The male redsided garter snakes exhibit a dissociated reproductive pattern in which their courtship

1
Utilizing Golgi Staining to Delineate Dendritic
Spine Density in the Neural Tissues of the Male
Red-Sided Garter Snake Adapting an Old
Technique to a Unique Model System.
Reem Mohammad and R.W. Krohmer Department of
Biological Sciences, Saint Xavier University,
3700 W. 103rd Street, Chicago, IL 60655
Abstract This investigation was initiated to
adapt (develop) an old histological technique,
the Golgi Stain, to work in a reptilian model
system. Investigations on avian and mammalian
model systems have demonstrated that neural
plasticity can be exhibited by changes in the
density of dendritic spines on the neurons that
comprise the pathways regulating specific
behaviors. In both birds and mammals, changes in
the density of dendritic spines appear to be in
response to internal and/or external stimuli. Of
interest to our lab is that changes in dendritic
spine density have also been demonstrated to
correlate to seasonal behaviors such as
territoriality, aggression and courtship and
mating. Therefore, use of this technique would
prove to be invaluable for examination of neural
tissues in red-sided garter snake.
Introduction The male red-sided garter
snakes exhibit a dissociated reproductive pattern
in which their courtship and mating behavior is
controlled by environmental factors not the level
of sex hormones present in their blood. The only
factor that determines the initiation of
courtship behavior is a period of low temperature
dormancy followed by an exposure to warm
temperatures. However, a non-dissociated
reproduction pattern is associated with the
circulating sex hormones in the blood, and
directly correlated with the activation of the
gonads. Red-sided garter snakes are good animal
models because they exhibit a dissociative
reproductive pattern in which their courting is
not associated with the activation of the gonads
or their hormones level. However, it is important
to note that the level of androgens are elevated
upon emergence from dormancy, the only direct
factors that affect courting and mating behavior
are environmental (Krohmer, 2004). Dendritic
spines, which are projections from the dendrites,
are the sites of termination for most excitatory
synaptic inputs in the brain. The spines of the
dendrites are known to undergo morphological
changes based on the physiological and behavioral
changes. Those changes constitute the density of
the spines as well as innervations (Gazzaley,
2002). It has been demonstrated that dendritic
spines can be used as an indicator of normal
development since clear abnormalities in
dendritic spine morphology can be a possible
marker of mental retardation. This process of
change in dendritic spines is an active process
which responds to changes in the sex hormones
such as estrogen and progesterone (Segal and
Murphy, 2001). However, it is not only the level
of the steroid hormones that might affect the
morphology of the spines seasonal changes,
learning, and courting behavior are all possible
stimuli that affect dendritic morphology.
Changes in the morphology of dendritic spines are
referred to as neural plasticity. (Gazzaley et
al., 2002). Using an old type of
histological technique that has been developed
since the early 1900s in order to view the
morphology of a neuron will be useful in studying
the dendritic spines. The Golgi stain method
shows the parts of the brain as black spots after
long immersion in solutions such as potassium
chromate and silver nitrate (Torres, 2006).
Therefore, studying the morphology of dendritic
spines using the Golgi stain in male red-sided
garter snakes under various environmental and
reproductive states will increase the database on
reproductive patterns.
solution D and E but double distilled water. The
slides were rinsed with distilled water again 2
times for 4 minutes each. Then dehydration took
place in which the slides were placed in 50,
75, and 95 ethanol for four minutes each. Then
the sections were dehydrated in absolute ethanol
for 4 times 4 minutes each. Lastly, the slides
were cleared in xylene 3 times 4 minutes each.
There was more than one experiment done and
in one, frozen courting brains were used which
means that those were placed in 20 sucrose
before freezing them. For the fresh brains, they
were placed in 20 sucrose right before
sectioning them. This, however, did not affect
our results. Summary Our preliminary
data has shown that in order to obtain the best
visualization of the dendrites, a 10-day
impregnation period is sufficient. A
significant difference was seen after comparing
the dendritic spines of courting and non-courting
male red-sided garter snakes. The spines of the
non-courting male were not dense at all and there
were barely any seen however, the spines of the
courting male were very dense and clearly seen
all over the dendrites. This indicates that the
plasticity of dendritic spines is affected by
environmental factors which is seen between the
non-courting and the courting behavior of male
red-sided garter snake. For the future
trials, we will be injecting estrogen and
testosterone in the male red-sided garter snake
for a minimum period of 2 weeks to see if that
will affect the morphology of the dendritic
spines. References Gazzaley, A, D.l Benson,
and S Kay. "Dendritic Spine Plasticity in
Hippocampus." Neuroscience 111 (2002) 853-862.
PubMed. 20 Jan.-Feb. 2007 lthttp//www.ncbi.nlm.nih
.gov/entrez/query.fcgigt. Krohmer, Randolph W.
"The Male Red-Sided Garter Snake (Thamnophis
Sirtalis Parietalis) Reproductive Pattern and
Behavior." ILAR 45 (2004). Segal, Menahem, and
Diane Murphy. "Estradiol Induces Formation of
Dendritic Spines in Hippocampal Neurons
Functional Correlates." Hormones and Behavior 40
(2001) 156-159. PubMed. 20 Jan.-Feb. 2007.
lthttp//www.ncbi.nlm.nih.gov/entrez/query.fcgigt.
Torres-Fernandez. "The Golgi Silver Impregnation
Method Commemorating the Centennial of the Nobel
Prize in Medicine (1906) Shared by Camillo Golgi
and Santiago Ramon Y Cajal." Biomedica 26 (2006)
498-508. Pubmed. 20 Jan.-Feb. 2007
lthttp//www.ncbi.nlm.nih.gov/entrez/query.fcgigt.

Arrows point to dendritic spines of a courting
male red-sided garter snake.
Arrows point to dendritic spines of non-courting
male red-sided garter snake.
Material and Methods Most of the material needed
for this research were obtained from the FD Rapid
Golgistain Kit which contained solutions labeled
as A, B, C, D, and E. The brains were
immersed in a solution mixture of equal amounts
of A and B for a period of ten days, two weeks,
and three weeks in different trials to see which
will yield the best results. This solution needed
to be replaced one day after immersion and then
the brains were stored in the dark at room
temperature for the designated period. After
that, the brains were transferred to solution C
for a minimum period of 48 hours for a week at
maximum. The solution needed to be replaced after
one day of immersion and stored at 4 Celsius in
the dark until ready to be sectioned. The brains
were then frozen on dry ice and sectioned using
the Cryostat. Some of the tissues were cut at 80
micrometers and some at a 100 micrometers. The
tissues were placed on gelatin-coated slides and
dried at room temperature. Then using a dropper,
solution C was added to the tissue and the excess
dried. After completely drying, the slides were
stored in a box until staining. As for the
staining procedure, the slides had to be washed
in distilled water 2 times, 2 minutes each. Then
placed in a mixture containing equal amounts of