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Naunyn-Schmiedeberg's Arch. Pharmacol. 305, 143-148 (1978)

Pharmacology 9 by Springer-Verlag1978

Effect of p-Chlorophenylalanine on Cerebral Serotonin Binding, Serotonin Concentration and Motor Activity in the Rat P. Steigrad, I. Tobler, P. G. Waser, and A. A. Borb~ly Pharmakologisches Institut der Universitfit Ziirich, Gloriastrasse 32, CH-8006 Ziirich, Switzerland

Summary. The effect of p-chlorophenylalanine (PCPA) on the specific binding of 3H-serotonin (5-HT) was investigated in crude membrane preparations of the rat forebrain. The level of cerebral 5-HT and 5-hydroxyindoleacetic acid were concomitantly determined. A single dose of PCPA (300 mg/kg i.p.) increased specific binding at a low concentration of aH-5-HT within 24 h by more than 100 %. The timecourse of the elevation of specific binding was mirrored by the reduction in the 5-HT level. However, 8 - 1 0 days after drug administration, specific binding was close to the control level, while 5-HT was still significantly reduced. A Scatchard analysis revealed that the apparent affinity was significantly increased (kD reduced) during the initial period after PCPA administration, whereas the number of binding sites (BM,x) was little changed. In contrast, after repeated injections of PCPA (3 injections of 300 mg/kg at 6 - 8 day intervals, or 100mg/kg/day for up to 12 days) leading to a prolonged 5-HT depletion, BMax was significantly increased, while k, tended to be restored towards the baseline level. Daily injections of PCPA (100 mg/kg) for 12 days resulted in a transitory increase in day-time motor activity with a maximum on drug day 3, and in a persistent reduction of night-time activity. It is concluded that the initial PCPA-induced decrease in kD was due to a reduced competition for binding sites by the endogenous 5-HT, whereas the increase of BMax after a prolonged 5-HT depletion may reflect a 5-HT receptor supersensitivity. Key words: Serotonin - Serotonin receptor p-Chlorophenylalanine - Motor activity.

-

Introduction Since the discovery by Koe and Weissman (1966) that p-chlorophenylalanine (PCPA) inhibits serotonin (5Send offprint requests to A.A. Borb~ly at the above address

HT) synthesis, many investigators have used this compound to study the relationship between the serotonergic system and behavior. PCPA-induced insomnia (Jouvet, 1972), aggressivity (Sheard, 1969), hypersexuality (Tagliamonte et al., 1969) and altered patterns of motor activity (Borb61y et al., 1973), have been attributed to the depletion of 5-HT in the brain. However, in various experiments with single or repeated doses of PCPA, a lack of temporal association between the cerebral 5-HT level and behavioral parameters such as sleep (Dement et al., 1972) or feeding and motor activity (Marsden and Curzon, 19"/6) has been observed. We considered therefore the possibility that the behavioral actions of PCPA might be more closely related to changes in 5-HT receptor sensitivity than to alterations in the 5-HT level, since the' latter may partly reflect a non-functional 5-HT pool. Altered sensitivity of catecholamine receptors has been repeatedly observed as a consequence of a prolonged treatment with agonists or antagonists (Burt et al., 1977; Banerjee et al., 1977, 1978), and could be even related to specific behavioral changes (Creese et al., 1977). However, chronic treatment with either an amino acid precursor, an uptake inhibitor or an antagonist of 5-HT failed to modify the specific 3H-5HT binding (Wirz-Justice et al., 1978). Similarly, no evidence for behavioral supersensitivity to a 5-HT precursor could be obtained after chronic administration of a 5-HT antagonist (Trulson et al., 1977). Increased specific 3H-5-HT binding has been observed only after administration of PCPA or reserpine (Bennett and Snyder, 1976). In analogy with the results obtained in raphe-lesioned animals, the authors interpreted their finding in terms of reduced competition for the receptors by endogenous 5-HT, and not as an increase in the number of binding sites. However, they did not support their hypothesis by presenting a Scatchard analysis of the data, nor did they investigate the timecourse of the changes. We decided therefore to measure both specific 3H-5-HT binding and 5-HT

0028-1298/78/0305/0143/$01.20

144 c o n c e n t r a t i o n s a f t e r single a n d r e p e a t e d d o s e s o f P C P A , a n d to a t t e m p t to r e l a t e t h e d a t a o f t h e m u l t i p l e d o s e s c h e d u l e to t h e effects o n m o t o r activity. M o r e o v e r , by s u b j e c t i n g o u r d a t a to a S c a t c h a r d analysis, we h o p e d to g a i n i n s i g h t i n t o t h e m e c h a n i s m s i n v o l v e d in t h e c h a n g e s o f 5 - H T b i n d i n g . W h i l e this m a n u s c r i p t was b e i n g p r e p a r e d , F i l l i o n a n d c o w o r k e r s (1978) r e p o r t e d t h e e x i s t e n c e o f t w o 3H5 - H T b i n d i n g sites t h a t a r e s e p a r a b l e b y s u b c e l l u l a r f r a c t i o n a t i o n . S i n c e in a c c o r d a n c e w i t h t h e s t u d y b y B e n n e t t a n d S n y d e r (1976) o n l y a single b i n d i n g site w a s a p p a r e n t in o u r c r u d e m e m b r a n e p r e p a r a t i o n , we d i d n o t a t t e m p t to i n t e r p r e t o u r results in r e l a t i o n to these n e w f i n d i n g s .

Naunyn-Schmiedeberg's Arch. Pharmacol. 305 (1978)

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Materials and Methods

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Adult male Sprague-Dawleyqvanovas rats (SIV 50 strain) weighing ca. 300 g were used. They were housed in transparent plastic cages (usually 2 animals per cage) and had free access to food and water. Temperature was maintained at 22~ and was controlled within a range of ___0.5 ~C. The animals were exposed to a 12 h light- 12 h dark cycle (dark from 0.00 to 12.00 h).

Experimental Procedure ExperimentL Forty-one animals were injected intraperitoneally with p-chlorophenylalanine-methylester (PCPA; from Sigma, St. Louis) at a dose of 376 mg per kg body weight (corresponding to 300 mg per kg PCPA acid; all doses in the text are expressed as acid). Uninjected animals were used as controls. The rats were killed at various time points after drug administration (see Fig. 1) for determination of forebrain 3H-serotonin (3H-5-HT) binding and serotonin (5-HT) concentrations. Specific binding of increasing concentrations of 3H5-HT was assayed in animals killed 2 days after PCPA-injection. The data were subjected to Scatchard analysis (Table 2).

ExperimentlL Twenty-four rats were divided in two equal groups and injected i.p. with 300 mg/kg PCPA. Both groups received a second injection of either PCPA (300 mg/kg) or saline 8 days after th e first injection. One group was killed after this second injection while the other received a third injection of either PCPA (300mg/kg) or saline 6 days after the second injection. Two additional groups of 4 rats received 2 or 3 saline injections respectively and served as drugfree controls. All animals were killed 2 days after the last injection for determination of 3H-5-HT binding, 5-HT and 5-hydroxyindoleacetic (5-HIAA) forebrain concentrations. Scatchard analysis was performed on the data of 4 rats that had received 3 injections of PCPA.

Experiment 11I. Five rats were kept individually in transparent plexiglas cages and housed in a separate room. The experimental room was entered daily at 11.30 h (30 min before light onset) when the animals were weighed and injected, and their food and water containers replenished. All manipulations were performed under dim red light. On days 1 - 3 the animals were injected with saline and on day 4 - 1 5 with PCPA (100mg/kg/day i.p.). The motor activity of these rats was continuously recorded by a force recorder under the cage (see Ruedin et al., 1978; Neuhaus and Borb61y, 1978). The recording device consisted of a platform resting on springs, with moving coils serving as mechano-electrical transducers. The vertical component of force changes induced corresponding voltage changes which were amplified and rectified, and then served as an input to an

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Fig. 1. Specific 3H-serotonin binding (upper curve) and serotonin concentration in rat forebrain (lower curve) determined at various timepoints after injection of p-chlorophenylalanine (PCPA; 300mg/kg i.p.). The points represent arithmetic mean values ( + SEM; 4 - 7 animals) and are expressed as a percentage of the daily control values that were determined concomitantly with the PCPA values. The absolute control values for specific 3H-5-HT binding at 2.5 nM are 1.4 + 0.10 (SEM) pmoles/g, and for the 5-HT-level 3.0 + 0.16 nmol/g (N= 7), Note the logarithmic time scale

integrator. The integrated signal of the motor activity was passed at 10-s intervals through an analog-to-digital converter and stored as a 7-bit word on paper tape. The information was then entered in an offline stage into a PDP 11/20 computer and analysed. Twelve additional animals (2 animals per cage) were kept under identical conditions without recording motor activity, and were injected with PCPA (100 mg/kg/day) for 3,6 or 9 days (4 animals per group). All animals were killed 24h after the last injection for determination of 3H-5-HT binding (Scatchard analysis), 5-HT and 5HIAA forebrain concentration.

Chemical Determination After killing the animal by decapitation, the brain was removed and dissected on an iced plateform. The right forebrain was stored at - 20 ~C until the 5-HT and 5-HIAA determination. The left forebrain was homogenized using a Potter-S (Braun Melsungen, Type 853 202) homogenizer, in 15ml ice-cold 0.05M Tris/HC1 buffer (pH7.7 at 21~ and centrifuged at 12,000g for 10rain. The pellet was rehomogenized in the same buffer, and centrifuged as before. This pellet was resuspended in I ml Tris/HC1 buffer (pH7.2 at 21~ containing 0.1 ~ 1-ascorbic acid, 101xM pargyline, 120raM NaC1, 2raM CaClz, 5mM KC1 and I mM MgCI2) per 17.brag original wet weight (corresponding to 1 mg protein), incubated for 10 min at 37 ~C, and then chilled on ice. 3H-5-HT (13.8 Ci.mmol-a experiment I and II, 15.2 Ci-mmol- 1 experiment III) binding was determined at 21 ~C by combining 0.90 ml of the above particulate suspension, with 3H-5HT-creatinine sulfate in 0.6ml Tris/HCl buffer (pH7.2). For Scatchard analysis concentrations of 3H-5-HT of 2 nM, 5 nM, 10 nM and 20 nM were used, for single binding points concentrations of 2.5 - 5 nM. The reaction mixture was incubated for 30 rain, cooled on ice for a h, and then filtered under reduced pressure through

P. Steigrad et al. : PCPA and 5-HT Binding

145

Table 1. Effects of p-chlorophenylalanine (PCPA) on specific 3H-serotonin (3H-5-HT) binding and concentration of serotonin (5-HT) and 5hydroxyindoleacetic acid (5-HIAA). Experiment 11: Intervals between 1st and 2nd, and 2nd and 3rd injections were 8 and 6 days, respectively; determinations were made 2 days after last injection. Experiment 111: Determinations were made 1 day after last injection. Mean values in % of control .+ SEM, N = number of animals

Sequence of injections

Experiment H (PCPA 300 mg/kg)

Experiment 111 (PCPA 100mg/kg/day)

N

3H-5-HT binding % control

5-HT

5-HIAA

% control

% control

Control A a (2 x NAG1) 1 x PCPA + NaC1 2 x PCPA

6

100 .+ 4.1

100 .+ 6.5

100 .+ 11.6

6 6

107 .+ 6.9 162 _+ 10.2

62 .+ 3.1 29 -+ 4.4

69 .+ 4,1 43 -+ 9.8

Contol B" (3 x NaC1) 2 x PCPA + NaC1 3 xPCPA

6 6 6

100 + 3.9 107 + 9.0 166+18.0

100 + 3.8 65 -+ 3,0 34.+6,1

100-+ 5.4 76 _+ 5.3 20.+ 1,3

ControP 3 x PCPA 6 x PCPA 9 x PCPA 12 x PCPA

8 4 4 4 5

100 +_ 3.1 218 + 16.7 219 +_ 10.4 192 _+20.2 196 +_ 13.4

100 + 3,1 18 -+ 4.8 11 _+0.7 18 +_ 1.8 18 + 2.8

100 _+ 7.2 25 + 9.3 60 _+ 14.2 10 +_ 1.7 22 _+ 7.4

absolute control values: Experiment H (Specific binding determined at 3 nM 3H-5-HT); Control A: specific 3H-5-HT binding: 2.8 + 0.12pmoles/g; 5-HT: 2.9 + 0.19 nmoles/g; 5-HIAA: 2.9 _+ 0.32nmoles/g. ControlB: specific 3H-5-HT binding: 2.6 ___ 0.11 pmoles/g; 5-HT: 2.98 ___ 0.11 nmoles/g; 5-HIAA: 3.0 +_ 0.16nmoles/g. Experiment 11I(specific binding determined at 5nM 3H-5-HT); specific 3H-5-HT binding: 4.1 ___0.13 pmoles/g; 5-HT: 4.2 + 0.90 nmoles/g; 5-HIAA: 3.1 +_ 0.14nmoles/g

Whatman glass fibre filters (GF/B). The filters were rinsed twice with 5 ml ice-cold Tris/HC1 buffer (pH 7.7) to remove most of the unbound radioactive ligand. The filter paper was placed in a counting vial containing 10 ml Insta Gel Packard and counted in a Packard Liquid Scintillation Spectrometer (Model 3375) at 37.7~ efficiency. Corrections were made for nonspecific accumulation by assaying parallel incubations which contained a large excess (50/aM) of 5-HTcreatinine sulfate. Specific binding, defined as the difference between total and nonspecific radioactivity was 3 0 - 80 % of the total binding. Each determination was made in quadruplicate. Brain proteins were determined by the method of Lowry (1951). 5-HT and 5-HIAA forebrain levels were determined after butanolheptane extraction according to a slightly modified fluorometric method of Curzon and Green (1970). Each sample was processed in duplicate. Flourescence was read on a Farrand Spectrofluorometer with excitation wavelength set at 360 nm and emission wave-length at 470 rim.

Results

Figure 1 shows the timecourse of specific 3H-5-HT binding and 5-HT-concentration after administration of a single dose of PCPA (300mg/kg; Experiment I). Within the first day 5-HT binding increased to more than 220 % of the control level, while the 5-HT-level decreased to approximately 30% of control. 5-HTconcentration continued to decrease to a minimum on day 3, whereas the binding values showed a slight trend towards the baseline level after the maximum had been reached on day 1. A discrepancy between the changes of

the two parameters was evident on day 8 when the value for specific binding (124 %) did not differ significantly from the control level, whereas the 5-HT-level (47 %) was still significantly lower (P < 0.001). By day 19 the 5HT-level (86 %) no longer differed significantly from control. Since both 5-HT binding and concentration were determined in the same animal, Spearman's correlation coefficient was computed for each time period. A high negative correlation between the binding values and the 5-HT-concentrations was seen for the determinations at 6 h (r = - 0 . 9 2 ) , 1 2 h (r = -0.94) and 24h (r = -0.87). Due to the larger number of animals, only the latter value reached the 0.05 significance level. However, the combined values from 6 - 2 4 h yielded a highly significant r ( - 0.88 ; P < 0.001). The correlation coefficients for the other timepoints, though with one exception all negative, were smaller and not significant

(P> 0.1). In Experiment II we administered PCPA (300 mg/kg) repeatedly to further investigate the dissociation between the changes of binding and 5-HTconcentration, as well as to check for a development of tolerance. In accordance with the findings obtained in Experiment I, the specific binding was no longer significantly elevated 10 days or 8 days after the last injection of PCPA, whereas the levels of 5 HT and 5-HIAA were still reduced (Table 1). The increase in specific binding

146

Naunyn-Schmiedeberg's Arch. Pharmacol. 305 (1978)

Table 2

Effects of p-chlorophenylalanine (PCPA) on equilibrium dissociation constants (kD) and maximal number of binding sites ( B ~ ) (estimated from Scatchard plots). The concentrations used for Scatchard analysis were 2nM, 5nM, 10riM and 20nM. BM~xis expressed as pmoles per g wet weight. Mean values + S.E,M., N = number of animals

N Control 1 x PCPA 3 x PCPA 3 x PCPA 6 x PCPA 9 x PCPA 12 x PCPA

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BM~

nM

pmoles, g- ~

11.3 + 0.8 4.3 + 0.6*** 9.6 _+2.3 4.1 + 0.4*** 4.9 -+ 1.2*** 5.8 + 0.8** 6.4_+0.5***

14.1 -+ 0.9 14.4_+ 1.3 20.2 _+2.4* 16.1 -+ 1.6 17.5 + 2.7 16.4 _+ 1.2 18.4_+0.7"*

8 4 4 4 4 4 5

300mg/kg" 300 mg/kg b 100 rng/kg/day ~ 100 mg/kg/day ~ 100 mg/kg/day ~ lOOmg/kg/day ~

" Determination 2 days after injection (Experiment I) b Interval between injections 8 and 6 days; determination 2 days after last injection (Experiment II) Determination 1 day after last injection (Experiment III) * P < 0.02, ** P < 0.01, ***P < 0.001, two-sided t-test 160

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Fig. 2. Scatchard plots for control animals (N = 8) and for animals killed 1 day after daily injection of PCPA (100 mg/kg) for 12 days (/7 = 5). Points represent arithmetic mean values of determinations made for individual animals; regression lines are based on mean values. Note that mean values ofko and BM~ in Table 2 are derived from Scatchard plots for individual animals, and not from the present plots

Fig. 3. motor activity during 3 control days (saline injections) and 12 days with daily injections of p-chlorophenylalanine (100 mg/kg/day). Solid line: total daily motor activity; dashed line: activity during light-phase; dotted line: activity during dark-phase. Values represent arithmetic means with SEM. For each animal the 1007o level (indicated by a horizontal line) was defined as the mean value of the control days. Motor activity was measured in arbitrary units

2 days after either the second or third injection of PCPA was, however, lower than after the first injection (209 ~, Fig. 1 ; differences not significant). On the other hand, t h e r e w a s no clear evidence for the development of tolerance during daily injections of PCPA (Table 1). Thus throughout the drug period, the binding values were at approximately 200 ~ of control, while the 5-HT-level was depressed by more than 80 ~. Scatchard analysis revealed that the PCPA-induced increase in binding was mainly due to an increase in the apparent affinity (Table2). Thus a single administration as well as daily injections of PCPA caused a marked, significant decrease in the kD-value, but only a minor increase in the number of binding sites (BMax)-

When the kD-values for successive 3-day intervals are considered (100 mg/kg/day PCPA), an increasing trend towards the baseline level is evident. In fact, kD after 12 injections was significantly higher than kD after only 3 injections (P < 0.01 ; two-sided t-test). A recovery upon repeated administration of PCPA is suggested also by the results that kD measured after 3 administrations o f 300 mg/kg was not significantly different from control. On the other hand, Buax was significantly increased by 3 injections of the high dose as well as after 12 injections of the low dose (Fig. 2). The effects of daily injections of PCPA on motor activity are illustrated in Fig. 3. After a transitory reduction on the first drug day (day 4), day-time activity increased to a maximum on the third drug day

P. Steigrad et al. : PCPA and 5-HT Binding (day 6), and then decreased gradually towards the baseline level on the subsequent days. In contrast, night-time activity remained continuously reduced to approximately 80 % of control. Total daily activity was consequently also decreased with the exception of day 6 when the increase in day-time activity compensated the depression of activity at night. The mean body weight on the animals was practically the same during the control days (304.2g +_ 5.74 SEM) and the PCPAperiod (302.8 _+ 12.71 SEM).

Discussion

Single administration as well as daily injections of PCPA increased the specific 3H-5-HT binding by 8 0 120 %, while repeated injections at 6 - 8 day intervals caused an increase of 60 - 70 % (Fig. 1, Table 1). These results are consistent with the original observations of Bennett and Snyder (1976) that PCPA enhanced 3H-5HT binding in various parts of the rat brain. These authors suggested that '[t]he enhanced binding observed at short intervals after 5-HT depletion would seem to result from depletion of endogenous 5-HT bound to receptors rather than from a proliferation of "supersensitive" receptors, which should display a longer time course.' Three aspects of the present study support the assumption that, at least in the initial period following the administration of PCPA, the increase in specific binding is a direct consequence of endogenous 5-HT depletion: (1) The timecourse of the changes in binding was mirrored to a large extent by the changes in 5-HT level (Fig. 1); (2) a high negative correlation was seen between binding and level of 5-HT 6 - 2 4 h after drug administration; (3) the enhanced binding was mainly due to an increase in the apparent affinity, and not to an increase in the number of binding sites. All these observations are compatible with the interpretation that the PCPAinduced depletion of endogenous 5-HT leads to a reduced competition for binding sites and hence to an increase in specific binding at low concentrations of 3H5-HT. However, this simple scheme seems to be inadequate to account for the observations made after a prolonged depletion of 5-HT. Thus the specific binding measured 8 - 10 days after administration of PCPA was no longer significantly elevated, whereas the 5-HT level was still lowered to 4 7 - 65 % of control (Fig. 1, Table 1). This could be explained by assuming that a functional 5-HT pool influencing the binding sites is more rapidly restored than a non-functional pool that contributes substantially to the total 5-HT level. In addition, the hypothesis could be considered that the 5-

147 HT receptors gradually develop a supersensitivity as a compensatory response to the prolonged 5-HT depletion. This hypothesis finds support in the observation that the number of binding sites (BMa~)was significantly increased in those animals that suffered the longest 5HT depletion (Table 2, Fig. 2). Moreover, a prolonged 5-HT depletion seemed to result in a restoration of the apparent affinity (kD) towards the control level. It would seem therefore that two processes underlie the PCPA-induced increase in specific binding at low concentrations of 3H-5-HT: (1) a rapidly developing increase in the apparent affinity reflecting the reduced competition for binding sites by the endogenous 5-HT; and (2) a slowly developing increase in the number of binding sites in response to a prolonged 5-HT depletion. Further experiments are in progress to substantiate the PCPA-induced increase in B~ax which so far constitutes the only evidence for a receptor supersensitivity in the serotonergic system. The behavioral concomitants of the daily injections of PCPA consisted in a transitory enhancement of daytime motor activity, and a persistent depression of night-time activity. The former effect corresponds to that observed in previous studies after a single injection of PCPA (Borb61y et al., 1973; Zucker et al., 1976). However, it is clear from the present experiments that the transitory day-time hyperactivity cannot be directly related to the 5-HT concentration in the brain since the latter was persistently reduced throughout the recording period. Thus the observations in this study add to the list of PCPA-induced behavioral changes (sleep, activity, feeding) that are outlasted by cerebral 5-HT depletion (Bond et al., 1972; Dement et al., 1972; Marsden and Curzon, 1976). Either a rapidly restored, but hitherto unidentified, functional 5-HT pool, or a general 'neurochemical disbalance' following the rapid depletion of 5-HT, might account for the timecourse of these behavioral changes. On the other hand, the continuous reduction of night-time activity indicates that the chronic administration of PCPA may cause not only transitory, but also persistent alterations in behavior. The present experiments did not disclose a simple relationship between PCPA-induced alterations in the serotonergic system and in behavior. On the contrary, various components emerged at both the neurochemical and behavioral level, exhibiting changes in opposite directions and varying with different timecourses. However, in spite of the high complexity, it is imperative to study the consequences of prolonged alterations in brain transmitters, since they may provide insights into the mechanisms of action of drugs used in psychopharmacotherapy. The present results indicate that a gradual change in receptor sensitivity which has been found during long-term pharmacological actions

148

on the catecholaminergic systems (see Introduction for references), may occur also in the serotonergic system. Slow modifications at the receptor level in response to chronic drug action may constitute therefore a common feature among various transmitter systems, and thus provide a promising avenue in the search for the neurochemical basis of behavior. Acknowledgement. We thank Dr. Anna Wirz-Justice, Mr. K. Kr/iuchi, PD Dr. H. Fcer (Psychiatric University Clinic Basel) and PD Dr. M. Schaub for constructive criticism and the advice in methodological problems. Mr. H. U. Neuhaus provided valuable help with data analysis..The assistance of Gabriella Costantini and Angela Gloor is gratefully acknowledged. The study was supported by the Swiss National Science Foundation, grants nr. 3.624-0.75 and 3.254-0.77, and by the Hartmann-Miiller Stiftung ffir Medizinische Forschung.

References Banerjee, S. P., Kung, L. S., Riggi, S. J., Chanda, S. K. : Development of fl-adrenergic receptor subsensitivity by antidepressants. Nature 268, 455--456 (1977) Banerjce, S. P., Sharma, V. K., Kung, L. S., Chanda, S. K. : Amphetanaine induces fl-adrenergic receptor supersensitivity. Nature 271,380-381 (1978) Bennett, J. P., Jr., Snyder, S. H.: Serotonin and lysergic acid diethylamide binding in rat brain membranes: Relationship to postsynaptic serotonin receptors. Mol. Pharmacol 12, 373- 389 (1976) Bond, V. J., Shillito, E. E., Vogt, M.: Influence of age and testosterone on the response of male rats to parachlorophenylalanine. Br. J. Pharmacol. 46, 46-55 (1972) Borb~ly, A. A., Huston, J. P., Waser, P. G.: Physiological and behavioral effects of parachlorophenylalanine in the rat. Psychopharmacologia (Berlin) 31, 131-142 (1973) Butt, D. R., Creese, I., Snyder, S. H.: Anfischizophrenic drugs: Chronic treatment elevates dopamine receptor binding in brain. Science 196, 326-327 (1977) Creese, I., Burt, D. R., Snyder, S. H. : Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity. Science 197, 596-598 (1977) Curzon, G., Green, A. R. : Rapid method for determination of 5hydroxytryptamine and 5-hydroxyindoteacetic acid in small regions of rat brain. Br. J. Pharmacol. 39, 653-655 (1970)

Naunyn-Scluniedeberg's Arch. Pharmacol. 305 (1978) Dement, W. C., Mitler, M. M., Henriksen, S. J. : Sleep changes during chronic administration of parachlorophenylalanine. Rev. Can. Biol. 31, Suppl. 239-246 (1972) Fillion, G. M. B., Rousselle, J. C., Fillion, M. P., Beaudoin, D. M. Goiny, M. R., Deniau, J. M., Jacob, J. J.: High-affinity binding of [3H]-hydroxytryptamine to brain synaptosomal membranes: Comparison with [3Hl-lysergic acid diethylamide binding. Mol. Pharmacol. 14, 50-59 (1978) Jouvet, M. : The role of monoamines and acetylcholine-containing neurons in the regulation of the sleep-waking cycle. Ergebn. Physiol. 64, 166-307 (1972) Koe, B. E., Weissman, A.: Parachlorophenylalanine: a specific depictor of brain serotonin. J. Pharmacol. Exp. Ther. 154, 4 9 9 516 (1966) 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) Marsden, C. A., Curzon, G. : Studies on the behavioral effects of tryptophan and parachlorophenylalanine. Neuropharmacology 15, 165-171 (1976) Neuhaus, H. U., Borbbly, A. A.: Sleep telemetry in the rat. II. Automatic identification and recording of vigilance states. Electroenceph. Clin. Neurophysiol. 44, 115 - 119 (1978) Ruedin, P., Bisang, J., Waser, P. G., Borbdly, A. A. : Sleep telemetry in the rat: I. A miniaturized FM-AM transmitter for EEG and EMG. Electroenceph. Clin. Neurophysiol. 44, 112 - 114 (1978) Sheard, M. H. : The effect of parachlorophenylalanine on behaviour in rats: relation to brain serotonin and 5-hydroxyindoleacetic acid. Brain Res. 15, 524-528 (1969) Tagliamonte, A., Tagliamonte, P., Gessa, G., Brodie, B. : Compulsive sexual activity induced by parachlorophenylalanine in normal and pinealectomized male rats. Science 166, 1433-1435 (1969) Trulson, M. E., Stark, A. D., Jacobs, B. L. : Lack of supersensitivity to L-5-hydroxytryptophan following chronic methysergide treatment. J. Pharm. Pharmacol. 29, 382-383 (1977) Wirz-Justice, A., Kr~iuchi, K , Lichtsteiner, M., Feer, H.: Is it possible to modify serotonin receptor sensitivity? Life Sci (in press, 1978) Zucker, I., Rusak, B., King, R. G.: Neural bases for circadian rhythms in rodent behavior. In: Advances in psychobiology, Vol 3. (A. H. Riesen and R. F. Thompson, eds.), pp. 35 - 74. New York: Wiley 1976

Received July 21/Accepted September 20, 1978