Genetic showed the destruction of cells and

Genetic
Disruption of Circadian Rhythms in the Suprachiasmatic Nucleus Causes
Helplessness, Behavioral Despair, and Anxiety-like Behavior in Mice

By Dominic Landgraf, Jaimie E. Long, Christophe D.
Proulx, Rita Barandas, Roberto Malinow, and David K. Welsh

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Abstract:  This was the study of depressive disorders
that is related to psychiatric disorders. 
This links mood disorders and circadian rhythm, they used mice that
faced scenarios of light/dark manipulation, mutated their clock genes, and
lesions in the brain that showed signs of changes in the circadian rhythm and
showed the destruction of cells and the eradication of neuronal connections (light
precepting pathways) these tests were performed by  injections of viral vectors that encoded for
short RNA’s to eliminate Bmal, essential clock gene, expressing to the brain’s
circadian clock, the suprachiasmatic nucleus (SCN), which did not show the
functions of circadian clocks affecting the regulation of moods.

Keywords: Circadian
clocks, suprachiasmatic nucleus, depression, Bmal, learned helplessness

Introduction:  Most species are
characterized by having a day that reoccurs due to the earth’s rotation around
the sun. All species have evolved to adjust their days around the sun, which
effects behavior, metabolism, and physiology. 
Most organisms need a timekeeping system, that is where the circadian clocks
come in.  The cells in animal tissues
also work within the circadian clocks which means they work in a 24-hour cycle.  The suprachiasmatic nucleus is what makes
this internal clock work within us and regulate the other clocks within bodies
such as the ones in brains and the rest of the body by harmonizing them to one
another in a 24-hour light and dark cycle.1  However, the SCN may not always work to
harmonize all these internal clocks since the peripheral tissues, such as the adrenals.  This may affect the rest of the body ife the
SCN is turned off or becomes nonfunctional.2

            Disorders, such as major depressive disorder (MDD) and
bipolar disorder, are usually related with the disruption of the circadian
rhythm which is shown by the disturbance of daily activity such as sleep,
appetite, and hormone release.3  This, however, has not been proved to conform
to how circadian rhythm and MDD affect one another.4  Rats on one hand  show stress when they deal with a slight
change in a change in their circadian rhythm, which seems to induce
depression-like comportment in their movement.5  In this study it shows whether or not MDD and
the circadian rhythm go hand in hand when the light manipulation is used to see
if there is any change in the subject (mice).

Methods/Materials:

Animals: This experiment
was performed using male mice that were from eight to fourteen weeks old with mPer2^Luciferase
(PER2::LUC), the wild type for the circadian clock
gene.  This gene replaced by the
homologous recombination of the gene, where a bioluminescent PER2::LUC protein
is fused for the Per2 that regulated this new addition to the gene.  The mice were also given SV40 polyadenylation
site that enhanced the gene expression and backcrossed to produce a congenic
C57BL/67 background.  Then the mice were
exposed to 12 hours of light and 12 hours of darkness at light levels of 200 lx.
Six days prior to the experiment the mice were injected with the stereotactic
adeno-associated virus (AAV) along with being kept alone with water and
food.  The experiment was conducted as
humanely as possible.

Virus Production: They made
a complementary DNA encoding of the shRNAs and AAV vectors by using a
polymerase chain reaction to increase a sequence that encodes the U6 promoter,
along with mixing the 19-nucleotide shRNA sequence, and the
cytomegalovirus–green fluorescent protein (CMV-GFP) cassette form the pLL3.7
vector (credited to Dr. Heberlein of University of California).  This part of the experiment placed the
messenger RNA to constantly mix the shRNA’s from Dr. Andrew C. Liu of
University of Memphis, which had no importance to the orientation with the mice
messenger RNA.

Virus Injection: They
then injected the mice with ketamine/dexmedetomidine which was in .9% saline
(about 70 mg/kg body weight of ketamine and .3 mg/kg body weight of
dexmedetomidine), that acted as anesthetic for surgery. Then they were injected
with a bilateral stereostatic injection of AAV particles in SCN, which was
injected in different doses into different parts of the body such as .46 in the
anteroposterior, .2 mm in the mediolateral, and 5.5 mm in the dorsoventral. The
mice were injected about .5 to .8 microliters of the virus in each of those
positions of the body in less than 30 minutes span using a Picospritzer6. All the mice got a
mixture of the two shRNAs: either both of the mixed shRNAs or both of the
Bmals, that was injected slowly in intervals of one to three minutes at the
locations mentioned earlier on the mice. Afterwards the anesthetic was reversed
by injecting .05 mg of atipamezole.

Behavioral Tests: To test
locomotion in the mice, the control and the SCN-Bmal1-KD mice were housed separate
with their own wheels and were closely monitored to see how much they would run
on the wheels when the lighting was changed. 
The mice were put in twelve hours of light and twelve hours of darkness
at 200 lx, then they were exposed to 13 days of complete darkness.  The mice were tested with the open field
test, sucrose preference, tail suspension test, and how much they portray
assert learned helplessness.  The sucrose
preference test was performed two times a week at 1% sucrose level and 6 days
before the AAV injections which was continued until the mice had perished.  Then the other behavioral tests that were
done six weeks after the mice were given the AAV injections, so the shots would
let Bmal1 shRNA to express fully. The test ran from the least to the most
stressful for the mice to start expressing learned helplessness.

Measurements/Data:  They took blood samples from the mice to
measure the concentration of corticosterone using an immunoassay kit. The mice
were moved to a room by themselves for a day before the blood sampling happened
and the room was put on the same 12-hour light and 12-hour dark cycle and the
light was kept off until the blood samples were taken.  The blood samples were taken in dim red
lighting every 4 hours and while the samples were taken the mice could move
freely.  Then the mice were also
restrained for 30 minutes and  blood
samples were taken to see how this effected the stress level of the mice.  This was also done in 10, 30, 60, and
90-minute restraint levels, then the blood samples were taken 6 hours to 9
hours after the light.  After the
behavioral test were done, they then put the mice under anesthesia and then
decapitated them to start the measurement process.  The mice’s brains then get parts sliced off
to measure PER2:LUC appearance patterns that were expressed using a LumiCycle
luminometer.  The data was collected
using GraphPad, LumiCycle, and Clocklab.

Results:
The
results showed that the rat, SCN-Bmal1-KD was more effected than the scrambled
rat, or the control rat.  During the tail
suspension test, the scrambled rat tended to show less movement than the rat
that did not have its brain altered with. 
When the rats were weighed during week 5, the Bmal rats tended to have
gained more weight, on average 15% more, than the Scrambled rat, only about 5%.  This corresponded with the fact that the Bmal
rats did tend to have more of an appetite than the Scrambled rats since they
tended to eat a couple grams more food on average.  During the light/dark box test the Bmal rats
preferred to be in the light part of the box since it seemed to give them more
anxiety than what they experienced in the dark, which is an average of about
160 seconds opposed to the control rat which was able to stay in the light box
for around a minute more.  During the learned
helplessness part of the experiment, the Bmal rat tended to fail more at
finding an escape opposed to the Scrambled rat. 
The only test that did not have a lot of variation between the rat was the
open field test, which tested spatial preference.  As for the difference in physiology the Bmal
mice brains did show a reduction in PER2::LUC in its expression but it did tend
to lengthen the period of time the circadian rhythm is in play, which did not
appear as much in the control group, scrambled mice.

Conclusion:  In conclusion, the
results show us that the mouse that had its circadian rhythm altered, Bmal mice,
did show a change in its appetite, behavior, and physiology.  They tended to have a slower reaction time,
slower problem-solving abilities, and a larger appetite which in turn made them
gain more weight than those mice that did not get their circadian rhythm altered.  The physiology of the rats did also tend to
vary from whether they got their circadian rhythms altered or not.

Discussion:  This study shows us that
circadian rhythm does affect our whole lively hood.  As the mice in the study show, the rats that were
in the control group (scrambled mice) did tend to have better reaction time and
they also did not tend to have abnormal behavior compared to the Bmal mice.  It also shows us that the circadian rhythm
does alter the genes of the mice, and in turn perhaps ours, since it has altered
their appetite, weight, and even the nature of this animal.  The light and dark model show us that the
mice with altered circadian rhythms effect the mice, how they cannot handle light
for a long period of time and they tend to show signs of learned helplessness a
lot more than the control mice.  These
mice also tend to be less friendly than the mice with unaltered circadian
rhythms, which may help us understand what MDD and the circadian rhythm may do
to affect each other.  This experiment
can easily be translated to human subjects in the future to understand the
relationship between circadian rhythm and mood disorders.

1 Welsh DK, Takahashi JS, Kay SA (2010):
Suprachiasmatic nucleus: Cell autonomy and network properties. Annu Rev Physiol
72:551–577.

2 .
Husse J, Leliavski A, Tsang AH, Oster H, Eichele G (2014): The lightdark cycle
controls peripheral rhythmicity in mice with a genetically ablated
suprachiasmatic nucleus clock. FASEB J 28:4950–4960.

3 Schnell
A, Albrecht U, Sandrelli F (2014): Rhythm and mood: Relationships between the
circadian clock and mood-related behavior. Behav Neurosci 128:326–343.

4 .
Landgraf D, McCarthy MJ, Welsh DK (2014): The role of the circadian clock in
animal models of mood disorders. Behav Neurosci 128:344–359.

5 Gorka
Z, Moryl E, Papp M (1996): Effect of chronic mild stress on circadian rhythms
in the locomotor activity in rats. Pharmacol Biochem Behav 54:229–234

6
Raimondo. Picospritzer. 2013

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