Repeated optogenetic stimulation of the suprachiasmatic nucleus (SCN) in mice at circadian time 15 resulted in chronic phase delays of the mice's circadian activity rhythms without affecting anxiety-like, depression-like, or locomotor behaviors. The study established a model for inducing chronic circadian phase delays and investigated the effects on mood-related behaviors, finding no changes to behaviors examined in tests of anxiety, depression, and locomotion.

1. I. Stimulation of the SCN at CT15 Delayed the Homecage Circadian Activity of the Mice
II. Repeated Stimulation of the SCN at CT15 Resulted in Repetitive Phase Delaying of the Homecage
Circadian Activity of the Mice
III. Two Weeks of Phase-Delaying SCN Optogenetic Stimulation Had No Effect on Anxiety-Like Behavior
in the Open Field
Animals: The study consisted of fourteen transgenic vesicular GABA transporter Cre recombinase (Vgat
Cre) X channelrhodopsin (ChR2) mice. Six mice received stimulation and six were no- stimulation handling
controls.
Stereotaxic Fiber Implantation: Mice received chronically implantable fibers (400 µm diameter core,
efficiency ≥85%) targeted above the SCN. (A/P= -0.1 mm, M/L= 0.0mm, D/L= -5.0 mm).
Housing and Software: Following optical fiber implantation surgeries, all mice were singly-housed in
Piezo Sleep boxes. The Piezo Sleep box software quantifies activity values in terms of the amount of
pressure applied and motion within the cage.
Stimulation: To first determine if our stimulation paradigm delayed the circadian activity of the mice, we
stimulated the mice after 5 days of free-running in constant darkness. Mice received a 10 ms blue light
(447-473 nm) pulse at a frequency of 8 Hz for 1 hr at circadian time (CT) 15. After establishing that we
could induce a significant phase delay, we started our chronic stimulation paradigm. Mice were
stimulated every 3 days for 16 days, and then we conducted behavioral tests.
Circadian Rhythm and Mood Disorders
• Association between phase delays, a type of circadian rhythm dysregulation, and major depressive
disorder [1,2].
• To investigate the causal relationship between circadian phase delays and mood disruptions, we
developed a rodent model of chronic phase-delay by selectively stimulating the suprachiasmatic
nucleus (SCN) and observing the mood-related behavioral effects in response to these phase delays.
Optogenetics
Channelrhodopsin: A
light-sensitive
protein from algae
Ion channel that opens in
response to blue light
Take the gene
that encodes for
this protein..
..And insert the DNA into specific
neurons in the brain
Neurons communicate by “firing”, or by sending an electrical signal created by
opening/closing ion channels.
Using optogenetics, we can use blue light to activate channelrhodopsin and cause neurons
to fire.
• Administration of a light pulse during the early
active phase of mice produced a robust phase
delay in mice with a similar genetic background [3].
• Dr. Jones and colleagues demonstrated that mice
entrain to repeated optogenetic stimulation of the
SCN [4]
The Effects of Phase-Delaying Optogenetic Stimulation
of the Suprachiasmatic Nucleus on Mood-Related Behaviors
Christine Heisler, Chelsea Vadnie Ph.D., Ryan Logan Ph.D., Colleen McClung Ph.D.
Translational Neuroscience Program, Department of Psychiatry, University of Pittsburgh, Pittsburgh PA
A Figure 1. Laser equipment setup. A
blue laser (473mm, Class IIIB,
100mw) (A) is connected via a
patch cord to the implantable
optical fiber (B).
(C) Coronal View of Fiber
implantation above SCN
B C
RESULTS IV. Two Weeks of Phase-Delaying SCN Optogenetic Stimulation Had No Effect on Anxiety-Like Behavior
in the Elevated Plus-Maze
• Chronic phase delaying optogenetic stimulation of the SCN resulted in delayed onsets of homecage
activity. Thus, we have established a system to investigate the effects of chronic SCN-mediated phase
delays in mice.
• We found no effect of our stimulation paradigm on depression- and anxiety- like behavior of this small
cohort. It is possible that we may see significant effects with a larger number of mice per group. In
addition, behavior testing was performed under dim red light and it is possible that we may see
different results if behavioral testing is performed in complete darkness to avoid stress-inducing
effects of red light.
• In future studies, we plan to examine the effect of chronic phase advancing on depression and
anxiety-like behavior. We investigated phase delays because of their association with major depressive
disorder, but phase advances may also have a great effect on mood since a phase advancing light
schedule has been shown to more greatly reduce the lifespan of aging mice [5].
1. Hickie et al. BMC Medicine. 11, 79, 2013
2. Lewy et al. J Clin Psychiatry.76 (5), e662–e664, 2015.
3. Honrado et al. J Comp Physiol A. 178, 563-570, 1996.
4. Jones et al. Nature Neuroscience. 18, 373-375, 2015.
5. Davidson et al. Curr Biol. 16(21): R914-R916, 2006.
I thank Dr. Chelsea Vadnie for acting as my mentor and including me on this project. We thank Dr. Yanhua
Huang and Dr. Ryan Logan for lending us the Piezo Sleep Boxes. We thank Kevin Donohue and Bruce
O’Hara at Signal Solutions for making rapid adjustments to the software for the Piezo Sleep Boxes.
Funding for my participation in the SSOE Research Internship Program was provided jointly by Dr. Colleen
McClung, the Swanson School of Engineering, and the Office of the Provost.
0 12 24
METHODS
STUDY AIMS
1) Induce a phase delay in mice by optogenetically stimulating at CT15 .
2) Create a chronic phase delay model.
3) Examine how chronic phase delays effect mood related behaviors in mice.
BACKGROUND
-4
-2
2
4
Phase Response Curve
Dead
Zone
Delay
Zone
Advance
Zone
Subjective
Day
PhaseShift(hr)
Subjective
Night
Circadian Time (hr)
Figure 2. The effect of a single optogenetic stimulation of
the SCN at CT15 on circadian activity. (A) The circadian
activity of a single stimulated mouse presented as a
double plotted actogram. The black vertical bars indicate
activity bouts and the height of the bars corresponds to
activity intensity. Gray shading indicates when the mouse
was placed in constant darkness. The day of stimulation
is marked with a red arrow. Stimulation resulted in a
delay of activity onset on the following day. (B). The
circadian activity of a control mouse. (C) The free-running
period of the two groups of mice were similar during
constant darkness. (D) Stimulation delayed the circadian
activity of the mice. *p < 0.05. n = 6.
Figure 3. The effect of repeated optogenetic
stimulations of the SCN on circadian activity. (A) The
circadian activity of a single mouse that received a
stimulation every three days at CT15. The red
arrows indicate the stimulation days. Circadian
activity onsets were delayed after each stimulation.
(B) The circadian activity of a control mouse. (C)
Chronic optogenetic stimulation of the SCN at CT15
resulted in chronic phase delaying relative to the
control animals. *p = 0.0001. n = 6.
Figure 4. Mice were placed in the
open field for 15 minutes to examine
the effects of chronic phase delaying
SCN stimulation on anxiety-like
behavior. There was no effect of the
simulations on the amount of time
mice spent in the center (A) or the
number of center entries (B). Mice
traveled a similar distance during the
test (C). n = 6.
Stimulated
0 12 012
Days
0 12 012
Control
Days
-50
0
50
100
Stimulation #
1 2 3 4 5
Stimulated
Control
*
23.0
23.5
24.0
24.5
25.0
Control Stimulated
Tau(hr)
Control Stimulated
0
40
80
100
PhaseDelay(min)
PhaseDelay(min)
ZT Time (hr)
ZT Time (hr)
0 12 012
Stimulated
Time (hr)
Days
0 12 012
Time (hr)
Control
Days
Control Stimulated
0
2
3
4
1
OpenArmEntries
Control Stimulated
0
20
30
40
10
Figure 4. Anxiety-like behavior was also
assessed by placing mice in the elevated plus
maze for 5 minutes, recording the time spent
in the open and closed arms of the maze.
There was no effect of stimulation on open
arm entries (A) or open arm time (B). n = 6.
IV. Two Weeks of Phase-Delaying SCN
Optogenetic Stimulation Had No
Effect on Locomotor Activity
IV. Two Weeks of Phase-Delaying SCN
Optogenetic Stimulation Had No
Effect on Depression-Like Behavior in
the Forced Swim Test
Figure 5. Chronic phase delaying
stimulation had no effect on the
locomotor activity of the mice during
a 1 hour test. n = 6.
Figure 6. Depressed-like behavior was assessed
by examining the immobility behavior of the
mice during a 6 minute forced swim test. There
was no effect of chronic phase delaying
stimulation on the amount of time that the
mice spent immobile (A) or the latency to
immobility (B). n= 6.
OpenArmTime(s)
Stimulated
Control
1500
2000
2500
0
0 10 20 30 40 50
1250
1500
2000
2500
Distance(cm)
Time (min)
Control Stimulated
0
100
150
200
50
LatencytoImmobility(s)
Control Stimulated
Immobility(s)
0
20
40
60
80
100
SUMMARY AND FUTURE DIRECTIONS
REFERENCES
ACKNOWLEDGEMENTS
A
B
C D
A
B
C
Control Stimulated
0
10
15
20
5
CenterDuration(s)
Control Stimulated
0
20
30
40
10
CenterEntries
Control Stimulated
0
20
40
60
80
100
Distance(cm)
A B C
A B
A
A B
*