MECHANISMS OF LEVODOPA ACTION IN THE CENTRAL NERVOUS SYSTEM Dissertation for'the Degree of Ph. D. MICHIGAN STATE UNIVERSIITY DAVID BUELL GDDDALE 1976 This is to certify that the thesis entitled MECHANISMS OF LEVODOPA ACTION IN THE CENTRAL NERVOUS SYSTEM presented by David Buell Goodale has been accepted towards fulfillment of the requirements for Ph.D. Pharmacology degree in KC??? Major professor Dan: February 26, 1976 0-7 639 ABSTRACT MECHANISMS OF LEVODOPA ACTION IN THE CENTRAL NERVOUS SYSTEM By David Buell Goodale L-Dihydroxyphenylalanine (L-Dopa) is presently the most effica- cious agent available for the treatment of Parkinson's disease. The antiparkinsonian action of this compound has been attributed to its ability to replenish the diminished concentrations of dopamine in the basal ganglia of patients with this disease. The objective of this study was to determine if the actions of L-dopa are totally dependent upon the conversion to dapamine, or if L-dopa pgr_§g_has an independent action. This was tested by comparing the actions of L-dopa to those of D-dopa, which is not metabolized to dopamine in the central nervous system. Analysis of the behavioral actions of D- and L-dopa were conducted in a simple model in which direct and indirect dopaminergic agonists can be differentiated. One week after the unilateral intrastriatal injection of 6-hydroxydopamine, which selectively destroys dopaminergic nerve terminals on the injected side, mice circle spontaneously toward the side of the lesion (ipsilateral circling). Indirect dopaminergic agonists increased the rate of ipsilateral circling. Administration of a David Buell Goodale direct-acting dopaminergic agonist (e.g. apomorphine) causes the mice to reverse the direction of circling towards the nonlesioned side (contralateral circling). Intraperitoneal administration of both L- and D-dopa elicited contralateral circling, but the L-isomer was approximately ten times more potent than that of D-isomer. Optical rotation analysis revealed that samples of D-dopa were not contaminated with L-dopa. Following the administration of doses of L- and D-dopa which produced approximately equal numbers of contralateral turns per two minutes (l0 and lOO mg/kg, respectively), the cerebral concentration of both isomers of dopa was approximately the same but the accumulation of dopamine occurred only after the administration of L-dopa. This experiment suggested that dopa may have direct dopaminergic receptor activating properties. The results of subsequent studies with de- carboxylase inhibitors revealed that this proposal was incorrect. Hydrazinomethyldopa, which blocks only peripheral aromatic-L- amino acid decarboxylase (AAAD) prolonged and enhanced the actions of both L- and D-dopa. Ro44602, an inhibitor of both peripheral and central AAAD activity, initially blocked and then enhanced the contralateral circling in response to both isomers of dopa. NSD lOlS, which also inhibits both central and peripheral AAAD, completely blocked contralateral circling in response to both L- and D-dopa, but not to apomorphine. In order to determine if a correlation exists between periods of active contralateral circling and increase cerebral conversion David Buell Goodale of 3H-dopa to 3H-dopamine, intravenous 3H-L-dopa was injected at selected time intervals following the administration of various AAAD inhibitors. During periods of increased L-dopa-induced contra- lateral circling in hydrazinomethyldopa or Ro44602 pretreated mice, there were concurrent increases in the concentrations of 3H-dopamine in the brain. When contralateral circling to dopa was blocked, following pretreatment with Ro44602 or NSD l0l5, the 3H-dopamine concentrations were significantly less than control. These results indicate that the administration of the centrally-active AAAD inhibitors effectively block jn_yjyg_conversion of dopa to dopamine and there is a correlation between the number of contralateral turns/2 minutes and the increase in brain dopamine. The ability of the AAAD inhibitors to block contralateral circling induced by both D- and L-dopa suggest that these compounds must be converted to dopamine before the dopaminergic receptors are activated. It has been reported that D-Dopa can be converted to L-dopa in the kidney. It is suggested that in the presence of an AAAD inhibitor L-dopa synthesized in the kidney from D-d0pa is reabsorbed and transported to the brain where small quantities are converted to d0pamine, which in turn activate the supersensitive dopaminergic receptors. It cannot be excluded that some undefined biochemical action of D- dopa plays some role in the D-dopa-induced contralateral circling. Systemic administration of L-dopa depletes the brain content of 5-hydrothryptamine (5-HT). The abilities of L- and D-dopa to deplete forebrain 5-HT were compared to ascertain whether d0pamine formation was essential for the depletion of 5-HT. Administration David Buell Goodale of L-dopa increased forebrain dopa and dopamine concentrations with a simultaneous reduction in the concentration of 5-HT. A ten fold higher dose of D-dopa resulted in smaller increases in dopa and dopamine concentrations than observed with L-dopa. The 5-HT concentration was significantly reduced only by the highest dose of D-dopa. The depletion of forebrain S-HT was better correlated with the increase in the dopamine concentration than with accumulation of cerebral dopa. L-Dopa-induced reduction of forebrain 5-HT was studied in mice with unilateral lesions of the dopaminergic nerve terminals. 6- Hydroxydopamine injection into the striatum markedly reduced the dopamine content without altering the 5-HT or dopa concentrations. In the nonlesioned hemiforebrain, L-dopa administration caused a dose-related increase in the concentration of dopa and dopamine with a concomitant reduction in the content of 5-HT. The only significant difference between control and lesioned hemiforebrains was a reduction of the dopamine accumulation on the lesioned side. These results support the contention that the depletion of 5—HT occurs independent of dopaminergic nerve terminals. MECHANISMS OF LEVODOPA ACTION IN THE CENTRAL NERVOUS SYSTEM By David Buell Goodale A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology T976 to my parents, for their inspirational faith, love, and guidance ii ACKNOWLEDGMENTS The author extends his deep appreciation to Dr. Kenneth E. Moore for his continued advice and encouragement during the course of this study. He acknowledges the constructive advice and criticism of Drs. Theodore M. Brody, Gerard L. Gebber, Richard H. Rech, John E. Thornburg, and Ralph A. Fax during the preparation of this disserta- tion. He is grateful for the collaborative efforts generously extended by Drs. Douglas E. Rickert and William H. Reusch in the studies concerning enantiomeric purity. He wishes to thank Mrs. Susan Stahl and Mrs. Mirdza Gramatins for their excellent technical assistance and many fond memories. iii TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES. INTRODUCTION. HISTORICAL BACKGROUND. LITERATURE REVIEW . A. Dopamine as a neurotransmitter for the nigrostriatal pathway. . L- -Dopa as a dopamine replenishing agent I . . . Evidence that L- -dopa acts independently of dopamine formation . L- -Dopa and displacement of brain 5- -hydroxytryptamine. 1. Competition for aromatic-L-amino acid decarboxy- lase . . . 2. Displacement of 5- hydroxytryptamine from storage granules by dopamine. . 3. Competition between L- dopa and L- -tryptophan for uptake into the brain . . D-Dopa literature review . l. Transport across the blood-brain-barrier. 2. Metabolism in the body. . Aromatic-L-amino acid decarboxylase (AAAD) . . l. Characterization. . . 2. Pharmacological inhibition . Turnover of 5- hydroxytryptamine in 6-hydroxydopamine- lesioned animals . . Utility of 5, 7- dihydroxytryptamine for producing . selective depletion of brain 5- hydroxytryptamine iv Page vii viii 12 22 24 24 24 28 30 3O 31 34 34 36 37 40 Page MATERIALS AND METHODS. . . . . . . . . . . . . 42 A. Methods. . . . . . . . . . . . . . . . 42 1. Mouse turning behavioral studies . . . . . . 42 2. Biochemical procedures. . . . . . . . . . 45 a. Analysis of dopa, dopamine, and S—hydroxy- tryptamine following ion-exchange chroma- tographic separation. . . . . . . . . 45 b. Separation of H-dopa and metabolites. . . . 5T c. Analysis of 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxytryptamine (5-HT), norepinephrine and dopamine following or- ganic extraction . . . . . . . . . . 52 3. Statistical analysis. .‘ . . . . . . . . . 55 a. Biochemical experiments. . . . . . . . . 55 b. Turnover rate comparison . . . . . . . . 55 c. Behavioral studies . . . . . . . . . . 55 B. Materials . . . . . . . . . . . . . . . 56 l. Buffers. . . . . . . . . . . . . . . 56 2. Reagents . . . . . . . . . . . . . . 57 3. Standards . . . . . . . . . . . . . . 58 4. Resin preparation . . . . . . . . . . . 59 5. Alumina purification . . . . . . . . . . 59 6. Drugs . . . . . . . . . . . . . . . 60 RESULTS . .. . . . . . . . . . . . . . . . 62 A. Behavioral rotation studies . . . . . . . . . 62 1. Dose response relationship between D- and L-dopa 62 2. Central decarboxylase inhibition and dopa- induced contralateral circling . . . . . . 66 a. Ro44602. . . . . . . . . . . . . . 66 b. NSD lOl5 . . . . . . . . . . . . . 69 3. Peripheral decarboxylase inhibition . . . . . 72 a. Hydrazinomethyldopa (HMD). . . . . . . . 72 b. NSD l055 (brocresine) . . . . . . . . . 72 B. Resolution of enantiomeric purity of D- and L-dopa. . 75 C. Biochemical studies . 1. 3H-L-Dopa metabolism. 2. D-Dopa metabolism in brain. 3. Depletion of 5—hydroxytryptamine. a. L-Dopa in 6-hydroxydopamine-lesioned mice. b. Comparison of D- and L-dopa 4. Turnover of 5-hydroxytryptamine in mice with unilateral 6-hydroxydopamine lesions of the striatum . . . . . . . 5. Utility of 5,7-dihydroxytryptamine for pro- ducing selective depletion of brain 5-HT DISCUSSION. A. D- -Dopa induced contralateral circling . B. Enantiomeric purity of D- and L- -dopa . . . C. Endogenous dopamine concentrations following L- or D- -dopa. . . . D. Peripheral decarboxylase inhibition and dopa E. Central decarboxylase inhibition and dopa induced . F. Conclusions on the dopaminergic receptor agonistic I‘D induced circling contralateral circling pr0perties of L- and D- dopa. . . . Depletion of 5- hydroxytryptamine by L- dopa in 6- hydroxydopamine- -lesioned mice . . Comparison of 5- hydroxytryptamine depletion by D- . and L-dopa . 5- Hydroxytryptamine turnover in 6- hydroxydopamine- lesioned animals J. Utility of 5, 7- dihydroxytryptamine for producing selective degeneration of serotonergic neurons . Prospective view into the mechanism of action of L-dopa. . BIBLIOGRAPHY . vi Page 78 78 82 82 82 85 9O 96 99 99 101 102 103 107 112 114 116 117 118 119 123 LIST OF TABLES Table Page l. Effects of NSD l055 (brocresine) on the contra- lateral circling behavior induced by L- and D—dopa . . . . . . . . . . . . . . . 76 2. Specific rotation of different samples of L- and D-dopa .. . . . . . . . . . . . . 77 3. Effects of decarboxylase inhibitors on the brain contents of 3H-dopa and 3H-dopamine following systemic administration of 3H-L-d0pa . . . . . 79 4. Effects of left intrastriatal injection of 5,7- dihydroxytryptamine on forebrain monoamine concentration. . . . . . . . . . . . . 97 vii LIST OF FIGURES Figure 1. 10. 11. Pathway for synthesis of dopamine and 5- hydroxytryptamine . Relationship of postsynaptic supersensitivity to the contralateral circling induced by apomorphine or L-dopa in mice with unilateral 6-hydroxy- dopamine lesions of the corpus striatum. Schematic representation of the theoretical mechanism by which cyclic AMP mediates dopaminer- gic transmission . Diagrammatic representation of the various metabolic pathways through which L—dopa may induce diffe- rent biochemical and behavioral effects. Major metabolic pathway of D-dopa in rat kidney. Sagittal view of the apparatus for making injections into the corpus striatum of the mouse brain Procedure for the analysis of dopa, dopamine and 5- hydroxytryptamine (5- HT) following ion- exchange chromatography. . . Procedure for the analysis of 5-hydroxyindoleacetic acid (5-HIAA) and 5-hydroxytryptamine (5-HT), norepinephrine and dopamine following organic extraction . . . . . Effects of L- and D-dopa on circling behavior in mice with unilateral 6-hydroxydopamine lesions Effects of peripheral and central aromatic-L-amino acid decarboxylase (AAAD) inhibition by Ro44602 on the contralateral circling behavior induced by L- and D-dopa Effects of peripheral and central aromatic-L-amino acid decarboxylase (AAAD) inhibition by NSD l0l5 on the contralateral circling behavior induced by L-dopa, apomorphine and D-dopa. viii Page 15 19 26 33 44 47 53 65 68 71 Figure 12. 13. 14. 15. 16. 17. Effects of peripheral aromatic-L-amino acid decarboxylase (AAAD) inhibition by hydrazino- methyldopa (HMD) on the contralateral circling behavior induced by L- and D-dopa . . . Effects of L- and D-dopa administration on the endogenous concentrations of dopa and dopamine in mouse forebrain . . . . . Comparison of L- -dopa- -induced depletion of 5- hydroxytryptamine (5- HT) in 6- -hydroxydopamine lesioned and control hemiforebrains . Comparison of high doses of L- and D-dopa on 5- hydroxytryptamine (5-HT) depletion in mouse forebrain . . . . . . . . . . . . 5-Hydroxytryptamine (5-HT) turnover in 6-hydroxy- dopamine-lesioned and control hemiforebrains 5-Hydroxytryptamine (5-HT) turnover in 6-hydroxy— dopamine-lesioned and control hemiforebrains following the administration of L-dopa ix Page 74 84 87 89 92 95 INTRODUCTION Over the last decade L-3,4-dihydroxyphenylalanine (L-dopa) has achieved prominence as the drug of choice in treating patients with Parkinson's disease (Hornykiewicz, T974, T975). The first clinical demonstration of the therapeutic efficacy of L-dopa was made indepen- dently by two groups of investigators, Birkmayer §t_al, (l96l) and Barbeau 95.91: (1961). The clinical efficacy of this amino acid was attributed to its ability to be decarboxylated to dopamine in the brain, thereby replenishing the diminished levels of this amine within the basal ganglia. At present most patients treated with L-dopa experience side effects and therefore this drug may not be viewed as an ideal thera- peutic agent. The purpose of this dissertation was to examine in detail various alternative mechanisms through which L-dopa might exert its pharmacological actions within the central nervous system. The major question which this dissertation has attempted to answer is whether or not L-dopa must be decarboxylated to d0pamine in order to produce its neurochemical and behavioral effects. The application of L-dopa to the treatment of parkinsonism is an excellent example of a therapeutic advance occurring as the direct result of basic research providing insight into neuropharma- cological mechanisms. A presentation of the historical development 2 of L-dopa as an effective antiparkinsonian agent will serve as a proper antecedent to a discussion on alternative mechanisms of action. HISTORICAL BACKGROUND James Parkinson (1817) in one of the classic papers of medical history, first recognized that certain neurological features were common to one disorder which he called shaking palsy or paralysis agitans. He described the disease as characterized by: involuntary tremulous motion with lessened musular power, in parts not in action and even when supported, with a propensity to bend the trunk for- ward, and to pass from a walking to a running pace; the senses and intellects being uninjured." Since tremor need not be present and true paralysis is not a con- sistent manifestation of this disease, Charcot in the middle of the nineteenth century proposed that paralysis agitans was a misnomer and that the disorder should be known as Parkinson's disease. Throughout this dissertation the term parkinsonism will be used in the broad sense to encompass a clinical syndrome composed of four features: tremor, rigidity, akinesia, and loss of postural re- flexes. After Parkinson's original description of the disorder, one century elapsed before a correlation was made between the neurolo- gical deficits and specific pathological changes within the brain. Trétiakoff (T919) studied the brain of nine patients with parkinson- ism. He found that there was a degeneration and reduction in the number of pigmented cells in the substantia nigra, together with cytoplasmic inclusions in some of the cells. Recently Bernheimer §t_ 4 a1, (1973) have confirmed and extended the original findings of Trétiakoff by comparing the neuropathological changes in the three major types of parkinsonism: postencephalitic, idiopathic (degene- rative), and arteriosclerotic (senile). No major qualitative differences in the morphological changes within the substantia nigra were found among these three types of parkinsonism. Following the suggestion by Trétiakoff that the substantia nigra was abnormal in parkinsonism, it was not until the mid-19505 that the functional significance of dopamine was appreciated. Prior to this time it had been considered simply a precursor to norepine- phrine. Carlsson (1957) suggested that the central effects of reserpine might be explained on the basis of dopamine depletion since the precursor DL-dopa dramatically antagonized the reserpine- induced behavioral depression. Subsequently, Carlsson (1958) de- veloped a fluorometric procedure for assaying dopamine which was much more sensitive and specific than previous procedures. Using this method he found the brain content of d0pamine and norepinephrine to be almost equivalent. The following year the regional distribu- tion of these two amines was found to be markedly different (Carlsson, 1959; Bertler and Rosengren, l959a,b). The striatum contained approximately 10 ug/g of dopamine and only 0.1 ug/g of norepinephrine, thus suggesting that the function of dopamine was not merely that of a precursor. In 1960 Ehringer and Hornykiewicz studied the brain content of dopamine and norepinephrine in patients with various neurological disorders. They found that in patients with Parkinson's disease 5 there was a marked reduction in the usually high levels of dopamine in the corpus striatum. This was not only direct evidence for a physiological role played by cerebral dopamine, but also suggested that the diminished levels of dopamine were causative in the genesis of the parkinsonian syndrome. Since it had been demonstrated that catecholamines would not effectively penetrate the blood-brain—barrier (Axelrod et_al,, 1959; Neil-Malherbe gt_al,, 1961; Samorajski and Marks, 1962), the immediate metabolic precursor of dopamine, L-dopa, became the logical candidate for clinical trials. In 1961 both Birkmayer and Hornykiewicz, and Barbeau §t_al, independently administered L-dopa intravenously and demonstrated a marked reduction in rigidity and akinesia with little or no effect on tremor. Cotzias §t_al, (1967, 1969), by gradually increasing and ultimately using large oral doses, demonstrated that L-dopa could be successfully used in the clinics as an antiparkinsonian agent. Further improvement of the therapeutic regimen was accomplished by inhibition of the peripheral route of L-dopa metabolism. In addition to 3'-O-methylation, the decarboxylation of L-dopa to dopamine has been shown to be a major route of metabolism both in the brain and in the periphery by Bartholini et_al, (1967), and Baldessarini and Chace (1972). The conversion of L-dopa to dopamine, known to be mediated by the enzyme arbmatic—L-amino decarboxylase (AAAD, EC 4.1.1.26) (Blaschko, 1939; Sourkes, 1966; figure 1), was demonstrated to be selectively inhibited in the periphery by drugs which did not penetrate the blood-brain-barrier such as: .mcwsmuaxguxxoccxc1m ucm mcwsmaou mo mwmmgucmm mg“ com szsqu .P mcamwu Ap:.nv uzifiififioS»: -o. z «121“:U1w; O: «zzgmzva: 2:15.09 2.3.9..— 1... <§14 ”mupx0¢o>z Zita—0.5:..— mm<4>x0~3>x 9.3.9.5. 25:02:: . ._ «321101f0? 2.305;... £21: :OOW1 1“: zoom 7 hydrazinomethyldopa (MK-486, HMD) or low doses ( AAAD DOPAMINE D_DOPA ....'l.é....> wo—tvrnnmx: mm>rn—CH2C|3HCOOH (D-DOPA) 2 Ola—amino acid oxidase HO-<:>-CH2CCOOH (DHPP) I transaminase HO \— HO—<_:>—CH2?HCOOH (rs-DOPA) (aromatic L-amino acid decarboxylase HO \— HO—< >—CH2CH2NH2 (dopamine) Figure 5. Major metabolic pathway of D-dopa in rat kidney (modified after Shindo and Maeda, 1974). 34 (500 mg/kg) no significant increase was seen in the endogenous brain dopamine content. Therefore, studies for this dissertation originally included D-dopa for the purposes of comparison to L-dopa, since the D- isomers had previously been considered not to be converted to dopamine. Thus, any similar behavioral or biochemical actions between L- and D-dopa would be considered indicative of dopa acting directly and not through a prerequisite conversion to dopamine. F. Aromatic-L-amino acid decarboxylase (AAAD) 1. Characterization In 1938 Holtz and coworkers discovered an enzyme in guinea pig kidney which converted dopa to hydroxytyramine and C02. The enzyme was subsequently referred to as aromatic-L-amino acid decarboxylase because it catalyzed the decarboxylation of many aromatic amino acids such as: L-dopa, tyrosine, 5-HTP, tryptophan and phenylalanine (Lovenberg, 1962). The stereospecificity of this enzymatic reaction was originally determined by measuring manometri- cally the C02 produced during incubation of d(+) and d(-) dopa with the enzyme (Blascko, 1942). Using purified enzyme preparations the stereospecificity of AAAD has been confirmed by determining spectro- fluorometrically (Lovenberg et_al,, 1962) or radiometrically (Sims gt_al,, 1973) the amount of dopamine formed during incubation of the enzyme with D- or L-dopa. Thus, it has been established that AAAD will catalyze the conversion of L-dopa to dopamine but will not convert D-dopa to dopamine. 35 Originally dopa decarboxylase (EC 1.1.1.26) and 5—HTP decar- boxylase (EC 1.1.1.27) were considered to be distinct enzymes (Clark gt_al,, 1954). More recently, Christenson et_al, (1972) have reported that cerebral dopa and 5-HTP decarboxylase enzymes were inhibited by antibodies against purified kidney AAAD. These authors suggested that one protein catalyzed the decarboxylation of both dopa and 5-HTP. However Sims §t_al, (1973) have demonstrated that dopa and 5-HTP decarboxylase activities have widely different kinetic optima for pH, temperature and substrate concentration. Furthermore, intraventricular 6-hydroxydopamine reduced dopa decar- boxylase without affecting 5-HTP decarboxylase activity (Sims and Bloom, 1973). However, Dairman gt_al, (1975) using 6-hydroxydopamine and 5,6-dihydroxytryptamine could not demonstrate a preferential decrease in either dopa or 5-HTP decarboxylase. Therefore, the question of whether dopa decarboxylase and 5-HTP decarboxylase represent distinct enzymes has not been completely resolved. Cerebral AAAD has been characterized as a "cytoplasmic" enzyme (Sims gt_al,, 1973) with the highest regional activity in the corpus striatum (Lloyd and Hornykiewicz, 1970b). The endothelium of cerebral capillaries has also been demonstrated to contain a substantial amount of decarboxylase activity (Bertler et_al,, 1966; Constantinidis, 1968; de la Torre, 1973). This decarboxylase activity may serve as an enzymatic blood-brain-barrier converting L-dopa to dopamine which does not penetrate into the brain parenchyma (Bertler, 1966). 36 l. Pharmacological inhibition Prior to the 19605 the best available inhibitors of AAAD were structural derivatives of cinnamic acid (Clark, 1959). Sub- sequently, the benzylhydrazines and benzoxyamines were demonstrated to be more potent inhibitors of decarboxylase activity jn_yjyg_ (Burkard gt_al., 1962; Hansson gt_al,, 1964). Ro44602, a benzyl- hydrazine, was demonstrated to inhibit the decarboxylase activity in the brain as well as in the periphery (Burkard gt_al,, 1962). Since these initial studies, the Ro44602-induced inhibition of cerebral AAAD has been more completely characterized than other AAAD inhibitors. The inhibition of Ro44602 was first shown to be maximal in the brain 30 minutes after intraperitoneal administration (Burkard et_ 21,, 1964). Subsequently, the decarboxylase activity slowly re- covered and at three hours after administration was approximately 40% of control activity. Small doses of Ro44602 (50 mg/kg) were shown to preferentially inactivate the peripheral decarboxylase activity (Bartholini, 1967). The larger doses of Ro44602 (100, 200, and 300 mg/kg) caused a dose related inhibition of decarboxylase to 75, 45 and 20% of control activity, respectively (Bartholini, 1967). The con- current inhibition of extracerebral decarboxylase and administration 14C—L-dopa resulted in a marked elevation of 14C-dopamine in the of brain without elevation of catecholamines in extracerebral tissues (Bartholini and Pletscher, 1968, 1969). Thus, the utility of 37 Ro44602 for inhibiting AAAD in both the peripheral and central nervous system was well established in the previous studies. In addition to inhibition of AAAD activity, Ro44602 has been demonstrated to inhibit catechol-O-methy1transferase (Burkard gt gl,, 1964; Baldessarini and Chace, 1972). Another action of Ro44602 was shown to be a small 20% reduction of endogenous norepinephrine and 5-HT content when administered at 700 mg/kg (Pletscher and Gey, 1963). Behaviorally, Ro44602 was demonstrated to not inhibit spon- taneous locomotor activity (Pletscher _t_gl,, 1964). However, when Ro44602 was used in combination with L-dopa there was a stimulation locomotor activity at low doses of Ro44602 (50 mg/kg) Bartholini gt gl,, 1968; Thut, 1970; Lotti and Porter, 1970). At higher doses (500 mg/kg) Ro44602 inhibited the locomotor stimulant actions of L- dopa (Bartholini gt_gl,, 1968; Lotti and Porter, 1970). Thus, Ro44602 is a potent inhibitor of cerebral AAAD and a valuable tool in studying the mechanism of action of L-dopa. G. Turnover of 5-hydroxytryptamine in 6-hydroxydopamine- lesioned animals An interaction, either direct or indirect, has been proposed between catecholamine and serotonergic neuronal systems (Blondaux gt_gl,, 1973). These investigators demonstrated that one week after the intracisternal administration of 6-hydroxydopamine to rats the 5-HT turnover rate was increased when compared to control rats. Peters gt_gl, (1974) have confirmed this finding following 38 the administration of 6-hydroxydopamine to newborn rats. This treatment resulted in an increased rate of 5-HT synthesis when brains from the adult rats were analyzed. 0n the other hand, Hery _t;gl, (1973) have reported that 6-hydroxydopamine pretreatment reduced the formation of 3H-5-HT during the light period and stimu- lated labeled 5-HT synthesis during the dark phase. In the studies by Blondaux gt_gl, (1973) and Hery gt_gl, (1973) 6-hydroxydopamine was administered into the cerebrospinal fluid and this route of administration has been shown to cause a greater degeneration of noradrenergic neurons than dopaminergic neurons (Bloom gt_gl,, 1969; Bell gt_gl,, 1970). Thus, no clear differentiation was made in the previous studies for which catecholamine neurons (noradre- nergic or dopaminergic) are mediating the alterations in 5-HT synthesis rates. Interneuronal regulation of 5-HT metabolism by catecholamine neurons has been demonstrated in only one nervous system area: the pineal gland (Brownstein, 1975). The activity of serotonin N- acetyltransferase, which converts 5-HT to melatonin in the rat pineal gland, was elevated after the administration of L-dopa (Deguchi and Axelrod, 1972). Furthermore, when the noradrenergic fibers innervating the pineal gland were severed chronically, L- dopa induced much more serotonin N-acetyltransferase than in the innervated pineal gland. Although subsequent studies have deter- mined that norepinephrine is controlling the serotonin N-acetyl- transferase activity (Deguchi and Axelrod, 1973a,b), no experimental 39 evidence is available demonstrating that a dopaminergic neuronal system controls 5-HT synthesis rate. The synthesis rate of 5-HT has been studied in the central nervous system as an indication of the functional status of the neuron. The turnover rate assumes that a steady state exists where the synthesis and transport of 5-HT into a metabolic pool equals its release and degradation (Tozer gt g1,, 1966). Several methods have been utilized to estimate the turnover rate of 5-HT by measur- ing: the initial accumulation (Millard gt_gl,, 1972) or subsequent decline (Neff gt§_l_., 1971) of ‘4 14 C—5-HT following an intravenous injection of C-tryptophan; the rate of 5-HIAA accumulation follow- ing administration of probenecid; the rate of 5-HT accumulation or 5-HIAA decline after inhibition of monoamine oxidase (Tozer gt_gl,, 1966); and most recently the accumulation of 5-HTP after decarboxy- lase inhibition (Carlsson gt_gl,, 1972). Experiments concerning 5- HT turnover in this dissertation have utilized the method of inhi- biting the metabolism of 5-HT through monoamine oxidase and quanti- fying the initial accumulation of 5-HT. Turnover studies have indicated that there is no end product inhibition of 5-HT synthesis at the tryptophan hydroxylase step (Lin gt_gl,, 1969a; Millard gt g1,, 1972. Thus, the inhibition of monoamine oxidase by pargyline administration has been demonstrated to be a reliable method for determining 5-HT synthesis rates jg_vivo (Morot-Gaudry, 1974). 40 H. Utility of 5,7-dihydroxytryptamine for producing selective depletion of brain 5-hydroxytryptamine Recently, 5,6-dihydroxytryptamine has been introduced as a pharmacological tool for producing selective degeneration of 5-HT- containing neurons in the brains of mammals (Baumgarten gt_gl,, 1971, 1972; Costa gt_gl,, 1972; Daly gt_gl,, 1973; Victor gt_gl,, 1974). However, the reduction of forebrain 5-HT was moderate and was accompanied by limited nonspecific tissue damage and degenera- tion of dopaminergic neurons close to the site of injection (Baum- garten and Schlossberger, 1973; Daly gt_gl,, 1973; Saner gt gl,, 1974). Subsequently, 5,7-dihydroxytryptamine has been studied and found to be neurotoxic to 5-HT neurons with some degeneration of noradrenergic neurons and little nonspecific damage to brain tissue (Daly gt_gl,, 1974; Baumgarten and Lachenmayer, 1972; ijrklund gt 21:: 1973; Baumgarten gt_gl,, 1973, 1975; Gershon and Baldessarini, 1974). Due to the lack of nonspecific tissue damage higher doses of 5,6-dihydroxytryptamine could be administered and therefore it was considered a potentially better drug for producing profound depletions of 5-HT. In contrast to the lack of regeneration by catecholamine neurons destroyed with 6-hydroxydopamine (Thoenen and Tranzer, 1973), 5-HT neurons regenerate following 5,6-dihydroxy- tryptamine (Baumgarten gt_gl,, 1974) and noradrenergic neurons regenerate following 5,7-dihydroxytryptamine (Bjdrklund gt_gl,, 1975). 41 Studies were designed to investigate the specificity of 5,7- dihydroxytryptamine after unilateral injection into the corpus striatum of the mouse brain. Following demonstration of the selective degeneration of 5-HT neurons, this experimental model would be further utilized to determine the role of 5-HT neurons in the mechanism of action of L-dopa. MATERIALS AND METHODS A. Methods 1. Mouse turning behavioral studies Male Swiss Webster mice (18-20 grams) obtained from Spartan Research Animal, Inc. (Haslett, Michigan) were used through- out these experiments. Groups of twelve mice were maintained in clear Plexiglass cages (45x12x13 cm). In addition to water and solid Lab Blox food (Allied Milk, Inc., Chicago, Illinois) the diets were supplemented with Sego (Very Chocolate) liquid diet food (Pet Inc., St. Louis, Missouri). The injection of selective cytotoxic agents into the striatum of the mouse brain was performed as described previously by Von Voigtlander and Moore (1973b). Following induction of anesthesia with methoxyflurane, the head of a mouse was placed in a plastic mold (see figure 6). Four microliters of redistilled water containing 0.8 micrograms (pg) sodium ascorbate and 10, 25, 50, or 100 ug 5,7-dihydroxytryptamine creatinine sulfate or 16 pg 6— hydroxydopamine were injected slowly over a 60-90 second time period. Since an inverse correlation has been demonstrated between apormophine-induced contralateral circling and depletion of striatal dopamine (Thornburg and Moore, 1975), the extent of dopamine de- pletion following 6-hydroxydopamine lesions was estimated seven days later by the subcutaneous injection of apomorphine (0.25 42 43 .:o_e;amo:mFmp mmzoe on» we Ezpmwgum mzacoo we“ mpcmmmcamc amen xmem mgp .mmcwczm mgu we mpvmw: mzp :o emsu copxc we“ eo zpmcm_ asp an cmumpsmmc wee mcowuomwcw asp Go mmpmcwucooo chmump use Lowemucm one .mmzos nmwwpmzummcm cm mo vow; on“ mm~w_wnoesw use muczoeesm mmem umgopmzimmoeo mg“ »n topmowv:w upoe cam; owpmmpa mgp .cemea wmaoe mzu mo Ezumwcum msaeoo mg» 0pc? mcowpomhcw mcwxms Low mspmeeaam meg we 3mm> Pmupwmmm .o mczmwm 44 .cweee emeee egg we Eeuewcum meeeee on» eye? mcewueeflcw mewxes Lew meueeeeee one we 3ew> weppwmem .m egemwm 45 mg/kg). Mice with contralateral circling responses of ten or more turns in 2 minutes were used in subsequent behavioral tests. The circling behavior of 6-hydroxydopamine-lesioned mice was quantified by placing the mouse in a 3 liter beaker housed in a sound-attenuating box. The box was illuminated from below and the behavior was observed through a window in the t0p of the box. The number of complete 360° rotations to the left or right was recorded for two minutes after placing the mouse in the beaker. The results, reported as net turns/2 min., represent differences between right and left turns. Positive (+) scores indicate net turns away from the lesioned side (contralateral); negative scores indicate net turns toward the lesioned side (ipsilateral). The time courses of each drug studied were determined in the following manner. A lesioned mouse was placed in the test beaker for 2 minutes and the spontaneous circling recorded (time=0 min.); the appropriate drug solution was then administered and the mouse retested at selected time intervals thereafter. 2. Biochemical_procedures a. Analygis of dopa, dqpamine, and Sshydroxytryptamine following ion-exchange chromatogrgphic separation Brain samples were analyzed for dopa, dopamine and 5-HT by ion-exchange and alumina adsorption chromatography using modifications of procedures described previously (Anton and Sayre, 1964; Laverty and Taylor, 1968; Moore and Rech, 1967; Barchas gt gl,, 1972; figure 7). In the experiments concerning either 3H-L- 46 Figure 7. Procedure for the analysis of dopa, dopamine and 5-hydroxytryptamine (5-HT) following ion-exchange chromatography. This illustration outlines the method used for the separation of dopa and dopamine in both the nonisotopic and isotopic studies. In the latter studies 50 uCi [3 H(G )]- L- dopa was intravenously injected 15 minutes prior to sacrifice by a whole body perfusion with approximately 50 m1 of saline. Subsequently one forebrain, consisting of a frontal cut just caudal to the corpora quadri emina, was extracted with perchloric acid. The H-dopa was analyzed by taking the 1.0 m1 of alumina eluate The H- -dopamine was analyzed by taking the 1.10 ml of alumina eluate (2). In the nonisotopic studies two hemiforebrains were pooled prior to extraction with perchloric acid (HC104). The alumina eluate (l) was analyzed for dopa while the column eluate was analyzed for both dopamine and 5—HT. 47 TISSUE EXTRACT (HCLO4) ION EXCHANGE CHROMATOGRAPHY (BIO-REX 70, Na , 200-400 mesh) pH 6.5 EFFLUENT ELUATE (HAc) dopa norepinephrine deaminated catechols dopamine 5-hydroxytryptamine ALUMINA ADSORPTION pH 8.4 ALUMINA ADSORPTION pH 8.4 EFFLUENT ELUATE 1. EFFLUENT ELUATE 2. O-methyldopa (HAc) O-methylated (HAc) dopa catecholamines dopamine norepinephrine Figure 7. Procedure for the analysis of dopa, dopamine and 5-hydroxytryptamine (5-HT) following ion-exchange chroma- tography. 48 dopa or D-dopa metabolism in brain, decapitation was preceded by perfusion of the whole body of each mouse through the left ventricle with approximately 50 m1 saline. Following decapitation the fore- brain was isolated by a frontal cut just caudal to the corpora quadrigemina and the left and right forebrains isolated by a mid- sagittal section. Two left or two right forebrains were pooled, weighed and placed in 4.0 milliliters (m1) of 0.4 N perchloric acid (containing 0.1% EDTA). Both tissue dissection and homogenization were performed on ice. Immediately following homogenization the samples were centrifuged (10,000 x g) at 4°C for ten minutes. The supernatants were decanted into ice cold tubes containing 1.0 ml 0.1 M phosphate buffer (pH 6.5, 0.1% EDTA) and nine drops of 2 N KOH. Final adjustment to pH 6.0-6.5 was made with 0.1 N KOH. The samples were then centrifuged at 800 x g for five minutes and the supernatants placed on columns containing 3.0 centimeters of a weak cation exchange resin (Bio-Rex 70) which had been washed previously with 4.0 ml 0.1 M phosphate buffer. The supernatant plus an additional 2.0 ml of 0.02 M phosphate buffer (pH 6.5, 1.0% EDTA) were collected as the column effluence which constitutes the dopa fraction. The dopa samples were con- centrated and further purified by adsorption onto aluminum oxide (Woelm Neutral, Waters Associates, Farmington, Massachusetts) as described previously (Anton and Sayre, 1962; Drell, 1970). Following the addition of 15 drops of alumina to the column effluence, the suspension was adjusted to pH 8.0-8.5 by the addition of 0.5 m1 49 1 M Trizma base. The tubes were shaken for 10 minutes in an Eber- bach horizontal tube shaker followed by a 5 minute centrifugation at 800 x 9. After discarding the noncatechol-containing supernatant, the alumina was rinsed twice with redistilled water and then eluted with 1.0 ml of 0.2 N acetic acid. The 1.0 ml of eluate was then analyzed for dopa content by oxidation producing the fluorescent dihydroxindole derivative (Kehr gt_gl,, 1972; Lindqvist gt_gl,, 1975). The oxidation of dopa was accomplished by the sequential addition of the following reagents at 2 minute intervals: 0.2 ml 0.1 M NazEDTA (pH 8.0) with l M NaAC); 0.2 ml 0.1 N iodine; 0.2 ml alkaline sulfite; 0.2 ml 5 N HAC. The tubes were then sealed with a marble, boiled for 5 minutes and then cooled in ice. Fluorescence was determined within 15 minutes in an Aminco-Bowman spectrophoto— fluorometer at activating-fluorescent wavelenghts of 320-380 mu. Tissue blanks for the iodine oxidation procedure were deter- mined in the following manner. The forebrain from a control mouse Was carried through the separation procedure as described previously. When oxidizing, all of the reagents were measured into an empty tube. Following the addition of 5 N acetic acid, the 1.0 ml of tissue blank was added and the remaining steps carried out. The reagent blank was carried through the separation and oxidation pro- cedure the same as a tissue sample. No difference in fluorescent readings were found between tissue and reagent blanks. The Bio-Rex 70 columns were washed with 15 m1 of 0.02 M phosphate buffer (pH 6.5, 1.0% EDTA) and twice with 3.0 ml 50 of redistilled water. Finally, 6.0 m1 of 0.5 N acetic acid were placed on the column and the first 3.0 ml collected constituting the fraction containing both dopamine and 5-HT (Barchas gt_gl,, 1972). The 5-HT content was analyzed by reacting 1.0 m1 of the column eluate with orthophthaldialdehyde (OPT) to produce the fluorescent derivative (Curzon and Green, 1970; Haubrich and Denzer, 1973; Atack and Lindqvist, 1973; Smith gt_gl,, 1975). The fluorescent product of 5-HT was produced by the addition of 2.0 ml of concen- trated HCl followed by 0.1 ml OPT to the tubes containing 1.0 ml sample. Subsequently the tubes were sealed with marbles, heated at 97-98°C for ten minutes and cooled in ice. Fluorescence was determined within 15 minutes in an Aminco-Bowman spectrophoto- fluorometer at activating-fluorescent wavelengths of 350-475 mu. Tissue blanks were carried through the separation procedure like the standards and samples. During the oxidation the concentrated HCl was added to the tissue blank which were then heated and cooled in ice. The OPT was added to the tissue blank immediately before reading the fluorescence. Dopamine content was analyzed by taking an additional 1.0 ml aliquot from the column eluate. To these samples were added 0.2 ml of 1.0 M acetate buffer (pH 8.0, 0.1 M EDTA) and the pH adjusted to pH 6.5 with 1 N NaOH, 0.1 N NaOH and 0.1 N HCl. Subsequently, the tubes were oxidized as described previously for dopa with one exception. The 0.2 ml of 1.0 M acetate buffer (pH 8.0, 51 0.1 M EDTA) was omitted from the first step of the timed sequence. The tissue and reagent blanks were determined as described for dopa. Recoveries for standards of dopa, 5-HT and dopamine expressed as the mean i one standard error, were 30.0i1.65%; 61.3i 5.5% and 88.6:4.3%, respectively. The unknown tissue concen- trations were determined from these standards. Groups of unilaterally-lesioned mice to be used in biochemical studies were sacrificed at least ten days after in- jection of 6-hydroxydopamine and at least three days after a test dose of apomorphine. b. Separation of 3H-dopa and metabolites Mice were injected intraperitoneally with different AAAD inhibitors or saline 30 minutes prior to an intravenous in- jection of 50 uCi of 3H-L-dopa in 0.25 ml of saline. Fifteen minutes later each mouse was perfused with saline through the left ventricle and the forebrain taken for analysis of 3H-dopa and 3H- 3H—dopa from 3H-dopamine dopamine. The biochemical separation of was accomplished by the detailed procedure given above (figure 7) with the following changes. The column eluate containing 3H- dopamine was concentrated by adsorption onto aluminum oxide with subsequent elution as described previously for dopa. Although this fraction is referred to as 3H-dopamine, it may also contain small amounts of 3H-norepinephrine. The alumina eluates (10 ml) for both dopa and dopamine were placed in 10 ml Phase Combining System (PCS) (Amersham/ Searle, Arlington Heights, Illinois) and disintegrations 52 per minute quantified by using an external standard ratio of 35.8 in a Beckman LS-lOO liquid scintillation system. c. Analysis of 5-hygroxyindole acetic acid (5-HIAA), 5- hygroxyttyptamine (5-HT), norepinephrine and dopamine following organic extraction Brain samples were analyzed simultaneously for norepinephrine, dopamine, 5-HIAA and 5-HT by organic extraction using modifications of procedures described previously (Curzon and Green, 1970; Haubrich and Denzer, 1973; figure 8). Following decapitation, the forebrain was isolated by a frontal cut just caudal to the corpora quadrigemina and the left and right fore- brains isolated by a midsagittal section. Two left or two right forebrains were pooled, weighed and placed in 4.0 ml of acidified butanol (10 mM HCl), (containing 0.1% EDTA). Both tissue dissection and homogenization were performed on ice. Following homogenization the tubes were kept on ice for 20 minutes and then centrifuged (10,000 x g, 9500 rpm) for 10 minutes at 4°C. The supernatants combined with 10 ml of heptane and 2.0 ml 0.01 N HCl were shaken for 5 minutes and centrifuged at 1000 x g for ten minutes. The organic phase combined with 1.0 ml 10 mM Tris HCl (pH 7.0) was shaken for 5 minutes and centrifuged at 1000 x g for 5 minutes. The following reagents were added sequentially to 0.5 ml of the supernatant: 0.05 ml 1% cysteine, 1.0 m1 concentrated HCl; 0.05 ml OPT (made fresh in methanol) and 0.05 ml 0.2% periodate solution. These samples were kept at room temperature for 30 minutes, boiled for 10 minutes and cooled in ice. Tissue blanks were prepared by 53 TISSUE EXTRACT (acidified butanol) EXTRACTION TO AQUEOUS PHASE (Heptane + 0.01N HCl) ORGANIC PHASE AQUEOUS PHASE (5-HIAA) norepinephrine dopamine (5-HT) EXTRACTION T0 AQUEOUS PHASE ALUMINA AASORPTION (10 mM TriS) pH 8.4 5-HIAA + OPT 5-HT + OPT ELUATE read 0 360/470 read @ 360/470 read @ 385/485 dopamine, boil read @ 320/380 Figure 8. Procedure for the analysis of 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxytryptamine (5-HT), norepinephrine and dopamine following organic extraction. 54 omitting the OPT until just before the fluorescence was determined. Fluorescence was determined in an Aminco-Bowman spectrophotofluoro- meter at activating-fluorescent wavelengths of 360—470 mu. The 5-HT content was analyzed by reacting 0.2 ml of the aqueous phase from the heptane mixture with 0.8 m1 OPT (in 10 N HCl). The samples were heated for 15 minutes at 95°C and the fluorescence read in an Aminco-Bowman spectrophotofluorometer at activating-fluorescent wavelengths of 360-470 mu. Tissue blanks were again prepared by adding the OPT just prior to the fluorescence reading. The norepinephrine and dopamine concentration were quantified following alumina adsorption of 1.0 ml of the aqueous phase from the heptane mixture. Ten drops of aluminum oxide were added to 1.0 ml of the aqueous phase and the suspension shaken for 10 minutes in an Eberbach horizontal tube shaker followed by a 5 minute centrifugation at 1000 x 9. After discarding the noncatechol- containing supernatant, the alumina was rinsed twice with redistilled water and then eluted with 1.0 m1 of 0.2 N acetic acid. The oxida- tion was accomplished by the sequential addition of the following reagents at 2 minute intervals: 0.2 ml 1.0 M acetate buffer (pH 8.0, 0.1 M EDTA); 0.2 m1 1 N iodine; 0.2 ml alkaline sulfite; 0.2 ml 0.5 N acetic acid. The samples were then left at room tempera- ture for 60 minutes and the fluorescence due to norepinephrine read at activating-fluorescent wavelengths of 385-485 mu. Tissue blanks were prepared by adding all reagents to an empty tube. After the 5 55 N acetic acid was added to the empty tube, the tissue blank was combined with reagents and mixed well. After reading the fluorescence for norepinephrine the samples were poured back into glass tubes, sealed with a marble, boiled for 5 minutes, and then cooled in ice. The fluorescence due to dopamine was read at activating-fluorescent wavelengths of 320-380 mu. After subtracting the blank values the concentrations of 5-HIAA, 5— HT, dopamine and norepinephrine were calculated directly from the regression line of the standards. 3. Statistical analysis a. Biochemical experiments The data are reported as the mean i one standard error of the mean (S.E.) obtained from the indicated number of experiments. Statistical comparisons were made with the Student's "t"-test for unpaired samples. P values of less than .05 were considered to be statistically significant (Goldstein, 1964). b. Turnover rate comparisons Linear regression analysis was performed by the method of least squares. The student's "t“ test was used to test the parallelism of the two slopes. c. Behavioral studies The data are reported as the mean i the standard error of the mean (S.E.). The same groups of lesioned mice were 56 used when comparisons between lesioned mice were made in one graph. Due to large differences in the variances, the Mann-Whitney U test was used for statistical determinations. Comparisons were made between the means of drug treated mice and the means of saline injected lesioned mice for the corresponding time point after injection. P values of less than 0.05 were considered indicative of statistical significance (Goldstein, 1964). B. Materials 1. Buffers 0.1 M 0.02M 1.0 M 0.2 M 1.0 M phosphate, pH 6.5-13.8 gm NaH2P04-H20 in 900 ml 0.1% EDTA. Adjust pH to 6.5 with 5 N NaOH and q.s. to 1000 ml. phosphate, 0.1% EDTA, pH 6.5-2.0 gm NaH2P04-H20 in 800 ml 0.1% EDTA. Adjust pH to 6.5 with 5 N NaOH and q.s. to 1000 ml. acetate, 0.1 M EDTA, pH 8.0-3.72 gm NaZEDTA in 80 ml hot redistilled water. Add 13.6 gm NaC2H302-3H20 to the cooled solution. Adjust pH to 8.0 with 5 H and 2 N NaOH and q.s. to 100 ml. acetate, pH 8.4-27.2 gm NaCZH302-3H20 (16.4 gm NaC2H302, anhydrous) Trizma base-3.025 gm Trizma base (Tris [Hydroxyl- methyl] aminomethane, Sigma Chemical Co.) in 250 ml redistilled water 57 10 mM Tris-HCl, pH 7.0 0.24 g Trizma base in 180 m1 H O 2 and adjust to pH 7.0 with 2.0 and 1.0 N HCl and q.s. to 200 ml. Reagents Disodium ethylene diamine tetracetic acid (EDTA) 0.1% w/v (Sigma Chemical Co., St. Louis, Missouri) 1 gm NaZEDTA in 1000 ml redistilled water-warm. Ortho-phthaldehyde (OPT) (Sigma) 1 mg OPT/2 ml methanol (Fischer, Fairland, New Jersey) prepared immediately after oxidation. Ortho-phthaldialdehyde (OPT in 10 N HCl) (Sigma) 12.5 mg OPT in 50 ml of 10 N HCl prepared 4-8 hours before use. Cysteine 1%, 290 mg L-cysteine hydrochloride-H20 (Aldrich Chemical Company, Milwaukee, Wisconsin) in 20 ml re- distilled water. Periodate solution 0.2%, 4 mg periodate NaIO4 (Mallinckrodt) in 20 m1 redistilled water. Iodine 0.1 N (Mallinckrodt, St. Louis, Missouri) 1.27 gm in 100 m1 100% ethanol. Alkaline sulfite 0.5 m1 NaZSO3 soln + 4.5 m1 5 N NaOH, NaZSO3: 2.5 gm Na2503 (Fischer) in 10 m1 redistilled water. Resin Bio-Rex 70, 200-400 mesh, sodium form (Bio-Rad Laboratories, Richmond, California). Methylcellulose (Fischer) 1.0%-10 gm in 1000 ml distilled hot water, stir and leave in cold for 12 hrs. 58 Sodium Metabisulfite (Baker) 0.01%-100 mg in 1000 ml distilled water. Aluminum Oxide (Woehm neutral, Eschwege, Germany). Acidified butanol (10 mM HCl) 0.85 ml of concentrated HCl/liter butyl alcohol (Matheson Coleman and Bell) or 0.4 ml concentrated HCl/pint butyl alcohol. Potassium hydroxide, 10 N KOH (Fischer) 56 gm KOH in 100 ml redistilled water. Sodium hydroxide, 5 N NaOH (Baker, Chemical Company, Phillipsburg, New Jersey) 50 gm NaOH in 250 ml cool redistilled water. ' Perchloric acid, 0.4 N HClO4 (Mallinckrodt) 3.54 ml of 70% HClO4 q.s. 100 ml redistilled water. Acetic acid, 5 N HAc (Mallinckrodt) 28.8 ml of glacial HAc q.s. 100 ml redistilled water. Hydrochloric acid, 2 N HCl (Baker) 190 ml concentrated HCl q.s. to 100 ml redistilled water. Standards Norepinephrine 1 mg/ml solution 18.87 mg norepinephrine bitartrate (Sigma) in 10 ml 0.2 N HAc. Dopamine 1 mg/ml solution 12.35 mg dopamine HCl (Sigma) in 10 ml 0.2 N HAc. 59 5-HT 100 mg/ml solution 2.30 mg 5-hydroxytryptamine creatinine sulfate monohydrate (Aldrich) in 10 ml 0.2 N HAc. DOPA 1 mg/ml solution 10 mg L-dihydroxyphenylalanine (Sigma) in 10 ml 0.1 N HCl. 4. Resin preparation Place 0.5 lb of Bio-Rex 70 (200-400 mesh, Na+) in a liter bottle and fill with distilled water. Adjust pH to 6.5 with con- centrated HCl under stirring. Hand shake, wait about 30 minutes until supernatant clear and decant the water from resin. Repeat following procedure eight times: add water, hand shake, wait and decant. Finally add 500 ml 0.1 M phosphate buffer (pH 6.5, 0.1% EDTA) to the washed Bio-Rex, keep refrigerated. 5. Alumina purification One half bottle of Woelm aluminum oxide (distributed by Waters Assoc. Inc., Farmington, Massachusetts) is placed in a liter bottle. A suspension of the alumina is shaken repeatedly with distilled water until supernatant becomes clear. Add 200 ml of 2.0 N HCl, shake for 30 minutes and decant the supernatant. Wash ten times with distilled water. Wash once with 0.2 N HAc and once with 0.02 N Na acetate (pH 8.4). Wash with distilled water ten times and then redistilled water five times. Store the alumina in a glass stoppered bottle containing sufficient water to thoroughly wet the alumina. 60 6.011193 6—Hydroxydopamine hydrobromide was obtained initially from Regis Chemical Company (Chicago, Illinois) and later from Sigma Chemical Company (St. Louis, Missouri). Apomorphine HCl, obtained from Eli Lilly and Company (Indianapolis, Indiana) was dissolved in 0.01% sodium metabisulfite. L-o-methyldopa hydrazine (HMD or MK 486), a gift of Dr. C. Stone, Merck Sharp & Dohme, was prepared for injection as a suspension in 1.0% methylcellulose. L- and D-dopa were obtained from both Nutritional Biochemicals Cor- poration (NBC; Cleveland, Ohio) and Sigma Chemical Company (St. Louis, Missouri) and were suspended in 1.0% methylcellulose. Meta- hydroxybenzylhydrazine (NSD 1015) was obtained from Aldrich Chemical Company (Milwaukee, Wisconsin). Methoxyflurane (Metofane) was obtained from Pitman-Moore, Inc. (Washington Crossing, New Jersey). 4-bromo-3-hydroxybenzyloxyamine dihydrogen phosphate (NSD 1055, brocresine) was a gift from Dr. Leon Ellenbogen at Lederle Labora- tories (Pearl River, New York). N-(DL-seryl)-N'-(2,3,4-trihydroxy- benzyl) hydrazine (Ro44602, Benserazid) was a gift from Dr. W. E. Scott at Hoffman-LaRoche, Inc. (Nutley, New Jersey). L-dopa for the optical purity studies was obtained from Calbiochem (Elk Grove Village, Illinois). [3H(G)]-L-3,4-dihydroxyphenylalanine (11.5 Ci/mmole, 3H-dopa) was purchased from New England Nuclear (Boston, Massachusetts). Pargyline HCl (Eutonyl) was obtained from Abbott Laboratories (Chicago, Illinois). 5,7-Dihydroxytryptamine creatinine sulfate was purchased from Regis Chemical Company (Chicago, Illinois). 61 Except where indicated all drugs were dissolved in saline. The drugs were weighed as the salts, prepared immediately before use and administered in a volume of l m1/lOO gm body weight. RESULTS A. Behavioral rotation studies 1. Dose-response relationship between D- and L-dopa Previous studies (Ungerstedt, l97la,b; Von Voigtlander and Moore, 1973; Fuxe and Ungerstedt, 1974; Mendez gt_gl,, 1975) have demonstrated that L-dopa induces contralateral circling in rodents with unilateral nigrostriatal pathway lesions. In order to provide further insight into the mechanism of action of L-dopa, the behavioral and biochemical properties of L-dopa were compared to those of its enantiomer, D-dopa. A comparison of the L- and D-dopa-induced circling behavior in mice with unilateral 6-hydroxydopamine striatal lesions is presented in figure 9. Prior to the testing of dopa, groups of ten lesioned mice were selected for equivalent contralateral circling responses to apomorphine; the means of the four groups of mice used in figure 9 were: +17.80:2.64; +16.90:2.28; +17.90t 2.30; +18.60:1.91. There were no significant differences between the latter apomorphine treatment groups as determined by the unpaired Student's t-test (p<.05). In figure 9 the solid symbols indicate those values different from methylcellulose-treated 6- hydroxydopamine-lesioned controls as determined by the Mann-Whitney U test (p<.05). 62 63 As demonstrated in figure 9A, L-dopa reversed the initial ipsilateral (negative) circling at time 0 minutes to produce contra- lateral (positive) circling. Administration of increasing doses of L-dopa (5, 10 and 20 mg/kg) resulted in a dose-related increase in the contralateral circling (figure 9A). If the contralateral circling induced by L-dopa is mediated by the formation of dopamine then the administration of D-dopa should not induce contralateral circling since it is not metabolized by the brain to dopamine (Shindo gt_gl,, 1973). However, figure 9B demonstrates that the intraperitoneal administration of D-dopa unexpectedly produced a marked increase in contralateral circling. Administration of D-dopa at 50, 100 and 200 mg/kg resulted in a dose-related increase in the circling away from the lesioned side. Furthermore, approximately ten times as much D-dopa had to be administered to produce contralateral circling rates equivalent to those produced by L-dopa (i.e., 10 mg/kg of L-dopa and 100 mg/kg of D-dopa). It is also noteworthy that following the high dose of D- dopa the duration of circling is greater when compared to the L- dopa-induced circling (compare figures 9A and B). The contralateral circling following D-dopa (200 mg/kg) is significantly elevated at 45 and 60 minutes in contrast to the L-dopa-induced circling which has subsided at these time points. 64 .Apeeepe_eeeceev eewm eecewme_ esp Eeew xeze mcwpeewe ucemeeeec wee—e> e>wewmee meeeecz A—eeeue__mewv eewm eecewmew ezu eeezee mewFULwe “cemeeeee epecweee exp :e meewe> e>wuemez .Aee.vev emo_=__eo_xeeee e_ new: eepeehcw eewe emecp Eeew uceeewwwe x_e:eewwwcmwm ece eecu meewe> eeeewecw mweesxm ewwem .Feesxm ece we meweee esp cece weep mw e:_e> esp cezz ueeexe Feesxm ceee :e mecww Feewuee> ecu we eepeewecw we geese eeeeceem eee .eeee-e Le ex\ee eom eee 00— .em eee eeoe-e we ex\ee em eee e_ .m Le seweebmeEee _eecefieeeeb:w e5 5.; eeeweeee meEe> eeeewecw U ece .<.O m_eeE>m ecw .eewe OF cw eeccheuee me Lecee eeeeeepm ece epecee mewa Feeweee> ecu ece mceeE ecu “cemeeeee m_eeE>m egw .maFeLwo Peeeueweeucee eeeeeewieeee1o ece 14 es» mcwxeepm cw new: eeez mcwpeewe eeeee2w1eewceees -eee we meeec pcewe>weee mcw>eg eewE we meeeem cued .Lew>e;ee oewweewe weeepeweeecee eeeeeCP1A.e.m mx\me mm.ov eewzeeeseee Lew eeumep eeez eews Eseeweem meeeee ecu eecw ecwEeeeeXxeeexsio we :ewpeencw Leewe exec :e>em .mcewmew eewEeeeUXxeeexgio weeepewwce new; eewe cw Lew>egee mew—eewe ce eeeeuo use 14 we mpeewwm .m eeemwm .mcewmew ecwEeeeeAxeeexc -o Feeeee_w:= eewz eewE cw eew>ecee mcwwecwe ce eeeeie ece 14 we meeewwm (30010 amt-4‘ mic. (801.. cmt< NEE. .EE aim man can we 8 m_. o .fiE R In“. Q1 Lfl 8 1 1 1 Id ‘ fi 65 sammw 11./swam 131v .m exemwd $310M“ Z/SNUOJ. 1.3M 66 2. Central decarboxylase inhibition and dopa-induced contralateral circling a. Ro44602 Previous studies by Ungerstedt (l97l) demonstrated that inhibition of the AAAD activity by high doses of Ro44602 would delay the contralateral circling response of lesioned rats to L- dopa. In order to determine if the delay in circling was related to decreased dopamine formation, the D-dopa-induced contralateral circling was studied following Ro44602. A comparison of the circling response to L- or D- dopa following Ro44602 is depicted in figure l0. The same group of lesioned mice was used for making comparisons within each graph. Subcutaneous administration of L-dopa (l0 mg/kg) produced the expected increase in contralateral circling (figure lOA). Following pretreatment with Ro44602, there was an initial delay followed by a prolonged period of circling as has been demonstrated previously (Ungerstedt, l97lb). The subcutaneous administration of D-dopa (100 mg/kg) did induce contralateral circling but the amount of circling was not as large as that following intraperitoneal administration. When the D-dopa injection followed pretreatment with Ro44602, the contra- lateral circling was initially blocked for 60 minutes. At 90 minutes an increase in contralateral circling occurred and was still elevated at 360 minutes after D-dopa (figure 103). Thus, the D-dopa-induced contralateral turning was unexpectedly delayed by an 67 .Amo.vav mmoP=F_muF»;pme &_ sow; capoahew move soc» ucmcmwmwu xppcmu_mwcmwm mew peep mm:_m> mpmowvcw mFonexm uwFom .xczpm maouuo mcu so» wows NF ccm xuapm maovue mcp Low wows N cw cmcwELmumc mm Loccm acmvcwpm wco mpocmu mmcwp _muwuem> ms“ use 285 we» 95$;qu 28E? of. 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Specific rotation of different samples of L- and D-dopa. SPECIFIC ROTATION EXPERIMENT l EXPERIMENT 2 INDIVIDUAL SAMPLES L-Dopa (Sigma) -ll.8O -ll.60 L-Dopa (NBC) ~12.15 -ll.65 L-Dopa (Calbiochem) -ll.65 -ll.55 D-Dopa (Sigma) +l2.00 +ll.95 D-Dopa (NBC) +12.55 ------ COMBINED SAMPLES (Sigma) 99% D-Dopa + l% L-Dopa ------ +ll.80 98% D-Dopa + 2% L-Dopa ------ +ll.45 97% D-Dopa + 3% L-Dopa ------ +ll.65 96% D-Dopa + 4% L-Dopa ------ +ll.00 95% D-Dopa + 5% L-Dopa ------ +ll.OO 90% D-Dopa + lO% L-Dopa +9.95 +9.95 80% D-Dopa + 20% L-Dopa +6.95 +7.05 REPORTED VALUES Corts and Koekkoek, l97l -l2.25 Appleby and Mitchell, l97l ~12.l0 Experiments were performed with a Perkin Elmer l4l Polarimeter at 20°C using a mercury lamp (A=587). Dopa was dissolved in l N HCl (20 mg/ml). . , , 20 ob ' spec1f1c rotation ([a]578) = (15§;Z$fle{2:%%;?31) 78 In order to determine the relationship between increasing amounts of L-dopa in individual D-dopa samples, a series of com- bined samples containing increasing concentrations of L-dopa were prepared and analyzed for changes in specific rotation. Increasing the amount of L-dopa (from 1 to 5%) contained in D-dopa samples resulted in a gradual reduction in the specific rotation. If the response to D-dopa was due to a contaminant of L—dopa, then the D-dopa sample would have to contain approximately l0% of the L-isomer as an impurity since the dose of L—dopa essential to produce an equivalent rate of circling is one tenth the dose of D- dopa (compare 0- and L-dopa dose responses in figure 9). One 0- dopa sample was prepared containing l0% L-dopa. The calculated value for specific rotation changed from +12.00 and +ll.95 to +9.95 emphasizing that our D-dopa samples were not contaminated with significant quantities of L-dopa (Table 2).- C. Biochemical Studies l. 3H-L-Dopa metabolism The effects of AAAD inhibitors on the brain content of 3H-dopa and 3H-dopamine following systemic administration of 3H-L- dopa are presented in Table 3. An intravenous injection of 3H-L- dopa l5 minutes prior to sacrifice resulted in an equivalent in— crease in forebrain concentrations of 3H-dopa and 3H-dopamine in mice pretreated with saline 30 or l95 minutes before the 3H-L-dopa. 79 .mweeewe mm_ ew mpee emz eww: eewEewweewee eewzo_Few eowewee -emeeee mewEween-Im we» eew eemexw mweeweee mewwwm newneeemmeeee we“ Eeew Amo.vev uewewwwwn awwwe -wemwewum mew: eewe: mewEween-I new ween-em we mmeww> ew nmewemme mpemEpwweu maen __< .eepweweew mmwwzxeeewemn w eww: Anepm emwm ew mews w epw: new mwwneem mewwwm eeee eew mews N ew nmewEewpmn mw Au.m P H ewme wee pemmweeme mmeww> .weweween-z new ween-z we mwmzwwew eew emewp ewweemeew wee new mewwwm we om xwmewewxeeeew euw: wwewepew> pwmm mew emeeeem nmmeweme mw: mmeee eewm we anee mwee: wee emeww mmpeewe emmewwe .wewwwm we mN.o ew ween-4-z we we: om we eewpewnew meeem>wepew ew ee eewee mmewp nmpwewnew wee aw mews ea .e.w nmemwmwewenw mmm: mewpwm ee meepweweew o<<< meewew> mw.mwmm.w: me.~ new.m~ . em ee\ee eem eeowwxewwe-e we.onN~.Pe mm.w “mm._w em ee\ee ee_ mme_ emz em._wee.m em.menep.ww mm. we.enee.e mw.mewem.mw em ee\me ee_ m_ew emz om.~wem.me em.e Hem.ew mm: mm.enwe.e me.m_wee.mm em ee\me eew Neeeeoe mm.ewew.om we.“ www.me - mew we.enee.mw we.e “mm.we ow me\me me we: em.ew_m.e ee._ He_.w mm_ e_.ewwm.e ew.: www.w em wew_wm wzezeaee eeee a coweemwew w en-4-: meewme eewee\weev emmwesze meme wzwewewewwee onH new ewme we» mpemmweeme ewe eewm we uemwwe mew .memeemweme nmeewmmw-mewsweenzxeenAe-o we» uemmweeme mewe nwwem mew new memeemweme nmeewmwweee wee wwwewnew mewe emeo .mewEwpexepxxeenxe-m new mewEween .ween we mwmxwwew meemewwweewm eew emewu mewweemeewwewe e:u new ween-4 we eewpemhew mew ewpww eeee mee nmewwweewm mew: mews mew .A.e.w me\me oom ee oowv ween-4 we eeweweumwewEnw meewme eeee mee xwmeemewu -=ee:m nwemmflew mw: Ame\me om .ozzv weenwxepmeeewNwenxr .pemswewexm mweu ew nmme mew: mmueewe N\meeep wwemewwweeeee awe meee ee emu eew: mews wmeew .A.e.m .me\me mm.ov meweeeeeeew eww: nmeemnew mew: mews .mewEweenxxeenze-n we eewuewwew wwuwweemwepew mew emeww emm: weo .mewweeweewwEme weeueee new nmeewmmw-mewEweenzxeenze-o ew Awe-my mewEweexeuxxeenxe-m we eewemwemn nmeenew-ween-4 we eemweweeeu .e_ meeewe 87 .mewweemeewwewe weeweee new nmeewmmw-wewEweenzxeenxe -o ew mewEweQAepxxeenxe-m we eewumwemn nmeenew-ween-4 we eemweweeeu .ww meemww e335 gee... we wwee OON OO— O CON 00. 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Law II. .m- .ee.w .w .. .8 Lee. . we . eew HI-m mz:2 w euw: newee mewp eewm pw nmpemmmeeme mw AOV ewweemeewweme nmeewmmweee we» ew new A3 nmeewmww mep ew mewEwpexeuxxeenxz-m we eeweweuemeeee eme mew .meenmeeee eewpewepxm ewewmee mew eew nwweee mewweemeewweme N new nm>eewe mw: ewweeweew mew .mewwweewm weewme mmeeewe om new ow .o xwwwmeeuwemewepew nmuemwew mw: Ame\me omwv mewwxmewe .xnepm mwep ew nmm: mew: mmueewe N\meeeu wees ee ewe euw: mews mmeew new weweeeeeeew epw: nmpmmp mew: mews .meweween -:xeenxe-o we eewpemmew wwpwwepmweeew wee mew:e_wew emm: meo .mewweemeewwEme weepeee new nmeewmmw-mewsweenxxeenxe-o ew em>eeeep Awe-my mewEwwexeexxeenxz-m .e_ mezmwe .90 .70 5-HYDROXYTRYPTAMINE (“I/gram) .60 Figure 16. 92 SYNTHESIS RATE .—. LESIONED 1.30 nmolcs/g/lw o— -0 NONLESIONED 1.71 «moles/Ill" * 0 10 20 TIME AFTER PARGYLINE (mlnules) 5-Hydroxytryptamine turnover in 6-hydroxydopamine- lesioned and control hemiforebrains. 93 to be different than zero. Furthermore, there was no statistical difference between the slopes of the lesioned versus the nonlesioned side when compared using the Student's "t" test (p<0.01). Thus, the 5-HT synthesis rates as determined from the slopes of the lines were not significantly different. The synthesis rates of 1.71 nmoles/gram/hour (nonlesioned hemiforebrain) and 1.30 nmoles/gram/ hour (lesioned hemiforebrain) were found to be in good agreement with Tozer et_al, (1966) and Lin gt_al, (l969b), but slightly lower than those reported by Morot-Gaudry et_al, (1974). An additional experiment was performed to determine if the synthesis rate of 5-HT neurons might be altered by the administration of L-dopa. Figure 17 is a comparison of the 5-HT synthesis rates in the lesioned and nonlesioned hemisphere after the administration of L-dopa (20 mg/kg). Following the injection of pargyline there was a linear increase in the endogenous 5-HT concentrations. The correlation coefficients as determined by least squares regressional analysis were r=.304 (nonlesioned hemi- sphere) and r=.4ll (lesioned hemisphere). The slopes of the lines for the lesioned and nonlesioned hemispheres were found to be different from zero but not significantly different from each other (p<0.05). The 5—HT synthesis rates of l.87 nmoles/gram/hour (non- lesioned hemisphere) and 2.09 nmoles/gram/hour (lesioned hemisphere) for the L-dopa treated mice were not significantly different from the synthesis rates in mice which did not receive L-dopa (figure 16) as determined by a comparison of the slopes of the lines (p<.05). 94 .wewewwwemwm wwwwewwmwwwwm mw eewwwwmeeeuw .mmeweem wmwmw we neewme mew xe nmwwweewwe mw mmeww eewmmmemme mew wemmmeewe mnwm nmeewmmw mew eew meww nmemwn mew new mnwm nmeewmmweee mew eew meww nwwem mew .mwwewew xwm ew nmeweemwmn mw eeeem newnewwm wee mewwwewnew meww wwewwem> w eww: wewee meww eewm ww nmwemmmeeme mw ewweemeewweme AOV nmeewmmweee new A3 nmeewmmw mew ew mewEwwexewxxeenxe-m we eewwwewemeeee ewme mew .mewwxmewe meewme mmweewe mp nmwemnew mw: Ame\ms omv ween-4 .mewwweewm meewme mmweewe om new ow .o xwwwmeewwemewewew nmwemnew mw: Ame\me omwv mewwxmewe .xnewm mwew ew nmm: mew: mmweews N\mee:w wwewwwwweweee wees ee emw eww: mews mmeew .A.e.m mx\me mm.ov meweeeeseew eww: nwwmmw mew: mews .mewEweenxxeenxe-o we eewwemhew wwwwwewmwewew mew emwww emw: meo .ween-4 we eewwwewmwewEnw mew mew:ewwew mewweemeewwEme weeweee new nmeewmww-mewEweenzxeenxe-n ew em>eeeew Awe-mv mewEwwexewxxeenxz-m .ww meemww 95 SYNTHESIS RATE ._. LESIONED 1.87 moles/allu- O - — O NONLESIONED 2.09 snacks/III" .70 5-HYDROXYTRYPTAMINE (ug [ya-am) .60 w 0 10 20 TIME AFTER PARGYLINEIInlnules) Figure 17. 5-Hydroxytryptamine (5-HT) turnover rate in 6- hydroxydopamine-lesioned and control hemiforebrains following the administration of L-dopa. 96 5. Utility of 5,7-dihydroxytryptamine for producing selective depletion of brain 5-HT As discussed previously, 5,7-dihydroxytryptamine has been proposed to represent an improvement over 5,6-dihydroxytryptamine in the chemical lesioning of serotonergic neurons (Baumgarten and Lachenmayer, 1972; Daly gt_gl,, 1974; Gershon and Baldessarini, 1974). Thus, a pilot study was initiated to determine the utility of 5,7-dihydroxytryptamine intracerebral injections for producing selective depletion of brain 5-HT. A comparison of different doses of 5,7-dihydroxytryptamine (lO-lOO pg) on the hemiforebrain concentration of monoamines is presented in Table 4. Although the data represent the mean of only two experiments, the injection of a low dose of 5,7-dihydroxytryp- tamine (10 ug) into the left striatum caused a reduction of 5-HT and 5-HIAA to approximately 67 and 59% of nonlesioned mice. At higher doses of 5,7-dihydroxytryptamine (25, 50 and 100 pg) 5-HT and 5-HIAA were reduced on the lesioned side but the 5-HT content of the nonlesioned hemiforebrain was also reduced. In other experi- ments, the injection of 5,7-dihydroxytryptamine in a volume of 2 pl also decreased 5-HT and 5-HIAA bilaterally. The reduction of 5-HT and 5-HIAA observed in Table 4 has been demonstrated previously by Jacoby gt_al, (1974). The nonspeci- ficity of this chemical lesioning technique was apparent by the reduction of dopamine content at the 10 pg dose and norepinephrine at the 25 ug dose (Table 4). The higher doses of 5,7-dihydroxy- tryptamine resulted in a further reduction of dopamine on the 97 .nwew ewwwewwwenewxxeenxe-m u < mew .memmww ewwee mew we eewwewewxm ewewmee mew:e_wew nmeeewewe mew: mmmxwwew wwewEmeeewe mew .mwweeeemw Eewnem m: m.o mewewwweee emww: nmwwwwmwn we memwwweeewe w mw: mwewem> eewwemnew mew .mmmzwwew Fwewemeeewe eew nmweee mem: mewweemeewweme wemwe e:w ee wwmw e:w new nmewwweewm mew: mews .Eewwwewm wwmw mew ewew sza-w.mv mewEwweaewzxeenaewn-w.m we eewwemnew mew mew:e__ew man emw m.nwm.ww N.w ww.mmw o.o ww.wm m.w wo.wn wemwe o.mwm.we m.o—wm.mm m.mwww.om m.o Hm._w wwmw wee-w.m a: cow o.owm.mm w.wmwm.www e.m ww.wm m.w ww.mm wemwe w.nwm.me m.~ wo.mm w.w Hm.mm m.opwn.Fm wwmp pro-m.m m: om —.mww.ww m.o wn.wm mm.m ww.mw m._wwm.nm wemwe N.Nww.wn o.w ww.Nw mo.w Hm.we m._ Hm.wm wwmw wee-w.m a: mm m.ewo.oow m.w wm.wow no.0 ww.mm w.w wm.ww wemwe m.mwm.oop w.m wo.—w mm.opwn.mm m.m wo.nm wwm— . hzo-w.m m: ow mszIemzHemmoz mzwz