In the previous Chapter, it was shown that feeding and locomotor rhythms of pinealectomized (P-X) pigeons can be entrained by physiological amounts of melatonin delivered in normal temporal patterns. These results show that a melatonin entrainable oscillator(s) exists in P-X pigeons and suggest that endogenous melatonin rhythms regulate behavioral rhythmicity in
pigeons. It has been previously shown that P-X pigeons have circadian oscillators that can sustain locomotor and blood melatonin rhythms in constant conditions but that bilateral enucleation (E-X) of P-X pigeons abolishes both of these rhythms (Ebihara et aI., 1984; Foa and Menaker, 1988). This leaves open the question as to whether behavioral rhythmicity can be restored to E XIP-X pigeons with melatonin infusions.
Transplantation of a pineal gland into arrhythmic house sparrows can restore its' locomotor rhythm (Zimmerman and Menaker, 1979). The results from a previous experiment have suggested that the pineal gland regulates circadian activity via the release of a diffusible substance (Zimmerman and Menaker, 1975). This diffusible substance has long been thought to be melatonin since this hormone is the major product of the avian pineal and is released rhythmically in vitro (Takahashi, 1981). While melatonin is a prime candidate as the hormonal output from a circadian oscillatory system responsible for the maintenance of behavioral circadian rhythms in birds, this hypothesis has not been directly tested. We chose to test this hypothesis using homing pigeons since the disappearance of behavioral rhythms has been correlated with the disappearance of its' blood melatonin rhythms (Ebihara et aI., 1984; Foa and Menaker, 1988). The purpose of this experiment was to determine if feeding and locomotor rhythms could be restored to pigeons made arrhythmic by combined P-x, E-X, with infusions of physiological amounts of melatonin delivered in normal temporal patterns.
Experimental Birds and Housing Conditions:
Homing pigeons (Columba livia; males and females, weight 400 - 600 g; n=13) were obtained as described in Chapter 2. These birds were previously used in an experiment testing the effects of physiologically significant levels of exogenous melatonin on P-X pigeons (Chapter 3). At least eight weeks elapsed between the previous experiment and the experiment described below during which the pigeons were held in a 12 hrs light/12 hrs dark (LD) cycle. Housing conditions were the same as described in Chapter 3.
Surgeries:
Pinealectomy (P-X)
Pigeons were P-X as previously described (Ebihara et al,1984; Chapter 2) several months before this series of experiments began.
Retinae Removal (E- X)
P-X pigeons were removed from their recording cages and anesthetized (60 mg/kg Ketamine followed by 10 mg/kg nembutal). Local anesthetic (lidocaine) was applied to the eyes and the eyelid comers surgically extended. A frontal section was then cut from the eye removing the lens, iris and a ring of sclera. The vitreous was removed with absorbent cotton swabs and the retina was gently teased away from the underlying pigment epithelium. A pair of iridectomy scissors was used to detach the retina at the pecten and at the optic nerve. After surgery the inside of the eye was inspected to verify that the retina had been completely removed. Often some of the pigment epithelium was removed as well. The amount of pigment epithelium removed varied with the individual (10 - 90%). The cavity of the eye was packed with Gel-foam (Upjohn, Kalamazoo, MI) and ophthalmic ointment (gentamicin sulfate). The eyelids were sutured shut and gentamicin sulfate applied externally. Pigeons were always allowed to recover on a heating pad before being returned to their cages in DD.
Cannulations and Infusions
Cannulations and infusions were performed as described in Chapter 3 except that, during infusions, the vehicle and melatonin/vehicle infusions were delivered at a rate of 31 ul/hr for 10 hours/timing cycle (dose = 0.93 ug melatonin/hr). The timing cycle of the infusions was controlled by two timers adjusted to have periods longer than 24 hours (24.51 and 24.65 hrs).
Blood Sampling, Extraction, Radioimmunoassay and Validation:
Blood sampling was performed by venipuncture as described in Chapter 3. The storage and extraction procedures were also described in the previous Chapter along with the methods to correct for extraction efficiency and interassay variation. The average extraction efficiency (7 extractions) was 81%. Intra-assay coefficient of variation (CV) for melatonin standards (63 pgl100 ul) in phosphate buffered saline was 8.6%. (7 assays). The inter-assay CV was 16% and the limit of detection was 19 pglml (for 100 ul samples). The values for 20%, 50%, and 80% binding on standard curves were 1700 + 128, 350 + 21, and 73 + 5 pglml respectively. This assay (melatonin antibody R1055) was previously validated as described in Chapter 3.
Data Analysis
When behavioral rhythmicity was clear by both visual inspection and periodogram analysis the period and phase of feeding rhythms were calculated by drawing a best eye-fit line through the behavioral offsets. The offsets (or
onsets) of free-running locomotor rhythms were not clear enough to accurately assess the period or phase. Chi-Square periodogram analyses were performed using a moving window (10 days/window) procedure on all segments (P-X/E-x, P-X/E-X/vehicle infused and P-X/E-X/melatonin infused) of the behavioral records. The significance of differences between means was determined using Student's t test (p < 0.05).
Representative behavioral records from a P-X bird that was bilaterally E-X and then subjected to periodic melatonin infusion are presented in Figure 1. Periodogram analyses of selected sections of the feeding and locomotor activity are presented in Figure 2. When this P-X pigeon, which was free-running in DD, was E-X and returned to DD, both feeding and locomotor rhythms were abolished (Figure 1,2 - section 1). When this bird was later subjected to rhythmic infusions of melatonin, rhythmic patterns of both feeding and locomotor activity were observed (Figure 1,2 - section 2).
The period of the feeding rhythm during melatonin infusion for this bird (Figure 1) was 24.49 hrs (period of infusion = 24.51 hrs). The average period of the feeding rhythms during infusions for all birds was similar to the period of melatonin infusion (24.52 + 0.03 hrs [n=6 pigeons] vs. 24.51 [infusion period] and 24.60 + 0.03 [n=8 pigeons] vs. 24.65 [infusion period]). In Figure 1, the main bout of feeding activity ended approximately 3.5 hours before (positive phase angle) melatonin infusion for this bird; the average phase angle for all birds was 2.1 + 0.6 hrs (SEM; n=14). Similar results of entrainment to and phase lead of melatonin infusions are presented in Figure 3a,b. When the melatonin infusions were discontinued, both feeding and locomotor rhythmicity persisted for several cycles (Figure 1,2 - section 3; Figure 3a,b). Later, significant rhythmicity was not observed (Figure 1,2 section 4).
Feeding and locomotor activities at times responded differently to melatonin infusions. While melatonin infusions generally restored both rhythms, in two cases only feeding rhythmicity was restored, and in one case only the locomotor rhythm was restored. Furthermore, while significant feeding rhythms persisted for up to 20 days in 3/6 pigeons allowed to free-run after melatonin infusion was stopped, significant locomotor rhythms persisted in only 1/6 of these pigeons. Additionally, feeding rhythms were usually clearer both by visual inspection and by periodogram analysis during, and immediately after, melatonin infusion. The average number of 10 day windows during melatonin infusion which indicated significant periodicity was 21.36 + 4.48 (n=l1) windows for feeding and 10.36 + 3.43 (n=l1) windows for locomotion (out of a possible 30 windows). These averages were significantly different (p < .001). The average free-running period of the feeding rhythm after melatonin infusions was stopped averaged 24.11 + 0.04 hrs (n=4) and was significantly different from the average period during melatonin infusion (p < .001).
While daily melatonin infusions were sufficient to restore both feeding and locomotor rhythmicity in P-X/E-X pigeons, daily infusions of vehicle alone were not. Representative feeding and locomotor activity records from a P XIE-X pigeon in DD which received vehicle infusions is presented in Figure 4. As is evidenced both by visual inspection and by periodogram analysis of these data (Figure 5), significant behavioral rhythmicity was not observed before and during vehicle infusion (Figure 4,5 - sections 1 and 2). Similar results were seen in all eightl other pigeons subjected to vehicle infusions (Figure 6a-d). However, in one individual transient [two 10 day windows out of a possible ten], significant; locomotor but not feeding rhythms were apparent during vehicle infusion. When melatonin infusions were later initiated after vehicle infusions, behavioral rhythmicity was restored (Figure 4,5 - section 3; 6a-d). While restoration of rhythmicity occurred within a few cycles (Figure 4; 6c,d) for some individuals (n=6), rhythmic activity was not manifest for 5 - 16 days (Figure 1; 3a,b; 6a,b) for others (n=7). The overall results are tabulated in Table 1.
P-X, P-X/E-X, and P-X/E-X/melatonin infused blood melatonin levels from an individual (top) and grouped birds are presented in Figure 7. As previously shown in Chapter 3, blood levels of P-X pigeons show clear circadian differences, the peaks of which are approximately 180 degrees out of phase with their locomotor and feeding activity peaks (Figure 7 - solid lines). The blood melatonin levels of P-XlE-X birds from samples taken immediately after melatonin infusion termination were low and arrhythmic (Figure 7 - dashed lines). When these birds were later infused with melatonin (0.93 uglhr for 10 hours), a blood melatonin rhythm again became apparent (Figure 7 - dotted lines). Importantly, the P-XlE-X peak melatonin levels during melatonin infusion were not higher than the peak P-X melatonin levels of these pigeons. The average blood melatonin peak values of P-X and P-XlE-Xlmelatonin infused birds were significantly higher than the average trough values (p < 0.001).
Our results demonstrate that melatonin infusions are sufficient to restore behavioral circadian rhythms to homing pigeons rendered arrhythmic by removal of the retinae and the pineal gland. Since vehicle infusions were not
sufficient to restore rhythmicity, these results indicate that melatonin itself was responsible for the restoration of rhythmicity. We also demonstrated that the blood melatonin levels of the P-XlE-X pigeons receiving melatonin infusions were within the physiological range. Oshima et aI., (1989a) recently presented evidence that daily melatonin injections can entrain locomotor rhythms in P-XIE-X pigeons. However, control injection data were not presented and the injections induced blood melatonin levels an order of magnitude higher than normal. Our results are therefore the first direct demonstration that physiological amounts of melatonin presented in a normal temporal pattern are sufficient to restore and maintain feeding and locomotor rhythmicity to P-XIE-X pigeons.
Melatonin infusions affect feeding and locomotor activity of P-XlE-X pigeons in a similar manner. Melatonin infusions restored both feeding and locomotor rhythms to most individuals. Some differences were observed however. In general feeding rhythms were clearer than locomotor rhythms during melatonin infusion and usually persisted for a longer period after melatonin infusions were terminated (Table 1). These findings are similar to previous results from a number of perturbational experiments in which these two behaviors were measured (Chapter 2).
While our results concerning the abolition of behavioral and blood melatonin rhythms are similar to previously published reports (Ebihara et aI., 1984; Foa and Menaker, 1988), there is a potentially significant methodological difference between our study and previous studies. While Ebihara et aI., (1984) and Foa and Menaker (1988) removed the whole eye (and presumably the Harderian gland as well) we removed only tissues inside the sclera. This suggests that the tissue responsible for the maintenance of locomotor rhythmicity is inside the eye, probably the retina since this tissue has been shown to contain high concentrations of melatonin in birds (Hamm et aI., 1983).
The Harderian gland has also been previously suggested to be a source of melatonin in the pigeon on the basis of a tissue content study (Vakkuri et aI., (1984). Our method of E-X which left the Harderian gland intact abolished both blood melatonin and behavioral rhythms. Therefore, the Harderian glands may not be capable of maintaining blood melatonin, feeding, or locomotor rhythms in pigeons in the absence of the retinae and the pineal gland. The persistence of rhythmicity in some individuals after melatonin infusion was stopped indicates that melatonin entrains an oscillator(s) in E-XIP-X pigeons. Previous results from Ebihara et aI., (1984) provided similar evidence for an oscillator in P-XlE-X pigeons which was entrainable by light. In the house sparrow, hypothalamic lesions (Takahashi and Menaker, 1982), that may have been large enough to include at least part of a lateral retinorecipient area as well as the more medial suprachiasmatic nuclei (SCN) (J. Takahashi, personal communication), abolished locomotor rhythmicity. Anatomical studies indicate the presence of retinorecipient areas (SCN) in the hypothalamus (Gamlin et aI., 1982) of the pigeon that are also labelled by iodinated melatonin (I-MEL; Oshima, Chabot and Menaker, unpublished observations). However, since hypothalamic lesion studies in the pigeon (Ebihara et aI., 1987) have yielded equivocal results, the extra-retinaVextra pineal structure( s) through which melatonin and light may exert their
circadian influence in the pigeon is unknown.
Our results strengthen the idea that melatonin pays a central role in the circadian system of this species. The direct demonstration that physiological amounts of infused melatonin delivered in normal temporal patterns restore feeding and locomotor circadian rhythms in P-XlE-X pigeons indicates an important role for blood melatonin rhythms in regulating the timing of these behaviors.