Effects of Medial Prefrontal Cortex Inhibition on Symptoms of Depression in Long Evans Rats

Anna Miller

Dr. James J. Cortright (Faculty Supervisor)

University of Wisconsin-River Falls

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Introduction

Depression is the most widespread disability on Earth affecting more than 350 million

people of all ages across the globe (World Health Organization, 2015). The pervasive nature of

this illness is testament to a need for better scientific understanding of its nature. It is known that

depression mostly affects women and can lead to self-injury, substance abuse, and even suicide

(World Health Organization, 2015). The gravity of these consequences indicates that depression

is a mental illness which can alter the fabric of an individual’s personality so completely that it

can cause extreme perversions of self-esteem or self-focus.

Self-focus (i.e. the process by which one engages oneself in self-referential processing) is

a core issue in the psychopathology of major depression (Lemogne, Delaveau, Freton, Guionnet,

& Fossati, 2012). When an individual becomes trapped within their possibly inaccurate

perception of self and self-situation it can lead to a sort of cognitive broken record which cycles

through depressing or negative self-referential thought. Previous studies have used functional

neuroimaging to identify that the cortical midline structures, including the medial prefrontal

cortex (mPFC), play a key role in self-referential processing in depressed subjects (Elliott, &

Dolan, 2003; Lemongne et al., 2012). The recency of these findings indicates room for further

research into the role of the mPFC in mediating depression.

Lemongne et al. found that the fMRI patterns of activity in the mPFC were inconsistent

among depressed subjects alternating between overactive and inactive with results suggesting

that self-focus in depression may emerge as a process competing for brain resources due to a lack

of inhibition of the default mode network, which is responsible self-referential thought, resulting

in detrimental effects on externally-oriented cognitive processes resulting in symptoms of

depression. Scopinho et al. (2010) examined the effects of acute reversible inactivation of the

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ventral mPFC in rats and found that inhibition induces antidepressant-like effects. These

conflicting findings indicate support for the proposed study which would examine how inhibition

specifically affects the mPFC and its interaction with symptoms of depression.

The proposed study holds significance in that it builds on research that has aimed to link

specific patterns of activity to specific areas of the PFC as mediating symptoms of depression

(Elliott & Dolan, 2003). The existing literature that has examined the role of the mPFC in

depression, as stated previously, has often found conflicting results in that this area shows both

over-activity and relative inactivity in depressed patients compared to control patients (Elliott &

Dolan, 2003). It should then be implied that further examination of the mPFC is warranted not

only as a possible precursor to the implication of its involvement in mediating depression, but

also in order to provide support for a dominant pattern of brain activity (inhibition) which

interacts with symptoms of depression.

The proposed study aims to provide support for the hypothesized link between the mPFC

and depression by using animal models of learned helplessness, lethargy, and anhedonia as

measures of self-referential processing in depression. In order to maintain high external validity

the proposed study would utilize female Long Evans rats in order to more accurately generalize

findings to the population of women which make up the majority of depressed individuals in

humans. Subjects will be tested for latency in regards to learned helplessness, for lethargy in a

radial arm maze and open field test, and for anhedonia using sucrose pellets. It is hypothesized

that a decrease in learned helplessness, lethargy, and anhedonia will be observed in animals that

have undergone inhibition of the mPFC as that is the area which has been determined to oversee

self-referential processing compared to animals which do not receive this treatment.

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Further examination of the effects of inhibition in areas of the PFC is needed in order to

assess its role mediating symptoms of depression. The proposed study aims to look at drug-

induced mPFC inhibition in animal models of depression in order to contribute to the body of

knowledge surrounding the mental processes of the mPFC.

Methods

Subjects

Female Long-Evans rats will be either housed in pairs or individually with food and

water available ad libitum in a 12 hour light/12 hour dark cycle room.

Drugs

Baclofen and muscimol will be obtained from Sigma (St. Louis, MO) and will be

dissolved in sterile saline (0.9% w/v).

Apparatus

Restrainer. Flat-bottomed restrainers (Braintree Scientific, Inc.) will be used to restrain rats.

The clear plastic restrainers (3.375 inches in diameter × 8.5 inches in length) are semi-cylindrical

with slots for ventilation.

Forced Swim. Plexiglas cylinders (20 centimeters in diameter) filled with 23 C water (30

centimeters deep) will be used to conduct the forced swim test.

Hot Plate. A hot plate (701012, Carolina Biological Supply Company, Burlington, NC) heated

to 55 ± 0.5 C with a glass cylinder (33 inches in height × 18 inches in diameter) placed on top

will be used for the hot plate test.

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Open Field. To assess levels of activity, each rat will be placed in the center of a wooden box

(32 × 32 × 24 inches) with an open top. The floor of the box is ruled into four squares of equal

area. These squares will be used for purposes of scoring activity.

Radial Arm Maze. An elevated, eight-arm radial arm maze (RAM) will be used to assess

spatial memory. The RAM consists of a central platform with eight identical arms extending out

from the center. Small food cups are located at the end of each arm.

Operant Chambers. Behavioral tests of anhedonia will be conducted in operant chambers

measuring 12 × 12 × 10 inches. Each chamber is constructed of Plexiglas and aluminum and is

equipped with a house light and food receptacle. A lever is centered above the receptacle on the

front wall. Lever presses and sucrose pellet rewards will be recorded and controlled via an

electrical interface by a computer using Med Associates software.

Procedure

Unpredictable Chronic Mild Stress. In order to induce depression, an unpredictable chronic

mild stress (UCMS) modified from Ortiz et al. (1996) and Shi et al. (2010) will be used.

Animals in the UCMS condition will be housed individually and receive stressors presented in a

pseudo-random order for 28 days. Stressors will include restraint for 6 hours, wet bedding for 12

hours, food and water deprivation for 12 hours, 16 hours of cage tilt (45), and forced swim tests.

A control group will be housed in pairs and left undisturbed.

Identification of Stage of Estrous Cycle. Due to the fact that there is a clear association

between depression and specific phases of estrous (Jenkins et al., 2001), vaginal lavages will be

performed daily to identify each rat’s stage of the estrous cycle. Saline will be used for each

procedure and unstained samples will be examined under a light microscope.

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Surgery. All surgical instruments (stereotaxic ear bars, surgical scissors, forceps, hemostats,

surgical needles for suturing, skull screw drill bits and handles, electric drill bits, spatulas,

screwdrivers, guide holders, bulldog clips, and scalpel handles) will be cleaned with liquinox and

water to remove any debris. After drying, they will be placed in a sterilization pouch along with

a sterility indicator. Additional surgical accessories such as suture thread, skull screws, gauze,

and cotton-tipped applicators will also be placed into a sterilization pouch along with a sterility

indicator. All surgical tools and accessories will then autoclaved at 121°C (steam sterilized for

30 minutes followed by a drying time of 20 minutes). The remainder of the stereotaxic apparatus

that cannot be autoclaved will be disinfected with CaviWipes. Dental cement mixing dishes will

be disinfected with CaviWipes. After autoclaving, all surgical tools and implants will remain in

the sterilization pouch until use. Upon use, the contents of the sterilization pouches will be

dumped onto a sterile drape so as not to touch the instruments. Between surgeries, all tools will

be washed with liquinox and water, dried, and then placed in the bead sterilizer (only the usable

tip) for approximately 10-20 seconds.

Following the chronic stress exposure procedure, rats will be anesthetized with a mix of

ketamine (100 mg/kg, IP) and xylazine (6 mg/kg, IP) and surgically implanted with chronic

bilateral guide cannulae targeting the mPFC using procedures described previously (Loweth et

al., 2013). The incision area will be shaven with a clipper. Puralube will be applied to the eyes.

The incision area will be scrubbed three times with betadine before an incision is made in the

skin. An incision will be made on the head and small bur holes (bilateral) will be made to the

exposed skull through which guide cannulae (22 gauge, Plastics One, Roanoke, VA) will be

lowered into the mPFC. These will be held in place with a dental cement cap anchored in place

by skull screws. The animal will be administered 10cc saline subcutaneously and Animax

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antibiotic ointment will be applied. Following the 45 – 60 minute surgery, 28 gauge Plastics One

obturators will be inserted into the guide cannulae and the animals will returned to their home

cages for a 7 – 10 day recovery period.

Medial Prefrontal Cortex Inhibition. Prior to each behavioral test the mPFC cortex will be

inhibited with a baclofen (0.3nmol/0.5µl/side) and muscimol (0.3nmol/0.5µl/side) cocktail based

on previous studies (McFarland & Kalivas, 2001; Rogers et al., 2008). Injection cannulae will

protrude 6 millimeters beyond the guide cannulae tips. Microinjections will be made over 10

minutes and the injection cannulae left in place for an additional 5 minutes to allow for diffusion

of the cocktail and absorption into brain tissue. Following removal of injection cannulae,

obturators will be re-inserted into the guide cannulae and remain in place until the end of the

experiment.

Behavioral Tests. A battery of behavioral tests will be used to assess depressive-like behaviors.

Animals will undergo behavioral testing in a counterbalanced design.

Open Field Test. Rats will be individually tested for six minutes in the open field. The

activity level of each animal will be scored to assess levels of depressive-like behavior. To

ensure accurate scoring of behavior, the open field test will be videotaped.

Radial Arm Maze Test. The end of each arm will be baited with a sucrose pellet. Rats

will be placed in the center of the RAM at the beginning of the every trial and be required to visit

each arm only once. If an arm is revisited during the trial, an error will be recorded. The time it

takes each subject to complete the trial will also be recorded. The RAM will be cleaned between

trials to eliminate olfactory cues.

Forced Swim Test. As described previously (Li et al., 2013), rats will be immersed in

Plexiglas cylinders (20 centimeters in diameter) filled with 23 C water (30 centimeters deep).

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Immobility will be scored with stopwatches for four, three minute time bins excluding the first

minute of the trial. Immobility will be defined as no movement other than those necessary to

keep the animal’s head above water.

Hot Plate Test. Similar to Shi et al. (2010), rats will be placed on a hot plate heated to 55

± 0.5 C with a glass cylinder (33 inches in height × 18 inches in diameter) placed on top. The

latency of the first hind paw withdrawal/licking will be measured in seconds as an index of

nociceptive threshold. To minimize tissue damage, a cut-off time of 30 seconds will be adopted.

Operant Responding for Sucrose. Reinforced lever presses will result in delivery of a

sucrose pellet. Testing will consist of 7 daily sessions. Rats will obtain sucrose pellets on a

fixed ratio one schedule of reinforcement (each reinforced lever press will result in a reward).

Number of pellets obtained will be recorded. In order to assess anhedonia in the final two

sessions, rats will be tested under a progressive ratio schedule. Under this schedule, the number

of responses required to earn each sucrose pellet will be determined by ROUND [5 × EXP (0.25

× reward number) – 5] to produce the following sequence of required lever presses: 1, 3, 6, 9, 12,

17, 24, 32, 42, 56, 73, 95, 124, 161, 208, etc (Richardson and Roberts, 1996). Daily progressive

ratio sessions will be terminated after two hours of after one hour elapses without a reward. The

number of lever presses and sucrose pellets obtained in each session will be recorded.

Euthanasia. Rats will be deeply anesthetized with a mix of ketamine (100 mg/kg, IP) and

xylazine (6 mg/kg, IP) and perfused via intracardiac infusion of saline and 10% formalin to allow

for fixing and removal of brains in order to verify cannulae placement.

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