Cis-flupenthixol Infusion In The Ventral Striatum On Amphetamine-Induced Behavior: Transition to Schizophrenia

Cis-flupenthixol Infusion In The Ventral Striatum On Amphetamine-Induced Behavior

Cis-flupenthixol Infusion In The Ventral Striatum

On Amphetamine-Induced Behavior:

Transition to Schizophrenia

Tyler S. Harikawa

University of California Santa Barbara

PSY 111L

Professor Szumlinski

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Abstract

Amphetamine-induced behavior show similar activities in the D1/D2 dopamine pathways in

schizophrenic individuals through the change of ventral striatal dopamine emission that disrupts the

dopamine transmission in subcortical sites/nucleus accumben. Through the infusion of cis-flupenthixol

into the ventral striatum of Sprague-Dawley rat subjects, amphetamine-induced behavior is expected to

be reduced through the blockade of the ventral striatal dopamine pathway so there would be no

disruption between the D1/D2 dopamine pathways. Unfortunately, the results came out as a failure in

methodological procedures due to the misplacement of the microinjector to the dorsal or anterior side

of the ventral striatum. As a result, the amphetamine-induced behavior wasn't reduced after the infusion

of cis-flupenthixol. Through the examination of studies by Kelly, Ahlenius, Bast, Moran, and Ikemoto

we were able to come to a result the drug is unable to spread ventrally into the ventral striatal region, it

only spread dorsally, where there is no mechanism that can alter the D1/D2 dopamine pathways.

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Introduction

Schizophrenia is a neuropsychiatric disease that has been linked to amphetamine-induced

activities due to the changes in the cortical and subcortical dopamine systems. In schizophrenic

individuals, the disruption of dopamine pathways in the subcortical sites caused by the reduced

dopamine conveyance from the medial prefrontal cortex (mPFC) leads to the development of

amphetamine-induced behavior (Bast, 2002). These changes in dopamine pathways are caused by the

use of dopamine agonist amphetamines such as cocaine or prescription stimulants. According to prior

research, early exposure to amphetamine prescriptions stimulants such as Adderall had been diagnosed

with types of psychosis, including schizophrenia (Moran, 2015). This emphasizes that younger

individuals that have been constantly taking amphetamine-induced prescription stimulants undergo an

oversensitivity of the striatum reduced dopamine transmission that can lead to a higher chance of

experiencing neuropsychiatric symptoms. Using a small dosage of cis-flupenthixol, which is a

antipsychotic used in schizophrenic patients, through bilateral injections of the brain, there should be

blocked stereotypies and hyperactivity in those amphetamine-induced behaviors (Ahlenius, 1986).

Precise microinjection to the ventral striatum is key to making the small dose of cis-flupenthixol work.

In rats depending on the dosages given, amphetamine injections would cause some range of

amphetamine-induced activity such as hyperactivity, locomotion, and augmented stereotyped

behaviors. Followed by the D1/D2 receptor antagonist cis-flupenthixol administration to the rat, there

should be a response in the ventral striatum that blocks the nucleus accumbal dompamine system,

which reduces the effect of amphetamine-induced behaviors. Assuming through this procedure will

bring upon those expectations, we will be looking at the effects of cis-flupenthixol in the ventral

striatum based on its enhancement or reduction of amphetamine-induced activities.

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Methods

Subjects

Sprague-Dawley strain of rats were housed in groups of two per home cage under standard

conditions. Food and water chambers are installed in each home cage for the rats for their availability

ad libitum. A rat was assigned to each groups of four undergraduate students for this study. In order for

the researchers to be accustomed to handling rats, each students carried the subject in their arms and

cared for them like you would for a pet. Cases in which the rats appeared jumpy, extra care was needed

in order for the subjects to be familiarized with the feeling of human handling. The rats were tested at

during the light phase of the 12-h day-12-h night cycle, starting at 1:00pm. With the approval of the

IACUC of the University of California Santa Barbara, the study obeyed procedures that is uniform with

NIH guidelines emphasized in the Guide for Care and Use of Laboratory Animals (2011).

Drugs

D-amphetamine (Sigma-Aldrich) at dosages of 4 mg/kg (1.0 ml/kg) or saline at dosage of 1.0

ml/kg were used for experimental use via subcutaneous injection. The amphetamine served as the

experimental injection, and the saline handled as the control injection. However there was some

amphetamine powder added to saline, which makes the control a placebo. Half the subjects were given

the saline while the other half was given the amphetamine injection. After that, cis-flupenthixol (10

mg/ml) at dosages of 1 µl per side of the brain was loaded through intracranial microinjection. Cis-

flupenthixol is a thioxanthene class antipsychotic drug that is used for individuals diagnosed with

schizophrenia that is also used as an antidepressant in low doses.

Apparatus

Locomotor activity of the subjects were analyzed in a black Plexiglas activity chambers

(22x43x33 cm) that was high enough so the Sprague-Dawley don't escape during experimentation. The

exclusion of the roof in the black cage allowed a better observation for stereotypy and repetitive head

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movement.

Surgery

Procedures for intracranial implantation of bilateral guide cannulae started with the use of

aseptic techniques to anesthetize the subjects. Banamine (2 mg/kg), a non-opioid analgesic, was

administered peri-operatively to maintain jurisdiction of the post-operative pain at a dosage of 1mg/kg

through subcutaneous microinjection. An intramuscular injection of Ketamine, a dissociative

anesthetic, and Xylazine, muscle relaxant, (in a 7.5:1 ratio) from 100mg/ml was administered. The

level of anesthesia given was checked through the examination of a hind leg withdrawal reflex after

pinching the toe. If the subject didn't have a reflex, then the subject has been anesthetized. Next, the rat

was placed into a stereotaxic instrument guide cannulae that keeps the head steady for the surgery.

Once that is confirmed, microinjector guide cannulae was planted in both sides of the brain 2 mm over

the nucleus accumbens core (NAC), and microdialysis guide cannulae was implanted in both sides of

the brain 3mm over the nucleus accumbens core (NAC) for neuroscience experimentation. The NAC

coordinates used for the anterior posterior (AP): +1 mm; dorsal ventral (DV): -6 mm. Using Paxinos

and Watsons (2000) rat brain atlas, we determined these coordinates. The AP coordinates are in point

junction to Bregma, as the DV coordinates were point junction to the surface of the skull. These

cannulae were screwed in with steel skull screws (Specialty Tool and Bolt, Goleta, CA, USA) and

followed with the applying of dental cement to keep the screws in a fixed position on the brain. 6 ml of

saline was administered to the subject to rehydrate. The animals were supervised for 4 days to observe

changes in health. Then behavioral testing began 7 days later.

Behavioral Scoring

We used two different rating scales in scoring the general stereotypy (GS) (Creese and Iversen

1972) and repetitive head movements (RHM) (Ujike et al. 1992) of the Sprague-Dawley subjects. The

GS scale was used to measure the locomotion, rearing and sniffing rate, while the RHM scale measured

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the amount of head movement in the test subjects. Using these scales, we measured the Sprague

Dawley behavior during its pre-treatment habituation period over the course of 30 minutes. During

every 5 minute intervals, the subject was observed for 15 seconds and behaviorally rated in terms of the

GS and RHM scale. For the post-treatment session, the same two scales were used per 5 minute

intervals with 15 seconds monitoring over the course of 60 minutes. After the experimental session, the

scores were added up and an average rating was taken for a final result for the two scales.

Tissue Collection/Histology

Before the tissue collection starts, the T.A.'s injects half the rats with 1 ml/kg of saline

subcutaneously (SC) and the other half with 4 mg/ml/kg of amphetamine SC. Prior to tissue extraction,

a 1-1.5 ml dosage of Euthasol was injected intraperitoneally (IP) which was done to euthanize the rat so

the tissue is still in a fixed structure. Once the subject is euthanized, we place the rat on its back and

proceed to open the ribcage and sternum to expose the heart. When the left ventricle is exposed, we

proceed to inject 0.1 ml of heparin into the left ventricle, which is done to prevent blood coagulation.

Now that the blood is easier to clear out, we inject two doses of 30 ml PBS phosphate buffer through

the left ventricle, which allows us to clear the blood out of the body. When that is done, we move on to

inject two doses of PFA paraformaldehyde through the same ventricle, which allows the tissues to be

fixated for extraction. Once we remove the brain from the skull, we place it in a jar with 20% sucrose

in phosphate buffer to preserve it and placed in dry ice for 30 seconds. Using the cryostat, we obtain

slices of the striatum and use Nissl/Cresyl Violet stain cell bodies of neurons to have a better

visualization of the placement of microinjectors in the dorsal striatum. This is done by chronologically

placing the slides of the striatum slices into different concentrations of ethanol (70%, 50% ETOH),

nano water, cresyl violet, different concentrations of ethanol again (70%, 95%, 95% + 15 drops of

acetic acid, 100% ETOH), and HemoDe (a clearing agent). The ethanols allows the slides to dehydrate,

nanowater serves to hydrate, the Cresyl violet allows the staining to occur, and the HemoDe serves as a

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clearing agent that has low toxicity levels which allows minimal tissue damage of the striatum slices.

Statistical Analysis

The average general stereotypy and repetitive head movement ratings made by 3-4 students

were averaged for each 5-min time-bin for each rat and these averages, as well as the average total

rating for both scales were used in the statistical analysis of the data from the habituation session. For

both variables, the data from the habituation session were analyzed using a Section (Tues, Wed, Thurs,

Fri) X Time analyses of variance (ANOVAs) with repeated measures on the Time factor (7, 5-min time-

bins). Significant interactions were followed-up by Least Significant Difference (LSD) post-hoc tests.

The data for the average total scores were analyzed by a one-way ANOVA, followed by LSD post-hoc

tests. To confirm that behavior habituated during the 30-min session, the data for the average of all of

the rats were also analyzed using a repeated measures ANOVA (7, 5-min time-bins), followed by paired

samples t-tests. For the amphetamine session, the data for both variables were analyzed using a Section

(Tues, Wed, Thurs, Fri) Pretreatment (saline vs. cis-flupenthixol) X Time analyses of variance

(ANOVAs) with repeated measures on the Time factor (13, 5-min time-bins) and the data for the

average total scores were analyzed by a Section X Pretreatment univariate ANOVA. As there were no

main effects of Section, the data for the rats in each section were collapsed for data presentation of

amphetamine-induced behavior. To determine whether or not a relation existed between baseline motor

activity and amphetamine-induced locomotion, Pearson correlational analyses were conducted on the

total scores for both stereotypy and repetitive head movements for all animals.

Results

Baseline motor activity

A Section by Time ANOVA conducted on the data for General Stereotypy indicated a significant

interaction between factors [F(6,72)=6.36, p<0.0001], but no main effect of Section or Section by Time

interaction (p’s>0.05). As illustrated in Figure 1A, the general stereotypy scores declined progressively

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throughout the 30-min habituation session. This observation was supported by the results of a linear

within-subjects contrast [F(1,12)=32.05, p<0.0001], and pair-wise t-test comparisons of the initial

stereotypy rating (time=0 min) with those subsequent indicated significantly lower ratings during the

25 min [t(15)=3.37, p=0.004] and 30 min [t(15)=6.48, p<0.0001] time-bins. In contrast, a similar

analysis conducted on the data for Repetitive Head Movements failed to indicate any change in

behavior across time during the habituation session (Figure 1B; Time effect and interaction: p’s>0.05)

and the ratings of head movements were consistent across sections (Section effect: p>0.05).

Cis-Flupenthixol effects upon amphetamine-induced motor activity

A Section by Pretreatment by Time ANOVA conducted on the data for both General Stereotypy

and Repetitive Head Movements failed to indicate any effect of intracranial pretreatment (Figure 2A)

[Time effect: F(12,96)=13.11, p<0.0001; Pretreatment effect and interactions, p’s>0.25; Section effect

and interactions, p’s>0.60] and an analysis of the total stereotypy score also did not support group

differences (Figure 2B) [Section X Pretreatment ANOVA, all ps’>0.60]. Likewise, comparable analyses

of the results for Repetitive Head Movements also failed to indicate an effect of cis-flupenthixol upon

the time-course of amphetamine-induced behavior (Figure 2C) [Time effect: F(12,168)=27.18,

p<0.0001; Pretreatment effect and interaction, p’s>0.45; Section effect and interaction, p’s>0.55] or

upon total behavior (Figure 2D) [Section X Pretreatment ANOVA, p’s>0.25].

Correlation between baseline and amphetamine-induced motor activity

As the results of the intracranial study failed to indicate any effect of cis-flupenthixol upon

amphetamine-induced behavior, the data for amphetamine-induced behavior was collapsed across

Pretreatment groups for correlational analyses. Despite the large sample size (n=16), correlational

analyses failed to indicate any relation between baseline behavior and amphetamine-induced behavior

(data not shown) [for Stereotypy, r=0.07, p=0.87; for Repetitive Head Movements, r=0.30, p=0.47].

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Discussion

Schizophrenia is caused by the reduced medial prefrontal cortex dopamine transmission that

antagonizes the dopamine emission in subcortical sites, which include the nucleus accumben and

ventral striatal region. The nucleus accumben or nucleus caudate is a key factor for amphetamine-

induced stereotypy (Kelly, 1975). Dopamine activities in schizophrenic patients are similar to

individuals with amphetamine-induced behaviors. Amphetamine-induced behavior was demonstrated

through the subcutaneous administration of d-amphetamine on the rats, which displayed hyperactivity

and increased stereotypy in the early minutes of the session and those behaviors decreasing overtime

(Fig. 1 [C]). From what the results presents, we were unable to uncover any findings on the cis-

flupenthixol effect in amphetamine-induced behaviors, which is unusual for a antipsychotic for

schizophrenia. The results indicate that after the cis-flupenthixol infusion into the brain, general

stereotypy (GS) and repetitive head movement (RHM) increased, which emphasizes that stereotypy

and locomotion did not help reduce amphetamine-induced behaviors. Perhaps the flaw lies in our

methodological misplacement of the microinjector or not a strong enough dosage of the drug.

In Fig. 1 (A), the graph shows that the Friday section groups experienced a higher stereotypy

score between the period of 10-20 minute of the session. Compared to the other sections, stereotypy

scores increased at the 10 minute mark compared to the other sections in which the stereotypy scores

decreases overtime. It is possible that the amphetamine had a delayed response in some particular case

due to an behavioral activation from the nucleus accumben core or the lateral/posterior tubercle

(Ikemoto 2002). Ikemoto states in his study of the ventral striatal anatomy of locomotor activity

induced by d-amphetamine, that a delayed response of the amphetamine is a result of a altered pathway

of the amphetamine. Normally the amphetamine will cause the medial ventral striatum to induce a

amphetamine-induced response. However the anesthetic action caused by the amphetamine allowed the

nucleus accumben core and the lateral/posterior tubercle to be more vulnerable than the medial ventral

striatum. Another possibility could be a simple methodological scoring error by the Friday session.

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Limitations of the Study

The histology of the brain tissue presents (Fig. 3) that the cis-flupenthixol microinjections

missed the ventral striatum and hit mostly on the dorsal or anterior end of the brain. For the experiment

to produce any results, cis-flupenthixol needed to be applied into the nucleus accumben in the ventral

region. We did have one successful injection in the ventral region, however that was for the saline

solution which wouldn't produce any changes in the spontaneous activity and amphetamine-induced

behaviors even when injected in the sweet spot. It can be assumed that the injection of cis-flupenthixol

showed no changes in the rats amphetamine-induced behavior because of misplacement of the

microinjector is too dorsal, which failed to block the dopamine D1/D2 receptor in the medial prefrontal

cortex.

Different amounts of the dosage of the cis-flupenthixol could not have played a factor in the

failed alteration in the amphetamine-induced behaviors. Based on prior studies of dopamine receptor

blockade in the rats medial prefrontal cortex, if the cis-flupenthixol microinjector hit anterior to the

ventral striatal region a higher dosage of cis-flupenthixol would not antagonize the effect in the D1/D2

receptors because the drug infusion spreads dorsally along the guide cannulae (Bast, 2002). Since our

microinjections were too dorsal, the drug isn't able to spread to the ventral striatal region. This

emphasizes that quality precision into the ventral region was necessary in order to inhibit

amphetamine-induced responses. In previous studies from Kelly, the nucleus accumben/ventral

striatum is a key point of general stereotypy and repetitive head movement. This is an implication that

the nucleus accumben as the primary part of the brain responsible for locomotor stereotypy in the

amphetamine-induced rats.

Future Study and Conclusion

Although this experience was a result of errors in methodological steps in the microinjection of

the amphetamines bilaterally into the ventral striatum, it does show the role of cis-flupenthixol in the

D1/D2 pathways when injected in the correct ventral striatal spot. It explains how schizophrenic

symptoms can be induced through the infusion of cis-flupenthixol in the nucleus accumben/ventral

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striatal region. Infusion in the dorsal/anterior regions causes no effect in the behavior due to the dorsal

spread of the drug. And in addition amphetamine responses differ depending on which part of the brain

the amphetamine spreads to. In order to gain a complete understanding of the mechanisms of the D1/D2

dopamine pathways, further research is needed in the effectiveness of cis-flupenthixol in various parts

of the brain. Dorsal infusion shows no change in amphetamine-induced behavior due to the drugs

failure to spread ventrally into the nucleus accumben, so further study is needed to conduct experiments

on the ventral striatum.

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References

Ahlenius, S., Hillegaart, V., Thorell, G., Magnusson, O., & Fowler, C. (1986). Suppression of

exploratory locomotor activity and increase in dopamine turnover following the local

application of cis-flupenthixol into limbic projection areas of the rat striatum. Brain Research,

402, 131-138.

Bast, T., Pezze, M.A., & Feldon, J. (2002). Dopamine receptor blockade in the rat medial prefrontal

cortex reduces spontaneous and amphetamine-induced activity and does not affect prepulse

inhibition. Behavioral Pharmacology, 13(8), 669-673. Retrieved from http://www.ovid.com

Hoogstraten-Miller, S., & Brown, Patricia. (2008). Techniques in Aseptic Rodent Surgery. NIH Public

Access, 1-27. doi:10.1002/0471142735.im0112s82

Ikemoto, S. (2002). Ventral striatal anatomy of locomotor activity induced by cocaine, D-amphetamine,

dopamine and D1/D2 agonists. Neuroscience, 113(4), 939-955. doi:10.1016/S0306-

4522(02)00247-6

Kelly, P., Seviour, Paul., & Iversen, S. (1975). Amphetamine and apomorphine responses in the rat

following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum. Brain

Research, 94, 507-522.

Medhus, S., Borger, E., Cand, R., Gossop, M., Holm, B., Morland, J., & Bramness, J. (2015).

Amphetamine-induced psychosis: Transition to schizophrenia and mortality in a small

prospective sample. The American Journal on Addiction, 7, 586-589. DOI: 10.1111/ajad.12274

Moran, L., Masters, Grace., Pingali, S., Cohem, B., Liebson, E., Rajararethinam, R.P., & Ongur, D.

(2015). Prescription stimulant use is associated with earlier onset of psychosis. Journal of

Psychiatric Research, 71, 41-47. doi:10.1016/j.jpsychires.2015.09.012

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Figure of Legend

Effect of subcutaneous infusion of amphetamines on rats based on general stereotypy score (GS) and repetitive head movement score (RHM). (A) & (D) is based on a minutes vs GS/RHM scores graph that illustrates the behaviors of the amphetamine-induced rats in 5 minute periods distinguished by different shapes and colors. ( C ) and (F) displays the average distribution. (B) & (E) is distributed in a total score basis of each section. The figure above indicates an average decline of the GS and RHM behaviors over the time-course of the amphetamine session.

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Effect of the saline and cis-flupenthixol bilateral microinjection into the ventral striatal region of the brain to amphetamine-induced rats. The scores are based on general stereotypy (GS) and repetitive head movement (RHM). (A) & ( C ) displayed in a GS/RHM score vs minute scale scored in 5 minute intervals. It depicts a linear relationship between the saline and cis-flu, which depicts an error in the experiment because cis-flu is supposed to reduce those stereotypy scores. (B) & (D) depicts the total score of the GS and RHM scale in terms of the saline and cis-flu infused subjects. Total score of the cis-flu subjects should be lower due to its function to block dopamine receptors.

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AMPH-SAL stands for the saline solution infused into the brain. AMPH-CIS stands for the cis-flupenthixol infusion. As show in the above, these infusion missed the microinjection point severely. Most of these injections are too dorsal or anterior to are objective point, which was the ventral striatum region near the ventricles of the brain. We had one successful saline microinjection, however that doesn't help show our desired outcome.

AMPH*CISAMPH*SAL

Fig. 3: Psych 111L F015

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Cis-flupenthixol Infusion In The Ventral Striatum On Amphetamine-Induced Behavior: Transition to Schizophrenia