Responses of dopaminergic and serotonergic systems to triethyllead intoxication

Neurotoxicologyand Teratology, Vol. 10, pp. 27%285. ©PergamonPress plc, 1988. Printedin the U.S.A.

0892-0362/88$3.00 + .00

Responses of Dopaminergic and Serotonergic Systems to Triethyllead Intoxication D. L. D E H A V E N - H U D K I N S , .1 D. W. S C H U L Z , .2 T. J. W A L S H $ 3 A N D R. B. M A I L M A N * ?

*Biological Sciences Research Center, and Curricula in Neurobiology and Toxicology ?Departments o f Psychiatry and Pharmacology, University o f North Carolina School o f Medicine Chapel Hill, N C 27514 and "~tLaboratory o f Behavioral and Neurological Toxicology National Institute o f Environmental Health Services, P.O. Box 12233 Research Triangle Park, N C 27709 R e c e i v e d 29 D e c e m b e r 1987 DEHAVEN-HUDKINS, D. L., D. W. SCHULZ, T. J. WALSH AND R. B. MAILMAN. Responses ofdoparninergic and serotonergic systems to triethyllead intoxication. NEUROTOXICOL TERATOL 10(4) 279-285, 1988.--Rats treated with triethyllead (TEL) exhibit a behavioral supersensitivity to challenge with dopamine agonists at 7 days following administration of TEL. In the present series of experiments, some neurochemical mechanisms which may affect this behavioral supersensitivity were detected. Administration of a single dose of TEL chloride (7.88 mg/kg, SC) to male Fischer-344 rats decreased the concentrations of dopamine in hippocampus, and of serotonin in olfactory tubercle, at Day 7 posttreatment. The ratio of 5-hydroxyindoleacetic acid/5-hydroxytryptamine (one estimate of serotonin turnover) was increased in nucleus accumbens (p<0.05), with a similar trend in olfactory tubercle and striatum (p<0.10). No changes were detected in binding of [3H]spiperone to D2 dopamine receptors in striatum or olfactory tubercle. However, although basal adenylate cyclase activity was unaltered in TEL-treated rats, the Vmaxfor dopamine-stimulated adenylate cyclase activity was significantly elevated in olfactory tubercle. Conversely, TEL at micromolar concentrations markedly attenuated both basal and dopamine-stimulated adenylate cyclase activity in vitro in striatal homogenates. These data suggest the hypothesis that administration of TEL to rats results in an up-regulation of D~ dopamine receptors in olfactory tubercle, and that the behavioral supersensitivity of TEL-treated animals to dopamine agonists may, in part, be a result of this receptor supersensitivity. Triethyllead

Dopamine

Serotonin

Adenylate cyclase

Receptors

daily 1.75 mg/kg doses of T E L to rats, concentrations of the major serotonin metabolite 5-hydroxyindoleacetic acid (5HIAA) were increased in striatum, hippocampus, frontal cortex and brainstem, while concentrations of 5-HT were also increased in hippocampus [17]. Unfortunately, it is difficult to reconcile these data into a single coherent picture. On the other hand, pharmacological studies have demonstrated that dopaminergic function is altered following treatment with TEL. For example, the antinociceptive effects caused by acute administration of T E L are not seen if rats are first lesioned with 6-hydroxydopamine. Moreover, the antinociception produced by T E L is enhanced by administration of dopaminergic agonists (Walsh et al., unpublished

T R I E T H Y L L E A D (TEL) is a toxic organometal that produces behavioral and neurological deficits in animals and man [41]. While the mechanism of the neurotoxicity of T E L is not known, several neurochemical studies suggest that dopaminergic and serotonergic function in the brain may be perturbed following treatment with TEL. For example, acute exposure to T E L in vivo resulted in enhanced uptake of dopamine (DA) into striatal Synaptosomes, in contrast to findings that in vitro uptake of DA and 5-hydroxytryptamine (5-HT) was inhibited after exposure to micromolar concentrations [22]. Micromolar concentrations of T E L in vitro also inhibited DA-stimulated adenylate cyclase activity in striatal homogenates [42]. One day following administration of five

1Requests for reprints should be addressed to D. L. DeHaven-Hudkins, Sterling Research Group, 9 Great Valley Parkway, Great Valley, PA 19355. 2Current address: Department of Pharmacology, Harvard University Medical School, 250 Longwood Avenue, Boston, MA 02115. aCurrent address: Department of Psychology, Rutgers University, Busch Campus, New Brunswick, NJ 08903.

279

280

DEHAVEN-HUDKINS, SCHULZ, WALSH AND MAILMAN ABBREVIATIONS

DA DOPAC HEPES 5-HIAA HPLC-EC 5-HT HVA NE TEL

dopamine 3,4-dihydroxyphenylacetic acid N-2-hydroxyethyl piperazine-N'-2-ethane sulfonic acid 5-hydroxyindoleacetic acid high-performance liquid chromatography with electrochemical detection 5-hydroxytryptamine homovanillic acid norepinephrine triethyllead

data). Administration of 7.88 mg/kg T E L results in a behavioral supersensitivity to challenge with amphetamine or apomorphine 7 days postdosing [40]. These potentiated psychopharmacological effects of amphetamine in TEL-treated rats were shown not to be due to altered distribution or accumulation of the drug [40], suggesting that T E L causes supersensitivity of dopaminergic systems by pharmacodynamic mechanisms. In the present report, we have examined the effects of T E L on the neurochemistry of dopaminergic systems, as well as other monoaminergic systems that are involved in the mediation of locomotor behavior and stereotypy [6, 9, 12]. METHOD

Animals Male Fischer-344 rats (Charles River Laboratories, Wilmington, MA), approximately 90-110 days of age and weighing 200-250 g at the time of T E L administration, were used. Animals were housed in groups of four in plastic cages, at controlled temperature (20°-+2°C) and humidity (50%_+ 10%). Rats were given free access to food (NIH diet No. 31) and tap water. A 12 hr light/dark cycle (lights on from 0700-1900) was in effect.

Treatment A single dose of T E L chloride (7.88 mg/kg as the salt) was administered subcutaneously (SC, 1 ml/kg) in a vehicle of distilled water. This dose was selected because previous studies had demonstrated that the behavioral responses to dopaminergic agonists were enhanced at 7 days following treatment with this dose [40]. Control animals received 1 ml/kg of vehicle by the same route of administration.

Tissue Preparation Animals were killed by decapitation 7 days after T E L administration, brains rapidly removed and placed in ice. Striatum, olfactory tubercle, nucleus accumbens, dorsomedial prefrontal cortex, hippocampus and cerebellum were dissected on ice by the methods of Glowinski et al. [15] and Heffner et al. [16]. Brain regions were weighed, immediately frozen on dry ice, and stored at -70°C until HPLC analysis was performed. For the radioligand binding and adenylate cyclase assays, fresh tissue was always used.

High Perjbrmance Liquid Chromatography (HPLC) Analysis and Protein Determination Quantification of the concentrations of DA, DOPAC,

HVA, 5-HT and 5-HIAA in various brain regions was per-

formed essentially using an HPLC method developed in this laboratory [21]. It utilizes N-methyl-5-hydroxytryptamine as an internal standard, and electrochemical detection after reverse-phase separation. N E was measured using a modification of the HPLC-EC procedure of Keller et al. [20] as described by Mailman et al. [26], which uses 3,4-dihydroxybenzylamine as the internal standard. Protein concentrations were determined by the method of Lowry et al. [25] adapted for use with a Technicon Autoanalyzer I (Tarrytown, NY).

Radioreceptor Assays pH]Spiperone binding in striatum and olfactory tubercle was accomplished by the following procedure. Tissues were homogenized with a Brinkmann Polytron in 100 volumes of cold 50 mM HEPES, pH 7.55 at 25°C. The suspension was centrifuged at 27,000 x g for 15 rain, the supernatant removed, and the pellet resuspended and centrifuged as above. The washed pellet was then resuspended in H E P E S at a concentration of 5 mg/ml and used. Each tube contained 2.5 mg of tissue (original wet weight) and pH]spiperone in concentrations ranging from 0.05 to 0.8 nM in a final assay volume of 1 ml. Nonspecific binding to dopamine receptors was determined by addition of a final concentration of 1 ~M unlabelled domperidone to half of the tubes. All tubes were incubated at 37°C for 20 rain and then filtered under vacuum and washed three times with cold HEPES buffer. Each data point was the average of three tubes. Filters were dried overnight, scintillation cocktail added, and radioactivity quantified by liquid scintillation spectroscopy at an efficiency of 45% for tritium. In striatum, KD and Bmax values were determined by Scatchard analysis [34] of individual animals. In olfactory tubercle, two separate experiments were conducted to determine KD and Bmax values using pooled tissue for each group. The procedure used for pH]5-HT binding was a minor modification of the method of Bennett and Snyder [2]. Briefly, hippocampus was homogenized in 40 volumes of cold 50 mM HEPES buffer (pH 7.7 at 25°C) with a Brinkmann Polytron (setting 5 for 20 sec) and centrifuged at 27,000 x g 1or 10 minutes. Tissue was resuspended and centrifuged once as described above. The washed pellet was then resuspended in an incubation buffer containing 50 mM H E P E S with 0.1% ascorbate, 4 mM CaCI._, and 10/~M pargyline (pH 7.2 at 25°C) and preincubated at 37°C for 10 minutes. Following this, the homogenate was centrifuged and the pellet was resuspended in incubation buffer at a concentration of 5 mg/ml tissue. Each incubation tube contained 2.5 mg of tissue (original wet weight) and various concentrations of pH]5-HT from 0.5 to 12 nM in a final assay volume of 2 ml, with nonspecific binding defined by addition of 10 ~M unlabelled 5-HT. All binding determinations were done in triplicate, and after incubation at 37°C for 15 minutes, the samples were filtered under vacuum and washed three times with cold HEPES buffer. Filters were dried overnight and counted as described above. Scatchard analysis [34] was performed on tissue from individual animals.

DA- and NE-Stimulated Adenylate Cyclase Activity Fresh tissue was homogenized manually in a Wheaton Teflon-glass homogenizer at a concentration of 20 mg/ml in 5 mM HEPES buffer (pH 7.5) containing 2 mM EGTA. An equal volume of 100 mM HEPES-2 mM EGTA was added and mixed with one additional stroke. A 20/~1 aliquot o f this homogenate was then added to a prepared reaction mixture,

T R I E T H Y L L E A D AND MONOAMINES

281

TABLE 1 EFFECTS OF TEL ADMINISTRATIONON CONCENTRATIONSOF NE, DA, DOPACAND HVA IN VARIOUS BRAINREGIONS AT DAY 7 POSTTREATMENT Region

Group

N

NE

DA

DOPAC

HVA

Striatum

Control TEL

18 18

ND ND

133.5 ± 3.7 129.8 -+ 5.8

19.6 ± 0.6 19.6 _ 0.9

8.2 ± 0.6 8.0 ± 0.5

Olfactory Tubercle

Control TEL

18 18

ND ND

104.8 _-4:_ 5.4 96.6 ± 10.5

28.3 ± 2.7 26.8 ± 2.3

5.4 ± 0.3 5.1 __ 0.4

Nucleus Accumbens

Control TEL

16 16

ND ND

78.5 --- 3.0 84.1 ± 6.4

23.6 ± 1.4 29.3 _ 2.9

6.9 ± 0.4 8.0 ± 0.7

Frontal Cortex

Control TEL

16 16

5.5 ± 0.8 5.2 ± 0.9

2.9 ± 0.4 2.6 ± 0.2

1.5 ± 0.1 1.4 ± 0.2

0.9 --- 0.1 0.9 ± 0.1

Hippocampus

Control TEL

18 18

8.8 ± 1.1 8.1 ± 1.2

0.8 ± 0.1 0.6± 0.1"

ND ND

ND ND

Rats received TEL (7.88 mg/kg, SC) on Day 0. Values represent the mean ± SEM in ng/mg protein. *Significantly different from control, p<0.05. ND=Not determined.

yielding a final volume of 100 ~1 containing 0.5 mM ATP, [a-32P]ATP (0.5/zCi), 1 mM cAMP, 2/xM MgCI2, 0.5 mM 3-isobutyl-l-methylxanthine, 0.7 mM HEPES buffer, 2 ~M GTP, 10 mM phosphocreatine, and 5 U creatine phosphokinase. As noted, dopamine (0 to 50 tzM) or norepinephrine (0 to 100 tzM) was added to various tissue preparations to activate adenylate cyclase. The reaction was initiated by placing the samples in a water bath at 30°C and terminated 10 minutes later by the addition of 100/xl of 3% sodium dodecyl sulfate. The [32P]cAMP was separated from other [32P]-containing nucleotides using an automated reverse-phase HPLC separation [35], and again quantified by liquid scintillation spectroscopy. Recovery was monitored by analyzing peak amplitudes of unlabelled cyclic AMP. The activity of adenylate cyclase is presented as pmoles cAMP formed per mg protein per minute. Eadie-Hofstee analyses were performed for individual brain regions from each animal in order to determine pseudo-Km and Vma x values for activation by dopamine in those cases. Otherwise, enzyme activation was expressed as the percent increases above a basal level. The effect of T E L on dopamine-stimulated adenylate cyclase in vitro was also evaluated. Striatal tissue from untreated animals was incubated as described above, except that T E L chloride in concentrations ranging from 1 to 30/.tM was included.

Materials T E L was purchased from Alfa Products (Danvers, MA). Scintiverse I was obtained from Fisher Scientific Co. (Pittsburgh, PA). The [ZH]spiperone (33.2 Ci/mmol) and [3H]5-HT (28.7 Ci/mmol) were bought from New England Nuclear, Inc. (Boston, MA). The [o~3~P]ATPwas synthesized according to the method of Johnson and Walseth [18]. Glass fiber filters (Gelman A/E) were from Gelman Sciences Inc. (Ann Arbor, MI). HEPES was a product of Research Organics Inc. (Cleveland, OH), domperidone was a gift from Janssen Pharmaceutical (New Brunswick, N J), and N-methyl-5-hydroxytryptamine was purchased from Aldrich

Chemical Co. (Milwaukee, WI). All other material was purchased from Sigma Chemical Co. (St. Louis, MO).

Statistical Analysis Our previous series of behavioral experiments [40] demonstrated that TEL-treated rats exhibited a behavioral supersensitivity when challenged with dopaminergic agonists, providing the hypothesis that receptor supersensitivity was present. Further, preliminary neurochemical studies indicated that T E L produced small, but consistent, increases in adenylate cyclase activity. Therefore, in the adenylate cyclase and [3H]spiperone binding studies, planned comparisons using one-tailed t-tests were used to test the hypothesis that administration of T E L would result in supersensitivity at dopaminergic receptors. All other comparisons between TEL-treated and control groups were performed using two-tailed t-tests. RESULTS

EJfects on Regional Concentrations of Biogenic Amines and Their Metabolites T E L induced no changes in dopamine and its metabolites, or norepinephrine, in most of the brain regions examined, the exception being a decrease in concentrations of DA in the hippocampus (p<0.05, Table 1). Concentrations of 5-HT were decreased in olfactory tubercle (.o<0.05, Table 2), and the ratio of 5-HIAA/5-HT was increased in nucleus accumbens (p<0.05, Fig. 1). It should be noted that the ratio of 5-HIAA/5-HT tended to be increased in both olfactory tubercle and striatum following treatment with T E L (Fig. 1), but did not reach statistical significance 6o<0.1).

Binding to [3H]Spiperone and [3H]5-HT Receptors The Bmax and Ko for [3H]spiperone binding in striatum and [ZH]5-HT binding in hippocampus were unaltered by treatment with T E L (Table 3). Similarly, Scatchard analysis of [aH]spiperone binding in pooled tissue from olfactory

282

DEHAVEN-HUDKINS,

SCHULZ, WALSH AND MAILMAN

15

TABLE 2

Control

~

TEL

14

EFFECTS OF TEL ADMINISTRATION ON CONCENTRATIONS OF 5-HT AND 5-HIAA IN VARIOUS BRAIN REGIONS AT DAY 7 POSTTREATMENT

13 ' 2 11

X X;

1

Region

Group

N

5-HT

5-HIAA

Striatum

Control TEL

18 18

12.2 ± 0.4 11.4 _+ 0.7

10.7 - 0.9 12.7 ± 1.4

Olfactory Tubercle

Control TEL

18 18

24.1 ± 0.9 20.7 ± 1.1"

12.9 _+ 0.7 12.5 -+ 0.4

Nucleus Accumbens

Control TEL

16 16

12.0 ± 0.4 11.7 _+ 0.7

10.9 ± 0.9 13.6 _+ 1.2

Frontal Cortex

Control TEL

16 16

14.1 ± 2.0 11.6 ± 1.4

4.8 ± 0.3 4.9 +_ 0.3

Hippocampus

Control TEL

18 18

10.5 ± 0.4 11.5 ± 0.7

10.3 _+ 0.3 9.4 _+ 0.4

09 08 07 06

xl ×!

05

Rats received TEL (7.88 mg/kg, SC) on Day 0. Values represent the mean --- SEM in ng/mg protein. *Significantly different from control, p<0.05.

04 03 02 01 0

STR

OT

NA

FC

HPC

FIG. 1. The effects of TEL administration on the ratio of 5-HIAA/5-HT in various brain regions at Day 7 posttreatment. Data are expressed as mean±SEM. N = 16-18 per group. Abbreviations: STR, striatum; OT, olfactory tubercle; NA, nucleus accumbens; FC, frontal cortex; HPC, hippocampus. *=Significantly different from vehicle, p<0.05.

TABLE 3 EFFECTS OF TEL ADMINISTRATION ON DOPAMINERG1C AND SEROTONERGIC RECEPTORS AT DAY 7 POSTTREATMENT

Bmax Ligand

Region

Group

N

(fmol/mg protein)

KD (nM)

Spiperone

Striatum

Control TEL

17 17

208 ± 33 219 ± 32

0.20 ± 0.03 0.24 +_ 0.03

5-HT

Hippocampus

Control TEL

6 6

158 _+ 20 146 _+ 18

2.26 _+ 0.31 2.79 _+ 0.36

Rats received TEL (7.88 mg/kg, SC) on Day 0. Values represent the mean _ SEM. Mean values for Bmax and KD were derived from Scatchard analysis of individual animals.

t u b e r c l e o f c o n t r o l a n d T E L - t r e a t e d rats did n o t r e v e a l a n y d i f f e r e n c e s b e t w e e n t h e t w o g r o u p s (Bmax=136 vs. 144 f m o l / m g p r o t e i n ; K D = 0 . 0 8 n M for b o t h groups).

larly, t h e r e w a s n o a p p a r e n t a l t e r a t i o n of e n z y m e a c t i v a t i o n b y n o r e p i n e p h r i n e in c e r e b e l l a r e m o v e d f r o m T E L - t r e a t e d r a t s (26_+4% vs. 27_+4% for control).

Activities of DA- and NE-Stimulated Adenylate Cyclase in Various Brain Regions

Effects of In Vitro TEL on DA-Stimulated Adenylate Cyclase in Striatum

T r e a t m e n t of r a t s w i t h T E L also did n o t affect b a s a l levels o f c A M P s y n t h e s i s in a n y o f t h e f o u r b r a i n r e g i o n s e x a m i n e d ( d a t a n o t s h o w n ) . H o w e v e r , as s h o w n in Fig. 2, t h e Vmax for d o p a m i n e - s t i m u l a t e d a d e n y l a t e c y c l a s e in o l f a c t o r y t u b e r c l e w a s i n c r e a s e d b y 38% ( p < 0 . 0 5 ) , w h i l e t h e a p p a r e n t Km w a s also i n c r e a s e d , b u t n o t significantly (Fig. 3). T h e e l e v a t i o n in t h e a p p a r e n t Km in o l f a c t o r y t u b e r c l e o f rats r e c e i v i n g T E L (Fig. 3) m a y b e r e l a t e d p r i m a r i l y to this c h a n g e in V . . . . since the percent stimulation of enzyme activity caused by 50/zM d o p a m i n e w a s i n c r e a s e d as a r e s u l t o f t r e a t m e n t w i t h T E L (126---15% vs. 102_+12%), while s t i m u l a t i o n b y 2.5 ~ M d o p a m i n e did n o t differ for t h e t w o g r o u p s (27.5_+4.6% vs. 27.6___3.6%). S t i m u l a t i o n o f a d e n y l a t e c y c l a s e b y d o p a m i n e in dorsomedial prefrontal cortex, however, was not affected by a d m i n i s t r a t i o n o f T E L ( 4 5 - 7% vs. 51 +- 6% f o r control). Simi-

A l t h o u g h a l t e r a t i o n s in b a s a l a d e n y l a t e c y c l a s e activity did n o t o c c u r following s y s t e m i c a d m i n i s t r a t i o n o f T E L , this w a s n o t t h e c a s e w h e n striatal h o m o g e n a t e s w e r e e x p o s e d to T E L in vitro. I n c u b a t i o n w i t h T E L (1 to 3 0 / ~ M ) c a u s e d a m a r k e d a t t e n u a t i o n o f b a s a l e n z y m e a c t i v i t y (Fig. 4), as well as a n a t t e n u a t i o n o f t h e ability o f D A to s t i m u l a t e the synthesis of cAMP. DISCUSSION W a l s h et al. [40] h a v e r e p o r t e d t h a t a d m i n i s t r a t i o n o f T E L c a u s e d a m a r k e d b e h a v i o r a l s u p e r s e n s i t i v i t y to challenge w i t h t h e d o p a m i m e t i c c o m p o u n d s a m p h e t a m i n e a n d a p o m o r p h i n e . In t h e c a s e o f a m p h e t a m i n e , this s u p e r s e n sitivity w a s n o t a t t r i b u t a b l e to d i f f e r e n c e s in u p t a k e or distrib u t i o n o f a m p h e t a m i n e in the b r a i n [40]. T h u s , t h e p r e s e n t

TRIETHYLLEAD AND MONOAMINES

240

~

Vehicle

(N=12)

283 12

TEL (IN=I I)

e

220 2O0

10

180

9

160 E

>E

~

Veh,cle

(N=12)

T

TEL (N=11)

11

E

\\\ \\\ \\\

8

140 120

6

100

5

80

4

60

3

40

2

20

1

\\\ \\\ \\\ \ \ \\\ \ \ \ \ \ \ \\\ \\\

\ \ \ \

0

strlotum

strloturn

olf tub

FIG. 2. The effect of administration of 7.88 mg/kg TEL on the V m a x for dopamine-stimulated adenylate cyclase activities in striatum and olfactory tubercle at Day 7 posttreatment. Mean values for Vmax were derived from Eadie-Hofstee analysis on individual animals. *=Significantly different from vehicle, p<0.05.

2'° l 200

190 t80 170 160 E

50

"6 140 &"

30 12O 110 t O0

J

f

9O 8O

,tl

T

--6

,

,

,

--5 6

--5 2

~

--48

,

--4 4

FIG. 4. The effect of various concentrations of TEL in vitro on dopamine-stimulated formation of cyclic AMP in striatum at Day 7 posttreatment. ([]) veh, (+) 1/xM TEL, (O) 3 p.M TEL, (A) 10/zM TEL, (x) 30 p.M TEL.

studies sought to determine if the mechanism for the increased sensitivity in TEL-treated rats involved changes in dopamine receptor sensitivity, dopaminergic neurotransmission or alterations in modulating monoamines. A single dose of T E L did, in fact, cause subtle effects on dopaminergic and serotonergic neurotransmission at 7 days following treatment. The changes were primarily limited to alterations of dopamine-sensitive adenylate cyclase, the class of receptors known as D1 [19]. While perturbation of dopaminergic function was not evidenced by changes in the concentrations of DA and its metabolites in striatum, olfactory tubercle, nucleus accumbens and frontal cortex, or by alterations in number or affinity of D2 dopaminergic receptors in striatum or olfactory tubercle, concentrations of DA were decreased in hippocampus. The hippocampus receives a relatively small dopaminergic innervation from the ventral tegmental area [31], and autoradiographic studies have demonstrated the presence of both D~ and Dz dopamine re-

olf tub

FIG. 3. The effect of administration of 7.88 mg/kg TEL on the Km for dopamine-stimulated adenylate cyclase activities in striatum and olfactory tubercle at Day 7 posttreatment. Mean values for Km were derived from Eadie-Hofstee analysis on individual animals.

ceptors in this region [11, 3l, 33]. The functional significance of the decrease in DA in this area is unknown, but may be related to the vulnerability of the hippocampus to toxic insult induced by exposure to T E L [41]. A consistent finding was that DA-stimulated adenylate cyclase activity was altered in olfactory tubercle of TEL-intoxicated rats. Alterations in serotonergic function, exhibited either as a decrease in 5-HT in olfactory tubercle or as an increase in turnover as reflected by 5-HIAA/5-HT ratios [28,32] in nucleus accumbens, were also observed. Although measurement of concentrations of NE are not a reliable indicator of other than dramatic alterations in noradrenergic function [12], the lack of effects on concentrations of N E in frontal cortex and hippocampus, or on NE-stimulated adenylate cyclase activity in cerebellum, do not support the involvement of noradrenergic systems in the sequelae to intoxication with TEL. The primary effect on dopamine-stimulated adenylate cyclase was an increase of Vmax, resulting in increased cAMP synthesis under all conditions. The lack of change in basal adenylate cyclase activity following T E L intoxication is in marked contrast to the results obtained with T E L in vitro. While there was some diminution in the response of adenylate cyclase to dopamine, there was a striking inhibition of basal enzyme activity. These data suggest that at high concentrations in vitro, T E L probably affects the guanine nucleotide binding protein and/or the catalytic subunit of the adenylate cyclase complex, rather than only the dopamine recognition site. These data are not readily reconciled with the data of Wilson [42], in which T E L was reported to be a purely competitive inhibitor of dopamine-sensitive adenylate cyclase in striatal homogenates. Further, although the concentrations of inorganic lead in brain were clearly in the range of neurotoxicity, there were no indications of neurochemical or behavioral effects that could be attributable to inorganic lead intoxication (see [43]), suggesting that the lead measured in brain was present in the organolead form. This hypothesis is supported by pharmacokinetic data demonstrating that concentrations of T E L in whole body of rat were stable in vivo for one week after a single intravenous administration of T E L [4]. One might hypothesize from the present data that T E L is

284

DEHAVEN-HUDKINS, SCHULZ, WALSH AND MAILMAN

present in brain at micromolar levels one week following intraperitoneal administration. This is substantiated by our data on the concentrations of lead in various regions of brain at 7 days following a single dose of 7.88 mg/kg T E L [40]. Based on concentrations of lead in striatum of 4.36 /xg/g tissue, and in olfactory tubercle of 4.17/xg/g tissue, the concentrations of TEL chloride present in these regions at day 7 following a dose of 7.88 mg/kg would be approximately 30 /xM. Therefore, the concentrations found in vivo per unit tissue at day 7 following a single dose of 7.88 mg/kg are roughly equivalent to the concentrations used in the in vitro experiment. The lack of effect of T E L administration in vivo on basal activity of dopamine-stimulated adenylate cyclase suggests that most of the T E L is not available at the receptor-cyclase complex, and that the effects on adenylate cyclase following in vivo administration of T E L may result from indirect or polyneuronal events. Our data indicate that the behavioral supersensitivity exhibited by TEL-treated rats in response to dopaminergic agonists is more closely related to changes in the DA receptor that is linked to adenylate cyclase, or the D, receptor. Although behaviors such as amphetamine-induced locomotion and apomorphine-induced stereotypy were formerly thought to be mediated exclusively by D2 dopaminergic receptors (cf. [9,10]), recent evidence indicates that these behaviors can be blocked by SCH 23390, a drug that is a selective antagonist of the D, receptor [7,27]. Autoradiographic studies have demonstrated high concentrations of D, dopamine receptors in the olfactory tubercle [11,33]. Increases in locomotion caused by administration of amphetamine or apomorphine are principally the result of interactions with mesolimbic dopaminergic terminals [8]. Furthermore, agonists which are specific for the D, receptor have been shown to possess behavioral activating properties, which include enhancement of locomotion and certain stereotypies [ 1, 29, 30]. The behavioral supersensitivity that results from treatment with T E L may therefore arise as a result of the increased activity of DA-stimulated adenylate cyclase in mesolimbic regions such as the olfactory tubercle. It should be noted that since both the Vmax and the Km for dopamine-stimulated adenylate cyclase were elevated following treatment with TEL, any behavioral changes which may reflect this neurochemical alteration should only be expected in situations which would elevate synaptic concentrations of dopamine to near-maximal levels. This may explain the enhancement of amphetamine-induced locomotion

caused by TEL, as well as the increase in apparent antinociception following placement of the rat on a hot-plate (Walsh et al., unpublished data), a stressful behavioral challenge which may increase dopamine release, as has been reported previously for foot shock stress [13,38]. Conversely, treatment with T E L does not cause alterations in baseline locomotor activity or stereotypy [40], consistent with the prediction that behavioral hyperactivity should not occur when synaptic dopamine concentrations are at normal levels. In agreement with data reported by Hong et al. [17], where a daily dosing regimen of 1.75 mg/kg/day was used, enhanced serotonergic neurotransmission as reflected as an increase in the ratio of 5-HIAA/5-HT was observed in dopaminergic terminal fields. The suggested hyperactivity of serotonergic systems following treatment with T E L may also contribute to the effects of this compound on behavior resulting from administration of dopaminergic agonists. Lee and Geyer [23,24] have demonstrated that the effects of apomorphine on serotonergic neurons projecting from the dorsal raphe are mediated through DA autoreceptors, and several groups have demonstrated a functional relationship between serotonin and dopamine in striatum and mesolimbic areas [5, 6, 14, 39]. Therefore, the slight increase in turnover of serotonin may result from secondary effects on this system that originate from functional alterations in dopaminergic neurons that terminate in striatum and mesolimbic regions. Contrary to the report of Hong and coworkers [17], we did not observe serotonergic alterations in hippocampus. The reasons for this are unclear but may be related to the different dosing regimens used in the two studies. In summary, treatment with 7.88 mg/kg TEL results in an increase of dopamine-stimulated adenylate cyclase activity in olfactory tubercle. This functional alteration at D, dopamine receptors may be related to the behavioral supersensitivity to dopaminergic agonists that is a consequence of T E L neurotoxicity. The recent availability of [aH]SCH 23390 [3, 36, 37] as a probe of D, dopamine receptors will assist in further studies of this problem. ACKNOWLEDGEMENTS This work was supported by USPHS grants ES-01104 and HD03110, and training grants ES-07126 and MH-14277. We thank Eddie Stanford, Stan Southerland, Laura Staples and Ronnie McLamb for technical assistance.

REFERENCES

1. Arnt, J. ; Hyttel, J. Differential inhibition by dopamine D-1 and D-2 antagonists of circling behaviour induced by dopamine agonists in rats with unilateral 6-hydroxydopamine lesions. Ear. J. Pharmacol. 102:349-354; 1984. 2. Bennett, J. P.; Snyder, S. H. Serotonin and lysergic acid diethylamide binding in rat brain membranes: Relationship to postsynaptic serotonin receptors. Mol. Pharmacol. 12:373-389; 1976. 3. Billard, W.; Ruperto, V.; Crosby, G.; lorio, L. C.; Barnett, A. Characterization of the binding of [3H]-SCH 23390, a selective D-I receptor antagonist ligand, in rat striatum. Life Sci. 35:1885-1893; 1984. 4. Bolanowska, W. Distribution and excretion of triethyUead in rats. Br. J. Ind. Med. 25:203-208; 1968.

5. Breese, G. R.; Mueller, R. A.; Hollister, A.; Mailman, R. Importance of dopaminergic pathways and other neural systems to behavior and action of psychotropic drugs. Fed. Proc. 37:2429-2433; 1978. 6. Carter, C. J.; Pycock, C. J. The effects of 5,7-dihydroxytryptamine lesions of extrapyramidal and mesolimbic sites on spontaneous motor behaviour, and amphetamineinduced stereotypy. Naunyn Schmiedebergs Arch. Pharmacol. 308:51-54; 1979. 7. Christensen, A. V. ; Arnt, J. ; Hyttel, J.; Larsen, J. J. ; Svendsen, O. Pharmacological effects of a specific dopamine D-1 antagonist SCH 23390 in comparison with neuroleptics. Life Sci. 34:1529-1540; 1984.

TRIETHYLLEAD

AND MONOAMINES

8. Costall, B.; Naylor, R. J. The behavioural effects of dopamine applied intracerebrally to areas of the mesolimbic system. Eur. J. Pharmacol. 32:87-92; 1975. 9. Costentin, J. ; Dubuc, I. ; Protais, P. Behavioral data suggesting the plurality of central dopamine receptors. In: Mandel, P.; De Feudis, F. V., eds. CNS receptors: From molecular pharmacology to behavior. New York: Raven Press; 1983:289-297. 10. Creese, 1.; Left, S. E. Dopamine receptors in the central nervous system. Int. Rev. Neurobiol. 23:255-301; 1982. 11. Dawson, T. M.; Gehlert, D. R.; McCabe, R. T.; Barnett, A.; Wamsley, J. K. D-1 dopamine receptors in the rat brain: a quantitative autoradiographic analysis. J. Neurosci. 6:2352-2365; 1986. 12. DeHaven, D. L.; Mailman, R. B. The interactions of behavior and neurochemistry, in: Annau, Z., ed. Behavioral toxicology. Baltimore, MD: The Johns Hopkins University Press; 1986:214-243. 13. Fadda, F.; Argiolas, A.; Melis, M. R.; Tissari, A. H.; Onali, P. L.; Gessa, G. L. Stress-induced increase in 3,4dihydroxyphenylacetic acid (DOPAC) levels in the cerebral cortex and in N. accumbens: Reversal by diazepam. Life Sci. 23:2219-2224; 1978. 14. Giambalvo, C. T. ; Snodgrass, S. R. Biochemical and behavioral effects of serotonin neurotoxins on the nigrostriatal dopamine system: Comparison of injection sites. Brain Res. 152:555-566; 1978. 15. Glowinskl, J.; lversen, L. L. Regional studies of catecholamines in the rat braln--l. The disposition of [3H]norepinephrine, [~H]dopamine and [3H]dopa in various regions of the brain. J. Neurochem. 13:655-669; 1966. 16. Heffner, T. G.; Hartman, J. A.; Seiden, L. S. A rapid method for the regional dissection of the rat brain. Pharmacol. Biochem. Behav. 13:453-456; 1980. 17. Hong, J. S.; Tilson, H. A.; Hudson, P.; Ali, S. F.; Wilson, W. E.; Hunter, V. Correlation of neurochemical and behavioral effects of triethyl lead chloride in rats. Toxicol. Appl. Pharmacol. 69:471-479; 1983. 18. Johnson, R. A,; Walseth, T. F. The enzymatic preparation of [alpha-32-P]-ATP, [alpha-32-P]-GTP, and [32-P]-cAMP, and their use in the assay of adenylate and guanylate cyclases and cyclic nucleotide phosphodiesterases. Adv. Cyclic Nucleotide Res. 10:135-166; 1979. 19. Kebabian, J. W.; Calne, D. B. Multiple receptors for dopamine. Nature 277:93-96; 1979. 20. Keller, R.; Oke, A.; Meffbrd, l.; Adams, R. N. Liquid chromatographic analyses of catecholamines: Routine assay Ibr regional brain mapping. Life Sci. 19:995-1004; 1976. 21. Kilts, C. D.; Breese, G. R.; Mailman, R. B. Simultaneous quantification of dopamine, 5-hydroxytryptamine and four metabolically related compounds by means of reversed-phase high-performance liquid chromatography with electrochemical detection. J. Chromatogr. 225:347-357; 1981. 22. Komulainen, H.; Pietarinen, R.; Tuomisto, J. Increase in dopamine uptake in rat striatal synaptosomes after an acute in vivo administration of organic and inorganic lead. Acta Pharmacol. Toxicol. 52:381-389; 1983. 23. Lee, E. H. Y.; Geyer, M. A. Similarities of the effects of apomorphine and 3-PPP on serotonin neurons. Eur. J. Pharmacol. 94:297-303; 1983. 24. Lee, E. H. Y.; Geyer, M. A. Indirect effects of apomorphine on serotoninergic neurons in rats. Neuroscience 11:437-442; 1984.

285

25. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. 26. Mailman, R. B.; Krigman, M. R.; Frye, G. D. ; Hanin, I. Effects of postnatal trimethyltin or triethyltin treatment on CNS catecholamine, GABA, and acetylcholine systems in the rat. J. Neurochem. 40:1423-1429; 1983. 27. Mailman, R. B.; Schulz, D. W.; Lewis, M. H.; Staples, L.; Rollema, H.; DeHaven, D. L. SCH-23390: A selective D1 dopamine antagonist with potent D2 behavioral actions. Eur. J. Pharmacol. 101:159-160; 1984. 28. Melamed, E.; Hefti, F.; Wurtman, R. J. Tyrosine administration increases striatal dopamine release in rats with partial nigrostriatal lesions. Proc. Natl. Acad. Sci. USA 77:4305-4309; 1980. 29. Molloy, A. G.; Waddington, J. L. Dopaminergic behaviour stereospecifically promoted by the D1 agonist R-SK & F 38393 and selectively blocked by the D1 antagonist SCH 23390. Psychopharmacology (Berlin) 82:409-410; 1984. 30. Molloy, A. G.; Waddington, J. L. Sniffing, rearing and locomotor responses to the D-I dopamine agonist R-SK & F 38393 and to apomorphine: Differential interactions with the selective D-1 and D-2 antagonists SCH 23390 and metoclopramide. Eur. J. Pharmacol. 108:305-308; 1985. 31. Oades, R. D.; Halliday, G. M. Ventral tegmental (A10) system: neurobiology. 1. Anatomy and connectivity. Brain Res. Rev. 12:117-165; 1987. 32. Robinson, T. E.; Becker, J. B. The rotational behavior model: Asymmetry in the effects of unilateral 6-OHDA lesions of the substantia nigra. Brain Res. 264:127-131; 1983. 33. Savasta, M.; DuBois, A.; Scatton, B. Autoradiographic localization of D~ dopamine receptors in the rat brain with [3H]SCH 23390. Brain Res. 375:291-301; 1986. 34. Scatchard, G. The attractions of proteins for small molecules and ions. Ann. NY Acad. Sci. 51:660--672; 1949. 35. Schulz, D. W.; Mailman, R. B. An improved, automated adenylate cyclase assay utilizing preparative HPLC: Effects of phosphodiesterase inhibitors. J. Neurochem. 42:764-774; 1984. 36. Schulz, D. W.; Stanford, E. J.; Wyrick, S. D.; Mailman, R. B. Binding of [aH]SCH23390 in rat brain: Regional distribution and effects of assay conditions and GTP suggest interactions at a D,-like dopamine receptor. J. Neurochem. 45:1601-1611 ; 1985. 37. Schulz, D. W.; Wyrick, S. D.; Mailman, R. B. [aH]SCH23390 has the characteristics of a dopamine receptor ligand in the rat central nervous system. Eur. J. Pharmacol. 106:211-212; 1985. 38. Thierry, A. M. ; Tassin, J. P. ; Blanc, G. ; Glowinski, J. Selective activation of the mesocortical DA system by stress. Nature 263:242-243; 1976. 39. Waldmeier, P. C. Serotoninergic modulation of mesolimbic and frontal cortical dopamine neurons. Experientia 36:1092-1094; 1980. 40. Walsh, T. J.; Schulz, D. W.; Tilson, H. A.; DeHaven, D. L. Acute exposure to triethyl lead enhances the behavioral effects of dopaminergic agonists: Involvement of brain dopamine in organolead neurotoxicity. Brain Res. 363:222-229; 1986. 41. Walsh, T. J.; Tilson, H. A. Neurobehavioral toxicology of the organoleads. Neurotoxicology 5:67-86; 1984. 42. Wilson, W. E. Dopamine sensitive adenylate cyclase inactivation by organolead compounds. Neurotoxicology 3:100--107; 1982. 43. Winder, C.; Kitchen, 1. Lead neurotoxicity: A review of the biochemical, neurochemical and drug induced behavioural evidence. Prog. Neurobiol. 22:59-87; 1984.