Capstone Paper Final

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-4aminobicylo[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 be

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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 5HT2 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 temperature­controlled room  and on a 12 hour light/dark cycle.  Food and water were available ad libitum.  For surgery, 

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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 3­5  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

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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 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 µL/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 µM 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 µL aliquots. On the day of an experiment, the LY379268 was further diluted in Dulbeco’s aCSF to 10 µM. Chromatography Dialysate samples were analyzed for DA content by reverse phase HPLC coupled 7

with electrochemical detection. Immediately following collection, 20 µL 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. 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 µm 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 + VEH DOI). 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 8

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.

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 + VEH DOI 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

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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 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 1. Abi-Dargham, A., Mawlawi, O., Lombardo, I., Gil, R., Martinez, D., Huang, Y., et al. (2002). Prefrontal Dopamine D1 Receptors and Working Memory in Schizophrenia. J. Neurosci., 22(9), 3708-3719. 2. Ambrosini, A., Bresciani, L., Fracchia, S., Brunello, N., & Racagni, G. (1995). Metabotropic glutamate receptors negatively coupled to adenylate cyclase inhibit N-methyl-D-aspartate receptor activity and prevent neurotoxicity in mesencephalic neurons in vitro. Mol Pharmacol, 47(5), 1057-1064. 3. Bessho, Y., Nawa, H., & Nakanishi, S. (1993). Glutamate and Quisqualate Regulate Expression of Metabotropic Glutamate Receptor mRNA in Cultured Cerebellar Granule Cells. Journal of Neurochemistry, 60(1), 253-259. 4. Bond, A., Jones, N. M., Hicks, C. A., Whiffin, G. M., Ward, M. A., O'Neill, M. F., et al. (2000). Neuroprotective Effects of LY379268, a Selective mGlu2/3 Receptor Agonist: Investigations into Possible Mechanism of Action In Vivo. J Pharmacol Exp Ther, 294(3), 800-809. 5. Cartmell, J., Salhoff, C. R., Perry, K. W., Monn, J. A., & Schoepp, D. D. (2000). Dopamine and 5-HT turnover are increased by the mGlu2/3 receptor agonist LY379268 in rat medial prefrontal cortex, nucleus accumbens and striatum. Brain Research, 887(2), 378-384. 6. Chow, E. W. C., Watson, M., Young, D. A., & Bassett, A. S. (2006). Neurocognitive profile in 22q11 deletion syndrome and schizophrenia. Schizophrenia Research, 87(1-3), 270-278. 7. Lewis, D. A., & Moghaddam, B. (2006). Cognitive Dysfunction in Schizophrenia: Convergence of {gamma}-Aminobutyric Acid and Glutamate Alterations. Arch Neurol, 63(10), 1372-1376. 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. 9. Moghaddam, B. (2003). Bringing Order to the Glutamate Chaos in Schizophrenia. Neuron, 40(5), 881-884. 10. Moghaddam, B., & Adams, B. W. (1998). Reversal of Phencyclidine Effects by a Group II Metabotropic Glutamate Receptor Agonist in Rats. Science, 281(5381), 1349-1352. 11. Nestler. (1997). Schizophrenia : An Emerging Pathology. Nature, 385(13). 12. Okamura, N., Hashimoto, K., Shimizu, E., Koike, K., Ohgake, S., Koizumi, H., et al. (2003). 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

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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 Nmethyl-d-aspartate stimulation of the ventral hippocampus in rats. Neuroscience, 132(1), 219232. 16. Roberts, G. W. (1991). Schizophrenia: a neuropathological perspective. The British Journal of Psychiatry, 158(1), 8-17. 17. Ryuichi, T. (1998). Glutamatergic Regulation of Basal and Stimulus-Activated Dopamine Release in the Prefrontal Cortex. Journal of Neurochemistry, 71(4), 1443-1449. 18. Saklayen, S. S., Mabrouk, O. S., & Pehek, E. A. (2004). Negative Feedback Regulation of Nigrostriatal Dopamine Release: Mediation by Striatal D1 Receptors. J Pharmacol Exp Ther, 311(1), 342-348. 19. Sawa, A., & Snyder, S. H. (2002). Schizophrenia: Diverse Approaches to a Complex Disease. Science, 296(5568), 692-695. 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. 21. Swanson, C., & Perry, K. (2004). The mGlu2/3 receptor agonist, LY354740, blocks immobilization-induced increases in noradrenaline and dopamine release in the rat medial prefrontal cortex. Journal of Neurochemistry, 88(1), 194-202. 22. Zhai, Y., George, C. A., Zhai, J., Nisenbaum, E. S., Johnson, M. P., & Nisenbaum, L. K. (2002). Group II Metabotropic Glutamate Receptor Modulation of DOI-induced c-fos mRNA and Excitatory Responses in the Cerebral Cortex. Neuropsychopharmacology, 28(1), 45-52.

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Figures

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

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 14

(±) 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 µL aliquots. On the day of an experiment, the LY379268 was further diluted in Dulbeco’s aCSF to 10 µM. Chromatography Dialysate samples were analyzed for DA content by reverse phase HPLC coupled with electrochemical detection. Immediately following collection, 20 µL 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. 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 µm 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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