Mating behavior and brain biogenic amine concentrations in chickens treated with parachlorophenylalanine (PCPA)

Physiology & Behavior, Vol. 32, pp. 603-607. Copyright©Pergamon Press Ltd., 1984.Printed in the U.S.A.

0031-9384/84$3.00 + .00

Mating Behavior and Brain Biogenic Amine Concentrations in Chickens Treated With Parachlorophenylalanine (PCPA) R. J. B A L A N D E R ,

S. J. B U R S I A N

Animal Science Department, 113 Anthony Hall, Michigan State University East Lansing, M I 48824

H. P. VAN K R E Y A N D P. B. S I E G E L Poultry Science Department, Virginia Polytechnic Institute and State University Blacksburg, VA 24061 R e c e i v e d 18 J a n u a r y 1982 BALANDER, R. J., S. J. BURSIAN, H. P. VAI~KREY AND P. B. SIEGEL. Mating behavior and brain biogenic amine concentrations in chickens treated with parachlorophenylalanine (PCPA). PHYSIOL BEHAV 32(4) 603-607, 1984.--Four experiments were conducted to determine the effect of parachlorophenylalanine (PCPA) on the male mating behavior of a line of chickens genetically selected for low mating frequency and to determine the effect on brain concentrations of 5-hydroxytryptamine, dopamine (DA) and norepinephrine (NE). Mating behavior was not affected by moderate levels (<64 mg/kg) of PqPA, and decreased significantly when higher levels (100 mg/kg) of the drugs were administered. Results suggest that elevated brain serotonin titers were not causing the low mating frequency associated with the selected line. Birds receiving higher levels of PCPA exhibited a lethargy which persisted for approximately ten days after withdrawal of the drug. Serotonin, DA, and NE were all significantly reduced in the brains of birds receiving PCPA (30 mg/kg). Mating behavior

PCPA

Parachlorophenylalanine

Chickens

THE neurotransmitter.serotonin is currently recognized as being inhibitory with respect to sexual behavior in several species of mammals. This view is partially a result of experiments utilizing parachlorphenylalanine (PCPA) which selectively and irreversibly inhibits tryptophan hydroxylase [16], the rate-limiting enzyme in the serotonin biosynthetic pathway. Thus, inhibition of tryptophan hydroxylase by PCPA results in depletion of neuronal serotonin stores. The administration of PCPA to male rats results in abnormally high levels of homosexual behavior [23,28]. PCPA also stimulates an increase in heterosexual behavior by increasing the percentage of animals that copulate with estrous females and/or by increasing the number of ejaculations per hour in males already exhibiting copulatory activity [18, 21, 29]. PCPA also causes significant increases in sexual behavior of rabbits [ll], which can become so extreme that treated males mount not only male and female rabbits, but other species of animals as well [26]. Furthermore, rabbits demonstrate this compulsive mounting behavior for three days or more after treatment with PCPA while rats lose their hypersexual drive in less than 24 hours.

Serotonin

The administration of PCPA to cats has had both positive and negative results with respect to increasing male sexual behavior. ,'Male cats given PCPA would not only compulsively mount anesthetized and passive male cats, but highly resistant toms as well [6,9]. Contrary to these results, PCPA was found to cause no change or even slight decreases in the sexual activity of some cats [3 l]. Results obtained with primates have been variable. A single large dose (400 mg/kg) of PCPA to male Rhesus monkeys increased sexual behavior, whereas chronic treatment had no effect (reviewed by Zitrin et al. [32]). In human studies concerned with the behavioral effects of PCPA, treated subjects did not report any feeling of increased libido, but complained of symptoms including tiredness, dizziness, nausea, uneasiness, headache, constipation and a tingling sensation of the skin [5]. Sicuteri et al. [24] "demonstrated that in patients complaining of migraine and sexual dysfunction, PCPA in combination with testosterone significantly increased sexual activity (measured by erections induced by erotic imagery)." In another study, PCPA had no effect on sexual behavior of healthy patients, nor did it have any therapeutic effect in sexually impotent patients [2].

~Michigan Agricultural Experiment Station Journal Article Number 11015.

603

604

BALANDER E i A i

Experiments investigating the effects of PCPA in birds are limited. PCPA significantly reduced whole brain serotonin levels in turkeys [8] and five day old chicks [22] whereas it significantly reduced both dopamine and serotonin levels in the brain of Coturnix quail [7]. The purpose of the experiments reported here was to investigate the role of serotonin in a line of chickens genetically selected for low mating activity, and to establish the effect of PCPA on several biogenic amines in the chicken brain. If high endogenous titers of serotonin were responsible for the relative lack of mating behavior in the low mating line males, the inhibitory action of the brain neurotransmitter in question could conceivably be diminished with PCPA therapy. METHOD

Three experiments were conducted to test the effect of PCPA on mating behavior of chickens. Experiment 1: The initial experiment utilized 30 low mating line males (LML) which had undergone 18 generations of selection (Sis) for a low cumulative number of completed matings [25] and 30 males from the Athens-Canadian Randombred (AC) control population. The LML chickens originated from the latter line. The males selected for experimentation were segregated from the large flocks and placed into individual floor pens (1.2×2.4 meters) several days prior to experimentation. The birds had been tested for sexual behavior approximately 6 weeks earlier, when 28-31 weeks of age. The mean number of completed matings at that time was 2.38 and 5.11 for the LML and AC lines, respectively (population means). Of the 30 birds from each line, 15 were treated with PCPA and 15 served as controls, The PCPA was prepared fresh daily as a suspension in 0.9% saline (30 mg/cc) to which 2-3 drops of polysorbate (Tween 80) had been added. The PCPA was injected intraperitoneally daily at a dose level of 100 mg/kg body weight. Control birds received intraperitoneal injections of saline containing Tween 80. Injections were given daily between 0900 and 1000 hours. The injection schedule was started four days prior to the mating trials and continued through the five days of testing for mating behavior (nine days total). Experiment 2: LML males also from the S~s generation were used in this experiment. The males were approximately 14 months of age when tested and were not the same birds used in the initial experiment. These males had also been tested for mating activity at 28-31 weeks of age and had not shown any mating activity at that time. Four different dosages of PCPA (0, 16, 32, 64 mg/kg body weight) were randomly assigned to 24 birds (6 birds/treatment). The birds received an injection daily for five consecutive days. Mating trials were conducted on alternate days following cessation of the injections such that a total of five trials were conducted over a ten-day period (no injections of PCPA were made during these ten days). Experiment 3:S~9 LML males, 14 months of age, were utilized to test four levels of PCPA (0, 30, 35, 40 mg/kg body weight). Other experimental procedures were as for Experiment 2, except seven birds per treatment were utilized in this experiment, and mating trials were run for five consecutive days following cessation of injections. Mating trials for Experiment 1 consisted of introducing a male singly for a period of 10 minutes into a 1.2×2,4 m pen in which a freshly sacrificed hen was positioned and restrained in a receptive posti~re [1]. Males were tested once daily and

TABLE t THE CUMULATIVE NUMBER OF COMPLETED MATINGS OF LOW MATING L I N E (LML) AND ATHENS-CANADIAN RANDOMBRED ~AC) COCKS T R E A T E D WITH P A R A C H L O R O P H E N Y L A L A N | N E (PCPA) OR SALINE Line

PCPA

S~lline

LML*

0~

2 ~'

ACt

2.7"

7.9 ~,

E x p e r i m e n t 1.

*Median values (analysis by Mann-Whitney-U Test). t Untransformed means (ANOVAon square root transformation). Any values in the same row with different superscripts are significantly different (p~<0.05). SPCPA dosage 100 mg/kg body weight.

the cumulative number of completed matings (CNCM) for the five trials was totaled. Mating trials for Experiments 2 and 3 were similar except each mating pen contained 6-8 receptive hens instead of a model. The LML data of Experiment 1 were analyzed by the Mann-Whitney-U Test because variances for the CNCMs of the saline and PCPA-treated brids were heterogenous and square root transformations did not eliminate the heterogeneity. The variances of the CNCMs for the PCPA and saline-treated AC birds were also heterogeneous, but the square root transformations of the data were homogenous and the transformed data were analyzed by analysis of variance. Mating data of Experiments 2 and 3 were analyzed by Chi-square analyses looking at two categories of maters versus non-maters. Experiment 4: A separate group of males was utilized to determine the effects of PCPA on serotonin (5HT), dopamine (DA), and norepinephrine (NE) in five brain areas. Five birds received daily injections of PCPA (30 mg/kg) for five consecutive days. Control brids received saline injections daily during the same time period. All birds were sacrificed on day five approximately one hour after the last injection. Brains were quickly excised and divided into the following parts: cerebellum, cerebral lobes, hypothalamus. midbrain, and medulla. Brain parts were frozen on dry ice and held at -20°C until assayed. The method of Jacobowitz and Richardson [15] was used for analysis of the monoamines. Data were analyzed by analysis of variance. RESULTS AND DISCUSSION

In Experiment 1, the PCPA-treated males mated significantly less than those receiving saline (Table 1). The response was, however, thought to be pharmacological in that males receiving PCPA developed a distinct lethargic condition as the experiment progressed. The lethargy was particularly evident when males were caught from the floor pens to administer the drug or for transport to the mating pens. The level of PCPA utilized in Experiment 1 (100 mg/kg body weight) was predicted upon what had been defined as an effective dose in mammalian sexual behavior studies. However, because of the lethargy, PCPA dose levels were reduced and graded in Experiment 2. It was for this reason also that injections of the drug were discontinued during the mating trials, and the trials were extended over a period of

MATING BEHAVIOR AND PCPA

605

TABLE 2

TABLE 3

THE NUMBER OF LOW MATING LINE (LML) COCKS MATING OR NOT MATING AFTER TREATMENT WITH VARYING LEVELS OF PARACHLOROPHENYLALANINE (PCPA)

THE NUMBER OF LOW MATING LINE (LML) COCKS MATING OR NOT MATING AFTER TREATMENT WITH VARYING LEVELS OF PARACHLOROPHENYLALAN INE (PCPA)

Number of Males

Number of Males Dosage of PCPA (mg/kg) 0 16 32 64 Total

Mating

Not Mating

Total

0 1 4 1 6

6 5 2 5 18

6 6 6 6

E x p e r i m e n t 2. X2(3)=7.21 (Not significant, 0.05).

ten days. The latter was to insure that a subtle narcoleptic effect was not precluding the expression of sexual behavior. The lethargy noted in Experiment 1 was less evident in Experiment 2. The males receiving the highest dose level (64 mg/kg) were relatively easier to capture, than the birds receiving lesser quantities of PCPA and exhibited what appeared to be normal avoidance actions. Despite these changes in experimental protocol, PCPA had no significant effect on copulatory behavior (Table 2). Nevertheless, the 32 mg/kg dose level appeared promising; four of the six males successfully completed matings, whereas none of these males had mated in earlier trials. Because of the promising results obtained with the 32 mg/kg dose level in Experiment 2, PCPA treatment was partitioned further in Experiment 3, As in the initial two experiments, however, PCPA therapy did not stimulate hypersexuality in the LML males (Table 3), nor was the lethargy noted in the earlier experiments evident in this experiment. The results of these three experiments are in sharp contrast to published results obtained with mammalian species where PCPA administration generally resulted in significant increases in sexual behavior. No other reports have been found to exist concerning PCPA and sexual behavior in avian species, making it difficult to discuss the findings on a relative basis. Whalen and Luttge [30], in discussing the PCPA-induced homosexual activity of male rats, suggested that the drug worked not by enhancing sexual motivation, but rather by altering the male's ability to adequately distinguish appropriate sexual partners. In the results reported here, both live hens (Experiments 2 and 3) and a female model (Experiment 1) were used to elicit sexual responses. The female model was utilized because it had been shown to significantly increase copulatory behavior of high and low mating males when compared to live hens [1]. Thus, it is doubtful that perceptual disorientation would account for the low responsiveness of PCPA that was noted in Experiment 1. As indicated, a PCPA-related listlessness was detected in some of the birds. It is difficult to state the cause of the lethargic condition, but it is possible that chronic PCPA administration and its resulting effects on monoamine concentrations (Experiment 4) precipitated a somnolent condition similar to that reported for humans [5]. It is interesting to note that in birds of the second experi-

Dosage of PCPA (mg/kg)

Mating

Not Mating

Total

0 30 35 40

1 0 1 0

6 7 6 7

7 7 7 7

Total

2

26

Experiment 3. ~(3)=2.15 (Not significant, 0.05).

ment receiving PCPA at a dose level of 64 mg/kg, the mild lethargy persisted through the ten days of mating trials, a time during which the drug was not being administered. In an earlier study [14] a single dose of PCPA (300 mg/kg) to rats entered the brain rapidly, but declined markedly after only one day. Tryptophan hydroxylase levels, however, remained undetected for four days and slowly returned to normal levels during the following week. Serotonin levels approximated the tryptophan hydroxylase levels. It is quite possible that the short period of chronic PCPA administration in this study had a similar long term effect on brain serotonin. The data of Experiment 4 show PCPA significantly decreased serotonin levels in four of five brain areas (Table 4). This is in general agreement with other avian reports [7, 8, 22] where whole brain analysis was utilized, and the plethora of reports of 5HT depletion by PCPA in mammalian species. Our data for DA are also in general agreement with that for the quail [7], but contrast for NE. While no change was observed in four brain areas, there was a significant decrease in NE in treated birds for the cerebral hemispheres. Two possible explanations are evident. First, that by utilizing whole brains for analysis (in the quail) there was a diluting effect which masked area differences that may have existed or, the quail and the chicken respond differently to PCPA with respect to NE. The role of DA in the regulation of male sexual behavior has been variable. While many reports indicate it to be stimulatory [10, 11, 17, 27] some reports indicate no change or even a decrease in libido [12,13]. These differences may be attributable to differencs in dgsage levels of L-DOPA and pre-treatment of the test animals. L-DOPA, the immediate precursor of DA, has been reported to have mild aphrodisiac effects in humans [3, 4, 19, 20]. These studies have all involved either Parkinsonian patients or sexually impotent males. As a result, the researchers point out that the increased libido may be the result of an overall general improvement rather than a direct result of L-DOPA. The role of DA or its action, in conjunction with serotonin, on sexual behavior in avian species is yet to be determined. Brain DA levels did not decrease after a 316 mg/kg injection of PCPA into rats [16], but DA levels were significantly reduced in Coturnix quail [7] and in two areas of the brain in chickens in this study. Norepinephrine levels were not affected in the two studies just cited, but were signifi-

606

B A L A N D E R t:i1 A I.. TABLE 4 MEAN -+ STANDARD ERROR FOR SEROTONIN, NOREPINEPHRINE, AND DOPAMINE CONCENTRATIONS (ng/g TISSUE) IN SELECTED BRAIN AREAS OF COCKS TREATED WITH PARACHLOROPHENYLALANINE (PCPA) (30 mg/kg) OR SALINE (CONTROLS)

SEROTONIN PCPA Control NOREPINEPHRINE PCPA Control DOPAMINE PCPA Control

Midbrain

Hypothalamus

Cerebellum

Medulla

Cerebral hemispheres

318.2 -+39.1 ~

244.5 -+68.0~

51.6 ± 7.3 a

391.2 -+51.8 a

248.6 _+19.2"

772.0 -+36.8 ~'

734.3 -+99.3 h

59.6 + 8.0a

742.6 ±34.7 b

603.0 --_11.5 t'

72.8 ±10.4 a

149.3 ±37.6 a

38.4 _ 2.54a

56.4 _+ 3.6a

189.8 ± 6.4 a

55.8 ± 3.1 a

115.8 ±17.6 a

42.6 + 1.69a

57.4 ± 6.8 a

273.4 _+20.3h

184.6 _+20.6a 169.8 _+12.2 a

--t

134.4 ± 7.7 a* 183.8 _+20.8a

160.2 _26.9 a 153.4 ± 19.0a

293.4 _+24.2a 396.0 _+26.0t,

--*

Values for any brain area within a monoamine which have different superscripts are significantly different from each other (p~<0.05). *Values are significantly different at p~0.06. tSamples accidentally destroyed.

c a n t l y r e d u c e d in the c e r e b r a l h e m i s p h e r e s o f t h e c h i c k e n s utilized here. It h a s b e e n p r o p o s e d [ 11, 28, 29] t h a t a r e c i p r o c a l c o n t r o l m e c h a n i s m for s e x u a l b e h a v i o r e x i s t s in t h e rat, w i t h catacholamines being stimulatory and 5HT being inhibitory. I f this also is t h e c a s e in a v i a n s p e c i e s , t h e r e d u c t i o n s o b s e r v e d for D A a n d N E m a y a c c o u n t for t h e lack o f m a t i n g in

o u r e x p e r i m e n t s . F u r t h e r e x p l o r a t i o n in t h e i n t e r a c t i o n o f t h e s e b i o g e n i c a m i n e s is b e i n g p u r s u e d . ACKNOWLEDGEMENTS Dr. Curtis E. Askelson and Mr. Nathan Belcher, Pfizer, Inc., Groton, CT for the PCPA.

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607 24. Sicuteri, F., E. Del Bene and C. Fonda. Treatment of impotence and hypersexuality by changing 5-hdyroxytryptamine turnover. Agents Actions 5:504 (abstract), 1975. 25. Siegel, P. Genetics of behavior: Selection for mating ability in chickens. Genetics 52: 1269-1277, 1965. 26. Sjoerdsma, A., W. Lovenberg, K. Engelman, W. T. Carpenter, R. J. Wyatt and G. L. Gessa. Serotonin now: Clinical implications of inhibiting its synthesis with para-chlorophenylalanine. Ann Intern Med 73: 607-629, 1970. 27. Tagliamonte, A., W. Fratta, M. Del Fiacco and G. L. Gessa. Evidence that brain dopamine stimulates copulatory behavior in male rats. Riv Farmacol Terap 4: 177-181, 1973. 28. Tagliamonte, A., P. Tagliamonte, G. L. Gessa and B. B. Brodie. Compulsive sexual activity induced by p-chlorophenylalanine in normal and pineal-ectomized male rats. Science 166: 1433-1435, 1969. 29. Tagliamonte, A., P. Tagliamonte and G. L. Gessa. Reversal of pargyline-induced inhibition of sexual behavior in male rats by p-chiorophenylalanine. Nature 230: 244-245, 1971. 30. Whalen, R. E. and W. G. Luttge. P-chlorophenylalanine methyl ester: an aphrodisiac? Science 169: 1000-1001, 1970. 31. Zitrin, A., F. A. Beach, J. D. Barchas and W. C. Dement. Sexual behavior of male cats after administration of parachiorophenylalanine. Science 170: 868-870, 1970. 32. Zitrin, A., W. C. Dement and J. D. Barchas. Brain serotonin and male sexual behavior. In: Contemporary Sexual Behavior: Critical Issues in the 1970's, Chapter 16, edited by J. Zubin and J. Money. Baltimore, MD: Johns Hopkins University Press, 1973.