Swarsattie Bhim
Cynthia Alejos
Dendritic Spine Plasticity in Transgenic Mice
A developing animal undergoes various changes neurologically. To explore further into
these changes, many new technological changes have occurred. One of these changes is the
transcranial photon imaging technique. This highlights the dendritic spines that were located in
layer 5 of the pyramidal neurons in the visual cortex of transgenic mice that were exposed to
YFS. YFS is a yellow fluorescent protein (Zuo et al, 2005).
H-Line Transgenic mice were used by overexpressing yellow fluorescent protein (YFP)
and observing them with transcranial two-photon microscope order to view their dendritic spines
on the apical dendrites. It was observed that mice that were only a month old had about 13%20% of spines eliminated and 5-8% formed. This occurrence took place over a course of 2 weeks
in the regions of the cerebral cortex. As animals develop, spinogenesis occurs which is defined
as the loss of dendritic spines.
The purpose of Holtmaat et al experiment was to examine where and when dendritic
plasticity is most prevalent in the mice brain. Holtmaat and is colleagues focused primarily on
the critical period of plasticity of a mice’s brain. The critical period of plasticity is the period in
time when the brain is most capable of change. The barrel cortex of the rat’s brain refers to the
dark staining of the four somatosensory cortex layers. The barrel cortex represents the whisker
barrels in the somatosensory cortex of rodents. Layer IV of the somatosensory cortex is the main
input layer from the thalamus. The whiskers are represented on the barrel cortex
topographically. Holtmaat et al imaged apical dendritic clusters in the adult somatosensory and
visual cortex to understand what influences dendritic and spine structural plasticity. Although
the brain’s ability to change and grow more neurons and build new connections is ideal at early
postnatal development, there is still substantial plasticity that occurs in the adult cortex. As
discussed by Holtmaat et al, plasticity is not only the means by which new synapses and neurons
are formed but also the means by which old synapses and neurons get eliminated as well. At the
cellular level studies have indicated that dendritic spines are not solely but largely responsible for
circuit plasticity. Dendritic spines are said to be predominantly excitatory and receives majority
of the brain’s synapses. Holtmaat et al experiment indicated that long term imaging of the striate
cortex shows that some spines grow while others die in an experience dependent manner. In
other words depending on how much the mice relies on a particular synapse will determine if the
synapse remain and gets stronger, whether new synapses for that same function is formed or
whether a synapse will become eliminated. The results from this study indicated that dendritic
spines plasticity is more evident in the somatosensory than in the visual cortex of the mice. In
majority of the series of experiment carried out by Holtmaat et al, transgenic mice were used
where all the neurons examined were reconstructed in vivo images or in fixed sections. Laser
scanning was used to determine whether GFP positive cells contained a normal subgroup of
cortical neurons.
Holtmaat and his colleagues concluded that GFP positive neurons have a functional
representative subgroup of cortical cells. A small glass window over the somatosensory or
visual cortex was used for imaging. In addition, 2-photon laser scanning microscopy was used to
repeatedly image the developmental capacities of the dendrites and their spines in the adult mice
brain. Young animals exhibited a bundle of filopodia in their early stage of life but as they
approached adulthood they began to disappear. Studies involving the visual cortex have showed
that dendritic spines become more stable as the animals mature which aids in long term
information storage. Mice of ages from 1-10 months were anesthetized and then had a surgical
incision in their scalp over the region of the visual cortex (Zuo et al, 2005). Pruning of dendritic
spines during development results in disappearance of the spines within days. Chronic imaging
was done in the third and fourth postnatal week. The rate at which spines were appearing and
disappearing was significantly different; however by week 4 the rate of addition was equaled to
the rate of subtraction. The fraction of spines surviving for at least 8 days was .35. These values
were obtained from a group of five experimental mice. This fraction is much less than that of
young adult mice thus it was concluded that spine plasticity and stability is regulated after the
closure of the critical period of plasticity.
In a follow up experiment, apical dendritic arbors of individual layer 5 beta neurons in
the striate cortex of mature adult mice were imaged. The spines in young adult mice the spines
appeared occasionally but were usually very thin. Spine survival fraction was used to calculate
the stability between different ages and varying cortical regions. Results indicate that 6-monthold mice had a significantly lower fraction of transient spines while the fraction of persistent
spines was significantly larger. The 6 month old mice were compared with other mice that were
5-11 weeks old.
Zuo and his colleagues acknowledged that the ratio of spines to dendritic spines changed
significantly in only a matter of two to four weeks. Dendritic spines would increase but remain
stable once young adulthood was reached. Little change was seen in adult mice over a two
month period in spine number and location. The long term stability of spines in the adult brain of
the mice has demonstrated the synaptic dynamics and how it can offer a basis for long term
information storage since they remain in the same location. The various changes in the
measurements of these spines do suggest that it could play a role in short term storage of
information as well.
There were substantial differences observed between the plasticity in the somatosensory
cortex and visual cortex. Holtmaat et al explained that the differences could be because of the
housing, handling or genotype of the animals. Spine structural plasticity was used to analyze the
differences between the primary somatosensory cortex and the primary visual cortex. Holtmaat
and his colleagues found that in the somatosensory cortex there were a lot of labeling in layer 5
beta, layer 6 and layer 2/3. In contrast in the visual cortex most labeling was found in layer 6
alone.
It was observed that spines grow faster in the somatosensory cortex than in the visual
cortex but the long term persistence between the two cortices is not significantly different. It is
also suspected that less spiny neurons are more plastic. Spine stability and plasticity in layer 5
beta and layer 2/3 was examined. It was found that in layer 2/3 the neurons have less elaborate
clusters than layer 5B. For this part of the experiment animals were grouped into 3 months or
older than three months. Holtmaat et al suggested that under stable sensory conditions spines are
either transient or persistent and generally persistent spines are thin. Long thin spines are more
likely to be found in the somatosensory cortex than in the visual cortex. This also explained by
the fact that mice use their sense of touch more than their sense of sight. Transient spines are
thin throughout their period of existence while persistent spines are relatively thick. Persistent
and transient spines in the neo-cortex can go through changes in seconds and minutes. Holtmaat
et al emphasized that persistent spines are rear and those spines that survive for 8 days are more
likely to survive for a month or even longer. A distinguishable morphological difference
between transient and persistent spines is that transient spines are small and thick while
persistent spines are thick and are shaped like mushrooms. Holtmaat et al concluded that the
increase in synaptic densities is likely due to neurons such as layer 2/3 which are born after layer
5 and constitute a less mature population of spines.
Additionally, Holtmaat et al explored whether the generation and loss of persistent spines
are enhanced by novel sensory experience. They confirmed that in layer 5B of the rat’s barrel
cortex, dendritic spines appear and disappear over days. For morphological purposes the
neurons were separated in either simple tuft cells or complex tuft cells. It was found that whisker
trimming stabilized new spines and destabilized previously persistent spines. It was also found
that new persistent spines always formed stable synapses. The number of new persistent and lost
persistent spines (after 8 days they disappeared) was a small ratio of the total number of
persistent spines.
In this follow up experiment, the rat’s whisker was trimmed contralaterally in such a way
that each deprived barrel column was surrounded by untrimmed barrel columns. This pattern of
trimming was referred to as chessboard deprivation. The whiskers on the ipsilateral whisker pad
were all trimmed. The trimming was preformed every 2 days under slight anesthesia. In this
experiment a total of 5333 spines were imaged over the entire imaging period. After trimming
the whiskers the density of spines did not change however after some time there was an increase
in the new persistent spine density. It was emphasized that different branches of the spines were
participating in distinct forms of plasticity. It was also observed that after whisker trimming,
persistent spines were more likely to disappear. After much speculation, Holtmaat et al
suggested that persistent spines might be the cause of changes in excitatory circuits which
extends to layer 5B of pyramidal cells. The authors of this paper suggested that spine volume
might be responsible for one of two functions. First, it might reflect increases and decreases in
synaptic strength or second it might be the signal for synapse formation or elimination.
In addition, Zuo et al, studied how synapse formation and elimination can happen
throughout a lifetime but yet the development of the dendritic spines has remained stable through
the cerebral cortex. These changes are vital because they explain how the nervous system
develops and works over the years. Mice that had already reached the adult stage, which is an
estimated time of 4-6 months, had 3%-5% of spines that were eliminated and formed over the
course of two weeks throughout the regions of the cerebral cortex. Even when mice were tested
around the time of 8 months they had 26% of their spines that had been elimated but at the same
time had 19% that had formed in the adult barrel cortex. This illustrates to researchers that there
is long term dendritic spine stability and that most of them remain in the cerebral cortex
throughout a lifetime. Results showed that the majority of spines can remain unchanged for a
span of up to 8 months in the area of the cortex (Zuo et al, 2005).
From Zuo et al experiment, Images show that a filopodium is converted to a spine line
protrusion within four hours. Four hours after that it disappears and is no longer present.
Filopodia give rise to dendritic spines making them less present as the mice mature.
Development in dendritic spines remains the same once adulthood is reached. This is witnessed
in the many regions of the cerebral cortex such as barrel, motor, or frontal. When 18 month old
mice were studied, it was seen that their dendritic branches showed spines that stayed at the same
place. They displayed dendritic spine stability for over 18-22 months.
For data interpretation a non-parametric bootstrap method was used to compare the
means and medians between the control and experimental groups. Further research must be
completed to further explore into synaptic dynamics to fully understand more about dendritic
spines. Even though it is unlikely that other spines differ, it is unknown how dendritic spines will
react if they are different neuronal types or not part of layer 5 pyramidal cells, or in other regions
not part of the visual cortex. Dendritic spines development is a subject that is still being
researched. Researchers suggest that the formation and elimination of filopodia is important for
the long term stability of dendritic spines
Figure 1: Transient and Persistent Spine in the Striate Cortex
Diagram 1a illustrates transient and persistent spines during development in the striate
cortex. This figure represents coronal sections from two yellow florescent protein mice.
Top diagram shows postnatal day 14 while the bottom diagram shows postnatal day 28.
This is a depiction of regulation of the yellow florescent protein expression in the striate
cortex. As evident the expression is brightest in Layer 5B at all ages.
Diagram 1b shows dendritic arbors in the yellow florescent protein animal at postnatal
day 16 and postnatal day 25. The bright dendrites belonging to one cell stands out but
several dim dendrites and axons are visible as well. The red arrow points at the same
branch point at both ages. At postnatal day 25 there are much more highlighted
dendrites than in postnatal day 16.
Diagram 1c shows a time lapse image of a dendritic branch of postnatal day 16-25. The
presence of persistent spines (shown by yellow arrowheads) that appear and disappear
over the imaging period is illustrated. The transient spines are shown in blue
arrowheads and new persistent spines by red arrowheads. In addition the filopodium is
illustrated by the white arrowheads. As age increases the persistent spines remained
the same but the number or new persistent spines increased. The transient spine also
disappeared by postnatal day 25.
Diagram 1D shows the fraction of spiny protrusions gained (squares) or lost (circles)
from day to day as a function of age. As age increased the amount of spiny protrusions
gained has decreased more than the amount of protrusions gained.
Figure 1E shows normalized spine density as a function of age. As age increases the
normalized spine density decreases. The decrease however, as illustrated is not
significant.
Figure 1F shows the survival function of spines in individual cells. As evident most of
the cell survival fraction starts at one after which they gradually decreases. Some
survival fraction decreases more than others but they all stay between .3 to .7
Figure 2. The Majority of Dendritic Spines in Adult Persist over 18 months
Figures A-D shows a time elapse image of the spines of both experimental and control animals
that demonstrate that dendritic spines still are present at the same location that they were 18
months ago. (E) This graph shows the difference in percentages of the spines over the course
of 18-22 months, that were formed (grey), eliminated (white) and stable (black). As illustrated
there were more stable spines than formed or eliminated spines. In addition there were also
more eliminated spines than formed ones. (F) This graph illustrates the correlation between the
percentage of total number of spines and age (1-24 months). Spines that developed in the
second view are represented by the arrowheads. As age increases the number of total spines
decreased significantly.