Involvement of N-methyl-D-aspartate receptors and intracellular signaling pathways in D2 dopamine receptor antagonistmediated gene expression.

Cortical afferents excite striatal efferent neurons through activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, which can be modulated by D2 dopamine receptors. The negative coupling of D2 dopamine receptors to adenylyl cyclase has led to the presumption that protein kinase A (PKA) plays a necessary role in D2/NMDA receptor interactions and in immediate early gene expression induced by D2 receptor blockade. It has been suggested from previous studies that activation of PKA by D2 receptor blockade may lead to NMDA receptor phosphorylation in the dendrites or phosphorylation of transcription factors in the nucleus. Thus, the levels and cellular localization of activated PKA may determine if D2 antagonist-mediated gene expression is dependent on NMDA receptor activation. However, this has never been directly demonstrated in vivo . This dissertation tested the overall hypothesis that immediate early gene expression induced by the D2 dopamine receptor antagonist eticlopride is dependent on PKA activation and that the levels of activated PKA will determine whether striatal immediate early gene induction in response to D2 receptor blockade is dependent on NMDA receptor activation in vivo . Specific aim 1 examined the effects of NMDA receptor antagonists on striatal gene expression after administration of low or high doses of eticlopride. The results showed that NMDA antagonists blocked induction by a low dose of eticlopride in all regions of the striatum, whereas induction by a higher dose of eticlopride was only blocked in the medial and central striatum. Fewer D2 dopamine receptors and thus less PKA activation in these regions might have explained why the expression was more sensitive to NMDA receptor blockade. Therefore, specific aim 2 examined the effects of PKA inhibition or activation on eticlopride-induced gene expression. The results suggest that PKA is not solely responsible for this gene expression. Specific aim 3 was designed to determine the effects of other signaling pathways on eticlopride-mediated gene expression. Inhibition of the mitogen-activated (MAP) and calcium/calmodulin-dependent (CaM) protein kinase pathways had no effect on eticlopride-induced expression. These findings suggest for the first time that PKA is not necessary for D2 antagonist-mediated gene induction and that multiple signaling pathways may be involved.

INVOLVEMENT OF N-METHYL-D-ASPARTATE RECEPTORS AND INTRACELLULAR SIGNALING PATHW A YS IN D2 DOPAMINE RECEPTOR ANT AGONIST -MEDIATED GENE EXPRESSION by AmyC. Adams A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience Department of Pharmacology and Toxicology University of Utah August 2000 Copyright © Amy C. Adams 2000 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Amy C. Adams This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. Chair: Kristen A. Keefe s/11& , f 4tLMIt 7~~Steve White Don K. Blumenthal THE UNIVERSITY OF UTAH GRADU ATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of Amy C. Adams in its [mal form and have found that (l) its format, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the fmal manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School. K isten A. Keefe Chair, Supervisory Committee Approved for the Major Department Eric M. Lasater CbairJDean Approved for the Graduate Council David S. Chapman Dean of The Graduate S:~ ABSTRACT Cortical afferents excite striatal efferent neurons through activation of N-methyl- D-aspartate (NMDA) and non-NMDA receptors, which can be modulated by D2 dopamine receptors. The negative coupling of D2 dopamine receptors to adenylyl cyclase has led to the presumption that protein kinase A (PKA) plays a necessary role in D2INMDA receptor interactions and in immediate early gene expression induced by D2 receptor blockade. It has been suggested from previous studies that activation of PKA by D2 receptor blockade may lead to NMDA receptor phosphorylation in the dendrites or phosphorylation of transcription factors in the nucleus. Thus, the levels and cellular localization of activated PKA may determine if D2 antagonist-mediated gene expression is dependent on NMDA receptor activation. However, this has never been directly demonstrated in vivo. This dissertation tested the overall hypothesis that immediate early gene expression induced by the D2 dopamine receptor antagonist eticlopride is dependent on PKA activation and that the levels of activated PKA will determine whether striatal immediate early gene induction in response to D2 receptor blockade is dependent on NMDA receptor activation in vivo. Specific aim 1 examined the effects of NMDA receptor antagonists on striatal gene expression after administration of low or high doses of eticlopride. The results showed that NMD A antagonists blocked induction by a low dose of eticlopride in all regions of the striatum, whereas induction by a higher dose of eticlopride was only blocked in the medial and central striatum. Fewer D2 dopamine receptors and thus less PKA activation in these regions might have explained why the expression was more sensitive to NMDA receptor blockade. Therefore, specific aim 2 examined the effects of PKA inhibition or activation on eticlopride-induced gene expression. The results suggest that PKA is not solely responsible for this gene expression. Specific aim 3 was designed to determine the effects of other signaling pathways on eticlopride-mediated gene expression. Inhibition of the mitogen-activated (MAP) and calciumlcalmodulin­dependent (CaM) protein kinase pathways had no effect on eticlopride-induced expression. These findings suggest for the first time that PKA is not necessary for D2 antagonist-mediated gene induction and that multiple signaling pathways may be involved. v TABLE OF CONTENTS ABSTRACT ................................................................................................................ iv LIST OF FIGURES .................................................................................................. viii ACKNOWLEDGMENT............................................................................................ x Chapter 1. INTRODUCTION Anatomy of the basal ganglia ........................................................................... l Modulation of intracellular signaling by dopamine receptors ............................ 4 Modulation of intracellular signaling by NMDA receptors ............................... 6 DopaminelNMDA interactions in the regulation of striatal neuron function ...... 8 In vivo evidence for PKA as a mediator of immediate early gene expression by dopamine receptor manipulations ......................................... 13 Research objectives ........................................................................................ 16 References ...................................................................................................... 18 2. DEPENDENCE OF ETICLOPRIDE-INDUCED IMMEDIATE EARLY GENE EXPRESSION ON NMDA RECEPTOR ACTN ATION: A ROLE FOR PKA? In.troduction .................................................................................................... 25 Methods ......................................................................................................... 32 Results ........................................................................................................... 37 Discussion ...................................................................................................... 42 References ...................................................................................................... 56 3. ETICLOPRIDE INDUCES STRIATAL IM:MEDIA 1E EARLY GENE EXPRESSION INDEPENDENT OF PR01EIN KINASE A ACTIVATION Introduction .................................................................................................... 60 Mefuods ......................................................................................................... 62 Results ........................................................................................................... 69 Discussion ...................................................................................................... 77 References ...................................................................................................... 94 4. INHIBITION OF MAP KINASE OR CAM KINASE DOES NOT AT1ENUA 1E ETICLOPRIDE-INDUCED IM:MEDIATE EARLY GENE EXPRESSION IN STRIATUM Introduction ................................................................................................... 101 Mefuods ........................................................................................................ 1 04 Results .......................................................................................................... 109 Discussion ..................................................................................................... 114 References ..................................................................................................... 124 5. DISCUSSION ...................................................................................................... 128 References ..................................................................................................... 146 Vll LIST OF FIGURES Figure Page 1.1 Basal gan.glia circuitry ....................................................................................... 2 1.2 Proposed model for the intran.euronallocalization of PKA after administration of low or high concentrations of forskolin or the D2 dopamine receptor antagonist eticlopride .................................................... 10 1.3 Proposed model for the signaling pathways involved in D2 dopamine an.d NMDA receptor interactions in the regulation of striatal immediate early gene expression ............................................................................................... 11 2.1 Effect of the NMDA receptor an.tagonists CGS 19755 an.d MK-801 on eticlopride-induced c-fos and zij268 expression in the striatum ........................ 29 2.2 Effect of the NMDA receptor antagonists CGS 19755 an.d MK-801 on c-fos expression induced by a low dose of eticlopride in the striatum ........................ 38 2.3 Effect of the NMDA receptor an.tagonists CGS 19755 an.d MK-801 on zij268 expression induced by a low dose of eticlopride in the striatum ........................ 40 2.4 Effect of IBMX on c-fos expression induced by a high dose of eticlopride in the striatum .................................................................. '" .............................. 43 2.5 Effect of IBMX on zij268 expression induced by a high dose of eticlopride in the striatum .................................................................................................. 45 2.6 Effect of CGS 19755 on c-fos expression induced in the striatum by the corrlbined administration of eticlopride and IBMX ........................................... 47 2.7 Effect of CGS 19755 on zif268 expression induced in the striatum by the combined administration of eticlopride and IBMX ........................................... 49 3.1 Effect of the PKA inhibitor H-89 on eticlopride-induced c-fos expression in ilie striatum .................................................................................................. 70 3.2 Effect of ilie PKA inhibitor H-89 on eticlopride-induced zif268 expression in ilie striatum .................................................................................................. 72 3.3 Effects of eticlopride, Sp-8-Br-cAMPs, and H-89 on PKA specific activity in ilie striatum .................................................................................................. 75 3.4 Effect of CGS 21680 on eticlopride-induced c-fos expression in ilie striatum .. 78 3.5 Effect of CGS 21680 on eticlopride-induced zif268 expression in ilie striatum 80 3.6 Effect of rolipram on eticlopride-induced c-fos expression in the striatum ....... 82 3.7 Effect of rolipram on eticlopride-induced zif268 expression in ilie striatum ..... 84 4.1 Effect of ilie MAP kinase inhibitor PD98059 on eticlopride-induced c-fos expression in ilie striatum ................................................................................ 110 4.2 Effect of ilie MAP kinase inhibitor PD98059 on eticlopride-induced zif268 expression in ilie striatum ................................................................................ 112 4.3 Effect of ilie CaM kinase inhibitor KN-93 on eticlopride-induced c-fos expression in ilie striatum ................................................................................ 115 4.4 Effect of ilie CaM kinase inhibitor KN-93 on eticlopride-induced zif268 expression in ilie striatum ................................................................................ 117 5.1 Multiple signal tranduction pailiways activated by D2 dopamine receptor blockade in striatal efferent neurons ................................................................ 143 ix ACKNOWLEDGMENTS Many people have been instrumental in the completion of this work. First and foremost, I would like to extend my sincere gratitude to my mentor, Dr. Kristen A. Keefe, for her support, patience, foresight, and encouragement throughout my graduate career. Her guidance and friendship have been more important to me than she can know. I would also like to thank the members of my dissertation committee for their invaluable contributions, advice, and encouragement: Dr. Donald K. Blumenthal, Dr. Glen R. Hanson, Dr. Mary T. Lucero, and Dr. H. Steve White. The members of the Keefe lab have taken on many roles in the course of my time at the University of Utah, including helpers, teachers, and most of all friends. I want to thank you for having been a support network when I really needed to smile: Anindita, David, Dave, Scot, Kamisha, and Aaron. To my friend and colleague, Dr. Anindita Ganguly, your thoughtfulness and kindness toward me over the last 4 years has meant so much. I value your friendship and wish you only success and happiness in the future. Additionally, I would like to acknowledge the student members of the neuroscience program and the pharmacology and toxicology students for their advice, support, and creativity. Finally and most importantly, I wish to thank my family. To Jim and Faye, thanks so much for your support, wisdom, and blessings. To Mom and Dad, you are two of the greatest people that I have ever known. I would not be who I am or where I am without your unconditional love and faith, and I am eternally grateful to have you in my life. Most of all, I would like to thank my husband and best friend, David. Your endless support, faith, guidance, and love have carried me through my most difficult moments. I dedicate this dissertation to you. Xl CHAPTER! INTRODUCTION Anatomy of the basal ganglia The main input nuclei of the basal ganglia are the caudate nucleus and putamen (striatum). These nuclei receive afferent glutamate projections from the cerebral cortex and thalamus (Kemp and Powell, 1971; Kitai et al., 1976; Kim et al., 1977; Dube et al., 1988) and dopamine input from the substantia nigra pars compacta (Homykiewicz et al., 1968). Cortical efferents excite striatal neurons through activation of N-methyl-D­aspartate (NMDA) and non-NMDA receptors (Cherubini et at, 1988). The glutamate input primarily synapses on the heads and the dopamine input on the necks of the dendritic spines of medium spiny striatal neurons (Somogyi et al., 1981; Kotter, 1994; Bouyer et aI., 1984). This anatomical arrangement suggests that interactions between dopamine and glutamate systems are likely to be inlportant for regulating striatal neuron function. The efferent proj ections of the striatum can be divided into two populations: striatonigral and striatopallidal (Fig 1.1). Striatonigral neurons directly send gamma- Indirect pathway Cortex (glutamate) Striatum Direct pathway entopeduncular nucleus Fig. 1.1. Basal ganglia circuitry 2 3 aminobutyric acid (GAB A) projections to the output nuclei of the basal ganglia, the substantia nigra pars reticulata and the entopeduncular nucleus (internal segment of the globus pallidus) (Vincent el aI., 1982; LeMoine et al., 1991). This pathway is referred to as the direct pathway. Striatopallidal neurons send GABA projections to the external segment of the globus pallidus, whose GABA projections in tum inhibit the subthalamic nucleus. The subthalamic nucleus projections use glutamate and innervate the output nuclei of the basal ganglia (Albin et aI., 1989; Alexander and Crutcher, 1990). The striatopallidal pathway is also referred to as the indirect pathway. Dopamine receptors can be classified based on their affinities for standard ligands and G protein coupling. The two major families of dopamine receptors, D 1 and D2, can be further subdivided into D1 and D5 receptors and D2, D3, and D4 receptors, respectively. In the striatum, D 1 dopamine receptors are located primarily on striatonigral neurons, whereas D2 dopamine receptors are located on striatopallidal neurons (Gerfen et aI., 1990; LeMoine et at, 1990; LeMoine et al., 1991). The NMD A receptor is a multimeric receptor composed of different subunits: NMDA1 (NR1) and NMDA2A-2D (NR2A-2D) (Moriyoshi et al., 1991; Monyer et al., 1992; Watanabe et al., 1993; Zukin and Bennett, 1995). The NR1 subunit is necessary for the fonnation of a functional receptor, whereas NR2 subunits couple with the NR1 to confer pharmacological specificity to NMDA receptors (Buller et at, 1994; Laurie and Seeburg, 1994). NR1, NR2A, and NR2B subunits are found on medium spiny 4 efferent neurons of the striatum (Standaert et aI., 1994; Landwehnneyer et aI., 1995). NR2A and NR2B subunits are expressed throughout the striatum; however, the NR2A subunit expression is greater in the lateral than in the medial region of the striatum (Watanabe et aI., 1993; Standaert et aI., 1994). Modulation of intracellular signaling by dopamine receptors The D1 dopamine receptor is coupled to a stimulatory G protein (Gs and Golf; Herve et aI., 1993). Stimulation of D 1 dopamine receptors increases the activity of adenylyl cyclase, increasing cyclic adenosine monophosphate (cAMP) formation and in tum activating protein kinase A (PKA) (Kebabian and CaIne, 1979; Nairn et aI., 1985; Monsma et aI., 1990; Kotter, 1994). PKA phosphorylates the transcription factor cAMP-response element-binding protein (CREB) at the amino acid residue Ser133 (Dash I et aI., 1991). CREB interacts with the cAMP-response element (CRE) site (Montminy and Bilezikjian, 1987; Konradi et aI., 1994) which is located in the promoter region of several genes. Upon phosphorylation, CREB binds to CREB-binding protein, recruiting RNA polymerase II to the promoter (Kwok et aI., 1994; Kee et al., 1996) and initiating transcription of CRE-containing genes, such as c-fos and zij268. Simpson and Morris (1995) have shown that application of the D 1 receptor agonist SKF 38393 results in elevated c-fos and zif268 mRNA levels in cultures of embryonic rat striatal neurons. The levels of c-fos mRNA induced by SKF 38393 were reduced by 45% after 5 pretreatment of the cells with the selective PKA inhibitor, KT5720, and by 87% after pretreatment with the selective PKC inhibitor, calphostin C (Simpson and Morris, 1995). The stimulation of zij268 mRNA levels by SKF 38393 was reduced by 90% after treatment with KT5720, whereas calphostin C did not significantly affect the expression levels (Simpson and Morris, 1995). Another study also implicates PKA as a primary constituent in D1 receptor-mediated CREB phosphorylation and c-fos induction, in that the PKA inhibitor H-89 blocked both the CREB phosphorylation and gene expression induced after D1 agonist application in PC12 cells (Chijiwa et al., 1990). Although these results support a role for PKA in D1-mediated c-fos and zif268 expression, other studies would suggest that PKA does not playa role in D1-mediated induction of immediate early genes. For example, Liu et al. (1995) have reported that in an organotypic striatal slice preparation, the D1 agonist-induced c-fos expression is abolished by the nonspecific serine/threonine protein kinase inhibitor H -7 but not by H- 89 (PKA inhibitor) or KN-62 (CaM kinase inhibitor). Together, these results from various in vitro studies are inconsistent in determining whether PKA is involved in D1 receptor-mediated gene expression. In addition, these same questions have not been addressed in vivo. Stimulation of the D2 dopamine receptor (coupled to an inhibitory G protein, Gi and to other G proteins, such as Go) acts antagonistically on adenylyl cyclase, inhibiting 6 cAMP fonnation and PKA activation. Antagonists of the D2 dopamine receptor have been shown to increase the expression of c-fos in striatum (Dragunow et aI., 1990; Miller, 1990; Robertson and Fibiger, 1992; Rogue and Vincendon, 1992; Ziolkowska and RoUt, 1993; Boegman and Vincent, 1996~ Keefe and Adams, 1998). Konradi and Reckers (1995) have shown that the antipsychotic haloperidol (a partial D2 dopamine receptor antagonist) induces c-fos via phosphorylation of CREB. This is presumably due to the elevation of PKA levels after blockade of the D2 dopamine receptor. In addition, haloperidol-induced c-fos expression is completely blocked in PKA knockout mice (Adams et aI., 1997). Thus, it is hypothesized that D2 dopamine receptor blockade results in the activation of PKA, phosphorylation of CREB on Ser133 , and subsequent induction of c-jos. Modulation of intracellular signaling by NMDA receptors Activation of the NMDA subtype of glutamate receptor occurs upon binding of ligand, presumably glutamate, and the co-agonist glycine. fu the presence of sufficient neuronal depolarization to remove the magnesium block of the receptor-associated channel, ligand binding results in calcium influx through the channel, strongly depolarizing the postsynaptic membrane. This has been suggested to trigger the opening of L-type voltage-sensitive calcium channels (Rajadhyaksha et aI., 1999). Calcium entry via the NMDA receptor and L-type channels presumably activates a 7 kinase cascade that leads to the phosphorylation of CREB and the induction of c-fos (Bading et aI., 1993; Bito et aI., 1997; Rajadhyaksha et aI., 1999). The activities of calcium/calmodulin-dependent (CaM) and mitogen-activated (MAP) protein kinases are increased by NMDA receptor activation (Bading et aI., 1993; Vincent et aI., 1998). These kinases can, like PKA, lead to the phosphorylation of CREB and gene induction (Dash et aI., 1991; Bito et aI., 1996; Impey et at, 1998). SeveraI previous studies have shown that CREB phosphorylation and striatal gene expression induced by various compounds are dependent on MAP kinase or CaM kinase activation. For example, Vincent et aI. (1998) have denlonstrated that treatment of primary striatal cell cultures with NMDA, the cAMP activator forskolin, or the D 1 dopamine receptor agonist SKF 38398 results in a rapid increase in the activity of MAP kinase. Furthermore, it has been shown previously that these same compounds also result in the phosphorylation of CREB and expression of c-fos (Konradi et at, 1994; Das et aI., 1997). In vivo electrical stimulation of the corticostriataI glutamate pathway also leads to the expression of c-fos and zij268 in striatum, and this expression is completely abolished by administration of the MAP kinase inhibitor PD98059 (Sgambato et aI., 1998). Finally, translocation of CaM kinase IV to the nucleus has been shown to result in phosphorylation of CREB in the hippocampus (Bito et at, 1996), and CaM kinase inhibition blocks calcium-dependent expression of c-fos and zif268 in PC12 cells (Enslen and Soderling, 1994). Thus, NMDA receptor activation 8 leading to the activation of various signaling kinases has an important role in the induction of immediate early genes. The serum response element (SRE) is another site within the promoter regions of the c-fos and zij268 genes (Treisman, 1986). The serum response factor recruits transcription factors such as Elk-1 to the SRE site for transcription of immediate early genes (Treisman, 1992). This additional Ca++-responsive site may also mediate NMDA receptor-mediated transcription (Bading et al., 1993). DopamineJNMDA interactions in the regulation of striatal neuron function It has been demonstrated that dopamine receptor manipulations can either potentiate or attenuate NMDA-induced currents in striatal neurons (Cepeda et aI., 1993). Specifically, D1 receptor activation potentiates and D2 receptor activation attenuates responses evoked by application of NMDA in striatal slice preparations. These data and the fact that glutamate afferents synapse on the heads and dopamine inputs on the necks of dendritic spines of striatal efferent neurons support interactions between dopamine and NMDA receptors in the regulation of striatal efferent neuron function. The degree of interaction between dopamine and NMDA receptors may depend, however, on the preparation used, the degree of receptor activation, and the dependent measure examined. Konradi and colleagues have examined the interactions 9 between Dl dopamine receptors and NMDA receptors in striatal cell cultures (Konradi et aI., 1996; Konradi, 1998; Rajadhyaksha et aI., 1998). They discovered differential effects of the NMDA receptor antagonist MK-801 on forskolin-mediated c-fos expression. When low concentrations of forskolin were applied to the cells, the levels of phosphorylated CREB and c-fos expression were increased. These effects were blocked in the presence of the NMDA receptor antagonist MK-801. However, when high concentrations of forskolin were applied, MK-801 was unable to block phosphorylation of CREB or c-fos induction (Rajadhyaksha et al., 1998). The model proposed by Konradi and colleagues for dopaminelNMDA interactions in the regulation of gene induction is thus based on the level of activation of PKA by the D 1 /cAMP pathway (Figs. 1.2 and 1.3; Konradi, 1998). Low levels of PKA activation in the dendrites by low forskolin or D 1 dopamine receptor stimulation phosphory lates the NMDA receptor (Rajadhyaksha et aI., 1998; Snyder et al., 1998), leading to the influx of calcium and a strong depolarization of the dendritic spine. This depolarization is thought to promote opening of voltage-sensitive L-type calcium channels. The resultant calcium influx is then thought to activate a signaling pathway that propagates to the nucleus, leading to the phosphorylation of CREB and the induction c-fos (Rajadhyaksha et al., 1999). Therefore, MK-801 is thought to effectively block c-fos induction by low concentrations of forskolin or by D 1 dopamine receptor stimulation by inhibiting Ca ++ Low forskolinl Low eticlopride Dendritic .- spines --... High forskolinl High eticlopride Fig. 1.2. Proposed model for the intraneuronal localization of PKA after administration of low or high concentrations of forskolin or the D2 dopamine receptor antagonist eticlopride (Adapted from Rajadhyaksha et aI., 1998) 10 11 Fig. 1.3. Proposed model for the signaling pathways involved in D2 dopamine and NMDA receptor interactions in the regulation of striatal immediate early gene expression. Blockade of the D2 dopamine receptor leads to the activation of protein kinase A (PKA). If the levels of activated PKA are great enough (administration of a high dose of eticlopride), the intracellular distribution of PKA is more widespread, allowing PKA to directly phosphorylate transcription factors such as CREB in the nucleus. In this case, immediate early gene expression induced by D2 receptor blockade would be independent of NMDA receptor activation. However, under conditions when less PKA is activated (low dose administration of eticlopride), the distribution of PKA is more confmed to the acute area of activation, the dendritic spine. PKA phosphorylation of the NMDA receptor leads to an increase in intracellular calcium concentrations via influx through the NMDA receptor channel and L-type voltage-sensitive calcium channels. The phosphorylation of transcription factors and subsequent induction of immediate early genes in this case is dependent on the activation of signaling pathways by NMDA receptor activation such as the calcium/calmodulin-dependent kinase pathway and the extracellular regulated kinase (a family of MAP kinases) pathway. NMDA receptor L-type VSCC receptor ~~yCamKII CalCaM P-ERK / ..• • •• ..• PERSK2 C-fos Elk-l-P I SRF-P & zij268 IsREl ~ ....... N 13 influx-mediated activation of signaling kinases. However, in the presence of high concentrations of forskolin or D 1 receptor stimulation, more cAMP is activated, leading to a more widespread distribution of active PKA throughout the neuron. In this case, PKA activated within the nucleus is thought to directly phosphorylate CREB, leading to c-fos induction that is independent of NMDA receptor activation. Thus, this effect is not blocked by NMDA receptor antagonist administration (Rajadhyaksha et al., 1998). While these data support a presumed role for PKA in dopamine-mediated immediate early gene expression and in determining the dependence of this expression on NMDA receptor activation, this role ofPKA has not yet been directly demonstrated. In vivo evidence for PKA as a mediator of immediate early gene expression by dopamine receptor manipulations The PKA enzyme consists of a regulatory subunit homodimer and two catalytic subunits. As noted above, the antipsychotic agent haloperidol, which has mixed D 11D2 receptor antagonist properties, induces the expression of immediate early genes in the striatum (Ziolkowska and Hollt, 1993; Konradi and Heckers, 1995; Boegman and Vincent, 1996). In mice bearing a targeted disruption of one of the isofonns of the PKA regulatory subunits, RII~, haloperidol-induced expression of c-fos in the striatum is completely abolished (Adams et aI., 1997). However, the density of D2 dopamine receptors in these mice is unchanged in the absence of the RII~ protein. This suggests 14 that immediate early gene expression in response to D2 receptor blockade by haloperidol is dependent on the activation of PKA. Studies conducted in our lab have examined the interactions between D 1 dopamine and NMDA receptors in the regulation of striatal neuron function. Results from these studies demonstrate that the full D1 dopamine receptor agonist SKF 82958 (1.0 mglkg, i. p.) induces the immediate early genes c-fos and zij268 throughout striatum in both intact (Keefe and Ganguly, 1998) and dopamine-depleted animals (Ganguly and Keefe, 1998). The NMDA receptor antagonist MK-801 (1.0 mglkg, i.p.) completely blocks this induction in intact animals (Keefe and Ganguly, 1998) but is unable to produce more than a 25% reduction in zij268 expression in dopamine-depleted animals (Ganguly and Keefe, 1998). We hypothesize that the differential sensitivity of the D1 response in this preparation reflects different levels of PKA activation, based on the model of Konradi (1998). As a consequence of dopamine-depleting brain lesions, D 1 dopamine receptors become supersensitive to ligand binding (Creese and Snyder, 1979) and therefore produce a potentiated response to dopamine when compared to nonlesioned animals. For example, D1 receptor stimulation has been shown to increase adenylyl cyclase activity in the dopamine-depleted striatum (Mishra et al., 1974). In addition, it has been demonstrated that CRBB phosphorylation is significantly greater in the striata of animals unilaterally depleted of dopamine after D 1 dopamine receptor stimulation on 15 the side ipsilateral to the lesion, but not in the contralateral side (Cole et aI., 1994). We hypothesize that the increased activation of PKA by D 1 dopamine receptor stimulation in the dopamine-depleted condition leads to direct phosphorylation of CREB, inducing immediate early genes independent of NMDA receptor activation as predicted by the findings of Konradi et al. (Konradi, 1998; Rajadhyaksha et aI., 1998). We have also observed regional differences in the sensitivity of striatal immediate early gene induction by the D2 dopamine receptor antagonist eticlopride to NMDA receptor antagonists in intact rats (Keefe and Adams, 1998). Eticlopride administered at a dose of 1.0 mg/kg induced c-jos and zif268 throughout striatum, with the highest levels apparent in lateral striatum. Pretreatment with CGS 19755 or MK- 801 attenuated eticlopride-induced c-fos and zif268 induction in medial striatum, while having no significant effect in the lateral third of striatum. We hypothesize that the regional differences occurred as a result of higher numbers of D2 dopamine receptors in the lateral striatum (Joyce et al., 1985; Robertson and Fibiger, 1992) and thus greater activation of PKA in that region. Presumably, less PKA was activated in the medial and central thirds of striatum, leading to a less direct phosphorylation of CREB by PKA. Rather, phosphorylation of the NMDA receptor by PKA, as proposed by Konradi (1998), presumably causes an increase in the influx of Ca++ through L-type channels, activation of signaling pathways leading to the phosphorylation of CREB, and 16 ultimately the induction of immediate early genes (Enslen and Soderling, 1994; Bito et aI., 1996; Vincent et aI., 1998). By blocking the NMDA receptor with CGS 19755 or MK-801, as in our studies, the D2 dopamine receptor-mediated induction of c-fos and zif268 is attenuated. Research objectives Although some of the second messenger systems have been delineated for the effects of dopamine and NMDA receptor activation on striatal function, the interactions between these two systems and the mechanisms proposed to be involved in the induction of immediate early genes have been identified primarily based on studies conducted in vitro. The extent to which these same processes occur in vivo, specifically as a consequence of D2 dopamine receptor blockade, has not yet been detennined. Therefore, we have tested in Chapters 2 and 3 the overall hypothesis that immediate early gene expression induced by the D2 dopamine receptor antagonist eticlopride is dependent on PKA activation. In addition, we have tested the hypyothesis that the levels of activated PKA will determine whether striatal immediate early gene induction in response to D2 receptor blockade is dependent on NMDA receptor activation in vivo. In addition, other signaling pathways, such as those activated by MAP kinase and CaM kinase protein kinases, have been implicated in mediating the expression of immediate early genes by dopamine and NMDA receptor manipulations (Enslen and Soderling, 17 1994; Bito et al., 1996; Das et aI., 1997; Vincent et aI., 1998; Sgambato et aI., 1998). Therefore, in Chapter 4, we address the roles that other signaling pathways may play in eticlopride-induced gene expression by administering inhibitors of the MAP kinase and CaM kinase pathways. The studies described in this dissertation will help to determine the signaling pathways involved in dopamine receptor-mediated immediate early gene expression and increase our understanding of the interactions between dopamine and NMDA receptors in the functioning of the basal ganglia. This information should aid in the design of new and more beneficial therapeutic strategies for disorders of the basal ganglia such as Parkinson's disease and schizophrenia. In addition, many of the antipsychotic drugs such as haloperidol exert their effects at D2 dopamine receptors in the striatum. 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Trends Neurosci 18:306-313. CHAPTER 2 DEPENDENCE OF ETICLOPRIDE-INDUCED IMMEDIATE EARLY GENE EXPRESSION ON NMDA RECEPTOR ACTIVATION: AROLEFORPKA? Introduction The striatum is the main input nucleus of the basal ganglia and is involved in motor and cognitive functions. The striatum receives afferent glutamate projections from the cerebral cortex and thalamus which act on N-methyl-D-aspartate (NMDA) and non-NMDA receptors and dopamine input from the substantia nigra pars compacta (Homykiewicz et aI., 1968; Kemp and Powell, 1971; Kitai et aI., 1976; Kim et aI., 1977; Dube et aI., 1988). Calcium entry via the NMDA receptor and L-type channels presumably activates a kinase cascade that leads to the phosphorylation of the transcription factor CREB (cAMP response elen1ent binding protein) and the induction of the immediate early genes c-fos and zij268 (Bading et aI., 1993; Bito et aI., 1997; Rajadhyaksha et aI., 1999). D2 dopamine receptors are negatively coupled to adenylyl cyclase, inhibiting the formation of cAMP and activation of protein kinase A (PKA; 26 Weiss et aI., 1985; Albert et aI., 1990). Thus, D2 receptor blockade presumably facilitates the activation of PKA. Additionally, it has been demonstrated that D2 receptor antagonists induce the expression of immediate early genes in striatum (Dragunow et al., 1990; Ziolkowska and Hollt, 1993; Boegman and Vincent, 1996; Keefe and Adams, 1998) and that this effect is completely blocked in mice bearing a targeted disruption ofPKA (Adams et al., 1997). Dopamine, acting through D 1 and D2 subtypes of dopamine receptors, modulates NMDA receptor-mediated responses in striatal slices (Cepeda et aI., 1993). In addition, NMDA receptor blockade has been shown to attenuate D2 dopamine receptor antagonist-mediated immediate early gene expression (Dragunow et aI., 1990; Ziolkowska and Hollt, 1993; Boegman and Vincent, 1996; Keefe and Adams, 1998). Such data suggest strongly that there are intracellular interactions between these two transmitter receptor systems. However, the signaling pathways responsible for the modulatory effects between these two neurotransmitter systems have not yet been fully explored particularly in vivo. Konradi and colleagues (Konradi, 1998; Rajadhyaksha et aI., 1998) have proposed that the levels of activated PKA may determine whether striatal immediate early gene expression is dependent on NMDA receptor activation. In primary striatal cell culture, the NMDA receptor antagonist MK-801 had no effect on phosphorylation of CREB or the induction of c-fos after the application of a high concentration of the 27 cAMP inducer forskolin (Konradi, 1998; Rajadhyaksha et al., 1998). However, MK- 801 did block CREB phosphorylation and c-fos expression induced by a low concentration of forskolin (Konradi, 1998; Rajadhyaksha et aI., 1998). The model proposed by Konradi and colleagues for dopaminelNMDA interactions in the regulation of gene induction is thus based on the level of activation of PKA by the Dl/cAMP pathway (Konradi, 1998). Low levels of PKA activation by low concentrations of forskolin or D 1 receptor stimulation are thought to remain localized to the dendrites and to phosphorylate the NMDA receptor (Rajadhyaksha et al., 1998; Snyder et al., 1998), leading to greater influx of calcium and a strong depolarization of the dendritic spine. This depolarization is thought to promote opening of voltage-sensitive L-type calcium channels. The resultant calcium influx is then thought to activate a signaling pathway that propagates to the nucleus, leading to the phosphorylation of CREB and the induction c-fos (Rajadhyaksha et aI., 1999). Therefore, MK-801 is thought to effectively block c-fos induction by low concentrations of forskolin or by D 1 dopamine receptor stimulation by inhibiting Ca2 + influx-mediated activation of signaling kinases. However, in the presence of high concentrations of forskolin or D 1 receptor stimulation, more PKA is activated, leading to a more widespread distribution of active PKA throughout the neuron. In this case, PKA activated within the nucleus is thought to directly phosphorylate CREB, leading to c-fos induction that is independent of NMDA 28 receptor activation. Thus, this effect is not blocked by NMDA receptor antagonist administration (Rajadhyaksha et aI., 1998). We have previously observed regional differences in striatal immediate early gene expression after administration of the D2 dopamine receptor antagonist eticlopride and the NMDA receptor antagonists CGS 19755 and MK-801 in intact rats (Keefe and Adams, 1998). Eticlopride administered at a dose of 1.0 mg/kg induced the immediate early genes c-fos and zij268 throughout striatum, with the highest levels apparent in lateral striatum (Fig. 2.1). Pretreatment with CGS 19755 or MK-801 attenuated eticlopride-induced c-fos and zij268 induction in medial striatum, while having no significant effect in the lateral third of striatum (Fig. 2.1). We think that the regional differences occurred as a result of higher numbers of D2 dopamine receptors in the lateral striatum (Joyce et aI., 1985; Robertson and Fibiger, 1992) and thus a more widespread activation of PKA throughout the neurons in that region. Presumably, less nuclear PKA was activated in the medial and central thirds of striatum, leading to a lesser degree of direct phosphorylation of CREB by PKA. Rather, phosphorylation of the NMDA receptor by PKA, as proposed by Konradi (1998), presumably causes an increase in the influx of Ca2 + through L-type channels, activation of signaling pathways leading to the phosphorylation of CREB, and ultimately the induction of immediate early genes (Enslen and Soderling, 1994; Bito et aI., 1996; Vincent et al., 1998). By 29 Fig. 2.1. Effect of the NMDA receptor antagonists CGS 19755 and MK-801 on eticlopride-induced c-fos and zij268 expression in the striatum. The NMDA receptor antagonists CGS 19755 ("CGS"; 10 mg/kg) and MK-801 ("MK"; 1 mg/kg) were administered intraperitoneally to animals treated with the D2 dopamine receptor antagonist eticlopride ("Etic"; 1 mglkg, i.p.). CGS 19755 was administered 1 hr before the administration of eticlopride. MK-801 was administered 30 min before the injection of eticlopride. Animals receiving eticlopride alone received a vehicle injection either 1 hr or 30 min before eticlopride administration. Control animals received two vehicle injections. Animals were sacrificed 40 min after the last injection. Values are mean gray values (±S.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the mid-striatum (approximately 0.5 mm anterior to bregma). Numbers in parentheses indicate the nUlTlber of animals per group. *Significantly different from control, p<0.05. +Significantly different from eticlopride alone, p<O. 05. ..-... 20 .U..I. -2 :::J a~s 15 ."..­. :s "as- "C"D" 10 :::J m > ~ e 5 C) c as CD :i 0 c-fos * * Control (5) Etic (4) 10 mg/kg CGS + Etic (5) 25 20 15 10 5 o • medial striatum • central striatum o lateral striatum * Control (5) Etic (3) 1 mg/kg MK + Etic (5) ow 080 == 370 .I~.­. 60 :c 50 I- ca ~40 ::::s "iii 30 > ~20 I- en c 10 ca ~ 0 * Control (5) Etic (4) zif268 10 mg/kg CGS + Etic (5) 80 70 60 Control (5) • medial striatum II central striatum o lateral striatum * Etic (3) 1 mg/kg MK + Etic (5) w,..... . 32 blocking the NMDA receptor with CGS 19755 or MK-801, the D2 dopamine receptor-mediated induction of c-fos and zif268 in the medial and central striatum therefore was attenuated. The present study was designed to further evaluate the hypothesis that the level of activation of PKA determines whether etic1opride-induced gene expression is dependent on NMDA receptor activation. We hypothesized that treatment with a low dose of etic10pride (0.5 mg/kg) would render immediate early gene expression susceptible to NMDA receptor blockade throughout the striatum due to lower levels of PKA activation, whereas a higher dose of etic10pride (1.0 mg/kg) had previously induced gene expression independent of NMDA receptor activation in the lateral striatun1 (refer to Fig. 2.1). In addition, we hypothesized that administration of the phosphodiesterase inhibitor IBMX to animals treated with etic10pride would increase the amount of PKA activation to the extent that NMDA receptor blockade would no longer have an effect on D2 antagonist-mediated gene induction in any region of striatum. Methods Animals and housing Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 225 to 300 g were used in all experiments. Rats were housed in groups of 33 four in hanging wire-mesh cages in a temperature-controlled room on a 12: 12 lightdark cycle. Rats had free access to food and water. All studies were approved by the Institutional Animal Care and Use Committee at the University of Utah and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Drugs CGS 19755 was donated by Ciba-Geigy Corporation (Summit, NJ). Eticlopride hydrochloride and MK-801 were obtained from Research Biochemicals International (Natick, MA) and IBMX was from Calbiochem (San Diego, CA). All drug doses were calculated as free bases with the exception of eticlopride hydrochloride, which was calculated as the salt. CGS 19755 and MK-801 were dissolved in phosphate-buffered saline, eticlopride in nonnal saline, and IBMX in dimethylsulfoxide (DMSO). All drugs were administered intraperitoneally in a volume of 1 mLlkg. Pharnlacological manipulations Determinin~ the effects of MK-801. CGS 19755. and IBMX on eticlopride-induced ~ene expression. Rats were taken from their home cages and placed in plastic tub cages (4 per cage). Rats were weighed and then injected with either MK-801 (1 mglkg), CGS 19755 (10 mglkg), IBMX (5.0 - 25.0 mglkg), or the appropriate vehicle solution. Sixty min (CGS 19755), 30 min (MK-801), or 15 min (IBMX) later, rats were 34 injected with the D2 dopamine receptor antagonist eticlopride (0.5 mg/kg, i.p.). Doses and times between injections were chosen based on previously published studies showing effective NMDA receptor blockade at these doses and within these time ranges (Carter et al., 1990; DeSarro and DeSarro, 1993; Koerner et aI., 1996; Cain et al., 1997). Animals were sacrificed 40 min after the injection of eticlopride. Previous studies have demonstrated that the expression of inunediate early genes in response to eticlopride administration is significant at this time point (H. Steiner, personal conununication). Control animals received two vehicle injections. Determining the effect of CGS 19755 on gene expression induced by the combined administration of eticlopride and IBMX. Rats were taken from their home cages, placed in plastic tub cages (4 per cage), and weighed. Rats were injected with CGS 19755 (10 mg/kg) or vehicle 45 min prior to the administration of IBMX (25 mg/kg) or vehicle. Fifteen min later, eticlopride was administered (1.0 mg/kg). Rats were sacrificed 40 min after the eticlopride injection. In situ hybridization histochemistry Rats were euthanized by exposure to CO2 (1 min) and decapitated. The brains were rapidly removed and frozen in isopentane chilled on dry ice. Brains were stored at -20°C until they were cut in 12-J.lm thick sections in a cryostat (Cryocut 1800, Cambridge Instruments, Germany) and thaw-mounted onto gelatin-chrome alum- 35 subbed slides. Slides were stored at -20°C. Once all brains from an experiment were sectioned, slides were postfixed in 4% paraformaldehyde/0.9% NaCI, acetylated in fresh 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCI (pH 8.0), dehydrated in an ascending series of alcohols, delipidated in chloroform, and rehydrated in a descending series of alcohols. Slides were air-dried and stored at -70°C. For detection of c-fos and zij268 mRNAs, full-length ribonucleotide probes complementary to the mRNAs for c-fos (Curran et aI., 1987) and zij268 (Milbrandt, 1987) were synthesized from the cDNAs using 35S_UTP and SP6 (c-fos) or T7 (zij268) RNA polymerase (Boehringer Mannheim, Indianapolis, IN). Labeled probes were diluted in hybridization buffer to obtain 2 x 106 cpml100 J,tL buffer. The ribonucleotide probe was mixed with nuclease-free water and RNA mix (final concentrations: 100 Jlg/mL salmon sperm DNA; 250 J,tg/mL yeast total RNA; 250 J,tg/mL yeast tRNA). The mixture was heated to 65°C for 5 min and then cooled on wet ice for 1 min. Dithiothreitol (100 mM, final concentration), sodium dodecyl sulfate (0.2% w/v, final concentration), sodium thiosulphate (0.1 % w/v, final concentration), and hybridization buffer were added to the ribonucleotide mixture. The hybridization buffer contained (final concentrations): Tris buffer (23.8 mM, pH 7.4), EDTA (1.2 mM, pH 8.0), NaCI (357 mM), dextran sulfate (11.9%, w/v), Denhardt's solution (1.2 x), and formamide (59.5%, v/v). Ninety JlL of probe in hybridization buffer was applied to each slide 36 containing four sections. Slides were coverslipped and hybridized overnight in humid chambers at 55°C. Slides were then washed at room temperature four times in 1 x saline-sodium citrate (SSC, 0.15 M NaCl/0.015 M sodium citrate, pH 7.2), incubated in ribonuclease A (RNase A; 5-20 1J,g/ml; Boehringer Mannheim) in buffer containing 0.5 M NaCl, 10 mM Tris (pH 8.0), and 0.25 mM EDTA (pH 8.0) for 15 min at room ten1perature, and washed 4 times in 0.2 x SSC at 60°C. Slides were rinsed briefly in deionized water, air dried, and apposed to Kodak Biomax x-ray film (Kodak Biomax MR, Eastman Kodak Co., NY) for 4 days to 2 weeks to obtain film autoradiograms. Data analysis Film autoradiograms were analyzed using the Macintosh-based image analysis program, Image (Wayne Rasband, NIH). Images of brain sections were captured with a video camera, digitized, and stored on computer. Images were captured under constant lighting conditions and within the linear range of the system response. Mean gray values were analyzed in medial, central, and lateral thirds of the right striatum from its dorsal aspect to the anterior commissure ventrally. The average gray value of the white matter was subtracted from the average gray value of the regions of interest to correct for background labeling. Data from film autoradiograms were analyzed with a one-way analysis of variance for medial, central, and lateral thirds of the striatum. Post hoc 37 analysis was perfonned with the Tukey-Kramer test. Statistical significance was set at p ~ 0.05. Results CGS 19755 and MK-801 block immediate early gene expression throughout striatum after administration of a low dose of eticlopride As noted in the introduction (see Fig. 2.1), the induction of c-fos and zij268 in striatum by 1 mg/kg eticlopride was not blocked in the lateral striatum by NMDA receptor antagonists. ill the present study, a lower dose of eticlopride (0.5 mg/kg) also induced c-fos and zif268 expression throughout the striatum, with greater expression in the lateral region. However, in this case the NMDA receptor antagonists CGS 19755 (10 mg/kg) and MK-801 (1 mg/kg) effectively attenuated eticlopride-induced gene expression in all regions of the striatum (p < 0.05) (Figs. 2.2 and 2.3). The nonselective phosphodiesterase inhibitor IBMX potentiates immediate early gene expression induced by eticlopride ill order better detennine the role of PKA in eticlopride-induced immediate early gene expression, we administered the phosphodiesterase inhibitor IBMX to animals treated with eticlopride. We hypothesized that IBMX, by inhibiting cAMP degradation, 38 Fig. 2.2. Effect of the NMDA receptor antagonists CGS 19755 and MK-801 on c-fos expression induced by a low dose of eticlopride in the striatum. The NMDA receptor antagonists CGS 19755 ("CGS"; 10 mglkg) and MK-801 ("MK"; 1 mglkg) were administered intraperitoneally to animals treated with the D2 dopamine receptor antagonist eticlopride ("Etic"; 0.5 mglkg, i.p.). CGS 19755 was administered 1 hr before the administration of eticlopride. MK-801 was administered 30 min before the injection of eticlopride. Animals receiving eticlopride alone received a vehicle injection either 1 hr or 30 min before eticlopride administration. Control animals received two vehicle injections. Animals were sacrificed 40 min after the second injection. Values are mean gray values (±S.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the mid-striatum (approximately 0.5 mm anterior to bregma). Numbers in parentheses indicate the number of animals per group. *Significantly different from control, p<0.05. +Significantly different from eticlopride alone, p<0.05. 010 :!c:: I ::;:, ~ 8~ ca -..t.--.. €ca 6 ""-"" (1) ~ 4 > .>c.a.a 2 C) c ca ~ 0 Control (4) * I *n Etic (4) CGS + Etic (4) I _ medial striatum I • central striatum I 0 lateral striatum MK + Etic (4) W 1..0 40 Fig. 2.3. Effect of the NMDA receptor antagonists CGS 19755 and MK-801 on zif268 expression induced by a low dose of eticlopride in the striatum. The NMDA receptor antagonists CGS 19755 ("CGS"; 10 mg/kg) and MK-801 ("MK"; 1 mg/kg) were administered intraperitoneally to animals treated with the D2 dopamine receptor antagonist eticlopride ("Etic"; 0.5 mg/kg, i.p.). CGS 19755 was administered 1 hr before the administration of eticlopride. MK-801 was administered 30 min before the injection of eticlopride. Animals receiving eticlopride alone received a vehicle injection either 1 hr or 30 min before eticlopride administration. Control animals received two vehicle injections. Animals were sacrificed 40 min after the second injection. Values are mean gray values (±S.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the mid-striatum (approximately 0.5 mm anterior to bregma). Numbers in parentheses indicate the number of animals per group. *Significantly different from control, p<0.05. +Significantly different from eticlopride alone, p<0.05. .m..-. 60 C :::J 50 ~ ca I- :.:c: 40 I- ca ~30 -c:::oJ > 20 c>a- '- tn10 c co ~ 0 Control (4) * * Etic (4) ..... medial striatum central striatum o lateral striatum + CGS + Etic MK + Etic (4) (4) ~ I--" 42 would potentiate eticlopride-induced gene expression. In fact, IBMX did potentiate the eticlopride-induced gene expression at the 25.0 mg/kg dose (p < 0.05; Figs. 2.4 and 2.5). CGS 19755 only partially attenuates c-fos expression and does not affect zif268 expression after the combined administration of eticlopride and IBMX As previously observed, the NMDA receptor antagonist COS 19755 significantly reduced the induction of c-fos and zij268 by 0.5 mg/kg eticlopride alone (Figs. 2.6 and 2.7). However, it did not block induction produced by the combined administration of eticlopride and IBMX (Figs. 2.6 and 2.7). The expression of c-fos was only partially attenuated by COS 19755 in the medial and central regions of the striatum, whereas zij268 expression was unaffected by the NMDA receptor blockade with COS 19755 in all regions of the striatum. Discussion Our previous data (Keefe and Adams, 1998) demonstrated that NMDA receptor blockade selectively attenuated immediate early gene expression induced by a high dose of eticlopride (1.0 mglkg) in the medial, but not the lateral aspect of the striatum. This was presumably due to greater numbers of D2 receptors and a more widespread activation of PKA in the lateral striatum. The results presented here demonstrate the 43 Fig. 2.4. Effect of IB}'dX on c-fos expression induced by a high dose of eticlopride in the striatum. The nonselective phosphodiesterase inhibitor IBMX (5, 10, and 25 mg/kg) was administered intraperitoneally to animals treated with the D2 dopamine receptor antagonist eticlopride ("Etic"; 1.0 mg/kg, i.p.). IBMX was administered 15 min before the administration of eticlopride. Animals receiving eticlopride alone received a vehicle injection 15 min prior to eticlopride administration. Control animals received two vehicle injections. Animals were sacrificed 40 min after the second injection. Values are mean gray values (±S.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the mid-striatunl (approximately 0.5 mm anterior to bregma). Numbers in parentheses indicate the number of animals per group. *Significantly different from control, p<0.05. +Significantly different from eticlopride alone, p<0.05. 0-..-..4 0 § 35 ~ m30 .I..­. :e 25 ca ~20 :::J 1>ti 15 iU10 l-t » c: 5 ca + * ~ OIT. II Control Etic (5) 5 mg/kg 10 mg/kg 25 mg/kg (6) IBMX + IBMX + IBMX + Etic (5) Etic (5) Etic (4) • medial striatum central striatum ~ o lateral striatum t 45 Fig. 2.5. Effect of IBMX on zif268 expression induced by a high dose of etic lop ride in the striatum. The nonselective phosphodiesterase inhibitor IBMX (5, 10, and 25 mg/kg) was administered intraperitoneally to animals treated with the D2 dopamine receptor antagonist eticlopride ("Etic"; 1.0 mg/kg, i.p.). IBMX was administered 15 min before the administration of eticlopride. Animals receiving eticlopride alone received a vehicle injection 15 nUn prior to eticlopride administration. Control animals received two vehicle injections. Animals were sacrificed 40 min after the second injection. Values are mean gray values (tS.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the mid-striatum (approximately 0.5 nun anterior to bregma). Numbers in parentheses indicate the number of animals per group. *Significantly different from control, p<0.05. +Significantly different from eticlopride alone, p<0.05. .0..-..1 20 c ;'100 I­m I- .:!c:: 80 I- m ~ 60 ::l (ij > 40 m>- l-t » 20 c m CD :E o Control (6) + + * Etic (5) 5 mg/kg 10 mg/kg 25 mg/kg IBMX + IBMX + IBMX + Etic (5) Etic (5) Etic (4) • medial striatum central striatum I'lIIXIIIlIID o lateral striatum ~ 0\ 47 Fig. 2.6. Effect of CGS 19755 on c-fos expression induced in the striatum by the combined administration of eticlopride and IBMX. The NMDA receptor antagonist CGS 19755 ("CGS"; 10 mg/kg) and the nonselective phosphodiesterase inhibitor IBMX (25 mg/kg) were administered intraperitoneally to animals treated with the D2 dopamine receptor antagonist eticlopride ("Etic"; 1.0 mg/kg, i.p.). CGS 19755 or vehicle was administered 45 min prior to IBMX or vehicle, which was administered 15 min before the administration of eticlopride. Animals receiving eticlopride alone received two vehicle injections 1 hr and 15 min prior to eticlopride administration. Control animals received three vehicle injections. Animals were sacrificed 40 min after the last injection. Values are mean gray values (tS.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the mid-striatum (approximately 0.5 mm anterior to bregma). Numbers in parentheses indicate the number of animals per group. *Significantly different from control, p<0.05. +Significantly different from eticlopride alone, p<0.05. #Significantly different from IBMX + eticlopride, p<0.05. 060 :t:: c: ;'50 1- ca 1- .:ta:: 40 1- ca ~30 :::J as > 20 c>a- 1- tD10 c: ca * * + * • medial striatum III central striatum o lateral striatum * G) 0 I :dfi :! Control Etic (7) 25 mg/kg 10 mg/kg CGS + (6) IBMX + eGS + IBMX + Etic (7) Etic (8) Etic (5) ~ 00 49 Fig. 2.7. Effect of CGS 19755 on zif268 expression induced in the striatum by the combined administration of eticlopride and IBMX. The NMDA receptor antagonist CGS 19755 ("CGS"; 10 mg/kg) and the nonselective phosphodiesterase inhibitor ffiMX (25 mg/kg) were administered intraperitoneally to animals treated with the D2 dopamine receptor antagonist eticlopride ("Etic"; 1.0 mg/kg, i.p.). CGS 19755 or vehicle was administered 45 min prior to IBMX or vehicle, which was administered 15 min before the administration of eticlopride. Anmlals receiving eticlopride alone received two vehicle injections 1 hr and 15 min prior to eticlopride administration. Control animals received three vehicle injections. Animals were sacrificed 40 min after the last injection. Values are mean gray values (±S.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the mid-striatum (approximately 0.5 rom anterior to bregma). Numbers in parentheses indicate the number of animals per group. *Significantly different from control, p<0.05. +Significantly different from eticlopride alone, p<0.05. #Significantly different from IBMX + eticlopride, p<0.05. • medial striatum central striatum IIIlI'IilIR o lateral striatum en 160., + L--+- ~ * * § 140 >- m 120 .I.-. :.c... 100 m ~ 80 ::J a>s 60 ~ 40 I-m c: 20 m CI) :! 0 Control Etic (7) 25 mg/kg 1 0 mg/kg CGS+ (6) IBMX+ CGS+ IBMX+ Etic (7) Etic (8) Etic (5) LIl 0 51 susceptibility of c-fos and zif268 expression to NMDA receptor blockade throughout striatum after the administration of a lower dose of eticlopride (0.5 mg/kg). We propose that the lower dose of etic10pride activated less nuclear PKA than the higher dose, rendering gene expression dependent on NMDA receptor activation even in the lateral third of the striatum. In this study, we have also shown that the nonselective phosphodiesterase inhibitor IBMX (25 mg/kg) potentiated eticlopride-induced gene expression to the extent that NMDA receptor blockade no longer affected the expression of zij268 and only partially suppressed c-fos expression. Although these studies do not directly assess the role of PKA in D2INMDA receptor interactions, they do indirectly support the model proposed by Konradi (1998) that the levels of activated PKA determine whether striatal gene expression is dependent on NMDA receptor activation. It has been demonstrated that D2 dopamine receptor stimulation leads to the activation of an inhibitory G protein (GJ (Vallar and Meldolesi, 1989), presumably inhibiting cAMP formation and the activation of PKA. Thus, D2 receptor blockade should disinhibit cAMP formation and PKA activation. PKA can phosphorylate CREB, a transcription factor bound to the cAMP response element (CRE) in the promoter region of genes such as c-fos and zif268 (Montminy and Bilezikjian, 1989). CREB phosphorylation recruits RNA polymerase II to the promoter region, leading to the transcription of immediate early genes (Kwok et aI., 1994; Kee et aI., 1996). It has also 52 been demonstrated that PKA phosphorylates the NMDA receptor in striatal neurons (Rajadhyaksha et aI., 1998; Snyder et al., 1998). NMDA receptor activation mediates a voltage-dependent influx of Ca++, strongly depolarizing the postsynaptic membrane. This can trigger the opening of L-type voltage-sensitive calcium channels (Rajadhyaksha et al., 1999). Calcium entry via the NMDA receptor and L-type channels then presumably activates a kinase cascade that leads to the phosphorylation of CREB and the induction of c-fos in striatal neurons (Rajadhyaksha et al., 1999). The induction of immediate early genes by D2 receptor blockade has been shown to be attenuated by NMDA receptor antagonists (Dragunow et aI., 1990; Ziolkowska and Hollt, 1993; Boegman and Vincent, 1996; Keefe and Adams, 1998), suggesting a modulatory interaction between these two receptors. For example, induction of c-fo s or Fos protein by the antipsychotic haloperidol is blocked by administration of the NMDA receptor antagonists MK-801 and CPP (Dragunow et al., 1990; Ziolkowska and Hollt, 1993; Boegman and Vincent, 1996). Boegman and Vincent (1996) showed a 68% blockade by MK-801 of Fos-positive innnunoreactivity in the dorsolateral region of the striatum following administration of a low dose of haloperidol (0.2 mglkg). Our data reported herein confinn and extend the findings of Boegman and Vincent (1996) in that MK-801 and CGS 19755 blocked c-fos expression induced by a low dose of etic10pride not only in the lateral striatum but also in the 53 medial and central regions. The expression of zij268 also was completely blocked by administration of MK-801 and CGS 19755 throughout striatum after administration of this lower dose of eticlopride. Furthermore, our findings extend those of previous reports in that we have shown that D2 dopamine receptor antagonist-mediated gene expression is differentially susceptible to NMDA receptor blockade depending on the dose of antagonist administered and the region of striatum examined (see Figs. 2.1, 2.2, and 2.3). TIris is presumably due to greater activation of PKA in the lateral striatum after administration of the higher dose of eticlopride. Interestingly, administration of a higher dose of haloperidol (1 mglkg) in the Boegman and Vincent study (1996) had a greater effect on the number of Fos-positive nuclei than did the lower dose, but data were not reported for the effect of MK-801 on Fos induction by the high dose of haloperidol. The model of Konradi (1998) suggests that the intracellular kinase mediating the interactions between NMDA and dopamine receptors is PKA. Therefore, we administered the nonselective phosphodiesterase inhibitor IBMX to animals treated with eticlopride in an attempt to potentiate the eticlopride-induced gene expression, presumably through an increase in PKA activation. The combined administration of IBMX plus eticlopride produced significantly greater gene induction than did eticlopride alone. Pretreatment with the NMDA receptor antagonist CGS 19755 had no 54 effect on the zij268 expression induced by the combined IBMX and eticlopride administration, whereas c-fos expression was partially attenuated. At present, the basis for the partial effect of CGS 19755 on c-fos expression is unknown. It has been previously demonstrated that IBMX administration alone can lead to the expression of c-fos in the striatum (Svenningsson et al., 1995). However, the induction by IBMX in that study was attributed to the antagonistic effect of IBMX on the adenosine A2a receptor (Fredholm et al., 1985) due to the lack of effect of other selective phosphodiesterase inhibitors on striatal c-fos expression (Svenningsson et aI., 1995). Adenosine A2a receptors are colocalized on striatopallidal neurons with D2 dopamine receptors (Schiffman et al., 1991). Adenosine A2a receptors are coupled to a stimulatory G protein (Gs) and activate adenylyl cyclase (Premont et al., 1979; Van Calker et aI., 1979). However, it has been demonstrated that A2a receptor antagonist administration leads to the induction of immediate early genes in striatum, presumably by promoting the release of calcium from microsomes of the endoplasmic reticulum (Verma et aI., 1992; Svenningsson et aI., 1995). Therefore, we cannot absolutely attribute the increase in immediate early gene expression reported herein to the phosphodiesterase properties of IBMX without also considering the A2a antagonist receptor properties of IBMX. Further experiments are necessary to differentiate these two possibilities. However, the present data clearly indicate that the degree of gene 55 induction and, presumably, intracellular signaling activity determines the extent to which D2 antagonist-induced gene expression is dependent on NMDA receptors. In conclusion, our data support the model proposed by Konradi (1998) that the levels of activated PKA determine whether D2 dopamine receptor antagonist-mediated irrnnediate early gene expression is dependent on NMDA receptor activation. It is important to note, however, that the contribution of PKA to the induction of irrnnediate early genes by etic10pride has not been directly tested. 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CHAPTER 3 ETICLOPRIDE INDUCES STRIA TAL IMMEDIATE EARLY GENE EXPRESSION INDEPENDENT OF PROTEIN KINASE A ACTIVATION Introduction The induction of immediate early genes by D2 dopamine receptor antagonists has been previously demonstrated (Dragunow et aI., 1990; Miller, 1990; Robertson and Fibiger, 1992; Rogue and Vincendon, 1992; Ziolkowska and HoUt, 1993; Keefe and Adams, 1998). This induction is thought to occur as a consequence of the activation of signaling pathways previously inhibited by ongoing D2 dopamine receptor activation. Stimulation of the D2 dopamine receptor leads to a decrease in adenylyl cyclase activity via an inhibitory G protein (Gj), reducing cAMP formation and protein kinase A (PKA) activation. Antagonists of the D2 receptor presumably relieve this inhibition, leading to the activation of PKA. PKA phosphorylates the cAMP-response element binding protein CREB (Dash et aI., 1991) which interacts with the cAMP-response element (CRE) site in the promoter region of several genes such as c-fos and zij268 (Montminy 61 and Bilezikjian, 1987; KonratH et at, 1994). Konradi and Heckers (1995) have shown that the antipsychotic haloperidol (a partial D2 dopamine receptor antagonist) induces the immediate early gene c-fos via phosphorylation of CREB. In addition, haloperidol-induced c-fos expression has been shown to be completely blocked in PKA knockout mice (Adams et al., 1997). Although the dependence of D2 dopamine receptor antagonist-mediated gene expression on the activation of PKA has been implicated, it has been directly examined in vivo only once (Adams et at, 1997). We have previously shown (Chapter 2) that the nonselective phosphodiesterase inhibitor IBMX (25 mg/kg) potentiated eticlopride-induced gene expression to the extent that NMDA receptor blockade no longer affected the expression of zij268 and only partially suppressed c-fos expression. Although this study did not directly assess the role of PKA in D2INMDA receptor interactions, the data indirectly support the model proposed by Konradi (1998) that the levels of activated PKA determine whether striatal gene expression is dependent on NMDA receptor activation. We therefore wanted to determine if eticlopride-mediated IEG expression was dependent on PKA activation. We administered the PKA inhibitor H- 89 to animals treated with a high dose of eticlopride (1.0 mg/kg) and hypothesized that H-89 would block eticlopride-induced immediate early gene expression in the striatum. In addition, we attempted to measure levels of activated PKA in the striatum after treatment with eticlopride alone or the combined administration of eticlopride and H-89. 62 We also administered either the adenosine A2a receptor agonist CGS 21680 or the phosphodiesterase-4 (PDE4) inhibitor rolipram to animals treated with etic1opride. Adenosine A2a receptors are colocalized with D2 dopamine receptors on striatopallidal neurons and are coupled to a stimulatory G protein (Gs), leading to the activation of adenylyl cyclase (Premont et al., 1977; Fink: et aI., 1992). Likewise, PDE4 is a PDE family regulated by cAMP that is found in striatum, and activation of PDE4s can increase the rate of cAMP breakdown (Beavo, 1995). Therefore, we hypothesized that if eticlopride-mediated innnediate early gene expression were dependent on PKA activation, CGS 21680 and rolipram would potentiate the gene expression induced by eticlopride. Our results show that eticlopride administration (1. 0 mg/kg) did not measurably activate PKA and that the induction of striatal c-fos and zij268 by eticlopride was not affected by PKA inhibition. In addition, the administration of either CGS 21680 or rolipram did not alter eticlopride-induced immediate early gene expression in striatum. These data suggest for the first time that PKA is not solely responsible for D2 dopamine receptor antagonist-mediated gene induction in vivo. Methods Animals and housing Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 225 to 300 g were used in all experiments. Rats were housed in groups of 63 four in hanging wire-mesh cages in a temperature-controlled room on a 12:121ight:dark cycle. Rats had free access to food and water. All studies were approved by the Institutional Animal Care and Use Committee at the University of Utah and were perfonned in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Drugs Eticlopride hydrochloride and sodium pentobarbital were obtained from Research Biochemicals International (Natick, MA) and cas 21680 from Sigma Aldrich (St. Louis, MO). H-89 and rolipram were from Calbiochem (San Diego, CA). All drug doses were calculated as free bases with the exception of eticlopride hydrochloride, which was calculated as the salt. Eticlopride was dissolved in nonnal saline. Sodium pentobarbital and cas 21680 were dissolved in deionized water and rolipram in dimethyl sulfoxide (DMSO). H-89 ·was dissolved in DMSO and diluted with artificial cerebrospinal fluid (aCSF) to obtain the appropriate concentrations. The aCSF consisted of (final concentrations): NaCl (144 mM), KCI (2 mM), KH2P04 (0.4 mM), CaCl2 (1.2 roM), and MgCl2 • 6 H20 (1.2 mM). All drugs were administered intraperitoneally in a volume of 1 mLlkg except for H-89, which was administered by intrastriatal infusion at a rate of 0.1 JlL/min (total volume, 7.0 JlL). 64 Surgical procedures For the intrastriatal infusion of H-89, rats were anesthetized with sodium pentobarbital (50 mglkg, i.p.) and placed in a stereotaxic instrument. A 26-gauge guide cannula with a 3.6 mm projection (Plastics One Inc., Roanoke, VA) was lowered into the brain just above the lateral striatum at the following coordinates relative to bregma (in mm): AP +0.7, ML -3.4, and DV -3.6. Cranioplastic cement and cranial screws were used to fix the guide cannula to the skull. A dummy cannula was tightened onto the guide cannula in order to prevent postoperative damage or cannula blockage during the 5-day recovery period. Pharmacological manipulations Determinin~ the effect of H-89 on eticlopride-induced striatal gene expression and PKA activity. Rats with intracranial guide cannulae were taken from their home cages, weighed, and placed individually in cylindrical plastic tubs (II" diameter, 15" high). Rats were hand held while the experimenter replaced the dummy cannula with a 33-gauge infusion cannula (1.9 mm projection from the bottom of the guide) connected to a fluid swivel and a 25-j.1L Hamilton syringe on a syringe pump. The syringe and connecting tubing were backfilled with a DMSO/aCSF mixture (1:2000) or the PKA inhibitor H-89. The DMSO/aCSF or H-89 was infused into the lateral striatum through the infusion cannula at a rate of 0.1 j.1L/min for the extent of the experiment. Thirty min 65 after beginning the infusion, rats were treated with eticlopride (1 mg/kg) or vehicle. Rats were sacrificed 40 min after the eticlopride injection for analysis of immediate early gene expression and 5 min after the eticlopride injection for the PKA activity assay (see below). In addition, levels of activated PKA were measured in a group of rats treated with the cAMP activator Sp-8-Br-cAMPs instead of eticlopride for comparison. These rats were infused with Sp-8-Br-cAMPs (10 rrlM) at a rate of 0.1 ~L/min for 35 min, at which time the animals were sacrificed. This concentration of Sp-8-Br-cAMPs has been shown to increase the phosphorylation of CREB and Fos innnunoreactivity in rats (Choe and McGinty, 1999). Determinin2 the effects of CGS 21680 and rolipram on eticlopride-induced striatal 2ene expression. Rats were transferred from their home cages to plastic tub cages and were weighed. Rats were injected with CGS 21680 (0.05 - 5.0 mg/kg), rolipram (4 mg/kg), or vehicle, followed 15 min (CGS 21680) or 30 min (rolipram) later by administration of eticlopride or vehicle. Forty min after the second injection, the rats were sacrificed. The highest dose of CGS 21680 that we administered has been shown to induce c-fos expression in the nucleus accumbens shell (Pinna et at, 1997). The lower doses of CGS 21680 have been shown to decrease open-field locomotor activity in rats 20 min after administration (Janusz and Berman, 1992) and protect against kainate-induced excitotoxicity in the hippocampus (Jones et at, 1998). Rolipram at 4 66 mg/kg has been shown to increase extracellular cAMP levels in the rat striatum 30 min after administration as measured by in vivo microdialysis (lyo et aI., 1996). In situ hybridization histochemistry Rats were euthanized by exposure to CO2 (1 min) and decapitated. The brains were rapidly removed and frozen in isopentane chilled on dry ice. Brains were stored at -20°C until they were cut in 12-l-!m thick sections in a cryostat (Cryocut 1800, Cambridge Instruments, Germany) and thaw-mounted onto gelatin-chrome alum-subbed slides. Slides were stored at -20°C. Once all brains from an experiment were sectioned, slides were postfixed in 4% paraformaldehyde/0.9% NaCl, acetylated in fresh 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl (pH 8.0), dehydrated in an ascending series of alcohols, delipidated in chloroform, and rehydrated in a descending series of alcohols. Slides were air-dried and stored at -70°C. For detection of c-fos and zij268 mRNAs, full-length ribonucleotide probes complementary to the mRNAs for c-fos (Curran et aI., 1987) and zij268 (Milbrandt, 1987) were synthesized from the cDNAs using 35S_UTP and SP6 (c-fos) or T7 (zij268) RNA polymerase (Boehringer Mannheim, Indianapolis, IN). Labeled probes were diluted in hybridization buffer to obtain 2 x 106 cpml100 I-!L buffer. The ribonucleotide probe was mixed with nuclease-free water and RNA mix (final concentrations: 100 ""g/rnL salmon spenn DNA; 250 I-!g/rnL yeast total RNA; 250 J,.lg/rnL yeast tRNA). The 67 mixture was heated to 65°C for 5 min and then cooled on wet ice for 1 min. Dithiothreitol (100 ruM, final concentration), sodium dodecyl sulfate (0.2% w/v, final concentration), sodium thiosulphate (0.1 % w/v, final concentration), and hybridization buffer were added to the ribonucleotide mixture. The hybridization buffer contained (final concentrations): Tris buffer (23.8 mM, pH 7.4), EDTA (1.2 mM, pH 8.0), NaCl (357 mM), dextran sulfate (11.9%, w/v), Denhardt's solution (1.2 x), and formamide (59.5%, v/v). Ninety J-lL of probe in hybridization buffer was applied to each slide containing four sections. Slides were coverslipped and hybridized overnight in humid chambers at 55°C. Slides were then washed at room temperature four times in 1 x saline-sodium citrate (SSC, 0.15 M NaClIO.015 M sodium citrate, pH 7.2), incubated in ribonuclease A (RNase A; 5-20 J-lg/ml; Boehringer Mannheim) in buffer containing 0.5 M NaCl, 10 roM Tris (pH 8.0), and 0.25 mM EDTA (pH 8.0) for 15 min at room temperature, and washed 4 times in 0.2 x SSC at 60°C. Slides were rinsed briefly in deionized water, air dried, and apposed to Kodak Biomax x-ray film (Kodak Biomax MR, Eastman Kodak Co., NY) for 4 days to 2 weeks to obtain film autoradiograms. PKA activity assay Rats were sacrificed 5 min after the eticlopride injection by immediate decapitation. Brains were rapidly removed and frozen in isopentane. A I-mm thick coronal section was taken from the area surrounding the infusion track. Striatal tissue 68 was dissected from the cut section and was inunediately added to cold homogenization buffer containing 25 mM Tris-HCI (pH 7.5),0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM ~-mercaptoethanol, 1 Jlg/mL leupeptin, 1 Jlg/mL aprotinin, and 5 rrIM sodium orthovanadate (final concentrations). Samples were homogenized and then centrifuged for 5 min at 14,000 x g at 5°C. Supernatants were assayed for PKA activity using a PKA assay kit from Calbiochem (San Diego, CA). Briefly, supernatants (5 JlL) were added to a PKA reaction mix containing AlP solution, PKA reaction buffer, 32p_ ATP, deionized water, and the biotinylated synthetic PKA substrate kemptide (LeuArgArgAlaSerLeuGly). The reaction was stopped after 5 min with trichloroacetic acid (50%) and bovine senun albumin (1 %). A neutralization solution was added to the samples, followed by the addition of an avidin solution. Twenty JlL of sample and 50 JlL of wash solution were added to centrifugal ultrafiltration units containing a reservoir and membrane for separation. Samples were centrifuged for 5 min at 14,000 x g. After 3 additional washes, reservoirs were transferred to liquid scintillation vials containing scintillation cocktail and 32p incorporation was measured using a scintillation counter. Data analysis Film auto radiograms were analyzed using the Macintosh-based image analysis program, Image (Wayne Rasband, NIH). Images of brain sections were captured with a video camera, digitized, and stored on computer. Images were captured under constant 69 lighting conditions and within the linear range of the system response. Mean gray values were analyzed in medial, central, and lateral thirds of the right striatum from its dorsal aspect to the anterior commissure ventrally. The area of the cannula track was excluded from analysis. The average gray value of the white matter was subtracted from the average gray value of the regions of interest to correct for background labeling. Data from film autoradiograms were analyzed with a one-way analysis of variance for medial, central, and lateral thirds of the striatum. Post hoc analysis was performed with the Tukey-Kramer test. Statistical significance was set at p S 0.05. Data obtained from the PKA activity assay were also analyzed with a one-way analysis of variance, followed by post hoc analysis using the Tukey-Kramer test. Statistical significance was set at p S 0.05. Results The PKA inhibitor H·89 did not block eticlopride-induced immediate early gene expression Eticlopride administration (1.0 mg/kg) led to the expression of the immediate early genes, c-fos and zij268 (p < 0.05), with the highest levels of expression in the lateral region of striatum. Intrastriatal infusion of the PKA inhibitor H -89 (100 nM-1 roM) did not block eticlopride-induced c-jos or zij268 expression in any region of the striatum (Figs. 3.1 and 3.2). We attempted to infuse a higher concentration ofH-89 into 70 Fig. 3.1. Effect of the PKA inhibitor H-89 on eticlopride-induced c-fos expression in the striatum. The PKA inhibitor H-89 (100 nM, 0.1 mM, and 1.0 mM) was infused into the mid-striatum (approxinlately 0.5 mm anterior to bregma) at a rate of 0.1 J...LL/min for the duration of the experiment (70 min). Eticlopride ("Etic"; 1.0 mg/kg, i.p.) was administered 30 min after beginning the infusion of H -89. Animals were sacrificed 40 min after the injection of eticlopride. Control animals were infused with vehicle followed 30 min later by an injection of 0.9% saline. Animals receiving eticlopride alone were infused with vehicle for the duration of the experiment. Val ues are mean gray values (±S.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the striatum. Numbers in parentheses indicate the number of animals per group. *Significantly different from control, p<0.05. 14 .......... UJ :c!:: 12 :::s ~10 .c.a. .~.c.. 8 ~ 6 (1) :::s ca 4 > .f.a. ' 2 e) c 0 m Control (6) Etic (6) :E - medial striatum • central striatum * o lateral striatum * 100 nM H- 0.1 mM H- 1 mM H-89 89 + Etic 89 + Etic + Etic (7) (6) (5) .-...J.. 72 Fig. 3.2. Effect of the PKA inhibitor H-89 on eticlopride-induced zij268 expression in the striatum. The PKA inhibitor H-89 (100 nM, 0.1 mM, and 1.0 InM) was infused into the mid-striatum (approximately 0.5 nun anterior to bregma) at a rate of 0.1 JlL/min for the duration of the experiment (70 min). Eticlopride ("Etic"; 1.0 mglkg, i.p.) was administered 30 min after beginning the infusion of H-89. Animals were sacrificed 40 min after the injection of eticlopride. Control animals were infused with vehicle followed 30 min later by an injection of 0.9% saline. Animals receiving eticlopride alone were infused with vehicle for the duration of the experiment. Values are mean gray values (tS.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the striatum. Numbers in parentheses indicate the number of animals per group. *Significantly different from control, p<0.05. 90 .~J-!! 80 I * 570 ~60 :.:.ct..... 50 :t... ~40 (I) .:! 30 ctS > 20 ~ f!10 C) c 0 m Control (6) Etic (6) :E * * IICIIIl'III:I medial striatum central striatum o lateral striatum 100 nM H- 0.1 mM H- 1 mM H-89 89 + Etic 89 + Etic + Etic (7) (6) (5) ....,) w 74 the striatum (100 mM); however the concentration ofDMSO required to dissolve the H- 89 at this concentration and the high acidic salt content of this concentration of H-89 induced gross and extensive tissue damage (data not shown), rendering the gene analysis difficult and interpretation of any changes unreliable. Eticlopride administration did not activate PKA The levels of activated PKA were measured after the administration of eticlopride, the PKA activator Sp-8-Br-cAMPs, or the combined administration of eticlopride and H-89. Eticlopride did not produce a measurable amount of activation of PKA. Therefore, we were not able to measure a decrease in activated PKA levels by the combined administration of eticlopride with 1 mM H-89 (Fig. 3.3). We were able to show, however, that intrastriatal administration of the direct cAMP activator Sp-8-Br-cAMPs increased levels of activated PKA to 3.5 times the amount measured in control animals (Fig. 3.3). The adenosine A2a receptor agonist CGS 21680 did not induce immediate early gene expression, nor did it potentiate the induction by eticlopride We administered the adenosine A2a agonist CGS 21680 to animals, hypothesizing that the positive coupling of the A2a receptor to adenylyl cyclase would induce gene expression and possibly potentiate the induction by eticlopride. However, 75 Fig. 3.3. Effects oj eticiopride, Sp-8-Br-cAMPs, and H-89 on PKA specific activity in the striatum. The PKA inhibitor H-89 (1.0 ruM), the cAMP activator Sp-8-Br-cAMPs (10 ruM), or vehicle was infused into the mid-striatum (approximately 0.5 nun anterior to bregma) at a rate of 0.1 J,.lL/min for the duration of the experiment (35 min). Eticlopride ("Etic"; 1.0 mglkg, i.p.) was administered 30 min after beginning the infusion ofH-89. Animals treated with Sp-8-Br-cAMPs received an injection of normal saline instead of eticlopride. Animals were sacrificed 5 min after the injection of eticlopride or saline. Control animals were infused with vehicle followed 30 min later by an injection of normal saline. Animals receiving eticlopride alone were infused with vehicle for the duration of the experiment. Values represent the PKA specific activity (pmole phosphate incorporated per minute per 5 J,.lL sample; ±S.E.M.). Numbers in parentheses indicate the number of animals per group. * Significantly different from control, p<0.05. 0.5 .~-0.4 ..>.-, (.) co 0.3 ....(-.- ..). (.) &0.2 fn « ~ 0.1 o Control (5) Etic (5) * Etic + 1 mM 10 mM Sp-8- H-89 (5) Br-cAMPs (5) -.....l 0\ 77 CGS 21680 (5.0 mg/kg) did not induce the expression of c-fos or zij268 in the striatum when administered alone, nor did it potentiate the induction of c-fos and zij268 by eticlopride (Figs. 3.4 and 3.5). The phophodiesterase-4 selective inhibitor rolipram did not potentiate eticlopride-induced immediate early gene expression We also administered the phosphodiesterase-4 selective inhibitor rolipram to animals in order to determine if inhibiting the hydrolysis of cAMP would potentiate eticlopride-induced gene expression by increasing the levels of PKA activation. Like the adenosine A2a receptor agonist, rolipram did not induce the expression of c-fos and zij268 in the striatum when administered alone, nor did it potentiate the induction by eticlopride (Figs. 3.6 and 3.7). In fact, rolipram slightly attenuated eticlopride-induced c-fos expression. Discussion The findings presented herein show that striatal immediate early gene expression induced by administration of the D2 dopamine receptor antagonist eticlopride is not altered by inhibition of PKA with H-89. In addition, the cAMP activator Sp-8-Br-cAMPs induces activation of PKA in the striatum, whereas eticlopride administration at a dose of 1 mg/kg either does not result in the activation of PKA or activates such low 78 Fig. 3.4. Effect of CGS 21680 on eticlopride-induced c-fos expression in the striatum. The adenosine A2a agonist CGS 21680 ("CGS"; 0.05, 0.5, and 5.0 mg/kg, i.p.) was administered systemically to animals treated with the D2 dopamine receptor antagonist eticlopride ("Etic"; 1.0 mg/kg, i.p.). CGS 21680 was administered 15 min before the administration of eticlopride. Animals receiving eticlopride alone received a vehicle injection 15 min prior to eticlopride administration. Control animals received two vehicle injections. Animals were sacrificed 40 min after the injection of eticlopride. Values are mean gray values (tS.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the mid-striatum (approximately 0.5 mm anterior to bregma). Numbers in parentheses indicate the number of animals per group. *Significantly different from control, p<0.05. 12- ~ f/J ~ ·c 10 ::s mI>I... S ...I.cI-..,. II.. 6 ..m......., Q) -::s 4 m > m~ 2 II.. D) * * * .... medial striatum central striatum o lateral striatum * * c 0 m Control Etic (5) 5 mg/kg 0.05 0.5 mgl 5.0 mgl :E (6) eGS (6) mg/kg kg eGS kg eGS eGS + + Etic (6)+ Etic (6) Etic (7) .....:J \0 80 Fig. 3.5. Effect of CGS 21680 on eticlopride-induced zij268 expression in the striatum. The adenosine A2a agonist CGS 21680 ("CGS"; 0.05, 0.5, and 5.0 mg/kg, i.p.) was administered systemically to animals treated with the D2 dopamine receptor antagonist eticlopride ("Etic"; 1.0 mg/kg, i.p.). CGS 21680 was administered 15 min before the administration of eticlopride. Animals receiving eticlopride alone received a vehicle injection 15 min prior to eticlopride administration. Control animals received two vehicle injections. Animals were sacrificed 40 min after the injection of eticlopride. Values are mean gray values (±S.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the mid-striatum (approximately 0.5 mm anterior to bregma). Numbers in parentheses indicate the number of animals per group. *Significantly different from control, p<0.05. 80 "........,. .J!-! 70 t: :::s 60 ~ --.rf.e.e-!.s. 5400 ~ :Q:s) 30 ~20 ~ f!10 C) * * .... medial striatum central striatum o lateral striatum t: 0 m Control Etic (5) 5 mg/kg 0.05 0.5 mgt 5.0 mgt :5 (6) eGS (6) mg/kg kg eGS kg eGS eGS + + Etic (6) + Etic (6) Etic (7) .0...0.. 82 Fig. 3.6. Effect of rolipram on eticlopride-induced c-fos expression in the striatum. The phosphodiesterase-4 selective inhibitor rolipram (4.0 mg/kg, i.p.) was administered systemically to animals treated with the D2 dopamine receptor antagonist eticlopride ("Etic"; 1.0 mg/kg, i.p.). Rolipram was administered 30 min before the administration of etic1opride. Animals receiving etic10pride alone received a vehicle injection 30 min prior to eticlopride administration. Control animals received two vehicle injections. Animals were sacrificed 40 min after the second injection. Values are mean gray values (±S.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the mid-striatum (approximately 0.5 mm anterior to bregma). Numbers in parentheses indicate the number of animals per group. *Significantly different from control, p<0.05. +Significantly different from eticlopride alone, p<0.05. 0.....4 0 '§ 35 ~30 .I...­. :c 25 I- ca ~20 Q) :::s c>a 15 ~10 I- ~ 5 ca G) 0 :E Control (4) tIIiIIIM.IB medial striatum central striatum o lateral striatum Etic (4) Rolipram (4) 4 mg/kg Rolipram + Etic (4) 00 w 84 Fig. 3.7. Effect of rolipram on eticlopride-induced zif268 expression in the striatum. The phosphodiesterase-4 selective inhibitor rolipram (4.0 mg/kg, i.p.) was administered systemically to animals treated with the D2 dopamine receptor antagonist eticlopride ("Etic"; 1.0 mg/kg, i.p.). Rolipram was administered 30 min before the administration of eticlopride. Animals receiving eticlopride alone received a vehicle injection 30 min prior to etic10pride administration. Control animals received two vehicle inj ections. Animals were sacrificed 40 min after the second injection. Values are mean gray values (±S.E.M.; arbitrary units) obtained from densitometric analysis of the medial, central, and lateral thirds of the mid-striatum (approximately 0.5 rnm anterior to bregma). Numbers in parentheses indicate the number of animals per group. *Significantly different from control, p<0.05. 90 .-s...-....a.. o 570 ~60 :.1.c.-. 50 1- ~40 G) .2 30 a1 > 20 >. ~10 C) ; 0 G) ~ Control (4) Etic (4) Rolipram (4) * 4 mg/kg Rolipram + Etic (4) • medial striatum • central striatum o lateral striatum 00 til 86 levels of PKA that it is not detectable by our PKA activity assay. Due to our inability to measure PKA activation after etic10pride administration, we were not able to detect a decrease in PKA activity by H-89. However, the doses of H-89 tested were approximately 2, 200, 20,000, and 2,000,000 times the ~ of the drug (Ki = 48 nM; Chijiwa et aI., 1990), suggesting that the doses that we directly infused into striatal tissue should have been sufficient to block PKA activity. Finally, the immediate early gene expression induced by eticlopride administration was unaffected by prior administration of the adenosine A2a receptor agonist CGS 21680 or the phosphodiesterase-4 selective inhibitor rolipram, both of which should increase PKA activity. Taken together, these findings suggest that activation of the PKA -mediated signaling pathway is not necessary for the induction of immediate early genes by acute D2 dopamine receptor blockade in vivo and that other signaling pathways must be involved in this gene expression. These results are in contrast to previous data demonstrating the involvement of PKA in dopamine-mediated gene expression (Adams et al., 1997). The activation of PKA by D2 dopamine receptor blockade and its role in D2 dopamine receptor antagonist-mediated striatal immediate early gene expression has been suggested due to the negative coupling of the D2 receptor to adenylyl cyclase activation (Weiss et aI., 1985; Albert et aI., 1990). In fact, it has been shown that gene expression induced by the partial D2 antagonist haloperidol is blocked in PKA 87 knockout mice (Adams et aI, 1997). However, haloperidol has affinity for other receptors such as ucadrenergic receptors, 5HT 2 serotonin receptors, and sigma receptors (Leysen et aI., 1993; Amt and Skarsfeldt, 1998). Therefore, the gene expression induced by haloperidol and the dependence of the expression on PKA may be a result of haloperidol acting through other receptor types on striatal neurons. Eticlopride is more selective for D2 dopamine receptors than haloperidol (Hall et a!., 1985; Kohler et a!., 1996), suggesting that the lack of effect of PKA inhibition on eticlopride-induced gene expression is due to very low levels of PKA activation upon D2 receptor blockade. Other studies have shown that D1 dopamine receptor stimulation, which is positively coupled to adenylyl cyclase, leads to gene expression in primary striatal cell culture and that this expression is sensitive to PKA inhibition (Simpson and Morris, 1995; Das et aI., 1997). However, conflicting data exist on the role that PKA has in dopamine-induced gene expression in more intact striatal preparations. For example, a study by Liu et a!. (1995) demonstrated in an organotypic striatal slice preparation that D 1 agonist-induced gene expression is not affected by PKA inhibition. Thus, the existing data from in vitro studies do not conclusively show that PKA is necessary for dopamine-mediated gene induction. Our data provide in vivo support to the study by Liu et aI. (1995) and suggest that PKA activation is not necessary or solely responsible for D2 antagonist-induced expression in vivo. 88 Compounds acting at the D2 dopamine receptor exert their intracellular effects through the coupling of the receptor to G proteins, namely, Gi and Go (Stoof and Kebabian, 1984). It has been demonstrated that D2 dopamine receptor stimulation leads to a decrease in adenylyl cyclase activity in striatal cells (Weiss et aI., 1985; Albert et al., 1990). As noted above, it has been presumed that the induction of immediate early genes by D2 receptor blockade is dependent on the disinhibition of adenylyl cyclase and subsequent PKA activation. However, our data do not support this idea, suggesting that D2 dopamine receptor-induced alterations in other cellular components may provide alternative pathways for D2 antagonists to induce gene expression. One effect of D2 dopamine receptor stimulation is to decrease voltage-activated calcium currents, specifically those carried through L-type and N-type voltage-sensitive calcium channels (Liu et aI., 1992; Lledo et aI., 1992; Van et al., 1997). It appears that the inhibition of calcium currents by dopamine acting at D2 receptors is mediated through a membrane-delimited pathway involving a Go protein (Lledo et al., 1992; Van et aI., 1997). In addition, voltage-dependent potassium (K+) channel currents are altered after D2 dbpamine receptor stimulation. Administration of the D2 receptor agonist BHT 920 activates K+ channels in pituitary neurons (Memo et aI., 1992), and D2 receptor stimulation of striatal neurons also activates voltage-dependent K+ channels (Freedman and Weight, 1988). Like voltage-sensitive calcium channels, the activation of K+ channels by D2 receptor stimulation is also thought to be membrane-delimited 89 and mediated by a Go protein (Chiodo et ai., 1992). Taken together, these data suggest that the activation of D2 dopamine receptors leads to a depression of neuronal activity via a reduction in adenylyl cyclase-mediated signaling, decreased calcium influx through voltage-sensitive calcium channels, and increased activation of K+ channels. Therefore, D2 antagonist administration may provide an excitatory drive for neuronal activity, increasing calcium influx and decreasing K+ efflux. Intracellular calcium can modulate multiple cellular pathways mediated by phospholipase C (PLC) , adenylyl cyclase, nitric oxide synthase, other phospholipases, and calcium/calmodulin (CaM) kinases (Clapham, 1995). Thus, D2 receptor antagonist administration may lead to the expression of immediate early genes by increasing intracellular calcium concentrations and subsequently activating other PKA-independent pathways. There have been contradictory reports concerning the effects of D2 dopamine receptor stimulation on the activation of protein kinase C (PKC) and mobilization of calcium from internal stores induced by the hydrolysis of phosphatidyl-inositol 4,5- bisphosphate (PIP2). PLC mediates the hydrolysis of PIP2 to inositol1,4,5-ttiphosphate (IP3) and diacylglycerol (DAG). IP3 binds to receptors on the surface of the endoplasmic reticulum, leading to the mobilization of calcium, whereas DAG activates PKC. Two studies have reported that D2 receptor stimulation inhibits the production of IP3 in the anterior pituitary and the striatum (Simmonds and Strange, 1985; Pizzi et al., 1987). However, other groups have not been able to verify the effects of D2 dopamine 90 receptor activation on the hydrolysis ofPIP2 (Kelly et aI., 1988; Rubinstein et at, 1989). The D 1 dopan1ine receptor may be coupled to PLC activation and the hydrolysis of PIP 2 as demonstrated in striatum (Undie and Friedman, 1990; Undie and Friedman, 1994; Wang et aI., 1995; Rosengarten and Friedhoff, 1998). Therefore, based on the knowledge that D1 and D2 receptors have opposing effects on adenylyl cyclase, we could speculate that D2 receptor blockade would activate PLC just as D1 receptor activation does. Although it has been proposed that the D2 receptor may be negatively coupled to PIP2 (see review by Vallar and Meldolesi, 1989), this has not been directly demonstrated in vivo. If D2 receptor blockade does cause the activation of PLC and subsequent hydrolysis of PIP 2' then we could also speculate that release of calcium from the endoplasmic reticulum might lead to the activation of other signaling pathways (see above) such as the CaM kinase-regulated pathway. It has been shown that activation of CaM kinase can lead to the expression of immediate early genes (Enslen and Soderling, 1994; Bito et aI., 1996). The role of the CaM kinase pathway in the induction of immediate early genes by D2 dopamine receptor antagonists has not been determined. Therefore, we examined the effects of a CaM kinase inhibitor on eticlopride-induced immediate early gene expression in Chapter 4. Additionally, hydrolysis of PIP 2 by D2 dopamine receptor blockade would lead to the activation of PKC by DAG. In fact, incubation of striatal synaptoneurosomes with D2 receptor antagonists has been shown 91 to increase PKC activity, whereas D2 receptor agonists decrease PKC activity (Giambalvo and Wagner, 1994). PKC has been shown to directly phosphorylate CREB (Xie and Rothstein, 1995). In addition, PKC can phosphorylate the NMDA receptor, potentiating NMDA-evoked currents in cultured hippocampal neurons (Xiong et aI., 1998). These data suggest that D2 dopamine receptor blockade may increase the activation of components of the PLC pathway induced downstream of PIP 2 hydrolysis (specifically activation of PKC) and that activation of these components may lead to the activation of non-PKA-mediated signaling pathways, providing an additional mechanism for D2 receptor blockade to induce immediate early gene expression. Finally, another study has also demonstrated that D2 receptor blockade induces the formation of cGMP in striatal neurons (Altar et al., 1990); however the mechanism by which this occurs is unknown. The enzyme guanylate cyclase converts GTP to cGMP, activating cGMP-dependent protein kinase, which can act directly on ion channels and phosphorylate receptors in the membrane (Wang and Robinson, 1997). In striatonigral nerve terminals, it has been demonstrated that phosphorylation of the protein phosphatase regulator dopanline- and cAMP-regulated phosphoprotein (DARPP-32) is mediated by the activation of cGMP-dependent protein kinase (Tsou et aI., 1993). DARPP-32 is found extensively throughout the striatum (Walaas and Greengard, 1984) and plays a critical role in the induction of immediate early genes by inhibiting the activity of protein phosphatases that directly mediate the 92 dephosphorylation of CREB (Hemmings et aI., 1984; Alberts et aI., 1994). Although the role of cGMP in striatal neuron function has not been deternrined, its activation does appear to be induced by D2 dopamine receptor blockade (Altar et aI., 1990). In addition, a recent study has demonstrated that glutamate input from the parafascicular thalamic nucleus acting through striatal NMDA receptors results in an increase in cGMP formation in medium spiny neurons (Consolo et al., 1999). Therefore, ongoing formation of cGMP by NMDA receptor activation may potentiate effects mediated by D2 receptor blockade. Cyclic GMP has been shown to activate mitogen-activated protein (MAP) kinase in neuroendocrine tissue through activation of cGMP-dependent protein kinase (Ho et al., 1999). Interestingly, it has been suggested that MAP kinase may have an important role in the induction of immediate early gene expression in striatal neurons and that MAP kinases may be activated by NMDA receptor stimulation (Ginty et al., 1994; Sgambato et al., 1998; Vincent et al., 1998). Therefore, the effect of administration of a MAP kinase inhibitor on eticlopride-induced gene expression was examined in Chapter 4. In vivo evidence for the activation of cGMP by D2 receptor blockade would provide further evidence that D2 antagonist-mediated gene expression may occur independently of PKA activation and requires further examination. 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