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Peptide hormone interactions with brain catecholamine metabolism

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Peptide hormone interactions with brain catecholamine metabolism
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Delanoy, Richard L., 1951-
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ix, 122 leaves : ill. ; 29 cm.

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Animal grooming ( jstor )
Antipsychotic agents ( jstor )
Catecholamines ( jstor )
Dosage ( jstor )
In vitro fertilization ( jstor )
Metabolism ( jstor )
Norepinephrine ( jstor )
Personal hygiene ( jstor )
Rats ( jstor )
Receptors ( jstor )
Catecholamines -- metabolism ( mesh )
Corticotropin ( mesh )
Dissertations, Academic -- neuroscience -- UF ( mesh )
Hormones ( mesh )
Neuroscience Thesis Ph.D ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis (Ph.D.)--University of Florida, 1979.
Bibliography:
Bibliography: leaves 108-121.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Richard L. Delanoy, Jr.

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Full Text
PEPTIDE HORMONE INTERACTIONS WITH
BRAIN CATECHOLAMINE METABOLISM
BY
RICHARD L. DELANOY, JR.
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA 1979




ACKNOWLEDGEMENTS
The author wishes to thank Dr. Adrian Dunn for his support, teaching, tolerance and sense of humor.
. . Ms. Leila Baz-Malcom, Mr. John Hockensmith,
Mr. Terry Moore, Ms. Gale Hunter, Ms. Laurie Puckett,
Mr. Harry Forster, Dr. Neal Kramarcy, and Ms.
Nancy Gildersleeve for their technical assistance
and in calming the panics, humoring the depressions,
sobering the insanities, reassuring the anxieties,
and enlivening the tedium which accompanied the
author's style of research.
. . Dr. Maartin Reith for the use of his amino
acid analyzer.
. . all my friends for their part in making my
time in Florida pleasureable.
. . Dr. Samuel Gurin for having convinced me that
I did not want to be a fireman.
The author was a National Science Foundation Predoctoral Fellow from July, 1976 to July, 1979. The research described was supported by USPHS AM-18399 and MH-25486.
ii




TABLE OF CONTENTS
SECTION PAGE
ACKNOWLEDGEMENTS ................................ ii
KEY TO ABBREVIATIONS .............................v
ABSTRACT. ........................................vi
I INTRODUCTION. ..................................... 1
Behavioral Effects of ACTH and LVP.............4
Behavioral Interactions of Catecholaminergic
Systems with ACTH and LVP Treatments......... 9
ACTH Effects on Catecholamine Metabolism......10 Effects of LVP on Catecholamine Metabolism.... 13
II HALOPERIDOL AND APOMORPHINE ALTER
CATECHOLAMINE SYNTHESIS IN VITRO BY
DIFFERENT MECHANISMS ..........................16
Introduction. ..................................16
Methods .......................................19
Materials. ...................................19
Procedures. ..................................21
Chromatography ..............................23
Quantification and Analysis................. 26
Results....................................... 27
Determination of Relevant Precursor Pool....27
K+-Induced Activation of the Accumulation
of [3H]Catecholamines. ....................30
Inhibition of [ H]Catecholamine
Accumulation from [3H]Tyrosine.. ......32
Accumulation of {3H]Protein from [H]-3
Tyrosine ..................................36
Liberation of [14C]CO2 from [14C]Tyrosine...40 Uptake of [14C]DA. ...........................40
Release and Metabolism of Preloaded
Labelled DA ...............................43
Discussion. ....................................49
Precursor Pool Determination ................49
Effects of Elevated K+ upon Synthesis,
Release, and Metabolism of DA............. 50
Effects of Apomorphine on DA Metabolism.....53
Effects of Haloperidol on Catecholamine
Synthesis and Release .....................56
Summary. .......................................68
iii




III ACTH AND LVP SELECTIVELY ACTIVATE
MESOCORTICAL DA SYNTHESIS .....................70
Introduction ..................................70
Methods .......................................72
Materials ................................... 72
Animals and Surgery. .........................73
Incubation Procedures .......................75
Chromatography ..............................76
Quantification and Analysis................. 78
Results.......................................79
Effects of icy ACTH and LVP on in vitro
[3H]Catecholamine Accumulation............ 79
Substantia Nigral Slices Incubated with
ACTH Analogs ..............................79
Frontal Cortical Slices Incubated with
ACTH and LVP ..............................82
Striatal Slices Incubated with ACTH and
LVP .......................................86
Discussion. ....................................90
IV CONCLUDING REMARKS .............................102
REFERENCES. .....................................108
BIOGRAPHICAL SKETCH ............................122
iv




KEY TO ABBREVIATIONS ACTH Adrenocorticotropic hormone aMPT a-methyl-para-tyrosine AVP Arginine vasopressin CNS Central nervous system CSF Cerebrospinal fluid DA Dopamine EDTA Ethylenediaminotetra-acetic acid GABA Y-aminobutyric acid icy Intracerebroventricular LVP Lysine vasopressin MSH Melanocyte stimulating hormone MIF Melanocyte stimulating hormone inhibiting factor (PLG)
PLG Prolyl-leucyl-glycinamide (MIF) NE Norepinephrine RSA Relative specific activity SA Specific Activity
V




Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PEPTIDE HORMONE INTERACTIONS WITH
BRAIN CATECHOLAMINE METABOLISM
By
Richard L. Delanoy, Jr.
December 1979
Chairman: Adrian J. Dunn
Major Department: Neuroscience
The accumulation of [ HIcatecholamines from [3H]tyrosine in frontal cortical, septal, striatal and hippocampal slices was examined following intracerebroventricular (icy) injections of ACTH1-24, lysine vasopressin (LVP) and saline. Both ACTH1-24 and LVP selectively increased the accumulation of [3H]dopamine (DA) in frontal cortical slices but did not affect [3H]norepinephrine (NE) accumulation. LVP also inhibited the accumulation of [3H]DA in striatal slices. Incubations with ACTH analogs or LVP failed to alter the rate of accumulation of [3H]catecholamines in striatal, substantia nigral and frontal cortical
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slices. Also, neither peptide modified the activation of catecholamine synthesis by 26 mM K+, nor did ACTH1-24 modify the inhibition of [ 3H]catecholamine accumulation caused by
0.3 IM apomorphine and 3 iM haloperidol. The selective activation of the mesocortical DA system by icy injections of peptides which also occurs in response to footshock stress suggests that endogenous ACTH and/or LVP may mediate the stress-induced mesocortical DA activation. Alternatively, icy injections of these peptides may be stressful and thus elicit a stress response.
A variety of control experiments was performed to
characterize the normal kinetics and the effects of elevated K apomorphine and haloperidol on catecholamine synthesis. Our experiments showed that (1) DA was preferentially synthesized from tyrosine recently taken up from the media. Kinetic data exhibited Michaelis-Menton kinetics only when corrected by media tyrosine specific activity and not tissue tyrosine specific activity. (2) K+-induced activation of DA synthesis in striatal slices occurred at concentrations that were insufficient to induce [3H]DA release, suggesting that synthesis activation is not consequent on release, but rather is a separate response to depolarizations.
(3) Apomorphine inhibited both [3H]DA and [3H]NE accumulation in striatal, cerebellar and substantia nigral slices in a dose-dependent manner. The potency of 10 pM apomorphine in inhibiting the accumulation of [3H]catecholamines was reduced by half when the media tyrosine concentration was vii




increased from 8.3 to 83 pM. K -induced activation of tyrosine hydroxylase (TH) did not diminish the potency of apomorphine to inhibit [3H]DA accumulation. Since activated TH is less sensitive to end-product inhibition, then apomorphine-induced inhibition of synthesis probably is not mediated by end-product inhibition of TH. Furthermore, 10 nM haloperidol increased K+ -stimulated synthesis of DA, perhaps reflecting an antagonism of the inhibition of DA synthesis by released DA. These findings suggest receptor-mediated inhibition of catecholamine synthesis. The ability of apomorphine to inhibit NE synthesis, together with the presence of presynaptic DAreceptors on NE-terminals, further argues that apomorphine regulates synthesis by acting on a presynaptic receptor, although one not specific to DA-receptors. (4) Haloperidol also inhibited [3H]DA accumulation from [3H]tyrosine in a dose-dependent manner, but apparently not by a receptormediated mechanism. Haloperidol (10 jM) elevated basal release of labelled DA and the formation of labelled 3,4 dihydroxyphenylacetic acid in striatal slices preloaded with labelled DA. These effects were K and Ca2+ independent, suggesting that the increased cytoplasmic DA concentration was not a consequence of increased release and reuptake. These effects of haloperidol were not observed with fluphenazine and chlorpromazine, perhaps reflecting properities specific to butyrophenones. Since reserpine was
viii




found to have effects identical to haloperidol on synthesis, release and catabolism, haloperidol is proposed to have reserpine-like properties at micromolar concentrations. Previous in vivo and in vitro experiments that have used haloperidol as a typical neuroleptic may have obtained results related to vesicular disruption and unrelated to dopamine receptor blockade.
ix




SECTION I
INTRODUCTION
Evidence has accumulated that within the central nervous system (CNS), ACTH and vasopressin may mediate behavioral responses. Intracerebroventricular (icy) injections of ACTH elicit excessive grooming in rats and mice (Gispen et al., 1975; Rees et al., 1976). This ACTH-induced grooming was antagonized with peripheral injections of haloperidol (Wiegant et al., 1977) and with injections of various dopaminergic agonists and antagonists into the striatum and nucleus accumbens (Cools et al., 1978), suggesting that the mesostriatal dopamine (DA) system functioned in the expression of grooming. Excessive grooming can also be induced with mildly stressful stimulation (Colbern et al., 1978), a response which was antagonized with icy ACTH antisera (Dunn et al., 1979). Similarly, icy injections of arginine vasopressin (AVP) and lysine vasopressin (LVP) facilitated the performance of rats in both active and passive avoidance tasks. AVP antisera (icy) impaired the performance of rats on these same tasks (van Wimersma Greidanus and de Wied, 1975). Since AVP is released into the cerebrospinal fluid of rats in response to stressful stimuli (Dogterom et al., 1978), a facilitation of task performance as a result of stressful stimulation may be mediated by AVP.
1




2
Recently, footshock was determined to selectively activate the mesocortical DA system by two separate research groups (Thierry et al., 1976; Fadda et al., 1978). Increases in both turnover and synthesis that were observed in frontal cortex and nucleus accumbens were not evident in NE terminals in these regions nor in DA terminals in the striatum.
In order to test the hypothesis that icy ACTH or LVP
might mimic a stress-induced change in catecholamine metabolism and that the mesostriatal DA system mediates ACTHinduced grooming as suggested by Wiegant et al. (1977), norepinephrine (NE) and DA synthesis rates were measured in frontal cortex, striatum, septal nuclei and hippocampus following icy injections of either ACTH or LVP. The accumulation of [3H]catecholamines from [3H]tyrosine was measured in brain slices dissected from animals receiving peptide or control injections.
ACTH competes with [3H]haloperidol for binding to
striatal membranes (Czlonkowski et al., 1978) and dopaminergic agonists and antagonists inhibit ACTH-induced grooming. Neuroleptics at low concentrations antagonize apomorphineinduced inhibition of DA synthesis in vivo (Kehr et al., 1972) and in vitro (Ebstein et al., 1974; Christiansen and Squires, 1974). At high concentrations, haloperidol inhibited DA synthesis in vitro (Christiansen and Squires, 1974). Since ACTH might have DA agonist properties, ACTH was also incubated with striatal and substantia nigral slices challenged with doses of apomorphine or haloperidol that inhibited DA synthesis by approximately 50%.




3
In vitro techniques admittedly have disadvantages.
In particular, changes in catecholamine metabolism may have such short half-lives that an effect of peptide administration may not be detectable by the time the assay period begins. Furthermore, the loss of homeostatic regulation due to the severing of modulating neuronal input probably alters baseline catecholamine metabolism. However, the use of an in vitro system reduces the possibility of precursor pool alterations which might artifactually influence synthesis rate measurements. Also, the use of an in vitro technique readily permitted an investigation of peptidergic effects to extend to the tissue level. By incubating slices with peptides and simultaneously measuring catecholamine synthesis, attempts were made to determine whether or not peptide reception occurred in the tissue being incubated, independent of interacting variables caused b1y changes in endocrine, peripheral, or remote neuronal functioning. Such a technique has successfully detected effects of ether stress on hypothalamic catecholamine synthesis (Hedge et al., 1976) and has assessed the effects of a variety of dopaminergic drugs (Uretsky and Snodgrass, 1977; Christiansen and Squires, 1974) as well as other peptides (e.g.,MIF, Friedman et al., 1973).
Following a general discussion of background literature on the behavioral and biochemical interactions of ACTH and LVP with the catecholamines, data derived from these experiments are presented as two manuscripts for publication. The first section contains control experiments characterizing




4
normal incubation parameters and the effects of elevated K+, apomorphine, and haloperidol on catecholamine metabolism. The second section reports on the effects of ACTH and LVP on catecholamine synthesis in vitro.
Behavioral Effects of ACTH and LVP
The possibility that various pituitary hormones might
be involved in CNS function was first suggested by the observation that hypophysectomized animals were impaired in the acquisition of a conditioned avoidance task. Replacement therapy with ACTH or ACTH4-10 was able to reverse this deficit (Applezweig and Baudry, 1955; de Wied, 1964: de Wied and Gispen, 1977). Also, if given shortly before training, ACTH and ACTH4-10 stimulated acquisition of a conditioned avoidance response in intact animals (de Wied and Gispen, 1977; Flood et al., 1976; Gold and McGaugh, 1977). Similarly, neurohypophysectomized rats (de Wied, 1965), Brattleboro rats, which have a genetic deficiency of vasopressin (de Wied et al., 1975), and rats given icy AVP antiserum all showed deficits in active and passive avoidance learning (van Wimersma Greidanus et al., 1975).
ACTH1-24, ACTH4-10, (de Wied, 1966) and LVP (de Wied,
1971) have been reported to delay the extinction of a conditioned avoidance response; ACTH4-10 and desglycinamide-LVP (DG-LVP) delayed the extinction of sexually motivated behavior in rats (Bohus et al.. 1975; Bohus, 1977); and ACTH410 or ACTH1-24 delayed the extinction of rewarded
4-10behavior (Garrud et al. 1974. However, while a sinle
behavior (Garrud et al., 1974). However, while a single




dose of LVP was effective in delaying the extinction for several days, ACTH analogs had to be administered daily to maintain their effect.
Finally, ACTH, ACTH4-10, and vasopressin, under certain conditions were all able to reverse the effects of amnestic agents on passive avoidance tasks (Flood et al., 1976: Rigter et al., 1975).
It was been argued in the past by de Wied and his colleagues that both ACTH and LVP are directly affecting memory formation or a retrieval mechanism (de Wied, 1966; van Wimersma Greidanus et al., 1975). However, the requirement of daily administrations of ACTH to maintain delayed extinction suggests that ACTH-induced delay of extinction does not reflect an affect on memory mechanisms, since improved memory following a single post-training injection would be evident more than one day later. Kastin et al. (1977) have claimed that ACTH4-10 and [N-acetyl Serl] ACTH1-13 (a-MSH) facilitated performance in tasks designed to measure attention in both humans and rodents. Taken together, these data argue against a direct action of ACTH on memory processes and instead suggest some change in attention or motivational state.
In the case of LVP, de Wied still maintains that it has a direct effect on memory processes. However, it can be argued that the effects of LVP closely parallel the effects of non-specific stimulants such as caffeine, nicotine or amphetamine. Caffeine, nicotine or amphetamine can reverse




6
retrograde amnesia (Flood et al., 1978; Barondes and Cohen, 1968). Nicotine and amphetamines can facilitate acquisition of active avoidance learning (Orsingher and Fulgeniti, 1971). Furthermore, since hypophysectomy results in elevated levels of vasopressin in the cerebrospinal fluid (Dogterom et al., 1977), the deficits in avoidance learning in neurohypophysectomized rats argue for a peripheral site of the influence of vasopressin on learning performance and not for a central one. This finding conflicts with the earlier findings of de Wied showing that intracerebroventricularly administered arginine vasopressin (AVP) antisera will impair retention of a passive avoidance task. This might indicate that the AVP antibodies are causing a deficit in AVP release from the neurohypophysis or once cleared from the central nervous system (CNS) through the arachnoid granulations, are binding to plasma AVP. However, until it is shown that neurohypophysectomy, instead of a full hypophysectomy, will also result in elevated vasopressin concentrations in the cerebrospinal fluid, the hypothesis that exogenous vasopressin alters memory performance via a central receptor remains viable. Conversely, the data derived through the use of AVP antisera do not necessarily support the hypothesis that vasopressin is an essential component of memory processes. Instead, AVP may be performing an essential physiological process necessary for optimal CNS activation state for adaptive behaviors.
A major problem with these data supporting a central section of systemically injected hormones is that the




7
peripheral effects of hormones so administered are indistinguishable from any possible central effects. Glucose metabolism, blood pressure, lipolysis, and steroidogenesis are all affected by exogenous administrations of ACTH or LVP. A possible way of bypassing many of these peripheral responses is to administer the hormones intracranially. De Wied (1976) has studied extensively the behavioral effects of vasopressin using this route and has found facilitation of performance on the pole-jump active avoidance task at remarkably low doses (2.5 ng arginine vasopressin). Moreover, the fact that rats receiving AVP antisera or that Brattleboro rats lacking endogenous AVP have impaired memory performance suggests that LVP may have a physiological role as a neuromodulator. This is further supported by the findings that vasopressin- and neurophysin-bearing neurons and terminals, as determined by immunohistology, are found in various nuclei in the brain (Buijs, 1978; Swanson, 1976). In addition, intraventricular injections of LVP in mice will elicit an increase in activity which is predominantly grooming or a mixture of grooming, foraging, scratching and squeaking, depending on the dose applied (Delanoy et al., 1978). Kruse et al. (1977) reported that icy LVP given to rats most often produced barrel rotations (rotation around the sagittal axis of the animal), and occasional episodes of "nest-building" activity. In contrast, barrel rotation in response to icy LVP was seldom observed in mice (Delanoy et al., 1978).




8
Intracerebral implantations of 10 Pg crystalline ACTH1-10 and [D-phe7]ACTH1_10 in the rostral mesencephalon and caudal diencephalon or in CSF produced behavioral effects similar to systemic injections of larger doses of these peptides (van Wimersma Greidanus and de Wied, 1971). Moreover, icy ACTH has other behavioral effects. Doses of 10 ug or less of ACTH1-24 elicit penile erection (Bertolini and Baraldi, 1975), excessive grooming (Gispen et al., 1975; Rees et al., 1976) and stretching and yawning (Baldwin et al., 1974). Moreover, Colbern et al. (1978) have observed increased grooming in animals when introduced to a novel environment, which has been found to be attenuated by hypophysectomy and by icy ACTH antisera (Dunn et al., 1979). This finding, as well as the probability that ACTH neurons are present in the CNS (Barchas et al., 1978) and that pituitary peptides may enter the CNS by retrograde blood flow in the hypophysis (Oliver et al., 1977) suggests that behaviors induced by icy ACTH are patterns also initiated by endogenous ACTH.
These data argue that receptors capable of recognizing ACTH and LVP do exist in the CNS and may mediate the facilitation of performance observed with peripheral administrations of these hormones. However, changes in spontaneous behavior observed with icy ACTH or LVP have never been reported following peripheral administrations. Such a lack of response might be reflecting the relative inability of peptides to cross the blood-brain barrier, or perhaps a quantitative difference in minimal dosages necessary to elicit changes in spontaneous behavior.




9
Behavioral Interactions of Catecholaminergic Systems with ACTH and LVP Treatments
Several lines of evidence predict that the hormones ACTH and LVP would have an effect on CNS catecholamine metabolism. Firstly, the release of ACTH (Ganong, 1974) and vasopressin (Sandman et al., 1973b) as well as the activation of the noradrenergic system (Stone, 1975) all appear to be consequences of stressful episodes. It is possible that the hormonal release and the noradrenergic activation are redundant, parallel systems that do not interact. However, anatomical studies show that ACTH and neurophysin (proteins released from terminals of oxytocinand vasopressin-bearing neurons) send projections from the hypothalamus to the areas of the locus coeruleus and nucleus solitarius (Barchas et al., 1978; Swanson, 1977). Segal (1977) has reported that inhibition of firing of hippocampal neurons by norepinephrine (NE) was antagonized by iontophoresed ACTH. Also, the facilitation of a passive avoidance response by AVP was attenuated by lesions of the dorsal noradrenergic bundle, which supplies NE to much of the forebrain (Kovacs et al., 1979).
ACTH and LVP also appear to interact in some way with dopaminergic neurons. Systemic haloperidol attenuates the excessive grooming induced by icy ACTH (Wiegant et al., 1977) and icy LVP (Delanoy et al., 1978). More specifically, intranigral injections, but not intrastriatal injections of ACTH,would elicit excessive grooming. In addition, melanocyte




10
release inhibiting factor (MIF, prolyl-leucyl-glycinamide), a peptide that affects avoidance learning in a manner similar to vasopressin and that comprises the C-terminal tripeptide fragment of oxytocin, potentiated the salivation, piloerection, and Straub tail phenomenon (stiffened and erect) induced by L-DOPA. It also antagonized the catalepsy induced by fluphenazine, a DA-receptor blocker (Plotnikoff et al., 1974; Voith, 1977).
ACTH Effects on Catecholamine Metabolism
An orderly body of evidence on the effects of ACTH has been obtained measuring the disappearance of NE following TH inhibition with aMPT. Peripheral injections of ACTH1-24 (Hokfelt and Fuxe, 1972), ACTH4-10 (Leonard et al., 1975; Versteeg, 1973) and MSH (Kostrzewa et al., 1975) have all been reported to increase NE disappearance (turnover) in the CNS. [D-phe7]ACTH4-10, a peptide with behavioral activity opposite to ACTH4-10 in active avoidance tasks,has been reported to increase NE disappearance (Leonard, 1974) or to have no effect at all (Versteeg, 1973). This contradiction might be explained by the different injection routes used in these two studies. Hypophysectomy, in contrast, caused a decrease in NE disappearance (Fuxe et al., 1973; Fuxe et al., 1970; Friedman et al., 1973) that was reversed by high concentrations of ACTH (Versteeg and Wurtman, 1975, cited as data to be published, method of analysis not known) but not with low doses (Fuxe et al., 1970). These changes in NE disappearance




are apparently not mediated by an adrenal factor since adrenalectomy caused a glucocorticoid reversible increase in NE disappearance (Javoy et al., 1968; Fuxe et al., 1973) and since glucocorticoids and mineralocorticoids only slightly, if at all, decreased NE disappearance in intact animals (Fuxe et al., 1973).
These manipulations affect DA disappearance after
aMPT-induced synthesis inhibition much less clearly. Hypophysectomy decreased DA disappearance (Versteeg et al., 1972; Friedman et al., 1973) while adrenalectomy appeared to have little affect (Fuxe et al., 1973). Furthermore, corticosterone and dexamethasone will increase DA turnover in hypophysectomized, but not in intact rats (Fuxe et al., 1970; Jonsson et al., 1972). However, while NE disappearance was clearly increased with peripheral ACTH administrations, ACTH124 given to hypophysectomized rats had either no effect or only slightly decreased DA turnover in the neostriatum and limbic forebrain (Fuxe et al., 1973).
Using a different estimate of turnover, Endroczi et al. (1976) investigated the effects of icy ACTH1-24 and ACTH4-10
3
on the rate of disappearance of preloaded [3H]NE from neocortex, hypothalamus and hippocampus. Either peptide (5-10 ig, icy) promoted an increase in 3H disappearance. Adrenalectomy caused a similar increase in turnover, while glucocorticoids given to intact animals had no effect on the disappearance rate. Thus, exogenous administrations of ACTH, and perhaps the increase in endogenous ACTH following adrenalectomy




12
due to decreased inhibitory feedback, appeared to stimulate NE turnover, seemingly confirming data derived using the measurement of disappearance rates of endogenous amines following synthesis inhibition. However, an important limitation with these results is that icy injected [3H]NE can be taken up by dopaminergic and serotoninergic neurons as well as noradrenergic (Snyder and Coyle, 1969). Thus, the disappearance of labelled NE reflects a complex average of the activation of these three neuronal systems.
Two studies have attempted to measure in vivo synthesis by measuring the accumulation of [3H]catecholamines from subcutaneously injected [3H]tyrosine. In the first study, Versteeg and Wurtman (1975) showed that ACTH4-10 increased the accumulation of total [3H]catecholamines. Moreover, this increase did not occur in hypophysectomized rats given ACTH4-10. The authors argued that these data indicated that the effects of ACTH4-10 might be mediated by an adrenal factor. Another alternative is that adrenalectomy caused a chronic increase in ACTH release which would mask any effects of exogenous injections. luvone et al. (1978) took this analysis a step further by differentiating the amount of label associated with each amine. They found that ACTH1-24, ACTH4-10, and [D-phe7]ACTH4-10 increased DA but not NE synthesis, while a-MSH, 6-MSH, or LVP were without effect. The effect of ACTH4-10 on DA synthesis was blocked by adrenalectomy, in agreement with Versteeg and Wurtman (1975).




13
These last two studies disagree markedly from the results determined with the QMPT method of turnover analysis. While ACTH activates DA synthesis without an effect upon NE synthesis, ACTH also activates NE disappearance with no consistent effect upon DA disappearance. This dichotomy of results might be due to an artifact of differential latencies of activation of synthesis and release, different times of assaying (10 minutes for synthesis, 2 or more hours for the turnover studies), or may in fact indicate a property of ACTH's interactions with the catecholamine populations.
Effects of LVP on Catecholamine Metabolism
Using the aMPT method of turnover analysis, Tanaka et al. (1977a) examined the effects of intraventricular arginine vasopressin on catecholamine disappearance. Looking at large brain regions, increased NE turnover was reported in the thalamus, hypothalamus and medulla. Increased DA turnover was only observed in the preoptic area, with no effect at all on basal ganglia DA. Later that year, Tanaka et al. (1977b) presented a similar analysis from microdissected pieces of tissue. They reported that NE disappearance was accelerated in dorsal septal nucleus, anterior hypothalamic nucleus, medial forebrain bundle, parafascicular nucleus, dorsal raphe nucleus, locus coeruleus, and the nucleus tractus solitarii. In contrast with their earlier publication, AVP was shown to increase the disappearance of DA in caudate nucleus several fold, as well as in median eminence,




14
dorsal raphe nucleus, and region A8. The inconsistency of the changes in striatal DA between the two tissue collection procedures is hard to explain but may be reflecting local areas within the basal ganglia which are preferentially activated by icy AVP injections. Nevertheless, an effect of icy AVP on striatal turnover appears to have been substantiated by these authors. AVP antisera reduces the disappearance rate of striatal DA (Versteeg et al., 1978). Furthermore, Brattleboro rats with an hereditary deficiency in vasopressin also have a reduced rate of striatal DA turnover (Versteeg et al., 1979). The only study to examine the effects of peripheral administrations of vasopressin on catecholamine synthesis was that by luvone et al. (1977), which did not find an effect.
The terminal tripeptide of oxytocin, MIF (PLG) has
behavioral potency in memory tasks (Sandman et al., 1973a; Stratton and Kastin, 1975) and like LVP will facilitate morphine dependence (van Ree and de Wied, 1976). However, icy MIF is ineffective as compared to LVP in terms of eliciting changes in spontaneous behavior (Delanoy et al., 1978). The effects of MIF on catecholamine metabolism are controversial at the moment. Using the aMPT method of measuring turnover, Versteeg et al. (1978) and Pugsley and Lippman (1977) report that MIF at 40 and 5 mg/kg body weight respectively caused an increase in DA turnover. Kostrzewa et al. (1974) and Plotnikoff et al. (1974) reported that MIF at 20 and 100 mg/kg respectively had no effect on turnover. DA




15
synthesis was reported to be increased in slices of striatum in intact but not hypophysectomized rats treated with 0.5 and 5 mg/kg MIF (ip). In view of reports that indicated that MIF potentiated the induction of salivation, piloerection and the Straub tail phenomenon by L-DOPA (Plotnikoff et al., 1974) and the reversed the induction of catalepsy by fluphenazine (Voith, 1977), it is likely that MIF is indeed affecting DA metabolism.




SECTION II
HALOPERIDOL AND APOMORPHINE ALTER CATECHOLAMINE SYNTHESIS IN VITRO BY DIFFERENT MECHANISMS Introduction
The experiments described in this report were initiated to test the effects of the pituitary peptides ACTH and lysine vasopressin (LVP) on catecholamine synthesis in vitro. ACTH has been shown to compete with [3H]haloperidol for binding to striatal membranes (Czlonkowski et al., 1978). Also, grooming induced by intracerebroventricular (icy) injections of ACTH was inhibited by haloperidol (Wiegant et al., 1977). Therefore, in preparation for studies examining possible interactions of haloperidol and the dopamine (DA) agonist apomorphine with ACTH and to test the validity of our slice preparation as a model of in vivo catecholamine synthesis, the effects of these two drugs on the accumulation of [ H]catecholamines from [3H]tyrosine were evaluated. Our initial experiments confirmed earlier reports that both haloperidol (Christiansen and Squires, 1974) and apomorphine (Christiansen and Squires, 1974; Ebstein et al., 1974) inhibited DA synthesis in vitro. The paradox that an agonist and an antagonist of DA receptors would have the same effect on synthesis prompted us to further examine the mechanisms by which these two agents exert their effects.
16




17
Iontophoretic administrations of DA or apomorphine into the substantia nigra inhibited the firing of dopaminergic neurons in the pars compacta of the substantia nigra (Skirboll et al., 1979; Aghajanian and Bunney, 1977; Groves et al., 1975). The inhibition of firing might have been due to a presynaptic facilitation of gamma-aminobutyric acid (GABA) release from terminals arising from cells in the striatum (Reubi et al., 1978). However, the existence of dendro-dendritic synapses between dopaminergic dendrites in the pars reticulata of the substantia nigra (Wilson et al., 1977) has supported the suggestion of Aghajanian and Bunney (1974) that DA receptors exist on dopaminergic neurons. Such receptors which mediate self-inhibition within a neuronal population have been collectively called autoreceptors. In noradrenergic neurons, dendritic autoreceptors inhibit cellular firing in response to adrenergic agonists (Aghajanian et al., 1977), as was observed in dopaminergic neurons. In addition, exogenous norepinephrine (NE) inhibited the K+-stimulated release of [3H]NE from noradrenergic terminals in vitro (Starke et al., 1977). Since the inhibition was reversed with a-adrenergic receptor blockers, a presynapic autoreceptor probably also exists.
The existence of a similar presynaptic autoreceptor on
DA terminals was supported by the finding that [3H]apomorphine binding was reduced following lesions of the nigrostriatal tract with the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA, Nagy et al., 1978). However, unlike NE presynaptic




18
receptors, most studies have not found an effect of dopamine agonists on DA release (Dismukes and Mulder, 1977; Seeman and Lee, 1975; Raiteri et al., 1978), although apomorphine has been reported to inhibit electrically (Farnebo and Hamberger, 1971) and K+ (Miller and Friedhoff, 1979) stimulated [3H]DA release by less than 20%. Also, DA receptor blockers inhibited the stimulated release of [3H]DA, an effect which would not be predicted by an action on dopaminergic presynaptic autoreceptors (Seeman and Lee, 1975; Raiteri et al., 1978). Thus, either DA autoreceptors do not modulate release, or they do so by mechanisms different from those found in noradrenergic terminals.
An alternative mechanism by which dopaminergic autoreceptors may regulate synaptic efficacy is by inhibiting DA synthesis. At micromolar concentrations, apomorphine strongly inhibited DA synthesis in striatal slices and synaptosomes, an effect reported to be reversible by neuroleptics (Christiansen and Squires, 1974; Westfall et al., 1976). However, haloperidol also inhibited DA synthesis in striatal slices at micromolar concentrations (Christiansen and Squires, 1974), a result which contradicts both the hypothesized regulation of DA synthesis by an autoreceptor, and the consistently reported finding that haloperidol activates DA synthesis in vivo (Kapatos and Zigmond, 1979; Carlsson et al., 1977; Lerner et al., 1977).
In a single incubation system, we have investigated these apparent contradictions by comparing the effects of




19
haloperidol and apomorphine on catecholamine synthesis and DA release, metabolism and uptake. Brain slices were employed rather than synaptosomes to maintain tissue integrity insofar as possible so that the chances of observing effects of ACTH and LVP would be improved. Characterizations of normal kinetics and the effects of elevated concentrations of K+ on the accumulation of [3H]catecholamines from [3H]tyrosine are also presented.
Our findings indicate that while the effects of
apomorphine on synthesis probably are mediated by a DA receptor, the effects of haloperidol on catecholamine metabolism in most in vitro studies may have been due to membrane disruption as originally suggested by Seeman et al. (1974).
Methods
Materials
Male CD-1 mice (Charles River, Wilmington, Mass.,
25-30 g), were used in all experiments; they were individually housed for the three days immediately prior to killing. Animals were maintained on a 7:00 a.m. 7:00 p.m. lighting schedule.
Drugs used in these experiments were as follows:
apomorphine (hydrochloride, Sigma), chlorpromazine (hydrochloride, Sigma), fluphenazine (hydrochloride, a gift from E.R. Squibb and Sons, Inc.), haloperidol (crystalline, a gift from McNeil Laboratories), and reserpine (crystalline, Sigma). Radioactive materials used were [2,6-3H]tyrosine




20
(34 Ci/mmol, The Radiochemical Centre, Amersham), [l-1C]tyrosine (58 mCi/mmol, New England Nuclear), and [l-1'C]dopamine hydrochloride (55 mCi/mmol, The Radiochemical Centre, Amersham). Other chemicals used in tissue preparations were HEPES (Ultrol, Calbiochem), TRIS (Trizma base, Sigma), PPO (Scintillar, Mallinkrodt), POPOP (Research Products International Corp.), and Triton X-100 (Scintillar, Mallinkrodt).
The medium used in all experiments was that described by Versteeg et al. (1974) and contained the following: 118 mM NaCl, 4.4 mM KC1, 2.6 mM CaC12, 1.3 mM MgSO4,
1.2 mM K2HPO4, 25 mM HEPES, and 12 mM glucose titrated to pH 7.3 with NaOH. Increases in KC1 were countered with an equivalent decrease in NaCl.
Apomorphine and haloperidol were dissolved in 0.2 M
acetic acid at 2 pg/jil and diluted to 0.25 ug/il with medium. Chlorpromazine and fluphenazine were dissolved in a drop of glacial acetic acid and diluted to 0.25 ig/pl with medium. Reserpine was solubilized in a drop of glacial acetic acid and diluted to 5.0 pg/l with medium. 12.1 4l of apomorphine, 15 ~Il of haloperidol, 17.1 pl of chlorpromazine or 14 ol of fluphenazine were added to a one ml final incubation volume to achieve a final concentration of i0-5 M. Twelve Pl of the reserpine stock solution was added to one ml of medium to achieve a i0-7 M concentration. Lower concentrations were produced by serial dilutions of the stock solutions with media. 'Saline' controls were composed of appropriate volumes of acetic acid and media in appropriate proportions.




21
Procedures
Mice were killed by decapitation. Brains were removed and striatum, substantia nigra, or cerebellum were dissected out on a chilled surface. Substantia nigra was obtained by the method of Westerink and Korf (1976). A coronal slab was removed by sectioning at the border of the pons and mesencephalon and at a plane 0.8 mm rostral. Substantia nigra, visible near the ventral surface of the slab, was removed along with the tegmental nucleus (A0lO) from the rest of the mesencephalon. In a typical 50 sample experiment, 10, 20, or 40 mice were used to generate slices of cerebellum, striatum, or substantia nigra, respectively. Dissected pieces were sliced in two dimensions with a McIlwain Tissue Chopper set at 300 pm. Slices were suspended and pooled in medium at 370C in 02:COZ2 (95:5) atmosphere. After 30 min the medium was aspirated and replaced with fresh, equilibrated medium for an additional 10 min.
Four kinds of experiments were performed. 1.) The
3
accumulation of [3H]catecholamines from [ H]tyrosine was measured. Prepared slices (0.5 ml) were added to a series of Corex round bottomed 15 ml centrifuge tubes containing 0.5 ml of equilibrated medium containing [2,6-3H]tyrosine, cold tyrosine, and appropriate drugs. Samples were incubated for 40 min. The reaction was stopped with 2 ml of ice-cold medium and centrifuged at 100 x g in a refrigerated (4C) Sorvall RC-3 Centrifuge. Media were decanted and




22
in some instances saved and processed for measurement of released metabolites. Another 2 ml of ice-cold medium was added to the samples and the centrifugation repeated. The media were once again decanted.
2.) The release and metabolism of labelled dopamine from preloaded slices were studied. Pooled slices were exposed to [3H]tyrosine (250 iCi/10 ml medium) for 10 min. Then [4C]dopamine (10 jiCi/10 ml medium) was added and the slices were incubated for a further 10 min with both labelled substrates. Slices were then washed four times with fresh equilibrated media. Slices preloaded in this manner with [3H]-and [14C]DA were added to Corex centrifuge tubes containing 0.5 ml of equilibrated medium containing appropriate drugs. Slices were incubated for 10 min and the reaction was stopped with 2 ml of ice-cold medium. Tissue and media were separated as described above.
3.) The uptake of [1-_1C]DA into striatal slices was examined. Prepared slices (0.5 ml) were added to Corex centrifuge tubes containing 0.5 ml medium, 0.1 yM [1-14C]DA, and drugs. Samples were incubated for 3 min. Uptake was terminated with 2 ml ice-cold medium.
4.) The liberation of [14C]CO2 from [l-14C]tyrosine was measured. Slices and media were prepared in the same manner as in the accumulation experiments, except that [l-4C]tyrosine was used in place of [3H]tyrosine. Once the slices were added to equilibrated media containing drugs and label, a plastic cup filled with glass wool, moistened




23
with 50 pl Soluene 350 (Packard Instrument Co) and suspended from a rubber stopper was inserted into the Corex centrifuge tube. After 20 min incubation, the reaction was stopped with 100 lil of 50% trichloroacetic acid (TCA) injected through the rubber stopper. After overnight refrigeration, each plastic cup was removed and placed in a scintillation vial with 10 ml of toluene based scintillator (14 g PPO, 0.4 g POPOP,
3.5 Z toluene).
Chromatography
Purification procedures were essentially those of Iuvone et al. (1977) and recoveries were similar. Rinsed slices were homogenized in 2 ml of 0.4 M perchloric acid with 0.5% sodium metabisulfite (w/v) in Corex centrifuge tubes with a teflon pestle machined to a diameter of 14.5 mm to fit the tubes tightly. The homogenate was centrifuged at 15000 x g in a refrigerated (4oC) Sorvall RC-2B centrifuge. Supernatants were loaded onto AG-50 x 4 cation exchange columns
H+
(6 x 20 mm, H form). The load volume contained tritiated water, 3,4-dihydroxyphenylacetic acid, and methylated, deaminated metabolites. Elutions with sodium acetate buffer (0.1 M sodium acetate, 0.1% sodium ethylenediaminetetraacetic acid (EDTA), titrated with 10 M NaOH to pH 7.0),
1 N HC1, and 4 N HC1 released tyrosine, norepinephrine, and dopamine, respectively. These fractions were further purified by alumina absorption or ion-exchange chromatography in the case of tyrosine.




24
The load volume, together with a 2 ml wash with
sodium acetate buffer, was collected in test tubes containing 200 mg of alumina prepared by the method of Anton and Sayre (1962) and moistened with 180 pl 0.1 M disodium EDTA. Samples were then titrated with 3 M Tris hydroxide (approximately 0.5 ml) to a pH of 7.9 and vortexed for five min. The alumina suspension was transferred to a glass wool stoppered pasteur pipette. The fraction not bound to the alumina, along with 0.5 ml of a water wash, was collected in a scintillation vial containing 10 ml of a Triton scintillator (10.68 g PPO, 0.12 g POPOP, 1 Z toluene, 1.4 k Triton X-100). This fraction contained tritiated water and methylated, deaminated metabolites of DA. The alumina was further washed with another 5.5 ml of water, and then catechols were eluted with 2 ml of
0.5 M HC1 into scintillation vials, and counted in 10 ml of Triton scintillator. Radioactivity in this fraction was determined to be 3,4-dihydroxyphenylacetic acid (DOPAC) by paper chromatography in n-butanol:acetic acid; H20, (120:30:50,v/v/v).
Once the load volume and a 2 ml sodium acetate buffer wash had been collected, the cation-exchange columns were washed with a additional 6 ml of sodium acetate buffer. Tyrosine was eluted with a second 6 ml of sodium acetate




25
buffer, titrated to pH 1.5 with 4 M HC1, and loaded onto standard AG-50 x 4 cation exchange columns charged with Na+. Following washes with 5 ml water and 8 ml 0.5 M HC1, tyrosine was eluted with 9 ml 0.1 M Na3PO. Of this volume,
0.9 ml aliquots were added to 0.1 ml 4 M HC1 and 10 ml Triton scintillator, while 1.8 ml aliquots were assayed for tyrosine using nitrosonaphthol as described by Udenfriend (1962). Fluorescence was measured on a Perkin-Elmer model 650-O10S Fluorescence Spectrophotometer.
After the tyrosine fraction was eluted from the original AG-50 x 4 columns, they were washed with 2 ml sodium acetate buffer and 2 ml of 1 M HC1. NE was then eluted with 4.75 ml of 1 M HC1 into test tubes containing 200 mg alumina. Following a further wash with 1.25 ml 1 M HC1, DA was eluted with 2.5 ml of 4 M HC1 into test tubes containing 200 mg alumina. NE and DA fractions were titrated to pH 7.9 with approximately 2.6 ml 3 M Tris and with 2.5 ml 3 M Tris and approximately 0.5 ml 10 M NaOH, respectively. Preparation, transfer, and filtration of alumina, as well as the elution of NE and DA from alumina,were described above for the purification of DOPAC.
In experiments in which release from the slices during incubation was analyzed, the media were titrated to pH 1.5 with 63 pl 4 M HC1 and loaded on to the AG-50 x 4 (H+ form) columns. Elution profile and secondary purifications were identical to those used for the homogenate supernatant, except that the sodium acetate buffer wash preceding the tyrosine elution was shortened to 4 ml instead of 6 ml.




26
The pellets formed by the centrifugation of the homogenate were dissolved in 2.5 ml of 0.3 M NaOH. Protein determinations were performed on duplicate 250 pl aliquots using the method of Bailey et al. (1967). Quantification and Analysis
Radioactivity was assayed using a Packard 2425 Liquid Scintillation Spectrophotometer. Correction factors for quenching and for emission spectrum overlap in double label experiments were determined with external standardization.
The disintegrations/minute (DPM) for each fraction (except media tyrosine) were divided by the amount of protein in that tissue sample to derive specific activities. Tyrosine specific activity was DPM/vg tyrosine. Blank values, determined in samples that received radioactivity but were chilled to 00C instead of being incubated, were subtracted from experimental values. Data for accumulation studies were expressed as moles of catecholamine accumulated/mg protein/minute (based on the tyrosine specific activity). Data from metabolism studies were expressed as percent total radioactivity recovered in fractions containing labelled dopamine or its metabolites.
One factor analyses of variance (ANOVA) and Student's
t-tests were performed on a Hewlett-Packard 9810A Calculator. An Amdahl 370, version 6 computer was used for quench and double label corrections and for two or more factor ANOVA's as programmed in the Statistical Analysis System (SAS).




27
Results
Determination of Relevant Precursor Pool
Striatal slices were incubated in media with various
concentrations of tyrosine as described in Fig. 2.1. Measurements of [3H]DA accumulation (DPM/pg protein) were divided by the initial (calculated) tyrosine specific activities. When plotted as a function of initial tyrosine concentration on a double reciprocal plot, normal MichaelisMenton kinetics were observed (Fig. 2.1a). However, because tyrosine was being exchanged between the media and the slices, the specific activities of both media and tissue would be somewhat different from the initial specific activities. Thus, we measured the specific activities of each pool at all concentrations of tyrosine and repeated the kinetic analyses. The measured concentrations of media tyrosine were markedly higher than those originally added to the media when the initial concentration was less than 1 ug/ml. This difference confirmed that tyrosine was being released from the tissue, diluting the medium tyrosine specific activity (Table 2.1). Similarly, initial media tyrosine concentrations of more than 1 ug/ml elevated tissue tyrosine concentrations.
The DA specific activity was normalized by either the measured media or the measured tissue tyrosine specific activity and subsequently plotted on double reciprocal plots as functions of measured media or measured tissue tyrosine concentrations, respectively. Despite the narrowing of the




28
A. CorreCted by viial
90 'mdio tyrosr
specific act.vty
705 10
10
_Io L
iritiol tyrosiwe (pg/mI)
B. Corrected by measured C. Corrected by .casred 18 medio tyrosine spgcific activity tim e tyroamne specific tiuiy
~4
S2
.5 0.5 1.0 1.5 .2 .4 .8 I I media tyrosine(pg/mil) tissue tyrose(pqg/mqg prote)i
Fig. 2.1. Kinetics of [3H]DA accumulations from [3H]tyrosine: effects of correcting with initial media, measured media, and measured tissue tyrosine specific activities. Striatal slices were incubated for 40 min with 4.17 pCi/ml [3H]tyrosine at 0.03, 0.1, 0.3, 1, 3, and 10 ig/ml tyrosine. DPM/mg protein was divided by tyrosine specific activity (DPM/ng tyrosine) and graphed as Lineweaver-Burke plots against tyrosine concentrations (pg/ml media). (A) [3H]DA accumulation normalized by initial tyrosine specific activity as a function of initial tyrosine concentration. (B) The same data normalized by measured media tyrosine specific as a function on measured media tyrosine and (C) by measured tissue tyrosine specific activity as a function of measured tissue tyrosine concentration. Each point represents the mean of 3 determinations.




29
Table 2.1. Relationship of Measured Media and Measured Tissue Tyrosine Concentrations to Initial Tyrosine Concentrations.
Tyrosine Concentration
Initial Measured Media Measured Tissue (vg/ml) (jig/ml) (pg/mg protein)
.03 .58.06 1.73.40 .10 .56.12 1.55.37 .30 .68.08 1.36.13
1.00 1.09.07 1.85.49 3.00 2.40.12 4.181.19 10.00 8.98.11 4.18.58




30
range of tyrosine concentrations in the media, the data corrected with the media tyrosine specific activities displayed Michaelis-Menton kinetics with a Vmax and a Km similar to those determined using the initial tyrosine specific activity. Data normalized with tissue tyrosine specific activity did not display normal kinetics. Although not shown, the labelling of protein with [3H]tyrosine also displayed Michaelis-Menton kinetics only when media tyrosine specific activities were used to normalize the data.
Since media tyrosine concentrations above 1 vg/ml were
not appreciably diluted by tyrosine released from the slices, subsequent experiments used 1.5 pg/l (8.3 pM) or 15 pg/ml (83 pM) tyrosine concentrations in the medium. The specific activity of media tyrosine, the pool which best reflects the relevant precursor, was assumed to be unaltered during incubation.
+ [3HCtcoaie K -Induced Activation of the Accumulation of [3H]Catecholamines
Various concentrations of KC1 were added to striatal
slices incubated in 83 pM tyrosine with [3H]tyrosine. [3H]DA was recovered from both slicesand media. As shown in Fig. 2.2, elevated K+ increased the formation and release of [3H]DA. Combined slice and media [3H]DA accumulations, presumably reflecting total synthesis, reached a maximum at 18 mM K+. Interestingly, accumulation of [3H]DA in the slices was increased by 14 mM K+ which had no significant effect on release. The accumulation of [3H]DA from [3H]tyrosine in striatal slices was elevated by 158%, 274%,




31
4 Combined
Slices
a 2....
X- *5*
c .. Media
U)
Eo
0.
I~ ~ ... X.......
' ... .. I I I
6 10 14 18 26 46 mM K
+
Fig. 2.2. Effects of elevated K on the accumulation of [3H]DA in striatal slices and media. Striatal slices were incubated at 83 iNM tyrosine and 4.17 pCi/ml [3H]tyrosine. Each point represents the mean and standard error of 4 determinations. Combined accumulation is the sum of slice and media accumulations.




32
and 322% by 26 mM K+ at 8.3, 27.4, 83, and 274 iM tyrosine, respectively. However, increased accumulation observed with elevated K was not consistently found in other regions.
3 3
[3H]NE accumulation from [3H]tyrosine was not facilitated in cerebellar slices incubated at 8.3 pM tyrosine. In substantia nigral slices, both [3H]NE and [3H]DA accumulations were facilitated at a medium concentration of 8.3 VM tyrosine, but not at 83 pM tyrosine (Fig. 2.3). Inhibition of [ H]Catecholamine Accumulation from [$HjTyrosine
Apomorphine, haloperidol and reserpine all potently inhibited the accumulation of [3H]DA in striatal slices (Fig. 2.4). Chlorpromazine (10 jM) caused a slight but significant inhibition (24%), while fluphenazine had no significant effect on [3H]DA accumulation.
Apomorphine and haloperidol were also tested at a range of doses in the presence of 26 mM K+ (Fig. 2.5). Generally, 26 mM K tended to reduce the effectiveness of haloperidol as compared to incubations at 6 mM K+, while it facilitated the effect of apomorphine. Furthermore, 10 n~I
3
haloperidol caused a slightly elevated [3H]DA accumulation in slices exposed to 26 mM K+. Similar, but nonsignificant increases were observed with 10 nM haloperidol and 0.1 nM reserpine at standard (6 mM) K+.
Reserpine, apomorphine and haloperidol all inhibited the accumulation of [ 3H]DA in substantianigral slices incubated at 8.3 iM tyrosine. Surprisingly, apomorchine




33
A. 8.3 pM tyrosine
Substantial nigra Cerebellum 9080 70
E
60
S
o 50
20. 6 E 30- CL
20 6 Z 4
C
10 2
O02
4 4 4 4 SAL 26mM K* SAL 26nM K, B. 83 pM tyrosine
Substantial nigra
DA
so80 NE
S60 *P <.01
E
40
20
8:8 8
SAL 18rM K' 26mM K'
Fig. 2.3. Effects of elevated K+ on the accumulation of [3H]DA and [3H]NE in substantia nigral and cerebellar slices. Open bars represent [3H]DA accumulation in slices. Shaded bars represent [3H]NE. Numbers at the base of each bar indicate the sample size for each group. SAL indicates standard incubation conditions (6 mM K ).
** indicates a significant difference from saline at p < .01 (ANOVA). (A) Substantia nigral and cerebellar slices incubated with 4.17 uCi/ml and 16.7 pCi [3H]tyrosine, respectively at 8.3 iJM tyrosine. (B) Substantia nigral slices incubated with 16.7 pCi/ml [3H]tyrosine at 83 ~iM tyrosine.




34
Reserpine e--Apomorphine o- -o Hatoperidol AChlorpromozine *---. Fluphenazine A- -A 120
100
o
0
.. 80
z
o 60
40
20
2 O **~l
1-10 o-9 6-8 10-7 1-6 10-5
DRUG CONCENTRATION (M)
Fig. 2.4. Inhibition of striatal [3H]DA accumulation by reserpine, apomorphine, haloperidol, chlorpromazine, and fluphenazine. Striatal slices were incubated with 4.17 UCi/ml [3H]tyrosine at 8.3 pM tyrosine and 6 mM K+. Each point represents the mean and standard error of 4 determinations, except for haloperidol, in which each point represents the mean of at least 8 determinations. The rate of [3H]DA accumulation for saline (100% value) was
0.89 pmoles/mg protein/min. ** represents a significant difference from saline at p < .01, *** at p < .001 (ANOVA).




35
Apomorphine
6mM K' o--o
26mM K" e---.
120- Holoperidol S26rm K' --,
I00
j *P <.05
0
O{2 80
z
o 60
40
20
10-10 10-9 10o8 1fo7 106 o-5 DRUG CONCENTRATION(M)
+
Fig. 2.5. Interactions of elevated K with haloperidoland apomorphine-induced inhibitions of striatal [3H]DA accumulation from [3H]tyrosine. Curves generated at 6 mM K+ are the same as presented in Fig. 2.4.+ The rate of [3H]DA accumulation for saline. at 26 mM K is 3.54 pmoles/ mg protein/min. Each point represents the mean and standard error of 4 determinations for apomorphine and at least 8 determinations for haloperidol. indicates a significant increase from saline at p < .05 (ANOVA).




36
and haloperidol, as well as reserpine, also inhibited the accumulation of [3H]NE (Fig. 2.6). [3H]NE accumulation was also inhibited by haloperidol and apomorphine in cerebellar slices as much as in substantia nigral slices, indicating that the effect was not an artifact of incomplete chromatographic separation of [3H]DA and [3H]NE (Fig. 2.7).
Like 26 mM K+, the effect of apomorphine on substantia nigral slices was diminished by 83 iM tyrosine (Fig. 2.6b). At a medium concentration of 8.3 pM tyrosine, 10 pM apomorphine inhibited [3H]DA (82%) and [3H]NE (65%) accumulations twice as much as observed at 83 VM tyrosine (41 and 30%, respectively). 10 pM haloperidol was equally effective at
8.3 and 83 pM tyrosine.
This decrease in the accumulation of [3H]DA and [3H]NE caused by the various drugs tested could be caused by nonspecific inhibition of cellular metabolism, a decrease in synthesis, a decrease in the reuptake of newly synthesized and released [3H]catecholamines, or a dramatic increase in the release and/or metabolism of the newly synthesized catecholamines.
Accumulation of [3H]Protein from [3H]Tyrosine
Since haloperidol and apomorphine affected the accumulation of both [3H]DA and [3H]NE, these agents might have a nonspecific effect upon cellular metabolism. However, the labelling of protein in the slices incubated with [3H]tyrosine was not affected by either haloperidol or apomorphine (Table 2.2).




37
A. 8.3 p M Tyrosine
g DA
*NE
30
*<.05
Ep<.OI
20
o
C.
I0" **
1 10SAL 106 10-5 10-6 10-5 t8 IO7 M HALOPERIDOL M APOMORPHINE U RESERPINE B. 83 pM Tyrosine
100
80
S60
E
% 40
E
20
SAL 107 10-I6 I0-5 107 106 iO5 M HALOPERIDOL U APOMORPHINE
Fig. 2.6 Inhibition of [3H]catecholamine accumulation from [3H]tyrosine in substantia nigral slices by haloperidol, apomorphine, and reserpine. Open and shaded bars represent [3H]DA and [3H]NE, respectively. ** indicates a significant difference from saline at p < .01, at p < .05 (ANOVA).
(A) Nigral slices were incubated with 4.17 Ci/ml [3H]tyrosine at 8.3 iM tyrosine and (B) with 16.7 pCi/ml [3H]tyrosine at 83 uM tyrosine.




38
12
oE
10
C
C 8
O
P 6
E *
04
E
2 *
4 4 4 4
SAL 10-6 10-5 10-6 10-5 M Apomorphine M Holoperidol
Fig. 2.7. Inhibition of [3H]NE accumulation from [3H]tyrosine in cerebellar slices by apomorphine and haloperidol. Cerebellar slices were incubated with 16.7 UCi/ml [3H]tyrosine at 8.3 pM tyrosine. Numbers at the base of each bar indicate the sample size. ** indicates a significant difference from saline at p < ..01 (ANOVA).




39
Table 2.2. The Accumulation of [3H]Protein from [3H]Tyrosine: Effects of Haloperidol and Apomorphine at 8.3 and 83 pM Tyrosine.
DPM/Pg Proteina
Treatment
Treatment 8.3 pM Tyrosine 83 vM Tyrosine
Saline 12.081.67 1.87.36
Haloperidol 12.081.03 2.13.23 (10 vIM)
Apomorphine 10.881.52 2.10.02 (10 IM)
a
a Homogenate pellets were dissolved in 2.5 ml of 0.3 M NaOH. One ml aliquots were added to 0.4 ml of 25% trichloroacetic acid (TCA). After chilling at 00C for 10 min, samples were centrifuged at 100 x g in a refrigerated (40C) Sorvall RC-3 centrifuge. The supernatant was decanted and replaced with 4 ml of 5% TCA. Once the centrifugation and decanting were repeated, the pellet was digested in
1.0 ml of Soluene 350 and added to 10 ml of toluene-based scintillation fluid.




40
Liberation of [1'4C]C02 from ["C]Tyrosine
In order to confirm an effect upon synthesis, the liberation of ['4C]C02 from striatal slices incubated with 8.3 VM [-14C]tyrosine was determined. Synthesis, as measured by the release of [4C]COz, was inhibited by both apomorphine and haloperidol (Fig. 2.8). Therefore, the decreased [3H]catecholamine accumulation probably reflected inhibition of DA and NE synthesis. For unknown reasons, both haloperidol and apomorphine were effective at doses lower than was found by measurements of accumulated [3H]DA.
Uptake of [14C]DA
Striatal slices were incubated with 0.1 pM [1-14C]DA
for a period of 3 min. Comparisons between 10 iM haloperidol and saline were made both with and without 12.5 pM nialamide and 100 pM EGTA, which were used to diminish subsequent release and metabolism of accumulated [14C]DA (Table 2.3). Under both conditions, haloperidol decreased the accumulation of [1 C]DA. However, haloperidol also increased the formation of [14 C]DOPAC. The increased [1C]DOPAC found in the media was probably formed by monoamine oxidase in dopaminergic terminals and,therefore, reflects additional [4 C]DA uptake by the slices. When the sum of [14C]DOPAC and [14C]DA is considered, the effect of haloperidol is reduced. Despite the fact that nialamide reduced [14C]DOPAC to near blank levels, an increase in [14C]DOPAC was still observed with haloperidol.




41
Apomorphine o--o 120
Haloperidol
100
o *
_j 80
C.,
o0 60o
40 -1 **
0
S40
p Ik
I I I I I I
10o 10 1o-9 10- I0-7 -6 10-5 DRUG CONCENTRATION (M)
Fig. 2.8. Inhibition of [1'C]C02 liberation from ['C]tyrosine by apomorphine and haloperidol. Striatal slices were incubated with 83 vCi/ml at 8.3 iiM tyrosine and 6 mM K+ for 20 min. Each point represents the mean and standard error of four determinations. The rate of [14C]C02 accumulation for saline was 8.75 pmoles/mg protein/min. indicates a significant difference from saline at p < .05, ** at p < .01 (ANOVA).




42
Table 2.3. Haloperidol inhibits the Accumulation of [14C]DA from Media, but not Total Uptake.
Treatment [14C]DAa 14C]DOPACb Totalc
Saline 1.09.08 .69.03 1.78.08
Haloperidol .85.06 .82.02 1.67+.07 (10 JIM)
-22% -6% With 12.5 PM
Nialamide and
100 pM EGTA:
Saline 1.69.06 .02.02 1.73.07
Haloperidol 1.48.06 .15.09 1.63.19 (10 JIM)
-12% -6%
Significance at p<.05 (two factor ANOVA, Duncan's) aFrom slices alone, DPM/pg protein bCombined from both slices and media, DPM/Pg protein c Sm 14 14 CSum of [14C]DA and [14C]DOPAC Note: Main effect of haloperidol in the ANOVA significant for DA at p<.02S and for DOPAC at p<.05.




43
Release and Metabolism of Preloaded Labelled DA
The effects of reserpine, apomorphine and the neuroleptics haloperidol, chlorpromazine and fluphenazine upon the disappearance of labelled DA were measured in two possible pools of DA, simultaneously. Pooled slices were incubated with both [3H]tyrosine and [14C]DA. Following a thorough washing to remove excess radioactivity, 0.5 ml of prepared slices were added to 0.5 ml of unlabelled media containing the pharmacological treatments. Radioactive DA in the media, DA remaining in the slices, total (slice and media combined) DOPAC, and total residual (tritiated water, homovanillic acid) radioactivity were measured (Fig. 2.9). Haloperidol (10 pM) and reserpine (0.1 pIM) caused a disappearance of between 30 and 50% of radioactive DA from the slices. Passive release (i.e. not induced by veratridine, elevated K+ or electrical stimulation) for both pools of radioactive DA was significantly elevated at 10 M haloperidol and 10 pM apomorphine. Passive release of ["C]DA, but not
3
[ H]DA was significantly elevated by 0.1 pM reserpine. However, the amount of radioactive DA recovered in the media accounted for only a small percentage of what was lost from the slices. More than 90% of the radioactive DA that had been lost from the slices as a result of haloperidol or reserpine treatments was found in the DOPAC fraction. More than 80% of the increased radioactive DOPAC was found in the media. Apomorphine also caused a significant elevation in radioactive DOPAC accumulation, but the effect was quite small in




Fig. 2.9. Effects of haloperidol, reserpine, apomorphine, chlorpromazine and fluphenazine on the release and metabolism of preloaded labelled DA in striatal slices. Pooled striatal slices were exposed to [3H]tyrosine (250 pCi/10 ml) for 10 min. Then 10 uCi/10 ml [' C]DA was added and the slices equilibrated for an additional 10 min. Slices were then washed 4 times. Aliquots of 0.5 ml of slices were added to 0.5 ml of media containing ionic and drug treatments and incubated for 10 min. Open and shaded bars represent the distribution of [3H] and [1C], respectively. DOPAC and Residual fractions were pooled from the slices and the media. The sum of slice DA, media DA, DOPAC, and residual equals 100% for each label. indicates a significant difference from saline for the same label at p < .05,
** at p < .01 (ANOVA for haloperidol and reserpine, Student's t-test for apomorphine, APO; chlorpromazine, CPZ; and fTuphenazine, FLU).




45
SLICE DOPAMINE
100
80 *
60
40 20
6 MEDIA DOPAMINE
5
4 3
2
O
0
40
0 S30
20
20 RESIDUAL
IO
0-6 3x-6 i0-5 10-8 3xO8 10-7 1O-5M -5M I-5M
SAL M HALOPERIDOL M RESERPINE AP O CPZ FLUo
SAL M HALOPERIDOL M RESERPINE APO CPZ FLU




46
comparison to reserpine and haloperidol, especially considering that no significant decline in slice radioactive DA was observed with 10 PM apomorphine. Fluphenazine and chlorpromazine both were without any effect on any fraction measured.
Because of the observed similarities of the effects of 10 uM haloperidol and 0.1 vM reserpine on synthesis, release, and metabolism of radioactive DA, comparisons were extended to include tests of Ca2+ and K dependency. Haloperidol and reserpine were tested in separate experiments against saline at 6 or 26 mM K and at 2.6 mM Ca2+ or 0 mM Ca2+ with 100 PM EGTA. Orthogonal comparisons within each experiment were made between treatment groups differing in only one treatment factor, using multiple Student's ttests. This was justified since the effect of K was so great that variance was not homogeneous, making the ANOVA an invalid test. Both [3H]- and [14C]DOPAC were greatly increased (more than 400%) by 10 pM haloperidol at both 6 and 26 mI K+ and with or without Ca2+, suggesting that the effect of haloperidol on DOPAC formation was independent of transmitter release (Fig. 2.10). As predicted, 0.1 uM reserpine generated a similar profile, although the effect was smaller than elicited by 10 pM haloperidol. In addition, 26 mM K+ caused a small increase of labelled DOPAC accumulation which was also observed in Ca2+-depleted media (p<.05).
As expected, K+ induced the release of both [3H]and [14C]DA from the slices. K -induced release was still




Fig. 2.10. Comparisons of the effects of haloperidol and reserpine on the release and metabolism of labelled DA in striatal slices. Slices were prepared as described in Fig. 2.9. Open and shaded bars represent [3H] and ["C], respectively. Using Student's t-tests, orthogonal comparisons were performed between treatment groups differing in only one variable. Significance levels presented indicate the effect of 10 vM haloperidol or 0.l M reserpine as compared against the saline value with the same label and at the same K+ and Ca2+ concentrations (i.e. [14C]DOPAC for 26 mM K+, 2.6 mM Ca2+ and haloperidol was compared to [14C]DOPAC for 26 mM K+, 2.6 mM4 Ca2+ and saline). indicates significance at p < .05, ** at p <.01, and *** at p < .001.




48
SLICE DOPAMINE
100
80 *
60 *
40 .
30
20
10*
50 MEDIA DOPAMINE
40
30 4
I-X
00
20
20 o- REIDA
60 DOPAC
I-- **
o ** *I-40 **
20 RESIDUAL
6mMK* 26mMK' 6mMK* 26mMK' 6mMK* 26mMK" 6mMK 26mMK 6m.MK 26mMK' 6mMK' 26mMK
SALINE HALOPERIDOL RESERPINE SALI NE HALOPERIDOL RESERPINE
2.6mM CaCl2 100 jiM EGTA




49
observable in Ca2+ depleted media, although the amount was considerably reduced from the 2.6 mM Ca2+ incubations for both [3H]- and [ C]DA (p < .01). Haloperidol not only elevated passive release, but also potentiated K+ -induced 2+ 2+
release in both Ca2+ maintained and Ca -depleted media. Reserpine significantly elevated passive release of [14C]DA and K+ -induced release for both [3H]- and [14C]DA at 2.6 mM Ca2+. Reserpine did not significantly elevate K+-induced release in Ca2+ depleted conditions, although the pattern of results was similar to that observed with haloperidol. No treatment combination had any consistent effect on the residual 3H and 14C. Changes in slice DA radioactivity inversely reflected the combined changes in media DA and DOPAC radioactivities.
Discussion
Precursor Pool Determination
The accumulation of [3H]DA from [3H]tyrosine in
striatal slices displayed Michaelis-Menton kinetics if the DA specific activity was corrected with initial or measured media tyrosine specific activity. Such was not the case when tissue tyrosine specific activity was used as a correction factor. Kapatos and Zigmond (1977) reported similar results measuring the liberation of [14C]C02 from [14C]tyrosine incubated with striatal synaptosomes. The dependence of normal kinetics upon the media tyrosine specific activity suggests that tyrosine hydroxylase (TH) might be linked to a




50
tyrosine uptake mechanism at the membrane. TH has been reported to exist in both soluble form and in a more active membrane bound form (Kuczenski and Mandell, 1972). However, in our samples we found that protein labelling with [3H]tyrosine also displayed Michaelis-Menton kinetics only if corrected with media tyrosine specific activity (not shown). Thus, the precursor pool for DA synthesis apparently is also reflected in protein synthesis. One possibile explanation is that areas with high metabolic activity, such as neuronal cell bodies and terminals, more rapidly exchange free tyrosine with the media than do glia and neuropil. Also, exchange of tyrosine with the medium may be higher in those terminals and cell bodies which are using more tyrosine as a precursor. Thus, dopaminergic terminals might exchange tyrosine with the media more freely than would terminals of other neurotransmitters. Such an explanation would explain why Kapatos and Zigmond found that tissue tyrosine in a synaptosomal preparation was not the relevant precursor pool for DA synthesis.
Effects of Elevated K+ upon Synthesis, Release, and Metabolism of DA
Depolarizing agents such as elevated K+ or veratridine induce release and activate synthesis of DA in vitro. Both the induction of release and the activation of synthesis are calcium dependent (Harris and Roth, 1971; Patrick et al., 1974; Bustos and Roth, 1979). The increase is correlated with an activation of TH to a form characterized by an




51
increased affinity for reduced pteridine cofactor and a decreased sensitivity to end-product inhibition by DA (Lovenberg et al., 1974; Bustos et al., 1974).
It has been presumed that the K -induced synthesis was a consequence of increased release. However, we observed that the activation of synthesis could be dissociated from the induction of release. Increased synthesis was apparent at 14 mM K+, a concentration at which no effect upon release was detected. K+ concentrationshigher than 18 mM produced an asymptotic effect on synthesis, while release increased linearly with increasing K+ concentrations up to at least 46 mM. The plateau of synthesis activation may reflect an inhibitory feedback upon DA synthesis mediated either by released DA stimulating a presynaptic receptor which would inhibit synthesis, or by end-product inhibition of TH. K -induced release and synthesis were previously reported by Bustos et al. (1974) to be dissociable: K -induced TH activation in striatal slices was suppressed by ethanol, while release was unaffected. Since TH activation by depolarization is calcium dependent (Simon and Roth, 1979), synthesis 2+
activation may occur subsequent to Ca2+ influx into the terminal. Whatever the mechanism, synthesis activation appears not to be a consequence of release, but independent of it. It is curious that K+ did not increase the accumulation of [3H]DA and [3H]NE in substantia nigral slices at 83 vM tyrosine, or of [3H]NE in cerebellar slices. Assuming that increased release of labelled catecholamines occurred




52
during incubation, an increase in synthesis may actually have taken place. However, amphetamine, which appears to activate DA synthesis in a manner similar to K+, also interacts with tyrosine, being effective at media concentrations of tyrosine above 10 jM (Uretsky and Snodgrass, 1977). Also, the effectiveness of apomorphine in inhibiting synthesis is reduced in the substantia nigra at 83 pM tyrosine (Fig. 7). It is possible that tyrosine at 83 PM may be toxic, thus masking synthesis changes. Alternatively, it may affect release. In either case, it is not clear why the K effect would be potentiated by increasing tyrosine concentrations in striatum, but would be suppressed at higher tyrosine concentrations in substantia nigral slices.
A significant percentage of the labelled [3H]DA released by K+ stimulation was found to be converted to the deaminated, nonmethylated metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), as was observed by Farah et al. (1977) for NE terminals. However, the metabolite accounted for only about 20% of the total released radioactivity. Cubeddu et al. (1979) found that reuptake blockers reduced the formation of DOPAC suggesting that the DOPAC was derived from labelled DA that had been released and subsequently taken up by the terminal. Our finding that the elevation of DOPAC formation caused by 26 mM K+ occurred independently of the presence of Ca2+ is somewhat at odds with this interpretation. However, if uptake mechanisms were saturated by the DA released by 26 M K+ +Ca+
26 mM K at 2.6 mM Ca then uptake might proceed at nearly




53
the same rate as in Ca2+-depleted media, making the amount of precursor available for monoamine oxidase fairly constant. Effects of Apomorphine on DA Metabolism
Electrophysiological results suggest that a DA receptor in the substantia nigra mediates inhibition of DA cell firing (Aghajanian and Bunney, 1977; Skirboll et al., 1979). It has been argued that this receptoris localized upon dopaminergic cells and is not presynaptic on terminals projecting to the substantia nigra. However, presynaptic autoreceptors regulating release of DA have not been generally accepted. Apomorphine has either weak inhibitory effects (Farnebo and Hamberger, 1971; Miller and Friedhoff, 1979) or no effect (Dismukes and Mulder, 1977; Raiteri et al., 1978) on DA release from striatal synaptosomes and slices. Furthermore, although neuroleptics do affect electrical field- and K stimulated release (Dismukes and Mulder, 1977; Seeman and Lee, 1975; Farnebo and Hamberger, 1971), the effect has been consistently reported to be inhibitory except at high doses (Perkins and Westfall, 1976), opposite to what would be predicted by DA autoreceptor blockade,
Since apomorphine potently inhibits DA synthesis both in vivo (Kehr et al., 1972) and in vitro (Fig. 2.4; Christiansen and Squires, 1974; Ebstein et al., 1974), several groups have proposed the alternative hypothesis that DA presynaptic autoreceptors may modulate synthesis and not release (Aghajanian and Bunney, 1974; Christiansen and Squires 1974). DA has been shown to inhibit synthesis in vitro




54
(Patrick et al., 1974). However, a controversy exists as to whether the site of action is on a DA receptor or on TH. DA-mediatedinhibition has been shown to be reversible by cocaine (Patrick et al., 1974), allegedly due to the ability of cocaine to block reuptake. If this is true, then at least part of the inhibition observed with exogenous DA in vitro might be due to end-product inhibition of TH. In contrast, both apomorphine- and DA-induced inhibitions of DA synthesis have been reported to be reversed with haloperidol or fluphenazine (Westfall et al., 1976; Christiansen and Squires, 1974). We were unable to replicate this finding at the dose of haloperidol used by Christiansen and Squires. However, 10 nM haloperidol significantly elevated the accumulation of [3H]DA from [3H]tyrosine in slices stimulated with 26 mM K+. This effect might be interpreted as being caused by a reversal of ongoing inhibition of DA synthesis. Synthesis would be suppressed at an autoreceptor by DA released by the elevated K concentration; this would be reversed by a DA receptor blocker. A similar hypothesis has been used to explain why striatal DA terminals display elevated synthesis following nigrostriatal tract lesions or impulse blockade with the GABA analogue gamma-butyrolactone (Walters et al., 1973).
Another possible site of action of apomorphine is TH. TH undergoes a conformational change following electrical or K+ stimulation, resulting in an increased affinity of the enzyme for pterin cofactor and reduced sensitivity to




55
end-product inhibition. Thus, if apomorphine was inhibiting TH by end-product inhibition, K+ activation should, relatively speaking, reduce the potency of apomorphine. Instead, we found that 26 mM K exacerbated the inhibition of [3H]DA accumulation by apomorphine. This result was probably not due to some change in release or metabolism since 10 v~M apomorphine did not cause a detectable depletion of [3H]DA from preloaded slices. Also, apomorphine either has no effect at all on K+ -stimulated release of labelled DA or it has a weak inhibitory effect. An inhibition of release should retain [3H]DA in slices during the incubation period, an effect we did not observe. Therefore, a mechanism other than direct inhibition of TH is responsible for the apomorphine-induced inhibition of DA synthesis.
We also found that apomorphine-induced inhibition of DA synthesis is dependent on the medium concentration of tyrosine in incubations of substantia nigral slices. The accumulation of [3H]DA in substantia nigral slices was inhibited 80% by 10 jM apomorphine when incubated at 8.3 pM tyrosine. Increasing the medium tyrosine concentration to 83 1M halved the effectiveness of apomorphine, but did not alter the inhibition by haloperidol (67% at 8.3 pM tyrosine, 65% at 83 pM tyrosine). This effect of tyrosine lacks a ready explanation, although similar interactions with medium tyrosine concentrations have been reported before. In particular, amphetamine stimulated DA synthesis in striatal slices at medium tyrosine concentrations of 10 jM or more,




56
whereas it had no effect at concentrations of tyrosine 1 PM or less (Uretsky and Snodgrass, 1977). Such findings might indicate a change in the K of TH for tyrosine. Alternam
tively, apomorphine might have impeded and amphetamine facilitated tyrosine uptake into a small pool of tyrosine with direct access to TH.
As speculated by Starke et al. (1977), we found that the inhibition of synthesis caused by apomorphine was not limited to DA terminals, but was also apparent in the accumulation of [3H]NE from [3H]tyrosine in substantia nigral and cerebellar slices. Their speculation arose from the repeated observation that DA inhibited release from NE terminals in some peripheral tissues (for review, see Starke et al., 1977), apparently by means of a presynaptic receptor similar to DA-receptors found in the CNS (Iversen et al., 1975). Therefore, the ability of apomorphine to inhibit both NE and DA synthesis in vitro does not necessarily argue against the hypothesis that inhibition is mediated by an autoreceptor. The same receptor might be present on both terminal populations. It does seem surprising, however, that a DA presynaptic receptor on NE terminals could modulate release and synthesis, while possibly the same receptor modulates only synthesis on DA terminals. Effects of Haloperidol on Catecholamine Synthesis and Release
We did not expect haloperidol to inhibit [3H]DA accumulation nearly as well as apomorphine. In vitro inhibition




57
of DA synthesis by haloperidol has only been briefly reported before; Christiansen and Squires (1974) commented that haloperidol at concentrations greater than 1 NM inhibited
3 3
[3H]DA synthesis from [3H]tyrosine in striatal synaptosomes. Such an inhibition of DA synthesis contradicts a model of autoreceptor regulation of DA synthesis. Such a model predicts that DA-receptor blockers would increase DA synthesis or have no effect at all. Also, while apomorphine clearly inhibits synthesis in vivo (Kehr et al., 1972) and in vitro (Ebstein et al., 1974; Christiansen and Squires, 1974), haloperidol dramatically increases DA synthesis in vivo (Kapatos and Zigmond, 1979; Lerner et al., 1977; Carlsson et al., 1977), contrary to the in vitro findings.
The mechanisms by which apomorphine and haloperidol
inhibit the accumulation of [3H]DA from [3H]tyrosine appear to be different. First, 26 mM K+ potentiated apomorphineinduced, while it lessened haloperidol-induced inhibition of DA synthesis. Also, apomorphine-induced inhibition of the accumulation of [3H]catecholamines in substantia nigral slices was halved by increasing the concentration of medium tyrosine, but no such effect was observed with haloperidol.
Attempts were also made to determine whether the
decreased accumulations of [3H]DA observed in the presence of haloperidol were due to decreased synthesis, increased spontaneous release, or decreased reuptake. By replacing [3H]tyrosine with [l-14C]tyrosine and by measuring the liberation of [1C]CO2, a more direct measure of DA synthesis




58
was obtained. In this case, haloperidol inhibited [14C]CO2 release as much as, if not more than, [3H]DA accumulation. Therefore, haloperidol inhibited DA synthesis.
However, incubations with 10 pM haloperidol also caused the loss of [3H]DA synthesized from [3H]tyrosine and of [1"C]DA preloaded in striatal slices. The amount of labelled DA found in the media was elevated, confirming several reports that haloperidol elevated the basal efflux of DA (Seeman and Lee, 1974; Dismukes and Mulder, 1977; Raiteri et al., 1978). However, the increased efflux of labelled DA accounted for less than 15% of the label lost from the slices; more than 80% of the missing label was located in a fraction containing DOPAC. Therefore, the total basal efflux of label induced by haloperidol in earlier studies has probably been incorrectly assumed to be associated with labelled DA. Paper chromatography of the fraction containing DOPAC revealed that over 80% of the [14C]materials from both saline and 10 iM haloperidol-treated slices co-chromatographed with standard DOPAC. A similar profile was apparent with [3HI labelled materials. Since the formation of labelled DOPAC was strongly inhibited by 12.5 iM nialamide (Table 2.3), the mechanism of synthesis was probably enzymatic and not due to spontaneous oxidation.




59
The inhibition of reuptake by haloperidol has been reported (Rio and Madronal, 1976; Seeman and Lee, 1974). We also found that haloperidol inhibited the apparent striatal accumulation of [14C]DA from the media. However, the decrement was much smaller than would be necessary to account for the decreased accumulation of [3H]DA from [3H]tyrosine. It has been suggested that apparent decreases in uptake might actually reflect enhanced release (Baumann and Maitre, 1976: Heikkila et al., 1975). When the sum of medium ['4C]DOPAC (presumably formed in DA terminals) and slice [14C]DA was compared, the effect of haloperidol was reduced, directly confirming this hypothesis.
Papaverine (Cubeddu et al., 1979a), Ro 4-1284 (Farah et al., 1977; Cubeddu et al., 1979b), and reserpine itself all cause patterns of release similar to those observed with haloperidol. Therefore, a variety of comparisons between haloperidol and reserpine were performed to further substantiate the possibility of a reserpine-like character for haloperidol. Like haloperidol, reserpine potently inhibited [3H]catecholamine accumulation in both striatum and substantia nigra, although at doses 100 times smaller than were needed for haloperidol. Similarly, reserpine increased the basal efflux of label from striatal slices preloaded with [3H]DA (Seeman and Lee, 1974; Fig. 2.9).




60
The increased formation of labelled DOPAC might also be ascribed to efficient reuptake and degradation of DA released into the medium. This possibility does not seem likely since the bulk of the radioactivity released by elevated K+ concentrations from striatal slices preloaded with labelled DA remains as DA (Fig. 2.9; Farah et al., 1977). We also found that the elevation of labelled DOPAC by 10 pM haloperidol and 0.1 i1M reserpine was independent of both K+ and Ca2+ concentrations, and therefore probably independent of vesicular release.
The effects of 0.1 IM reserpine and 10 vM haloperidol on release were complex but essentially identical. Both agents increased basal efflux of labelled DA and facilitated the release of DA induced by 26 mM K+. Haloperidol also elevated the Ca 2+-independent, K -induced release of labelled DA. Reserpine did not significantly elevate the release of labelled DA in Ca2+ depleted media, although the pattern of results was similar to that observed with haloperidol. The facilitation of basal release by haloperidol has been reported before (Seeman and Lee, 1974; Dismukes and Mulder, 1977). However, most studies of field-stimulated (Seeman and Lee, 1975; Dismukes and Mulder, 1977), K -induced (Miller and Friedhoff, 1979) and veratridine-induced (Raiteri et al., 1978) release of preloaded labelled DA indicated that haloperidol reduced the amount of label released. The only exception was the report of Perkin and Westfall (1976) which indicated that 10 pM haloperidol, the dose used in this study,




61
facilitated stimulated release while lower doses inhibited release.
Reported similarities of the effects of reserpine and neuroleptics are numerous. Reserpine has potency in the treatment of schizophrenia and like other neuroleptics used in this study, causes parkinsonian-like symptoms (Crow et al., 1976; Bleuler and Stoll, 1955). All of these agents induce catalepsy at high doses (Janssen et al., 1966). These agents have an anesthetic property as defined by the ability to block impulses along peripheral nerves (Seeman et al., 1974). Reserpine and haloperidol both inhibit catecholamine synthesis in vitro and activate catecholamine synthesis in vivo (Kapatos and Zigmond, 1979; Mueller et al., 1969). In contrast, however, reserpine causes a profound depletion of monoamine concentrations. Haloperidol has been reported to cause a moderate depletion of striatal DA (Massoti, 1977), although most reports have not found this result (Kapatos and Zigmond, 1979; Lerner et al., 1977).
The effects of haloperidol and reserpine observed in
this study to not appear to be receptor-mediated. The ranked efficacies of reserpine, haloperidol, chlorpromazine and fluphenazine (listed in decreasing order of potency) does not correlate with any of the widely studied measures of DAreceptor antagonist efficacy (Creese et al., 1976). Fluphenazine is typically as effective as haloperidol, while chlorpromazine is less potent by an order of magnitude, in reversing apomorphine-induced stereotypy, in competing with




62
labelled DA for binding (Creese et al., 1976) and in causing extrapyramidal side effects (Crow et al., 1976). In contrast, fluphenazine has little potency in our preparations. Also, reserpine does not reverse apomorphine-induced stereotypy (Janssen et al., 1966), but it is potent in inhibiting the accumulation of [3H]catecholamines (Fig. 2.4).
Seeman and Lee (1975) expanded their initial findings of neuroleptic mediated anesthesia to include an evaluation of field-stimulated release of labelled DA from striatal slices. They too found that fluphenazine was as potent as haloperidol or reserpine in inhibiting stimulated release, as well as inducing anesthesia. Once again, this result does not agree with our findings, indicating that changes in membrane solubility caused by these agents was not the cause of haloperidol- and reserpine-induced inhibition of synthesis. However, this pattern was not replicated by Dismukes and Mulder (1977). Attempting to use similar stimulation parameters, they found that haloperidol and spiroperidol effectively inhibited the stimulated release of DA from striatal slices. However, fluphenazine and chlorpromazine were without effect, correlating very well with our current results. They also found that stimulated [3H]GABA release was also inhibited to a similar extent as [3H]DA, compatible with our finding that NE synthesis was inhibited as well as DA synthesis. Finally, the concentration of haloperidol which resulted in a 50% inhibition of release (IC50) was 3 iM (Dismukes and Mulder, 1977), the dose determined




63
to be the IC50 of K -induced release of [3H]DA from striatal slices (Miller and Friedhoff, 1979) and of catecholamine synthesis as reported in this study. Seeman and Lee (1975) found an IC50 of 0.1 vM for haloperidol-mediated inhibition of stimulated release. This large inconsistency may reflect some qualitative difference in experimental procedures which might explain the qualitative differences in ranked neuroleptic dose efficacy. Despite these differences, the close correlation of our data with those of Dismukes and Mulder (1977) as well as with Miller and Friedhoff (1979) suggests that the inhibition of synthesis we observed with haloperidol is accomplished by the same mechanism that inhibited stimulated release in their systems.
A hint of the special properties of the butyrophenones as compared to other neuroleptics arose from the original Seeman et al. (1974) paper examining nerve impulse-blocking with neuroleptics. They found that the nerve impulse-blocking potency of the phenothiazines and neuroleptics in general correlated quite well with their partition coefficient for the membrane. The butyrophenones, haloperidol and trifluoperidol, however, were much more effective in blocking impulses than their partition coefficients would have predicted. They speculated that the butyrophenones were probably specifically bound to Na+-conductance channels in addition to nonspecifically altering membrane fluidity. Similarly, haloperidol was much less effective than other neuroleptics in inhibiting DA-sensitive adenylate cyclase




64
than its clinical potency would predict (Davis, 1974). Thus, haloperidol and perhaps the butyrophenones in general, appear to have properties distinct from other neuroleptics.
These distinct properties of haloperidol may be related to its reserpine-like properties of vesicular disruption and catecholamine synthesis inhibition. These properties might be related to the blockade of Na+-conductance channels, although this mechanism seems inadequate to explain the apparent release of DA from vesicles into the cytoplasm. In any event, neither simple anesthesia caused by changes in membrane fluidity nor DA receptor blockade appear to explain our results or the findings of Dismukes and Mulder (1977).
The relationship between synthesis inhibition and
vesicular disruption is uncertain. Vesicular disruption might elevate the cytoplasmic concentration of DA sufficiently to produce end-product inhibition of TH. The inhibitory potency of haloperidol on the accumulation of [3H]DA from [3H]tyrosine was ameliorated by the activation of synthesis (and presumably of TH) induced by 26 mM K Such a result correlates with the reduced susceptibility of TH to endproduct inhibition following allosteric activation, arguing that increased cytoplasmic DA concentrations mediated the haloperidol-induced inhibition of DA synthesis. Unfortunately, the reduction of K+ -induced release by haloperidol reported by Miller and Friedhoff (1979) would tend to increase the amount of [3H]DA retained in the slices in a synthesis study, and thereby confound interpretations.




65
Another possibility is that haloperidol may in some
way alter the relationship of TH to the cell membrane. The membrane-bound form of TH has been reported to be more active than the soluble form (Kuczenski and Mandell, 1972). Thus, membrane disruption might dislodge the enzyme from the membrane and consequently reduce its activity. Also, membrane disruption might interfere with precursor uptake, although no competitive interactions between haloperidolinduced synthesis inhibition and media tyrosine concentrations were observed in substantia nigral slices.
The mechanism by which neuroleptics exert their effects in vivo are difficult to relate to in vitro observations. The effectiveness of fluphenazine and haloperidol in reversing apomorphine-induced emesis and stereotypy suggests that neuroleptics are DA-receptor blockers. However, at progressively higher doses, anesthesia (Groves et al., 1975; Seeman and Lee, 1974) or vesicular disruption may occur. Waddington (1979) presented evidence that 0.4 mg/kg haloperidol attenuated by 30-40% the contralateral turning induced by intranigral injections of muscimol or baclofen. This effect of muscimol or baclofen was not reversible by 6-OHDA lesions of the ascending nigrostriatal fibers. Furthermore, 0.4 mg/kg totally blocked intranigral apomorphine-induced turning, suggesting that DA systems were not involved in the muscimolor baclofen-induced turning. Waddington concluded that while DA receptors were blocked by haloperidol at this dose, another effect of haloperidol was also evident.




66
Thus, a future source of difficulty in investigating the action of neuroleptics will lie in trying to determine at what doses DA-receptor-mediated effects of neuroleptics give way to anesthetic and/or reserpine-like properties. All three activities tend to decrease synaptic efficacy, consequently decreasing feedback inhibition and increasing DA cell firing rates. The increase in DOPAC concentrations detected in vivo following haloperidol treatments might be due either to increased release (Roth et al., 1976) or to vesicular disruption. Finally, the increases in DA synthesis and TH activation might be due to increased firing of DA cells or to a depletion of terminal DA. Kainic acid lesions of the striatum have been reported to suppress the activation of TH by haloperidol treatments, but not the activation of synthesis (Di Chiara et al., 1977, 1978; Tissari et al., 1978). This finding implicates a postsynaptic receptor and long-loop feedback as the means of regulating enzyme activation. However, the maintained increase of DA synthesis in the striatum suggests that haloperidol also affects the terminal directly. By transiently inhibiting synthesis or depleting amine concentration, haloperidol might cause a subsequent activation of synthesis in vivo which would be evident at 60-90 minutes following injection. A time course of synthesis rates following haloperidol injections will be necessary for confirmation. It would also be of interest to know whether the increased DA synthesis following kainic acid lesions would be elicited by neuroleptics which do not have reserpine-like effects in vitro.




67
Similarly, while sensory input causes reciprocal
changes in metabolism and synthesis of caudate and nigral DA (Nieoullen et al., 1977), haloperidol increased in vivo DA synthesis in both structures simultaneously (Argiolas et al., 1979a). Also, while haloperidol increased DOPAC concentrations within substantia nigra (mesostriatal system), it did not affect the ventral tegmental area (mesocortical system, Argiolas et al., 1979b). This differential effect was suggested to reflect the selective activation of the mesocortical system following footshock stress. However, both frontal cortical and striatal DOPAC concentrations were elevated to the same extent with haloperidol. If the normal metabolic responses of striatum and substantia nigra or of striatum and frontal cortex are reciprocal, parallel effects of haloperidol on these structures suggest a site of action on the synthetic mechanism within the terminals,and not on afferent processes or dopaminergic cell dendrites. The lack of an effect in the ventral tegmental area may reflect a lack of vesicularmediated release, making it resistant to the effects of haloperidol or reserpine.
In contrast, functional isolation of DA terminals with nigrostriatal lesions (Kehr et al., 1972), with gammabutyrolactone (Kuczenski, 1978) or with baclofen (Wuerthele et al., 1979) blocks the in vivo increase of DA synthesis by haloperidol. These findings suggest that haloperidol did not have a direct effect on DA terminals. However, the




68
functional isolation of DA terminals may have only prevented a rebound in synthesis. A transient inhibition of synthesis, which would normally cause a rebound increase in synthesis, might still have occurred.
Summary
1. Medium tyrosine appears to be the relevant precursor pool for DA synthesis.
2. Activation of DA synthesis commences at K concentrations relatively ineffective in inducing release, suggesting that activation of synthesis is concurrent with, but not consequent on K+-induced release.
3. Haloperidol, apomorphine, and reserpine inhibited DA synthesis in a dose-dependent manner.
4. We interpret our findings to concur with earlier
indications that a DA autoreceptor regulates DA synthesis. K -induced activation of TH did not diminish the potency of apomorphine to inhibit [3H]DA accumulation. Since activated TH is less sensitive to end-product inhibition, then apomorphine-induced inhibition of synthesis probably is not mediated by end-product inhibition of TH. The ability of apomorphine to inhibit NE synthesis, together with the presence of presynaptic receptors capable of regulating NE release, further argues that apomorphine regulates synthesis by means of an autoreceptor, although one not specific to DA terminals.




69
5. Most of our effects of haloperidol on in vitro
DA metabolism and synthesis do not appear to be associated with a receptor mechanism. Instead, ves.icular disruption and synthesis inhibition suggest that haloperidol acts as a membrane disruptor by a mechanism more specific than simple membrane solubility, perhaps as a blocker of Na channels as suggested by Seeman and Lee (1975). The single exception was that 10 nM haloperidol caused a small facilitation of [3H]DA accumulation from [3H]tyrosine in striatal slices incubated at 26 mM K possibly reflecting an antagonism of autoreceptor-regulated inhibition of DA synthesis.
The possibility exists that many experiments that have
used haloperidol to examine changes in catecholamine release and metabolism have produced results unrelated to DA receptor blockade.




SECTION III
ACTH AND LVP SELECTIVELY ACTIVATE MESOCORTICAL DA SYNTHESIS
Introduction
Intracranial administrations of the pituitary peptides ACTH and LVP have potent behavioral effects. Pellets of ACTH1_10 (10 jig) implanted in rostral mesencephalon and caudal diencephalon (van Wimersma Greidanus and de Wied, 1971) and doses of icy LVP as low as 2.5 ng (de Wied, 1976) delayed the extinction of avoidance responses in rats. Also, ACTH (icy, 1 ig) in rats and mice and LVP (icy,10-100 ng) in mice induce bouts of excessive grooming (Gispen et al., 1975; Rees et al., 1976; Delanoy et al., 1978). Higher doses of LVP (1 pg) produced barrel rotation (rotation around the animal's longitudinal axis) in rats and a hyperactivity characterized by scratching, foraging, and vocalizing in mice (Kruse et al., 1978; Delanoy et al., 1978). Ranked efficacies of various analogs in altering memory performance and in inducing spontaneous behavior do not correlate well, suggesting that more than one receptor exists for both ACTH and LVP (Greven and de Wied, 1978; Delanoy et al., 1979).
A variety of arguments suggests that ACTH and LVP may exert their effects through catecholaminergic systems. ACTH (Ganong, 1974) and vasopressin (Sandman et al., 1973b) as well as the catecholamines (Stone, 1975) are released in
70




71
response to stressful stimuli. Anatomical reports have indicated that some hypothalamic projections to areas containing catecholamine cell populations have been labelled with antisera believed to be specific to ACTH (Barchas et al., 1978) or LVP (Buijs, 1978). ACTH (icy) increased the in vivo disappearance of [3H]NE from the forebrain of rats preloaded with icy administered [3H]NE (Endroczi et al., 1976), while LVP (icy) has been found to exert a varied effect on the disappearance of endogenous catecholamines following synthesis inhibition with aMPT (Tanaka et al., 1977a, 1977b). Behaviorally, the grooming response to either ACTH or LVP can be suppressed with systemic injections of the neuroleptic haloperidol (Wiegant et al., 1977). Moreover, ACTH has been found to compete with [3H]haloperidol for binding to striatal membranes (Czlonkowski et al., 1978).
Recently, traumatic stressors have been reported to
activate DA synthesis in frontal cortex but not in striatum, as assessed by changes in DA and DOPAC concentrations (Lavielle et al., 1979; Fadda et al., 1978). Since ACTH and LVP are released as consequences of stressful stimuli, because peptide induced grooming is antagonized by haloperidol, and since preliminary biochemical evidence suggests that icy injections of ACTH and LVP alter catecholamine turnover, we have examined the effects of ACTH and LVP on the activation of catecholamine synthesis in various central nervous system structures. Using a slice technique described in an earlier report (Section II), we measured the in vitro




72
accumulation of [3H]catecholamines from [3H]tyrosine in brain tissues dissected from animals receiving ACTH or LVP. In vitro accumulation was also measured in slices incubated with ACTH or LVP. Because the peptides might alter accumulation by means of modulating release and metabolism, we tested both peptides in the presence of 26 mM K+. Finally, because of its known interactions with the neuroleptic haloperidol, we tested whether ACTH, when incubated with striatal or substantia nigral slices, might alter haloperidol- or apomorphineinduced inhibitions of synthesis.
Methods
Materials
ACTH1-24 (Cosyntropin, 100 U/mg), and purified ACTH4-10 and [D-phe7]ACTH4-10 were provided by Dr. Henk Greven and Dr. Henk Van Riezen of Organon International B.V. Apomorphine (hydrochloride salt) and LVP (1000 U/5.Smg) were obtained from Sigma Chemical Co. Crystalline haloperidol was provided by McNeil Laboratories. [2,6-3H]tyrosine (34 Ci/mmol,
1 mCi/ml, lyophilized before use) was obtained from the Radiochemical Centre, Amersham, Other chemicals used in tissue preparations were HEPES (Ultrol, Calbiochem), Tris hydroxide (Trizma base, Sigma), PPO (Scintillar, Mallindkrodt), POPOP (Research Products International Corp.) and Triton X-100 (Scintillar, Mallindkrodt).
The medium used in all experiments was that described by Versteeg et al. (1974) and contained the following: 118 mM NaCI, 4.4 mM KC1, 2.6 mM CaCla2, 1.3 mM MgSO4, 1.2 M




73
K2HPO4, 25 mM HEPES, and 12 mM glucose. The solution was titrated to pH 7.3 with NaOH. Increases in KC1 concentration were countered with an equivalent decrease in NaCl.
Apomorphine and haloperidol were dissolved in 0.2 M
acetic acid at 2 mg/ml and diluted to 0.25 mg/ml with media. 12.1 P1 of apomorphine or 15 pl of haloperidol stock solutions were added to one ml incubation volumes to achieve a final concentration of 10 PM. Lower concentrations were produced with serial dilutions of the stock solutions with media. 'Saline' controls were composed of appropriate volumes of acetic acid and media.
For icy preparations, ACTH and LVP were dissolved with saline to a concentration of 0.5 Pg/Pl. For experiments in which the peptides were incubated with brain slices, ACTH and LVP were initially dissolved in media containing radioactive tyrosine (16.6 or 166 pM) to a concentration of 200 pM. Concentrations of 20, 2, and 0.2 pM were obtained by serial dilutions with labelled media. The addition of an equal volume of slices in unlabelled medium resulted in final concentrations of 8.3 or 83 PM tyrosine and 100, 10, 1, and 0.1 pM peptide.
Animals and Surgery
Male CD-1 mice (Charles River, Wilmington, Mass., 25-30 g) were maintained on a 7:00 am 7:00 pm lighting schedule and were kept at temperatures between 22 and 280C. Animals not receiving icy injections were individually housed for the three days immediately prior to killing.




74
Animals receiving icy injections were provided with injection ports, injected and observed as described by Delanoy et al. (1978). Briefly, jewellers' screws (1/8 in x 0080) lubricated with vacuum grease were screwed into holes stereotaxically placed over the lateral ventricles (0.4 mm caudal to bregma, 1.6 mm lateral). Screws were inserted only as far as needed to bind to the cranium (the screw head surface set at 2.8 mm above the cranium), leaving the dura unpenetrated. Dental cement was applied around the screw up to the base of the head of the screw. As the dental cement hardened, threads matching those of the screws formed in the dental cement, allowing subsequent screw removal. Animals were housed individually for the following six days prior to killing. For all experiments in which icy ACTH was used, the screws were removed on the day prior to injection and killing. Of five experiments performed examining the effects of icy LVP, two were performed with screws removed 5 days before killing, two were performed with screws removed on the morning of the experiment and one was performed in which half of the animals had screws removed at five days and half had screws removed immediately before injection. No significant effects of the different times of screw withdrawal were found in a multiple factor ANOVA.
For icy injections, 27 ga needles 15 mm long (Unimetrics model 2040) were sleeved with 22 ga cannula tubing, leaving
4.4 mm of 27 ga needle exposed. These were fitted onto 10 il Unimetrics syringes (model 5010R). Saline or 1 iig of ACTH or LVP were injected into the lateral ventricles in 2 il volume (1 .1/ventricle).




75
The spontaneous behavior of each animal was scored and recorded every 30 sec for 30 min. An inclusive list of scores was as follows: quiet, moving, grooming, eating, drinking, foraging, squeaking, and scratching. For experiments in which ACTH was tested, the sum of grooming and scratching was tabulated. Similarly, the sum of scratching, foraging and squeaking was tabulated when icy LVP was tested. Animals were sacrificed immediately after the last observation.
Incubation Procedures
Two basic types of experiments were performed. In the
first, tissues obtained from animals receiving icy injections of peptides or saline were dissected and chopped in two dimensions with a McIlwain Tissue Chopper set at 300 4m. Tissues from each animal were suspended in 15 ml Corex round bottomed centrifuge tubes containing 2 ml of fresh equilibrated medium (370C, in an 02:C02, 95:5 atmosphere). This medium was immediately aspirated and replaced with 0.5 ml of medium containing 8.3 vM tyrosine and 3.75 pCi [2,6-3H]tyrosine, except the striatal slices, which received 0.57 pCi. Slices were incubated for 20 minutes. The reaction was then stopped with 2 ml of ice cold media.
In the second type of experiment, sliced tissues were pooled and equilibrated in medium for 30 min. This medium was aspirated and replaced with fresh medium, after which the slices were equilibrated for an additional 10 min. Aliquots of pooled slices (0.5 ml) were added to 15 ml Corex




76
round bottomed centrifuge tubes containing 0.5 ml of equilibrated media with labelled tyrosine, peptide treatments, and additional drug treatments. Samples were incubated for 40 minutes and the reaction was stopped with 2 ml of ice cold media.
In both types of experiments, once chilled, the slices were centrifuged at 100 x g in a refrigerated (40C) Sorvall RC-3 centrifuge for 5 min. The media were then decanted. In experiments in which the specific activity of media tyrosine was measured, the decanted media were saved for later analysis. Another 2 ml of ice cold mediumwere added to each sample and the centrifugation was repeated. Chromatography
Purification procedures and recovery efficiencies were essentially those reported by luvone et al. (1977). Slices were homogenized in the Corex centrifuge tubes with a teflon pestle specially machined to a diameter of 14.5 mm to tightly fit the Corex tubes. The homogenate was centrifuged at 15000 x g for 10 min in a refrigerated (4oC) Sorvall RC_2B centrifuge. Supernatants were .loaded onto AG-50 x 4 cation exchange columns (Bio-Rad, 200-400 mesh, 6mm x 20 mm, H+ form). Once loaded, the cation exchange columns were washed with 16 ml of a sodium acetate buffer (0.1 M sodium acetate,
0.1% disodium EDTA, titrated with NaOH to pH 7.0). Following a wash with 2 ml 1 M HC1, NE was eluted with 4.75 ml of 1 M HC1 into test tubes containing 200 mg alumina. Alumina was prepared by the method of Anton and Sayre (1962) and was




77
moistened prior to use with 100 Ipl of 0.1 M disodium EDTA. The columns were washed with an additional 1.25 ml of 1 M HC1 and DA was eluted with 2.5 ml of 4 M HC1 into test tubes containing prepared alumina. NE and DA fractions were titrated to pH 7.9 with approximately 2.6 ml 3 M Tris hydroxide and with 2.5 ml of 3 M Tris hydroxide and approximately 0.5 ml of 10 M NaOH, respectively. Titrated suspensions of aluminabound catecholamines were transferred to glass wool stoppered pasteur pipettes. When the alumina suspension had been filtered through the glass wool, the alumina retained by filtration was washed with 6 ml water. NE and DA were eluted from the alumina into scintillation vials with 2 ml of 0.5 M HC1. Samples were counted in 10 ml of triton-based scintillation fluid (10.68 g PPO, 0.12 g POPOP, 1 Z toluene and 1.4 Z Triton X-100).
In experiments which required the analysis of media tyrosine specific activity, the media were titrated to pH
1.5 with approximately 65 pl of 4 M HC1 and loaded onto the AG-50 x 4 cation exchange columns (H+ form). The columns were then washed with 6 ml of sodium acetate buffer. Tyrosine was eluted with another 6 ml of sodium acetate buffer, titrated to pH 1.5 with 4 M HC1, and loaded onto the standard AG-50 x 4 columns (Na+ form). Following washes with 5 ml water and 8 ml
0.5 MN HC1, tyrosine was eluted with 9 ml of 0.1 M Na3PO4. Of this volume, 0.9 ml aliquots were added to 100 il of 4 M HCl and 10 ml of triton based scintillator, while 1.8 ml aliquots were assayed for tyrosine using nitrosonaphthal




78
as described by Udenfriend (1962). Fluorescence was measured on a Perkin-Elmer model 650-10S Fluorescence Spectrophotometer. In one experiment, 0.3 ml aliquots of the initial incubation media were lyophilized and tyrosine content was measured on an amino acid analyzer by Dr. Maartin Reith at the New York Institute for Neurochemistry and Drug Addiction.
Pellets formed by the centrifugation of the homogenate were dissolved in 2.5 ml of 0.3 M NaOH. Protein determina. tions were performed on duplicate 250 ul aliquots using the method described by Bailey et al. (1967).
Quantification and Analysis
Radioactivity was assayed using a Packard 2425 Liquid Scintillation Spectrophotometer. Correction factors for sample quenching were determined with external standardization. The disintegrations per min (DPM) for NE and DA were divided by the amount of protein in tissue samples to derive specific activities. Tyrosine specific activity was DPM/mg tyrosine. Blank values, determined in parallel samples that received radioactivity but were chilled to 00C instead of being incubated, were subtracted from experimental values. Data were expressed as moles of catecholamines accumulated/mg protein/ minute (based on the tyrosine specific activity).
One factor analyses of variance (ANOVA), Student's t-tests and Mann-Whitney U tests were executed on a Hewlett Packard 9810A Calculator. An Amdahl 370, version 6 computer was used for quench corrections and for two or more factor ANOVA's and regression analyses as programmed in the Statical Analysis System (SAS).




79
Results
Effects of icy ACTH and LVP on in vitro [3H]Catecholamine Accumulation
The number of grooming scores recorded during a 20 min period from 10 to 30 min following icy injections of 1 vg ACTH1-24 was elevated to 20/40 from a baseline median of 6/40 (p < .05, Mann-Whitney U test). One ig LVP increased the summed scores of scratching, foraging, and squeaking during the total 30 min period following injection from a median of 3/60 to 44/60 (p < .001, Mann-Whitney U test).
Both 1 pg LVP and 1 pg ACTH1-24 (Fig.'s 3.1 and 3.2) increased by approximately 18% the accumulation of [3H]DA
3
from [ 3H]tyrosine in frontal cortical slices. LVP also decreased [3H]DA accumulation in striatal slices by 15%. Because several animals injected with ACTH did not show excessive grooming, multiple regression analyses of grooming and [3H]catecholamine accumulation were performed. Under no conditions were the correlations of grooming and 1[3H]catecholamine accumulation close to significance, suggesting that the changes in [3H]DA accumulation were unrelated to the grooming response. Substantia Nigral Slices Incubated with ACTH Analogs
Since intranigral, but not intrastriatal,injections
of ACTH1-24 elicit excessive grooming (Wiegant et al., 1977) and because changes in frontal cortex or striatum should be reflected in the cell regions A9 and A10, we first examined the accumulation of 1[3H]catecholamines from [ 3H]tyrosine in




80
120
110
I 10
Uii
Z0
-J
9 0
80
4242 3 35 27 27 272 43 4
FRO NE FRO DA SEP NE SEP DA STR DA
Fig. 3.1. Effects of icy LVP on the in vitro accumulation of [3H]catecholamines from [3H]tyrosine. Tr o6ntal cortical, septal and striatal slices were obtained from animals injected with either icy LVP or saline. Following suspension and rinsing, slices were incubated in 0.5 ml of media with 8.3 pM tyrosine for 20 min. Striatal slices were incubated with 1.14 pCi/ml [3H]tyrosine, while frontal cortical and septal slices were incubated with
7.50 yCi/ml 13H]tyrosine. Open and cross-hatched bars represent icy saline and 1 pg LVP, respectively. Numbers at the base of each bar indicate the total sample size over 5 experiments. ANOVA's for each amine in each region were performed with experiments and treatments as variables for each amine in each region. ** indicates a significant difference from saline at p < .01.




81
120
IIO
w /
z100
~IOO -"
J
90
80
28 28 2829 2828 2324
FRO NE FRO DA HIP NE STR DA
Fig. 3.2 Effects of icy ACTH1-.24 on the in vitro accumulation of [3H]catecholamines from [3T]tyrosine. Slice procedures, statistics and presentation are the same as in Fig. 3.1. Open and cross-hatched bars represent icy saline and 1 pg ACTH1-24, respectively. The combined results of 4 experiments are presented. indicates a significant difference from saline at p < .05.




82
substantia nigral slices containing both cell regions. Substantia nigral slices incubated with 100 pm ACTH1-24 at 8.3 pM tyrosine demonstrated decreased accumulations of both [3H]NE and [3H]DA (Fig. 3.3A).. [D-phe7]ACTH4-10 has been reported to induce excessive grooming (Gispen et al., 1975), while ACTH4-10 has behavioral potency on a variety of learning and memory tasks (de Wied, 1966; Flood et al., 1976). In a second experiment, incubations of nigral slices with these peptides at 8.3 pM tyrosine did not affect [3H]NE and [3H]DA accumulations (Fig. 3.3A). ACTH1-24 had no effect when the media tyrosine concentrations were increased to 83 pM. This lack of effect was observed under standard conditions or when the slices were challenged with 26 mM K+ or with 3 pM haloperidol. Frontal Cortical Slices Incubated with ACTH and LVP
Frontal slices were incubated with 1, 10, and 100 pM ACTH or LVP at 8.3 MM tyrosine. As was seen in substantia nigral slices at 8.3 1M tyrosine, ACTH1-24 significantly
3
decreased the accumulations of both [3H]NE and [3H]DA. 100 1M LVP significantly decreased [3H]NE accumulation (Fig. 3.4).
ACTH1-24 contains two tyrosine residues while LVP contains one residue at position 2. Peptidase-mediated degradation might release free tyrosine in sufficient quantities to decrease the specific activity of media tyrosine, the relevant precursor pool for DA synthesis (Kapatos and Zigmond, 1977; Fig. 2.1). Such a decrease would cause




Fig. 3.3. Effects of ACTH analogs on the accumulation of [3H]catecholamines from [3H]tyrosine in substantia nigral slices. Open and shaded bars represent [3H]DA and [3H]NE, respectively. Numbers at the base of each pair of bars indicate the total number of samples used. ** indicates a significant decrease from saline at p < .01, *** at p < .001 (ANOVA). (A) For ACTH analog comparisons, nigral slices were incubated at
8.3 1M tyrosine with 4.17 pCi/ml [3H]tyrosine.
(B) Nigral slices were incubated with ACTHl-24 at 6 mM K+, at 26 mM K+ and at 3 VM haloperidol (HAL) and 6 mM K+. Tyrosine was set at 83 iM with 16.7 UCi/ml [3H]tyrosine. Asterisks indicate a significant effect of 3 uM haloperidol.




84
A. 8.3 pM tyrosine
80
S
E
0.
? 40 .
0
20
9 9 9 7 71 7 7 7 7 4
-7 -6 -5-4 -6 -5-4 -6 -5-4
SAL 10- 106 IO5 104 SAL 106 10-5 104 106 10 10-4
M ACTHI-24 M ACTH410 M ACTH4_10(D-pe)
B. 83 pM tyrosine
100
.c 80
E
N,
ITI
S60
E N *
m 40
o
E
4)1
20
0 6 16 6 16"1 9 1 1 10 4 4 4
-6 -5 -4 -6 -5 -4 -6 -5 -4 M ACTHI24SAL 106 10 10-4 SAL 106 105 10-4 SAL 106 105 10-4
SALINE 26 mM K' 3X 10-6M HAL




85
12
N.
E 10- d
4) 8
Cx
E 6
E
4
2 K
4 4 4 44
SAL -6 -5 -d4 -6 -5 -4 10 10 I0 10 10 10 M ACTH I- 24 M LVP
Fig.,3.4. Effects of ACTH1 4 and LVP on the accumulation of [3H]catecholamines from [H]tyrosine in frontal cortical slices. Open and shaded bars represent [3H]DA and T3H]NE, respectively. Frontal cortical slices were incubated with
8.3 pM tyrosine at 16.7 pCi/ml [3H]tyrosine. indicates a significant decrease from saline at p < .05, ** at p < .01 (ANOVA).




86
an artifactual decrease in [3H]catecholamine accumulation. Measurements of media tyrosine concentrations at the end of the incubation period indicated that dilution had occurred (Table 3.1). Using the measured specific activity as a correction factor to determine the NE and DA relative specific activities (RSA's) instead of the initial specific activity correction normally used, the effect of LVP was eliminated. However, ACTH significantly elevated the NE RSA. Assuming that peptide degradation and accumulation of [3H]catecholamines are linear throughout the incubation period, the average percentage change in specific activity should be half of what was observed at the end of the incubation. Using this revised tyrosine specific activity, the apparent effects of ACTH were abolished.
Striatal Slices Incubated with ACTH and LVP
Striatal slices were incubated with 10 and 100 -M
ACTH1-24 at 8.3, 83, and 830 uM tyrosine. As was observed in substantianigra and frontal cortex, ACTH1-24 decreased the accumulation of [3H]DA only at 8.3 viM tyrosine (Fig. 3.5). Final tyrosine concentrations were measured at 100 vM ACTH and saline at 8.3 pM tyrosine by two methods: fluorescence assay of the tyrosine fraction and amino acid analysis of the media. By both methods, a significant decline in specific activity was observed (Table 3.2).
Using 83 vM tyrosine to mask any tyrosine dilution effects of peptide degradation, striatal slices were incubated with ACTH1-24 in combination with 6 or 26 mM K+ and with 0.3 uM




Table 3.1. Changes in Media Tyrosine Specific Activity (SA) Caused by ACTH1-24 and Their Effects on Catecholamine Relative Specific Activities (RSA).
Correction Factor
Treatment Media Tyrosine SA Measured Tyrosine SA Averaged Tyrosine SAa
(DPM/ng) DA RSA NE RSA DA RSA NE RSA
Saline 20260719 .061.005 .120.013 .061 .006 .120.013 ACTH124
1 PM 20150643 .072*.008 .115.004 .072*.008 .115 .004
10 pM 17933307 .066.003 .107.001 .062*.003 .100.002
100 PM 10177,194 .076.006 .175 .006 .050*.004 .117*.005 LVP
1 PM 20380*590 .057.009 .115.004 .057*.009 .115.004 10 PM 20620310 .053*.003 .109.005 .053.003 .109t.005 100 pM 17330*280 .062*.006 .104.008 .058*.008 .102+.004
Significance at p<.01, at p<.001
aThe average of the measured tyrosine SA for each treatment and of the measured saline tyrosine specific activity, providing an approximation of the average specific activity during the total incubation period.
00




88
7
E6
5 5
0
E
S3
0
n5RIR 5511511 151 1
M ACTHI-24 SAL 10-5 10-4 SAL 10-5 10-4 SAL 10-510-4
8.3 pM TYR 83 pM TYR 830 pM TYR
Fig. 3.5. Effects of ACTH1-24 on the accumulation of [3H]DA from [3H]tyrosine in striatal slices at 8.3, 83, and 830 vM tyrosine. Striatal slices were incubated at 4.17 pCi/ml [3H]tyrosine. Numbers at the bottom of each bar indicate the sample size. ** indicates a significant decrease from saline at p < .01 (ANOVA).




Table 3.2. Changes in Media Tyrosine Specific Activity (SA) Caused by ACTI11-24.
Fluorescence Assay Amino Acid Analysis Treatment
Tyrosine Tyrosine SA Tyrosine Tyrosine SA
(Pg) (DPM/ng) (pg) (DPM/ng) Saline 1.45.07 6220550 .63.13 155502300
100 vM ACTH1-24 3.55.10 2750*150 1.08.10* 93251000*
-56% -40%
Significance at p<.05 (t-test)
***Significance at p<.001
O0




90
apomorphine, 3 pM haloperidol, or saline. 26 mM K elevated [3H]DA accumulation by approximately 200% while apomorphine and haloperidol inhibited [ H]DA accumulation by approximately 50%. ACTH1-24 had no effect under any combination of treatments (Fig. 3.6).
Similarly, striatal slices were incubated with LVP at
83 uM tyrosine and at both 6 and 26 mM K+. Once again, there was no peptide effect (Fig. 3.7).
Discussion
Both ACTH and vasopressin are released into the peripheral circulation in response to stressful stimuli (Ganong et al., 1974; Sandman et al., 1973b). Only in recent years has evidence suggested that these hormones might also subserve a stress response in the CNS. Changes in behavior following icy administrations of ACTH or LVP have indicated that receptors exist in the CNS which are capable of recognizing these peptides. Intracranial placements of 10 pg pellets of ACTH 1-10 in caudal diencephalon and rostral mesencephalon were shown to facilitate performance in avoidance tasks (van Wimersma Greidanus and de Wied, 1971). Lesions of this area were effective in disrupting ACTHinduced facilitation of task performance. Similarly, icy injections of as little as 2.5 ng LVP delayed the extinction of a conditioned pole jump avoidance (de Wied, 1976). Excessive grooming can be induced by either icy ACTH24
(Gisen et al. 19751 Rees et al.-24 1976 or low doses of (Gispen et al., 1975; Rees et al., 1976) or low doses of




91
A. 6mM K'
1.0
_.8
C
*.6
E 41 E
.2 J
101 11O1 1 0 101 4 4 4 4 8 8 8 8
B. 26mM K'
4
C
. 3
.
l **
4 4 4 4 4 4 4 4 4 4 4
M ACTH-. SAL 106 65 d4 SAL 06 10 5 1o-4 SAL Id6 10 10'
SALINE 3 X 1 7APO 3 X FO6 HAL
Fig. 3.6. Effects of ACTH1-24 on the accumulation of I3H]DA from 13H]tyrosine in striatal slices challenged
with 0.3 iM apomorphine and 3 pM haloperidol at 6 and 26 mM K Striatal slices were incubated as described
in Fig. 3.5. ** indicates significant effects of haloperidol and apomorphine at p < .01 (ANOVA).




Full Text

PAGE 1

PEPTIDE HORMONE INTERACTIONS WITH BRAIN CATECHOLAMINE METABOLISM BY RI CHARD L. DEL.Ai"'IOY, JR. A DISSERTATION PRESENTED TO THE GRADUATE COUN CIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIRiMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY U N I VERSITY OF FLORIDA 1979

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ACKNOWLEDGEMENTS The author wishes to thank Dr. Adrian Dunn for his support, teaching, tolerance and sense of humor Ms. Leila Baz-Malcom, Mr. John Hockensmith, Mr. Terry Moore, Ms. Gale Hunter, Ms. Laurie Puckett, Mr. Harr y Forster, Dr. Neal Kramarcy, and Ms. Nancy G ildersleeve for their technical assistance and in calming the panics, humoring the depressions, sobering the insanities, reassuring the anxieties~ and enlivening the tedium which accompanied the author's style of research Dr. Maartin Reith for the use of his amino acid analyzer all my friends for their part in making my time in Florida pleasureable. Dr. Samuel Guri n for having convinced me that I did not want to be a fireman. The author was a National Science Foundation Predoctoral Fellow f rom July, 1976 to July, 1979. The research described was supported b y USPHS AM-18399 and MH-25486. 11

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SECTION I II TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS ................................ ii KEY TO ABBREVIATIONS ............................. v ABSTR.L\.CT ........................................ vi INTRODUCTION ..................................... 1 Behavioral Effects of ACTH and LVP ............. 4 Behavioral Interactions of Catecholaminergic Systems with ACTH and LVP Treatments ......... 9 ACTH Effects on Catecholamine Metabolism ...... 10 Effects of LVP on Catecholamine Metabolism .... 13 HALOPERIDOL AND APOMORPHINE ALTER CATECHOLAMINE SYNTHESIS IN VITRO BY DIFFERENT MECHANISMS .... -...................... 16 Introduction .................................. 16 Methods ....................................... 19 Materials ................................... 19 Procedures .................................. 21 Chromatography .............................. 23 Quantification and Analysis ................. 26 Results ....................................... 27 Determination of Relevant Precursor Pool .... 27 K+-Induced Activation of the Accumulation of [3H]Catechqlamines ..................... 30 Inhibition of [JH]Catecholamine Accumulation from [3H]Tyrosine ............ 32 Accumulation of {3H]Protein from [3H]-Tyros ine .................................. 36 Liberation of [14C]COz from [14C]Tyrosine ... 40 Uptake of [14C]DA ........................... 40 Release and Metabolism of Preloaded Labelled DA ............................... 43 Discussion .................................... 49 Precursor Pool Determination ................ 49 Effects of Elevated K+ upon Synthesis, Release, and Metabolism of D A ............. SO Effects of Apomorphine on DA Metabolism ..... 53 Effects of Haloperidol on Catecholamine Synthesis and Release ..................... 56 Summary ....................................... 68 iii

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III ACTH AND LVP SELECTIVELY ACTIVATE MESOCORTICAL DA SYNTHESIS ..................... 7 0 Introduction .................................. 7 0 Methods ....................................... 72 Materials ......... ...... .................... 72 Animals and Surgery ......................... 73 Incubation Procedures ....................... 7 5 Chromatography .............................. 76 Quantification and Analysis ................. 78 Results ....................................... 79 Effects of icv ACTH and LVP on in vitro [3H]Catecholamine Accumulatiori-:-........... 79 Substantia Nigral Slices Incubated with ACTH Analogs .............................. 79 Frontal Cortical Slices Incubated with ACTH and LVP .............................. 82 Striatal Slices Incubated with ACTH and L V P ....................................... 86 Discussion .................................... 90 IV CONCLUDING REMARKS ............................. 102 REFERENCES ..................................... 108 BIOGRAPHICAL SKETCH ............................ 12 2 lV

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ACTH aMPT AVP CNS CSF DA EDTA GABA lCV LVP MSH MIF PLG NE RSA SA KEY TO ABBREVIATIONS Adrenocorticotropic hormone a-methyl-para-tyrosine Arginine vasopressin Central nervous system Cerebrospinal fluid Dopamine Ethylenediaminotetra-acetic acid y-aminobutyric acid Intracerebroventricular Lysine vasopressin Melanocyte stimulating hormone Melanocyte stimulating hormone inhibiting factor (PLG) Prolyl-leucyl-glycinamide (MIF) Norepinephrine Relative specific activity Specific Activity V

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Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PEPTIDE .HORMONE INTERACTIONS WITH BRAIN CATECHOLAJv:IINE METABOLISM By Richard L. Delanoy, Jr. December 1979 Chairman: Adrian J. Dunn Major Department: Neuroscience The accumulation of [ 3H]catecholamines from [ 3H]tyrosine 1n frontal cortical, septal, striatal and hippocampal slices was examined following intracerebroventricular ( i c v ) injections of ACTH1 24 lysine vasopressin (LVP) and saline. Both ACTH1 24 and LVP selectively increased the accumulation of [3H]dopamine (DA) in frontal cortical slices but did not affect [3H]norepinephrine (NE) accumulation. LVP also inhibited the accumulation of [3H]DA in striatal slices. Incubations with ACTH analog s or LVP failed to alter the rate of accumulation of [3H]catechola mi nes in striatal, substantia nigral and frontal cortica l vi

PAGE 7

slices. Also, neither peptide modified the activation of catecholamine synthesis by 26 ~1 K+, nor did ACTH1 2 4 modify the inhibition of [3H]catecholamine accumulation caused by 0.3 M apomorphine and 3 M haloperidol. The selective activation of the mesocortical D A system by icv injections of peptides which also occurs in response to footshock stress suggests that endogenous ACTH and/or LVP may mediate the stress-induced mesocortical D A activation. Alternatively icv injections of these peptide s may be stressful and thus elicit a stress response. A variety of control experiments w a s performed to characterize the normal kinetics and the effects of elevated + K, apomorphine and haloperidol on catecholamine s ynthesis. Our experiments showed that ( 1 ) D A was preferentially s ynthesized from t yrosine recently taken up from the media. Kinetic data exhibited Michaelis-Menton kinetics only when corrected b y media t yrosine specific activity and not tissue t yrosine specific activity (2) K+-induced activation o f D A s ynthesis in striatal slices occurred a t concentrations tha t were insufficient to indu~e [3H]DA release, suggesting that synthesis activation is not consequent on release, but rather is a separate response to depolarizations. ( 3 ) Apomorphine inhibited both [3H]DA and [3H]N E accumulation i n striatal, c erebellar and substantia nigral slices in a dose-dependent manner. The potency o f 1 0 M apomorphine i n inh ibiting the accumulation o f [3H]cate c holamines w a s reduced b y half when t h e m edia t yrosine concentration was vii

PAGE 8

increased from 8.3 to 83 M. + K -induced activation of tyrosine hydroxylase (TH) did not diminish the potency of apomorphine to inhibit [ 3H]DA accumulation. Since activated TH is less sensitive to end-product inhibition, then apomorphine-induced inhibition of synthesis probably is not mediated by end-product inhibition of TH. Further-+ more, 10 nM haloperidol increased K -stimulated synthesis of DA, perhaps reflecting an antagonism of the inhibi-tion of D A synthesis by released DA. These findings suggest receptor-mediated inhibition of catecholamine s ynthesis. The ability of apomorphine to inhibit NE synthesis, together with the presence of presynaptic D A receptors on NE-terminals, further argues that apomorphine regulates synthesis by acting on a presynaptic receptor, although one not specific to DA-receptors. ( 4) Haloperidol also inhibited [ 3H]DA accumulation from [ 3H]tyrosine in a dose-dependent manner, but apparently not b y a receptormediated mechanism. Haloperidol (10 M) elevated basal release of labelled DA and the formation of labelled 3,4 dihydroxyphenylacetic acid in striatal slices preloaded with labelled D A + 2 + These effects were K and C a indepen-dent, suggesting that the increased cytoplasmic D A concentration was not a consequence of increased release and reuptake. These effects of haloperidol were not observed with fluphenazine and chlorpromazine, perhap s reflecting properities spetific to butyrophenones. Since reserpine was viii

PAGE 9

found to have effects identical to haloperidol on synthesis, release and catabolism, haloperidol is proposed to have reserpine-like properties at micromolar concentrations. Previous in vivo and in vitro experiments that have used haloperidol as a typical neuroleptic may have obtained results related to vesicular disruption and unrelated to dopamine receptor blockade. lX

PAGE 10

SECTION I INTRODUCTION Evidence has accumulated that within the central nervous system (CNS), ACTH and vasopressin may mediate behavioral responses. Intracerebroventricular (icv) injections of ACTH elicit excessive grooming in rats and mice (Gispen et al., 1975; Rees et al., 1976). This ACTH-induced grooming was antagonized with peripheral injections of haloperidol (Wiegant et al., 1977) and with injections of various dopaminergic agonists and antagonists into the striatum and nucleus accumbens (Cools et al., 19 78), suggesting that the mesostriatal dopamine (DA) s ystem functioned in the expression of grooming. Excessive grooming can als o be induced with mildly stressful stimulation (Colbern et al., 1978), a response which was antagonized with icv ACTH antisera (Dunn et al., 1979). Similarly icv injections of arginine vasopressin (AVP) and l ysine vasopressin (LVP) facilitated the performance of rats in both active and passive avoidance tasks. AVP antisera (icv) impaired the performance of rats on these same tasks (van Wimersma Greidanus and de Wied, 19 75). Since AVP is released into the cerebrospinal fluid of rats in response to stressful stimuli (Dogterom et al., 19 78) a facilitation of task performance as a result of stressful stimulation may be mediated by AVP. 1

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2 Recently, footshock was determined to selectively activate the mesocortical DA system by two separate research groups (Thierry et al., 1976; Fadda et al., 1978). Increases in both turnover and s ynthesis that were observed in frontal cortex and nucleus accumbens were not evident in NE terminals in these regions nor in DA terminals in the striatum. In order to test the hypothesis that icv ACTH or LVP might mimic a stress-induced change in catecholamine metabolism and that the mesostriatal DA system mediates ACTHinduced grooming as suggested by Wiegant et al. (1977), norepinephrine (NE) and DA synthesis rates were measured in frontal cortex, striatum, septal nuclei and hippocampus following icv injections of either ACTH or LVP. The accumulation of [ 3H]catecholamines from [ 3H]tyrosine was measured in brain slices dissected from animals receiving peptide or control injections. ACTH competes with [ 3H]haloperidol for binding to striatal membranes (Czlonkowski et al., 1978) and dopaminergic a gonists and antagonists inhibit ACTH-induced grooming. Neuroleptics at low concentrations antagonize apomorphineinduced inhibition of DA synthesis in vivo (Kehr et al., 1972) and in vitro (Ebstein et al., 1974; Christiansen and Squires, 1974). At high concentrations, haloperidol inhibited DA synthesis in vitro (Christiansen and Squires, 19 74) Since ACTH might have DA agonist properties, ACTH was also incubated with striatal and substantia nigral slices challenged with doses of apomorphine or haloperidol that inhibited DA synthesis by approximately 50%

PAGE 12

In vitro techniques admittedly have disadvantages. In particular, changes in catecholamine metabolism may have such short half-lives that an effect of peptide administration may not be detectable by the time the assay period begins. Furthermore, the loss of homeostatic regulation due to the severing of modulating neuronal input probably alters baseline catecholamine metabolism. However, the use of an in vitro system reduces the possibility of precursor pool alterations which might artifactually influence synthesis rate measurements. Also, the use of an in vitro technique readily permitted an investigation of peptidergic effects 3 to extend to the tissue level. By incubating slices with peptides and simultaneously measuring catecholamine synthesis, attempts were made to determine whether or not peptide reception occurred in the tissue being incubated, independent of interacting variables caused b y changes in endocrine, peripheral, or remote neuronal functioning. Such a technique has successfully detected effects o f ether stress on hypothalamic catecholamine s ynthesis ( Hed g e et al., 19 7 6 ) and has assessed the effects of a variety of dopaminergic drug s (Uretsky and Snodgrass, 19 77; Christiansen and Squires, 19 74) as well as other peptides (e.g.,MIF, Friedman et al., 1973 ) Following a general discussion of background literature on the behavioral and biocRemical interactions of ACTH and L V P with the catecholamines, data derived from these experiments are presented as t w o manuscripts for publication. The first section contains control experiments characterizing

PAGE 13

normal incubation parameters and the effects of elevated K+ apomorphine, and haloperidol on catecholamine metabolism. The second section reports on the effects of ACTH and LVP on catecholamine synthesis in vitro. Behavioral Effects of ACTH and LVP The possibility that various pituitary hormones might 4 be involved in CNS function was first suggested by the observation that hypophysectomized animals were impaired in the acquisition of a conditioned avoidance task. Replacement therapy with ACTH or ACTH4 10 was able to reverse this deficit (Applezweig and Baudry, 1955; de Wied, 1964: de Wied and Gispen, 1977). Also, if given shortly before training, ACTH and ACTH4 10 stimulated acquisition of a conditioned avoidance response in intact animals (de Wied and Gispen, 1977; Flood et al., 1976; Gold and McGaugh, 1977). Similarly, neurohypophysectomized rats (de Wied, 1965), Brattleboro rats, which have a genetic deficiency of vasopressin (de Wied et al., 1975), and rats given icv AVP antiserum all showed deficits in active and passive avoidance learning (van Wimersma Greidanus et al., 1975). ACTH1 24 ACTH4 10 (de Wied, 1966) and LVP (de Wied, 1971) have been reported to delay the extinction of a conditioned avoidance response; ACTH4 10 and desglycinarnide-LVP (DG-LVP) delayed the extinction of sexually motivated behavior in rats (Bohus et al., 1975; Bohus, 1977); and ACTH4 10 or ACTH1 _24 delayed the extinction of rewarded behavior (Garrud et al., 1974). However, while a single

PAGE 14

dose of LVP was effective in delaying the extinction for several days, ACTH analogs had to be administered daily to maintain their effect. Finally, ACTH, ACTH4 10 and vasopressin, under certain conditions were all able to reverse the effects of amnestic agents on passive avoidance tasks (Flood et al., 1976: Rigter et al., 1975). It was been argued in the past by de Wied and his colleagues that both ACTH and LVP are directly affecting memory formation or a retrieval mechanism (de Wied, 1966; van Wimersma Greidanus et al., 1975). However, the requirement of daily administrations of ACTH to maintain delayed extinction suggests that ACTH-induced delay of extinction does not reflect an affect on memory mechanisms, since improved memory following a single post-training injection would be evident more than one day later. Kastin et al. (1977) have claimed that ACTH4 10 and ~-acetyl Ser~ ACTH1 13 5 (a-MSH) facilitated performance in tasks designed to measure attention in both humans and rodents. Taken together, these data argue against a direct action of ACTH on memory processes and instead suggest some change in attention or motivational state, In the case of LVP, de Wied still maintains that it has a direct effect on memory processes. However, it can be argued that the effects of LVP closely parallel the effects of non-specific stimulants such as caffeine, nicotine or amphetamine. Caffeine, nicotine or amphetamine can reverse

PAGE 15

retrograde amnesia (Flood et al., 1978; Barondes and Cohen, 1968). Nicotine and amphetamines can facilitate acquisition of active avoidance learning (Orsingher and Fulgeniti, 6 19 7 1 ) Furthermore, since hypophysectomy results in elevated levels of vasopressin in the cerebrospinal fluid (Dogterom et al., 197 7), the deficits in avoidance learning in neurohypophysectomized rats argue for a peripheral site of the influence of vasopressin on learning performance and not for a central one. This finding conflicts with the earlier findings of de Wied showing that intracerebroventricularly administered arginine vasopressin (AVP) antisera will impair retention of a passive avoidance task. This mi ght indicate tha t the AVP antibodies are causing a deficit in A V P release f rom the neurohypophysis or once cleared from the central nervous system ( C N S ) through the arachnoid granulations, are binding to plasma AVP. However, until it is shown that neurohypophysectomy instead of a full h ypophy sectomY, will also result in elevated vasopressin concentrations in the cerebrospinal fluid, the h ypothesis that exogenous vasopressin alters memor y performance via a central receptor remains viable. Conversely the data derived through the use o f AVP antisera do not necessarily support the h ypothesis that vasopressin is an essential component o f memory processes. Instead, AVP may be performing an essential physiol ogical process necessary for optimal C N S activation state for adaptiv e behav iors. A major prob lem with these data supporting a centra l section of systemically injected hormones is that the

PAGE 16

peripheral effects of hormones so administered are indistinguishable from any possible central effects, Glucose metabolism, blood pressure, lipolysis, and steroidogenesis are all affected by exogenous administrations of ACTH or LVP. A possible way of bypassing many of these peripheral responses is to administer the hormones intracranially. 7 De Wied (1976) has studied extensively the behavioral effects of vasopressin using this route and has found facilitation of performance ori the pole-jump active avoidance task at remarkably low doses (2,5 ng arginine vasopressin). Moreover, the fact that rats receiving AVP antisera or that Brattleboro rats lacking endogenous AVP have impaired memory performance suggests that LVP may have a physiological role as a neuromodulator. This is further supported by the findings that vasopressin-and neurophysin-bearing neurons and terminals, as determined by immunohistology, are found in various nuclei in the brain (Buijs, 1978; Swanson, 1976). In addition, intraventricular injections of LVP in mice will elicit an increase in activity which is predominantly grooming or a mixture of grooming1 foraging, scratching and squeaking, depending on the dose applied (Delanoy et al,, 19 78) Kruse et al. (1977) reported that icv LVP given to rats most often produced barrel rotations (rotation around the sagittal axis of the animal), and occasional episodes of "nest-building" activity. In contrast, barrel rotation in response to icv LVP was seldom observed in mice (Delanoy et al., 1978).

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8 Intracerebral implantations of 10 g crystalline ACTH1 10 and [D-phe7]ACTH1 10 in the rostral mesencephalon and caudal diencephalon or in CSF produced behavioral effects similar to systemic injections of larger doses of these peptides (van Wimersma Greidanus and de Wied, 1971). Moreover, icv ACTH has other behavioral effects. Doses of 10 g or less of ACTH1 _24 elicit penile erection (Bertolini and Baraldi, 1975), excessive grooming (Gispen et al., 1975; Rees et al., 1976) and stretching and yawning (Baldwin et al., 1974). Moreover, Colbern et al. (1978) have observed increased grooming in animals when introduced to a novel environment, which has been found to be attenuated by hypophysectomy and by icv ACTH antisera (Dunn et al., 1979). This finding, as well as the probability that ACTH neurons are present in the CNS (Barchas et al., 1978) and that pituitary peptides may enter the CNS by retrograde blood flow in the hypophysis (Oliver et al., 1977) suggests that behaviors induced by icv ACTH are patterns also initiated by endogenous ACTH. These data argue that receptors capable of recognizing ACTH and LVP do exist in th~ CNS and may mediate the facilitation of performance observed with peripheral administrations of these hormones. However, changes in spontaneous behavior observed with icv ACTH or LVP have never been reported following peripheral administrations. Such a lack of response might be reflecting the relative inability of peptides to cross the blood-brain barrier, or perhaps a quantitative difference in minimal dosages necessary to elicit changes in spontaneous behavior.

PAGE 18

Behavioral Interactions of Catecholamineraic S stems with ACTH an LVP Treatments Several lines of evidence predict that the hormones ACTH and LVP would have an effect on CNS catecholamine metabolism. Firstly, the release of ACTH (Ganong, 1974) and vasopressin (Sandman et al., 1973b) as well as the activation of the noradrenergic system (Stone, 1975) all appear to be consequences of stressful episodes. It is possible that the hormonal release and the noradrenergic activation are redundant, parallel systems that do not interact. However, anatomical studies show that ACTH and neurophysin (proteins released from terminals of oxytocinand vasopressin-bearing neurons) send projections from the hypothalamus to the areas of the locus coeruleus and nucleus solitarius (Barchas et al., 1978; Swanson, 1977). Segal (1977) has reported that inhibition of firing of hippocampal neurons by norepinephrine (NE) was antagonized by iontophoresed ACTH. Also, the facilitation of a passive avoidance response by AVP was attenuated by lesion s of the dorsal noradrenergic bundle, which supplies NE to m uch of the forebrain (Kovacs et al., 1979)~ ACTH and LVP also appear to interact in some way with dopaminergic neurons. Systemic haloperidol attenuates the excessive grooming induced by icv ACTH (Wiegant et al., 9 1977) and icv LVP (Delanoy et al., 1978). More specifically, intranigral injections, but not intrastriatal injections of A CTH,would elicit excessive grooming. In addition, melanocyte

PAGE 19

10 release inhibiting factor (MIF, prolyl-leucyl-glycinamide), a peptide that affects avoidance learning in a manner similar to vasopressin and that comprises the C-terminal tripeptide fragment of oxytocin, potentiated the salivation, piloerection, and Straub tail phenomenon (stiffened and erect) induced by L-DOPA. It also antagonized the catalepsy induced by fluphenazine, a DA-receptor blocker (Plotnikoff et al., 1974; Voith, 1977). ACTH Effects on Catecholamine Metabolism An orderly body of evidence on the effects of ACTH has been obtained measuring the disappearance of NE following TH inhibition with aMPT. Peripheral injections of ACTH1 _24 (Hokfelt and Fuxe, 1972), ACTH4 10 (Leonard et al., 1975; Versteeg, 1973) and aMSH (Kostrzewa et al., 1975) have all been reported to increase N E disappearance (turnover) in the CNS. [D-phe7JACTH4 10 a peptide with behavioral activity opposite to ACTH4 10 in active avoidance tasks, has been reported to increase N E disappearance (Leonard, 1974) or to have no effect at all (Versteeg, 19 73). This contradiction mi ght be e xplained b y the different injection routes used in these t w o studies. H ypophysectomy, in contrast, caused a decrease in NE disappearance (Fuxe et al., 19 73; Fuxe et al., 1970; Friedman et al., 1973) that was reversed by high concentrations of ACTH (Versteeg and Wurtman, 19 75, cited as data to be published, method of analysis not known ) but not with low doses (Fuxe et al., 19 70). These changes in N E disappearance

PAGE 20

11 are apparently not mediated by an adrenal factor since adrenalectomy caused a glucocorticoid reversible increase in NE disappearance (Javoy et al., 1968; Fuxe et al., 1973) and since glucocorticoids and mineralocorticoids only slightly, if at all, decreased NE disappearance in intact animals (Fuxe et al., 1973). These manipulations affect DA disappearance after aMPT-induced synthesis inhibition much less clearly. Hypophysectomy decreased DA disappearance (Versteeg et al., 1972; Friedman et al., 1973) while adrenalectomy appeared to have little affect (Fuxe et al., 1973). Furthermore, corticosterone and dexamethasone will increase DA turnover in hypophysectomized, but not in intact rats (Fuxe et al., 1970; Jonsson et al., 1972). However, while NE disappearance was clearly increased with peripheral ACTH administrations, ACTH1 _24 given to hypophysectomized rats had either no effect or only slightly decreased DA turnover 1n the neostriatum and limbic forebrain (Fuxe et al., 1973). Using a different estimate of turnover, Endroczi et al. (1976) investigated the effects of icv ACTH1 _24 and ACTH4 10 on the rate of disappearance of preloaded [ 3H]NE from neocortex, hypothalamus and hippocampus. Either peptide (5-10 g, icv) promoted an increase in 3 H disappearance. Adrenalectomy caused a similar increase in turnover, while glucocorticoids given to intact animals had no effect on the disappearance rate. Thus, exogenous administrations of ACTH, and perhaps the increase in endogenous ACTH following adrenalectomy

PAGE 21

due to decreased inhibitory feedback, appeared to stimulate N E turnover, seemingly c o nfirming data derived using the measurement of disappearance rates of endogenous amines following s ynthesis inhibition. However, an important limitation with these results is that icv injected [ 3H]NE can be taken up by dopaminergic and serotoninergic neurons as well as noradrenergic ( Sn yder and Coyle, 1969) Thus, the disappearance of labelled NE reflects a complex average of the activation of these three neuronal s ystems. Two studies have attempted to measure 1n vivo s ynthesis --., b y measuring the accumulation o f [JH]catecholamines from subcutaneously injected [ 3H]tyrosine. In the first study Versteeg and Wurtman (197 5 ) showed that ACTH4 _10 increased the accumulation of total [ 3H]catecholamines. Moreover, 12 this i ncrease did not occur in h ypophysectomized rats give n ACTH4 _10. The authors argued that these data indicated that the effects of ACTH4 _10 mi ght be mediated b y an adrenal factor. Another alternative is that adrenalectomy caHsed a chronic increase in ACTH release which would mask any effects of e x o genous injections. Iuvone et al. (1978) took this analysis a step further b y differentiating the amount of label associated with e ach amine. They found that ACTH1 _24, ACTH4 _10, and [D-phe7 ] ACTH4 _10 increased D A but not N E s ynthesis, while a M S H B M S H, or L V P were without effect, The e ffect of ACTH 4 _1 0 on D A s ynthesis was blocked b y adrenalectomy in agreement with Versteeg and Wurtma n ( 1 97 5 )

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These last two studies disagree markedly from the results determined with the aMPT method o f turnover analysis. While ACTH activates DA synthesis without an effect upon NE synthesis, ACTH also activates NE disappearance with no consistent effect upon DA disappearance. This dichotomy of results might be due to an artifact of differential latencies of activation of synthesis and release, different times of assaying (10 minutes for synthesis, 2 or more hours for the turnover studies), or may in fact indicate a property of ACTH's interactions with the catecholamine populations. Effects of LVP on Catecholamine Metabolism 13 Using the aMPT method of turnover analysis, Tanaka et al. (197 7a) examined the effects of intraventricular arginine vasopressin on catecholamine disappearance. Looking at large brain regions, increased NE turnover was reported in the thalamus, hypothalamus and medulla. Increased DA turn over was only observed in the preoptic area, with no effect at all on basal ganglia DA. Later that year, Tanaka et al. (1977b) presented a similar analysis from microdissected pieces of tissue. They reported that NE disappearance was accelerated in dorsal septal nucleus, anterior hypothalamic nucleus, medial forebrain bundle, parafascicular nucleus, dorsal raphe nucleus, locus coeruleus, and the nucleus tractus solitarii. In contrast with their earlier publication, A V P was shown to increase the disappearance of DA in caudate nucleus several fold, as well as in median eminence,

PAGE 23

dorsal raphe nucleus, and region A8. The inconsistency of the changes in striatal DA between the two tissue collection procedures is hard to explain but may be reflecting local areas within the basal ganglia which are preferentially activated by icv AVP injections. Nevertheless, an effect 14 of icv AVP on striatal turnover appears to have been substantiated by these authors. AVP antisera reduces the disappearance rate of striatal DA (Versteeg et al., 1978). Furthermore, Brattleboro rats with an hereditary deficiency in vasopressin also have a reduced rate of striatal DA turnover (Versteeg et al., 1979). The only study to examine the effects of peripheral administrations of vasopressin on catecholamine synthesis was that by Iuvone et al. (197 7 ) which did not find an effect. The terminal tripeptide of oxytocin, MIF (PLG) has behavioral potency in memory tasks (Sandman et al., 1973a; Stratton and Kastin, 1975) and like LVP will facilitate morphine dependence (van Ree and de Wied, 1976). However, icv MIF is ineffective as compared to LVP in terms of eliciting changes in spontaneous behavior (Delanoy et al., 19 7 8) The effects of MIF on catecholamine metabolism are controversial at the moment. Using the aMPT method of measuring turnover, Versteeg et al. (1978) and Pugsley and Lippman (19 7 7) report that MIF at 40 and 5 mg/kg body weight respectively caused an increase in DA turnover, Kostrzewa et al. (1974) and Plotnikoff et al. (1974) reported that MIF at 20 and 100 mg/kg respectively had no effect on turnover. DA

PAGE 24

synthesis was reported to be increased in slices of striatum in intact but not hypophysectomized rats treated with 0.5 15 and 5 mg/kg MIF (ip). In view of reports that indicated that MIF potentiated the induction of salivation, piloerection and the Straub tail phenomenon by L-DOPA (Plotnikoff et al., 1974) and the reversed the induction of catalepsy by fluphenazine (Voith, 1977), it is likely that MIF is indeed affecting DA metabolism.

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SECTION II HALOPERIDOL AND APOMORPHINE ALTER CATE.CHOLAMINE SYNTHESIS I N VITRO BY DIFFERENT MECHANISMS Introduction The experiments described in this report were initiated to test the effects of the pituitary peptides ACTH and lysine vasopressin (LVP) on catecholamine synthesis in vitro. ACTH has been shown to compete with [ 3H]haloperidol for binding to striatal membranes (Czlonkowski et al., 1978). Also, grooming induced by intracerebroventricular (icv) injections of ACTH was inhibited by haloperidol (Wiegant et al,, 1977). Therefore, in preparation for studies examining possible interactions of haloperidol and the dopamine (DA) agonist apomorphine with ACTH and to test the validity of our slice preparation as a model of in vivo catecholamine synthesis, the effects of these two drugs on the accumulation of [ 3H]catecholamines from [~H]tyrosine were evaluated. Our initial experiments confirmed earlier reports that both haloperidol (Christiansen and Squires, 19 74) and apomorphine (Christiansen and Squires, 1974; Ebstein et al., 19 7 4) inhibited DA synthesis in vitro. The paradox that an agonist and an antagonist of DA receptors would have the same effect on synthesis prompted us t o further examine the mechanisms by which these two agents exert their effects. 16

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Iontophoretic administrations of DA or apomorphine into the substantia nigra inhibited the firing of dopaminergic netirons in the pars compacta of the substantia nigra (Skirboll et al., 1979; Aghajanian and Bunney, 1977; Groves et al., 1975). The inhibition of firing might have been due to a presynaptic facilitation of gamma-aminobutyric acid (GABA) release from terminals arising from cells in the striatum (Reubi et al., 1978). However, the existence of dendro-dendritic synapses between dopaminergic dendrites in 17 the pars reticulata of the substantia nigra (Wilson et al., 1977) has supported the suggestion of Aghajanian and Bunney (1974) that DA receptors exist on dopaminergic neurons. Such receptors which mediate self-inhibition within a neuronal population have been collectively called autoreceptors. In noradrenergic neurons, dendritic autoreceptors inhibit cellular firing in response to adrenergic agonists (Aghajanian et al., 1977), as was observed in dopaminergic neurons. In addition, exogenous norepinephrine (NE) inhibited the K+-stimulated release of [ 3HJNE from noradrenergic terminals in vitro (Starke et al., 1977). Since the inhibition was reversed with a-adrenergic receptor blockers, a presynapic autoreceptor probably also exists. The existence of a similar presynaptic autoreceptor on DA terminals was supported by the finding that [ 3H]apomorphine binding was reduced following lesions of the nigrostriatal tract with the catecholaminergic neurotoxin 6-hydroxydopamine (6 -0HDA, Nagy et al., 1978). However, unlike NE presynaptic

PAGE 27

receptors, most studies have not found an effect of depamine agonists on DA release (Dismukes and Mulder, 1977; Seeman and Lee, 19 75; Raiteri et al., 19 7 8 ) although apomorphine has been reported to inhibit electrically (Farnebo and Hamberger, 19 71) and K+ (Miller and Friedhoff, 19 7 9 ) stimulated [ 3H]DA release by less than 20% Also, DA receptor blockers inhibited the stimulated release of [ 3 H]DA, an effect which would not be predicted by an action on dopaminergic presynaptic autoreceptors (Seeman and Lee, 19 75; Raiteri et al., 19 78). Thus, either DA autoreceptors do not modulate release, or they do so b y mechanisms different from those found in noradrenergic terminals. A n alternative mechanism by which dopaminergic autoreceptors may regulate synaptic efficacy is b y inhibiting DA s ynthesis. A t micromolar concentrations, apomorphine strongly inhibited DA synthesis in striatal slices and s ynaptosomes, an effect reported to be reversible by neuroleptics (Christiansen and Squires, 19 74; Westfall et al., 19 7 6 ) However, haloperidol also inhibited D A s ynthesis in striatal slices at micromolar concentrations (Christiansen and Squires, 19 7 4 ) a result which contradicts both the hypothesized regulation of DA synthesis by an autoreceptor, and the consistently reported finding that haloperidol activates DA s ynthesis in vivo (Kapatos and Z i g mond, 1979; Carlsson et al., 1977; Lerner et al., 197 7) In a single incubation s ystem, we have investigated these apparent contradictions b y comparing the e ffects of 18

PAGE 28

haloperidol and apomorphine on catecholamine synthesis and DA release, metabolism and uptake. Brain slices were employed rather than synaptosomes to maintain tissue integrity insofar as possible so that the chances of observing effects of ACTH and LVP would be improved. Characterizations of normal kinetics and the effects of elevated concentrations of K+ on the accumulation of [ 3H]catecholamines from [ 3H]tyrosine are also presented. Our findings indicate that while the effects of apomorphine on synthesis probably are mediated by a DA receptor, the effects of haloperidol on catecholamine metabolism in most in vitro studies may have been due to membrane disruption as originally suggested by Seeman et al. (1974). Methods Materials Male CD-1 mice (Charles River, Wilmington, Mass., 19 25-30 g), were used in all experiments; they were individually housed for the three days immediately prior to killing. Animals were maintained on a 7:00 a.m. 7:00 p.m. lighting schedule. Drugs used in these experiments were as follows: apomorphine (hydrochloride, Sigma), chlorpromazine (hydrochloride, Sigma), fluphenazine (hydrochloride, a gift from E.R. Squibb and Sons, Inc.) haloperidol (crystalline, a gift from McNeil Laboratories), and reserpine (crystalline, Sigma). Radioactive materials used were [ 2,6-3H]tyrosine

PAGE 29

(34 Ci/mmol, The Radiochemical Centre, Amersham), [l-14C]tyrosine (58 mCi/mmol, New England Nuclear), and [l-14C]dopamine hydrochloride (55 mCi/mmol, The Radiochemical Centre, Amersham). Other chemicals used in tissue preparations were HEPES (Ultrol, Calbiochem), TRIS (Trizma base, Sigma), PPO (Scintillar, Mallinkrodt), POPOP (Research Products International Corp.), and Triton X-100 (Scintillar, Mallinkrodt). The medium used in all experiments was that described by Versteeg et al. (1974) and contained the following: 118 mM NaCl, 4.4 mM KCl, 2.6 rnM CaC1 2 1.3 mM M gS0 4 1.2 mM K2HP04, 25 mM HEPES, and 12 mM glucose titrated to pH 7.3 with NaOH. Increases in KCl were countered wit~ an equivalent decrease in NaCl. Apomorphine and haloperidol were dissolved 1n 0.2 M acetic acid at 2 g/l and diluted to 0.25 g/l with medium. Chlorpromazine and fluphenazine were dissolved in a drop of glacial acetic acid and diluted to 0.25 g/l with medium. Reserpine was solubilized in a drop of glacial acetic acid 20 and diluted to 5.0 g/l with medium 12.1 l of apomorphine, 15 l of haloperidol, 1 7.1 l of chlorpromazine or 14 l of fluphenazine were added to a one ml final incubation volume to achieve a final concentration of 10-5 M Twelve l of the reserpine stock solution was added to one ml of medium to achieve a 10-7 M concentration. Lower concentrations were produced by serial dilutions of the stock solutions with media. 'Saline' controls were composec of appropriate volumes of acetic acid and media in appropriate proportions.

PAGE 30

Procedures Mice were killed by decapitation. Brains were removed and striatum, substantia nigra, or cerebellum were dissected out on a chilled surface. Substantia nigra was obtained by the method of Westerink and Korf (1976). A coronal slab was removed by sectioning at the border of the pons and mesencephalon and at a plane 0.8 mm rostral. Substantia nigra, visible near the ventral surface of the slab, was removed along with the tegmental nucleus (AlO) from the rest of the mesencephalon. In a typical 50 sample experi-ment, 10, 20, or 40 mice were used to generate slices of cerebellum, striatum, or substantia nigra, respectively. Dissected pieces were sliced in two dimensions with a Mcilwain Tissue Chopper set at 300 m. Slices were suspended and pooled in medium at 37C in 02:C02 (95:5) atmosphere. After 30 min the medium was aspirated and replaced with fresh, equilibrated medium for an additional 10 min. Four kinds of experiments were performed. 1.) The accumulation of [ 3H]catecholamines from [ 3H]tyrosine was measured. Prepared slices (0.5 ml) were added to a series of Corex round bottomed 15 ml centrifuge tubes containing 0 5 ml of equilibrated medium containing [2,6-3H]tyrosine, cold tyrosine, and appropriate drugs. Samples were incu bated for 40 min. The reaction was stopped with 2 ml of ice-cold medium and centrifuged at 1 0 0 x gin a refrigerated ( 4C ) Sorvall RC-3 Centrifuge. Media were decanted and 21

PAGE 31

in some instances saved and processed for measurement of released metabolites. Another 2 ml of ice-cold medium was added to the samples and the centrifugation repeated. The media were once again decanted. 22 2.) The release and metabolism of labelled dopamine from preloaded slices were studied. Pooled slices were exposed to [ 3H]tyrosine (250 Ci/10 ml medium) for 10 min. Then [14C]dopamine (10 Ci/10 ml medium) was added and the slices were incubated for a further 10 min with both labelled substrates. Slices were then washed four times with fresh equilibrated media. Slices preloaded in this manner with [ 3H]and [14C]DA were added to Corex centrifuge tubes containing 0.5 ml of equilibrated medium containing appropriate drugs. Slices were incubated for 10 min and the reaction was stopped with 2 ml of ice-cold medium. Tissue and media were separated as described above. 3 ) The uptake of [l-14C]DA into striatal slices was examined. Prepared slices (0.5 ml) were added to Corex centrifuge tubes containing 0.5 ml medium, 0.1 M [l-14C]DA, and drugs. Samples were incubated for 3 min. Uptake was terminated with 2 ml ice-cold medium 4.) The liberation of [14C]C02 from [l-14C]tyrosine was measured. Slices and media were prepared in the same manner as in the accumulation experiments, except that [l-1 4C]tyrosine was used in place of [ 3H]tyrosine. Once the slices were added to equilibrated media containing drugs and label, a plastic cup filled with glass wool, moistened

PAGE 32

with SO l Soluene 350 (Packard Instrument Co) and suspended from a rubber stopper was inserted into the Corex centrifuge tube. After 20 min incubation, the reaction was stopped 23 with 100 of 50% trichloroacetic acid (TCA) injected through the rubber stopper. After overnight refrigeration, each plastic cup was removed and placed in a scintillation vial with 10 ml of toluene based scintillator (14 g PPO, 0.4 g POPOP, 3.5 t toluene). Chromatography Purification procedures were essentially those of Iuvone et al. (1977) and recoveries were similar. Rinsed slices were homogenized in 2 ml of 0.4 M perchloric acid with 0.5% sodium metabisulfite (w/v) in Corex centrifuge tubes with a teflon pestle machined to a diameter of 14.S mm to fit the tubes tightly. The homogenate was centrifuged at 15000 x g in a refrigerated (4C) Sorvall RC-2B centrifuge. Supernatants were loaded onto AG-SO x 4 cation exchange columns + ( 6 x 20 mm, H form) The load volume contained tritiated water, 3,4-dihydroxyphenylacetic acid, and methylated, deaminated metabolites. Elutions with sodium acetate buffer (0.1 M sodium acetate, 0.1% sodium ethylenediaminetetraacetic acid (EDTA), titrated with 10 M NaOH to pH 7.0), 1 N HCl, and 4 N HCl released tyrosine, norepinephrine, and dopamine, respectively These fractions were further purified by alumina absorption or ion-exchange chromatography in the case of tyrosine.

PAGE 33

The load volume, together with a 2 ml wash with sodium acetate buffer, was collected in test tubes containing 200 mg of alumina prepared by the method of Anton and Sayre (1962) and moistened with 180 0.1 M disodium EDTA. Samples were then titrated with 3M Tris hydroxide (approximately 0.5 ml) to a pH of 7.9 and vortexed for five min. The alumina suspension was transferred to a glass wool stoppered pasteur pipette. The fraction not bound to the alumina, along with 0.5 ml of a water wash, was collected in a scintillation vial containing 10 ml of a Triton scintillator (10.68 g PPO, 0.12 g POPOP, 1 toluene, 1.4 Triton X-100). This fraction contained tritiated water and methylated, deaminated metabolites of DA. The alumina was further washed with another 5.5 ml of water, and then catechols were eluted with 2 ml of 0.5 M HCl into scintillation vials, and counted in 10 ml of Triton scintillator. Radioactivity in this fraction was determined to be 3,4-dihydroxyphenylacetic acid (DOPA C ) b y paper chromatography in n-butanol:acetic acid; H 20, (120:30:50,v/v/v). Once the load volume and a 2 ml sodium acetate buffer wash had been collected, the cation-exchang e columns were washed with a additional 6 ml of sodium acetate buffer. T yrosine was eluted with a second 6 ml of sodium acetate 24

PAGE 34

buffer, titrated to pH 1.5 with 4 M HCl, and loaded onto standard AG-50 x 4 cation exchange columns charged with 25 + Na. Following washes with 5 ml water and 8 ml 0.5 M HCl, tyrosine was eluted with 9 ml 0.1 M NajP04 Of this volume, 0.9 ml aliquots were added to 0.1 ml 4 M HCl and 10 ml Triton scintillator, while 1.8 ml aliquots were assayed for tyrosine using nitrosonaphthol as described by Udenfriend (1962). Fluorescence was measured en a Perkin-Elmer model 650-lOS Fluorescence Spectrophotometer. After the tyrosine fraction was eluted from the original AG-50 x 4 columns, they were washed with 2 ml sodium acetate buffer and 2 ml of 1 M HCl. NE was then eluted with 4 .75 ml of 1 M HCl into test tubes containing 200 mg alumina. Following a further wash with 1.25 ml 1 M HCl, DA was eluted with 2.5 ml of 4 M HCl into test tubes containing 200 mg alumina. NE and DA fractions were titrated to pH 7.9 with approximately 2 6 ml 3M Tris and with 2 5 ml 3M Tris and approximately 0.5 ml 10 M NaOH, respectively. Preparation, transfer, and filtration of alumina, as well as the elution of N E and DA from alumina,were described above for the purification of DOPAC. In experiments i n which release from the slices during incubation was analyzed, the media were titrated to pH 1.5 + with 63 l 4 M HCl and loaded on to the AG-SO x 4 (H form) columns. Elution profile and secondary purifications were identical to those used for the homogenate supernatant, except that the sodium acetate buffer wash preceding the tyrosine elution was shortened to 4 ml instead of 6 ml.

PAGE 35

The pellets formed by the centrifugation of the homogenate were dissolved in 2.5 ml of 0.3 M NaOH. Protein determinations were performed on duplicate 250 l aliquots using the method of Bailey et al. (1967). Quantification and Analysis Radioactivity was assayed using a Packard 2425 Liquid Scintillation Spectrophotometer. Correction factors for quenching and for emission spectrum overlap in double label experiments were determined with external standardization. The disintegrations/minute (DPM) for each fraction (except media tyrosine) were divided by the amount of 26 protein in that tissue sample to derive specific activities. Tyrosine specific activity was DPM/ g tyrosine. Blank values, determined in samples that received radioactivity but were chilled to 0C instead of being incubated, were subtracted from experimental values. Data for accumulation studies were expressed as moles of catecholamine accumulated/mg protein/minute (based on the tyrosine specific activity). Data from metabolism studies were expressed as percent total radioactivity recovered in fractions containing labelled dopamine or its metabolites. One factor analyses of variance ( A NOVA) and Student's !-tests were performed on a Hewlett-Packard 9810A Calculator. An Amdahl 3 70, version 6 computer was used for quench and double label corrections and for two or more factor ANOVA's as programmed in the Statistical Analysis System (SAS).

PAGE 36

Results Determination of Relevant Precursor Pool Striatal slices were incubated in media with various concentrations of tyrosine as described in Fig. 2.1. Measurements of [ 3H]DA accumulation (DPM/g protein) were 27 divided by the initial (calculated) tyrosine specific activities. When plotted as a function of initial tyrosine concentration on a double reciprocal plot, normal MichaelisMenton kinetics were observed (Fig. 2.la). However, because t yrosine was being exchanged between the media and the slices, the specific activities of both media and tissue would be somewhat different from the initial specific activities. Thus, we measured the specific activities of each pool at all concentrations of tyrosine and repeated the kinetic analyses. The measured concentrations of media tyrosine were markedly higher than those originally added to the media when the initial concentration was less than 1 g/ml. This difference confirmed that tyrosine was being released from the tissue, diluting the medium tyrosine specific activity (Table 2.1). Similarly, initial media tyrosine concentrations of more than 1 g/ml elevated tissue tyrosine concentrations. The DA specific activity was normalized by either the measured media or the measured tissue tyrosine specific activity and subsequently plotted on double reciprocal plots as functions of measured media or measured tissue tyrosine concentrations, respectively. Despite the narrowing of the

PAGE 37

I A. Contcted by 1 .,, ,01 g,:, l'T'ledi o t1rosine S,Qecifi c oct,vity 3 10 initial tyrosine ()Jg/rnll B. CorreciN by measured C Corrected by meo,urp,d 18 med i a tyrosir,e specific act i vity tissue tyt"C)Slfte SQecific acnritJ 2 5 I I 0 5 1.0 1.5 2 4 -~ 8 l'nedio tyrosine (g/ml) Fig. 2.1. Kinetics of [ 3HJDA accumulations from [3H]tyrosine: effects of correcting with initial media, measured media, and measured tissue tyrosine specific activities. Striatal slices were incubated for 4 0 min with 4.17 Ci/ ml [3H]tyrosine at 0.03, 0.1, 0.3, 1, 3, and 10 g/ml tyrosine. DPM/mg protein was divided by tyrosine specific activity (DPM/ng tyrosine) and graphed as Lineweaver-Burke plots against tyrosine concentrations (g/ml media). (A) [3H]D A accumulation normalized by initial tyrosine specific activity as a function of initial tyrosine concentration. ( B ) The same data normalized by measured media t yrosine specific as a function on measured media tyrosine and (C) by measured tissue tyrosine specific activity as a function of measured tissue t yrosine concentration. Each point represents the mean of 3 determinations. 28

PAGE 38

Table 2.1. Relationship of Measured Media and Measured Tissue Tyrosine Concentrations to Initial Tyrosine Concentrations. Initial (g/ml) .03 10 30 1. 00 3.00 10.00 Tyrosine Concentration Measured Media (g/ml) .58.06 .56.12 .68,08 1.09.07 2.40.12 8 .98.11 Measured Tissue (g/mg protein) 1.73.40 1.55.37 1.36.13 1.85.49 4.18.19 4.18.58 29

PAGE 39

range of tyrosine concentrations in the media, the data corrected with the media tyrosine specific activities displayed Michaelis-Menton kinetics with a V and a max Km similar to those determined using the initial tyrosine specific activity. Data normalized with tissue tyrosine specific activity did not display normal kinetics. Although not shown, the labelling of protein with [ 3H]tyrosine also displayed Michaelis-Menton kinetics only when media tyrosine specific activities were used to normalize the data. 30 Since media tyrosine concentrations above 1 g/ml were not appreciably diluted by tyrosine released from the slices, subsequent experiments used 1.5 g/1 (8 3 M) or 15 g/ml (83 M) tyrosine concentrations in the medium. The specific activity of media t yrosine, the pool which best reflects the relevant precursor, was assumed to be unaltered during incubation. K+ -Induced Activation of the Accumulation of [ 3H]Catecholamines Various concentrations of KCl were added to striatal slices incubated in 83 M tyrosine with [ 3H]tyrosine. was recovered from both slicesand media. A s shown in Fig. 2 .2, elevated K+ increased the formation and release of [ 3 H]DA. Combined slice and media [ 3H]DA accumulations, presumably reflecting total s ynthesis, reached a maximum at 18 ~Vi K+. Interestingly, accumulation of [ 3H]DA in the slices was increased by 14 mM K+ which had no significant effect on release. The accumulation of [ 3H]DA from [ 3H] t yrosine in striatal slices was elevated by 158 % 274 %

PAGE 40

4 CD :, C 3 E C C1) -0 '2 a. 0, C (I) Cl) 0 E C. 6 ... I ... I 1 0 14 18 Combined ...... Slices ...... --......... J 1 ........... Media 46 Fig. 2 .2. Effects of elevated K+ on the accumulation of [3H]DA in striatal slices and media. Striatal slices were incubated at 83 M tyrosine and 4.17 Ci/ml [3HJ t yrosine. Each point represents the mean and standard error of 4 determinations. Combined accumulation is the sum of slice and media accumul~tions. 31

PAGE 41

and 322 % by 26 mM K+ at 8.3, 27.4, 83, and 274 M tyrosine, respectivBly. However, increased accumulation observed 32 with elevated K+ was not consistently found in other regions. [ 3H]NE accumulation from [ 3H]tyrosine was not facilitated in cerebellar slices incubated at 8.3 M tyrosine. In substantia nigral slices, both [ 3H]NE and [ 3H]DA accumulations were facilitated at a medium concentration of 8.3 M tyrosine, but not at 83 M tyrosine (Fig. 2.3). Inhibition of [ 3H]Catecholamine Accumulation from [ jH]Tyros ine Apomorphine, haloperidol and reserpine all potently inhibited the accumulation of [ 3H]DA in striatal slices (Fig. 2.4). Chlorpromazine (10 M) caused a slight but significant inhibition (24%) while fluphenazine had no significant effect on [ 3H]DA accumulation. Apomorphine and haloperidol were also tested at a range of doses in the presence of 26 mM K+ (Fig. 2.5). Generally, 26 mM K+ tended to reduce the effectiveness of haloperidol as compared to incubations at 6 mM K+, while it facilitated the effect of apomorphine. Furthermore, 10 ~1 haloperidol caused a slightly elevated [ 3H]DA accumulation in slices exposed to 26 mM K+. Similar, but nonsignificant increases were observed with 10 nM haloperidol and 0.1 nM + reserpine at standard (6 mM) K Reserpine, apomorphine and haloperidol all inhibited the accumulation of [ 3H]DA in substantianigral slices incubated at 8.3 M t yrosine. Surprisingly, apomorphine

PAGE 42

-C e ..... C .; 0 "' E ..... .. ., 0 E C E ..... C 90 80 70 60 50 40 30 20 10 8 0 .:; 60 e a. c,, E 4 0 ., g -20 A 8 3 /J M t yros ine Substont i o nigro ;';, 4 SAL 2 6mM K B 83 JJM t yros ine S u bs t ont i o n igro 26mM K Cerebellum C ~ ..... C 10 .; e 8 a. "' E 6 ..... Lu z 4 .. ., 0 2 E -Fig. 2 3 Effeet s o f elevated K+ o n the accumulation of [3H]DA a n d [3H]N E in s ub s t antia nigr a l and cerebellar slices. Open bars represe n t [3H]DA accumulation in slice s Shaded bars represent [3H] N E N umbe r s a t the base of each bar i ndicat e the sample size for each grou p SAL indicates standard incubation conditions (6 mM K + ) ** indicates a significant difference from saline at p < 01 CANOVA). (A) Subst antia nigral and cerebellar slices incubated with 4.1 7 Ci/ml and 16,7 Ci [3HJt yrosine, respectively at 8 3 M tyrosine. (B) Substantia nigral slices incubated with 1 6 7 Ci/ml [3H]tyrosine at 83 ~! tyrosine. 33

PAGE 43

120 100 __J 0 er 80 .... z 0 u i.. 0 60 0 4 0 20 Reserpi ne Apomorphine o --o Holoperidol .._____... Chlorpromozine F l uphenazine 1:1.--t:i. ~~~---* DRUG CONCENTRATION ( M) Fig 2.4. Inhibition of striatal [ 3H]DA accumulation b y reserpine, apomorphine, haloperidol, chlorpromazine, and fluphenazine. Striatal slices were incubated with 4 1 7 Ci/ml [3H]tyrosine at 8 3 M t yrosin e and 6 m M K +. Each point represents the mean and standard error of 4 determinations, except for haloperidol, in which each point represents the mean of at least 8 determinations. The rate of [3H]D A accumulation for saline ( 100 % value) was 0 8 9 pmoles/mg protein/min. ** represent s a significant difference from saline a t p < .01, *** at p < .001 CANOVA). 34

PAGE 44

...J 0 a: Iz 0 u LL. 0 ::,!! 0 35 Apomorphine 6mM K o--o 26mM K .-120 Holoperidol 6mM K A--A 26mM K .__. 100 p <.05 80 60 40 20 DRUG CONCENTRATION(M) Fig. 2.5. Interactions of elevated K+ with haloperidol-and apomorphine-induced inhibitions of striatal [3H]DA accumulation from [3H]tyrosine. Curves generated at 6 mM K! are the same as presented in Fig. 2.4. + The rate of [~H]DA accumulation for saline. at 26 mM K is 3 .54 pmoles/ mg protein/min. Each point represents the mean and standard error of 4 determinations for apomorphine and at least 8 determinations for haloperidol. indicates a significant increase from saline at p < .05 CANOVA).

PAGE 45

and haloperidol, as well as reserpine, also inhibited the accumulation of [ 3H]NE (Fig. 2.6). [ 3HJNE accumulation was also inhibited by haloperidol and apomorphine in cerebellar slices as much as in substantia nigral slices, indicating that the effect was not an artifact of incomplete chromatographic separation of [ 3HJDA and [ 3H]NE (Fig. 2 7) Like 26 mM K+, the effect of apomorphine on substantia nigral slices was diminished by 83 M tyrosine (Fig. 2.6b). At a medium concentration of 8.3 M tyrosine, 10 M apomorphine inhibited [ 3H]DA (82 % ) and [ 3H]NE (65 % ) accumulations twice as much as observed at 83 M tyrosine (41 and 30 % respectively). 10 M haloperidol was equally effective at 8 3 and 83 M tyrosine. This decrease in the accumulation of [ 3HJDA and [ 3H]NE caused by the various drugs tested could be caused by nonspecific inhibition of cellular metabolism, a decrease 36 in synthesis, a decrease in the reuptake of newly synthesized and released [ 3H]catecholamines, or a dramatic increase in the release and/or metabolism of the newly synthesized catecholamines. Accumulation of [ 3H]Protein from [ 3H]Tyrosine Since haloperidol and apomorphine affected the accumulation of both [ 3HJDA and [ 3 HJNE, these agents might have a nonspecific effect upon cellular metabolism. However, the labelling of protein in the slices incubated with [ 3H]tyrosine was not affected by either haloperidol or apomorphine (Table 2.2)

PAGE 46

3 0 C e ~ 20 e Q. c,, E Kl 10 0 1 0 0 C: 80 C .; 0 a. 60 e "' "a 40 E -20 A. 8 3 )J M Tyrosin e SAL 166 105 M HALOPERIOOL B. 8 3 !JM T y r o sine SAL M HALOPERIDOL ODA I NE p<.05 lp<.01 1 0 6 105 108 ,o 7 M APOMORPHIN E Y RESERPINE 101 1 0 6 ,o5 M APOMORPHINE 37 Fig 2.6 Inhibition of [3H]catecholamine accumulation from [3H]tyrosine in substantia nigral slices by haloperidol, apomorphine, and reserpine. Open and shaded bars represent [3H]DA and [3H]NE, respectively ** indicates a significant difference from saline at p < 01, at p < O S CANOVA). (A) Nigral slices were incubated with 4.17 Ci/ml [3H]tyrosine at 8 3 M t yrosine and (B) with 16 7 Ci/m l [3H]tyrosine at 83 M t yrosine.

PAGE 47

38 12 10 C e 8 C v 0 ... 0. Cl 6 E Cl) 4 0 E -* 2 4 4 4 4 SAL 10-6 1 05 ,o-6 ,o-5 M Apomorphine M Haloperi dol Fig 2 7 Inhibition of [ 3H]N E accumulation f rom [ 3 H] t yrosine in cerebellar slices b y a p omor phine and haloperidol. Cerebellar slices were incubated with 16 7 Ci/ ml [ 3H] t yrosine at 8 3 M t yrosine. Numbers a t the base of each bar indicate the sample siz e ** indicates a significant difference from saline at p < 01 (ANOVA).

PAGE 48

Table 2.2. The Accumulation of [ 3H]Protein from [ 3H]Tyrosine: Effects of Haloperidol and Apomorphine at 8.3 and 83 M Tyrosine. Treatment Saline Haloperidol (10 M) Apomorphine (10 M) DPM/g Proteina 8.3 M Tyrosine 83 M Tyrosine 12.08.67 1.87.36 1 2.08.03 2.13.23 10.88.52 2.10.02 a Homogenate pellets were dissolved in 2.5 ml of 0.3 M NaOH. One ml aliquots were added to 0.4 ml of 25% trichloroacetic acid (TCA). After chilling at 0C for 10 39 min, samples were centrifuged at 100 x gin a refrigerated (4 C) Sorvall RC-3 centrifuge. The supernatant was decanted and replaced with 4 ml of 5 % TCA. Once the centrifugation and decanting were repeated, the pellet was digested in 1.0 ml of Soluene 350 and added to 10 ml of toluene-based scintillation fluid.

PAGE 49

Liberation of [1 4C]C02 from [14C]Tyrosine In order to confirm an effect upon synthesis, the liberation of [14C]C02 from striatal slices incubated with 8.3 M [l-14C]tyrosine was determined. Synthesis, as measured by the release of [14C]C02 was inhibited by both apomorphine and haloperidol (Fig. 2.8). Therefore, the decreased [ 3H]catecholamine accumulation probably reflected inhibition of DA and NE synthesis. For unknown reasons, both haloperidol and apomorphine were effective 40 at doses lower than was found by measurements of accumulated [ 3H]DA. U ntake of [14C]DA Striatal slices were incubated with 0.1 M [l-14C]DA for a period of 3 min. Comparisons between 10 M haloperidol and saline were made both with and without 1 2.5 M nialamide and 100 M EGTA, which were used to diminish subsequent release and metabolism of accumulated [14C]DA (Table 2.3). Under both conditions, haloperidol decreased the accumulation of [14C]DA. However, haloperidol also increased the formation of [14C]DOPAC. The increased [14C]DOPAC found in the media was probably formed by monoamine oxidase in dopaminergic terminals and, therefore, reflects additional [14C]DA uptake by the slices. When the sum of [14C]DOPAC and [14C]DA is considered, the effect of haloperidol is reduced. Despite the fact that nialamide reduced [14C]DOPA C to near blank levels, an increase in [14C]DOPA C was still observed with haloperidol.

PAGE 50

...J 0 a: f-2 0 u t... 0 <> 120 100 80 60 4 0 20 DRUG CONCENTRATION (M) Apomorphine o-o Holoperido l b--6 Fig. 2 8 Inhibition of [14C]C02 liberation fro m [14C] tyrosine by apomorphine and haloperidol. Striatal slices were incubated with 83 Ci/ml at 8 3 M tyrosine and 6 mM 41 K+ for 20 min. Each point represents the mean and standard error of four determinations. The rate of [ 14C] CO2 accumulation for saline was 8.75 pmoles/mg protein/min. indicates a significant difference from saline at p < .OS, ** at p < .01 CANOVA}

PAGE 51

Table 2 .3. Haloperidol inhibits the Accumulation of [ 14 c ] DA from Media, but no t Total Uptake. Treatment Saline Haloperidol (10 M) With 12 5 M Nialamide and 100 M EGTA: Saline Haloperidol ( 10 M) 1.09. 08 .85.06 -22% 1.69,06 1.48, 06 -12% .69.03 .82,02 .02.02 .15.09 C Total 1.78,08 1.67. 07 -6% 1.73.07 1.63. 19 -6% Significance at p < .05 ( two factor A NOVA, Duncan's) aFrom slices alone, DPM/ g protein bCombined from both slices and media, DPM / g protein cSum of [ 14 c]DA and [ 1 4 c]D0PAC N ote: Main effect of haloperido l in the ANOVA significant for DA at p<. 025 and f o r DOPAC at p<.05. 42

PAGE 52

Release and Metabolism of Preloaded Labelled DA The effects of reserpine, apomorphine and the neuroleptics haloperidol, chlorpromazine and fluphenazine upon the disappearance of labelled DA were measured in two possible pools of DA, simultaneously. Pooled slices were incubated with both [ 3H]tyrosine and [14C]DA. Following a thorough washing to remove excess radioactivity, 0.5 ml of prepared slices were added to 0.5 ml of unlabelled media containing the pharmacological treatments. Radioactive DA 43 in the media, DA remaining in the slices, total (slice and media combined) DOPAC, and total residual (tritiated water, homovanillic acid) radioactivity were measured (Fig. 2.9), Haloperidol (10 M) and reserpine (0.1 M) caused a disappearance of between 30 and 50 % of radioactive DA from the slices. Passive release (i.e. not induced by veratridine, elevated K+ or electrical stimulation) for both pools of radioactive DA was significantly elevated at 10 M haloperidol and 10 M apomorphine. Passive release of [1 4C]DA, but not [ 3H]DA was significantly elevated by 0,1 M reserpine. However, the amount of radioactive DA recovered in the media accounted for only a small percentage of wha t was lost from the slices. More than 90 % of the radioactive DA that had been lost from the slices as a result of haloperidol or reserpine treatments was found in the DOPAC fraction. More than 80% of the increased radioactive DOPAC was found in the media. Apomorphine also caused a significant elevation in radioactive DOPAC accumulation, but the effect was quite small 1n

PAGE 53

Fig. 2.9. Effects of haloperidol, reserpine, apomorphine, chlorpromazine and fluphenazine on the release and metabolism of preloaded labelled DA in striatal slices. Pooled striatal slices were exposed to [3H]tyrosine (250 Ci/10 ml) for 10 min. Then 10 Ci/10 ml [14C]DA was added and the slices equilibrated for an additional 10 min. Slices were then washed 4 times. Aliquots of 0.5 ml of slices were added to 0 5 ml of media containing ionic and drug treatments and incubated for 10 min. Open and shaded bars represent the distribution of [3H] and [14C), respectively. DOPAC and Residual fractions were pooled from the slices and the media. The sum of slice D A media D A D O P A C, and residual equals 100 % for each label. indicates a significant difference from saline for the same label at p < .05, ** at p < .01 (ANOVA for haloperidol and reserpine, Student's t-test for apornorphine, APO; chlorpromazine, CPZ; and fiuphenazine, FLU).

PAGE 54

>.... > .... u ct 0 0 100 80 60 40 20 6 5 4 3 2 SLICE DOPAMINE MEDIA DOPAMINE ct DOPAC er so .J 40 0 .,_ 30 0 20 10 20 RESIDUAL 10 SAL ,o6 3xto6 10-5 M HALOPERIDOL [b IO-S 3xl0-B I0-7 M RESERPINE 45 tO

PAGE 55

46 comparison to reserpine and haloperidol, especially considering that no significant decline in slice radioactive DA was observed with 10 M apomorphine. Fluphenazine and chlorpromazine both were without any effect on any fraction measured. Because of the observed similarities of the effects of 10 M haloperidol and 0.1 M reserpine on synthesis, release, and metabolism of radioactive DA, comparisons were extended to include tests of Ca2 + and K+ dependency. Haloperidol and reserpine were tested in separate experi-+ 2+ ments against saline at 6 or 26 nu~ K and at 2.6 mM Ca or 0 mM Ca2 + with 100 M EGTA. Orthogonal comparisons within each experiment were made between treatment groups differing in only one treatment factor, using multiple Student's ttests. This was justified since the effect of K+ was so great that variance was not homogeneous, making the ANOVA an invalid test. Both [ 3H]and [14C]DOPAC were greatly increased (more than 400%) by 10 M haloperidol at both 6 and 26 nu~ K+ and with or without Ca2 +, suggesting that the effect of haloperidol on DOPAC formation was independent of transmitter release (Fig. 2.10). As predicted, 0.1 M ~eserpine generated a similar profile, although the effect was smaller than elicited by 10 M haloperidol. In addition, 26 mM K+ caused a small increase of labelled DOPAC accumulation which was 2+ also observed in Ca -depleted media (p<.05). As expected, K+ induced the release of both [ 3H] and [14C]DA from the slices. K+-induced release was still

PAGE 56

Fig. 2.10. Comparisons of the effects of haloperidol and reserpine on the release and metabolism of labelled DA in striatal slices. Slices were prepared as described in Fig. 2.9. Open and shaded bars represent [3H] and [14C], respectively. Using Student's t-tests, orthogonal comparisons were performed between treatment groups differing in only one variable. Significance levels presented indicate the effect of 10 M haloperidol or 0.1 M reserpine as compared against the saline value with the same label and at the same K+ and Ca2+ concentrations ( i. e [ 1 4 C ] DOPA C for 2 6 mM K + 2 6 mM Ca 2 + and ha 1 ope r i do 1 was compared to [14C]D0PAC for 26 rnM K+, 2.6 rnl'-1 Ca2+ and saline). indicates significance at p < .OS, ** at p <.01, and*** at p < .001.

PAGE 57

> I-> .:: u
PAGE 58

observable in ca2 + depleted media, although the amount was considerably reduced from the 2.6 mM ca2 + incubations for both [ 3H]and [14C]DA (p < .01). Haloperidol not only elevated passive release, but also potentiated K+-induced release in both ca2 + maintained and Ca2+-depleted media. Reserpine significantly elevated passive release of [14C]DA and K+-induced release for both [ 3H]and [14C]DA at 2.6 mM ca2 +. Reserpine did not significantly elevate K+-induced release in Ca2 + depleted conditions, although the pattern of results was similar to that observed with haloperidol. No treatment combination had any consistent effect on the residual 3 H and 14C Changes in slice DA radioactivity inversely reflected the combined changes in media DA and DOPAC radioactivities. Discussion Precursor Pool Determination The accumulation of [ 3H]DA from [ 3H]tyrosine in striatal slices displayed Michaelis-Menton kinetics if the DA specific activity was corrected with initial or measured media tyrosine specific activity. Such was not the case 49 when tissue tyrosine specific activity was used as a correction factor. Kapatos and Zigmond ( 19 77) reported similar results measuring the liberation of [14C]C02 from [14C}tyrosine incubated with striatal synaptosomes. The dependence of normal kinetics upon the media tyrosine specific activity suggests that tyrosine h ydroxylase (TH) might be linked to a

PAGE 59

tyrosine uptake mechanism at the membrane. TH has been reported to exist in both soluble form and in a more active membrane bound form (Kuczenski and Mandell, 1972), However, so in our samples we found that protein labelling with [ 3H]tyrosine also displayed Michaelis-Menton kinetics only if corrected with media tyrosine specific activity (not shown). Thus, the precursor pool for DA synthesis apparently is also reflected in protein synthesis. One possibile explanation is that areas with high metabolic activity, such as neuronal cell bodies and terminals, more rapidly exchange free tyrosine with the media than do glia and neuropil. Also, exchange of tyrosine with the medium may be higher in those terminals and cell bodies which are using more tyrosine as a precursor. Thus, dopaminergic terminals might exchange tyrosine with the media more freely than would terminals of other neurotransmitters. Such an explanation would explain why Kapatos and Zigmond found that tissue tyrosine in a synaptosomal preparation was not the relevant precursor pool for DA synthesis. + Effects of Elevated K upon S ynthesis, Release, and Metabolism of DA Depolarizing agents such as elevated K+ or veratridine induce release and activate synthesis of DA in vitro. Both the induction of release and the activation of synthesis are calcium dependent (Harris and Roth, 1971; Patrick et al., 1974 ; Bustos and Roth, 1979) The increase is correlated with an activation of TH to a form characterized by an

PAGE 60

increased affinity for reduced pteridine cofactor and a decreased sensitivity to end-product inhibition by DA (Lovenberg et al., 1974; Bustos et al., 1974). It has been presumed that the K+-induced synthesis was a consequence of increased release. However, we observed that the activation of synthesis could be dissociated from the induction of release. Increased synthesis was apparent + at 14 mM K, a concentration at which no effect upon release was detected. K+ concentrations higher than 18 mM produced an asymptotic effect on synthesis, while release increased linearly with increasing K+ concentrations up to at least 46 mM. The plateau of synthesis activation may reflect an inhibitory feedback upon DA synthesis mediated either by released DA stimulating a presynaptic receptor which would inhibit synthesis, or by end-product inhibition of TH. K+-induced release and synthesis were previously reported by Bustos et al. (1974) to be dissociable: K+ -induced TH activation in striatal slices was suppressed by ethanol, 51 while release was unaffected. Since TH activation by depolarization is calcium dependent (Simon and Roth, 19 7 9 ) s ynthesis activation may occur subsequent to ca2+ influx into the terminal. Whatever the mechanism, synthesis activation appears not to be a consequence of release, but independent of it. It is curious that K+ did not increase the accumula-tion of [3H)DA and 83 M t yrosine, or [ 3H)NE i n 3 of [ H]NE substantia nigral slices at in cerebellar slices. Assuming that increased release of labelled catecholamines occurred

PAGE 61

52 during incubation, an increase in synthesis may actually have taken place. However, amphetamine, which appears to activate DA synthesis in a manner similar to K+, also interacts with tyrosine, being effective at media concentrations of tyrosine above 10 M (Uretsky and Snodgrass, 1977). Also, the effectiveness of apomorphine in inhibiting synthesis 1s reduced in the substantia nigra at 83 M tyrosine (Fig. 7). It is p0ssible that tyrosine at 83 M may be toxic, thus masking synthesis changes. Alternatively, it may affect release. + In either case, it is not clear why the K effect would be potentiated by increasing t yrosine concentrations in striatum, but would be suppressed at higher t yrosine concentrations in substantia nigral slices. A significant percentage of the labelled [ 3H]DA released b y K+ stimulation was found to be converted to the deaminated, nonmethylated metabolite 3,4-dihydroxyphenylacetic acid ( DOPAC), as was observed by Farah et al. (1977 ) for N E terminals. However, the metabolite accounted for only about 20% of the total released radioactivity Cubeddu et al. (1979) found that reuptake blockers reduced the formation of DOPA C suggesting that the DOPAC was derived from labelled DA that had been released and subsequently taken up by the terminal. Our finding that the elevation of DOPAC formation + caused by 26 mM K occurred independently of the presence of ca2+ is somewhat at odds with this interpretation. However, if uptake mechanisms were saturated b y the D A released b y + ? + 26 m M K a t 2.6 mM ca, then uptake might proceed at nearly

PAGE 62

53 2+ the same rate as in Ca -depleted media, making the amount of precursor available for monoamine oxidase fairly constant. Effects of Apomorphine on DA Metabolism. Electrophysiological results suggest that a DA receptor 1n the substantia nigra mediates inhibition of DA cell firing (Aghajanian and Bunney, 1977; Skirboll et al., 1979). It has been argued that this receptor is localized upon dopaminergic cells and is not presynaptic on terminals projecting to the substantia nigra. However, presynaptic autoreceptors regulating release of DA have not been generally accepted. Apomorphine has either weak inhibitory effects (Farnebo and Hamberger, 19 71; Miller and Friedhoff, 1979) or no effect (Dismukes and Mulder, 1977; Raiteri et al., 1978) on DA release from striatal synaptosomes and slices. Furthermore, although neuroleptics do affect electrical field-and K+ stimulated release (Dismukes and Mulder, 1977; Seeman and Lee, 1975; Farnebo and Hamberger, 1971), the effect has been consistently reported to be inhibitory except at high doses (Perkins and Westfall, 1976), opposite to what would be predicted by DA autoreceptor blockade, Since apomorphine potently inhibits DA synthesis both in vivo (Kehr et al., 1972) and in vitro (Fig. 2,4; Christiansen and Squires, 1974; Ebstein et al., 1974), several groups have proposed the alternative hypothesis that DA presynaptic autoreceptors may modulate synthesis and not release (Aghajanian and Bunney, 1974 ; Christiansen and Squires 1974). DA has been shown to inhibit synthesis in vitro

PAGE 63

(Patrick et al., 1974). However, a controversy exists as to whether the site of action is on a DA receptor or on TH. DA-mediated inhibition has b~en shown to be reversible by cocaine (Patrick et al., 1974), allegedly due to the ability of cocaine to block reuptake. If this is true, then at least part of the inhibition observed with exogenous DA in vitro might be due to end-product inhibition o f TH. In contrast, both apomorphine-and DA-induced inhibitions of DA synthesis have been reported to be reversed with haloperidol or fluphenazine (Westfall et al., 1976; Christiansen and Squires, 1974). We were unable to replicate this finding at the dose of haloperidol used by Christiansen and Squires. However, 10 nM haloperidol significantly elevated the accumulation of [ 3H]DA from [ 3H]tyrosine in slices stimulated with 26 mM K+. This effect might be interpreted as being caused by a reversal of ongoing inhibition of DA synthesis. Synthesis would be suppressed at ~11 autoreceptor by DA released by the elevated K+ concentration; this would be reversed by a DA receptor blocker. A similar hypothesis has been used to explain why striatal DA terminals display elevated synthesis following nigrostriatal tract lesions or impulse blockade with the GABA analogue gamma-butyrolactone (Walters et al., 1973), Another possible site of action of apomorphine is TH. TH undergoes a conformational change following electrical or K+ stimulation, resulting in an increased affinity o f the enzyme for pterin cofactor and reduced sensitivity to 54

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55 end-product inhibition. Thus, if apomorphine was inhibiting TH by end-product inhibition, K+ activation should, relatively speaking, reduce the potency of apomorphine. Instead, we found that 2 6 mM K+ exacerbated the inhi~ition of [ 3H]DA accumulation b y apomorphine. This result was probably not due to some change in release or metabolism since 10 M apomorphine did not cause a detectable depletion of [ 3H]DA from preloaded slices. Also, apomorphine either has no effect at all on K+-stimulated release of labelled DA or it has a weak inhibitory e ffect. A n inhibition of release should retain [ 3H]DA in slices during the incubation period, an effect we did not observe. Therefore, a mechanism other than direct inhibition of TH is responsible for the apomorphine-induced inhibition of DA s ynthesis. We also found that apomorphine-induced inhibition o f D A s ynthesis is dependent on the medium concentration of t yrosine in incubations of substantia nigral slices. The accumulation of [ 3H]DA in substantia nigral slices was inhibited 8 0 % b y 10 M apomorphine when incubated at 8.3 M t yrosine. Increasing the medil.Ilil tyrosine concentration to 83 M halved the effectiveness of apomorphine, but did not alter the inhibition by haloperidol (67 % at 8,3 M tyrosine, 65% at 83 M tyrosine). This effect of t yrosine lacks a ready explanation, although similar interactions with medium t yrosine concentrations have been reported before. In particular, amphetamine stimulated DA s ynthesis in striatal slices at medium t yrosine concentrations of 1 0 Mor more,

PAGE 65

56 whereas it had no effect at concentrations of tyrosine 1 M or less (Uretsky and Snodgrass, 1977). Such findings might indicate a change in the K of TH for tyrosine. Alterna-m tively, apomorphine might have impeded and amphetamine facilitated tyrosine uptake into a small pool of tyrosine with direct access to TH. As speculated by Starke et al. (1977), we found that the inhibition of synthesis caused by apomorphine was not limited to DA terminals, but was also apparent in the accumulation of [ 3HJNE from [ 3H]tyrosine in substantia nigral and cerebellar slices. Their speculation arose from the repeated observation that DA inhibited release from N E terminals in some peripheral tissues (for review, see Starke et al., 197 7), apparently by means of a presynaptic receptor similar to DA-receptors found in the CNS (Iversen et al., 1975). Therefore, the ability of apomorphine to inhibit both N E and D A synthesis in vitro does not necessarily argue a gainst the hypothesis that inhibition is mediated by an autoreceptor. The same receptor mi ght be present on both terminal populations. It does seem surprising however, that a D A presynaptic receptor on N E terminals could modulate release and synthesis, while possibly the same receptor modulates only synthesis on DA terminals. Effects of Haloperidol on Catecholamine Synthesis and Releas e W e did not expect haloperidol to inhibit [ 3HJDA accumulation nearly as well as apomorphine. In vitro inhibition

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57 of DA synthesis by haloperidol has only been briefly reported before; Christiansen and Squires (1974) commented that haloperidol at concentrations greater than 1 M inhibited [ 3H]DA synthesis from [ 3H]tyrosine in striatal synaptosomes. Such an inhibition of DA synthesis contradicts a model of autoreceptor regulation of DA synthesis. Such a model predicts that DA-receptor blockers would increase DA synthesis or have no effect at all. Also, while apomorphine clearly inhibits synthesis in vivo (Kehr et al., 1972) and in vitro (Ebstein et al., 1974; Christiansen and Squires, 1974), haloperidol dramatically increases DA synthesis in vivo (Kapatos and Zigmond, 1979; Lerner et al., 1977; Carlsson et al., 1977), contrary to the in vitro findings. The mechanisms by which apomorphine and haloperidol inhibit the accumulation of [ 3HJDA from [ 3H]tyrosine appear to be different. First, 26 mM K+ potentiated apomorphineinduced, while it lessened haloperidol-induced inhibition of DA synthesis. Also, apomorphine-induced inhibition of the accumulation of [ 3HJcatecholamines in substantia nigral slices was halved by increasing the concentration of medium tyrosine, but no such effect was observed with haloperidol. Attempts were also made to determine whether the decreased accumulations of [ 3HJDA observed in the presence of haloperidol were due to decreased synthesis, increased spontaneous release, or decreased reuptake. By replacing [ 3H]tyrosine with [l-14C]tyrosine and by measuring the liberation of [1 4C]C02 a more direct measure of DA synthesis

PAGE 67

was obtained. In this case, haloperidol inhibited [14C] C02 release as much as, if not more than, [ 3H]DA accumulation. Therefore, haloperidol inhibited DA synthesis. However, incubations with 10 M haloperidol also caused the loss of [ 3H]DA synthesized from [ 3H]tyrosine and of [14C]DA preloaded in striatal slices. The amount of labelled DA found in the media was elevated, confirming several reports that haloperidol elevated the basal efflux of DA (Seeman and Lee, 1974; Dismukes and Mulder, 1977; Raiteri et al., 1978). However, the increased efflux of labelled DA accounted for less than 15 % of the label lost from the slices; more than 80% of the missing label was located in a fraction containing DOPAC. Therefore, the total basal efflux of label induced by haloperidol in earlier studies has probably been incorrectly assumed to be associated with labelled DA. Paper chromatography of the fraction containing DOPAC revealed that over 80% of the [14C]materials from both saline and 10 M haloperidol-treated slices co-chromatographed with standard DOPAC. A similar profile was apparent with [ 3H] labelled materials. Since the formation of labelled DOPAC was strongly inhibited by 12.5 M nialamide (Table ~3), the mechanism of synthesis 58 was probably enzymatic and not due to spontaneous oxidation.

PAGE 68

The inhibition of reuptake by haloperidol has been reported (Rio and Madronal, 1976; Seeman and Lee, 19 7 4 ) We also found that haloperidol inhibited. the apparent striatal accumulation of [14C]DA from the media. However, the decrement was much smaller than would be necessary to account for the decreased accumulation of [ 3H]DA from [ 3H]tyrosine. It has been suggested that apparent decreases in uptake might actually reflect enhanced release (Baumann and Maitre, 19 76: Heikkila et al., 19 75). When the sum of medium [14C]DOPAC (presumably formed in D A terminals) and slice [14C]DA was compared, the effect of haloperidol was reduced, directly confirming this h ypothesis. Papaverine (Cubeddu et al., 19 79a), Ro 4-1284 (Farah et al., 1977; Cubeddu et al., 19 7 9b) and reserpine itself all cause patterns of release similar to those observed with haloperidol. Therefore, a variety of comparisons between haloperidol and reserpine were performed to further substantiate the possibility of a reserpine-like character for haloperidol. Like haloperidol, reserpine potently inhibited [ 3H]catecholamine accumulation in both striatum and substantia nigra, although at doses 100 times smaller than were needed for haloperidol. Similarly, reserpine increased the basal efflux of label from striatal slices preloaded with [ 3H]DA (Seeman and Lee, 19 74; Fig 2 9) 59

PAGE 69

The increased formation of labelled DOPAC might also be ascribed to efficient reuptake and degradation of DA released into the medium. This possibility does not seem likely since the bulk of the radioactivity released b y elevated K+ concentrations from striatal slices preloaded 60 with labelled DA remains as DA (Fig. 2.9; Farah et al., 1977) We also found that the elevation of labelled DOPAC by 10 M haloperidol and 0.1 M reserpine was independent of both K+ 2+ and Ca concentrations, and therefore probably independent of vesicular release. The effects of 0.1 M reserpine and 10 M haloperidol on release were complex but essentially identical. Both agents increased basal efflux of labelled DA and facilitated the release of DA induced by 26 mM K+. Haloperidol also elevated the ca2+-independent, K+-induced release of labelled DA. Reserpine did not significantly elevate the release of labelled DA in ca2 + depleted media, although the pattern of results was similar to that observed with haloperidol. The facilitation of basal release by haloperidol has been reported before (Seeman and Lee, 19 74; Dismukes and Mulder, 1977). However, most studies of field-stimulated ( Seeman and Lee, 1975; Dismukes and Mulder, 1977), K+-induced (Miller and Friedhoff, 1979) and veratridine-induced (Raiteri et al., 19 7 8) release of preloaded labelled DA indicated that haloperidol reduced the amount of label released. The only exception was the report of Perkin and Westfall ( 1976 ) which indicated that 10 M haloperidol, the dose used in this study

PAGE 70

facilitated stimulated release while lower doses inhibited release. Reported similarities of the effects of reserpine and neuroleptics are numerous. Reserpine has potency in the treatment of schizophrenia and like other neuroleptics used 61 in this study, causes parkinsonian-like symptoms (Crow et al., 1976; Bleuler and Stoll, 1955). All of these agents induce catalepsy at high doses (Janssen et al., 1966). These agents have an anesthetic property as defined by the ability to block impulses along peripheral nerves (Seeman et al., 1974). Reserpine and haloperidol both inhibit catecholamine synthesis in vitro and activate catecholamine synthesis in vivo (Kapatos and Zigmond, 1979; Mueller et al., 1969). In contrast, however, reserpine causes a profound depletion of monoamine concentrations. Haloperidol has been reported to cause a moderate depletion of striatal DA (Massoti, 1977), although most reports have not found this result (Kapatos and Zigmond, 1979; Lerner et al., 1977). The effects of haloperidol and reserpine observed in this study to not appear to be receptor-mediated. The ranked efficacies of reserpine, haloperidol, chlorpromazine and fluphenazine (listed in decreasing order of potency) does not correlate with any of the widely studied measures of DAreceptor antagonist efficacy (Creese et al., 1976). Fluphenazine is typically as effective as haloperidol, while chlorpromazine is less potent by an order of magnitude, 1n reversing apomorphine-induced stereotypy, 1n competing with

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62 labelled DA for binding (Creese et al., 1976) and 1n causing extrapyramidal side effects (Crow et al., 1976). In con-trast, fluphenazine has little potency in our preparations. Also, reserpine does not reverse apomorphine-induced stereotypy (Janssen et al., 1966), but it is potent in inhibiting the accumulation of [ 3H]catecholamines (Fig. 2.4). Seeman and Lee (1975) expanded their initial findings of neuroleptic mediated anesthesia to include an evaluation of field-stimulated release of labelled DA from striatal slices, They too found that fluphenazine was as potent as haloperidol or reserpine in inhibiting stimulated release, as well as inducing anesthesia. Once again, this result does not agree with our findings, indicating that changes in membrane solubility caused by these agents was not the cause of haloperidol-and reserpine-induced inhibition of synthesis. However, this pattern was not replicated by Dismukes and Mulder (1977). Attempting to use similar stimulation parameters, they found that haloperidol and spiroperidol effectively inhibited the stimulated release of DA from striatal slices. However, fluphenazine and chlorpromazine were without effect, correlating very well with our current results, They also found that stimulated [ 3H]GABA release was also inhibited to a similar extent as [ 3 H]DA, compatible with our finding that NE synthesis was inhibited as well as DA synthesis. Finally, the concentration of haloperidol which resulted in a 50% inhibition of release (IC50) was 3 M (Dismukes and Mulder, 1977), the dose determined

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63 to be the rc50 of K+-induced release of [ 3H]DA from striatal slices (Miller and Friedhoff, 19 7 9 ) and of catecholamine s ynthesis as reported in this study. Seeman and Lee ( 1 97 5 ) found an rc50 of 0,1 M for haloperidol-mediated inhibition of stimulated release. This large inconsistency may reflect some .qualitative difference in experimental procedures which might explain the qualitativ e differences in ranked neuroleptic dose efficacy. Despite these differences, the close correlation of our data with those of Dismukes and Mulder ( 19 77) as well as with Miller and Friedhoff ( 19 7 9 ) suggests that the inhibition of synthesis we observed with haloperidol is accomplished by the same mechanism that inhibited stimulated release in their s ystems. A hint of the special properties of the butyrophenones as compared to other neuroleptics arose from the original Seeman et al. ( 19 74) paper examining nerve impulse-blocking with neuroleptics, They found that the nerve impulse-blocking potency of the phenothiazines and neuroleptics in general correlated quite well with their partition coeffi cient for the membr ane. The butyrophenones, haloperidol and triflu o peridol, however, were much more effectiv e in blocking impulses than their partition coefficients would have predicted. The y speculated that the butyrophenones were + probably specifically bound to N a -conductance channels in addition to nonspecifically altering membrane fluidity Similarly haloperidol was much less effective than other neuroleptics in inhibiting D A-sensitive adenylate c yclase

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than its clinical potency would predict (Davis, 1974). Thus, haloperidol and perhaps the butyrophenones in general, appear to have properties distinct from other neuroleptics, These distinct properties of haloperidol may be related to its reserpine-like properties of vesicular disruption and catecholamine synthesis inhibition. These properties might + be related to the blockade of Na -conductance channels, although this mechanism seems inadequate to explain the apparent release of DA from vesicles into the cytoplasm. In any event, neither simple anesthesia caused by changes in membrane fluidity nor DA receptor blockade appear to explain our results or the findings of Dismukes and Mulder (1977). The relationship between synthesis inhibition and vesicular disruption is uncertain. Vesicular disruption 64 might elevate the cytoplasmic concentration of DA sufficiently to produce end-product inhibition of TH,. The inhibitory potency of haloperidol on the accumulation of [ 3H]DA from [ 3H]tyrosine was ameliorated by the activation of synthesis (and presumably of TH) induced by 26 mM K+. Such a result correlates with the reduced susceptibility of TH to endproduct inhibition following allosteric activation, arguing that increased cytoplasmic DA concentrations mediated the haloperidol-induced inhibition of DA synthesis. Unfortunately, the reduction of K+-induced release by haloperidol reported by Miller and Friedhoff ( 19 79) would tend to increase the amount of [ 3H]DA retained in the slices in a synthesis study, and thereby confound interpretations.

PAGE 74

Another possibility is that haloperidol may in some way alter the relationship of TH to the cell membrane The membrane-bound form of TH has been reported to be more active than the soluble form (Kuczenski and Mandell, 1972). Thus, membrane disruption might dislodge the enzyme from 65 the membrane and consequently reduce its activity. Also, membrane disruption might interfere with precursor uptake, although no competitive interactions between haloperidolinduced synthesis inhibition and media tyrosine concentrations were observed in substantia nigral slices. The mechanism by which neuroleptics exert their effects 1n vivo are difficult to relate to in vitro observations. The effectiveness of fluphenazine and haloperidol in reversing apomorphine-induced emesis and stereotypy suggests that neuroleptics are DA-receptor blockers. However, at progressively higher doses; anesthesia (Groves et al., 1975; Seeman and Lee, 1974) or vesicular disruption may occur. Waddington (1979) presented evidence that 0.4 mg/kg haloperidol attenuated by 30-40% the contralateral turning induced by intranigral injections of muscimol or baclofen. This effect of muscimol or baclofen was not reversible by 6-0HDA lesions of the ascending nigrostriatal fibers. Furthermore, 0.4 mg/kg totally blocked intranigral apomorphine-induced turning, suggesting that DA systems were not involved in the muscimolor baclofen-induced turning. Waddington concluded that while DA receptors were blocked by haloperidol at this dose, another effect of haloperidol was also evident.

PAGE 75

Thus, a future source of difficulty in investigating the action of neuroleptics will lie in trying to determine at what doses DA-receptor-mediated effects of neuroleptics give way to anesthetic and/or reserpine-like properties. All three activities tend to decrease synaptic efficacy, consequently decreasing feedback inhibition and increasing 66 DA cell firing rates. The increase in DOPAC concentrations detected in vivo following haloperidol treatments might be due either to increased release (Roth et al., 1976) or to vesicular disruption. Finally, the increases in DA synthesis and TH activation might be due to increased firing of DA cells or to a depletion of terminal DA. Kainic acid lesions of the striatum have been reported to suppress the activation of TH by haloperidol treatments, but not the activation of synthesis (Di Chiara et al., 1977, 1978; Tissari et al., 1978). This finding implicates a postsynaptic receptor and long-loop feedback as the means of regulating enzyme activation. However, the maintained increase of DA s ynthesis in the striatum suggests that haloperidol also affects the terminal directly. By transiently inhibiting synthesis or depleting amine concentration, haloperidol might cause a subsequent activation of synthesis in vivo which would be evident at 60-90 minutes following injection. A time course of s ynthesis rates following haloperidol injections will be necessary for confirmation. It would also be of interest to know whether the increased D A s ynthesis following kainic acid lesions would be elicited by n euroleptics which do not have reserpine-like effects in vitro.

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Similarly, while sensory input causes reciprocal changes in metabolism and synthesis of caudate and nigral DA (Nieoullen et al., 19 77), haloperidol increased in vivo DA synthesis in both structures simultaneously (Argiolas et al., 19 79a). Also, while haloperidol increased DOPAC concentrations within substantia nigra (mesostriatal system), it did not affect the ventral tegmental area (mesocortical system, Argiolas et al., 1979b). This differential effect was suggested to reflect the selective activation of the mesocortical system following footshock stress. However, both frontal cortical and striatal D OPAC concentrations were elevated to the same extent with haloperidol. If the normal metabolic responses of striatum and substantia nigra or of striatum and frontal cortex are reciprocal, parallel effects of haloperidol on these structures suggest a site of action on the s ynthetic mech anism within the terminals, and not on afferent processes or dopaminergic cell dendrites. The lack o f an effect in the ventral tegmental area may reflect a lack of vesicular mediated release, making it resistant to the e ffects o f haloperidol or reserpine. In contrast, functional isolation of D A terminals with nigrostriatal lesions (Kehr et al., 19 7 2 ) with gammabutyrolactone (Kuczenski, 19 7 8 ) or with baclofen (Wuerthele et al., 19 7 9 ) blocks the in vivo increase of D A synthesis b y h aloperidol. These findings suggest that h aloperidol did not have a direct effect on D A terminals. H o wever, the 67

PAGE 77

68 functional isolation of DA terminals may have only prevented a rebound in synthesis. A transient inhibition of synthesis, which would normally cause a rebound increase in synthesis, might still have occurred. Summary 1. Medium tyrosine appears to be the relevant precursor pool for DA synthesis. 2 + Activation of DA synthesis commences at K concen-trations relatively ineffective in inducing release, suggesting that activation of synthesis is concurrent with, but not consequent on K+-induced release. 3. Haloperidol, apomorphine, and reserpine inhibited DA synthesis in a dose-dependent manner. 4. We interpret our findings to concur with earlier indications that a DA autoreceptor regulates DA synthesis. K+-induced activation of TH did not diminish the potency of apomorphine to inhibit [ 3H]DA accumulation. Since activated TH is less sensitive to end-product inhibition, then apomorphine-induced inhibition of synthesis probably is not mediated by end-product inhibition of TH. The ability of apomorphine to inhibit NE synthesis, together with the presence of presynaptic receptors capable of regulating NE release, further argues that apomorphine regulates synthesis by means of an autoreceptor, although one not specific to DA terminals.

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5. Most of our effects of haloperidol on in vitro DA metabolism and synthesis do not appear to be associated with a receptor mechanism. Instead, vesicular disruption and synthesis inhibition suggest that haloperidol acts as a membrane disruptor by a mechanism more specific than + simple membrane solubility, perhaps as a blocker of Na channels as suggested by Seeman and Lee (1975). The single exception was that 10 nM haloperidol caused a small facilitation of [ 3H]DA accumulation from [ 3H]tyrosine in striatal + slices incubated at 26 mM K, possibly reflecting an an-t agonism of autoreceptor-regulated inhibition of DA s ynthesis. 69 The possibility exists that many experiments that have used haloperidol to examine changes in catecholamine release and metabolism have produced results unrelated to DA receptor blockade.

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SECTION III ACTH AND LVP SELECTIVELY A CTIVATE MESOCORTICA L DA SYN THESIS Introduction Intracranial administrations of the pituitary peptides ACTH and LVP have potent behavioral effects. Pellets of ACTH1 10 (10 g) implanted in rostral mesencephalon and caudal diencephalon (van Wimersma Greidanus and de Wied, 1971 ) and doses of icv L V P as low as 2.5 ng (de Wied, 1976 ) delayed the e xtinction of avoidance responses in rats. Also, ACTH (icv, 1 g) in rats and mice and L V P (icv, 10-100 n g) in mice induce bouts of excessive grooming (Gispen et al., 19 75; Rees et al., 1976; Delanoy et al., 19 7 8 ) Hi gher doses o f LVP ( 1 g) produced b arrel rotation (rotation around the animal.'s longitudinal axis) in rats and a hyperactivity characterized by scratching foraging and vocalizing in mice (Kruse et al., 1 978 ; Delanoy et al., 19 7 8 ) Ranked e fficacies of various analog s in altering memor y performance and in induci n g spontaneous behavior do not correlate well, suggesting that more than one receptor exists for both ACTH and L V P ( Greven and de Wied, 19 78; Delanoy et al., 19 79). A variety o f arguments suggests that ACTH and L V P may exert their effects throug h catecholaminergic s ystems. ACTH (Ganong 1 97 4 ) and vasopressin ( S andma n et al., 1 973 b ) as well as the catecholamines (Stone, 1975 ) are released in 70

PAGE 80

71 response to stressful stimuli. Anatomical reports have indicated that some hypothalamic projections to areas con~ taining catecholamine cell populations have been labelled with antisera believed to be specific to ACTH (Barchas et al., 1978) or LVP (Buijs, 19 78). ACTH (icv) increased the in vivo disappearance of [ 3H]N E from the forebrain of rats preloaded with icv administered [ 3H]NE (Endroczi et al., 19 76), while LVP (icv) has been found to exert a varied effect on the disappearance of endogenous catecholamines following s ynthesis inhibition with aMPT (Tanaka et al., 1977a, 1977b ) Behaviorally, the grooming response to either ACTH or LVP can be suppressed with systemic injections of the neuroleptic haloperidol (Wiegant et al., 1977). Moreover, ACTH has been found to compete with [ 3H]haloperidol for binding to striatal membranes (Czlonkowski et al., 19 78) Recently, traumatic stressors have been reported to activate DA synthesis in frontal cortex but not in striatum, as assessed by changes in DA and DOPAC concentrations (Laviell e et al., 1979; Fadda et al., 19 78) Since ACTH and LVP are released as consequences of stressful stimuli, because peptide induced grooming is antagonized b y haloperidol, and since preliminary biochemical evidence suggests that icv injections of ACTH and LVP alter catecholamine turnover, we have examined the effects of ACTH and L V P on the activation of catecholamine synthesis in various central nervous system structures. Using a slice technique described in an earlier report (Section II), we measured the in vitro

PAGE 81

accumulation of [ 3H]catecholamines from [ 3H]tyrosine in brain tissues dissected from animals receiving ACTH or LVP. In vitro accumulation was also measured in slices incubated 72 with ACTH or LVP. Because the peptides might alter accumu lation by means of modulating release and metabolism, we tested both peptides in the presence of 26 mM K+. Finally, because of its known interactions with the neuroleptic haloperidol, we tested whether ACTH, when incubated with striatal or substantia nigral slices, might alter haloperidol-or apomorphineinduced inhibitions of s ynthesis. Methods Materials ACTH1 _24 (Cosyntropin, 100 U/mg ) and purified ACTH4 10 and [D-phe7JACTH4 10 were provided by Dr. Henk Greven and Dr. Henk Van Riezen of Organon International B.V Apomorphine ( h ydrochloride salt) and LVP ( 1000 U/5.Smg) were obtained from Sigma Chemical Co. Cr ystalline haloperidol was pro-vided by M c Neil Laboratories. [ 2,6-3 H]tyrosine (34 Ci/ mmol, 1 mCi/ml, lyophilized before use) was obtained from the Radiochemical Centre, Amersham, Other chemicals used in tissue preparations were HEPES (Ultrol, Calbiochem) Tris h ydroxide (Trizma base, Sigma), PPO (Scintillar, Mallindkrodt), POPOP (Research Products International Corp.) and Triton X-100 (Scintillar, Mallindkrodt) The medium used in all experiments was tha t described by Versteeg et al. (1974 ) and contained the following : 11 8 m M NaCl, 4.4 m M KCl, 2 6 ml\! CaC12 1.3 mM MgS04 1.2 mM

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K2HP04, 25 mM HEPES, and 12 mM glucose. The solution was titrated to pH 7.3 with NaOH. Increases in KCl concentration were countered with an equivalent decrease in NaCl. 73 Apomorphine and haloperidol were dissolved in 0.2 M acetic acid at 2 mg/ml and diluted to 0.25 mg/ml with media. 12.1 l of apomorphine or 15 l of haloperidol stock solutions were added to one ml incubation volumes to achieve a final concentration of 10 M. Lower concentrations were produced with serial dilutions of the stock solutions with media. 'Saline' controls were composed of appropriate volumes of acetic acid and media. For icv preparations, ACTH and LVP were dissolved with saline to a concentration of 0.5 g/l. For experiments in which the peptides were incubated with brain slices, ACTH and LVP were initially dissolved in media containing radioactive tyrosine (16.6 or 166 M) to a concentration of 200 M. Concentrations of 20, 2, and 0.2 M were obtained by serial dilutions with labelled media. The addition of an equal volume of slices in unlabelled medium resulted in final concentrations of 8.3 or 83 M tyrosine and 100, 10, 1, and 0.1 M peptide. Animals and Surgery Male CD-1 mice (Charles River, Wilmington, Mass., 25-30 g) were maintained on a 7:00 am 7:00 pm lighting schedule and were kept at temperatures between 22 and 28C Animals not receiving icv injections were individually housed for the three days immediately prior to killing

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Animals receiving icv injections were provided with injection ports, injected and observed as described by Delanoy et al. (1978). Briefly, jewellers' screws (1/8 in x 0080) lubricated with vacuum grease were screwed into holes stereotaxically placed over the lateral ventricles (0.4 mm caudal to bregma, 1.6 mm lateral). Screws were inserted only as far as needed to bind to the cranium (the screw head surface set at 2.8 mm above the cranium), leaving the dura unpenetrated. Dental cement was applied around 74 the screw up to the base of the head of the screw. As the dental cement hardened, threads matching those of the screws formed in the dental cement, allowing subsequent screw removal. Animals were housed individually for the following six day s prior to killing. For all experiments in which icv ACTH was used, the screws were removed on the day prior to injection and killing. Of five experiments performed examining the effects of icv LVP, two were performed with screws removed 5 days before killing, two were performed with screws removed on the morning of the experiment and one was performed in which half of the animals had screw s removed a t f i v e day s and half had screws removed immediately before injection, N o significant effects of the different times of screw withdrawal were found in a multiple factor A NOVA. For icv injections, 27 g a needles 15 m m long (Unimetrics model 2 040 ) were sleeved with 2 2 ga cannula tubing leaving 4.4 mm of 2 7 ga needle exposed. These were fitted onto 10 u l Unimetrics s yringes (model SOlOR) Saline or 1 g of ACTH or L V P were injected into the lateral ventricles in 2 u l volume ( 1 U l /ventricle)

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The spontaneous behavior of each animal was scored and recorded every 30 sec for 30 min, An inclusive list of 75 scores was as follows: quiet, moving, grooming, eating, drinking, foraging, squeaking, and scratching. For experiments in which ACTH was tested, the sum of grooming and scratching was tabulated, Similarly, the sum of scratching, foraging and squeaking was tabulated when icv LVP was tested. Animals were sacrificed immediately after the last observation. Incubation Procedures Two basic types of experiments were performed. In the first, tissues obtained from animals receiving icv injections of peptides or saline were dissected and chopped in two dimensions with a Mcilwain Tissue Chopper set at 300 um. Tissues from each animal were suspended in 15 ml Corex round bottomed centrifuge tubes containing 2 ml of fresh equilibrated medium (37C, in an 02:C02 95:5 atmosphere). This medium was immediately aspirated and replaced with 0.5 ml of medium containing 8.3 M tyrosine and 3.7 5 Ci [2,6-3H]tyrosine, except the striatal slices, which received 0,57 Ci. Slices were incubated for 20 minutes. The reaction was then stopped with 2 ml of ice cold media. In the second type of experiment, sliced tissues were pooled and equilibrated in medium for 30 min. This medium was aspirated and replaced with fresh medium, after which the slices were equilibrated for an additional 10 min. Aliquots of pooled slices (0.5 ml) were added to 15 ml Corex

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round bottomed centrifuge tubes containing 0,5 ml of equilibrated media with labelled tyrosine, peptide treatments, 76 and additional drug treatments. Samples were incubated for 40 minutes and the reaction was stopped with 2 ml of ice cold media. In both types of experiments, once chilled, the slices were centrifuged at 100 x gin a refrigerated (4C) Sorvall RC-3 centrifuge for S min. The media were then decanted. In experiments in which the specific activity of media tyrosine was measured, the decanted media were saved for later analysis. Another 2 ml of ice cold medium w e -re added to each sample and the centrifugation was repeated. Chromatography Purification procedures and recovery efficiencie s were essentially those reported b y Iuvone et al. ( 19 77) Slices were homogenized in the Corex centrifuge tubes with a teflon pestle specially machined to a diameter of 1 4.S mm to tightly fit the Corex tubes. The homogenate was centrifuged at 15000 x g for 10 min in a refrigerated (4C) Sorvall RC, 2 B centrifuge. Supernatants were _loaded onto AG-SO x 4 c ation + exchang e columns (Bio-Rad, 200-400 mesh, 6mm x 2 0 mm, H f orm ) Once loaded, the cation exchange columns were washed with 16 ml of a sodium acetate buffer (0.1 M sodium acetate, 0.1% disodium EDTA titrated with N a O H to pH 7.0) Following a wash with 2 ml 1 M HCl, N E was eluted with 4.7 5 ml of 1 M HCl into test tubes containing 200 m g alumina. Alumina was prepared b y the method o f Anton and Sa yre ( 1 962) and was

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moistened prior to use with 100 l of 0.1 M disodium EDTA. The columns were washed with an additional 1.25 ml of 1 M HCl and DA was eluted with 2.5 ml of 4 M HCl into test 7 7 tubes containing prepared alumina. N E and DA fractions were titrated to pH 7.9 with approximately 2.6 ml 3 M Tris hydroxide and with 2.5 ml of 3 M Tris hydroxide and approximately 0,5 ml of 10 M NaOH, respectively. Titrated suspensions of aluminabound catecholamines were transferred to glass wool stoppered pasteur pipettes. When the alumina suspension had been filtered throug h the glass wool, the alumina retained by filtration was washed with 6 ml water. N E and DA were eluted from the alumina into scintillation vials with 2 ml of 0.5 M HCl. Samples were counted in 10 ml of triton-based scintillation fluid (10.68 g PPO, 0,12 g POPOP, 1 i toluene and 1.4 i Triton X-100). In experiments which required the analysis of media t yrosine specific activity, the media were titrated to pH 1.5 with approximately 65 l of 4 M HCl and loaded onto the + AG-50 x 4 cation exchange columns ( H form) The columns were then washed with 6 ml of sodium acetate buffer. T yrosine was eluted with another 6 ml o f sodium acetate b uffer, titrated to pH 1.5 w ith 4 M HCl, and loaded onto the standard A G-SO x 4 + columns (Na form). Following washes with 5 ml water a nd 8 m l 0.5 M HCl, t yrosine was elute d with 9 ml o f 0.1 M N a3P04 O f this volume, 0.9 ml aliquots were added to 100 l o f 4 M H Cl a nd 1 0 ml of triton based scintillator, while 1.8 ml aliquots were assayed for t yrosine using nitrosonaphtha l

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78 as described by Udenfriend (1962). Fluorescence was measured on a Perkin-Elmer model 650-lOS Fluorescence Spectrophotometer. I n one experiment, 0.3 ml aliquots of the initial incubation media were lyophilized and tyrosine content was measured on an amino acid analyzer by Dr. Maartin Reith at the New York Institute for Neurochemistry and Drug Addiction. Pellets formed by the centrifugation of the homogenate were dissolved in 2.5 ml of 0.3 M NaOH. Protein determinations were performed on duplicate 250 l aliquots using the method described by Bailey et al. (1967). Quantification and Analysis Radioactivity was assayed using a Packard 2425 Liquid Scintillation Spectrophotometer. Correction factors for sample quenching were determined with external standardization. The disintegrations per min (DPM) for NE and DA were divided by the amount of protein in tissue samples to derive specific activities. Tyrosine specific activity was DPM/~g tyrosine. Blank values, determined in parallel samples that received radioactivity but were chilled to 0C instead of being incubated, were subtracted from experimental values, Data were expressed as moles of catecholamines accumulated/mg protein/ minute (based on the tyrosine specific activity). One factor analyses of variance CANOVA), Student's t-tests and Mann-Whitney U tests were executed on a Hewlett Packard 9810A Calculator. An Amdahl 370, version 6 computer was used for quench corrections and for two or more factor ANOVA's and regression analyses as programmed in the Statical Analysis System (SAS)

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Results Effects of icv ACTH and LVP on in vitro [ 3H]Catecholamine Accumulation The number of grooming scores reco~ded during a 20 min period from 10 to 30 min following icv injections of 1 g ACTH1 _24 was elevated to 20/40 from a baseline median of 79 6/40 (p < .OS, Mann-Whitney U test). One g LVP increased the summed scores of scratching, foraging, and squeaking during the total 30 min period following injection from a median of 3/60 to 44/60 ( p < .001, Mann-Whitney U test) Both 1 g LVP and 1 g ACTH1 _24 (Fig. 's 3.1 and 3.2) increased by approximately 18 % the accumulation of [ 3H]DA from [ 3H]tyrosine in frontal cortical slices. LVP also decreased [ 3H]DA accumulation in striatal slices by 15 % Because several animals injected with ACTH did not show excessive grooming, multiple regression analyses of grooming and [ 3H]catecholamine accumulation were performed. Under no conditions were the correlations of grooming and [ 3H]catecholamine accumulation close to significance, sug gesting that the changes in [ 3H]DA accumulation were unrelated to the grooming response. Substantia Nigral Slices Incubated with ACTH Analogs Since intranigral, but not intrastriatal, injections of ACTH1 _24 elicit excessive grooming (Wiegant et al., 1977) and because changes in frontal cortex or striatum should be reflected in the cell regions A9 and AlO, we first examined the accumulation of [ 3H]catecholamines from [ 3H]t yrosine in

PAGE 89

120 110 w z 100 _J
PAGE 90

120 110 w 2 100 ...J
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substantia nigral slices containing both cell regions. Substantia nigral slices incubated with 100 m ACTH1 _24 at 8.3 M tyrosine demonstrated decreased accumulations of both [ 3H]NE and [ 3H]DA (Fig. 3.3A). [D-phe7]ACTH4 10 has been reported to induce excessive grooming (Gispen et al., 1975), while ACTH4 10 has behavioral potency on a variety of learning and memory tasks (de Wied, 1966; Flood et al., 1976). In a second experiment, incubations of nigral slices with these peptides at 8.3 M tyrosine did not affect [ 3H]NE and [ 3H]DA accumulations (Fig. 3,3A). ACTH1 _24 had no effect when the media tyrosine concentrations were increased to 83 M. This lack of effect was observed under standard conditions or when the slices were challenged with 26 mM K+ or with 3 M haloperidol. Frontal Cortical Slices Incubated with ACTH and LVP Frontal slices were incubated with 1, 10, and 100 M ACTH or LVP at 8.3 M tyrosine. As was seen in substantia nigral slices at 8.3 M tyrosine, ACTH1 _24 significantly decreased the accumulations of both [ 3H]NE and [ 3 H]DA. 100 M LVP significantly decre?sed [ 3H]NE accumulation (Fig. 3. 4) ACTH1 _24 contains two tyrosine residues while LVP contains one residue at position 2. Peptidase-mediated degradation might release free tyrosine in sufficient quantities t o decrease the specific activity of media tyro sine, the relevant precursor pool for DA synthesis (Kapatos and Zigmond, 1977; Fig. 2.1). Such a decrease would cause 82

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Fig. 3.3. Effects of ACTH analogs on the accumulation of [3H]catecholamines from [3H]tyrosine in substantia nigral slices. Opeu and shaded bars represent [3H]D A and [3H]NE, respectively. Numbers at the base of each pair of bars indicate the total number of samples used. ** indicates a significant decrease from saline at p < .01, *** at p < .001 (ANOVA) (A) For ACTH analog comparisons, nigral slices were incubated at 8.3 M tyrosine with 4.17 Ci/ml [3H]tyrosine. (B) Nigral slices were incubated with ACTHl-24 at 6 mM K+, at 26 mM K+ and at 3 M haloperidol (HAL) and 6 mM K+. Tyrosine was set at 83 M with 16.7 Ci/ml [3H]tyrosine. Asterisks indicate a significant effect of 3 M haloperidol.

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80 C g C 60 ~ -0 ... a. Cl E 40 VI 0 E -20 100 -~ 80 C a, e so a. c,, A. 8.3 M tyrosine f ,. t 1 SAL io-6 ,o-5 ,o-4 M ACTH4_1Q(D-phe} B. 83 M tyrosine E 40 a, 0 E 20 M ACTH 1-24 ~SAL ,o-6 ,o-5 ,o-4 SALINE -6 -5 -4 SAL 10 10 10 26 mM K -6 -5 -4 SAL 10 10 IO 3 X J0-6 M HAL 84

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. s E S '1> -0 ... Q. OI E U) '1> 0 E ..... 12 10 8 6 4 2 SAL 106 K55 164 M ACTH 1 _24 10-6165 104 M LVP Fig. 3 4 Effects of A C THl -24 and LVP on the accumulation of [3H ]catecholam i nes from [3H]tyrosine i n frontal cortical slices. Open a n d shaded bars rep r e s ent [3H] D A and T3HJ N E respect i vely Frontal cortical slice s were intubated with 8 3 M t yrosine at 1 6 7 Ci/m l [ 3H]tyrosine. indicate s a significant decrease from saline at p < 05, ** a t p < 01 (.ANOVA). 8 5

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an artifactual decrease in [ 3H]catecholamine accumulation. Measurements of media tyrosine concentrations at the end of the incubation period indicated that dilution had occurred (Table 3.1). Using the measured specific activity as a correction factor to determine the NE and D A relative specific activities (RSA's) instead of the initial specific activity correction normally used, the effect of LVP was eliminated. However, ACTH significantly elevated the N E 86 RSA Assuming that peptide degradation and accumulation of [ 3H]catecholamines are linear throughout the incubation period, the average percentag e change in specific activity shoul d be h alf of what was observed at the end o f the incubation. Using this revised t yrosine specific activity the apparent e ffects of ACTH were abolished. Striatal Slices Incubated with ACTH and L V P Striatal slices were incubated with 10 and 100 M ACTH1 _24 at 8.3, 83, and 830 M t yrosine. A s was observed in substarrtia nigra and frontal cortex ACTH1 _24 decreased the accumulation of [ 3 H]DA only at 8.3 M t yrosine (Fig. 3.5) Final t yrosine concentrations were measured at 10 0 M ACTH and saline at 8.3 M tyrosine b y two methods: fluorescence assay of the t yrosine fraction and amino acid analysis of the media. B y both methods, a significant decline in specific activity was observed (Table 3.2). Using 8 3 M t yrosine to mask a n y t yrosine dilution effects of peptide de gradation, striatal slices were incubated with ACTH1 _24 in combination with 6 or 2 6 m M K + and with 0.3 M

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Table 3.1. Changes in Media Tyrosine Specific Activity (SA) Caused by ACTH1 _24 and Their Effects on Catecholamine Relative Specific Activities (RSA) Correction Factor Treatment Media Tyrosine SA (DPM/ng) Measured Tyrosine SA Averaged Tyrosine SAa Saline ACTH1 _24 1 M 10 pM 100 M LVP 1 M 10 M 100 M 20260719 20150 ** 17933 *** 10177 20380 20620310 ** 17330280 ** *** DA RSA .061.005 .072 .008 .066.003 .076.006 .057.009 .05 3 .003 .062.006 Significance at p < .01, at p <.001 NE RSA DA RSA NE RSA .120.013 .061.006 .115.004 .072.008 .107.001 .062.003 ** .175 .006 .050.004 .115.004 .109 .005 .104.008 .057.009 .053.003 .058.008 .120.013 .115.004 .100.002 .117.005 .115.004 .109,005 .102.004 aThe average of the measured tyrosine SA for each treatment and of the measured saline tyrosine specific activity, providing an approximation of the average specific activity during the total incubation period.

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7 C E 6 ...... C 5 0 ~4 O> E ";;; 3 Q) 2 Q. 5 M ACTH1 _24SAL 10-5 10-4 8.3 M TYR 5 5 5 SAL 10-5 10-4 83 M TYR 5 5 5 SAL ,o-5 ,o-4 830 M TYR Fig. 3.5. Effects of A CTHl-24 on the accumulation of [3H]DA from [3H]tyrosine in striatal slices at 8.3, 83, and 830 M t yrosine. Striatal slices were incubated at 4.17 Ci/ml [3H]tyrosine. Numbers at the bottom of each bar indicate the sample size. ** indicates a significant decrease from saline a t p < 01 CANOVA). 88

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Table 3.2. Changes in Media Tyrosine Specific Activity (SA) Caused by ACTH1 24 Fluorescence Assay Amino Acid Analysis Treatment Tyrosine Tyrosine SA Tyrosine Tyrosine SA (]Jg) (DPM/ng) (]Jg) (DPM/ng) Saline 1.45.07 6220550 .63.13 15550 100 M ACTI\_ 2 4 *** *** 1.08.10* 9325* 3 55 .10 2750150 -56% -40% Significance at p< .05 (!-test) ***Significance at p <.001 00 I.!)

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90 apomorphine, 3 M haloperidol, or saline. 26 mM K+ elevated [ 3H]DA accumulation by approximately 200 % while apomorphine and haloperidol inhibited [ 3H]DA accumulation by approximately 50%. ACTH1 _24 had no effect under any combination of treatments (Fig. 3.6). Similarly, striatal slices were incubated with LVP at 83 M tyrosine and at both 6 and 26 mM K+. Once again, there was no peptide effect (Fig. 3.7), Discussion Both ACTH and vasopressin are released into the peripheral circulation 1n response to stressful stimuli (Ganong et al., 1974; Sandman et al., 1973b). Only in recent years has evidence suggested that these hormones might also subserve a stress response in the CNS. Changes in behavior following icv administrations of ACTH or LVP have indicated that receptors exist in the CNS which are capable of recog-nizing these peptides, Intracranial placements of 10 g pellets of ACTH1 10 in caudal diencephalon and rostral mesencephalon were shown to facilitate performance in avoidance tasks (van Wimersma Greidanus and de Wied, 197 1 ) Lesions of this area were effective in disrupting ACTHinduced facilitation of task performance. Similarly, icv injections of as little as 2.5 ng LVP delayed the extinction of a conditioned pole jump avoidance (de Wied, 1976), Excessive grooming can be induced by either 1cv ACTH1 _24 (Gispen et al. 1975; Rees et al., 1976) or low doses of

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1 0 ~ 8 ..... C: .:; f .6 "' E ..... "' "5 .4 E Q. 2 4 C: e :S 3 ., Q. "' E ';;; 2 ., g Q. L. A. 6mM K 10 10 10 10 8 26mM K ,= 4 4 4 4 -6 -5 4 M ACTHSAL 10 10 1 0 SALINE 4 4 4 4 4 4 4 4 -6 5 -4 SAL 1 0 10 10 3 X 10 7 APO 8 8 8 8 4 4 4 4 SAL 1 06 105 I0-4 3 X 106 HAL ;1g. 3.6. Effects of ACTH1-24 on the a ccumulation of I3H]DA from I3H]tyrosine in striatal slices challenged with 0.3 M apomorphine and 3 M haloperidol a t 6 and 26 mM K +. Striatal slices were incubated a s described i n Fig. 3.5. ** indicates significant effects o f haloperidol and apomorphine at p < 01 CANOVA). 91

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C E 3 C V -0 'a. c,, 2 E 1/) 0 E a. 6 6 6 6 M LVPSAL 1 6 6 1 0 5 o -4 6mM K + 1 6 12 1 8 18 1 8 SAL o -7 106 ,o-5 ,o4 26 mM K .. Fig. 3 7 Effects of LVP on the accumulation of [ 3HJD A f rom [3H]tyrosine in striatal slices at 6 and 26 mM K+. Incubations and presentation are as described in Fig. 3.5. 92

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93 icv LVP (Delanoy et al., 1978). At higher doses, LVP elicits a complex of behaviors in mice including foraging, squeaking, and vigorous scratching. Similar doses in rats in-duce rotations around the animals' longitudinal axes (Kruse, et al., 1977). Immunohistochemical techniques have shown that ACTH and AVP are probably endogenous to the CNS. Antisera, believed to be specific for these peptides, labelled neurons 1n the hypothalamus which project to among other areas regions of the brain stem contai~ing noradrenergic cell groups (Buijs, 1978; Barchas et al., 1978). Specific antisera administered 1cv also disrupt behaviors which have been associated with normal responses to stress. Rats treated with icv AVP antisera displayed a faster rate of extinction of a conditioned pole jump avoidance task and poorer retention of a passive avoidance task, effects opposite to what was observed with icv vasopressin (Van Wimersrna Greidanus and de Wied, 1975). Since cerebrospinal fluid concentrations of vasopressin were elevated in rats subjected to stressful stimuli (Dogterom et al., 1977), the performance improvement observed with icv LVP may reflect normal stress adaptation. In parallel, increased grooming in rats caused by the introduction of a novel environment can be blocked by ACTH antisera (Dunn et al., 1979). Since hypophysectomy also blocked the grooming response, the principal source of ACTH mediating stress-induced grooming might not be ACTH neurons. Instead, ACTH released into the h ypophyseal portal

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system may be transported back to the median eminence and introduced into the CNS (Oliver et al., 1977). The mesocortical DA system has been shown to be selectively activated by footshock stress in two separate systems. Thierry et al. (1976) have demonstrated increased disappearance of endogenous DA from frontal cortex and 94 nucleus accumbens following TH inhibition with aMPT. In creased turnovers were not observed for NE or striatal and olfactory tubercle DA. Nearly identical results were found with measurements of DA and DOPAC concentrations (Fadda et al., 1978; Lavielle et al., 1979). DOPAC concentrations were elevated i n frontal cortex and nucleus accumbens, but not in striatum. Since DA concentrations remained constant, an increase in DA synthesis was assumed to have occurred 1n frontal cortex and nucleus accumbens. LVP and ACTH1 _24 (icv) appear to alter catecholamine synthesis in a similar pattern. LVP, and probably ACTH1 _24, increased the accumulation of [ 3H]DA in frontal cortex in vitro. LVP also decreased striatal [ 3H]DA accumulation. A parallel decrease in the mean accumulation of striatal [ 3H]DA was also observed followin~ icv ACTH1 _24 although the effect was not significant Measures of regional [ 3HJ2-deoxyglucose uptake following icv LVP injections have also indicated a selective inhibi~ tion of frontal cortical metabolism (Dunn, unpublished data). The similar activation of mesocortical DA by both footshock and icv peptide administrations, together with the probable peptidergic participation in a stress response in

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the CNS, suggests that the changes in mesocortical DA acti vity following footshock might be mediated by endogenous ACTH and/or LVP. Alternatively, peptide administrations may be causing uncomfortable sensations that are stressful. Consequently, LVP or ACTH would be initiating a general stress response that footshock would also activate, perhaps involving steroidogenesis or peripheral catecholamines. 95 Regardless of which situation actually exists, an extensive investigation of slices incubated with various concentrations of ACTH analogs or LVP failed to provide evidence for direct effects on DA or NE in substantia nigral, frontal cortical, and striatal slices, Brain slice incubations with ACTH, and to a lesser extent LVP, reduced the accumulation of [ 3H]catecholamines. However, the changes were believed to be artifactual; high concentrations of ACTH and LVP caused increases in tyrosine media concentra. tions, perhaps due to enzymatic degradation of the peptides o r due to an increased cellular proteolysis caused by the peptides. A variety of evidence corroborates this explana tion. High doses of ACTH consistently depressed both [ 3H]N E and [ 3H]DA accumulations in ali regions tested, a situation not observed following in vivo administrations of peptides. Second, the effect of both ACTH and LVP was never evident at 83 M tyrosine. Finally, ACTH analogs that do not contain tyrosine residues, but that can induce grooming or facilitate conditioned avoidance performance had no effect on substantia nigral [ 3H]catecholamine accumulation at 8.3 M t yrosine

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96 The effects of ACTH as determined in this investigation do not agree w~ll with previous reports that employed peripheral administrations of ACTH and measured the disappearance of endogenous catecholamines following TH inhibition with aMPT. Peripheral administrations of ACTH1 _24 (Hokfelt and Fuxe, 1972), ACTH4 10 (Leonard et al., 1975; Versteeg, 1973) and a-MSH (Kostrzewa et al., 1975) have been reported to increase the disappearance (turnover) of NE. a peptide with opposite behavioral activity in active avoidance tasks, has been reported to increase NE disappearance (Leonard, 1974), or to have no effect at all (Versteeg, 1973). Hypophysectomy caused a decrease in NE disappearance (Fuxe et al., 1973; Fuxe et al., 1970; Friedman et al., 1973) that was reversed by high concentrations of ACTH (Versteeg and Wurtman, 1975, cited as data to be published) but not with low doses (Fuxe et al., 1970). These changes in NE disappearance are apparently not mediated by an adrenal factor, since adrenalectomy caused a glucocorticoid reversible increase in NE disappearance (Javoy et al., 1968; Fuxe et al., 1973) while glucocorticoids and mineralocorticoids only slightly, if at all, decreased NE disappearance in intact animals (Fuxe et al., 1973). Using another analysis of turnover and icv administrations, Endroczi et al. (1976) investigated the effects of icv ACTH1 _24 and ACTH4 10 on the rate of disappearance of preloaded [ 3HJNE from neocortex, hypothalamus and hippocampus. Once again, NE turnover was increased by ACTH. However, since

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NE can be taken up by dopaminergic or serotonergkneurons (Snyder and Coyle, 1969), the activities of NE neurons are not clearly distinguishable. 97 These manipulations altered DA disappearance much less clearly. Hypophysectomy decreased DA disappearance (Versteeg et al., 1972; Friedman et al., 1973),while adrenalectomy appeared to have very little effect (Fuxe et al., 1973), Furthermore, corticosterone and dexamethasone will increase DA turnover in hypothysectomized, but not intact rats (Fuxe et al., 19 70; Jonsson et al., 1972). Intravenous ACTH given to hypophysectomized rats has been reported to have no effect or to only slightly decrease DA turnover in the neostriatum and limbic forebrain (Fuxe et al., 1973) In contrast, studies of in vivo accumulation of [ 3 H] catecholamines from [ 3H]tyrosine indicate a selective involvement of DA systems following peripheral administrations of ACTH. Versteeg and Wurtman (1975) reported that ACTH4 10 increased the accumulation of total [ 3H]catecholamines. Moreover, this increase did not occur in h ypophysectomized and adrenalectomized animals g~ven ACTH4 10 The authors suggested that these data indicated that the effects of ACTH4 10 might be mediated by an adrenal factor. Another alternative is that adrenalectomy caused an increase in ACTH release that would mask any effects on exogenous injections. Iuvone et al. ( 1978) took this analysis a step further by differentiating the amount of label associated with each amine and found that ACTH analogs increased D A but not N E s ynthesis. The effect of ACTH4 10 on D A

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synthesis was blocked by adrenalectomy, in agreement with Versteeg and Wurtman (1975). While Iuvone et al. described an increase in [ 3H]DA accumulation for the whole brain, we have found increased [ 3H]DA only in the mesocortical system in response to icv ACTH. Nevertheless, while ACTH consistently facilitated the disappearance of NE with no effect on DA, ACTH has been reported to alter DA but not NE synthesis, regardless of the route of administration. 98 This dichotomy of results might be due to different properties of NE and DA neurons, a consistent artifact of either turnover or synthesis studies, or may in fact indicate a property of ACTH's interactions with these two catecholamines. Using the aMPT method of analyzing catecholamine turnover, Tanaka et al. (1977a, 1977b) demonstrated a complex pattern of effects following icv AVP. Most relevant to this study, they found that striatal DA disappearance was enhanced by icv AVP when a micropunch technique was used for sample collection (Tanaka et al., 1977b). However they did not find an increase in DA disappearance when the basal ganglia was assayed as a whole. The difference in these results is difficult to explain, but may indicate that only local areas within the basal ganglia are activated by icv AVP. Nevertheless, an effect of icv AVP on striatal DA turnover appears to be substantiated by these authors. Decreases in DA disappearance were obtained in animals receiving AVP antisera or in rats with hereditary deficiencies of vasopressin (Versteeg et al., 1978, 19 79). This apparent activation of the rnesostriatal s ystem is in contradiction to our

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findings, which demonstrated a decrease in striatal [ 3H]DA accumulation with icv LVP. Several differences between these two studies should be considered. In particular, while measurements of turnover were performed in rats over a 3 hour span of time, measurements of synthesis were performed in mice over a 20 min period beginning 30 min after injection. In addition, aMPT has been shown to be steroido genie, an effect which may interact with the effects of LVP, which has corticotrophin releasing activity. Whatever the explanation, both studies implicate striatal DA as being affected by icv LVP. ACTH injections into the substantia nigra, but not 99 into the striatum elicit excessive grooming. Either systemic or intrastriatal injections, but not intranigra~ injections of haloperidol prevent the ACTH-induced grooming. These results have implicated DA neurons as essential for the expression of the grooming response. The activation of frontal DA synthesis may support this hypothesis although only with qualifications. Immediately following a period of intense grooming induced by ACTH1 _24, we observed no change in striatal DA synthesis, arguing that the mesocortical and not the mesostriatal DA system reflected the expression of grooming. Also, incubations of substantia nigral slices with ACTH analogs did not significantly alter accumulation of [ 3H]catecholamines beyond an artifactual decrease apparently caused by precursor pool dilution. Nor was ACTH effective when substantia nigral slices were challenged

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with 26 rnM K+ or with 3 M haloperidol. Therefore, either the slice preparation does not retain the ability to interact with ACTH, or reception takes ~lace outside of the substantia nigra and is communicated trans-synaptically. This latter possibility is likely since ACTH containing neurons project to the periaqueductal gray region of the brain stern. Furthermore, although she used very hig h doses (50-100 g), Jacquet (19 78) has documented behavioral responses (explosive motor behavior) to injections of ACTH into the periaqueductal gray Thus, ACTH reception that mediates grooming may take place in the periaqueductal gray However, the correlation coefficient relating grooming scores and frontal cortical [ 3H]DA accumulation was not significant. Moreover, a variety of other a gents which activate D A s ystems, such as amphetamine and MIF, do not induce grooming (Delanoy et al., 19 7 8). Evidence for a role of DA in the expression of grooming arises from the disruption of grooming b y intracranial administrations of doparninergic a gonists and antagonists. However, intrastriatal injections of small volumes would produce hig h local concentrations of drug which might cause behavioral abnormalities such a s stereoty p y 100 or catalepsy Such abnormalities mi ght mask the expressi o n of a variety of behaviors, including grooming and not be indicating a role o f D A in the expression of grooming

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Similarly, the use of haloperidol-induced disruption of grooming (Wiegant et al., 1977) as a criterion for DA involvement is hazardous, since behavioral effects unrelated to DA function have been reported at doses as low as 0.4 mg/kg (Waddington, 1979). 101 In summary, icv injections of LVP and ACTH1 _24 selectively activated the accumulation of [ 3H]DA in frontal cortex, correlating with earlier reports of mesocortical activation following footshock stress. It is unclear whether the peptide effect reflects a role of endogenous peptides mediating an effect of footshock, or is simply causing another form of stress which.is indirectly activating DA synthesis. The induction of grooming with moderately stressful stimuli and the disruption of such grooming with ACTH-antisera suggests that endogenous ACTH participates in a CNS stress response and may be mediating the activation of the mesocortical DA system in response to stress. Our findings in general do not support an hypothesis of an obligatory role of DA in the expression of ACTH-induced grooming as proposed by Wiegant et al. (1977). We did find an increase in mesocortical D A s ynthesis following icv ACTH1 _24. However, this change did not correlate with an increased grooming response. The lack of an effect of ACTH when incubated with substantia nigral slices and the weak effect of icv administrations of ACTH1 _24 suggests that ACTH reception is somewhat remote from the substantia nigra and is communicated trans-synaptically or through an endocrine intermediary

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SECTION IV CONCLUDING REMARKS The findings of this dissertation contribute to the testing of the two hypotheses presented in the introduction. The first hypothesis concerned the obligatory role of DA neurons in mediating ACTH-or LVP-induced grooming. The most convincing data defending this position were those presented i n a report by Cools et al. (1978). Their approach exploited previous findings of Cools et al. (1977) indicating that two separate populations of DA receptors overlapped in the basal ganglia. An excitation-mediating DA system (DAe) with terminal areas predominantly in the striatum and an inhibition-mediating DA system (DAi) with terminals pre-dominantly in the nucleus accumbens were identified on the basis of differential responses to a variety of intracranially injected DA agents. Cools et al. (1978) found that agonists or antagonists effective in DAe areas would block ACTH-induced grooming when injected into the striatum. The same pattern was found for agonists and antagonists effective in DA. 1 areas injected into nucleus accumbens. They concluded that a ". specific balance between the activity of DAe and DAi systems underlies ACTH-induced grooming." The principal flaw in their data is that they do not report on the effects of the agents injected intracranially on spontaneous behavior. 102

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103 For example, 5 g of the DA agonist apomorphine, injected e intracranially, results in high local concentrations of drug. Most likely, a concentration capable of inducing stereotypy would have transiently been present. Thus, the induction of stereotypy, and perhaps even the induction of increased locomotor activity at lower concentrations of apomorphine, might be overriding the expression of grooming. Similar arguments can be applied to haloperidol and the other agents used in the Cools et al. study. By these arguments, ACTHinduced grooming would not have been prevented because of the blockade of DA systems, but instead because behavioral abnormalities induced by DA agents would be masking the grooming response. The authors had also published that peripheral injections of 0.2 mg/kg haloperidol would block ACTH-induced grooming. This dose has only mildly sedative properties, suggesting that the effect of haloperidol was on DA receptors and not due to a less specific mechanism. However, the hazards of using haloperidol-induced inhibition as a criterion for DA-mediated behaviors was discussed by Waddington ( 19 79) and in the first manuscript of this dissertation. Although DA antagonist properties were clearly evident at the 0.4 mg/kg dose of haloperidol used by Waddington, he also observed additional effects that apparently did not involve DA transmission. In vitro, the threshold foranesthesiaand synthesis inhibition of haloperidol, properties unrelated to DA transmission, is about 1 M.

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If dopaminergic systems were mediating grooming, then activation of the system by other agents should also elicit grooming. This has not been found. Apomorphine, ampheta mine, melanocyte stimulating hormone inhibiting factor and a variety of other agents which affect dopaminergic systems do not elicit grooming. Our data also do not support the hypothesis of a role 104 of the mesostriatal DA systems in mediating ACTH-induced grooming. Neither ACTH1 _24 or [D-phe7]ACTH4 1W peptides with potency in the induction of grooming,caused any changes in [ 3H]catecholamine accumulation when incubated with substantia nigral slices. This lack of an effect may have been due to opposite reactions of cell bodies giving rise to the differentially activated mesocortical and mesostriatal systems. In fact, such a possibility is predicted from the divergent effects of both ACTH and LVP on catecholamine metabolism in these two regions. However, the simpler interpretation is that ACTH reception does not take place in the substantia nigra. The very small changes and the large variance in the response to icv ACTH and LVP suggests that the peptide reception does not occur directly on catecholamine neurons. Wiegant et al. (1977) found that intranigral, but not intrastriatal, injections of ACTH would induce grooming. The possibility exists that backwashing of peptide along the outside of the cannula may have introduced ACTH dorsal to the cannula tip along the cannula tract in the central gray region or the reticular formation. The central gray area

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105 is the location described by Barchas et al. (1978) as receiving ACTH projections from hypothalamus, Furthermore, although very high doses of ACTH were used, Jacquet (1978) has reported behavioral effects of ACTH injected into the central gray of rats. Cools et al. (1978) reported that the injections of DA agent s into the mesostriatal terminal areas of striatum and nucleus accumbens attenuated the grooming response to icv ACTH. No activation of striatal synthesis was observed following icv ACTH in mice. If changes in synthesis reflect changes in transmitter utilization, then only the activation of the mesocortical DA system correlates with the ACTH-induced grooming. Even this correlation is not very strong. Although the activation of synthesis by icv ACTH was significant the correlation of grooming and catecholamine synthesis changes was not. Also, preliminary evidence suggests that ACTH-induced grooming is not reversible with diazepam (Dunn, unpublished observations). In contrast, diazepam reversed the mesocortical activation of synthesis and metabolite accumulation following footshock stress (Lavielle et al., 1978; Fadda et al., 19 78) Two possible explanations can account for this disparity. The changes in DA synthesis following ACTH may be part of a parallel stress response and not reflect the grooming response. Otherwise, the changes in DA synthesis caused by ACTH are only coincidentally similar to changes caused by footshock stress and would not be reversible with diazeparn. In light of the hazards of using behaviorally

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106 potent agents as a criterion for dopaminergic involvement in a behavioral response, since a variety of agents that activate DA metabolism such as amphetamine and MlF do not elicit grooming, and because no significant correlation between grooming and DA synthesis was found, then ACTH-induced grooming probably is not mediated by DA systems. The second hypothesis discussed in the introduction was the possible role of ACTH or LVP in mediating a selective change in mesocortical DA metabolism following footshock episodes. DOPAC concentrations were elevated in frontal cortex and in nucleus accumbens without any change in DA concentrations, suggesting increased synthesis as well as release (Lavielle et al., 1979; Fadda et al., 19780. AVP was found to be released into cerebrospinal fluid of rats following footshock (Dogterom et al., 1977). Furthermore, endogenous AVP apparently contributes to the performance of rats in stressful avoidance tasks (van Wimersma Greidanus and de Wied, 1975). Similarly, endogenous ACTH may mediate stress-induced grooming (Dunn et al., 1979). Thus, because ACTH and LVP presumably are released into the central nervous system in response to stressful stimuli, the changes in mesocortical activation following footshock might be in part regulated by ACTH or LVP. The results presented in this report do not contradict this possibility: both icv ACTH and icv LVP selectively elevated DA synthesis in the frontal cortex, paralleling the effects of footshock. However, [ 3H]catecholamine accumulation in slices of frontal

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107 cortex and substantia nigra incubated with ACTH or LVP did not change in response to either peptide, and thus failed to indicate a direct peptidergic .effect on catecholamine neurons. If ACTH or LVP reception relevant to mesocortical activation does not take place on dopaminergic neurons, then two possibilities remain. The first possibility is that footshock causes the release of ACTH or AVP which in turn initiate neuronal responses that trans-synaptically activate the mesocortical system. Alternatively, in the doses administered, icv ACTH or icv LVP may be as stressful as the footshock used (six 160 msec shocks of 1.6 mA during 2 sec, every 10 sec for one hour, Lavielle et al., 1979) and initiate a stress response at the level of sensory input. In simpler terms, do icv ACTH and LVP mimic endogenous peptides that mediate stress, or are they stressful themselves? The discrimination of these two alternatives will not be easy. The most obvious experiment would be to test the mesocortical response to footshock in hypophysectomized animals. However, while hypophysectomy probably decreases CNS ACTH concentrations (hypophysectomy prevents stressinduced grooming, Dunn et al., 1979), AVP concentrations in the cerebrospinal fluid increase following hypophysectomy (Dogterom et al., 1977). Another possible, but very expensive, approach would be to selectively deplete either or both peptides with specific antisera. Until subsequent analyses are performed, the peptidergic mediation of stress-induced mesocortical DA activation remains an attractive hypothesis.

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BIOGRAPHICAL SKETCH The author was born in Columbus, Indiana. on September 11, 1951, to Richard L. and Grace Hayes Delanoy. After having attended elementary school in Endicott, New York state and junior high school in Huntsville, Alabama, he graduated from hig h school from Springbrook High School of Silver Spring, Maryland, in 1969. He attended Wake Forest University in Winston-Salem, North Carolina, under a Carswell Academic Scholarship for three years. He received a Bachelor of Arts in biology 1n 1973. After working for the Whitney Marine Laboratories as a technician, he entered the Department of Neuroscience of the University of Florida in 1975. He has been supported b y a fellowship from the National Science Foundation. 12 2

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. I.. Adrian J. Dunn, Chairman Associate Professor of Neuroscience I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. William G. Lutug~ Associate Professor of Neuroscience I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope ~nd quality, as a dissertation for the degree of Doctor of Philosophy. '; '": --:z..,.~-c!..-..-z Philip Posner Associate Professor of Physiology

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. of Neuroscience This dissertation was submitted to the Graduate Faculty of the College of Medicine and to the Graduate Council, and was accepted as partial fulf:llment of the requirements for the degree of Doctor of Philosophy. December 1979