The Role of Metabotropic Glutamate Receptor Agonists in 5-HT2 Agonist Induced

Prefrontal Cortex Dopamine Release

Sameed S. Shaikh, Tara Byrd, and Elizabeth A. Pehek

Departments of Biochemistry(S.S.S),Psychiatry and Neurosciences (E.A.P) , Case Western Reserve University,

Cleveland, OH, 44106; and Louis Stokes Cleveland DVA Medical Center, Cleveland, OH, 44106

Abstract

A variety of factors have been implicated in the physiopathology of

schizophrenia. Current treatments all decrease dopamine function by blocking D2-like

dopamine receptors. Previous research has shown an increase in dopamine levels linked

to increased glutamate and serotonin release in the prefrontal cortex. We examined the

neurocircuitry of these systems by measuring dopamine release in vivo using

microdialysis in the prefrontal cortex of the rat. Rats were treated with 1-(2,5

dimenthoxy-4-iodophenyl)-2-aminopropane (DOI), a 5-HT2A/2C agonist and (-)-2-oxa-4-

aminobicylo[3.1.0]hexane-4,6-dicarboxylate (LY379268), a Glu2/3 agonist. Our research

so far is only preliminary and the findings have yet to be conclusive. We have reaffirmed

our laboratories’ previous finding that DOI administration increases prefrontal cortex

dopamine release. With further research we hope to develop a more comprehensive

knowledge of the circuitry involved in normal and abnormal neural function for a variety

of clinical applications, including schizophrenia.

Introduction

Schizophrenia is a neurological disorder characterized by impairment in higher

order brain function (Moghaddam, 2003). This includes four basic categories of function;

(1) information and sensory processing, (2) – abnormal mood and affect, (3) – cognitive

impairment, including memory and attention, and (4) – movement abnormalities

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(Moghaddam, 2003). It is general stated that schizophrenic symptoms are both “positive”,

in the sense that delusions and hallucinations are added to normal perception, and

“negative”, including social withdrawal and apathy (Sawa, 2002). In addition,

schizophrenics exhibit cognitive defects, including a decrease in working memory.

Presently, no conclusive model exists for the physiological cause of

schizophrenia, although a variety of hypotheses have been presented. A variety of

structural changes have been identified in the brains of schizophrenics, ultimately finding

a 6% reduction in total brain mass with 21% reductions in the temporal lobe gray matter

(Roberts, 1991). This is associated with an increase in ventricular size. (Roberts, 1991).

Recent genetic inquiry into schizophrenia has found many genes that may be involved in

the development of the disease. Like cancer, it is suggested that mutations and

modifications to multiple genes may compound to have some role in the development of

the disorder (Chow, 2006). The deletion of the gene 22q11, and the resulting “22q11

Deletion Syndrome” (22q11DS), has been shown to correlate to schizophrenia. Between

25 to 30% of patients with 22q11DS develop full blow schizophrenia, accounting for

about 2% of the total schizophrenic population (Chow, 2006).

The hypotheses of schizophrenia with the greatest clinical application have been

neurobiological. During the 1950s, the drug chlorpromazine, a neuroleptic, was found to

alleviate many of the positive symptoms of schizophrenia (Sawa, 2002). Further research

showed neurolepics to be D2 dopamine receptor antagonists. Around the same time it

was found that amphetamine, which triggers the release of dopamine (DA), exacerbated

symptoms of schizophrenia. The result was the “Dopamine Hyperactivity Hypothesis” of

schizophrenia, which attributes the symptoms of the disease to an increase in DA levels

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(Sawa, 2002). Since that time until the later 1990s, research and drug development

focused on D2-like dopamine systems (D2, D3, and D4 subtypes) (Nestler, 1997). It

should also be noted that some of the neuroleptics that were developed also showed a

high potency at 5-HT2 receptors, suggesting a possible role for serotonin in the

development of schizophrenia (Sawa, 2002).

With positron emission tomography (PET), researchers were able to identify a

similar expression of D2-like receptors in schizophrenics and non-schizophrenics, but

found a sharp reduction of D1-like DA receptors (D1 and D5) in the PFC of

schizophrenics (Okubu, 1997). D1-like receptors have been found to be the most

expressed DA receptors in the dorsolateral PFC and studies have shown the activation of

the D1-like receptors to be crucial for development of working memory in non-human

primates (Abi-Dargham, 2002). Alterations in PFC activation have been observed in

schizophrenics completing working memory tasks (Nestler, 1997).

The NMDA-receptor (NMDAR) is an ionotropic receptor for glutamate, which is

the most abundant excitatory neurotransmitter in the central nervous system (CNS).

Nearly all cortical efferent pathways are glutamatergic, implying glutamate irregularity

may lead to the total higher level dysfunction typical of schizophrenia (Moghaddam,

2004). Early research, dating to the 1970s, has shown irregular glutamate levels in the

cerebrospinal fluid of schizophrenics but was not extended upon until recently

(Moghaddam, 2003). It has also been found that phencyclidine (PCP), a drug that creates

effects similar to schizophrenia, is a NMDAR antagonist (Moghaddam, 2003). Through

this research the “Glutamate Hypothesis” of schizophrenia emerged. NMDARs, however,

are located throughout the CNS so direct drug targeting of the receptor is considered to

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be a risky choice at best. Luckily, sequencing of the human genome brought to light

another class of glutamate receptors. The metabotropic receptors (mGluRs) are G-protein

coupled and modulate the activity related to NMDA activation (Lewis, 2006).

Specifically, mGlu5 has been shown to modulate the sensitivity of the NMDAR itself

through a phospholipase-C linked pathway. The Group II mGluRs, mGlu2 and mGlu3,

have been shown to attenuate the release of glutamate from presynaptic neurons through

an adenyl cyclase linked pathway (Moghaddam, 2004). The highly selective nature of the

mGluRs has made them an ideal drug target, when compared to the ubiquitous NMDA

receptor (Moghaddam, 2004). The mGlu5 receptor has been shown to desensitize rapidly,

making it a poor drug target, but current literature suggests that mGlu2 and 3 may be an

appropriate target for glutamate control (Moghaddam, 2004). The general hypothesis

regarding the role of mGlu2/3 agonists in treatment of schizophrenia is based on a series

of findings within the last decade. NMDA antagonists, and presumably schizophrenics,

have increased glutamate levels in the prefrontal cortex while over-activating glutamate

neurons with non-NMDA receptors (AMPA and Kainate receptors are also ionotropic

receptors stimulated by glutamate).

As mentioned before, many antipsychotics bind 5-HT2 receptors with a high

affinity, implicating a role for serotonin in the development and/or treatment of

schizophrenia. The mesocortical DA pathway, which connects the ventral tegmental area

(VTA) to the cerebral cortex, is innervated by serotonergic neurons (Pehek, 2006). 1-(2,5

dimenthoxy-4-iodophenyl)-2-aminopropane (DOI) is a 5-HT2A/2C agonist and powerful

hallucinogen. Administration of DOI in animals has shown to produce behaviors similar

to schizophrenia (Zhai, 2003). It has been shown that administration of DOI in rats

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increases glutamergic transmission in the prefrontal cortex (Marek, 2000). Studies have

also suggested that increased glutamate levels in the PFC result in an increased DA

activity (Takahata, 1998). Our lab has also previously shown an increase in cortical DA

levels resulting from systemic injection of DOI (Pehek, 2006). On the basis of these

findings, numerous studies have investigated group II mGluR agonists and their effect on

DOI symptoms. Administration of (-)-2-oxa-4-aminobicylo[3.1.0]hexane-4,6-

dicarboxylate (LY379268), a GluR2/3 agonist, along with DOI, shows a decrease in the

expression of the gene c-fos, which is normally increased with DOI along (Zhai, 2003).

LY379268 has also been found to inhibit DOI-associated head shakes and excitatory

postsynaptic potentials (EPSPs) in the rat cortex, in a dose dependent manner

(Klodzinska, 2002).

Based on the aforementioned research, we hypothesize that the effect of the 5-

HT2 agonist, DOI, is dependent on local glutamate release in the PFC. We tested this

relationship by injecting rats with DOI and using in vivo microdialysis, coupled with high

pressure liquid chromatography (HPLC) with electrochemical detection, to measure DA

in the PFC. We then examined the ability of the mGluR2,3 agonist LY379268 to decrease

glutamate-depended DA release by DOI .

Methods & Materials

Animals & Surgery

Male Sprague-Dawley rats weighing between 250 and 350g at the time of surgery

were used. Before surgery, rats were housed in pairs at the Veterans Affairs Louis Stokes

Medical Center Animal Research Facility. Housing was in temperature-controlled room

and on a 12 hour light/dark cycle. Food and water were available ad libitum. For

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surgery, rats were administered a mixture of ketamine (70 mg/kg) and xylazine (6

mg/kg), injected intramuscularly. Additional ketamine was administered, if needed, to

ensure the rat was unconscious. The top of the rat’s head was shaved and then mounted

in a stereotaxic frame. After swabbing the skin with betadine and injection of 2 mL

marcaine subcutaneously, an initial incision was made. A hole was then drilled in the

skull above the prefrontal cortex. After puncture of the dura, a guide cannula (CMA

microdialysis) was lowered into the prefrontal corex (+0.32 mm AP, ±0.07 mm ML, from

bregma according to Paxinos and Watson, 1998). In order to control for potential,

hemispheric differences, half of the cannula were placed on the left side and half on the

right. Three screws were inserted into the skull, one in each quadrant, and were held in

place with cranioplastic cement. This served to anchor the assembly surrounding the

guide cannula. A clamp for a tether was also installed into the cement, but was not

implanted into the skull of the rat. Animals were housed individually after the surgery,

and were given 3-5 days to recover before the experiment was conducted. After each

microdialysis experiment was complete, the rat was euthanized with 2 mL pentobarbital,

and the brain was extracted for histology. All animal use procedures were in strict

accordance with the NIH Guide for the Care and Use of Laboratory Animals and were

approved by the local animal care committee.

Microdialysis

The day before the micodialysis experiment was conducted, each rat was placed

in a plexiglass chamber (Harvard Apparatus, Holliston, MA) with bedding and

food/water available ad libitum. A microdialysis probe (CMA microdialysis) was inserted

into the cannula and a tether was attached to the clamp on the skull. This tether was

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attached to a swivel and a counterbalance arm (Instech, Plymouth Meeting, PA) that

allowed movement around the chamber. This setup can be seen in figure 1.

On the day of the experiment, a modified Dulbeco’s artificial cerebrospinal fluid

(aCSF) buffer solution (137 mM NaCl, 3 mM KCl, 1.2 mM MgSO, 0.4 mM KHPO, with

1.2 mM CaCl and 10 mM glucose; pH 7.4) was perfused through the probe via a

microinfusion pump (PHD 2000TM, Harvard Apparatus) and liquid swivel (Instech). Each

sample was collected over a 30-minute period at 1.5 L/minute, and analyzed

immediately for DA using HPLC. Samples were collected repeatedly throughout the day.

After a stable baseline reading was achieved, usually within three hours, the pump was

manually switched to a similar aCSF solution (control) or aCSF containing 10 M

LY379268. After the first 30 minute sample on the drug treatment, rats were injected

subcutaneously with water (vehicle control) or a DOI solution (2.5 mg/kg/mL). A total of

six samples were collected for each animal once treatment was started.

Drugs

() DOI hydrochloride was obtained from Sigma-Aldrich (St Louis, MO) and

was diluted in water. LY379268 was obtained from Tocris Cookson. LY379268 was

initially prepared as a 10 mM stock solution and stored into 20 L aliquots. On the day of

an experiment, the LY379268 was further diluted in Dulbeco’s aCSF to 10 M.

Chromatography

Dialysate samples were analyzed for DA content by reverse phase HPLC coupled

with electrochemical detection. Immediately following collection, 20 L of dialysate was

injected into a 2 mm Phenomenex column (UltracarbTM, 3 µm particle size, ODS 20).

The HPLC’s mobile phase was 6.7 g citric acid, 7.4g sodium acetate, 25g EDTA, 0.05

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octane sulfonic acid, pH = 4.19. A BAS LC-4C electrochemical detector with a glassy

carbon electrode, maintained at a potential of +0.60 V relative to an Ag/AgCl reference

electrode, was employed.. ChromPerfect (Justice Laboratories, CA) software was used to

collect and analyze all samples.

Histology

After each experiment was complete, the rat was euthanized with 2 mL

pentobarbital solution, and the brain was extracted. Probe placement was verified

histologically with a cryostat, using 20 m slices. Only animals whose probe placements

were verified to be in the PFC were included in this study.

Experimental Design

Four groups of treatments were used for this experiment. The first set of rats was

given both a DOI injection and LY379268 through aCSF (LY + DOI). The second group

received DOI but no LY379268 (VEHLY + DOI). The third group was injected with

water, as a control for DOI, and received LY379268 through aCSF (LY + VEHDOI). The

final group was given a control injection for DOI and control aCSF (VEHLY + VEHDOI).

For all groups, the injection (DOI or water) was given 30 minutes after switching to the

drug treatment (LY379268/aCSF or aCSF).

Data Analysis

The quantity of DA (pg/20 µl) was expressed as a percentage of the last three

baseline samples preceding drug treatment. This preliminary study will be continued by

our lab. In our final study, we will include a 2-way ANOVA with time as a repeated

measures (within group) factor and drug treatment as a between group factor.

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Results

From the many animals we have run, 13 had both successful chromatography and

histology. 3 animals were in the LY + DOI group, 3 in the LY + VEHDOI group, 3 in the

VEHLY + DOI group, and 4 in the VEH/VEHDOI group. Mean dopamine quantities, as a

percentage of the three previous baseline samples, are illustrated in figure 2. At time 0,

lines were switched to LY or VEHLY. DOI or VEHDOI were injected subcutaneously at 30

minutes.

Discussion

Presently, we find that intracortical infusions of rats with the potent group II

mGluR agonist LY379268 did not decrease PFC DA levels in rats treated with DOI.

Infusions of LY379268 also did not decrease basal DA levels. We were able to reconfirm

an increase in PFC DA with DOI treatment.

This data is in no way conclusive, as we are reporting here on preliminary trials.

A variety of factors may be responsible for our results. An upward trend in DA levels

was observed in both DOI groups before administration of the drug, a random factor that

may be controlled with an increase in the number of animals. The sharp decrease in DA

levels in the VEHLY+DOI group at time 60 minutes does not match either literature or this

lab’s previous findings. We believe that continued trials will prove the data points

contributing to low VEHLY+DOI to be outliers.

Despite our continuing effort into this study, a number of possible variables have

been identified to increase the informational value of further studies. Primary factors we

wish to examine in future work include modulation of LY379268 concentration and time

between administration of LY379268 and injection of DOI. We used previous literature

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as our basis for choosing the concentration of LY379268 in our study. It is possible that

with our specific probe setup, a higher concentration may be needed in the aCSF in order

to cross the microdialysis membrane and elicit an affect. Since LY379268 targets a

metabotropic receptor, it is possible that an increased time may be needed to observe the

results.

A variety of neurobiological factors have been implicated in both normal and

abnormal function. Glutamate, dopamine, and serotonin have all been shown to

contribute to schizophrenia in an elegant and complicated way. Our study focuses on a

hypothesis of behavior that takes all these factors into consideration. With additional

data, we may be able to assert with more confidence whether our hypothesized

neurocircuitry has a clinical application. With further research concerning the interaction

of these three, and possibly more, factors, it may at some point be possible to create a

comprehensive model of neurological function and dysfunction.

Acknowledgements

I would like to thank Tara Byrd for her extensive help in training and problem

solving during the course of this research. I would also like to thank Dr. Elizabeth Pehek

for her guidance and the ability to work in her lab. Finally, I would like to thank the

Louis Stokes VA staff for their hospitality and assistance. This research was made

possible by a grant from the Department of Veterans Affairs.

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References

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8. Marek, G. J., Wright, R. A., Schoepp, D. D., Monn, J. A., & Aghajanian, G. K. (2000). Physiological Antagonism between 5-Hydroxytryptamine2A and Group II Metabotropic Glutamate Receptors in Prefrontal Cortex. J Pharmacol Exp Ther, 292(1), 76-87.

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Protective effect of LY379268, a selective group II metabotropic glutamate receptor agonist, on dizocilpine-induced neuropathological changes in rat retrosplenial cortex. Brain Research, 992(1), 114-119.

13. Palucha-Poniewiera, A., Klodzinska, A., Stachowicz, K., Tokarski, K., Hess, G., Schann, S., et al. (2008). Peripheral administration of group III mGlu receptor agonist ACPT-I exerts potential antipsychotic effects in rodents. Neuropharmacology, 55(4), 517-524.

14. Pehek, E. A., Nocjar, C., Roth, B. L., Byrd, T. A., & Mabrouk, O. S. (2005). Evidence for the Preferential Involvement of 5-HT2A Serotonin Receptors in Stress- and Drug-Induced Dopamine Release in the Rat Medial Prefrontal Cortex. Neuropsychopharmacology, 31(2), 265-277.

15. Peleg-Raibstein, D., Pezze, M. A., Ferger, B., Zhang, W. N., Murphy, C. A., Feldon, J., et al. (2005). Activation of dopaminergic neurotransmission in the medial prefrontal cortex by N-

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methyl-d-aspartate stimulation of the ventral hippocampus in rats. Neuroscience, 132(1), 219-232.

16. Roberts, G. W. (1991). Schizophrenia: a neuropathological perspective. The British Journal of Psychiatry, 158(1), 8-17.

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20. Scruggs, J. L., Patel, S., Bubser, M., & Deutch, A. Y. (2000). DOI-Induced Activation of the Cortex: Dependence on 5-HT2A Heteroceptors on Thalamocortical Glutamatergic Neurons. J. Neurosci., 20(23), 8846-8852.

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Figures

Figure 1. Rat microdialysis setup. Probe is inserted into the prefrontal cortex. A clamp, in the back, holds the mouse in the cage.

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Figure 2. The effect of LY379268 on DOI-induced dopamine release in the prefrontal cortex. The micodialysis line was pumped with articifical cerebrospinal fluide (aCSF) or aCSF containing LY379268. Rats were treated with the 5-HT2 Agonist DOI or a water control injection at T=30. Both DOI groups show an increase in dopamine levels. The drug appears to have no effect at reducing dopamine levels. Note that the sharp drop in dopamine in VEH+DOI group at T=90 does not match previous literature.

MATERIALS & METHODS

Animals & Surgery•Male Sprague-Dawley rats weighing between 250 and 350g at the time of surgery were used. •The top of the rat’s head was shaved and then mounted in a stereotaxic frame. •A hole was then drilled in the skull above the prefrontal cortex. After puncture of the dura, a guide cannula (CMA microdialysis) was lowered into the prefrontal corex (+0.32 mm AP, ±0.07 mm ML, from bregma)Microdialysis•Neurotransmistters diffuse into artificial cerebrospinal fluid passing through inner membrane of microdilaysis probe.•Samples collected every 30 minutes at 1.5μμ

Drugs() DOI hydrochloride was obtained from Sigma-Aldrich (St Louis, MO) and

was diluted in water. LY379268 was obtained from Tocris Cookson. LY379268 was initially prepared as a 10 mM stock solution and stored into 20 L aliquots. On the day of an experiment, the LY379268 was further diluted in Dulbeco’s aCSF to 10 M.

Chromatography Dialysate samples were analyzed for DA content by reverse phase HPLC coupled

with electrochemical detection. Immediately following collection, 20 L of dialysate was injected into a 2 mm Phenomenex column (UltracarbTM, 3 µm particle size, ODS 20). The HPLC’s mobile phase was 6.7 g citric acid, 7.4g sodium acetate, 25g EDTA, 0.05 octane sulfonic acid, pH = 4.19. A BAS LC-4C electrochemical detector with a glassy carbon electrode, maintained at a potential of +0.60 V relative to an Ag/AgCl reference electrode, was employed.. ChromPerfect (Justice Laboratories, CA) software was used to collect and analyze all samples.

HistologyAfter each experiment was complete, the rat was euthanized with 2 mL

pentobarbital solution, and the brain was extracted. Probe placement was verified histologically with a cryostat, using 20 m slices. Only animals whose probe placements were verified to be in the PFC were included in this study. Experimental Design

Four groups of treatments were used for this experiment. The first set of rats was given both a DOI injection and LY379268 through aCSF (LY + DOI). The second group received DOI but no LY379268 (VEHLY + DOI). The third group was injected with water, as a control for DOI, and received LY379268 through aCSF (LY + VEHDOI). The final group was given a control injection for DOI and control aCSF (VEHLY + VEHDOI). For all groups, the injection (DOI or water) was given 30 minutes after switching to the drug treatment (LY379268/aCSF or aCSF). See my Neuropsychopharmacology paper in 2006

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