addition, we investigated whether the found increase in synapse density due to enrichment (Van Praag et al., 2000) is caused by an increased production in the early stages of development, or by a reduction in pruning in the later phase. Therefore, we studied the time course of the development of connectivity by using mice in the range of 1 to 12 weeks. A significant decrease in soma surface was observed in de standard condition between 1 and 12 weeks. This is surprising because it has been shown in rats that the cell body surfaces of non-pyramidal cells in the visual cortex and relay neurons in the dorsal lateral geniculate nucleus increase during development (Parnavelas and Uylings, 1980 and Parnavelas et al., 1977). In the enriched environment, the soma surface was larger at 8 and 12 weeks. This is in line with a rat study of Diamond et al. (1975), where enrichment resulted in an increase in soma surface of neurons in the occipital cortex. In terms of total branch length, there was a significant increase in the first 4 weeks in the standard condition. After that, pruning seemed to occur between 6 and 8 weeks at both the apical and basal dendrite, although not significant. The non significance of the data could be due to the relatively small sample size. The results support earlier indications for the occurrence of pruning in the rabbit CA1 and the human cortex (Schwartzkroin, 1986, Huttenlocher and Dabholkar, 1997, respectively). The increased branch length of the apical dendrite is most likely the result of differences in mean terminal segment length. For the basal dendrite, the mean terminal segment root length and mean terminal segment root distance are most likely responsible for the changes. Surprisingly, we observed a second increase in dendrite length after the pruning period in both the standard and the enriched condition. This does not match previous literature and hypotheses about pruning (Keuken and Ramakers, 2008, Ramakers, 2005). When pruning is thought to be necessary to optimize cognitive performance (Ramakers, 2005), it is at least surprising that the dendrite length increases after this period. The relatively small sample size could be responsible for these remarkable findings. The enriched environment showed a larger dendrite length compared to the standard condition at 6, 8 and 12 weeks in both the apical and the basal dendrites. Furthermore, the number of branch points ended up equally in the EE and the standard condition in both the apical and the basal dendrites. This is not in line with earlier findings, where no increase in dendrite length was found, but an increase in dendrite branches occurred (Reviewed in Van Praag et al., 2000). The most intriguing finding, matching neither of our two hypotheses, is that dendrite length in the enriched mice seemed to reach a maximum later in time than in the standard mice. This indicates that the window of neuronal development is lengthened due to enrichment or, alternative, that the pruning is delayed. In a study by Shaw et al. (2006), it was shown that children with superior intelligence (IQ range 121–149) demonstrate a prolonged phase of prefrontal cortical increase, which yields to equally vigorous cortical thinning by early adolescence. They conclude that the formation and selective pruning of synapses, may contribute to the changes in cortical dimensions. The prolonged phase of cortical development in the superior intelligent might reflect an extended „critical‟ period for the development of high-level cognitive cortical circuits, according to the authors. The findings could perhaps be, indirectly, linked to enrichment studies of mice, including our research. The human research showed that the people with a higher IQ had a prolonged phase of development. In mice studies the enriched environment seems to make mice „smarter‟ (according to previous literature). Our research data indicate that the window of development in the mice CA1 is lenghtened due to enrichment. The possible link is then that IQ correlates with a prolonged network development, although this is speculative. To conclude, the findings suggest that there is an overshoot of dendrite length in the hippocampal CA1 region of WT mice. Indications for pruning were found. Furthermore, the data indicate that the enriched environment causes a higher dendrite length and that the window of development is lengthened due to enrichment, although an enrichment group before the age of weaning is lacking. Therefore, we don‟t know what the influence of EE is on the whole window of development. It would therefore be interesting for future research to use age groups younger than 3 weeks for the EE, to see what happens before those weeks. 6. Limitations of this study Due to time limitations, we were not able to look at the spine development, although this is an essential aspect of neuronal network formation. We recommend future studies to look at the development at the level of spines. Furthermore, the total N is small in certain age groups, especially at 6 weeks; there was only 1 mouse in the standard condition and there were 2 mice in the enriched condition. So we would also advise future studies to work with a larger N, which makes it possible to generalize the conclusions to a larger population of animal models. Furthermore, the unexpected increase between 8 and 12 weeks should be replicated with a larger N too see if the same effect occurs. References Alpar, A., Ueberham, U., Bruckner, M.K., Seeger, G., Arendt, T., & Gartner, U. (2006). Different dendrite and dendritic spin alterations in basal and apical arbors in mutant human amyloid precursor protein transgenic mice. Brain research, 1099, 189-198. Chugani, H.T. (1998). A critical period of brain development: studies of cerebral glucose utilization with PET. Preventive medicine, 27, 184-188. Diamond, M.C., Johnson, R.E., Ingham, C., Rosenzweig, M.R., & Bennett, E.L. (1975). Effects of differential experience on neuronal nuclear and perikarya dimensions in the rat cerebral cortex. Behavioral Biology, 15, 107-111. Faherty, C.J., Kerley, D., & Smeyne, R.J. (2003). A golgi-cox morphological analysis of neuronal changes induced by environmental enrichment. Developmental brain research, 141, 55-61. Van Galen, E.J.M., Ramakers, G.J.A. (2005). Rho proteins, mental retardation and the neurobiological basis of intelligence. Progress in Brain research, 147, 295-317. Van Huizen, F., Romijn, H.J., Habets, A.M.M.C., & Van den Hooff, P. (1987). Accelerated neural network formation in rat cerebral cortex cultures chronically disinhibited with picrotoxin. Experimental neurology, 97, 280-288. Van Huizen, F., Romijn, H.J., & Habets, A.M.M.C. (1985). Synaptogenesis in rat cerebral cortex cultures is affected during chronic blockade of spontaneous bioelectric activity by tetrodotoxin. Developmental Brain Research, 19, 67-80. F. VAN HUIZEN, H. J. ROMIJN and A. M. M. C. HABETS Huttenlocher, P.R., Dabholkar, A.S. (1997). Regional differences in synaptogenesis in human cerebral cortex. The Journal of Comparative Neurology, 387, 167-178. Huttenlocher, P.R. (1991). Dendritic and synaptic pathology in mental retardation. Pediatric Neurology, 7, 79-85. Image pro, version 5, Media Cybernetics, Silver Spring, USA Image-Pro Plus Neurodraw; developed by K. de Vos, J. van Heerikhuize and C. W. Pool, Netherlands Institute for Brain Research, Amsterdam, The Netherlands. Keuken, M.C., Ramakers, G.J.A. (2008). Creating a baseline for temporal and qualitative development in the CA1 hippocampal area in wild-type mice. Unpublished internship at the Netherlands Institute for Neuroscience. Parnavelas, J.G, Bradford, R., Mounty, E.J., & Lieberman, A.R. (1977). Postnatal growth of neuronal perikarya in the dorsal lateral geniculate nucleus of the rat. Neuroscience Letters, 5, 33-37. Parnavelas, J.G., & Uylings, H.B.M. (1980). The growth of non-pyramidal neurons in the visual cortext of the rat: a morphometric study. Brain Research, 193, 373-382. Van Praag, H., Kempermann, G., & Gage, F.H. (2000). Neural consequences of environmental enrichment. Neuroscience, 1, 191-198. Ramakers, G.J.A. (2005). Neuronal network formation in human cerebral cortex. Progress in brain research, 147, 3-14. Ramakers, G.J.A., Winter, J.T.M., Hoogland, M.B., Lequin, P., van Hulten., van Pelt, J., & Pool, C.W. (1998). Depolarization stimulates lamellipodia formation and axonal but not dendritic branching in cultured rat cerebral cortex neurons. Developmental brain research, 108, 205-216. Rampon, C., Tang, Y., Goodhouse, J., Shimizu, E., Kyin, M., & Tsien., J.Z. (2000). Enrichment induces structural changes and recovery from nonspatial memory deficits in CA1 NMDAR1-knockout mice. Nature neuroscience, 3(3), 238-244. Rosenzweig, M. R., Bennett, E. L., Hebert, M. & Morimoto, H. (1978). Social grouping cannot account for cerebral effects of enriched environments. Brain Research, 153, 563– 576. Rosenzweig, M. R., Krech, D., Bennett, E. L. & Diamond, M. C. (1962). Effects of environmental complexity and training on brain chemistry and anatomy. Journal of Comparative & Physiological Psychology 55, 429–437. Schwartzkroin, P.A. (1982). Development of rabbit hippocampus: Physiology. Developmental Brain Research, 2, 469-486. Segal, M., Korkotian, E., & Murphy, D.D. (2000). Dendritic spine formation and pruning: common cellular mechanisms? Trends Neuroscience, 23, 53–57. Shaw, P., Greenstein, D., Lerch, J., Clasen, L., Lenroot, R., Gogta, N., Evans, A., Rapoport, J., & Giedd, J. (2006). Intellectual ability and cortical development in children and adolescents. Nature, 440, 676-679 Walsh, R.N. (1980). Effects of environmental complexity and deprivation on brain chemistry and physiology: A review. International Journal of Neuroscience, 1, 77-89. Appendix A Golgi-Cox procedure – Embedding Brains are immersed in Golgi-Cox solution for 21. The volume of the solution must be at least ten times the volume of the brain. Day 1 10.00 a.m.: - Rinse the brains four times 5 minutes with Milli-Q (MQ). - Dehydrate in 70% ethanol for +/- 24h. Day 2 10.00 a.m.: Embed in 96% ethanol for +/- 24h. Day 3 10.00 a.m.-6 pm.: - Transfer to 100% ethanol (ethanol absolute) for 8 hours. -Afterwards (at 6 p.m.) put the brains in ethanol/ether 1:2 for 16 hours (o/n). Day 4 10.00 a.m.: Embed the brains in 3% celloidine for +/- 24h. Day 5 10.00 a.m.: Embed in 6% celloidine for +/- 72h. Day 8 10.00 a.m.: Embed in 12% celloidine for +/- 72h. Day 11/12 5 p.m.: - Put the brains in small stronger cardboard boxes filled with celloidine - Put weights on top of the boxes. - Add chloroform to cure the celloidine for 16 hours. When the celloidine stays in chloroform longer than 16 hours, the celloidine will fall apart very easily!!! Day 12/13 9 a.m.: - Discard the chloroform and add 70% ethanol. - Stick the embedded brains on top of black cubes with 12% celloidine in 70% ethanol. Keep the embedded brains in 70% ethanol and in a cold room (4°C, dark) for +/24h (but for no longer than 1 month!!!) before further processing. Staining procedure after cutting 5 min. in Milli-Q. When the sections float to the top, gently push them down into their wells. 30 min. in 16% ammonia. This will “develop” the Golgi stain. The sections should turn darker. 2 min. in Milli-Q. This acts as a rinse for the ammonia. 7 min. in 1% sodium thiosulphate. This will “fix” the sections so they do not continue to develop and the stain will remain as it is. 5 min. in Milli-Q. 5 min. in fresh Milli-Q. 5 min. in 70% ethanol. This is the start of the dehydration process. 5 min. in 96% ethanol. 5 min. in butanol. 5 min. in histoclear. This is the final step to the staining procedure. Leave the sections here while mounting. alles in verleden tijd! Vermijd vage begrippen of omschrijvingen; wees exact en zo veel mogelijk concreet; vermijd informeel taalgebruik, let op de logica, let op de opbouw, onderbouw je statements met referenties, maak een duidelijk verschil tussen bewezen en veronderstelde beweringen. Beter letten op juist gebruik van lidwoorden. Relevante zaken: connectivity essential of info processing (e.g. mental retardation). Wiring/connectivity is dependent on interaction of genome and environment during development