Parachlorophenylalanine Retards Tail Regeneration in the Gekkonid Lizard Hemidactylus Flaviviridis Exposed to Continuous Light

A physiological role for serotonin (5-HT) in the regulation of gonadotrophin secretion in vertebrates has frequently been suggested (see Vitale et al. 1986). The distribution of serotonergic fibres in the median eminence (Villar et al. 1984) and their spatial relationship to luteinizing hormone-releasing hormone (LHRH) fibres (Jennes et al. 1982) provide neuroanatomical support for the conclusion that 5-HT can be involved physiologically in the release of LHRH from the median eminence through an action on axon terminals (Vitale et al. 1984).

The large number of studies supporting a neurohormonal role for 5-HT in the central nervous system accounts for the continuing interest in drugs capable of selectively depleting brain 5-HT, either by a selective release mechanism or by inhibiting 5-HT biosynthesis (see Costa et al. 1962a,b). p-CPA is reported to deplete the 5-HT stores in the brain, peripheral tissues and blood in rats and dogs. The 5-HT content of the brain, in particular, is reduced to very low levels, although brain norepinephrine and dopamine concentrations are only slightly decreased (Sloviter et al. 1978). The injection of p-CPA, an inhibitor of tryptophan hydroxylase (Koe & Weisman, 1966; Walker, 1982), is reported to increase luteinizing hormone (LH) levels and suppress prolactin (PRL) levels of broody turkeys, resulting in ovarian growth (El Halawani et al. 1983). Blockage of 5-HT synthesis by p-CPA completely inhibits the rise in PRL that is normally associated with the return of broody turkeys from cages to the nest (El Halawani et al. 1980). p-CPA, as well as the 5-HT antagonists methysergide, SQ10631 and cyptoheptadine, have been shown to decrease basal PRL levels in male chickens (Rabii et al. 1981).

There are reports indicating the influence of the pineal and PRL in the regeneration of amphibian appendages (see Maier & Singer, 1981). Our recent observations have shown that exogenous PRL improves tail regeneration in lizards exposed to continuous darkness (P. I. Ndukuba & A. V. Ramachandran, in preparation). The aim of the present investigation was to determine the effect, if any, on the regenerative performance of lizards exposed to continuous light with physically intact pineals, but deprived of their ability to synthesize 5-HT by the injection of p-CPA.

Adult Hemidactylus flaviviridis of both sexes weighing 10±lg (±S.D.) and measuring 80 ± 5 mm (±S.D., snout-vent length) were obtained from a commercial supplier (M/S Zoophyton, Baroda, India) and maintained on a diet of cockroaches ad libitum for a period 7 days prior to experimentation, for acclimation to the laboratory conditions. 30 lizards were used for the investigation, and they were divided into three groups of 10 lizards each and exposed to continuous light (24h:0h L:D) of 2500 lx intensity.

The first group of 10 lizards received a daily intraperitoneal injection of 200 μg kg⁻¹p-CPA (low dose) 5 days before and 30 days after tail autotomy. Food and water were provided ad libitum.

A second group of 10 lizards received daily intraperitoneal injection of 400 μg kg⁻¹p-CPA (high dose) 5 days before and 30 days after tail autotomy. Food and water were provided ad libitum.

The third group of 10 lizards, which served as the control, received a daily intraperitoneal injection of 0·6 % sterile saline 5 days before and 30 days after tail autotomy. Food and water were provided ad libitum.

Parachlorophenylalanine, p-CPA (Sigma chemical company, St Louis, USA), was dissolved in 0·6% (w/v) NaCl and brought to pH 6·0 by the addition of 5 mol I⁻¹ Na₂HPO₄·0·6g of reagent grade sodium chloride (NaCl) was dissolved in 100 ml of redistilled water and stored in a refrigerator for daily use.

All the experimental animals were exposed to continuous light of 2500 lx intensity. The cages housing the animals measured 46 cm × 38 cm × 25 cm with one side made of transparent glass and ventilation on three sides. Each cage housed 10 lizards, five males and five females, and the animals selected were of similar size to eliminate any possible error in the comparative analysis of the regeneration process due to sex and size differences. The three cages housing the animals were placed (glass surface up) under suspended 40 W fluorescent lamps, facing the source of illumination. The inside of the wooden cage was lined with aluminium foil so that lighting was direct as well as reflected. The distance from the fluorescent lamp to the glass surface of the cage was 38 cm and to the floor level 63 cm. The light intensity was measured at the floor level using a luxmeter (Weston Electrical Instrument Corporation, New Jersey, USA). To obtain the high light intensity of 2500 lx needed for the experiment, four fluorescent lamps were fixed and beamed together. We employed a high light intensity in this investigation because we have earlier demonstrated that the regeneration process is markedly enhanced by the length of photoillumination as well as its intensity (P. I. Ndukuba & A. V. Ramachandran, in preparation).

Tail autotomy was performed by pinching off the tail at the third segment from the vent. The length of tail removed from the animals varied from 50 to 60 mm. The length of new growth (regenerate), in mm, was measured daily with a meter rule and recorded at fixed intervals of 10, 15, 20, 25 and 30 days after caudal autotomy. The recorded readings were used later for morphometric calculations and Student’s t-tests were used in determining the statistical significance. This investigation was conducted during the summer month of May and the average daily temperature at the level of the animals was 30°C. Differences at the P < 0·05 level were considered to be statistically significant.

The regeneration blastema appeared in saline-treated animals and those treated with 200 μg kg⁻¹p-CPA by day 5 and in those injected with 400 μg kg⁻¹p-CPA by the tenth day after tail autotomy (Table 1). The high dose of p-CPA retarded the regeneration process more than the low dose. The total lengths of tail regenerated by day 30 in control lizards and lizards injected with 200 and 400 μg kg⁻¹p-CPA were 27·7mm, 26·3 mm and 13·2 mm, respectively, which corresponded to a replacement of 52·8 %, 50·5 % and 25·7 % (Figs 1,3). The pattern of growth rate (Fig. 2) indicates a linear increase up to 15–20 days in animals treated with 400 μg kg⁻¹p-CPA. The saline-injected lizards showed a biphasic growth rate curve, with the first phase lasting up to 10 days and the second occurring between 20 and 30 days, whereas the lizards treated with 200 μg kg⁻¹p-CPA did not show the second phase.

Comparisons (total length of tail regenerated and total percentage replacement) between the three groups of animals (Student’s t-test) revealed no statistically significant difference between the saline and 200 kg⁻¹p-CPA groups. However, comparisons between the control and 400μgkg⁻¹p-CPA groups and between 200 μg kg⁻¹p-CPA and 400 μg kg⁻¹p-CPA groups were statistically significant at the 5% level (Student’s t-test).

These results show that tail regeneration in the gekkonid lizard Hemidactylus flaviviridis was significantly retarded with daily intraperitoneal injection of 400 μg kg⁻¹p-CPA (high dose) but only insignificantly so with a low dose (200 μg kg⁻¹) of p-CPA (Table 1, Figs 1, 3). This finding demonstrates that in Hemidactylus the retardation effect of p-CPA is dose-dependent, with the high dose producing a marked effect. The mechanism of action of p-CPA in higher vertebrates has been demonstrated previously. p-CPA is a neutral amino acid and can compete with tyrosine for uptake into catecholamine neurones (Wurtman, 1975). It has been shown that p-CPA selectively decreases the concentration of 5-HT in the brain without altering the concentration of noradrenaline or dopamine. This selective action is probably effected by inhibition of the enzyme tryptophan hydroxylase (Koe & Weisman, 1966; Walker, 1982).

The perception of light provides important information for the organism about its environment. For this purpose, most animals possess well-developed photoreceptors and neuronal networks in the retina of the lateral eyes. Interestingly, even in species with highly organized ocular photoreceptors, additional photoreceptive structures - extraocular photoreceptors - are utilized in the transmission of photic information about the day-night schedule and seasonal photoperiodic changes. Considerable evidence supports the view that the pineal organ is the principal site of extraocular photoreception in lower vertebrates (see Meissl & Dodt, 1981). The pineal system (pineal organ and parietal eye) has been shown to be light-sensitive on the basis of neurophysiological and cytological evidence (Wurtman et al. 1968). Recent studies from our laboratory have demonstrated that continuous light stimulates tail regeneration in the lizard, H. flaviviridis, whereas continuous darkness depresses it (P. I. Ndukuba & A. V. Ramachandran, in preparation) and, further, that the lateral eyes, or retinae, do not participate in this photoperiodic response as blinded lizards regenerated their lost (automized) tails as effectively as did their sighted counterparts exposed to the same experimental photoperiodic conditions (Ndukuba & Ramachandran, 1988). It has been shown that the pineal organ is the principal site of extraretinal photoreception in Hemidactylus, since pinealectomy, as well as light deprivation to the pineal, abolished the stimulatory influence of continuous illumination and significantly retarded the regeneration process (Ramachandran & Ndukuba, 1988), and also tail regeneration was stimulated by exogenous PRL in lizards kept in continuous darkness (P. I. Ndukuba & A. V. Ramachandran, in preparation). The present report shows that the initiation of regeneration, the daily growth rate, the total length of new growth (regenerate) produced at the end of regeneration, and the p-CPA retards tail regeneration in Hemidactylus 241 total percentage replacement of the lost (autotomized) tails in lizards exposed to continuous light were all significantly retarded by a daily intraperitoneal injection of 400μgkg⁻¹p-CPA. The results obtained here were similar to those obtained earlier with pinealectomized lizards exposed to continuous illumination (see Figs 1,3; Ramachandran & Ndukuba, 1988).

PRL has been established as a growth promoter in developing organisms (Crim, 1975) and in regenerating systems (Maier & Singer, 1981; P. I. Ndukuba & A. V. Ramachandran, in preparation) and has been shown to stimulate protein synthesis in developing tadpoles (Yamaguchi & Yasumasu, 1977). Depletion of hypothalamic catecholamines by compounds that inhibit their synthesis resulted in a rise in serum PRL level (Donoso et al. 1971). In contrast, pharmacological procedures that enhance the amine levels in brain, the injection of monoamine oxidase inhibitors or L-dopamine, inhibit PRL release (Lu & Meites, 1971). In addition to the vast literature implicating dopamine in the control of PRL secretion, some studies suggest that 5-HT is a neurotransmitter involved in the stimulation of PRL release. Kamberi et al. (1971) induced PRL release by injecting 5-HT into the third ventricle, and Lawson & Gala (1976) stimulated PRL release by systemic administration of 5-HT. The 5-HT precursor 5-hydroxytryptophan (5-HTP) has been shown to induce PRL release in rats (Chen & Meites, 1975). The above reports are consistent with a stimulatory role for 5-HT in the control of PRL secretion. In the present investigation, the marked retardation in tail regeneration in lizards treated with p-CPA indicates that 5-HT neurones may be mediating the stimulatory effect of continuous illumination by way of PRL secretion during tail regeneration in lacertilians (P. I. Ndukuba & A. V. Ramachandran, in preparation).

Our recent observations have shown that half the tail is replaced, irrespective of the light factor, since lizards exposed to continuous darkness regenerated 50 % of their lost tails (P. I. Ndukuba & A. V. Ramachandran, in preparation). This study, together with that of Ramachandran & Ndukuba (1988), demonstrated that continuous light can increase both the rate and the extent of tail regeneration and that pinealectomy can totally abolish these light-induced effects. Apparently, the intact pineal is the photoreceptor which mediates the favourable influence of light on tail regeneration in H. flaviviridis. The present study further reveals that lizards with physically intact pineals, but deprived of their ability to synthesize 5-HT by the injection of p-CPA, failed to show the positive influences of continuous light on tail regeneration. This sequence of observations leads to the conclusion that the pineal is not only the photoreceptor but also the essential synchronizer which transduces and translates the photic information into favourable regenerative growth in lacertilians. Hence, it may be tentatively surmised that the purported serotonergic mechanism of PRL release (Clemens et al. 1977) may be the operative mechanism in lizards, triggered by continuous light, and that such a release of PRL can be blocked at the level of the enzyme tryptophan hydroxylase by its inhibitor, p-CPA, leading to the depletion of 5-HT from the brain. However, pince p-CPA inhibits only the first step in the synthesis of 5-HT, it is possible to bypass its blocking action, and thereby re-establish the concentration of 5-HT, by injecting the direct precursor of 5-HT following the injection of p-CPA. A study of this is now in progress in our laboratory, employing the direct precursor of 5-HT, 5-HTP, which readily crosses the blood-brain barrier. The observation that p-CPA did not completely inhibit tail regeneration in Hemidactylus (only 50 % retardation was obtained) strengthens our earlier inference that 50% tail replacement is an innate ability which is independent of photoperiodism and associated neuroendrocrine mechanisms and, apparently, occurs under basal levels of PRL secretion (Ramachandran & Ndukuba, 1988).

The authors wish to acknowledge the facilities provided by the University Grants Commission of India sponsored Departmental Research Support in Developmental Physiology.