on activity rhythms in the house finch (Carpodacus mexicanus). Jannon L. Fuchs*. Department of Neuroscience, University of California, San Diego, La Jolla, ...
Journal of Comparative Physiology. A
J Comp Physiol (1983) 153:413~419
9 Springer-Verlag 1983
Effects of pinealectomy and subsequent melatonin implants on activity rhythms in the house finch (Carpodacusmexicanus) Jannon L. Fuchs* Department of Neuroscience, University of California, San Diego, La Jolla, California 92093, USA Accepted July 1, 1983
Summary. Activity patterns were analyzed in house finches which were pinealectomized and subsequently implanted with melatonin capsules. Most pinealectomized house finches showed loss of circadian perch-hopping rhythms in constant darkness (DD). Total amount of activity in DD was about 175% higher in pinealectomized than in intact finches. In a light-dark cycle (LD) with 8 h L and 16 h D, activity in pinealectomized birds was redistributed such that an increase in activity during phase lead counter-balanced a decrease during L. Mean duration of phase lead increased from 1/2 h in intact finches to about 31/2 h in pinealectomized birds. Melatonin capsules implanted in pinealectomized finches shortened phase lead in LD and lengthened phase trail, suggesting a role for melatonin levels in setting the phase of the activity rhythm apart from phasic changes in melatonin release. Melatonin did not restore circadian rhythmicity in pinealectomized finches and instead was associated with a decline in signs of residual, damped rhythmicity. Amount of 24-h activity was positively correlated with percentage of total activity occurring during daytime; both of these were inversely correlated with melatonin dosage. In pinealectomized finches, the amount of DD activity correlated positively with phase lead activity in LD, but not with amount of activity during L. The data are interpreted in terms of the hypothesis that phase lead represents a component of activity rhythms governed by a damped oscillator of nonpineal location but influenced by melatonin. * Present address: Department of Psychology, University of Illinois, Champaign, Illinois 61820, USA Abbreviations: L D light-dark cycle; D D constant darkness; L
light portion of the light-dark cycle; D dark portion of the light-dark cycle
Introduction Gaston and Menaker (1968) found that surgical removal of the pineal gland from the house sparrow (Passer domesticus) results in loss of free-running circadian activity rhythms in constant darkness (DD). Subsequent research has supported the hypothesis that the sparrow pineal is a 'master' circadian pacemaker which drives other rhythms through its rhythmic output of a hormone, perhaps melatonin (Gaston 1971; Binkley et al. 1972; Menaker and Zimmerman 1976). The interpretation that the pineal is a pacemaker utilizing a hormonal signal is particularly useful in explaining transfer of rhythmicity and phase to a pinealectomized host sparrow upon transplantation of a pineal to the anterior chamber of the eye (Zimmerman and Menaker 1979). However, it could be hypothesized that the pineal is not a pacemaker, but is the source of a substance necessary to permit expression of circadian rhythmicity generated elsewhere. Thus, for example, a transplanted pineal would allow rhythmicity to be restored as soon as the pineal substance(s) reached levels sufficient for a continuously active bird to enter a rest period. In a model developed by Wever (1980), conditions such as light intensity change a bird's oscillatory level with respect to the threshold between activity and rest. It is known that constant light above a certain intensity yields continuous activity in intact sparrows (Menaker 1968); since light suppresses melatonin synthesis (Binkley et al. 1977), it is conceivable that both pinealectomy and constant light abolish signs of circadian rhythmicity in a similar manner (Menaker 1968). Wever predicted that tonic melatonin administered to a pinealectomized bird could return the bird's circadian oscillatory level to a value needed to reestablish rhythmicity (Wever 1980).
J.L. Fuchs: Effects of pinealectomy and melatonin on activity rhythms
In recent studies of bird species other than sparrow, most pinealectomized individuals did retain s e l f - s u s t a i n e d c i r c a d i a n r h y t h m s in D D ( s t a r l i n g , G w i n n e r 1978; c h i c k e n , M a c B r i d e 1973; q u a i l , S i m p s o n a n d F o l l e t t 1981), w i t h p i n e a l e c t o m y aff e c t i n g p a r a m e t e r s s u c h as l a b i l i t y , p e r i o d l e n g t h , and clarity of division between rest and activity ( G w i n n e r 1978). A n a d d i t i o n a l a v i a n c i r c a d i a n pacemaker candidate has recently been identified: ablation of the hypothalamic suprachiasmatic nucleus produces arrhythmicity (house sparrow, Takahashi and Menaker 1979b; Java sparrow, Ebihara and Kawamura 1981). T h e s e o b s e r v a t i o n s h a v e l e d to a r e v i s e d m o d e l o f a v i a n p a c e m a k e r f u n c t i o n , in w h i c h m u t u a l l y c o u p l e d n o n - p i n e a l o s cillators normally driven by the pineal may generate rhythmicity; the pineal influences coupling strength to various degrees depending on the species ( G w i n n e r 1978; T a k a h a s h i a n d M e n a k e r 1979a). Because a key feature of circadian pacemaker function likely involves melatonin-mediated signals from the pineal to non-pineal components o f t h e r h y t h m - g e n e r a t i n g s y s t e m , it is p a r t i c u l a r l y i m p o r t a n t t o i n v e s t i g a t e t h e effects o f m e l a t o n i n in pinealectomized birds. In the present study, melatonin implants did not restore rhythmicity to house finches made arrhythmic by pinealectomy, a result which favors the importance of a rhythmic property of the pineal. M e l a t o n i n h a d c o n s p i c u o u s effects o n t h e d i s t r i b u t i o n p a t t e r n a n d level o f a c t i v i t y i n p i n e a l e c t o mized birds.
Materials and methods Subjects. Subjects were adult house finches (Carpodacus mexicanus) of both sexes, trapped near San Diego, California. Each finch was housed in a separate cage (about 40 x 20 x 20 cm) equipped with a perch. Each time the bird sat on its perch, a microswitch closed, activating the pen of an Esterline-Angus Event Recorder. Food and water were available continuously and were changed approximately every 10 days. All subjects were visually isolated from one another. Eight of the 20 cages were further isolated in individual enclosures to minimize acoustic interaction among the birds.
Pinealectomy and histology. The pinealectomy procedure is described elsewhere (Binkley et al. 1971). For histological examination, finches were perfused with Bouin's fixative. The dorsal brain and overlying skull were examined for obvious pineal remnants. Brains were cut sagittally in 30 gm serial sections which were then stained with cresyl violet.
Melatonin implants. Capsules made of Silastic tubing (Dow Corning No. 602-235, 1.47 mm i.d., 1.96 mm o.d.) were filled with crystalline melatonin (Sigma) and sealed at both ends with Silastic material. Capsules were placed intraperitoneally. Mela-
tonin release under such conditions is about 4 pg per day per 5 mm capsule length (Turek et al. 1976). A capsule between 5 and 10 mm is here designated a 'low' dosage. A 'high' dosage is two or more 8-11 mm capsules, with total dosages ranging from 16 to 71 mm (mean, 41 ram).
Lighting schedules. The lighting schedules were LD 8:16 (8 h of light, 16 h of darkness) and DD (constant darkness). 'Darkness' was actually very dim light (mean, 0.1 lux), as finches in total darkness seldom leave their perch, egen for food and water. The mean light intensity in L for the various cages was 91 lux. Incandescent lighting was used. Activity was recorded over a 9-month period from late December through September. Intact finches were exposed to LD for 3 weeks and DD for 4 weeks, and were then pinealectomized. Birds which did not survive were replaced by newly pinealectomized birds without pre-operative activity records. Pinealectomized birds were typically kept in LD for 4 weeks and DD for 4 weeks, and then in LD for 3 weeks. Finches which showed arrhythmicity in constant dim light were then implanted with a melatonin capsule of length between 5 and 10mm, while birds with persisting free-running activity rhythms were autopsied and replaced by newly pinealectomized birds. After approximately 2 weeks in LD and 8 weeks in DD, all finches which were arrhythmic in DD were implanted with high doses of melatonin (16-71 mm). The pinealectomized finches with high dosages were exposed to LD for 2 weeks and DD for 7 weeks, and were then sacrificed for histology. Complete or nearly complete records were obtained for 16 intact house finches. Post-operative records were obtained for 12 successfully pinealectomized finches, 6 of which also had pre-operative records. Six of the 12 pinealectomized birds then received low melatonin doses. These 6 together with 4 additional pinealectomized finches then received high melatonin doses.
Data analysis. Each subject's 24-h records were cut and glued onto a chart, one below the other in chronological sequence. Presence or absence of free-running circadian rhythmicity in DD was judged by visual inspection of the charts. The onset of phase lead (heavy activity preceding L) was indicated on the chart by drawing a best-fit line, after a fairly constant phase had been achieved (usually beginning after the first week of LD). Similar criteria were applied for phase trail, which is the continuation of the active period after lights-off. Limits of phase lead and phase trail were generally distinct, except that in pinealectomized subjects with high melatonin dosages the end of phase trail was frequently ambiguous. Individual perch-hops (pen deflections) were counted, using an estimate of 6 hops per 10-min interval where the marks were too closely spaced to be resolved (Hendel and Turek 1978). Counts for each condition were averaged over at least 7 days chosen at regular intervals over the appropriate span of weeks. To measure activity during subjective L in DD, a best-fit line was drawn through the midpoints of the active periods of successive days; 6 h either side of this line were then included i n ' subjective L '. Subjective L was extended beyond 8 h because the active period of free-running birds was typically much longer than 8 h, as was the active period in subjects which showed phase lead. The 12-h window was sufficient to encompass the active period for all but 2 subjects. 'Clarity' of rhythms in DD was defined as the percentage of 24 h activity occurring during subjective L. Clarity of rhythms in LD was measured similarly, with 12 h of daytime activity centered around the midpoint of the 8-h L period.
J.L. Fuchs: Effectsof pinealectomyand melatonin on activityrhythms Results
'Pinealectomized' birds are those without melatonin capsules, unless otherwise specified. Pinealectomized birds used in the statistical tests include only those judged to be completely pinealectomized, but exclude the 2 of these 12 which retained free-running circadian rhythms. Means are given with standard errors. Differences between groups were evaluated using the Mann-Whitney U-test, and correlations were measured using Spearman's rank correlation coefficient, r'. All P-values are for 2-tailed tests.
Effects of pinealectomy Pinealectomized finches typically showed absence of free-running circadian activity rhythms. Figure 1 shows activity records from a house finch in which pinealectomy resulted in loss of circadian activity rhythms in DD. Of the 12 finches judged by histological criteria to be completely pinealectomized, 2 showed persisting free-running circadian activity rhythms in DD. The additional 6 with incomplete pinealectomies showed some free-running rhythmicity in DD.
Pinealectomized finches showed longer phase lead in LD. Phase lead increased from 0.5-+0.3 h ( n = 16) in intact finches to 3.5 -+ 0.9 (n = 10) in pinealectomized finches (P < 0.01, U-test). Pinealectomy did not substantially alter total LD activity, but redistributed it such that phase lead activity increased, while activity during L decreased. In pinealectomized finches, an average of 35% of total daily perch hops in LD (154+_ 13; n = 10) ocCurred during phase lead, while in intact birds this average was only 8% of the total of 159_+7 ( n : 16). Perch hops during L (of LD) declined 32% from 146-+ 8 (n = 16) in intact birds, to 100_+ 9 (n = 10) in pinealectomized birds (P