Cerebral Cortex June 2005;15:802--808
doi:10.1093/cercor/bhh181
Advance Access publication September 15, 2004
Experience-induced Changes of Dendritic
Spine Densities in the Prefrontal and
Sensory Cortex: Correlation with
Developmental Time Windows
The present study provides evidence for the hypothesis that the
extent and the direction of experience-induced synaptic changes in
cortical areas correlates with time windows of neuronal as well as
endocrine development. Repeated brief exposure to maternal
separation prior to the stress hyporesponsive period (SHRP) of
the hypothalamic-pituitary-adrenal (HPA) axis induced significantly
reduced dendritic spine density (--16%) in layer II/III pyramidal
neurons of the anterior cingulate cortex (ACd) of 21-day-old rats,
whereas separation after termination of the SHRP resulted in
increased spine densities (116%) in this neuron type. In addition,
rats of both groups displayed elevated basal plasma levels of
corticosterone at this age. Separation during the SHRP (postnatal
days 5--7) did not influence spine density in the ACd, and basal
corticosterone levels remained unchanged. In contrast, pyramidal
neurons in the somatosensory cortex (SSC) displayed significantly
enhanced spine densities (up to 52% increase) independent from
the time of separation. These results indicate that alterations in the
synaptic balance in limbic and sensory cortical regions in response
to early emotional experience are region-specific and related to the
maturational stage of endocrine and neuronal systems.
Keywords: development, endocrine systems, limbic, prefrontal, stress
Introduction
Juvenile stressful experience, such as the separation of a new-
born animal from its mother or parents, interferes with the
maturation of endocrine and behavioral function in rodents and
primates, including man (Heim and Nemeroff, 2001; Pryce and
Feldon, 2003), and thereby can affect cognitive as well as
emotional capacities throughout life. The observed behavioral
alterations are most likely a consequence of synaptic changes
within functional neuronal networks. Recent studies in rodents
have shown that repeated periods of traumatic experiences
during the first weeks of life induce alterations of synaptic
connectivities in the limbic anterior cingulate cortex and
hippocampus (Helmeke et al., 2001a,b; Ovtscharoff and Braun,
2001; Poeggel et al., 2003).
For sensory systems it has been shown that the functional
maturation of cortical circuits is characterized by develop-
mental time windows, during which the impact of sensory
experience on synaptic reorganization is particularly strong
(Fox, 1994; Katz and Shatz, 1996; Yuste and Sur, 1999; Kral et al.,
2001). Immature developing brain regions, in particular, areas of
the limbic system, are considered to undergo ‘experience-
expectant’ developmental time windows, during which ade-
quate social, emotional, perceptual and cognitive stimulation
is required for normal development (Greenough et al., 1987;
Joseph, 1999; Andersen, 2003). Such time windows of enhanced
synaptic plasticity on one hand serve to fine-tune and adapt the
Cerebral Cortex V 15 N 6 Ó Oxford University Press 2004; all rights reserved
Jörg Bock, Michael Gruss, Susann Becker
and Katharina Braun
Institute of Biology, Department of Zoology and
Developmental Neurobiology, Otto von Guericke Unversity,
Brenneckestr. 6, 39118 Magdeburg Germany
synaptic network according to the individual’s environment, but
they also represent phases of high vulnerability in cases when
no or adverse experiences interfere with the development of
brain circuits.
Phases of pronounced synaptic reorganization, i.e. prolifera-
tion and elimination of synaptic connections, have also been
described for sensory and prefrontal cortical areas of human and
non-human primates (Wolff and Missler, 1993; Bourgeois et al.,
1994; Huttenlocher and Dabholkar, 1997). However, it is still
rather unclear if the described experience driven synaptic
alterations, especially in the limbic system, are correlated to
distinct developmental time windows and which cellular and
endocrine factors define such time windows of cortical devel-
opment. The aim of the present study was to identify a correlation
between the developmental time windows of endocrine stress
systems and experience-induced synaptic changes. To test this
hypothesis, the impact of repeated periods of emotional stress,
evoked by maternal separation, prior to, during or after the stress
hyporesponsive period (SHRP) of the hypothalamic-pituitary-
adrenal (HPA) axis, on the development of dendritic spines in
a limbic and a sensory cortical area was quantitatively analyzed.
The SHRP, which is characterized by low basal levels of stress
hormones and a relative non-responsiveness to external stressors
(Rosenfeld et al., 1992; Levine, 2001), was proposed to protect
the juvenile brain against the deteriorating effects of high levels
of stress hormones (Meaney et al., 1991). If endocrine function is
related to the stress-induced synaptic changes, different changes
of spine densities should be expected during these three time
windows.
Material and Methods
Subjects
Male White-Wistar rat pups (Leibniz Institute for Neurobiology,
Magdeburg, Germany) were used. The date of birth was designated as
postnatal day (PND) 0. After birth, litters were culled to 12 pups (six
males and six females) and housed together with their mother in
standard rat cages (56 3 34 3 25 cm) in an air-conditioned room with
controlled temperature (22 ± 2°C) and a 12/12 h light/dark cycle. Fresh
drinking water and rat pellets were available ad libitum. During the
entire experiment the cages were not cleaned to avoid handling and
stress-related disturbance of the family. All experimental protocols were
approved by the ethical committee of the government of the state of
Saxony-Anhalt according to the German guidelines for the care and use
of animals in laboratory research (§8, Abs. 1, 25.05.1998). All experi-
ments were performed in accordance with the European Communities
Council Directive of November 1986 (86/609/EEC).
Maternal Separation
The pups were removed from their home cage and individually isolated
for 1 h in incubators under controlled temperature (35 ± 2°C) and
humidity. During isolation no visual or tactile, only auditory and
olfactory communication was possible between the siblings. After
isolation, the pups were placed back in their home cage.
Experimental Groups
To test whether separation-induced changes are possibly linked to
distinct developmental time-windows, pups were randomly allocated to
one of the following four experimental groups (n = 16, four per
experimental group, all animals per goup from four different litters).
Naive Control Animals (NC)
These animals were reared undisturbed with their mother.
Maternal Separation prior to SHRP (MS 1--3)
Pups were individually separated from the dam for 1 h per day on PND
1--3.
Maternal Separation during SHRP (MS 5--7)
Pups were individually separated from the dam for 1 h per day on PND
5--7.
Maternal Separation after SHRP (MS 14--16)
Pups were individually separated from the dam for 1 h per day on PND
14--16.
Golgi-Cox Impregnation of Neurons and Quantitative Analysis
After decapitation, the brains were rapidly removed and immersed in
50 ml of Golgi--Cox solution for 14 days. Thereafter brains were
dehydrated and embedded in 8% Celloidin. Serial transverse 150 lm
sections were collected, mounted on glass slides and processed using
a modified Golgi--Cox protocol by Glaser and Van der Loos (1981). Spine
densities and dendritic length of layer II/III pyramidal neurons of the
anterior cingulate (Fig. 1) and somatosensory cortex were analyzed
using the image analysis system NEUROLUCIDA (MicroBrightField,
Figure 1. Representative Golgi-impreganted pyramidal neuron in the ACd, black
arrows indicate basal dendrites, white arrow indicates apical dendrite. The inset shows
a dendritic segment of an apical branch. All visible protrusions (two representative
examples are indicated with arrows) were counted as spines.
Colchester, VT), which allows quantitative three-dimensional analysis
of complete dendritic trees. Only neurons that were impregnated in
their entirety and displaying complete dendritic trees were selected.
Thirty-two pyramidal neurons per experimental group were analyzed.
The length of the dendritic trees was measured by tracing the entire
dendrite while counting dendritic spines. However, it is notable that
dendritic spines do not represent all synaptic contacts of the analyzed
neurons. Mean spine frequencies (number of visible spines per lm) and
the length of the dendritic branches were calculated and tested for
significant differences between the groups using a Kruskal--Wallis
One-Way analysis of variance (ANOVA) followed by a two-tailed Mann--
Whitney U-test. All measurements were done by an experimenter blind
to the experimental conditions of the animals. An attempt to correct for
hidden spines (Feldman and Peters, 1979) was not made, since the use
of visible spine counts for comparison between different experimental
conditions had been validated previously (Horner and Arbuthnott,
1991).
Corticosterone Measurements
Stress Treatment and Blood Sampling
Subjects used in this experiment were from different litters than the
animals used for the morphological analysis. Blood samples were taken
at PND 21 from male pups of the four experimental groups described
above applying the following treatments (n = 72):
1. Basal levels (n = 24, six per experimental group, all animals per
group from six different litters): the hormone levels of this group
are considered as ‘relative’ baseline values, to which the effect of
the conditions described under (2) and (3) can be compared,
rather than values of ‘absolute’ resting conditions, since catching
the animals per se might act as a stressor. Animals were decapitated
within 10 s after removal from the home cage and trunk blood was
collected in chilled tubes containing EDTA (BD Vacutainer, Becton
Dickinson, UK); samples were centrifuged to separate plasma,
which was stored at –80°C until assayed for corticosterone.
2. Stress challenge (n = 24, six per experimental group, all animals per
group from six different litters): the animals were individually
placed into an unfamiliar environment (open field arena 50 3 50 3
40 cm) for 30 min, after which the blood samples were taken and
handled as described for (1).
3. Stress recovery (n = 24, six per experimental group, all animals per
group from six different litters): after open field experience as
described under (2), the animals were returned to their family in
the home cage for a recovery period of 45 min, after which the
blood samples were taken and handled as described for (1).
Corticosterone Assays
Commercially available radioimmunoassys were used for the determin-
ation of plasma levels of cortiocosterone (Diagnostic Systems Labora-
tories Inc., Webster, TX). The intra and inter-assay coefficients of
variation for corticosterone were 4.2 and 10.0%, respectively.
Statistics
Data were analysed by two-way ANOVA with the age at isolation and
stress treatment paradigm as experimental factors, followed by post hoc
Tukey tests (SigmaStat 2.0, Jandel, Germany), with a level of significance
set at P < 0.05. Corticosterone levels had a log normal distribution, and
a logarithmic transformation was therefore applied to the data for
statistical analysis.
Results
Spine Frequencies
Anterior Cingulate Cortex
The anterior cingulate cortex (ACd) of MS 1--3 rats displayed
significantly lower spine frequencies on basal (--17%, P = 0.019)
Cerebral Cortex June 2005, V 15 N 6 803
as well as apical dendrites (--16%, P = 0.012) compared with
naive control animals (Figs 2a and 3). In contrast, MS 14--16 rats
showed higher overall spine frequencies on basal dendrites
(+16%, P = 0.05) and no changes on the apical dendrites. In the
MS 5--7 rats no effects on spine densities neither on basal nor on
apical dendrites were found (Figs 2a and 3).
Somatosensory Cortex
In the somatosensory cortex (SSC) increased spine frequencies
were measured on the basal as well as on the apical dendrites of
all three maternal separation groups compared with the con-
trols, irrespective of the time window of separation (Figs 2b and
3). The most pronounced separation-induced spine increase
was observed in the MS 5--7 (apical: +52%, basal: +40%, P <
0.001) and MS 14--16 (apical: +48.5%, basal: +38%, P < 0.001)
animals, and to a lesser extent in the MS 1--3 group (basal: +17%,
P = 0.003).
Dendritic Length
Experience-induced changes of dendritic length showed no
correlation to the time period of maternal separation (Fig. 4),
but subtle differences between the two cortical areas were
detected. In the ACd the length of apical dendrites was
significantly decreased only in MS 1--3 rats (P = 0.005) compared
with the naive control animals, whereas no changes were
observed in the other two maternal separation groups. In the
SSC no changes of apical dendritic lengths were detected. Basal
dendrites in the ACd showed significantly increased dendritic
lengths in rats separated during (PND 5--7) (P = 0.009) and after
Figure 2. Mean spine frequencies (± SEM) in layer II/III pyramidal neurons from 21-
day-old rats. (a) Apical and basal dendrites in the anterior cingulate cortex (ACd). (b)
Apical and basal dendrites in the somatosensory cortex (SSC).
804 Spine Changes and Developmental Time Windows d
Bock et al.
(PND 14--16) (P = 0.028) the SHRP, whereas dendritic length
remained unchanged in rats separated prior the SHRP. The same
tendencies were found in the SSC, where basal dendrites
displayed significantly (P < 0.001) elongated dendrites in rats
separated during (PND 5--7) and after the SHRP (PND 14--16).
Body and Brain Weights
Measurements of body and brain weights did not show signifi-
cant differences between the experimental groups.
Blood Corticosterone
Measurements of stress hormones revealed an influence of
maternal separation on basal stress hormone levels, since sig-
nificant differences in the basal levels of corticosterone were
observed between the four experimental groups. MS1--3 (+71%)
and MS 14--16 (+56.8%) rats showed significantly enhanced
basal levels of corticosterone at PND 21 compared with naive
control animals (P < 0.05), whereas MS 5--7 rats showed cortico-
sterone levels comparable to the naive animals (Fig. 5a).
The HPA-axis response to stress challenge and stress recovery
was similar in all groups [F (2,71) = 97.4, P < 0.001], i.e. all groups
showed significant elevations of corticosterone immediately
after maternal separation at PND 21 (stress challenge)
(P < 0.05) and a strong reduction after reunification with the
family (recovery) (P < 0.05) (Fig. 5b).
Discussion
Dendritic spines are the main target for excitatory inputs and
play a key role in the expression of synaptic plasticity (Koch
and Zador, 1993; Harris and Kater, 1994; Segal, 2002). Multiple
studies in different systems have shown that these postsynaptic
structures are quite sensitive to developmentally, experience or
pharmacologically induced synaptic plasticity (Bock and Braun,
1999a,b; Goldin et al., 2001; Segal, 2002; Trachtenberg et al.,
Figure 3. Representative images of Golgi-impreganted segments from basal
dendrites of the four experimental groups. The upper row shows dendritic segments
of the anterior cingulate cortex (ACd), the lower row dendritic segments of the
somatosensory cortex (SSC). Scale bar, 5 lm.
Figure 4. Mean dendritic length (± SEM) of layer II/III pyramidal neurons from 21-
day-old rats. (a) Apical and basal dendrites in the anterior cingulate cortex (ACd). (b)
Apical and basal dendrites in the somatosensory cortex (SSC).
2002; Lieshoff and Bischof, 2003). It has been shown in
neonatal birds and rodents that positive or negative emotional
experiences alter synaptic connectivities in limbic regions
(Wallhausser and Scheich, 1987; Bock and Braun, 1998;
Helmeke et al., 2001a,b; Poeggel et al., 2003). Thus, quite
comparable to the experience-driven maturation of sensory and
motor systems (Turner and Greenough, 1985; Rosenzweig
and Bennett, 1996), the synaptic development of limbic cortical
regions is sensitive towards environmental conditions, in par-
ticular emotional experience.
Synaptic Changes in the Prefrontal Cortex Induced by
Early Emotional Stress are Dependent on the Time Point
of Stress Experience during Early Developmental Phases
The present results clearly demonstrate that the extent and the
direction of experience-induced alterations of spine synapses
in the limbic ACd and sensory SSC are critically dependent on
the developmental time window during which the stressful
experience is encountered. Furthermore, our results also reveal
region specific differences between the analysed cortical areas.
In the ACd spine changes only occurred when maternal
separation was applied prior or after the SHRP of the HPA-axis,
whereas maternal separation during the SHRP had no influence
on dendritic spine densities. Moreover, maternal separation
prior or after the SHRP resulted in opposite changes of spine
densities. In contrast to the findings in the ACd, pyramidal
neurons in the SSC displayed elevated spine densities, irrespec-
tive of the time point of separation. The strongest effects were
Figure 5. Plasma levels (± SEM) of corticosterone in 21-day-old rats of the four
morphologically analyzed experimental groups. (a) Corticosterone levels during basal
conditions. (b) Corticosterone levels during stress challenge and after reunification
with the family (recovery). *P \ 0.05.
found in the MS 5--7 and MS 14--16 animals and to a lesser extent
in the MS 1--3 animals.
Factors that might be reponsible or causally linked to this
phase-dependent and cortex--specific spine changes include (i)
the maturity and activity of endocrine systems including neuro-
nal mineralo- and glucocorticosterone expression; (ii) the
maturity of the sensory systems, which determine the complex-
ity of environmental stimuli perceived by the animal when it is
removed from its familiar environment; (iii) the maturity of pre-
existent synaptic connections; and (iv) the plasticity potential
of the neurons and their synaptic contacts, determined by
molecular events.
Correlation with Endocrine Time Windows?
The findings for spine development on layer II/III pyramidal
neurons of the ACd are in line with the hypothesis that the time
windows for experience-induced synaptic fine-tuning correlate
with developmental time periods of endocrine functions. How-
ever, although similarily elevated basal corticosterone levels
were found in the MS 1--3 and MS 14--16 rats at PND21, spine
densities in the ACd changed in opposite directions in these
two groups. Thus, the contrasting spine changes might be
linked to different developmental profiles of glucocorticoid and
mineralocorticoid receptor expression during these two time
windows. Prefrontal cortical areas have been shown to be a
target for glucocorticoids in rodents and primates (Meaney and
Aitken, 1985; Sanchez et al., 2000), where the glucocorticoid
Cerebral Cortex June 2005, V 15 N 6 805
receptor mRNA is particularly enriched in the cortical layers II/
III and V (Patel et al., 2000). However, the developmental time
profile for receptor expression for these layers has not been
examined in detail. The expression of glucocorticoid receptors
is regulated by early environmental manipulations (Meaney
et al., 1996; Avishai-Eliner et al., 1999; Ladd et al., 2004), and
corticosterone treatment can induce dendritic reorganization
and alterations of spine density in the hippocampus and medial
prefrontal cortex of adult rats (Woolley et al., 1990; Wellman,
2001; Seib and Wellman, 2003).
Correlation with Sensory Development?
Following the concept that synaptic development is regulated
by the complexity of patterned and converging synchronous
neuronal activity, the degree and direction of the synaptic
changes in the ACd and the SSC is most likely also determined
by preexisting synaptic connections, e.g. the functional matur-
ity of the sensory and modulatory input systems. Animals in the
oldest separation group (MS 14--16), ending up with elevated
spine densities in the ACd and SSC, were able to detect more
complex sensory features associated with the stress situation
than the animals from the youngest separation group (MS 1--3),
ending up with reduced spine densities in the ACd. The higher
spine densities in the ACd of the oldest maternal separation
group is in line with the findings in the precocious rodent
Octodon degus, whose sensory systems are already functional at
birth. In this species elevated spine densities have been found
after periodic parental separation (Helmeke et al., 2001a,b;
Poeggel et al. 2003). It seems that in the more mature animals
the separation from the familiar environment might partly act
as ‘enriched environment’, which has been shown to result in
elevated synaptic densities (Turner and Greenough, 1985;
Rosenzweig and Bennett, 1996). Furthermore, the observed
changes of endocrine function and synaptic density after
maternal separation may on the one hand have been caused
by the separation itself, but on the other hand there is
convincing evidence that changes in the quality of maternal
care after separation are a critical factor (Levine, 2002; Huot
et al., 2004). In addition, the differential, phase-specific changes
in the limbic ACd and sensory SSC might be explained by their
unimodal (SSC) and associative (ACd) properties. The input into
the SSC is mainly confined to somatosensory stimulation, e.g.
tactile stimulation during handling, exploration of the unfamil-
iar environment and maternal behaviors such as grooming and
licking. In contrast, the associative properties of the ACd
integrate polysensory inputs to achieve much more complex
environmental information.
The present study also revealed changes in dendritic length
in the ACd as well as in the SSC. In line with findings in the
hippocampus of adult rats and primates, where dendritic
atrophy has been described in the hippocampal formation and
prefrontal cortex after stress exposure (Watanabe et al., 1992;
Magarinos et al., 1996; Radley et al., 2004), decreased dendritic
length of the apical dendrites of layer II/III pyramidal neurons
in the ACd was observed in the youngest separation group (MS
1--3). The other two groups of neonatally stressed rats, however,
displayed elongated basal dendrites in both the ACd and the SSC.
These differential, phase-dependent differences between apical
and basal dendritic lengths, especially in the ACd, are suggestive
of a role of differential afferent input activity and different
expression of transmitter receptors on the two dendritic arbors.
806 Spine Changes and Developmental Time Windows d
Bock et al.
Alterations in the Stress Hormone Systems are
Correlated to the Same Developmental Time Windows
as the Experience-induced Synaptic Changes
In addition to the neuronal changes, stress hormone systems
are also altered after repeated juvenile stress experience and
also depend on specific time windows. Only the MS 1--3 and MS
14--16 rats displayed significantly enhanced basal levels of cor-
ticosterone at PND 21, whereas MS 5--7 rats had corticosterone
levels comparable to the naive animals. In contrast, acute stress-
induced corticosterone elevations during the stress-challenge
experiment were similar in all groups at PND 21, which is not
in line with studies where a single period of 24 h maternal
separation was used as paradigm (Lehmann et al., 2002). In the
cited study, 24 h maternal separation during different phases
of the SHRP did not induce altered basal stress hormone levels
but did result in a significant increase of the corticosterone
response to restraint stress in adult animals. These changes
were independent of the ontogenetic/SHRP status of the pup.
In human and non-human primates, disrupted mother--infant
attachment and premature separation have been considered to
impair socio-emotional and cognitive competence and to pro-
mote vulnerability to major psychiatric illnesses (Suomi, 1997;
Agid et al., 1999; Heim and Nemeroff, 2001). Early separation
paradigms in rats and non-human primates provide experi-
mental approaches to identify and characterize the cellular and
molecular mechanisms that may underlie the development of
mental disorders (Ellenbroek et al., 1998; Nestler et al., 2002).
The medial prefrontal cortex is connected to limbic areas such
as the nucleus accumbens, the hippocampus and the amygdala
(Heidbreder and Groenewegen, 2003), regions which are crit-
ical for emotional behavior as well as for learning and memory
formation. Moreover, there is evidence that subregions of the
prefrontal cortex might be directly involved in the regulation of
HPA function (Sullivan and Gratton, 2002), and dysfunctions of
HPA-axis regulation have been suggested to be causally related
to depression (Holsboer, 2001). Taken together, our results
indicate that the synaptic circuits within the limbic ‘reward’
system, which plays a critical role in emotional regulation,
learning and memory formation, are adapted to the animal’s
early postnatal environment and shape the network capacities
for species-specific behavioural, socio-emotional and cognitive
functions in adolescent and adult animals.
Acknowledgements
We would like to thank Sabine Westphal and Claus Luley for meas-
urements of blood samples and Aileen Schröter for help with the
illustrations. This work was supported by grants from the German
Science Foundation (SFB 426) and the Volkswagenstiftung.
Address correspondence to Dr Jörg Bock, Institute of Biology,
Department of Zoology and Developmental Neurobiology, Otto von
Guericke Unversity, Brenneckestr. 6, 39118 Magdeburg Germany. Email:
joerg.bock@nat.uni-magdeburg.de.
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