Download Synaptogenesis in the human cortex occurs between - UvA-DARE

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