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University Microfilms International 300 N. ZEEB RD., ANN ARBOR, Ml 48106 8129093
S c h w a r z , R o y D .
PROSTAGLANDIN MODULATION OF DOPAMINE-MEDIATED NEUROTRANSMISSION IN THE CENTRAL NERVOUS SYSTEM
The Ohio State University Ph.D. 1981
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University Microfilms International PROSTAGLANDIN MODULATION OF DOPAMINE-MEDIATED
NEUROTRANSMISSION IN THE CENTRAL NERVOUS SYSTEM
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Roy D. Schwarz, B.S., M.S.
The Ohio State University
1981
Reading Commitee: Approved By:
Dr. Joseph R. Bianchine
Dr. Norman J. Uretsky
Dr. Richard H. Fertel Advisor
Dr. Sarah Tjioe Department of Pharmacology ACKNOWLEDGMENTS
The author wishes to express his sincere appreciation to his advisor, Dr. Joseph Bianchine, for his support and guidance through out his entire graduate career. This support has allowed me to work independently and pursue those areas which I might not have been able to do so otherwise.
My future success as a researcher will to a large extent be due to Dr. Norman J. Uretsky. His expertise in the CNS area and approach to scientific questions in general have helped me to develop as a scientist. I have enjoyed working with him both professionally as well as personally.
The author would like to thank Dr. Richard Fertel for his enthu siastic approach to science and his willingness to discuss any subject at any time. Working in his laboratory is something every graduate student in this department should do during their career.
To my parents and sister, this degree is a result of our close family and the support it has always given me in all my decisions.
To Deb's family for making me feel like a member of the family and their continued support for both of us.
To Deb, who has made all this work and effort worth it. I couldn't have done it without her love and support. She deserves this degree as much as I do. This one's for you! VITA
February 20, 1949 ...... Born - Dover, N.J., U.S.A.
1971 ...... B.S., Marietta College Marietta, Ohio
1972 - 1977 ...... CIBA-Geigy Corporation Summit, N.J. Associate Scientist Scientist I
1975 ...... M.S., Fairleigh Dickinson University Teaneck, N.J.
1977 - 1981 ...... Graduate Research Associate The Ohio State University Columbus, Ohio
AWARDS
1981 ...... Chauncey D. Leake Award
1981 ...... Jack Van Fossen Award
1980 ...... Clayton S. Smith Award
PUBLICATIONS
Journal Articles:
Schwarz, R.D., J.W. Stein, P. Bernard, (1978), Rotometer for re cording rotation in chemically or electrically stimulated rats, Physiol. Behav. 20: 351-354.
Fertel, R.H., J.E. Greenwald, R.D. Schwarz, L. Wong, J.R. Bianchine (1980), The opiate receptor binding and analgesic effects of the tetra- hydroisoquinolines salsolinol and tetrahydropapaveroline, Res. Comm. Chem. Path, and Pharm. 27: 3-18.
Schwarz, R.D., N.J. Uretsky, J.R. Bianchine, (1980), The relation ship between the stimulation of dopamine synthesis and release produced by amphetamine and high potassium in striatal slices, J. Neurochem. 35: 1120-1127. iii Fertel, R.H., J.Z. Yetiv, M.A. Coleman, R.D. Schwarz, J.E. Greenwald, J.R. Bianchine, (1981), Chicken egg yolk: A new model for the production of antibody for radioimmunoassay, submitted for publication.
Schwarz, R.D., N.J. Uretsky, J.R. Bianchine, (1981), Prostaglan din inhibition of amphetamine-induced circling in mice, submitted for publication.
Schwarz, R.D., N.J. Uretsky, J.R. Bianchine, (1981), Prostaglan din inhibition of apomorphine-induced circling in mice, submitted for publication.
Book Chapters:
Bianchine, J.R., D.A. Brys, R.D. Schwarz, D.I. Eneanya, D.O. Duran, J.E. Greenwald, B.D. Andresen, (1980), Clinical correlates of changes in receptors during aging, Neural Regulatory Mechanisms During Aging, Alan R. Liss Inc, N.Y., 129-141.
Abstracts:
Greenwald, J.E., R.H. Fertel, L.K. Wong, R.D. Schwarz, J.R. Bianchine, (1979), Salsolinol and tetrahydropapaveroline bind opiate receptors in the rat brain, Fed. Proc. 38: 379.
Schwarz, R.D., N.J. Uretsky, J.R. Bianchine, (1979), Comparison of amphetamine and high potassium on the synthesis and release of dop amine, Pharmacologist 21: 180.
Schwarz, R.D*. N.J. Uretsky, J.R. Bianchine, (1980), Prostaglan din modulation of ^H-dopamine release from striatal slices, Fed. Proc. 39: 529.
Schwarz, R.D., N.J. Uretsky, J.R. Bianchine, (1980), Prostaglan din alteration of amphetamine-induced circling in mice, Pharmacologist 22: 293.
Schwarz, R.D., J.R. Bianchine, N.J. Uretsky, (1980), Modulation of dopamine-mediated neurotransmission in vivo and in vitro, Soc. Neurosci. Abstr. Vol. 6 : 625.
Yetiv, J.Z., R.H. Fertel, M.A. Coleman, R.D. Schwarz, J.E. Greenwald, J.R. Bianchine, (1980), Chicken Egg Yolk: A new source of antibody for radioimunoassay, Clin. Res. 28: 818A.
Schwarz, R.D., N.J. Uretsky, J.R. Bianchine, (1981), Postsynap- tic activity of prostaglandins as shown by the inhibition of apomor phine-induced circling in mice, Fed. Proc. 40: 316. FIELDS OF STUDY
or field of study: Neuropharraacology
Autonomic Pharmacology - Dr. S. Tjioe
Neurochemistry - Dr. L. Horrocks
Neuroanatomy - Dr. Humbertson
Gas Chromatography/ - Dr. L. Wong; Dr. C. Hammar Mass Spectometry
Toxicology - Dr. D. Couri TABLE OF CONTENTS
Page ACKNOWLEDGMENTS ...... ii
VITA ...... iii
LIST OF TABLES ...... xi
LIST OF FIGURES ...... xii
INTRODUCTION ...... 1
A. Historical Background ...... 1
B. Nomenclature ...... 2
C. Biosynthesis ...... 4
D. Metabolism ...... 6
E. Prostaglandin Inhibitors ...... 6
F. Prostaglandins in the CNS ...... 7
1. Biosynthesis ...... 7
2. Metabolism ...... 8
3. Suggested Roles ...... 9
G. Anatomy of Central Dopamine Pathways ...... 11
H. Role of Dopamine in Motor Function...... 15
I. Involvement of Dopamine in Disease States ...... 17
J. Statement of Problem...... 19
CHAPTER
I. MEASUREMENT OF BRAIN PROSTAGLANDINS
A. Introduction ...... 20
vi B. Methods ...... 21
1. Coupling of prostaglandin to KLH ...... 21
2. Generation of antibodies for radioimmunoassay.. 21
3. Sample preparation for radioimmunoassay ...... 21
4. Prostaglandin radioimmmunoassay ...... 22
C. Results ...... 22
1. Cross-reactivity of generated antibodies...... 22
2. Sensitivity of prostaglandin directed anti bodies ...... 23
3. Measurement of prostaglandins in whole brain of rats and mice ...... 23
D. Discussion ...... 24
II. PROSTAGLANDIN INHIBITION OF AMPHETAMINE-INDUCED CIRCLING IN MICE
A. Introduction ...... 30
B. Methods ...... 31
1. 6-Hydroxydopamine lesion in mice ...... 31
2. Testing protocol for circling ...... 32
3. Temperature measurement in mice ...... 33
4. Statistics ...... 33
C. Results ...... 33
1. Effect of intraventricularly administered prostaglandins on amphetamine-induced circling in mice ...... 33
2. Effect of intrastriatal administration of prostaglandins on amphetamine-induced circling in mice ...... 34
3. Modulation of body temperature by prostaglan dins ...... 35
D. Discussion ...... 36
vii III. PROSTAGLANDIN INHIBITION OF APOMORPHINE-INDUCED CIRCLING IN MICE
A. Introduction ...... 53
B. Methods ...... 54
1. 6-Hydroxydopamine lesion in mice ...... 54
2. Electrolytic lesion in mice .•...... 55
3. Testing protocol for circling ...... 55
4. Statistics ...... 55
C . Results ...... 56
1. Effect of intraventricular administration of prostaglandins on apomorphine-induced circling in mice ...... 56
2. Effect of intrastriatal administration of prostaglandins on apomorphine-induced circling in mice ...... 56
3. Effect of indomethacin pretreatment upon apo morphine-inducedcircling in mice ...... 58
D. Discussion ...... 58
IV. PROSTAGLANDIN MODULATION OF DOPAMINE RELEASE AND SYNTHESIS
A. Introduction ...... 74
B. Methods ...... 75
1. Dopamine release in rat striatal slices ...... 75
2. Dopamine synthesis in rat striatal slices ...... 76
3. Statistics ...... '...... 76
C. Results ...... 76
1. ^H-Dopamine release after varying K+ con centrations ...... 76
2. Effect of prostaglandins on basal release of dopamine ...... 77
3. Effect of prostaglandins on K+-stimulated release of dopamine ...... 77
viii 4. Effect of prostaglandins on basal dopamine synthesis ...... 77
5. Effect of prostaglandins on K+-stimulated dopamine synthesis ...... 78
D. Discussion ...... 78
V. PROSTAGLANDIN MODULATION OF STRIATAL CYCLIC AMP
A. Introduction ...... 91
B. Methods ...... 92
1. Assay of cyclic AMP in mouse striatal slices ... 92
2. Measurement of cyclic AMP by radioimmunoassay .. 93
3. Measurement of protein ...... 93
4. Statistics ...... 94
C. Results ...... 94
1. Effect of dopamine and apomorphine on cyclic AMP formation ...... 94
2. Effect of ethanol on cyclic AMP formation ..... 94
3. Effect of ethanol on dopamine-stimulated cyclic AMP formation ...... 94
4. Effect of prostaglandins on cyclic AMP forma tion ...... 95
5. Effect of prostaglandins on dopamine-stimulated cyclic AMP formation ...... 95
D. Discussion ...... 95
VI. EFFECT OF PROSTAGLANDINS ON 3 H-ACEYLCHOLINE RELEASE IN VITRO
A. Introduction ..... 109
B. Methods ...... 110
1. In vitro 3 H-Ach release from striatal slices ... 110
2. Statistics ...... Ill
C. Results ...... Ill
ix 1. Effect of varying K+ concentrations of 3 H-Ach release ...... Ill
2. Effect of Ca++ on K+-stimulated release of 3H-Ach ...... Ill
3. Effect of time of incubation on K+-stimulated 3H-Ach release ...... 112
4. Effect of varying aporaorphine conentrations on 3H-Ach release ...... Ill
5. Effect of apomorphine on K+-stimulated release of 3H-Ach ...... 112
6 . Effect of prostaglandins on basal 3H-Ach release ...... 112
7. Effect of prostaglandins on K+-stimulated release of 3H-Ach ...... 112
8 . Effect of prostaglandins on apomorphine inhibition of K+-stimulated release ...... 113
D. Discussion...... 113
VII. SUMMARY...... 134
LIST OF REFERENCES ...... 138
x LIST OF TABLES
TABLE PAGE
1. CROSS-REACTIVITY OF PROSTAGLANDIN-DIRECTED ANTIBODIES GENERATED FOR PROSTAGLANDIN RADIOIMMUNOASSAY ...... 28
2. MEASUREMENT OF PROSTAGLANDINS IN WHOLE BRAIN OF RATS AND MICE ...... 29
3. EFFECT OF INTRAVENTRICULARLY INJECTED PROSTAGLANDINS ON BODY TEMPERATURE IN MICE ...... 52
xi LIST OF FIGURES
FIGURE PAGE
1. MAJOR METABOLIC PATHWAYS IN THE CONVERSION OF ARACHIDONIC ACID IN THE HUMAN ...... 3
2. HORIZONTAL PROJECTION OF THE ASCENDING NE AND DA PATHWAYS.. 13
3. LATERAL VIEW OF THE ASCENDING DA PATHWAYS ...... 14
4. SENSITIVITY OF PROSTAGLANDIN-DIRECTED ANTIBODIES ...... 27
5. EFFECT OF INTRAVENTRICULARLY (ICV) AND INTRASTRIATALLY (IS) INJECTED PGD2 ON AMPHETAMINE-INDUCED CIRCLING IN MICE... .. 43
6 . EFFECT OF INTRAVENTRICULARLY (ICV) AND INTRASTRIATALLY (IS) INJECTED PGE2 ON AMPHETAMINE-INDUCED CIRCLING IN MICE... .. 45
7. EFFECT OF INTRAVENTRICULARLY (ICV) AND INTRASTRIATALLY (IS) INJECTED PGF2oj ON AMPHETAMINE-INDUCED CIRCLING IN MICE__ __ 47
8 . EFFECT OF INTRAVENTRICULARLY (ICV) THROMBOXANE B2 (TxB2) AND INTRASTRIATALLY (IS) INJECTED TxB2 AND 13, 14-DIHYDRO- -15-KETO-PGE2 ON AMPHETAMINE-INDUCED CIRCLING IN MICE .. 49
9. DOSE RESPONSE OF PROSTAGLANDINS ON AMPHETAMINE- INDUCED CIRCLING IN MICE ...... 51
10. EFFECT OF INTRAVENTRICULARLY (ICV) INJECTED PROSTAGLANDINS ON APOMORPHINE-INDUCED CIRCLING IN MICE ...... 63
11. EFFECT OF INTRASTRIATAL ADMINISTRATION OF PGD2 ON APOMORPHINE-INDUCED CIRCLING IN MICE ...... 65
12. EFFECT OF INTRASTRIATAL ADMINISTRATION OF PGE2 OR 13, 14-DIHYDRO-15-KETO-PGE2 ON APOMORPHINE-INDUCED CIRCLING IN MICE ...... 67
13. EFFECT OF INTRASTRIATAL, ADMINISTRATION OF PGF2t* ON APOMORPHINE-INDUCED CIRCLING IN MICE ...... 69
14. DOSE RESPONSE OF PROSTAGLANDINS ON APOMORPHINE- INDUCED CIRCLING IN MICE ...... 71
xii EFFECT OF INDOMETHACIN PRETREATMENT ON APOMORPHINE- INDUCED CIRCLING IN MICE ...... 73
EFFECT OF VARYING K+ CONCENTRATIONS ON DOPAMINE RELEASE ___ 82
EFFECT OF PROSTAGLANDINS ON BASAL RELEASE OF DOPAMINE ..... 84
EFFECT OF PROSTAGLANDINS ON K+-STIMULATED DOPAMINE RELEASE ...... 86
EFFECT OF PROSTAGLANDINS ON BASAL DOPAMINE SYNTHESIS ...... 88
EFFECT OF PROSTAGLANDINS ON K+-STIMULATED DOPAMINE SYNTHESIS ...... 90
EFFECT OF DOPAMINE AND APOMORPHINE ON CYCLIC AMP FORMATION ...... 100
EFFECT OF ETHANOL ON BASAL CYCLIC AMP FORMATION ...... 102
EFFECT OF ETHANOL ON DOPAMINE-STIMULATED CYCLIC AMP FORMATION ...... 104
EFFECT OF PROSTAGLANDINS ON BASAL CYCLIC AMP FORMATION ...... 106
EFFECT OF PROSTAGLANDINS ON DOPAMINE-STIMULATED FORMATION OF CYCLIC AMP ...... 108
EFFECT OF VARYING IC+ CONCENTRATION ON 3 H-ACH RELEASE ...... 117
EFFECT OF TIME OF INCUBATION ON K+-STIMULATED 3 H-ACH RELEASE ...... 119
EFFECT OF VARYING APOMORPHINE CONCENTRATIONS ON 3H-ACH RELEASE ...... 121
EFFECT OF APOMORPHINE ON K+-STIMULATED RELEASE OF 3-ACH ___ 123
EFFECT OF PROSTAGLANDINS ON BASAL 3 H-ACH RELEASE ...... 125
EFFECT OF PROSTAGLANDINS ON K+-STIMULATED RELEASE OF 3h -a c h ...... 127
EFFECT OF PGD2 ON APOMORPHINE INHIBITION OF K+-STIMULATED 3 H-ACH RELEASE ...... 129
EFFECT OF PGE2 ON APOMORPHINE INHIBITION OF K+-STIMULATED 3 H-ACH RELEASE ...... 131
EFFECT OF PGF2er ON APOMORPHINE INHIBITION OF K+-STIMULATED 3 H-ACH RELEASE ...... 133
xiii INTRODUCTION
HISTORICAL BACKGROUND
The report by Kurzrck and Lieb (1930) that uterine strips taken
from sterile women contracted in response to human semen, while strips
from fertile women relaxed, suggested that semen contained a biologic
ally active substance which was later identified as prostaglandin (PG)-
like material. Several years later, Goldblatt (1933) and von Euler
(1934), working independently, found that extracts of seminal plasma
or human semen caused contraction of the isolated uterus and a lowering
of blood pressure. This activity appeared to be present in acidic
lipid fractions and von Euler, believing that the active substances were produced in the prostate gland, named them PGs (von Euler, 1935).
The next development was the isolation and characterization of the
PGs, which was accomplished by Bergstrom and others at the Karolinska
Institute in Sweden (1962). Working independently, Bergstrom (1964) and van Dorp (1964) demonstrated that the precursors to the PGs are
the polyunsaturated fatty acids: dihomo-gamma-linolenic acid, arachi- donic acid, and eicosapentaenoic acid. Corey and others (1968) at
Harvard then synthesized the PGs from these precursors and consequently synthetic PGs became widely available for research. In 1971, Vane and others discovered that nonsteroidal anti-inflammmatory drugs, such as aspirin and indomethacin, inhibited PG biosynthesis and suggested that this inhibition was responsible for their anti-inflammatory action
(Ferreira, et al., 1971). The first unstable endoperoxide (PGH2 ) was
isolated by Hamberg and Samuelsson, (1973) who showed that PGH2 was an
an intermediate in the pathway from arachidonic acid to the stable PGs.
Having a half-life of 5 min at 37°C, the endoperoxides were found to
decompose into the major PGs and also to have unique biological effects
by themselves, particularly on platelets and vascular and respiratory
smooth muscle. The recognition of a platelet aggregating factor deriv
ed from arachidonic acid, which was different from the endoperoxides
and the stable PGs, led to the discovery of a new compound, thromboxane
A2 . This had previously been referred to as rabbit aorta contracting
substance (Hamberg and Samuelsson, 1974) . In the search for thrombox
ane-forming enzymes, it was found that the endoperoxides could be con
verted into a substance with vasodilator and antiaggregating properties
that were opposite to thromboxane A2 . Originally called PGX, the com
pound was renamed prostacyclin and given the abbreviation of PGI2 .
(Moncada, et al., 1976). In 1978, it was reported that arachidonic acid was metabolized by the lipoxygenase enzyme to form lipid peroxides which in turn formed substances involved in allergic reactions. These
substances have been named leukotrienes and one of them, leukotriene C, may be slow reacting substance of anaphylaxis (SRS-A) (Murphy, et al.,
1979).
NOMENCLATURE
PGs are oxygenated and unsaturated 20 carbon fatty acids that con tain a cyclopentane ring. They are considered derivatives of the hypo thetical prostanoic acid skeleton. The nomenclature and structures can IH »^^COOH HOQ. V>N-^Ng/S/* lipo>»8,n*1' Aiichiaonic acid **»«SS7« /— na^ C ^ cc t — - » ^-^COOn h P E I E Fatly add cyda-oiygtnatt \ ”CT- 0" h£t£ i . C C ^ £00H — 'COOH 6oh -COOH p g g , P G H , H
- « ^ s C 0OH
PGDj
>N^«^^COOrt r-^— »-N^sC0OH *** OH V v y w f-fceio~PGF|4 n o Ah in POEj PGA)
HV v - ^ - ^ - scooh TV*'— "'v-'NCOOH COOH y ~ i — lE-kalo-ll.UHj-PGFj,, IS-hald-U.UHj-PCE]
4 iAidcftjat HV r ^ c°OH V^^COOH A ^ . ^ ^ ^ cqoh ho'^crs»»Y^^-' h a o OH Ed, ?d-dih|r Major metabolic pathways in the conversion of arachidonic acid in the human. (Granstrom, et al., 1978) Fig. 1 be seen in Fig. 1. The type of primary PGs (designated by A, B, etc.) is based on structural differences in the cyclopentane ring. For ex ample, PGEs are B-hydroxyketones, the PGFs are 1,3-diols, and the PGAs are B-unsaturated ketones. The PGs are further classified by the de gree of unsaturation in the side chains, with the number of double bonds indicated as a subscript (e.g. Dj, D2 , and D3 ). They are fur ther subdivided into stereoisomers, with alpha and beta designations assigned according to the configuration at the Cg position on the cy clopentane ring with alpha = cis and beta = trans configurations (Andersen and Ramwell, 1974). BIOSYNTHESIS PGs are biosynthesized from polyunsaturated fatty acids with di- homo-gamma-linolenic acid (C20:3), arachidonic acid (C20:4), and eico- sapentaenoic acid (C20:5) giving rise to mono-, bi-, and trienoic PGs respectively. Arachidonic acid, the most common precursor, is found in membrane phospholipids and can be obtained directly from the diet or by desaturation and chain elongation of linoleic acid (C18:2). Fig. 1 is a schematic representation of arachidonic metabolism and illustrates the formation of the major PGs as well as their structures. Arachidonic acid can be released from cell membranes by the action of phospholipases, with phospholipase A2 being the most commmon. It has been suggested that this step may be the rate limiting step in PG synthesis. Once re leased, arachidonic acid is metabolized by two types of enzymes. One is the lipoxygenase enzyme which peroxidizes arachidonic acid forming un stable hydroperoxides (HPETEs) which break down to the stable hydroxy- acids (HETEs) or are further transformed into substances labelled as leukotrienes. The other enzyme is a cyclo-oxygenase (or PG synthetase) that forms the PG endoperoxide PGG2 which is converted to PGH2 . PGH2 then breaks down enzymatically or non-enzymatically to the stable PGs, thromboxane A2 , PGI2 , 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and malondialdehyde. The PG synthetic system studied in bovine vesicular glands was classified into two components with the enzyme, PG cyclo-oxygenase, con verting precursor acids to endoperoxides and a second specific enzyme (either an isomerase or reductase) converting PGH2 to one of the stable PGs. (For reviews on PG biosynthesis see: Samuelsson, et al., 1975, Maclouf, et al., 1977, Samuelsson, et al., 1978, and Lands, 1979). PG cyclo-oxygenase requires O2 , heme, and an aromatic compound such as tryptophan. The presence of hydroquinone prevents auto-destruction and protects the enzyme from inactivation by H2O2 . Different enzymes are involved in the formation of the different stable PGs from the endo peroxides. To form PGEs, the PG endoperoxide E isomerase enzyme acts on both PGG2 and PGH2 with the rate of PGG2 isomerization being only half of that of PGH2 . Glutathione serves as a cofactor. PGD2 is formed through the action of another isomerase which also requires the presence of glutathione. A PG endoperoxide reductase has been reported to act in PGF production. In contrast to PGE synthesis neither reduced glutathione nor NADPH stimulated PGF2 « synthesis. In the presence of Cu++, PG production by sheep vesicular gland shifts from PGEs to PGFs. Further enhancement of PGF production also occured in the presence of dithiols. Thromboxane A2 isomerase (synthetase), the enzyme that converts PG endoperoxides to thromboxane, has been studied most often in platelets. PG endoperoxide I isomerase has been shown to be necessary for PGI2 production. Large quantities of this enzyme are found to occur in vascular tissue. METABOLISM PGs are metabolized by four major types of transformations: 1. oxidation of the alcohol group at C15 to the 15-keto-PGs by an NADH dependent 15-hydroxy-PG dehydrogenase which is present in the cyto plasm. 2. The deltaq3~trans double bond is reduced by a specific re ductase which is present in the cytoplasm. 3. Beta-oxidation to form C18 (dinor-) and (tetranor-) PGs, which has been demonstrated to occur in liver mitochondria. 4. Microsomal omega-oxidation which transforms the partial metabolites into dicarboxylic acids. (Wolfe, 1975; Lands, 1979; and Samuelsson, 1975). PROSTAGLANDIN INHIBITORS A variety of substances have been shown to inhibit PG synthesis in biological tissue (Flower, 1974). Corticosteroids have been shown to reduce PG biosynthesis by inhibiting release of precursor fatty acids through suppression of phospholipase A2 . Nonsteroidal anti-in flammatory drugs, such as aspirin and indomethacin, reduce PG synthesis by inhibiting PG endoperoxide synthetase (cyclo-oxygenase). All fatty acids inhibit cyclo-oxygenase by competing with the fatty acid precur sors of the PGs. Fatty acids which have been shown to be more potent inhibitors of PG biosynthesis are: 5,8,11,14-eicosatetraynoic acid, 8,12,14-eicosatriienoic acid, and 5,8,11,14-eicosatetraenoic acid. The first fatty acid named may also inhibit the lipoxygenase enzyme. Several thromboxane synthetase inhibitors which have been reported in clude: imidazole, dipyridamole, NO 164, and L-8027. PROSTAGLANDINS IN THE CNS BIOSYNTHESIS PGs in the brain appear to follow the same biosynthetic sequence as described previously. Galli, et al., (1978) have shown that large quantities of arachidonic acid can be derived from inositol phosphogly- ceride. However, since choline phosphoglyceride is more abundant in brain, it may represent a larger source for arachidonic acid. Once liberated from the phospholipid, aracidonic acid can be converted by either lipoxygenase to HETE compounds or cyclo-oxygenase to the endo peroxides and then to the PGs. The yield of labelled PGs from labelled exogenous arachidonic acid has been low since exogenous arachidonate does not appear to gain acess to cyclo-oxygenase due to saturation of the enzyme by endogenous fatty acids which are released during decapi tation and dissection. Because of this low yield of PG, the interpre tation of experiments using this technique are unclear (Wolfe, et al., 1976). All the stable PGs have been found to be synthesized in the CNS as shown by direct measurement of PGs. Biosynthetic capacity to form both PGE2 and PGF201 have been shown to be present in both rat slice and ho- mogenate preparations with formation being greatly stimulated by the catecholamines, apomorphine, and adrenochrome (Wolfe, et al., 1976; Leslie, 1976). TXB2 has also been shown to be formed in guinea pig and rat cerebral cortex in amounts equal to or higher than E2 or F2 > while Abdel-Halim, et al., (1977) found PGD2 in highest concentrations 8 in all brain regions of the rat. PGI2 , as measured by 6 -keto-PGFjot also has been found in brain, but appears to be formed mainly in cere bral vessels rather than neural tissue (Chaplin and Hillier, 1979). HETE was also measurable in both rat cortex (Sautebin, et al., 1978) and gerbil cortex (Spagnuolo, et al., 1979) indicating that lipoxy genase is also present in brain tissue. METABOLISM The mechanism of PG inactivation within the brain is currently not clear. Anggard, et al., (1971) initially showed that porcine brain contained both 15-hydroxy-dehydrogenase (15-PGDH) and delta^-reductase (PGR), but subsequent studies did not confirm these results (Nakano, et al., 1972). A third enzyme, PGE 9-keto-reductase, which converts PGEs to PGFs has been shown to be present in brain (Leslie and Levine 1973), but Wolfe, et al., (1976) failed to see any significant conver sion of E to F. One explanation for these negative findings is that whole brain analysis may mask the action of small, discrete cell groups which do contain the metabolizing enzymes. Thus, Siggins, et al., (1971) showed that Purkinje cells within the cerebellum had high 15- PGDH activity, while analysis of the whole cerebellum showed no activ ity. Bito and Wallenstein (1977) showed that PGs are removed from brain extracellular fluid by a saturable, carrier-mediated transport system which could be inhibited by organic acid transport inhibitors. Thus, the action of PGs could be terminated through their transport from the extracellular fluid into the general circulation. Another possibility is that the endoperoxides (PGG2 and PGH2 ) are the biologi cally active compounds and transformation of endoperoxides to the stable PGs represents a deactivation (Coceani and Pace-Asciak, 1976). In summary, it is presently unclear as to the exact mechanism of inac tivation of PGs within the CNS. If PG activity is related to neuronal activity, more rapid mechanisms of inactivation would be expected. SUGGESTED ROLES FOR PROSTAGLANDINS IN THE CNS There are many suggested roles for PGs in the CNS. Among the more accepted roles are: 1 . neuromodulation of transmitter release; 2 . con trol of thermoregulation; 3. release of anterior pituitary hormones; 4. control of cerebral circulation; 5. control of pain; and 6 . initiation and maintenance of migraine headache (Wolfe, 1975) . One of the roles which was ascribed to the PGS, modulation of neurotransmission, was first shown in the peripheral nervous system. The Hedqvist theory states that PGEs control autonomic neurotransmis sion by feedback inhibition of NE release from nerve terminals, while PGFs facilitate NE release (Hedqvist, 1977). A similar role has been suggested for PGs in the CNS, but there is both evidence for and against this hypothesis (Bergstrom, et al., 1973; Reimann, et al., 1980). Considerable evidence has shown that PGs mediate fever produced by bacterial endotoxins but do not alter normal body temperature (For re view see Veale, et al., 1978). Evidence supporting this concept are: 1 . PGE2 is a normal constituent of hypothalamic tissue and injection of PGE2 into the hypothalamus produces fever; 2 . PGE2 acts on neurons that are also responsive to pyrogens; 3. pyrogen fever is associated with elevated levels of a PGE-like material; and 4. antipyretic and anti inflammatory drugs inhibit PG synthesis (Vane, et al., 1971). However, there is also contradictory evidence. In newborn animals, injection 10 of PGE into the hypothalamus does not produce fever and in rabbits, salicylate which has been shown to inhibit PG synthesis, has no effect on the febrile response (Wolfe and Goceani, 1979). Thus, there may be PG dependent and independent types of fever produced. PGs have been shown to have marked effects on the anterior pitui tary resulting in the release of hormones. Hormones which have been released due to an action of the PGs are: LH/FSH, prolactin, ACTH, and TSH (Wolfe, 1975). Release of these hormones may result from a direct action of the PGs or by an indirect action. The indirect action may be due to the stimulation of cAMP production which in turn releases gonadotropins or through the alteration of catecholamine release which may alter neuronal activity in the anterior pituitary (Behrman, 1979). The action of PGs on blood vessels through-out the body has been the subject of intensive study. PGs, which can be synthesized in the walls of blood vessels, exert marked effects on vascular tone. PGI2 appears to be one of the most potent vasodilators, while TxB2 is a potent constrictor (Wolfe and Coceani, 1979). PGs are also formed in cerebral vessels with PGD2 and PGE2 dilating cerebral vessels, while PGF20L produces cerebral vasoconstriction (Ellis, et al., 1979; Welch, et al., 1974). Cerebral vasospasm and brain ischemia following head injury have been two pathological conditions in which PGs may play a causative role by altering vessel hemodynamics. Thus, the role of PGs in controlling circulation may be as important in the brain as it is in the rest of the body (Wolfe and Coceani, 1979). The perception of pain may also involve the synthesis of PGs. There are many types of pain, but pain resulting from headache, 11 inflamation and direct tissue damage appear to be associated with PGs (Wolfe, 1975). The use of aspirin for relief of headache and indometh- acin or aspirin for treatment of inflammation, are due to their ability to inhibit PG synthesis. Tissue damage has also been shown to result in the release of fatty acids and the production of PGs (Galli, 1978). Collier and Roy (1974) first speculated that the action of morphine was mediated through PGs by inhibiting the PGE-stimulated formation of cAMP. Thus, treatment of pain may involve PG activity at sites which mediate perception of pain. Control of cerebral circulation may also be responsible for the action of PGs in the initiation and maintenance of migraine headache. However, there is little direct evidence suggesting that PGs have a crucial role in initiating migraine attacks, since PG inhibitors fail to control attacks and no evidence has been given to suggest that PG inhibitors act prophylactically. Barrie and Jowett (1967) did find a lipid material in the CSF of migraine patients which appeared to be PG-like, however this has not been substantiated (Gross, et al., 1977). The work discussed in this thesis examines the role PGs play in central DA pathways. The following sections will examine the current understanding of the location and physiological function of the DA sys tem and also disease states where DA is involved. ANATOMY OF CENTRAL DOPAMINE PATHWAYS There are several major dopaminergic (DA) pathways within the CNS. Reviews by Moore and Bloom (1978) and Lindvall and Bjorklund (1978) have outlined the major DA pathways which have been elucidated by a variety of anatomical techniques such as histo-fluorescence 12 using formaldehyde or glyoxyic acid, horseradish peroxidase tracing, and immunohistochemical visualization. DA neurons in the upper mesencephalon are divided into two main systems, the mesostriatal and the mesocortical systems. As shown in Fig. 2 and 3 the mesostriatal system has two sets of cell bodies, with one set in the substantia nigra, pars compacta (A9 designation of Dahlstrom and Fuxe). Axons leaving the cell body region of the nigra (nigrostriatal pathway) course rostrally in the brain, passing through the lateral hypothalamus and internal capsule to terminate in the caudate putamen (striatum) and the globus pallidus. The second set of cell bodies localized in the ventral tegmental area (Aiq) also have axons which course rostrally to terminate predominantly in the nucleus accumbens (mesolimbic pathway). The mesocortical system also has cell bodies in both substantia nigra and tegmental area, but axons from these areas project to allocortical areas (olfactory tuberacle, septum, interstitial nucleus of the stria terminalis, and amygdala) and neo- cortical areas (frontal cingulate, entorhinal, and perirhinal areas). Another major dopamingergic pathway is the tubero-hypophyseal (tuberoinfundibular) system. In this pathway cell bodies in the arcu ate nucleus of the hypothalamus (A]^) project to the median eminence, the stalk, the neural lobe and the pars intermedia of the adenohypo physis. The incertohypothalamic system (An and A1 3 ) is also located in the hypothalamus. Cell bodies in the zona incerta, posterior hypothal amus send short projections into the dorsal hypothalamus and septum. 13 NORADRENALINE DOPAMINE n-accumbens septum tub. otfactor luin Cingulum n-caudatus stria term.- hypothal. n.amygdaloid centralis amygdala- dorsa bundle substantia ventral nigra bundle subst.grisea centralis Horizontal projection of the ascending NA and DA pathways. Reproduced from Ungerstedt (1971). Fig. 2 14 septal & frontal' areas net accunbena > tuberctAim oNactonum striatum ■ Lateral view of the ascending DA pathways to (a) striatum: nigrostriatal; (b) nucleus acumbens and tuberculum olfactorium:mesoltmbic; (c) septum, frontal and ringulate cortex: mesocortical. Kindly supplied by Ungerstedt. Fig. 3 15 There is a periventricular system which contains DA fibers and originates in the periventricular and periaqueductal gray of the medulla. Other fibers containing NE also originate in this same area and contribute fibers to this system as well. The DA fibers project to the periaqueductal gray, medial thalamus, and hypothalamus. There appears to be a retinal DA system located mainly in the inner nuclear layer of the retina and which sends processes into the inner plexiform layer. The periglomerular DA system consists of DA cells in the olfactory bulb. Periglomerular cells project to mitral cells of olfactory glo meruli. ROLE OF DOPAMINE IN MOTOR FUNCTION Three important discoveries in the 1960's focused the attention of researchers on DA-mediated pathways. First, Carlsson (1959) devel oped biochemical assays for quantifying brain levels of catecholamines. Second, Dahlstrom and Fuxe (1965) using the Falck-Hillarp histological technique were able to map catecholamine pathways through-out the brain. Third, Erhinger and Hornykiewicz (1960) observed that patients who had died with Parkinson's disease showed degeneration of the nigro striatal pathway. The association of a specific motor disorder and abnormal DA transmission led to pharmacological research on drugs which could alleviate neurogical disorders (iversen, 1975). In order to study the role of DA in motor fuction, several animal models of behavior have been developed (See Kelly (1975) for reviews of these models). Three which are widely used for studying drug-induced motor behavior are: locomotor activity, stereotyped behavior and 16 drug-induced circling. Locomotor activity is influenced not only by drugs but a wide variety of genetic, developmental, hormonal, motiva tional, and enviornmental factors. In regard to the DA system, indi rect acting agents such as amphetamine, appear to release DA from nerve terminals resulting in locomotor activity. This conclusion is based on the effect of altering DA transmission by inhibiting DA syn thesis, blocking DA receptors, or destroying DA neurons. While the exact site is unclear, there is evidence which strongly implicates the mesolimbic DA system in drug-induced locomotion. Stereotyped behavior, produced by high doses of amphetamine, is seen in rats as licking, biting, with gnawing behavior and stereotyped behavior having been ob served as part of the human schizophrenic syndrome. In contrast to locomotor activity, stereotyped behavior appears to result mainly from the release of DA from nerve endings of the nigrostriatal pathway which terminate in the striatum. Other brain areas may also be involved, with the globus pallidus, olfactory tubercle, and amygdala being shown to influence drug-induced stereotypy. Drug-induced circling (rotational behavior) is the result of an asymmetry in the activity of neurons in volved in motor function on one side of the brain compared to the other and may be used to study functional relationships within the basal ganglia and between the basal ganglia and other areas of the brain. DA neurons of the mesolimbic and nigrostriatal systems seem to be in timately involved in circling behavior. The relative activity of the nigrostriatal DA neurons on each side of the brain determines the di rection of circling, while the activity of the mesolimbic DA system determines the degree of circling (Kelly, 1975). 17 Other types of behavior are also controlled by forebrain DA path ways. Iversen (1975) reviews the role of DA in unconditioned and con ditioned behaviors. Unconditioned behaviors include: spontaneous motor activity, eating, drinking, mating and aggression, while conditioned behaviors include: avoidance behavior, intracranial self-stimulation, appetitive learning, and memory consolidation. Spontaneous motor ac tivity is used as an index of the animal's overall physiological state and responsiveness to the environment and is also a prerequisite be havior for motivated behaviors such as feeding and drinking. Depletion of DA by the use of reserpine or alpha-methylparatyrosine inhibits this behavior as do DA receptor blockers. Selective lesioning studies have further shown that the nucleus accurabens (mesolimbic system) plays a major role in this type of behavior. Conditioned behaviors are also blocked by inhibiting DA neuronal function. Of the behaviors listed above, all appear to be dependent upon the release of newly synthesized DA, but since behavior is the integration of many neuronal circuits, several different neuronal pathways can control these behaviors. INVOLVEMENT OF DOPAMINE IN DISEASE STATES DA has been implicated in major disease states, most of which are characterized by a change in motor function. Among these motor dis eases, evidence that DA is involved is strongest in: Parkinsonism, tardive dyskinesia, and Huntington's chorea. Schizophrenia (psychosis) although not a disease of motor dysfunction, involves DA systems, since the treatment of choice are drugs which are DA receptor blockers. Bianchine (1980) recently reviewed the pathological changes and drug therapy for Parkinsonism. Characterized by a striatal DA 18 deficiency, Parkinsonism results in the clinical symptoms of: tremor, bradykinesia, rigidity and postural defects. Drugs that deplete or block the effects of DA often cause Parkinsonian-like syndromes, while drugs which enhance DA activity, such as L-dopa, alleviate the symp toms. Acetylcholine (Ach) is also involved, since drugs which decrease central cholinergic activity are effective when used alone or in con junction with L-dopa therapy to relieve the symptoms of this disease. Tardive dyskinesia is a neurological disorder characterized by ab normal oral, facial, and tongue movements, as well as choreic and ath etotic movements of the trunk and extremities. The disease is associ ated with prolonged exposure to neuroleptic (antipsychotic) drugs (Paulson, 1975; Baldessarini, 1980). In contrast to Parkinsonism, tardive dyskinesia may be due to an excess in DA function. Prolonged neuroleptic therapy may make the DA receptor supersensitive and/or cause changes in affinity of DA for the receptor (Baldessarini, 1980). At present, it is unclear whether these effects result from alteration of DA neurons within the nigrostriatal or mesolimbic pathways since DA receptor blockers act on both systems. Huntington's chorea is another disease which shows a dysfunction of brain motor systems. Hyperkinetic locomotion and other*behaviors seen may be the result of excessive DA activity in the basal ganglia. More recent evidence suggests that destruction of Ach- and GABA-con- taining neurons within the striatum may be more responsible for this disease than alterations in the activity of DA neurons. However, these striatal interneurons are innervated by DA neurons and thus, there is a strong relationship between all three transmitter substances and the disease (Bianchine, 1980; Coyle and Schwarcz, 1976). Schizophrenia is not a disease of motor function, but of thought and perception. This disease is treated by administering DA receptor blocking agents, such as chlorproraazine or haloperidol (Snyder, et al., 1974). The anatomy of the DA systems shows that DA neurons from mesolimbic and mesocortical tracts innervate the limbic system and parts of the cerebral cortex, areas of the brain involved in thought and emotion. Recent studies support this hypothesis with evidence that mesolimbic DA pathways are altered in schizophrenia (Stevens, 1979). Thus, in addition to controlling motor function, DA pathways are involved in cognitive thought processes and emotional behavior. STATEMENT OF THE PROBLEM The overall question which is being examined in this thesis is: what is the role of PGs within central pathways mediated by DA neuro transmission? Since PGs have been shown to be neuromodulators and DA has been shown to control motor function, a more specific question is how do PGs modulate DA pathways which control motor function? Since the nigrostriatal DA pathway has been best characterized among the different DA pathways by anatomical, biochemical, and pharmacological techniques, activity of PGs on this pathway will be examined. It is hoped that the results will contribute to the understanding of DA function in both normal and abnormal states and also enlarge upon the knowledge of PG function within the CNS. MEASUREMENT OF BRAIN PROSTAGLANDINS Since the early observation by Samuelsson (1964) that ox brain contained a PGF-like material, prostaglandins (PGs) have been identi fied in the CNS of every species examined (Wolfe, 1975). All major PGs have been identified in neural tissue, but the PG in greatest abundance is different from species to species. For example, PGD2 has been measured in greatest amounts in rodent brain (Abde1-Halira, et al., 1977) while high amounts of PGE2 are found in cat brain (Abdel-Halim, et al., 1979). In addition, there is a regional distri bution within the brain, with limbic areas and cerebral cortex showing much higher levels of PGs than cerebellum (Abdel-Halim, et al., 1979). Currently, there exists four major means for measuring PGs: bio assay, gas chromatography coupled to mass spectometry (GC/MS), HPLC, and radioimmunoassay (RIA). Bioassay, while highly sensitive, requires a different type of tissue for each PG measured and can not separate PGs according to the number of double bonds within a PG series (e.g. PGE^ vs PGE2 vs PGE3 ) (Bergstrom, 1968; Moncada, et al., 1978). Chromato graphic separation coupled with mass spectroraetric analysis provides the most definitive measurement since this procedure identifies compounds according to molecular composition (Green, et al., 1978). However, it is expensive, time consuming, and only a small number of samples can be processed per day. HPLC may also provide exact measurement, but the equipment is expensive and it may lack sensitivity necessary for samples containing low levels of PGs. RIA allows the rapid processing of a 20 21 a large number of samples with a high degree of sensitivity if highly specific antibodies (Abs) can be generated for the assay (Granstrom, et al., 1978). The following chapter details the development of PG RIAs. Abs were generated in a novel system, the chicken egg yolk. The resultant Ab allowed the rapid and sensitive measurement of PGs in whole brains of rats and mice, the two species used for experimentation. METHODS A. Coupling of Prostaglandin to Keyhole Limpet Hemacyanin. Ten mg of PG was dissolved in 2 ml NaPO^ buffer (0.2M, pH 6.5) and added to a mixture of 40 mg of keyhole limpet hemocyanin (KLH) and 20 mg of carbodiimide in 2 ml H2 O (pH maintained at 5.0-5.2). This solution was incubated for 24 hours at room temperature, followed by dialysis against distilled H2O for at least 24 hours at 4°C. B. Generation of Antibodies for RIA. The coupled PGs from the above procedure were diluted 1:2 with distilled H2O and then mixed with an equal amount of Freund's complete adjuvant. White, leghorn pullet hens were then each injected with 1.5 ml of this solution. Three to 4 weeks later chickens were given booster injections of this same solution and thereafter at 4 weeks intervals. Eggs were collected after the first booster injection and the yolk containing the Ab fraction was checked for the ability to bind the appropriate %-PG. A dilution of the yolk generated Abs was used to obtain 30 - 60% binding of ^h-pg. This dilution was subsequently used for all RIAs. 22 C* Sample Preparation. Mice or rats were sacrificed by microwave irradiation. This procedure has been shown to inactivate enzymes by rapidly elevating internal tissue temperature (Guidotti, et al., 1974). After cooling on ice the brain was removed and weighed (approximately 250-350 mg tissue) and homogenized in 3 ml IN formic acid. Following a centri fugation (500 x g, 10 min) the supernatant was extracted with ethyl acetate (10 ml, 3x). The organic phase was evaporated with a rotovap and then reconstituted in 3 ml of 1:1 methanolrethyl acetate. This was blown dry under N2 and the samples were stored in 1 ml of ethanol at 4°C until assayed for PG content by RIA. Per cent recovery was greater than 75% as measured by recovery of % - P G added to duplicate tubes run in parallel to tissue samples. At the time of assay, an aliquot was evaporated under N2 and then diluted to the appropriate concentration with 50mM Tris buffer (pH 7.3). B. Prostaglandin RIA. A lOOul aliquot of sample was added to 100 ul of appropriate Ab and 100 ul of -^H-PG (approx. 6000cpm) in 10 x 75 tubes. The samples were incubated overnight at 4°C, dextran-charcoal solution (0.5ml) was added, incubated for 10 rain (4°C), and finally centrifuged for 20 min at 500 x g. The supernatant was then taken for scintillation counting. For the standard curve, instead of sample, known amounts of authentic PG was added to the tubes in varying concentrations. RESULTS A. Cross-reactivity of Generated Antibodies. As can be seen in Table 1, the Abs generated by chickens were 23 highly specific. Eight substances with related molecular structures to the PGs were examined for their ability to displace ^H-PG from Abs directed against the respective PG. Ab directed against 6-keto-PGF^pC was the most specific with only a 0.16% cross-reaction seen with PGAi. Thromboxane B2 (TXB2 ) showed no greater than a 1.0% cross-reactivity with PGD2 > while PGE2 cross-reacted with PGFj at 3.2%. PGF2«. showed the the highest cross-reactivity of any Ab generated in chicken egg yolk with a 15% cross-reactivity with PGFjot. PGD2 , a gift from Dr. R. Gorman and Dr. F.A. Fitzpatrick, Upjohn, Co., was obtained from rabbit antisera. It cross7 reacted with both PGFjcX (15.8%) and PGF2ot (3.1%). Also shown in Table 1 is the dilution necessary to obtain 25-35% binding to -^H-PG. The Ab in greatest concentration in chicken egg yolk was 6-ketoPGFitf*. which obtained this percentage of binding at a 1 :10,000 dilution. B. Sensitivity of Prostaglandin-Directed Antibodies. The ability of authentic PGs to displace their respective ^H-PG from Ab generated against that PG is shown in Fig. 4. It can be seen that each displacement curve is sigmoidal in shape. The linear portion in the middle of each curve shows the ability of non-radioactive (cold) PG to displace ^H-PG in a highly specific fashion. One index of sensi tivity is the amount of cold PG causing 50% displacement. The 50% displacements for PGD2 , PGE2 , PGF2<*, 6-keto-PGF]©£, and TxB2 under these conditions are: 120pg, 260pg, 63pg, 48pg, and 47pg respectively. G. Mesurement of Prostaglandins in Whole Brain of Rats and Mice. As can be seen in Table 2, the development of PG RIAs allowed the measurement of PGs in whole brains of both rats and mice, the two 24 species used for experimentation. In the rat, PGF2«ti and TxB2 appear ed to be present in greatest quantities, with PGE2 levels higher than those of the PGI2 metabolite, 6-keto-PGF]^. Measurement of PGs in mouse whole brain showed that PGs were approximately the same as in rat brain. (PGD2 antisera was unavailable at the time the measure ments were made in rat brain). DISCUSSION RIAs must meet the criteria of being specific for the substance being assayed and also sensitive enough to measure the quantities of the desired substance found in biological tissue. The development of an RIA for PGs which meet these two requirements allows the precise measurement of specific PGs. In addition, a large number of tissue samples can be measured in a relatively short amount of time. Accuracy or the per cent cross-reactivity of the Ab for different substances is shown in Table 1. The Abs generated in chicken egg yolk are highly specific with only PGF26J. showing a high degree of cross-reactivity with PGFjctC15%). However, this should not present a problem since PGs of the 1-series are found in low concentrations throughout the body, and virtually not at all in the brain since the precursor fatty acid (di- homo-gamma- lino lenic acid) has not been shown to be present in brain tissue (Galli,et al., 1978). Displacement curves (Fig. 4) show that all PGs were highly sensi tive with linear portions of the curves extending to the picogram range. The 50% displacements, one index used for sensitivity, also indicated that all PGs can be assayed with a high degree of sensitivity. Measurement of whole brain PGs in rats and mice after microwave irradiation showed that in normal animals, endogenous levels of PGs are extremely low (Table 2). The processes of decapitation and dissection of the brain have been found to cause the release of PG fatty acid pre cursors and subsequent PG synthesis (Borsisio, et al., 1976). Thus, most previous measurements have been on a stable pool of PGs which forms after dissection and is in the high nanogram to microgram range (Abdel-Halim and Anggard, 1979). However, changes in endogenous levels may be masked by measuring such a large pool. One method of inactiva ting synthetic enzymes is by freezing the tissue with liquid nitrogen. However, freezing of brain tissue makes dissection of discrete areas difficult. A second method, microwave irradiation, inactivates enzymes by heat, yet allows dissection. It has also been shown that irradiation prevents the formation of PGs from arachidonic acid (Borsisio, et al., 1976). Thus, the low levels of PGs measured following irradiation ap pears to more accurately represent basal levels of PGs than the levels in nonirradiated animals and allows changes to be seen which might be undetected when measuring large stable pools forming post mortem. The ability to measure very low levels of PGs by RIA, plus the use of microwave irradiation, will allow small, but significant changes in PG levels to be seen. Thus, Abs generated from chicken egg yolk will be highly beneficial to an examination of PGs in the brain. FIGURE 4 SENSITIVITY OF PROSTAGLANDIN-DIRECTED ANTIBODIES Antibody (Ab) directed against a specific PG was added to equal amounts of 3H-PG and unlabelled PG, incubated overnight at 4°C and separated by the dextran-charcoal method. Bound Ab-3H-PG was counted in a scintillation counter and results are graphed as the amount of PG bound vs. quantity of unlabelled PG present. 26 100 80 P 60 40 20 0.001 0.01 0.1 1.0 10 100 ngm/final volume N5 Fig. 4 TABLE 1 CROSS-REACTIVITY OF PROSTAGLANDIN-DIRECTED ANTIBODIES FOR RIA PER CENT CROSS-REACTIVITY dilution for 25 - 35 % Arach. AB binding ac id Aj d2 e2 F1 oi ?2oL 6K-F]0£ TxB£ PGD2 1:160 <.01 <.01 100 <.01 15.8 3.1 <.01 <.01 PGE2 1:300 < .01 .16 1.6 100 3.2 .75 .05 .41 PGF2oc 1:6000 <.01 <.01 <.01 .02 15.0 100 .14 .06 6K-PGFiot 1 :10,000 <.01 .16 .03 .03 .03 100 .03 Tx B2 1:1200 <.01 .05 1.0 .08 <0.1 .25 .06 100 to 00 TABLE 2 MEASUREMENT OF PROSTAGLANDINS IN WHOLE BRAINS OF RATS AND MICE pgrams/mg wet weight PG Measured Rata Moused PGD2c 18.8 +6 .4 PGE2 1.6 11.5 _+0.6 +1 .3 PGF2oe 8.8 7.1 +1.9 +2.1 6-keto-PGFio< 1.0 3.9 _+0 .2 +1.4 TxB2 14.0 11.7 + 4.8 +5.8 a n = 6 b n = 4 c rabbit antisera for PGD2 was a gift from Dr. R. Gorman and Dr. F.A. Fitzpatrick, Upjohn Co., Kalamazoo, Mich. PROSTAGLANDIN INHIBITION OF AMPHETAMINE-INDUCED CIRCLING IN MICE Since Sarauelsson (1964) first identified PGF2ct in ox brain, prostaglandins (PGs) have been identified in the CNS of all species examined, including man. PGs are unevenly distributed throughout the brain and specific PGs are more abundant in some species than others (Abdel-Halim et al., 1979). Suggested roles for the PGs found in the CNS include: modulation of transmitter release, initiation of fever, release of anterior pituitary hormones, control of cerebral blood flow, control of pain, and initiation of migraine headache (Wolfe, 1975). Although PGs have been shown to alter CNS function, their exact mechanism of action within the nervous system is currently unclear. One possibility is that they act to modulate transmitter release. In the peripheral nervous system, PGs of the E series inhibit norepi nephrine (NE) release and PGF201 facilitates its release (Brody and Kadowitz, 1974; Hedqvist, 1977). A similar role has been suggested for PGs in the CNS, but the experimental results supporting this concept are equivocal (Bergstrom, et al, 1973; Reimann, et al., 1980). Both indirect and direct evidence suggest that PGs may modulate central dopamine (DA)-mediated neurotransmission. High concentrations of PGs have been measured in the caudate nucleus, the area of the brain richest^in DA (Abdel-Halim et al., 1977). The administration of PGs to rats has been shown to produce catalepsy (Horton, 1964) and block conditioned avoidance responding (Potts et al., 1973) in a 30 31 manner similar to DA receptor blockers, such as chlorpromazine and haloperidol. A decrease in food intake is produced in rats lesioned along the DA nigrostriatal pathway (Ungerstedt, 1971a) as well as in animals injected centrally with PGs (Scaramuzzi et al., 1971). In add- . . . . 3 ltion, PGE2 has been shown to inhibit the release of H-DA from field- stimulated striatal slices (Bergstrom et al., 1973). Thus, these re sults suggest that PGs may play a modulator role in central DA systems. If PGs are active on dopaminergic neuronal pathways, then the central administration of PGs should alter motor function controlled by these pathways. The rodent circling model has been used to evaluate the effect of drugs on motor function which is dependent upon DA neuro transmission. Amphetamine (amph) has been shown to produce circling behavior in rodents previously lesioned unilaterally in the striatum with 6-hydroxydopamine. This circling is caused by the release of DA from nerve terminals in the intact striatum resulting in the unilat eral stimulation of DA receptors. The following experiments were designed to test whether PGs could modulate amph-induced circling in mice and whether indirect changes in body temperature were responsible for these behavioral effects. METHODS A. 6-Hydroxydopamine Lesions in Mice. Swiss-Webster, male mice (20 - 30 gm) were injected in the left caudate nucleus with 6-hydroxydopamine (6-OHDA). The 6-OHDA was dis solved in ice-chilled 0.9% saline containing 1.6 ug of ascorbic acid and 4 ul of this solution was injected over 4 min. Coordinates for 32 the injection site were: 5 mm anterior to the occipital suture (lambda), 2.1 mm lateral to the midline, and 3.5 mm below the surface of the skull. Five to seven days after surgery, all mice were injected with d-amph (4 mg/kg, ip) and only those mice circling at a rate of greater than 10 turns/min were retained for further testing. Visual measure ment of circling was carried out in 2 liter round bottom flasks and both the direction and number of 360° turns were counted. B. Testing Protocol for Circling. Mice were injected with amph (4 mg/kg, ip) and 10 min later received an injection of either PG or saline directly into the lateral ventricle or into the striatum. Turns/min were counted for 1 or 2 min periods beginning 10 min after the central injection (20 min after amph administration). A "free hand" method for the central injection of PG or saline into the brain was employed (Pycock, et al., 1975). Under light, halothane anesthesia, mice were injected into either the lateral ventricle (Coordinates: from bregma, 1.8 mm lateral and 3.5 mm below the surface of the skull, or the striatum (same coordinates as for striatal lesion, but drug was injected on the unlesioned side). A 10 ul Hamilton syringe, having a polyethylene cuff allowing only the distal portion of the needle to be exposed, was used for all injec tions. Two ul were injected over a 45 sec period with the needle being held in place for an additional 15 sec. The incision was then closed by a wound clip, with animals recovering from the anesthesia within 2 - 4 min after the injection. The site of the injection was veri fied at the end of the experiment by examining the location of dye after the injection of bromthymol blue into the same sites as the PGs. 33 C . Temperature Measurements in Mice. Rectal temperatures were measured in mice with a Tele-thermo meter, Yellow Springs Insturment Co., Yellow Springs, Ohio (Model 43TD). Temperatures were taken in mice, injected intraventricularly with PG or saline, either alone or after the administration of amph. D. Statistics. Where appropriate, the data was analyzed by the Dunnett test for multiple comparisons. RESULTS A. Effect of Intraventricularly Administered Prostaglandins on Amphetamine-Induced Circling in Mice. In results similar to those reported by others (Von Voitlander, et al., 1973; Pycock, 1980) d-amph administration to mice lesioned with 6-OHDA produced circling towards the side of the lesion (ipsilateral direction). Mice were injected intraventricularly (icv) with 0.9% saline 10 min after the ip injection of amph (4 mg/kg, ip). Twenty min later (30 min after amph administration), they circled at a maximum of app roximately 15 turns/min. Thereafter, the circling slowly decreased with time. This can be seen in the upper portion of Fig. 5 - 8 marked as control (icv). As seen in the upper portion of Fig. 5, the injection of 1.0 nmole/gm of PGD2 significantly inhibited amph-induced circling when compared to the saline control. A dose of 0.3 nmole/gm inhibited circling for 30 min while 0.01 nmole/gm did not inhibit circling. Nei ther saline nor PGD2 when administered without amph, produced signifi cant circling in either direction (data not shown). The top portion of Fig. 6 (icv administration) shows the effect of PGE2 on amph-induced 34 circling. As with PGD2 , a dose of 1.0 nmole/gm significantly reduced the circling produced by amph. However, PGE2 at 0.1 nmole/gm caused the mice to circle in a contralateral direction (or away from the side of the lesion). At 0.03 nmole/gm, there was no difference from control animals. PGE2 admininistered in the absence of amph also did not pro duce any circling behavior. PGF2tft inhibited the circling response at 1.0 nmole/gm as did PGD2 and PGE2 (top of Fig. 7). However, neither 0.03 nmole/gm nor 0.01 nmole/gm of this compound significantly inhibi- ited circling, and in addition, PGF2«t did not produce circling when in jected by itself. In order to determine whether a compound with a sim ilar structure to to the major PGs would produce inhibition of amph- induced circling the stable metabolite of thromboxane A2 » TxB2 , was injected intraventricularly at 1.0 nmole/gm (top of Fig. 8 ). It did not alter amph-induced circling and did not produce circling when injected by itself. B. Effect of Intrastriatal Administration of Prostaglandins on Amphetamine-Induced Circling in Mice. Ten minutes after amph administration, mice injected intrastri- atally with 0.9% saline circled in an ipsilateral direction and slowly increased the number of turns with a maximum of 22 turns/2 min seen 30 min later (bottom of Fig. 5 - 8 marked as control, is). PGD2 at 0.1 nmole/ gm significantly inhibited the circling caused by amph (Fig. 5, bottom portion). A dose of 0.03 nmole/gm also inhibited the circling response at all times, but 0.01 nmole/gm was only effective at the first time period examined. PGE2 (bottom of Fig. 6 ) also inhibited amph- induced circling in a dose range of 0.01 - 0.1 nmole/gm. Doses of 0.1 nmole/gm and 0.3 nmole/gra showed inhibition at all time periods examin ed, while 0.01 nmole/gm was significantly different from control only at 10 and 30 min. Fig. 7 (bottom portion) shows that intrastriatal in jection of PGF2tx inhibited araph-induced circling at 0.1 nmole/gm. Nei ther 0.03 nmole/gm nor 0.01 nmole/gm produced statistically different values from saline control. PGD2 , PGE2 , and PGF2©* did not produce any circling when injected alone by the intrastriatal route of administra tion. In contrast to its inactivity when injected by the intraventric- ular route of administration, TXB2 at 0.1 nmole/gm was able to inhibit amph-induced circling at the first two time periods examined (Fig. 8 , bottom portion). However, the major PGE2 metabolite, 13, 14-dihydro- 15-keto-PGE2, did not alter circling at this dose (bottom of Fig. 8 ). Fig. 9 shows the dose response curves of the three PGs for both intraventricular and intrastriatal injections. In general, with in creasing doses, there was a greater inhibition of circling. For both routes of administration, PGE2 appears to be most potent of the PGs. C . Modulation of Body Temperature by Prostaglandins. PGs were tested for their ability to alter body temperature fol lowing intraventricular administration in order to determine whether alteration in behavior was due to a change in body temperature. As shown in Table 3, saline injected into the ventricle initially de creased body temperature at 10 and 20 min after injection, but 30 min post injection, temperatures had returned to baseline values. Admin istration of amph (4 mg/kg, ip) 10 min prior to the central saline injection did not alter this effect of saline. PGD2 and PGF20* when injected icv at a dose of 1.0 nmole/gm did not produce a significant 36 change in body temperature compared to saline control. Although PGE2 (1 nmole/gm) significantly increased body temperature in normal ani mals, there was no significant increase in body temperature in the presence of peripherally administered amph. DISCUSSION The results from these experiments show that PGs can alter motor function when injected directly into the CNS. As shown previously the systemic administration of amph to unilaterally lesioned mice resulted in marked circling toward the side of the lesion. This is thought to be due to the release of DA from intact nerve terminals in the striatum and the subsequent stimulation of DA receptors (Ungerstedt, 1971; Von Voitlander and Moore, 1975; Pycock, 1980). The administra tion of PGD2 , PGE2 , and PGF2 Since all of the PGs tested inhibited amph-induced circling, it is possible that fatty acid compounds in general could nonspecifically inhibit circling behavior. However, the stable metabolite of throm boxane A2 , TxB2 , failed to significantly inhibit circling after intra ventricular administration at a dose of 1 nmole/gm. In contrast when the PGs were administered at this dose, circling was almost completely inhibited. Similarly, when the PGE2 metabolite, 13, 14-dihydro-15- 37 keto-PGE2 was injected directly into the striatum, there was no signif icant inhibition although the PGs at the same dose produced a marked inhibition. In addition, there was a difference in potency among the PGs. For both the intraventricular and intrastriatal administration, the order of potency was: PGE2 > PGD2 PGF2*h . This difference in potency further suggests that inhibition of amph-induced circling is due to a selective action of the PGs and not a nonspecific effect related to the fatty acid nature of these compounds. The reduction in amph-induced circling by drugs is thought to be due to the inhibition of the effects of amph on the intact side of the brain (Pycock, 1980; Fung and Uretsky, 1980). After intraventricular injection, the PGs inhibited amph-induced circling. However, PGE2 at a dose of 0.1 nmole/gm produced a change in the direction of circling in the presence of amph; the animals now turned toward the intact side of the brain. Since circling behavior is thought to be caused by an asymmetrical activity of neurons involved in motor function on one side of the brain compared to the other (Glick, et al., 1976), PGE2 at 0.1 nmole/gm may have changed the direction of circling by producing a greater inhibition of neuronal function on the intact side than on the lesioned side. A higher dose of PGE2 , 1 nmole/gm, did not result in significant net circling, suggesting that at this dose neuronal function on both sides of the brain is equally inhibited. Our results do not indicate the exact sites in the brain where neuronal function is inhibited by PGE2 after intraventricular injection. Although the intraventricular injection of PGs changed the re sponse to amph, it was not clear whether this alteration could be due 38 to an effect on DA pathways. PGs traversing the ventricular spaces could interact with several neuronal pathways, any one of which could alter DA function. Therefore, PGs were injected into the striatum, the major site of DA synapses, in order to determine whether PGs might directly alter function. When administered by this route, PGD2 , PGE2 , and PGF2ot again inhibited amph-induced circling and the doses of PGs producing inhibition were lower after intrastriatal injection than in traventricular administration. The PGs also showed the same order of potency in the striatum as in the ventricle, with PGE2 ^ PGD2 > PGF2ct • This is consistant with the idea that PGs can, in part, alter circling behavior after intraventricular injection by acting in the striatum. The intrastriatal injection of PGE2 , unlike the intraventricular in jection, produced no reversal in the direction of circling. This observation suggests that the sites involved in the reversal of the direction of circling by PGE2 are not located in the striatum. After intrastriatal injection, TxB2 produced a small inhibition of amph- induced circling at 0.1 nmole/ gm, in contrast to its ineffectiveness by the intraventricular route of administration. However, the major PGE2 metabolite, 13, 14-dihydro-15-keto-PGE2, when injected by this route failed to alter amph-induced circling at 0.1 nmole/gm, a dose at which all three PGs inhibited amph-induced circling. The chemical structure of this compound is closer to the major PGs than is TxB2 and therefore, it may be a better control for specificity of action. It is possible that the activity of TxB2 when injected by the intrastri atal route is due to a specific interaction with neurons involved in motor function. 39 Although our results show that PGs alter behavior dependent upon DA neurotransmission, it is possible that the PGs act through indirect mechanisms. Potts and East (1972) showed that intraventricular injec tion of E series PGs produced a marked elevation in body temperature in rodents. Thus, a change in body temperature could in part, be re sponsible for the alteration in behavior seen in our experiments, par ticularly after intraventrucular injection of PGs. Therefore, the effect of PGs on body temperature was studied in order to determine whether the changes in body temperature corresponded to the changes produced in amph-induced circling. Since the hypothalamus has been shown to be the central site of temperature regulation (Cranston, 1978), PGs were administered by the intraventricular route in order to have the highest concentration reach that site. PGD2 and PGF20C administered intraventricularly to amph treated animals at a dose which inhibited circling, did not significantly change body tempera ture from that produced by intraventricular saline injection. Injec tion of PGE2 alone, as previously shown by others, significantly increased body temperature. However, while the doses of PGE2 tested inhibited amph-induced circling at all times examined after PGE2 administration (10 - 40 min; Fig. 6 , top portion), the rise in temperature in the presence of amph was not significantly different from saline treated animals (Table 3). Thus, the inhibition of amph- induced circling by PGs does not appear to be due to an alteration in body temperature. A second indirect mechanism by which PGs may alter behavior is by producing general CNS depression. Early work showed that the PGs 40 of the E series had a sedative-tranquilizer action and at high doses produced catelepsy (Horton, 1964). Further, PGEs were found to decrease exploratory behavior and locomotion (Poddubuik, 1976) and potentiate hexobarbitone-induced sleep time (Raviprakash and Sabir, 1978). In our studies, we observed that the intraventricular injec tion of PGs at 1.0 nmole/gm decreased general locomotion. However, the doses which inhibited circling were clearly not cateleptic. In addi tion PGs injected directly into the striatum did not appear to either decrease locomotor activity or produce catalepsy at doses that inhibi ted circling behavior. Thus, general depression of CNS function appears not to be responsible for PG-induced inhibition of circling. A third indirect mechanism through which PGs could act is by a change in cerebral circulation. Although we did not directly test the action of PGs on cerebral blood flow, indirect evidence suggests that the PGs tested did not inhibit circling by altering cerebral circula tion. PGD2 , PGE2 , and PGF2e*-all inhibited amph-induced circling. However, PGD2 and PGE2 are potent cerebral vasodilators (Ellis et al., 1979), while PGF2 1974). Since the direction of the behavioral changes by these PGs were the same, changes in circulation should also be in the same direction if this were the underlying mechanism. Thus, a change in cerebral blood flow does not appear responsible for the inhibition of amph-induced circling in mice, although it can not be completely ruled out at this time. Preliminary results from our laboratory indicate that the PGs also block circling produced by apomorphine. This suggests that PGs 41 may have postsynaptic as well as presynaptic sites of action within DA neuronal pathways. In summary, the present experiments suggest that PGs which are normally found in the brain can actively modulate DA- mediated neurotransmission. The ability of PGs to block amph-induced circling suggests that they may have a role in modulating motor func tion governed by normal DA neuronal pathways within the brain. FIGURE 5 EFFECT OF INTRAVENTRICULARLY (ICV) AND INTRASTRIATALLY (IS) INJECTED PGD2 ON AMPHETAMINE-INDUCED CIRCLING IN MICE Mice, previously lesioned with 6-hydroxydopamine in the left striatum, were administered d-amphetamine (4mg/kg, i.p.) followed 10 min later by the central injection of either PGD2 or saline. The number and direction of 360° turns were counted for either 1 or 2 min periods beginning 10 min after the central injection. Each point represents the mean +_ S.E. of 3 - 6 animals. 42 TURNS PER MINUTE TURNS PER 2 MINUTES o cn O o O min -6 control o o o o o 3 4> OJ FIGURE 6 EFFECT OF INTRAVENTRICULARLY (ICV) AND INTRASTRIATALLY (IS) INJECTED PGE2 ON AMPHETAMINE-INDUCED CIRCLING IN MICE Mice, previously lesioned with 6-hydroxydopamine in the left striatum, were administered d-amphetamine (4 mg/kg, i.p.) followed 10 min later by the central injection of either PGE2 or saline. The number and direction of 360° turns were counted for either 1 or 2 min periods beginning 10 min after the central injection. Each point represents the mean +_ S.E. of 4 - 10 animals. 44 E t- 3 z a 111 2 E Z 3 lu 10 15 TURNS PER10 2 MINUTES 20 25 15 110 20 i. 6 Fig. TIME TIME min min , 0 3 0 3 0 4 03 .0 0 0.01 control control 03 .0 0 lev PGE 45 FIGURE 7 EFFECT OF INTRAVENTRICULARLY (ICV) AND INTRASTRIATALLY (IS) INJECTED PGF2<* ON AMPHETAMINE-INDUCED CIRCLING IN MICE Mice, previously lesioned with 6-hydroxydopamine in the left striatum, were administered d-amphetamine (4 mg/kg, i.p.) followed 10 min later by the central injection of either PGF2 or saline. The number and direction of 360° turns were counted for either 1 or 2 min periods beginning 10 min after the central injection. Each point represents the mean _+ S.E. of 3 - 10 animals. 46 Z 3 i t- in o)20 & a 0 Ul GC 3 z TURNS PER MINUTE ) 15 10 25 15 10 5 0 10 10 20 20 i. 7 Fig. TIME I E TIM min min 0 3 0 3 0 4 0 4 control 0.01 03 .0 0 0.1 nmoles/gm control 03 .0 0 0.1 P G F 2tt 2tt F G P lev rGF2a 1.0 1.0 nmoles/gm I. s. 47 FIGURE 8 EFFECT OF INTRAVENTRICULARLY (ICV) INJECTED THROMBOXANE B2 (TxB2> AND INTRASTRIATALLY (IS) INJECTED TxB2 AND 13, 14-DIHYDRO-15-KETO- PGE2 ON AMPHETAMINE-INDUCED CIRCLING IN MICE Mice, previously lesioned with 6-hydroxydopamine in the left striatum, were administered d-amphetamine (4 mg/kg, i.p.) followed 10 min later by the central injection either TxB2 » 13, 14-dihydro-15-keto-PGE2, or saline. The number and direc tion of 360° turns were counted for either 1 or 2 min periods beginning 10 min after the central injection. Each point represents the mean S.E. of 3 - 10 animals. 48 TURNS PER MINUTE 5 z (0 0C1O 0C1O M C A ( Q. III ^ ^15 III 10 15 s 20 25 10 10 20 20 i. 8 Fig. TIME min TIME min 30 0 3 40 0 4 control control 15-K ETO -PG E2 E2 -PG ETO 15-K -DIHYDRO' 4 ,1 13 0.1 , B x T lev T control . moles/gm 0.1 nm x B 2 1.0nmotos/gm 1.0nmotos/gm 2 49 FIGURE 9 DOSE RESPONSE OF PROSTAGLANDINS ON AMPHETAMINE-INDUCED CIRCLING IN MICE The per cent inhibition of amphetamine-induced circling is compared to the dose injected by intraventricular (icv) and intrastriatal (is) routes of administation. The total num ber of turns of each animal administered PG was taken as a per cent of the mean total number of turns of saline control animals. 50 51 100 ICV TxB. 6 0 6 0 O “ 40 Z o o 20 0.01 0.1 1.0 20 100 13,14, DIHYDRO 8 0 • 15-KETO-PGEj o T x B , E 6 0 O 4 0 20 PGE 0 . 0 1 0 . 0 3 0.1 DOSE [nmol«a/gm| Fig. 9 52 TABLE 3 EFFECT OF INTRAVENTRICULARLY (ICV) INJECTED PROSTAGLANDINS ON BODY TEMPERATURE IN MICE Body Temperature (°C) Time After ICV Time After ICV Admin- Administration istration in the Presence of Amph O' 1 0 ' 2 0 ' 30' O' 10' 2 0 ' 30' Saline 38.8 36.3 37.6 38.3 38.3 36.5 37.6 38.2 +0.1 +0A _+0.4 ^0.5 +0.1 +0.3 j+,0.4 + 0 .3 pgd2 38.4 36.5 37.0 37.4 38.0 36.5 37.4 37.8 i 0 -1 +0.3 +0.2 +0.1 +0.2 +0.5 jtO.4 +0.4 pge2 38.5 38.0 39.la 39.7a 38.7 37.6 38.6 39.2 i 0 -1 +0.2 jfO.3 +0.3 +0.1 +0.2 jtO.5 +0.5 PGF21* 38.7 37.3 37.8 38.3 38.6 37.2 37.6 38.4 +0.2 +0.3 +0.2 +0.2 +0.1 jtO.3 +0.5 +0.3 a p = 0.05 when compared to saline control Rectal temperatures were taken in mice previously lesioned unilaterally in the striatum with 6-OHDA. Time indicated refers to the time after the ICV injection of either saline or PG (1 nmole/gm). 01 was taken immediately before the central injection. When amph (4mg/kg, ip) was given in combination with the central injection, it was administered 10 min prior to the central injection. Each value is the mean + S.E. of 3 - 6 animals. Prostaglandin Inhibition of Aporaorphine-Induced Circling in Mice Although Prostaglandins (PGs) have been shown to be present in the CNS and to alter CNS activity (Wolf, 1975), their exact role within neuronal pathways controlling motor function is currently unclear. One possibility is that they modulate transmitter release (Hedqvist, 1977). Our laboratory is currently studying whether PGs modulate motor func tion governed by dopamine (DA)-mediated neurotransmission. In the last chapter it was seen that centrally injected PGs had the ability to inhibit amphetamine (amph)-induced circling in mice. PGD2 , PGE2 j and PGF2ot inhibited circling when administered either intraventricularly or intrastriatally with the order of potency being: PGE2 ^ PGD2 >“ PGF2«u In addition, specificity of action was further shown by the inability of intraventricularly injected TxB2 , or intra- striiatally injected 13, 14-dihydro-15-keto-PGE2 (a major PGE2 metab olite) to inhibit amph-induced circling (Schwarz, et al, 1980, 1981). Circling produced by amph has been shown to be the result of dopamine (DA) release from intact nerve terminals in unilaterally lesioned animals (Pycock, 1980). Inhibition of DA release is one possible explanation for the decrease in amph-induced circling pro duced by PG administration. This idea is consistant with experimental results obtained in the peripheral nervous system showing that PGs of the E series inhibit norepinephrine (NE) release (Hedqvist, 1977). Additionally, PGE2 has also been reported to inhibit the release of % - D A from field-stimulated striatal slices (Bergstrom, et al., 1973; 53 54 Westfall, 1975) and from K+-stimulated striatal slices in vitro (Schwarz, et al., 1980). However, another explanation for the PG inhibition of circling produced by amph, is that the PGs act at sites postsynaptic to the DA synapse. It is known that direct acting DA agents such as apomorphine (apo) cause circling in unilaterally lesioned animals by direct stim ulation of the DA receptor. The ability of agents to inhibit apo- induced circling in mice would therefore suggest a postsynaptic site of action. The following results are the first report giving direct evidence for the idea that within the DA neuronal pathway, PGs can act at sites postsynaptic to the DA synapse. METHODS A. 6-Hydroxydopamine Lesion in Mice. Swiss-Webster, male, mice (20 - 30 gm) were lesioned in the left striatum with 6-hydroxydopamine (6-OHDA). The 6-OHDA was dissolved in ice-chilled 0.9% saline containing 1.6 ug of ascorbic acid and 4 ul of this solution was injected over 4 min. Coordinates for the injec tion site were: 5 mm anterior to the occipital suture (lambda), 2.1 mm lateral to the midline and 3.5 mm below the surface of the skull. Five to 7 days after surgery, all mice were injected with d-amph (4 mg/kg, ip) and only those mice that circled toward the side of the lesion at a rate of 10 turns/min were retained for further testing. Circling was measured visually by placing the mice in a 2 liter round bottom flask and recording both the direction of turning and number of 360° turns within consecutive 5 min time periods. 55 B. Electrolytic Lesion in Mice. In another group of mice, an electrolytic lesion was made in the left striatum at the same coordinates as the 6-OHDA lesion (see above) with a current of 2.5 ma applied through a stainless steel insect pin for a period of 20 sec. The animals were allowed 3 - 4 days to recover and then tested for a turning response in a manner similar to the 6-OHDA lesioned animals. C . Testing Protocol for Circling. The mice were injected centrally with either PG or saline and 10 min later administered apo (1.0 mg/kg, ip). The number of turns/5 min was counted beginning 5 min after the administration of apo. A "free hand" method similar to one used by Pycock, et al., (1975) was employed for the central injection of PG or saline into the brain . Under light, halothane anesthesia, mice were injected with PG or saline into the lateral ventricle using a 10 ul Hamilton syringe and needle which had a polyethylene cuff that allowed only the distal portion of the needle to be exposed. The 2 ul injections were made over a 45 sec period with the needle being held in place for an additional 15 sec. The incision was then closed with a wound clip, with the animals recovering from the anesthesia within 2 - 4 min after the injection. The site of the injection was verified at the end of the experiment by examining the location of dye after the injection of bromthymol blue into the same site as the PG injection. *D. Statistics. Where appropriate, the data was analzyed by the Dunnett Test for Multiple Comparisons or paired t-test. 56 RESULTS A. Effect of Intraventricular Administration of Prostaglandins on Apomorphine-Induced Circling in Mice. Apomorphine (1 mg/kg, ip) administration resulted in contralateral circling in mice previously lesioned unilaterally with 6-OHDA in the left striatum. Five minutes after apo, mice circled at a maximum rate of 25 turns/5 min and subsequently the circling decreased with time. At a dose of 1.0 nmole/gm all PGs tested significantly inhibited apo- induced circling when compared to saline control animals, with PGF20C appearing to be the most potent (Fig. 10). PGs administered alone did not produce net circling in either direction. B. Effect of Intrastriatal Administration of Prostaglandins on Apomorphine-Induced Circling. Preliminary studies had shown that mice lesioned with 6-OHDA in the striatum and then reinjected with saline into the same site as the lesion did not circle. It appeared that the lesioned striatum did not function in the same manner as an intact stiatum. Therefore, mice were electrolytically lesioned in the left striatum. With this lesion, pre- as well as postsynaptic elements are destroyed and the administration of apo systemically causes circling towards the side of the lesion (ipsilateral direction), since only DA receptors on the intact side are available for stimulation. As shown in Fig. 11 - 13 the administration of apo (1 mg/kg, ip) to animals which had received a unilateral intra striatal saline injection to the intact side of the brain (controls) resulted in ipsilateral circling which was maximal 5 min after apo injection, and thereafter decreased with time. In Fig. 12 it can be 57 seen that the effect of the PGE2 metabolite, 13, 14-dihydro-15-keto- PGE2 , was not significantly different from saline control animals as measured by the amount of circling. As shown in Fig. 11, intrastriatally injected PGD2 at 0.1 nmole/ gm resulted in the mice circling away from the lesioned side of the brain (contralateral direction). A dose of 0.03 nmole/gm produced a small, but not significant decrease in circling, while the dose of 0.01 nmole/gm had little affect on circling compared to saline controls. Intrastriatal PGE2 injection also resulted in inhibition of apo- induced circling with 0.1 nmole/gm causing almost a total inhibition (Fig. 12). A dose of 0.03 nmole/gm was significantly different from control at 5 and 10 min after apo administration, while 0.01 nmole/gm was significantly different only at the first time period examined. Fig. 12 also shows inability of the major PGE2 metabolite (13, 14-dihy- dro-15-keto-PGE2) to inhibit apo-induced circling at 0.1 nmole/gm, a dose at which the major PGS inhibited circling. Like the other two major PGs tested, PGF2o£ inhibited apo-induced circling behavior (Fig. 13). As shown previously for PGD2 , the dose of 0.1 nmole /gm produced circling in the contralateral direction. In contrast to both PGD2 and PGE2 » 0.03 nmole/gm of PGF2«. resulted in a significant reduction in circling at all three time periods examined. In addition, the lowest dose tested, 0.01 nmole/gm, inhibited circling at 5 and 10 min post apo administration. Thus, it appears that PGF20C was the most potent of the PGs in its ability to inhibit apo-induced circling. 58 Fig. 14 shows the dose response curves of the three PGs and 13, 14-dihydro-15-keto-PGE2 injected by the intrastriatal route of adminis tration. In general, with increasing doses, there was greater inhibi tion of circling, with PGF20Cappearing to be the most potent of the PGs. C. Effect of Indomethacin Pretreatment Upon Apomorphine-Induced Circling in Mice. The effect of a 20 min pretreatraent with indomethacin on apo- induced circling was examined in mice previously lesioned electroly- tically lesioned in the left striatum. As shown in Fig. 15, animals given 20 mg/kg indomethacin, i.p., circled slightly, but not signifi cantly more than control animals. However, animals given 45 mg/kg in domethacin circled at significantly higher rates than control animals. DISCUSSION Previous studies indicate that PGs might affect DA neurotrans mission. Central PG administration has been shown to produce catelepsy (Horton, 1964) and block conditioned avoidance responding (Potts, et al., 1973) in a manner similar to DA receptor blockers, such as halo- peridol and chlorpromazine. In addition, the central administration of PGs has been shown to produce a decrease in food intake (Scaramuzzi, et al., 1971), an effect that may have features in common with the decrease in food intake following lesioning of the DA nigrostriatal pathway (Ungerstedt, 1971a). The finding that PGs have been measured in the caudate nucleus, the area of the brain richest in DA synaptic connections (Abdel-Halira, et al., 1977) suggested that endogenous PGs may play a physiological role in the modulation of motor function 59 regulated by dopaminergic neurotransmission. Our initial observation that PGs inhibited amphetamine-induced circling further indicated PGs could affect DA-mediated neurotransmission (Schwarz, et al., 1980, 1981). The results reported in this paper provide the first direct evidence showing that PGs act at sites postsynaptic to the DA synapse within central DA-mediated pathways. When injected intraventricularly PGD2 , PGE2 , and PGF2©iall inhib ited apo-induced circling at a dose of 1.0 nmole/gm, with PGF2«tbeing the most potent. PGs were injected intrastriatally in order to deter mine whether the inhibition of circling could be due to the inhibition of the effects of DA receptor stimulation. After intrastriatal injec tion all three major PGs tested inhibited apo-induced circling in the dose range of 0.01 - 0.1 nmole/gm. In contrast the PGE2 metabolite, 13, 14-dihydro-15-keto-PGE2, at a dose of 0.1 nmole/gm, did not inhibit apo-induced circling suggesting that the inhibtion of circling was not due to a nonspecific action of fatty acid compounds in general. Simi larly, in previous studies we have shown that the intrastriatal injec tion of 13, 14-dihydro-15-keto-PGE2 did not inhibit amph-induced circ ling at a dose in which the major PGs produced marked inhibition (Schwarz, et al., 1980, 1981). A second similarity with the inhibition of amph-induced circling was the ability of lower doses of PGs to in hibit apo-induced circling when injected intrastriatally than when in jected intraventricularly. These results suggest that PGs alter motor function after intraventricular injection by acting in the striatum to inhibit the effects produced by DA receptor stiumulation. 60 The administration of PGD2 and PGF2« a t 0.1 nmole/gm, the high est dose of PG administered intrastriatally, caused electrolytically lesioned animals treated with apo to circle toward the intact side of the brain. Although the mechanism of this change in direction is not known, circling behavior is generally thought to be due to a greater activity of neurons involved in motor function on one side of the brain compared to the other side. After electrolytic lesions of one striatum, net circling movements were not observed at 3 days following the lesion. This is presumably because neurons on both sides of the brain adjust to the lesion by balancing their activity. Apo adminis tration enhanced DA transmission on the intact side of the brain, creating an imbalance of neuronal activity and produced net circling toward the lesioned side of the brain. The PGs, when injected into the intact striatum inhibited the effects of DA receptor stimulation by apo as measured by the inhibition of circling behavior. It is possible that a high dose of PG may produce such a complete inhibition of DA receptor stimulation on the intact side, that an imbalance between the two sides is created and the activity of neurons involved in motor function on the lesioned side of the brain then becomes dominant. This could cause net circling behavior toward the intact side of the brain, and explain the change in direction of circling produced by high doses of PGs administered after apo. The effectiveness of exogenously injected PGs in producing an in hibition of apo-induced circling suggests that endogenous PGs might function to inhibit the effects produced by DA receptor stimulation. Indomethacin has been shown to inhibit the the enzyme, cyclooxygenase, 61 which converts arachidonic acid to the endoperoxides (PGG2 and PGH2) which in turn are converted to the major PGs (Flower, 1964). Although pretreatment with 20 mg/kg of indomethacin produced no significant change in apo-induced circling, 45 mg/kg, i.p., a dose used previously to inhibit PG formation in the brain (Huidobro-Toro, et al., 1980), significantly increased the amount of circling seen after peripheral apo administration. These results support the hypothesis that endoge nous PGs modulate motor function regulated by DA receptor stimulation. Since our results suggest a postsynaptic site of PG alteration of DA neuronal mechanisms, further study into the exact site of action is warranted. Recently, it has been proposed (Kebabian, et al., 1972) that there are two types of DA receptors with the DA} receptor linked to adenylate cyclase and the formation of cyclic AMP. It is possible that since PGs alter cyclic nucleotides in other cell systems (Kuehl, et al., 1973), they may inhibit circling behavior through inhibition of DA-stimulated adenylate cyclase. In addition, PGs may act at syn apses of non-dopaminergic neurons in the striatum which interact with dopaminergic neurons such as cholingergic, gabergic, or enkephalinergic neurons. In summary, our results show that PGs injected by either the intraventricular or intrastriatal route of administration inhibits apo-induced circling. This is the first direct evidence of PGs acting at sites postsynaptic to the DA synapse. FIGURE 10 EFFECT OF INTRAVENTRICULARLY (ICV) INJECTED PROSTAGLANDINS ON APOMORPHINE-INDUCED CIRCLING IN MICE Mice, previously lesioned with 6-hydroxydopamine in the left striatum, were administered PGs (1 nraole/gra) intraventricularly followed 10 min later by apomorphine (1 mg/kg, ip). The number and direction of 360° turns were counted for 5 min periods be ginning 5 min after apo injection. Each point represents the mean + S.E. of 3 - 6 animals. 62 63 TIME [mini le v 10 15 20 PGE 74 PGD, w 15 20 _A control 25 30 I.Onmole/gm Fig. 10 FIGURE 11 EFFECT OF INTRASTRIATAL ADMINISTRATION OF PGD2 ON APOMORPHINE- INDUCED CIRCLING IN MICE Mice, previously electrolytically lesioned in the left striatum, were injected in the right striatum with PGD2 followed 10 min later by apomorphine (1 mg/kg, ip). The number and direction of 360° turns were counted for 5 min periods beginning 5 min after apo administration. Each point represents the mean S.E. of 3 - 8 animals. 64 TURNS PER 5 MINUTES 10 12 6 8 TIME Fig. 11 10 (min) 15 0.1 0.1 nmole/gm 0.01 control 0.03 PGD i.s. 65 » FIGURE 12 EFFECT OF INTRASTRIATAL ADMINISTRATION OF PGE2 OR 13, 14-DIHYDRO- 15-KETO-PGE2 ON APOMORPHINE-INDUCED CIRCLING IN MICE Mice, previously electrolytically lesioned in the left striatum were injected in the right striatum with PGE2 or 13, 14-dihydro- 15-keto-PGE2 followed 10 rain later by apomorphine (1 mg/kg, ip). The number and direction of 360° turns were counted for 5 min periods beginning 5 min after apo administration. Each point represents the mean + S.E. of 3 - 8 animals. 66 67 PGE i.s. 10 (/) UJ H control 13,14, DIHYDRO in . 15-KETO-PGE, a: in s . [0<1] a 0.01 cn 0.03 z tc 3 H 0.1nmole/gm 10 TIME (mini Fig. 12 FIGURE 13 EFFECT OF INTRASTRIATAL ADMINISTRATION OF P G F ^ ON APOMORPHINE- INDUCED CIRCLING IN MICE Mice, previously electrolytically lesioned in the left striatum were injected in the right striatum with PGF201 followed 10 min later by apomorphine (1 mg/kg, ip). The number and direction of 360° were counted for 5 min periods beginning 5 min after apo administration. Each point represents the mean S.E. of 3 - 8 animals. 68 TURNS PER 5 MINUTES 10 5 TIME Fig. 13 10 (mini 15 0.03 A 0.1 nmole/gm 0.01 control i.s. GF pG 2 « 69 FIGURE 14 DOSE RESPONSE OF PROSTAGLANDINS ON APOMORPHINE- INDUCED CIRCLING IN MICE The per cent inhibition of apomorphine-induced circling in mice is compared to the dose injected by the intrastriatal route of administation. The total number of turns of each animal administered PG was taken as a per centage of the mean total number of turns of saline control animals. 70 CONTROL -20 100 40 0 4 20 0 6 80 0.01 i. 14 Fig. (nmoles/gm) OE \ DOSE . \ 3 0.0 0.1 15-KETO-PGEo 13,14 DIHYDRO 71 FIGURE 15 EFFECT OF INDOMETAHCIN PRETREATMENT ON APOMORPHINE-INDUCED CIRCLING IN MICE Mice previously electrolytically lesioned in the left striatum were pretreated with indomethacin (20 and 45 mg/kg, sc) 20 min prior to the administration of apomorphine (1 rag/kg, ip). The number and direction of 360° turns were counted for 5 rain periods beginning 5 min after apo administration. Each bar represents the mean S.E. of 5 animals. *p = 0.05 when compared to control group. 72 AVE TOTAL TURNS/20 MIN 20 40 30 10 50 60 0 45 20 C INDOMETHACIN gk mg/kg mg/kg i. 15 Fig. 73 PROSTAGLANDIN MODULATION OF DOPAMINE SYNTHESIS AND RELEASE It has been hypothesized that prostaglandins (PGs) may regulate the release of neurotransraitters from nerve terminals. Tliis hypothe sis was derived from work originally done in the sympathetic nervous system which showed that norepinephrine (NE) release from nerve term inals was inhibited by PGs of the E series and facilitated by PGF2<» (Hedqvist, 1977; Westfall, 1977; Stjarne, 1979). Similar studies have been carried out in the CNS, but the results have been inconsistent, ranging from no effect of PGs, to inhibition of NE release in a manner similar to that reported in the sympathetic nervous system. Circling behavior produced by amphetamine (amph) in unilaterally lesioned animals appears to be caused by both the release of DA and also the stimulation of DA synthesis which ensures sufficient DA avail able for release (Ungerstedt, 1971; Pycock, 1980). Thus, the inhibi tion of amph-induced circling by PGs shown previously (Schwarz, et al. 1981) could be due to an effect of PGs on the release or synthesis of DA. There is little information on the effects of PGs on DA release mechanisms. A reduction in ^h-DA release from field stimulated slices was seen with 3 x 10”^M PGE2 (Bergstrom, et. al., 1973) and confirmed by Westfall and Kitay (1977). However, Roberts and Hillier (1976) failed to see a reduction in ^H-DA release from rat cortical and hypo thalamic synaptosomes, as did Von Voitlander (1976) in an in vivo study using cats. More recently, Reimann, et al., (1980) also failed 74 75 to see an alteration in DA release by PGs in both rat and rabbit striatal slices. Thus, the following experiments were designed to examine whether PGs could alter either DA release or synthesis and whether this alter ation could be responsible for changes in behavior previously seen. METHODS A. Dopamine release in rat striatal slices. Male, Sprague-Dawley rats were pretreated with indomethacin (20 mg/kg, i.p.) 60 min prior to being stunned and decapitated in order to prevent the endogenous synthesis of endogenous PGs in the brain. The striata were dissected out according to the procedure of Glowinski and Iversen (1966). The tissue was weighed, sliced into 0.3 mm x 0.3 mm sections on a Mcllwain tissue chopper, and dispersed in ice-chilled Krebs-Ringer Hepes buffered medium. This was composed of: NaCl (119mM), KC1 (4.7), CaCl2 (1.2mM), KH2 PO4 (1.2mM), MgS04 (1.2mM), Hepes (22mM), and d-glucose (10mM). The pH was adjusted to 7.2 with 3.0M Tris. Following a centrifugation at 500 g for 2 min, the supernatant was discarded and the slices resuspended in a volume of medium such that 0 . 2 ml aliquots of this suspension would be equivalent to at least 15 - 20 mg of tissue. The aliquots were added to flasks containing ^H-DA (50Ci/mmol) and incubated for 15 min at 37°C. The slices were washed 4x with normal media and then incubated with PG present for 15 min. After a quick separation, the slices were further incubated for 15 min with PG present in normal media or media containing 12.5mM K+ as a depolarizing agent. (To insure that isotonicity was maintained, 76 NaCl was lowered by a corresponding amount). Tissue and media were separated by centrifugation and placed on ice and 1 ml of perchloric acid (0.2M) was added to the tissue and 0.5 ml to the media. In addi tion, 0.2 ml of 20% sodium metabisulfite in 4% EDTA was added to both tissue and media samples. ^h-dA was assayed by strong cation-exchange chromatography and liquid scintillation counting as previously describ ed (Uretsky, 1974). Release of ^H-DA is expressed as -%-DA in medium as a percentage of ^H-DA present in tissue and media. B. Dopamine Synthesis in Rat Striatal Slices. Rat striatal slices were prepared exactly as in the release studies. The flasks containing the slices were preincubated for 5 min at 37°C with PG present in either normal or 12.5mM K+ media. L-(side chain-2, 3, % ) tyrosine, at a final concentration of lOuM, was then added and the slices were allowed to incubate for 15 min. The incu bation was ended by placing the flasks on ice. Tissue blanks were prepared in the same manner as samples, but remained on ice during the incubation period. ^H-catechols in both the tissue and media were assayed using alumina adsorption chromatography as described previously (Uretsky and Snodgrass, 1977). C. Statistics. Statistical comparisons of the appropriate data were made by Student's t test or the Welch Test for unequal variances. RESULTS A. ^H-Dopamine Release After Varying Kt Concentrations. As seen in Fig. 16, a 15 min incubation with 10-15 mg of tissue 77 resulted in a basal release of 13.0% of total ^h -DA present. Increas ing K+ from 5mM in normal media to lOmM, 12.5mM, 20mM, 40mM, and 60mM resulted in increasing amounts of ^H-DA released with 15.2%, 31.0%, 56.7%, 71.1%, and 80.8% being released respectively. B . Effect of Prostaglandins on Basal Release of Dopamine. The effect of PGS on basal release was examined by compar ing the amount of ^h -DA released into the media when PG was present to the amount released in the absence of PG. As can be seen in Fig. 17 PGD2 added to normal media significantly increased basal release at —7 • . *3 10 M with PGE2 also significantly raising the amount of H-DA released — 8 . — 8 -5 into the media at 10 M. At concentrations of 10 - 10 M, PGF2©t failed to significantly alter basal ^H-DA release from striatal slices. C . Effect of Prostaglandins on Kjj-Stimulated Dopamine Release. Addition of 12.5mM K+ to normal media resulted in an approximate doubling of ^H-DA released from striatal slices after a 15 min incuba tion. When compared to release caused by 12.5mM K+ , PGD2 significantly increased K+-stimulated release at 10“®M (Fig. 18). In contrast to this increase, both PGE2 (10-8M) and (10-^M and 1Q"‘^M) signifi- cantly inhibited the ability of 12.5mM K+ to stimulate ^H-DA release D. Effect of Prostaglandins on Basal Dopamine Synthesis. Since the processes of transmitter release and synthesis appear to be coupled, the effect of PGs on DA synthesis was examined. Fig. 19 shows that while PGD2 appeared to increase basal synthesis at 10”^ M, it was not statistically significant. In addition, both PGE2 and ^ F2«.at concentrat*-ons IQ' 8 “ 10”^M failed to significantly alter basal DA synthesis. 78 E. Effect of Prostaglandins on Kit-Stimulated Dopamine Synthesis. The effect of 12.5mM K+ on DA synthesis was similar to its effect on DA release, in that it produced a two-fold increase in effect. While both PGD2 and PGF2<5tappeared to decrease the ability of K+ to stimulate DA synthesis, only PGF2 ei produced a statistically significant decrease at 10 (Fig. 20). PGE2 had no effect on K+-stimulated synthesis at concentrations of lO”? - 10-5m. DISCUSSION As shown in previous studies, depolarization of striatal slices by K resulted in increased release of H-DA. PGE2 (10 M) and PGF2 0S (10~6 and 10~7m) significantly decreased K+-stimulated % - D A release • • j. O while PGD2 increased both basal and K -stimulated H-DA release at 10” 7 and 10”®M respectively (Fig. 17 and 18). These results confirm those of Bergstrom, et al., (1973) and Westfall and Kitay (1977) on the ability of PGE2 to inhibit depolarized H-DA release. However, the present results show PGF2^inhibiting K+-stimulated release, and PGD2 increasing DA release, while those same authors showed no effect of PGF2 «. and did not examine the effects of PGD2 . Release and synthesis appear to be coupled processes in catechol amine-containing neurons. The work of Dismukes and Mulder (1977) sug gested that the release of DA from nerve terminals may be governed not by presynaptic receptors, but indirectly through modulation of DA bio synthesis. Our results showed no significant change in basal DA syn thesis by PGs and only PGF2 ^ at 10 ^M (Fig. 20) significantly decreas ing the ability of K+ to stimulate synthesis. There are reports of PGs altering DA turnover in vivo. Poddubiuk and Kleinrok (1976) showed 79 that both PGE2 and PGF2 «x enhanced DA turnover in rats after intraven- tricular administration. Brus, et al., (1978) also showed higher DA content in the striatum following intraventricularly injected PGE2 and PGF2 ««. However, Nielsen, et al., (1980) saw decreases in DOPAC, HVA, and MHPG following administration of PGF2etthrough a push-pull cannula implanted in the ventricle. The administration of 3 mg/kg (ip) of indomethacin, which would inhibit PG synthesis, depressed HVA produc tion in rat striatum (Abdel-Halim, et al., 1979). Thus, results con cerning the ability of PGs to alter DA synthesis and release are con flicting and interpretation of results is difficult. Release from peripheral synpathetic neurons appears to be con trolled by presynaptic receptors (or autoreceptors). Activation of alpha-adrenergic presynaptic receptors by NE, or NE agonists, results in a decrease of NE outflow, while blockade of these same receptors prevents the feedback inhbition of NE release (Westfall, 1977). The Hedqvist theory relates the modulatory effects of PGs on peripheral sympathetic neurons with alpha-adrenergic presynaptic receptors sen sitive to PGE2 decreasing NE release and presynaptic receptors sensi tive to PGF20Iincreasing NE release (Brody and Kadowitz, 1974; Hedqvist, 1977; and Westfall, 1977). Experiments have shown that there may also be PG sensitive release mechanisms in central NE neurons. Bergstrom, et al., (1973) showed that 1 uM PGE2 reduced the stimulation induced out-flow of 3h-NE from rat cortical slices. These results were con firmed in rat hypothalamic synaptosomes by Wendel and Strandhoy (1978); rat corical slices by Hillier and Templeton (1980), Shenoy and Ziance (1980) and Reiman, et al., (1980). However, Roberts and Hillier (1976) 80 reported that PGE2 facilitated NE release from rat brain synaptosomes while Reiman, et al., (1980) showed that NE neurons of rabbit cortex failed to respond to PGs. Thus, while the majority of evidence demon- strats that PGs may alter central NE release, not all species may pos sess PG-sensitive mechanisms. In summary, experimental results suggest that PGs may play a sig nificant role in altering NE release from central and peripheral neurons. However, while some evidence indicates PGs may alter central DA release and synthesis, conflicting evidence makes it difficult to draw firm conclusions on their exact role. It appears that PGs may only at best, weakly modulate DA function by presynaptic control of release and syn thesis . FIGURE 16 EFFECT OF VARYING K+ CONCENTRATIONS ON DOPAMINE RELEASE Rat striatal slices (10 - 1-5 mg) were incubated for 15 min at 37° with ^h-DA, washed 4x with normal media, and then incubated for an additional 15 min with varying K+ concentrations. was assayed by strong cation-exchange chromatography and liquid scintillation counting. The percentage of ^H-DA released is expressed as a percentage of ^h -DA present in tissue and media. 81 % RELEASE 10 ■tk 0> 00 o o o o o o 3 H* tn S oM PR ON + o o> O 00 f O FIGURE 17 EFFECT OF PROSTAGLANDINS ON BASAL RELEASE OF DOPAMINE Rat striatal slices (10 - 15mg) were incubated for 15 min at 37° with ^H-DA, washed 4x with normal media, and then incubated for an additional 15 min with prostaglandins present at concen trations of 10“® - 10“^M. ^H-DA was then assayed by strong cat ion-exchange chromatography and liquid scintillation counting. The percentage of ^H-DA released is expressed as a percentage of ®HDA present in tissue and media. *p = 0.05 when compared to control. 83 CONTROL 100 125 50 25 OCN F G |M| PG OF CONCEN Fig. 17 10 PGD • PGF2oc F G P ° PGE ■ 5 84 FIGURE 18 EFFECT OF PROSTAGLANDINS ON K+-STIMULATED DOPAMINE RELEASE Rat, striatal slices (10 - 15mg) were incubated for 15 min at 37° with ®H-DA, washed 4x with normal media, and then incubated for an additional 15 min with prostaglandins at concentrations of 10“® -10”^M added to media containing 12.5mM K+. ®H-DA was assayed by strong cation-exchange chromatography and liquid scintillation counting. The percentage of ®H-DA released is expressed as a percentage of ^h -DA present in tissue and media. * p = 0.05 when compared to control. 85 K+ CONTROL 100 200 150 50 OCN F PG OF CONCEN i. 18 Fig. 10 6 PGD PGE 5 86 FIGURE 19 EFFECT OF PROSTAGLANDINS ON BASAL DOPAMINE SYNTHESIS Rat, striatal slices (20 -30mg) were preincubated in normal media for 5 min at 37° with prostaglandins present at concentrations of 10”® -10~^m. ^H-tyrosine (10uM) was added and slices were allowed to further incubate for 15 min. ^H-catechols £n both tissue and media were assayed using alumina adsorption chrorao- tography. 87 CONTROL 100 125 50 25 75 OCN F PG OF CONCEN i. 19 Fig. 7 10 " 6 PGE * PGD • 5 88 FIGURE 20 EFFECT OF PROSTAGLANDINS ON K+-STIMULATED DOPAMINE SYNTHESIS Rat, striatal slices (20 - 30mg) were preincubated in media con taining 12.5mM K+ for 5 min at 37° with prostaglandins present at concentrations of 10”® - 10”-*M. ®H-tyrosine (lOuM) was added and slices were allowed to further incubate for 15 min. ®H- catechols werre assayed using alumina adsorption chromotography. * p = 0.05 when compared to respective control group. 89 K+ CONTROL 100 125 50 25 75 r 8 OCN F PG OF CONCEN i. 20 Fig. r7 r 6 PGD • * PGE -5 90 PROSTAGLANDIN MODULATION OF RAT STRIATAL CYCLIC AMP Since Steinberg, et al., (1964) first showed that PGE^ blocked the action of lipolytic agents, which increase cAMP levels in fat cells, evidence has accumulated showing an intimate relationship between PGs and cyclic nucleotides in neuronal as well as non-neuronal systems (Kuehl, et al., 1973; Gorman, et al., 1975). Early work in the brain by Berti, et al., (1973) and Dismukes and Daly (1975) showed that PGs of the E-series elicited increases in cAMP in vitro, while Wellman and Schwabe (1973) showed that PGE2 , PGEj, and PGF2ctall increased cAMP in vivo. In clonal cell lines of CNS origin, PGs have also been shown to be potent stimulators of cAMP formation (Ortman, 1978). In the DA sys tem within the striatum, PGE2 has been shown to significantly elevate cAMP in both homogenate and slice preparations (Havemann and Kuschinsky, 1978). There is also evidence suggesting an action of PGs on catechol- amine-stimulate adenylate cyclase. Siggins, et al., (1971) showed that PGE2 inhibited the NE-stimulated formation of cAMP which in turn normally hyperpolarized Purkinje cells in the cerebellum. Thus, it would appear that the effects of PGs within the CNS may in part be a result of alterations in cAMP formation. At present, radioligand binding studies have suggested that there are several types of dopamine receptors with some evidence suggesting as many four types (Seeman, 1981). Work by Kebabian and Greengard, (1972) in addition to others, has indicated that one of these receptors is linked to adenylate cyclase (DA^ receptor). Occupation of the DAi 91 92 receptor by dopamine (DA) activates adenylate cyclase which catalyzes the formation of cyclic AMP. This alteration in cAMP formation has been suggested to be responsible for changes in motor function con trolled by DA (iversen, 1975). Apomorphine (apo) has been shown to cause circling in unilaterally lesioned animals by direct activation of DA receptors (Pycock, 1980). Previous results have shown that PGs can inhibit apo-induced circling in unilaterally lesioned mice (Schwarz, et al., 1981). This was the first evidence to suggest an action for the PGs at sites which are postsynaptic to the DA synapse. PGD2 , PGE2 , and PGF2«tall inhibi ted apo-induced circling when injected either intraventricularly or intrastriatally, with the order of potency being: PGF2e£ > PGE2 > PGD2 . The different potencies of the PGs plus the inability of the PGE2 me tabolite, 13,14-dihydro-15-keto-PGE2, to inhibit apo-induced circling, suggested that this inhibition was caused by a selective action of the PGs and not a nonselective action of fatty acid compounds in general. It seemed reasonable to assume that since PGs alter cyclic nucleo tides in DA neuronal systems, that the inhibtion of apo-induced circling might be due to the inhibition of DA-stimulated formation of cyclic AMP The following experiments were designed to test this hypothesis. METHODS A. Assay of Cyclic AMP in Mouse Striatal Slices. The striatum of male mice (Swiss-Webster, 20-30 gm) was dissected out and cut into cuboidal 0.26mm x 0.26mm slices on a Mcllwain tissue chopper. These slices were then suspended in a Krebs-Ringer Hepes buffered media which was composed of: NaCl (119mM), KCl (4.7mM), CaCl2 93 (1.2mM), KH2PO4 (1.2mM), MgS0 4 (1.2mM), Hepes (22mM), and d-glucose (10 mM). The pH was adjusted to 7.2 with 3.0M Tris. Following a centrifu gation at 500 x g for 2 min, the supernatant was discarded and the slices were incubated in 10 ml media at 37°C for 60 min with media changes at 20, 40, and 60 min. Aliquots, containing 15 mg tissue (approx. 1 mg protein) were added to 10 x 75 tubes containing the phos phodiesterase inhibitor, ImM isobutyl-methylxanthine (IBMX), in the presence or absence of DA (ImM) and varying PG concentrations (10“® - 1 0 “ 4 m) in a final volume of 0.5 ml. The tubes were incubated for 15 min at 37°C and the reaction was terminated by placing the tubes on ice and adding 0.5 ml of 10% trichloroacetic acid (TCA). Tissue and media were homogenized and samples were frozen until assayed for cyclic nucleotide content by radioimmunoassay (RIA). B. Measurement of Cyclic AMP by RIA. Samples were extracted with water saturated, ethyl ether in order to remove the TCA (3x with 5ml ether). Following 10 min in 50°C water to evaporate the ether, the pH was adjusted to 6.5 by adding 100 ul of 1 M Na+-acetate buffer. For the cAMP assay 100 ul of sample was acety- lated by 10 ul of a mixture of 2 parts acetic anhydride to 5 parts of triethylamine. Added to this solution was 50 ul of cAMP-directed Ab and 50 ul of Tubes were incubated overnight at 20°C and following an ammonium sulfate (60%) precipitation, the precipitate containing the Ab-Ag complex was counted in a Beckman gamma counter. (Unverferth, et al., 1981). C. Measurement of Protein. Protein was measured according to the method of Lowry et al.,(1951). 94 D. Statistics. Where appropriate the data was analyzed by Student's t-test or the Dunnett test for multiple comparisons. RESULTS A. Effect of Dopamine and Apomorphine on Cyclic AMP Formation. As reported by Forn, et al., (1974), addition of DA results in the stimulation of cAMP formation. Fig. 21 shows that 0.1, 0.3, and l.OmM DA produced a stimulation of 130%, 173%, and 142% respectively. To show that the system was sensitive to other DA agonists, apomor phine (apo) was added to the incubation solution. It can be seen in Fig. 1 that 0.1, 0.3, and l.OmM apo also significantly stimulated the formation of cAMP. B. Effect of Ethanol on Cyclic AMP Formation. Since the PGs were stored in 95% ethanol and diluted with buffer to the appropriate dilution, we examined the effect of different ethan ol concentrations on basal cAMP formation. Fig. 22 shows that the addi tion of varying ethanol concentrations significantly increased the amount of cAMP formed with the greatest effect seen at 17mM ethanol. C . Effect of Ethanol on Dopamine-Stimulated Cyclic AMP Formation. As can be seen in Fig. 23 addition of ethanol at concentrations of 0.17 - 170mM, did not significantly alter the ability of DA to stimu late cAMP formation, while a concentration of 1.7M ethanol significantly inhibited DA stimulation (data not shown). In the absence of ethanol, DA (ImM) increased cAMP by 187 5.9%, while the presence of 0.17, 1.7, 17.0, and 170 mM ethanol increased cAMP formation by 201 _+ 16.0%, 188 _+ 14.3%, 156 +_ 17.1% and 200 _+ 20.6% respectively. 95 D. Effect of Prostaglandins on Cyclic AMP Formation. The results shown in Fig. 24 confirm previous reports on the abil ity of PGE2 to increase the formation of cAMP in striatal slices with a significant increase seen at 10 ^M. PGF2et also significantly in- —5 —8 —4 creased cAMP at 10 M. However, PGD2 at concentrations of 10 - 10 M failed to significantly increase striatal cAMP, although a slight but non-significant increase was seen at 10“^M. E. Effect of Prostaglandins on Dopamine-Stimulated Cyclic AMP Formation. Addition of PGD2 , PGE2 , and PGF2et at concentrations of 10"® - lO'^M failed to significantly alter the ability of DA to stimulate the forma tion of cAMP, although as seen above both PGE2 and PGF2 etsignificantly increased cAMP (Fig. 25). DICUSSION The previous results of Havemann and Kuschinsky (1978) showed that PGE2 stimulated the formation of cAMP in rat, striatal slices. Our results show that in another species, the mouse, PGE2 also had the ability to significantly elevate cAMP in striatal tissue. In addition PGF2 Halim, 1977), we also examined the ability of PGD2 to stimulate cAMP formation. While there was a slight increase at 10_^M, PGD2 failed to significantly increase striatal cAMP at concentrations of 10“® - 10“^M. Since occupation of the DA^ receptor by DA or DA agonists such as apo results in cAMP formation, we tested the ability of PGs to alter 96 DA-stimulated cAMP formation. Neither PGD2 , PGE2 , nor PGF2©c altered the ability of ImM DA to stimulate the production of cAMP. The com bination of PG plus DA results in a stimulation of cAMP formation equal to DA alone. There are several possibilities for the lack of an additive effect. One possibility is that ImM DA results in a maximum stimulation of cAMP and the PGs cannot stimulate above this maximum level. A second possibility is that DA and PGs are stimulating a common pool of adenylate cyclase and maximum stimulation by one com pound does not allow stimulation above that of the first by the second compound. Apo causes circling in unilaterally lesioned animals by direct activation of DA receptors. We have previously shown that PGs can inhibit apo-induced circling in unilaterally lesioned mice. Thus, it was hypothesized that the inhibition of apo-induced circling in mice might be due to the inhibition of DA-stimulated formation of cAMP. However, none of the PGs tested inhibited DA-stimulated cAMP. The PGs acted more similar to DA agonists since cAMP formation was increased. The lack of inhibition by PGs argues that PGs do not inhibit apo-induc ed circling through blockade of DA stimulation of adenylate cyclase. Early work had suggested that adenylate cyclase was the DA re ceptor (Kebabian and Greengard, 1972). However, radioligand binding studies indicated that there were multiple DA receptor sites, only one of which was linked to adenylate cyclase (Seeraan, 1981). The idea of multiple binding sites was in part due to the observation that ergot alkaloids, such as bromocriptine and lisuride, failed to stimulate cAMP formation, in contrast to DA and apo, in cell-free preparations. In 97 fact, these compounds antagonized the stimulation of adenylate cyclase activity induced by DA (Saiani, 1979). However, when these same ergot alkaloids were tested in slice preparations, a stimulation of cAMP for mation was seen. These results suggested that there were multiple forms of adenylate cyclase with tissue homogenization affecting the coupling of the enzyme to the receptor. Localization studies, using selective lesioning techniques, suggested that DA-stimulated adenylate cyclase may be present in both postsynaptic striatal neurons and also glial cells. It is therefore difficult to determine which pool of adenylate cyclase mediates a particular physiological function (Spano, et al., 1980). The results from pharmacological studies would tend to suggest that all major DA-related drug effects in the striatum occur at DA2 receptors (non-adenylate cyclase linked), with the DA^ sites being "receptors in search of a function" (Snyder and Goodman, 1980). The failure of PGs to inhibit DA-stimulated adenylate cyclase may add additional evidence to the idea that DA^ receptors are functionally unimportant. PGs may however, play an important role in non-dopaminergic sys tems which depend upon the formation of cAMP in order to initiate or maintain a physiological process. Recently, it has been shown that activation of alpha-adrenergic receptors in the cerebral cortex or hypothalamus by NE results in the formation of cAMP (Skolnick, et al., (1976). This accumulation of cAMP was markedly reduced after incubation with the PG synthetase inhibitor, indoraethacin, with addition of low concentrations of PGE2 restoring the NE-stimulated formation of cAMP. The authors conclude that PGs of the E series are required for the expression of alpha-adrenergic receptor mediated activation of cAMP formation in brain tissue (Partington, et al., 1980). A second area of research where it has been shown that PGs and cAMP formation are inter-related is in the mechanism of morphine analgesia. However, the experimental results are not consistent. Collier and Roy (1974) first suggested that the analgesic effect of morphine was elicited through the inhibition of PGE-stimulated adeny late cyclase. In addition, the morphine antagonist, naloxone, complet ly antagonized this biochemical action of morphine. However, Tell, et al., (1975) failed to confirm the ability of PGE^ or PGE2 to stimulate adenylate cyclase and morphine did not modify adenylate cyclase activ ity in the presence or absence of PGE^. In a rat brain mince system, Katz and Catravas (1977) found that morphine did not prevent the PGE].- stimulated cAMP formation. These negative findings were attributed by Havenamm and Kuschinsky (1978) to the fact that release of endogenous cAMP activators, such as adenosine, may mask the inhibitory effect of opiates on PG-induced stimulation of cAMP. Thus, PGs may be involved in the action of morphine and other opiate-lilce compounds. In summary, PGs appear to actively stimulate the formation of cAMP. This stimulation may or may not result in changes of physio logical function. In testing our original hypothesis, PGs do' not inhibit the ability of DA to stimulate the formation of cAMP. Thus, it does not appear that this is the biochemical mechanism responsible for the inhibition of apo-induced circling in mice. FIGURE 21 EFFECT OF DOPAMINE AND APOMORPHINE ON CYCLIC AMP FORMATION Striatal slices were incubated for 60 min at 37° C in normal media with media changes every 20 min. Aliquots of tissue (15 mg) were added to incubation tubes containing IBMX (ImM) and varying concentrations of DA or apo and allowed to further in cubate for 15 min. Cyclic AMP content was measured by RIA. 99 % STIMULATION 200 100 150 50 . 03 1.0 0.3 0.1 CONCEN CONCEN Fig. 21 mM ■ ----- DA • APO ■ 100 FIGURE 22 EFFECT OF ETHANOL ON BASAL CYCLIC AMP FORMATION Striatal slices were incubated for 60 min at 37°C in normal media with media changes every 20 min. Aliquots of tissue (15 mg) were added to incubation tubes containing IBMX (ImM) and varying concentrations of ethanol and allowed to incu bate for an additional 15 min. Cyclic AMP content was mea sured by RIA. 101 pmoles cAMP/ mg protein ro oi *si o 0 1 o 0 1 o 1 — = — ^7 FIGURE 23 EFFECT OF ETHANOL ON DOPAMINE-STIMULATED CYCLIC AMP FORMATION Striatal slices were incubated for 60 min at 37°C in normal media with media changes every 20 min. Aliquots of tissue (15 mg) were added to incubation tubes containing IBMX (ImM) and varying conentrations of ethanol in the presence of ImM DA and allowed to further incubate for 15 min. Cyclic AMP content was measured by RIA. 103 CONTROL 200 150 100 50 —i H 0 0 0.17 M ETHANOL mM 1.7 i. 23Fig. 17.0 170 104 FIGURE 24 EFFECT OF PROSTAGLANDINS ON BASAL CYCLIC FORMATION Striatal slices were incubated for 60 min at 37°C in normal media with media changes every 20 min. Aliquots of tissue (15 mg) were added to incubation tubes containing IBMX (ImM) and varying concentrations of prostaglandins and allowed to further incubate for 15 min. Cyclic AMP content was measured by RIA. *p - 0.05 when compared to control group. 105 CONTROL 100 150 125 25 50 75 0 17 0 15 104M 105 106 107 108 OCN F PG OF CONCEN i. 24 Fig. 106 FIGURE 25 EFFECT OF PROSTAGLANDINS ON DOPAMINE-STIMULATED FORMATION OF CYCLIC AMP Striatal slices were incubated for 60 min at 37°C in normal media with media changes every 20 min. Aliquots of tissue (15 mg) were added to incubation tubes containing IBMX (ImM) and varying concentrations of prostaglandins in the presence of ImM DA and allowed to further incubate for an additional 15 min. Cyclic AMP content was measured by RIA. 107 CONTROL 100 125 25 50 75 8 OCN F PG OF CONCEN 10 r7 i. 25Fig. 10 6 r 0 1 PGE PGD 108 EFFECT OF PROSTAGLANDINS ON STRIATAL ACETLYCHOLINE RELEASE IN VITRO Neuroanatomical studies have shown that dopamine (DA) axons of the nigrostriatal pathway originate from cell bodies within the substantia nigra and course rostrally to terminate in the striatum (Moore and Bloom, 1978). DA nerve terminals in the striatum appear to innervate cholinergic interneurons at this site and inhibit their activity (Bar- tholini, 1980). This hypothesis is supported by the observations that systemically administered DA receptor agonists inhibit the release of of acetylcholine (Ach) in the striatum, while DA receptor antagonists, enhance Ach release (Bartholini, et al., 1976). In addition, it has been shown in vitro that DA, or the DA agonist, apomorphine (apo), can inhibit the depolarization-induced release of ^H-Ach from striatal slices (Stoof, et al., 1979). These data indicate that the activity of striatal cholinergic interneurons is inhibited by the activation of a DA receptor. Present evidence suggests that this DA receptor is different from that coupled to DA-sensitive adenylate cyclase and is a DA2 receptor (Euvrard, et al., 1979). Recently we showed that PGs can inhibit apo-induced circling in unilaterally lesioned mice (Schwarz, et al., 1981). This was the first evidence to suggest an action of PGs at sites that are postsynaptic to the DA synapse. Further investigation revealed that this inhibition of apo-induced circling was not due to an effect of the PGs on the DA receptor linked to adenylate cyclase (DA^ receptor) as shown by the failure of PGs to inhibit the DA-stimulated formation of cAMP. 109 110 Thus, the following experiments were designed to examine whether PGs can alter the release of ^H-Ach from striatal slices or reverse the inhibition by apo of depolarization-induced release of ^H-Ach. This functional assay would presumably test the activity of PGs at the the DA2 receptor within the striatum. METHODS 'A. In Vitro jjH-Acetylcholine Release in Mouse Striatal Slices. Male mice (Swiss-Webster, 20-30 gm) were stunned, decapitated, and the striatum was carefully dissected out. Cuboidal slices were cut (0.3mm x 0.3mm) on a Mcllwain tissue chopper and dispersed in ice- chilled Krebs-Ringer Hepes buffered media composed of: NaCl (119mM), KC1 (4.7mM), CaCl2 (1.2mM), KH2PO4 (1.2mM), MgS04 (1.2mM), Hepes (22mM), and glucose (10mM). The pH was adjusted to 7.4 with 3.0 M Tris. Following a quick centrifugation at 500 x g, the supernatant was dis carded and the slices incubated with ^H-choline (15 Ci/mmole; final concentration of lO-^M) in 10 ml for 15 min at 37°C. The slices were washed 3x with 5 ml media and then resuspended in a volume of media such that 0.2 ml aliquots of this suspension would be equivalent to at least 10 - 15 mg of tissue. The slices were incubated in a final vol ume of 3 ml in the presence or absence of 12.5mM K+ , apo (luM), or PG for 10 min at 37°C. In those experiments where apo was present, all samples contained 0.09uM ascorbic acid. The reaction was terminated by placing the samples on ice with the media being separated from the tissue by rapid centrifugation. The media was placed dirctly into scintillation vials while tissue was homogenized in 1 ml 0.4 N per chloric acid and 2 ml of H2O, centrifuged, and the resultant Ill supernatant placed in scintillation counting vials. Results were ex pressed as percentage of radioactivity in the media compared to total radioactivity of tissue plus media. As described previously (Stoof, et al., 1979; Hadhazy and Szerb, 1977), the depolarization-induced release of radioactivity from slices previously incubated with ^H-choline is a valid reflection of the release of ^H-Ach. Moreover, this release occurs by a Ca++-dependent process and gives further evidence that ves icular release of ^H-Ach is occuring (Mulder, et al., 1974). B . Statistics. Where appropriate, the data was analyzed by Student's t test or the Dunnett test for multiple comparisons. RESULTS A. Effect of Varying Kl. Concentrations on .^H-Ach Release. As seen in Fig. 26 during a 15 min incubation increasing amounts of K+ resulted in increasing amounts of 3R-Ach being released. Basal release was 18.2%, with lOmM, 12.5mM, 20mM, and 40mM K+ producing 31.9%, 36.6%, 57.7%, and 81.6% ^n-Ach release respectively. B . Effect of Ca** on Rjj-Stimulated Release of jfl-Ach. Depolarization-induced release from vesicular stores has been shown to require extracellular Ca++ (Rubin, 1973). K+ at a concen- of 12.5mM produced a greater release of ^H-Ach when the medium con tained 1.25mM Ca++ than in the absence of Ca++. At 1.25raM Ca++, the K+-stimulated release was 36% while at 0 Ca++, K+-stimulated release was only 6.7% (n = 4 for both groups; data not shown). C. Effect of Time of Incubation on Ki-Stimulated iLl-Ach Release. Fig. 27 shows that release by 12.5mM K+ of ^n-Ach appears to occur 112 in the first 10 min of incubation. A 5 min incubation resulted in a 9.7% increase of release over the basal level, while at 10 min there was a 17.8% increase and an 18.2% increase at 15 min. Basal, or con trol release, was 10.1%, 13.4%, and 18.2% for 5, 10, and 15 min respectively. Later experiments were run at the 10 min incubation period. D. Effect of Varying Apomorphine Concentrations of jjH-Ach Release. While concentrations of 10”^ - 10“^M, apo caused a slight decrease at 10~^M, but no concentration significantly alterated basal ^n-Ach release (Fig. 28). D. Effect of Apomorphine on Kl.-Stimulated Release of jH-Ach. In results similar to that of Stoof, et al., (1979) apo at con centrations of 10“7 - 10~5m inhibited the ability of K+ to stimulate the release of ^H-Ach from striatal slices. As see in the bottom of Fig. 29 the greatest inhibition occured at 10“^ M apo, with there being a 40% decrease. This dose was subsequently used for later PG experi ments . E. Effect of Prostaglandins on Basal jH-Ach Release. PGD2 , PGE2 , and PGF2C* were tested for their ability to alter basal 3 * Q release of H-Ach. As can be seen m Fig. 30 at concentrations of 10 - 10“^M, none of the PGs tested significantly altered basal ^H-Ach release from striatal slices. F. Effect of Prostaglandins on K—-Stimulated4* Release of £H-Ach. Q The addition of 12.5mM K+ resulted in a doubling in the amount of ^H-Ach released from striatal slices. In order to test whether PGs altered K+-stimulated release, varying concentration of PGs (10-®-10--’M) 113 were added to the incubation flasks (Fig. 31). Neither PGD2 , PGE2 , + ^ nor PGF2^. significantly altered the ability of K to increase H-Ach release. G. Effect of Prostaglandins on Apomorphine-Inhibition of Kjj-Stimulated Release. The addition of luM apo results in a decreased ability of K+ to stimulate ^H-Ach release (Fig. 32-34). PGs were tested for an ability to reverse the inhibition caused by apo, since this could explain the inhi bitory effect of PGs on circling produced by DA agonists. None of the PGs tested inhibited the ability of apo to decrease the K+-stimulated release of ^H-Ach. In fact, PGD2 at 10-^M significantly increased the decline of release produced by apo (Fig. 32). DISCUSSION Experimental evidence both in vivo and in vitro suggests that striatal cholinergic interneurons are under an inhibitory dopaminergic control (Bartholini, 1980). Since we have shown that PGs can inhibit circling produced by the DA agonist, apo, in unilaterally lesioned mice (Schwarz, et al., 1981), we wanted to test whether this inhibition by apo was a result of altering DA activity mediated through DA receptors located on striatal cholinergic interneurons. Presumably, these DA receptors appear to be of the DA2 type (non-adenylate cyclase linked) (Euvrard, et al., 1979). Our results in striatal slices from mice, are similar to those of Stoof, et al., (1979) in rats and Hertting, et-al., (1980) in rabbits. Depolarization of striatal slices resulted in the release of newly synthesized ^n-Ach with a concentration of 12.5mM K+ producing a 114 doubling of release of radioactivity (Fig. 26). Moreover, this release was a Ca++ dependent process which suggests that release was from vesi cular stores. When luM apo was added, there was an average 40% decrease in ^n-Ach released, an amount similar to that seen by Stoof, et al., (1979) in rats. These results in mice add additional evidence that striatal cholinergic neurons are under an inhibitory dopaminergic con trol. When PGs were tested for their ability to alter ^H-Ach release, neither PGD2 , PGE2 j nor PGF2oe significantly altered either basal or K+-stimulated release. In addition, PGs did not inhibit the ability of apo to reverse K+-stimulated release. The trend of the PGs was to increase apo's inhibitory effect with PGD2 significantly increasing this ability. It is difficult to reconcile this effect which resembles that of a DA agonist and the behavioral effects of the PGs which are to inhibit the effects of dopamine receptor stimulation. We can con clude however, that PGs do not modulate behavior mediated by DA at DA2 receptor sites which are linked to Ach release. There have been reports of PGs interacting with central choliner gic mechanisms. Bodzenta and Wisniewski, (1977) showed that intraven- tricularly administered PGEj and its precursor, dihomo-gamma-linolenic acid, increased the depressant action of eserine on locmotion. Unfor tunately the significance of this finding is not clear since PGE^ is not synthesized in the brain (Galli, 1978). Later, Grbovic and Radraanovic (1979) showed that PGE2 and PGF2ot inhibited acetylcholinesterase in some areas of the cat brain. This result was consistant with the ob servations of Poddubiuk and Kleinrok (1976) who reported that PGEj and PGF20C.increased the total brain concentrations of Ach in rats. Thus, biochemical studies may show that PGs interact with central cholinergic neurons, but this interaction does not explain the inhibi tion of the effects of DA receptor stimulation as shown in the behav ioral studies. FIGURE 26 EFFECT OF VARYING K+ CONCENTRATION ON 3H-ACH RELEASE Mouse, striatal slices were incubated with 3H-choline for 15 min, washed 3x, and then further incubated with varying K+ concentrations for 15 min. Radioactivity was measured in both tissue and media by scintillation counting and results are expressed as per cent radioactivity in media compared to that in tissue and media. 116 RELEASE 100 25 50 75 1 1. 2 40 20 12.5 10 C ■h M K+ mM i. 26Fig. 117 FIGURE 27 EFFECT OF TIME OF INCUBATION ON K+-STIMULATED 3H-ACH RELEASE Mouse, striatal slices were incubated with 3H-choline for 15 min, washed 3x, and then further incubated in media containing 12.5mM K+ for 5, 10, or 15 min. Radioactivity was measured in both tissue and media by scintillation counting and results are expressed as per cent radioactivity in media compared to that in tissue plus media. 118 c* RELEASE 20 10 * 0 15* 108 5* TIME i. 27Fig. 119 FIGURE 28 EFFECT OF VARYING APOMORPHINE CONCENTRATIONS ON 3H-ACH RELEASE Mouse, striatal slices were incubated with ^H-choline for 15 min, washed 3x, and then further incubated in normal media containing 10”? - 10~5m apomorphine for 15 min. Radioactivity was measured in both tissue and media by scintillation counting and results are expressed as per cent radioactivity in media compared to that in tissue plus media. 120 C 10 7 10® 10® M APOMORPHINE Fig. 28 FIGURE 29 EFFECT OF APOMORPHINE ON K+-STIMULATED RELEASE OF 3H-ACH Mouse, striatal slices were incubated with 3H-choline for 15 min, washed 3x, and then further incubated in media containing 12.5mM K+ and 10” ^ - 10”3M apomorphine for 15 min. Radioactivity was measured in both tissue and media by scintillation counting and results are expressed as per cent radioactivity in media compared to that in tissue plus media. *p = 0.05 when compared to control. 122 RELEASE 20 0 1 K+ to i. 29Fig. 7 APOMORPHINE 6 0 1 105M 123 FIGURE 30 EFFECT OF PROSTAGLANDINS ON BASAL 3H-ACH RELEASE Mouse, striatal slices were incubated with 3H-choline for 15 min, washed 3x, and then further incubated in normal media containing prostaglandins (10-® - 10“3M) for 15 min. Radio activity was measured both tissue and media by scintillation counting and results are expressed as per cent radioactivity in media compared to that in tissue plus media. 124 100 CONTROL 125 25 50 G CONCEN PG i. 30 Fig. 6 ~ PGD ♦ PGE * 125 FIGURE 31 EFFECT OF PROSTAGLANDINS ON K+-STIMULATED RELEASE OF 3H-ACH Mouse, striatal slices were incubated with 3H-choline for 15 min, washed 3x, and further incubated in media containing 12.5mM K+ and prostaglandins (10-3 - 10“3M) for 15 min. Radioactivity was measured in both tissue and media by scin tillation counting and results are expressed as per cent radioactivity in media compared to that in tissue plus media. 126 CONTROL 100 125 50 25 75 G CONCEN PG i. 31 Fig. PGD • PGE * 5 127 FIGURE 32 EFFECT OF PGD2 ON APOMORPHINE-INHIBITION OF K+-STIMULATED 3H-ACH RELEASE Mouse, striatal slices were incubated with 3H-choline for 15 min, washed 3x, and further incubated in media containing . _0 _ C 12.5mM K , luM apomorphine, and 10 - 10 M PGD2 for 15 min. Radioactivity was measured in both tissue and media by scintillation counting and results are expressed as per cent radioactivity in media compared to that in tissue and media. *p = 0.05 when compared to K+ + apo group. 128 RELEASE 20 10 rl- K apo + K+ i. 32 Fig. 108 io 7 PGD< 10 6 rh 105M 129 FIGURE 33 EFFECT OF PGE2 ON APOMORPHINE INHIBITION OF K+-STIMULATED 3H-ACH RELEASE Mouse, striatal slices were incubated with ^H-choline for 15 min, washed 3x, and further incubated in media containing 12.5mM K+, luM apomorphine, and 10~® - 10~3M PGE2 for 15 min. Radioactivity was measured in both tissue and media by scin tillation counting and results are expressed as per cent radioactivity in media compared to that in tissue and media. 130 RELEASE 20 10 ■ $ 0 1 1 1 M 10 10 10 108 $ K apo i. 33 Fig. e g p 2 131 FIGURE 34 EFFECT OF PGF2£* ON APOMORPHINE INHIBITION OF K+-STIMULATED 3H-ACH RELEASE Mouse, striatal slices were incubated with ^H-choline for 15 min, washed 3x, and further incubated in media containing 12.5mM K+ , luM apomorphine, and 10”® - 10”% PGF2otfor 15 min. Radioactivity was measured in both tissue and media by scintillation counting and results are expressed as per cent radioactivity in media compared to that in tissue and media. 132 RELEASE 20 10 ' • • K apo K* + i. 34Fig. 8 0 1 107 PGF 106 a 2 105M 133 134 SUMMARY The specific question examined in this thesis is: how do PGs modulate DA pathways which control motor function? In order to answer this question, studies were performed using behavioral as well as bio chemical techniques. If PGs play a physiological role in brain fucntion, one criterion which must be met is that PGs should be found in measureable amounts within the brain. The development of an RIA for PGs by a novel system, chicken egg yolk, provided the means to measure PGs within the brain. Abs generated by this system were highly specific and also allowed great sensitivity in PG measurement with endogenous PG levels in rat amd mouse whole brain found to be in the low picogram range. Thus, the use of RIA has shown that PGs are present in the brain. Since PGs are found within the brain, injection of exogenous PGs should alter behavior controlled by DA pathways. One behavior which has been shown to be dependent upon DA neurotransmission is amph-induced circling in rodents. PGD2 , PGE2 , and PGF201 all inhibited amph-induced circling in unilaterally lesioned mice when injected into either the CSF or directly into the striatum with the order of potency being: PGE2 > PGD2 > PGF2«. The failure of injections of intraventricular TxB2 or intrastriatal 13, 14-dihydro-15-keto-PGE2 (PGE2 metabolite) at doses which the major PGs inhibited circling, plus the difference in potency, suggests that there is a specificity of action by the PGs. The inhibition of circling was not a nonspecific effect related to the fatty acid nature of these compounds. In addition, inhibition of 135 circling was not due to alterations in body temperature caused by the PGs. These results, together with the observation that PGs are found in striatal tissue, support the concept that PGs in the striatum func tion to modulate behavior regulated by DA-mediated neurotransmission. Since one role suggested for PGs in the CNS is the control of neurotransmitter release, it seemed possible that the inhibition of amph-induced circling might be due to PG alteration of DA release or DA synthesis. Using an in vitro slice preparation, it was seen that PGE2 (10~^M) and PGF2et(10-^ and 10-7M) significantly decreased K+- 3 + stimulated H-DA release, while PGD2 increased K -stimulated release (10_7M). Only PGF2flC (10-7M) significantly decreased the ability of K+ to stimulate DA synthesis. While the ability of PGE2 and PGF2 ej to inhibit amph-induced circling may in part be due to their inhibi tion of release and synthesis, the effect of PGD2 can not be explain ed by alteration DA release or synthesis. These results, in addition to those already in the literature, suggest that PGs may only at best, weakly modulate DA function by presynaptic control of release and synthesis. Another explanation for PG inhibition of amph-induced circling, is that the PGs are acting at sites postsynaptic to the DA synapse. Direct acting agents, such as apo cause circling in unilaterally lesioned animals by direct stimulation of the DA receptor. Injection directly into the CSF or striatum of PGD2 , PGE2> or PGF2ot inhibited apo-induced circling while the PGE2 metabolite failed to inhibit circling at a similar dose. The ability to block apo-induced 136 circling suggests that PGs are acting at sites postsynaptic to the DA synapse. Pretreatment with indomethacin, a PG synthesis inhibitor, significantly increased apo-induced circling. These results support the hypothesis that endogenous PGs modulate motor function regulated by DA receptor stimulation. Recent evidence has suggested that the DA^ receptor is linked to adenylate cyclase with occupation of the receptor resulting in the subsequent formation of cAMP. Since PGs alter cyclic nucleotides in a variety of systems, it was possible that the inhibition of apo- induced circling was due to the inhibition of DA-stimulated formation of cAMP at DAj receptors. PGD2 , PGE2> and PGF2 at concentrations of 10"8 - 10"4M failed to inhibit the DA-stimulated formation of cAMP. In fact, both PGE2 and PGF2 at 10 significantly increased the formation of cAMP. Thus, it does not appear that the PG inhibition of apo-circling is due to an inhibition of DA-stimulated formation of cAMP. Many pharmacololgical effects within the striatum appear to be the resulc of alterations occuring at DA2 receptors. These receptors are not linked to adenylate cyclase. It has been shown that DA nerve terminals in the striatum innervate cholinergic interneurons and inhibit their activity. Apo can inhibit the depolarization-in duced release of 8H-Ach from striatal slices _in vitro. 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