Light and Biological Rhythms in Man

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 Preface
T H E OBJECTIVE of this b o o k is to s u m m a r i z e the k n o w l e d g e of light as a
regulator of biological r h y t h m s in m a n in relation to h e a l t h a n d disease.
T h e first scientific meeting o n biological r h y t h m s in Sweden was held in
1937 in the small spa of R o n n e b y in the s o u t h of Sweden. A b o u t twenty
scientists w h o called themselves " R h y t h m s e n t h u s i a s t s " g a t h e r e d a n d
established a Society for Biological R h y t h m Research. T h e second meeting
o n biological r h y t h m s in Sweden was held in 1955 in S t o c k h o l m , still with
a small g r o u p of scientists.
T h e p r e s e n t a t i o n s published in this b o o k u p d a t e w h a t has h a p p e n e d
d u r i n g the last four decades a n d focus in particular o n h o w light is
influencing biological r h y t h m s . In 1959 A a r o n Lerner a n d c o w o r k e r s
discovered the time keeping substance m e l a t o n i n . In o n e c h a p t e r in this
b o o k Lerner r e c o u n t s the history of his efforts for four years to find the
melanin tonizing h o r m o n e (melatonin) which b l a n c h e d frog skin, until
they found the s u b s t a n c e a n d it could be said "This is it!". F r o m there on
m a n y speculations a b o u t the m e d i a t o r of biological r h y t h m regulation
have b e c o m e scientifically testable h y p o t h e s e s . T h e results b a s e d o n
Lerner's w o r k h a v e led t o solid d a t a o n which m u c h of m o d e r n biological
r h y t h m research is based.
T h a t A a r o n Lerner's historical aspects are included in this v o l u m e , as
well as all the o t h e r p r e s e n t a t i o n s will we h o p e benefit y o u n g e r generations
of students a n d scientists for w h o m the research frontiers of t o d a y have a
s t r o n g b e a r i n g o n t h e clinical practice of t o m o r r o w a n d well b e y o n d the
year 2000.
In different ways research is confronted with the fact t h a t m a n y of the
fundamental r h y t h m processes are n o t easily observable a n d m u s t m a n y
times be reconstructed from preliminary d a t a . A n interesting m o d e l t o
explain the origin of biological r h y t h m s is the o n e of Erik O d e b l a d from
University of U r n e a , Sweden, w h o h a s p r o p o s e d t h a t it is b a s e d o n the
presence of collective vibrations ( p h o n o n s ) in l o o p - s h a p e d molecules or
molecular aggregates. V i b r a t i o n a l waves a l o n g a closed l o o p will give rise
to self-interference as the basis for r h y t h m s . F u r t h e r experiments t o
measure p h o n o n s in tissues a n d o r g a n s are called for t o test this
hypothesis.
V
vi Preface

As illustrated in this b o o k , interfacing disciplines progress steadily

t h r o u g h experimentation, observation a n d theoretical interpretation, each
one helping the advances of the other. T h e biological r h y t h m s covered in
this b o o k range from short (infradian) a n d a b o u t 2 4 - h o u r r h y t h m s
(circadian) to longer r h y t h m s (ultradian) present in all m a m m a l i a n
species.
D u r i n g the last decade it has b e c o m e clear t h a t h u m a n s also are in m a n y
ways influenced by biological r h y t h m s . T i m e structures are present o n
several levels from p o p u l a t i o n , individuals, o r g a n s (e.g. b r a i n a n d h e a r t ) to
tissues, cells a n d subcellular o r g a n i z a t i o n . A n i n t r o d u c t o r y c h a p t e r
identifies the genetic m a c h i n e r y , including the light influenced proteins,
which governs the clocks t h a t regulate us all.
T h e new findings a b o u t the effect of light o n biological r h y t h m s has
already lead to therapeutic trials in different conditions. T h e use of light to
regulate m e n s t r u a l cyclicity m a y have clinical value for r h y t h m c o n t r a c e p -
tive m e t h o d s a n d for t r e a t m e n t of infertility. T h e further studies of the
effect of light o n the m e n s t r u a l cycle m a y lead to new views of reproductive
endocrinology. In s o m e c h a p t e r s , specialists in light perception a n d light
reception have presented d a t a o n light t r e a t m e n t as a n antidepressant in
seasonal affective disorder. It is clear t h a t clinical testing is needed to
determine the o p t i m a l intensities, the timing a n d d u r a t i o n of light
t r e a t m e n t for p r o d u c i n g beneficial effects.
In the r e p o r t of placebo-controlled studies it is stated t h a t it will require
further studies to establish h o w light t r e a t m e n t c o m p a r e s with p l a c e b o .
Such w o r k is also i m p o r t a n t to help u n d e r s t a n d the m e c h a n i s m s a n d
etiology of depression a n d other c o n d i t i o n s .
T o u n d e r s t a n d the r h y t h m regulating effects of light it is necessary t o
examine the ocular m e c h a n i s m s which m e d i a t e the p h o t i c effect t h r o u g h
the eye. W h e n light reaches the retina, there are individual differences in
the sensitivity of the photoreceptivity a n d the ability of the m e l a t o n i n
r h y t h m generating system t o integrate p h o t i c stimuli, temporally.
T h e overall a i m in the present v o l u m e is to provide a basis, b o t h for
scientific as well as clinical perspectives to establish the specific m o d e s a n d
measures which mediate therapeutic a n d physiologically beneficial effects
of light as a regulator of biological r h y t h m s .
I a m truly indebted to all of the c o n t r i b u t o r s to this v o l u m e a n d very
grateful for the s u p p o r t a n d help of D a v i d O t t o s o n a n d J o h a n Beck-Friis.
T h e generous financial c o n t r i b u t i o n of the W e n n e r - G r e n C e n t e r F o u n d a -
tion a n d the Swedish Medical Research Council is a c k n o w l e d g e d .

Stockholm, 1993
LENNART WETTERBERG
1
Biological Rhythms: From Gene
Expression to Behavior*
J O S E P H S. T A K A H A S H I

NSF Center for Biological Timing, Department of Neurobiology and

Physiology, Northwestern University, Evanston, IL 60208-3520, USA

Abstract
C i r c a d i a n r h y t h m s r e g u l a t e t h e function of living s y s t e m s at virtually every level of
o r g a n i z a t i o n . In t h e last d e c a d e , o u r u n d e r s t a n d i n g of t h e cellular a n d m o l e c u l a r p r o c e s s e s
involved in t h e g e n e r a t i o n a n d r e g u l a t i o n of c i r c a d i a n r h y t h m s h a s a d v a n c e d c o n s i d e r a b l y .
N e w m o d e l s y s t e m s for s t u d y i n g c i r c a d i a n o s c i l l a t o r s h a v e b e e n d e v e l o p e d , a p o t e n t i a l
r e g u l a t o r y role for cellular i m m e d i a t e - e a r l y genes in c i r c a d i a n b e h a v i o r h a s b e e n d i s c o v e r e d ,
a n d critical p e r i o d s of m a c r o m o l e c u l a r synthesis for p r o g r e s s i o n of t h e c i r c a d i a n clock
t h r o u g h its cycle h a v e b e e n defined. T h e s e findings a r e of p a r t i c u l a r interest b e c a u s e
i n d e p e n d e n t a p p r o a c h e s suggest t h a t a n i m p o r t a n t role for m a c r o m o l e c u l a r s y n t h e s i s exists
at all levels of t h e c i r c a d i a n s y s t e m .

Introduction
Circadian r h y t h m s regulate the behavior, physiology a n d3 biochemistry , 1 505 , 3 8of
m o s t living systems: from c y a n o b a c t e r i u m to m a n . ' Among
animals, m u c h is k n o w n a b o u t the physiology of circadian r h y t h m s , a n d in
all cases, circadian control is exerted by structures 4 2 t 4h a5,t c255
1o n9t a i n circadian
p a c e m a k e r s within the central nervous s y s t e m . ' ' ' O n the basis of
a wide variety of evidence, the m e c h a n i s m of the circadian clock a p5p 5 e1a54
2r s to
be cell a u t o n o m o u s a n d to involve periodic gene e x p r e s s i o n . ' ' In
addition, signal t r a n s d u c t i o n p a t h w a y s into the clock m e c h a n i s m are
present for conveying e n v i r o n m e n t a l information for e n t r a i n m e n t of the
clock. F u r t h e r m o r e , the clock m e c h a n i s m has diverse o u t p u t p a t h w a y s for
exerting circadian control at all levels of organismal biology. A n u m b e r of
generalizations can be m a d e a b o u t circadian biology. C i r c a d i a n r h y t h m s
are a p r o p e r t y of all e u k a r y o t i c a n d s o m e p r o k a r y o t i c o r g a n i s m s a n d are
entrained primarily by e n v i r o n m e n t a l cycles of light a n d t e m p e r a t u r e .
Circadian r h y t h m s are genetically determined a n d single-gene clock
* This paper is dedicated to D r A a r o n B. Lerner for his seminal work on melatonin.

3
4 Light and Biological Rhythms in Man

m u t a n t s h a v e been found in six organisms including m a m m a l s . Circadian

oscillations are r e m a r k a b l y precise a n d can have a variation in cycle length
of less t h a n o n e p a r t in a t h o u s a n d . T h e period length of the oscillation is
t e m p e r a t u r e - c o m p e n s a t e d a n d usually varies less t h a n 2 0 % for each 10°C
change in t e m p e r a t u r e (Q1Q= 0.8-1.2). Finally, the biochemical m a -
chinery responsible for generating circadian r h y t h m s can be expressed at
the level of individual cells. T h u s , circadian rhythmicity is a fundamental
organizing feature of virtually all o r g a n i s m s . T h e properties of these
r h y t h m s are u n i q u e a n d widely conserved a m o n g living systems.
This c h a p t e r will be organized in t w o p a r t s . First, a brief review of
vertebrate circadian o r g a n i z a t i o n will be presented. Second, a s u m m a r y of
o u r o w n w o r k on p h o t i c e n t r a i n m e n t a n d regulation of i m m e d i a t e early
genes in m a m m a l s will follow.

C o m p o n e n t s of circadian systems
All circadian systems c o n t a i n at least three elements: (1) a n i n p u t p a t h w a y
or set of i n p u t p a t h w a y s t h a t convey e n v i r o n m e n t a l information t o the
circadian p a c e m a k e r for e n t r a i n m e n t ; (2) a circadian p a c e m a k e r t h a t
generates the oscillation; a n d (3) a n o u t p u t p a t h w a y or set of o u t p u5t8
p a t h w a y s by which the p a c e m a k e r regulates its various o u t p u t r h y t h m s .
In all systems, a p h o t i c e n t r a i n m e n t p a t h w a y is present as a n input. It is
already clear from the diversity of p h o t o p i g m e n t s a n d p h o t o t r a n s d u c t i o n
p a t h w a y s t h38a t p h o t i c e n t r a i n m e n t p a t h w a y s differ m a r k e d l y in different
o r g a n i s m s . At the receptor level, the diversity spans the spectrum from
p h y t o c h r o m e in plants t o m e m b e r s of the r h o d o p s i n family in a n i m a l s .
T h e r e a p p e a r s to be c o m p a r a b l e diversity at the second messenger level in
p h o t i c signal t r a n s d u c t i o n p a t h w a y s . At the o u t p u t level, diversity is even
m o r e extreme. Again, this type of regulation spans the clock control of
photosynthesis in plants to e n d o51c r i n e a n d behavioral r h y t h m s in animals.
As I have argued p r e v i o u s l y , the i n p u t a n d o u t p u t p a t h w a y s of the
circadian clock within each o r g a n i s m a p p e a r to be specific to each system.
Despite these differences in the coupling p a t h w a y s (inputs a n d o u t p u t s ) of
the circadian clock, the core m e c h a n i s m of the p a c e m a k e r a p p e a r s to be
fundamentally similar in all o r g a n i s m s . W h e t h e r these similarities in
p a c e m a k e r m e c h a n i s m s will ultimately be found to be functionally
a n a l o g o u s or phylogenetically h o m o l o g o u s remains to be seen.

Physiological organization of v e r t e b r a t e circadian

systems
Circadian p a c e m a k e r s , which control the behavior a n d physiology of
animals, 4have 5 55
, 4been
8 localized as discrete structures within the n e r v o u s
system. ' At the physiological level, b o t h "oscillator" a n d "pace-
m a k e r " function have been defined. A "circadian oscillator" is a structure
 Biological Rhythms: From Gene Expression to Behavior 5

t h a t expresses a self-sustained oscillation u n d e r c o n s t a n t conditions (the

absolute m i n i m u m n u m b e r of cycles is two). Circadian oscillators have
been localized by isolating the tissue in question in vitro a n d then
d e m o n s t r a t i n g the persistence of circadian oscillations in the isolated
tissue u n d e r c o n s t a n t c o n d i t i o n s . In vertebrates, three diencephalic
structures h a v e been s h o w n to c o n55t a i n circadian oscillators, the pineal
gland of birds, reptiles a n d f 2i s h29 4, the h y p o t h a l a m i c s u p r a c h i a s m a t i c
4 37
nucleus ( S C N ) of m a m m a l s , ' a n d the retinas of a m p h i b i a n s a n d
b i r d s . ' A "circadian p a c e m a k e r " is a circadian oscillator t h a t has been
shown to drive a n d therefore control some overt r h y t h m i c process such as
l o c o m o t o r behavior. P a c e m a k e r function has been experimentally d e m o n -
strated by t r a n s p l a n t i n g the structure in question a n d then showing t h a t
the phase or period of the recipient r h y t h m is regulated by the t r a n s p l a n t .
This result has been d e m o n s t r a t e d in vertebrates for the pineal gland of
A2>A5 for the optic lobes
s p a r r o w s a n d the S C N of h a m s t e r s , a n d in invertebrates
of cockroaches a n d the brain of Drosophila.
A l t h o u g h a n o c u l a r - S C N - p i n e a l axis underlies vertebrate circadian
o r g a n i z a t i o n , it is difficult to m a k e simple generalizations at the
physiological level. T h e clearest division is a m a m m a l i a n versus n o n -
m a m m a l i a n d i c h o t o m y . In m a m m a l s , the organization is the simplest. T h e
d o m i n a n t circadian p a c e m a k e r is located in the S C N , the p h o t o r e c e p t o r s
for e n t r a i n m e n t are exclusively retinal, a n d o u t p u 2t4 p a t h w a y s such as the
pineal r h y t h m of m e l a t o n i n are well d e f i n e d . 3 45
In 5 non-mammalian
vertebrates, the o r g a n i z a t i o n is m o r e c o m p l e x ; ' a n d , multiple
oscillators a n d p h o t o r e c e p t o r s are involved. T h e pineal gland, the S C N
a n d the eyes can each play a d o m i n a n t role in circadian behavior of birds
depending on the species. F o r example, in passerine birds, such as the
h o u s e s p a r r o w , the pineal gland plays a d o m i n a n t p a c e m a k e r role;
whereas, in gallinaceous birds, such as chickens a n d quail, the pineal gland
is n o t essential. T h e avian S C N a p p e a r s to be necessary in all species
examined, b u t its role as a p a c e m a k e r has n o t been determined. Finally, in60
J a p a n e s e quail, the eyes play a d o m i n a n t role in circadian o r g a n i z a t i o n .
In a d d i t i o n to the multiplicity of oscillatory centers, multiple p h o t o r e c e p -
tors for61 e n t r a i n m e n t are located in the retina, the pineal gland a n d the
brain. In s u m m a r y , n o n - m a m m a l i a n vertebrates a p p e a r to have
distributed circadian oscillators with local photoreceptive input; whereas,
m a m m a l s a p p e a r to have retained only a subset of these c o m p o n e n t s .

The m a m m a l i a n suprachiasmatic nucleus

A wealth of experimental evidence strongly argues t h a t the h y p o t h a l a m i c
S C N is the 2site
24 49of48
5a circadian p a c e m a k e r t h a t drives overt r h y t h m s in
m a m m a l s . ' ' ' Six lines of evidence s u p p o r t this conclusion. First,
the S C N receives direct i n p u t from the retina t h r o u g h a specialized visual
6 Light and Biological Rhythms in Man

p a t h w a y , the r e t i n o h y p o t h a l a m i c tract, which is required for e n t r a i n m e n t

to light cycles. Second, a wide variety of circadian r h y t h m s in r o d e n t s are
disrupted by S C N lesions, ruling o u t the possibility of a highly specific,
limited effect of lesions o n a particular subsystem, for instance, l o c o m o t o r
behavior. T h i r d , the S C N expresses circadian r h y t h m s of multi-unit
electrical activity t h a t persist after neural isolation in vivo. F o u r t h , S C N
expiants c o n t i n u e to express circadian r h y t h m s of single-unit electrical
activity, vasopressin release a n d m e t a b o l i c activity in vitro. Fifth, circadian
rhythmicity can be restored to a r r h y t h m i c SCN-lesioned animals by
t r a n s p l a n t a t i o n of fetal tissue containing S C N cells. Sixth, t r a n s p l a n t a t i o n
of S C N tissue derived from Tau m u t a n t h a m s t e r s , which express short
period r h y t h m s , d e m o n s t r a t e s t h a t the genotype of the d o n o r S C N
determines the period of the restored r h y t h m . T a k e n together, these results
d e m o n s t r a t e t h a t the S C N plays a d o m i n a n t role in the generation of
circadian r h y t h m s in m a m m a l s .

F u n c t i o n a l p r o p e r t i e s o f p h o t i c e n t r a i n m e n t in
mammals
P h o t i c e n t r a i n m e n t in m a m m a l s is m e d i a t e d by retinal 29p h o t o r e c e p t o r s
t h a t project to the S C N via the r e t i n o h y p o t h a l a m i c t r a c t . W e have used
light-induced p h a s e shifts of the circadian r h y t h m of wheel-running
activity to m e a s u r e the p h o t i c sensitivity of the circadian system of the
golden h a m s t e r . Previously we showed t h a t the spectral sensitivity
function for phase-shifting m53 a t c h e s a n opsin-based p h o t o p i g m e n t with a
peak ( A m )a a xr o u n d 500 n m . A l t h o u g h the p e a k sensitivity is similar to
t h a t of r h o d o p s i n , t w o features of this photoreceptive system are u n u s u a l :
the threshold of the response is high, especially for a r o d - d o m i n a t e d retina
like t h a t of the h a m s t e r , a n d the reciprocal relationship 33 between intensity
a n d d u r a t i o n holds for extremely long d u r a t i o n s . Figure 1 shows the
phase-shifting response to 300 sec light pulses at circadian time (CT) 19.
T h e phase-shifting response increases with light intensity a n d can be fit
with a four p a r a m e t e r logistic function. T h e sensitivity to stimulus duration
was assessed by m e a s u r i n g the m a g n i t u d e of phase-shift responses to
photic stimuli of different irradiance a n d d u r a t i o n (Figure 2). T h e h a m s t e r
circadian system is m o r e sensitive to the irradiance of longer d u r a t i o n
stimuli t h a n to t h a t of briefer stimuli. T h e system is maximally sensitive to
the irradiance of stimuli of 300 sec a n d longer in d u r a t i o n (Figure 3A). As
s h o w n previously the threshold for p h o t i c stimulation of the h a m s t e r
circadian p a c e m a k e r is high. T h e threshold irradiance (the m a x i m u m
irradiance necessary 11 to induce-2statistically 1 significant responses) is
approximately 1 0 photons c m s e c " for o p t i m a l stimulus d u r a t i o n s .
2
This threshold is equivalent t o a l u m i n a n c e at the cornea of a b o u t 0.1
c d - m ~ . W e also m e a s u r e d the sensitivity of this visual p a t h w a y to the
 Biological Rhythms: From Gene Expression to Behavior 1

y/-

' — — • — • — • — . — • — • — · — • — - J
Dark 8 9 10 11 12 13 14 15 16 17

Irradiance (log p h o t o n s · cm~~2 . — 1 )

s
F I G . 1. M a g n i t u d e of p h a s e shift of h a m s t e r activity r h y t h m in r e s p o n s e t o 300 sec
503 n m light pulses of different i r r a d i a n c e at c i r c a d i a n t i m e 19 ( C T 1 9 ) . T h e p o i n t s
33
r e p r e s e n t t h e m e a n ± S E M . T h e c o n t i n u o u s line is a modified N a k a - R u s h t o n
function fitted t o t h e d a t a . ( F r o m N e l s o n a n d T a k a h a s h i , c o p y r i g h t by J.
Physiol. (Lond.).)

Dark 9 10 11 12 13 14 15

Log (irradiance)

F I G . 2. M a g n i t u d e of p h a s e shift of h a m s t e r activity r h y t h m in r e s p o n s e to 503 n m

light pulses of v a r y i n g d u r a t i o n s a n d i r r a d i a n c e s a t C T 1 9 . O p e n circles a r e 3,600
sec; closed circles a r e 300 sec; o p e n t r i a n g l e s a r e 30 sec; a n d closed t r i a n g l e s a r e 3
33
sec s t i m u l i . C u r v e s a r e best fit modified N a k a - R u s h t o n f u n c t i o n s . ( F r o m N e l s o n
a n d T a k a h a s h i , c o p y r i g h t b y J. Physiol. (Lond.).)

total number of p h o t o n s in a stimulus (Figure 3B). Surprisingly, the system

is maximally sensitive to p h o t i c stimuli between 30 a n d 3,600 sec in
d u r a t i o n . T h e m a x i m u m q u a n t u m efficiency of p h o t i c integration occurs
in 300 sec stimuli.
T o s u m m a r i z e , the p h o t i c t h r e s h o l d for e n t r a i n m e n t , even u n d e r
o p t i m a l c o n d i t i o n s , is relatively insensitive a n d c o r r e s p o n d s t o thresholds
r e p o r t e d for cone p h o t o r e c e p t o r s . In a d d i t i o n , t h e system can integrate
light over very long d u r a t i o n s . F r o m a functional perspective, these
characteristics are clearly a d a p t i v e . T h e threshold is j u s t a b o v e the level of
full m o o n l i g h t a n d t h u s e n t r a i n m e n t w o u l d n o t be disrupted by this
i n a p p r o p r i a t e light source. F u r t h e r m o r e , the o p t i m u m for long d u r a t i o n
pulses would also render the system insensitive to very intense b u t brief
8 Light and Biological Rhythms in Man

0 1 2 3 4
Stimulus duration (log s)
F I G . 3. Sensitivity c u r v e s t o i r r a d i a n c e a n d t o t a l n u m b e r of p h o t o n s in a p u l s e
m e a s u r e d a t C T 1 9 . A, t h e relative sensitivity t o i r r a d i a n c e is p l o t t e d as a function
33
of s t i m u l u s d u r a t i o n . B, t h e relative sensitivity t o t o t a l p h o t o n s as a function of
stimulus duration. (From Nelson and T a k a h a s h i , c o p y r i g h t b y J. Physiol.
(Lond.).)

sources of light such as lightening. T h u s , b o t h the threshold a n d

integration characteristics of the p h o t i c e n t r a i n m e n t p a t h w a y in h a m s t e r s
m a k e this system optimally t u n e d to r e s p o n d to the daily l i g h t - d a r k cycle,
while at the same time r e m a i n i n g unresponsive to e n v i r o n m e n t a l "noise"
t h a t w o u l d interfere with stable e n t r a i n m e n t . T h e n a t u r e of the
p h o t o r e c e p t o r s m e d i a t i n g p h o t i c e n t r a i n m e n t r e m a i n to be established;
however, it is already clear t h a t specialized retinal elements m u s t exist. F o r
example, experiments using the rd m u t a t i o n in mice strongly argue 1t0h a t
r o d p h o t o r e c e p t o r s are n o t essential for the phase-shifting r e s p o n s e . In
a d d i t i o n , recent experiments from o u r l a b o r a t o r y show t h a t the spectral
sensitivity for phase-shifting in a n o t h e r species, the D j u n g a r i a n h a m s t e r ,
has a peak62 a r o u n d 475 n m which is clearly different from t h a t of
r h o d o p s i n . T a k e n together these results suggest t h a t the "circadian
p h o t o r e c e p t o r " is n o t a r o d a n d is either a cone or some other unidentified
p h o t o r e c e p t o r class in the retina.

Role o f m a c r o m o l e c u l a r synthesis in m a m m a l i a n
circadian rhythms

It has been k n o w n for a n u m b e r of years t h a t inhibitors of protein synthesis

o n 80S ribosomes p r o d u c e changes in the period length or p h a s e of
 Biological Rhythms: From Gene Expression to Behavior 9

circadian r h y t h m s of m i c r o o r g a n i s m s a n d invertebrates, including11,18,22,32,46,58,63

Eug-
lena, Acetabularia, Gonyaulax, Neurospora a n d Aplysia.
M o r e recently, we have d e m o n s t r a t e d t h a t the acute a d m i n i s t r a t i o n of t w o
protein synthesis inhibitors (anisomycin a n d cycloheximide), with t w o
different m e c h a n i s m s of action, are capable of inducing p r o n o u n c e d p h a s e
shifts in the circadian clock of h a m s t e r s , a n d t h a t the site of action of 1these 56 64
6
inhibitors a p p e a r s to be o n cells within the S C N r e g i o n . ' '
Interestingly, the p h a s e response curves for anisomycin a n d cycloheximide
in h a m s t e r s are similar to those m e a s u r e d for protein synthesis inhibitors
in m i c r o o r g a n i s m s a n d invertebrates, suggesting t h a t the biochemical
m e c h a n i s m s generating circadian oscillations in m a m m a l s m a y share
c o m m o n features with those found in very distantly related phylogenetic
groups.
In a d d i t i o n to causing p h a s e shifts by themselves, protein synthesis
inhibitors have also been s h o w n to block 20A2 the phase-shifting effects of light
pulses in Aplysia a n d Neurospora. W e have also found t h a t
light-induced p h a s e shifts can be blocked in h a m s t e r s suggesting t h a t the
signal t r a n s d u c t i o n p a t h w a y s for p h o t i c e n t r a i n m e n t m a y involve
m a c r o m o l e c u l a r synthesis ( T a k a h a s h i et al, unpublished results). Indeed,
as described below, light does induce the expression of a set of genes in the
SCN.
A l t h o u g h a requirement 6 1 424 for new R N A synthesis has been inferred from
genetic e x p e r i m e n t s , ' ' this issue has only recently been addressed with
the use of the reversible R N A synthesis inhibitor, 5,6-dichloro-l-/?-D- 41
ribofuranosylbenzimidazole ( D R B ) , in the Aplysia e y e . Pulses of D R B
caused p h a s e - d e p e n d e n t delays at phases between circadian time (CT) 20
a n d 10, a n d h a d n o effects from C T 1 0 to C T 2 0 . C o n t i n u o u s t r e a t m e n t with
D R B caused a d o s e - d e p e n d e n t lengthening of the circadian period. Similar36
effects of D R B o n p h a s e a n d period have been found in chick pineal c e l l s .
T h e D R B experiments suggest t h a t a critical period for transcription of
specific genes involved in the generation of circadian r h y t h m s occurs from
C T 2 0 to C T 1 0 in Aplysia, a n d from C T 1 8 to C T 1 9 in chick pineal cells.
While the inhibitor pulse experiments suggest t h a t a critical period for
m a c r o m o l e c u l a r synthesis exists within the circadian cycle, these experi-
ments d o n o t provide evidence t h a t such t r e a t m e n t23 s can arrest or s t o p the
m o t i o n of the circadian p a c e m a k e r . K h a l s a et al h a v e addressed this
issue by applying very long (5-44 h o u r s ) inhibitor pulses of cycloheximide
to Bulla eyes in vitro. Shorter pulses t h a t d o n o t extend past C T O have
minimal effects o n the r h y t h m . H o w e v e r , longer pulses t h a t extend b e y o n d
C T O delay the p h a s e of the subsequent r h y t h m precisely by the d u r a t i o n
t h a t the inhibitor pulse extends past C T O . T h e results are consistent with
the interpretation t h a t the m o t i o n of the circadian p a c e m a k e r is arrested
d u r i n g these long inhibitor pulses. By e x t r a p o l a t i n g the phases of the
r h y t h m s " b a c k w a r d s " t o w a r d s the end of the inhibitor pulses, it a p p e a r s
10 Light and Biological Rhythms in Man

t h a t the critical period for protein synthesis in Bulla begins in the late
subjective night (near C T O ) . By analogy to w o r k in the cell cycle, the
" S T A R T " of the circadian cycle therefore a p p e a r s to be n e a r C T O . T h u s ,
b o t h the inhibitor p h a s e shift a n d the circadian cycle progression
experiments are c o n c o r d a n t a n d suggest t h a t the circadian clock requires
m a c r o m o l e c u l a r synthesis d u r i n g a critical period for progression t h r o u g h
the cycle. In addition to inhibitor experiments, there is n o w direct evidence
t h a t the Drosophila period gene p r o d u c t s express circadian oscillations 1 34 , 1
which a p p e a r critical for the expression of behavioral r h y t h m s .

P h o t i c r e g u l a t i o n o f i m m e d i a t e - e a r l y g e n e s in t h e
SCN

A n u m b e r of g r o u p s have r e p o r t e d t h a t the p r o d u c t s of the25 p r o t o -

oncogene, c-fos, are stimulated by light in the S C N of r o d e n t4s7. In the
h a m s t e r26there is a p r o f o u n d induction of F o s i m m u n o r e a c t i v i t y a n d c-fos
mRNA in the S C N following light exposure d u r i n g the "subjective"
night. T o begin to address whether F o s mediates transcriptional events
involved in phase shifting of the circadian p a c e m a k e r , we have further
characterized the p h o t i c induction of c-fos m R N A utilizing in situ
hybridization techniques. By using defined light stimuli for which the
effects on the hamster's circadian r h y t h m of activity have been m e a s u r e d ,
26
we have c o m p a r e d the p h o t i c induction of c-fos m R N A a n d the behavioral
phase shifts p r o d u c e d by l i g h t . Specifically, we examined whether the
photic induction of c-fos was quantitatively correlated with p h a s e shifting
of l o c o m o t o r activity. Because F o s m u s t act in concert with a m e m b e r of
the J u3n 0 family of p r o t o - o n c o g e n e s to form the transcription factor,
A P - 1 27, we have also examined whether J u n a n d A P - 1 are induced by
light.

Photic induction of c-fos and j u n - B mRNA in the

SCN

Light exposure of h a m s t e r s d u r i n g the night, at C T 1 9 , causes a d r a m a t i c

induction of c-fos m R N A levels in the S C N (Figure 4). T h e increase in c-fos
m R N A levels illustrated here was induced by a 5 m i n u t e light pulse, after
which the a n i m a l was r e t u r n e d to d a r k n e s s for 25 minutes before sacrifice.
T h e dark-field p h o t o m i c r o g r a p h shows t h a t hybridization to c o m p l e m e n -
tary R N A p r o b e s for c-fos is localized mainly in the ventrolateral p o r t i o n
of each S C N , a n d extends dorsally from the S C N t o w a r d the 2periventricu-
7
lar region. Similar results are seen with jun-B m R N A l e v e l s . In c o n t r a s t
to the d r a m a t i c e n h a n c e m e n t of jun-B m R N A expression, light exposure at
C T 1 9 causes only a m o d e s t increase of c-jun m R N A hybridization in the
S C N . N o other effect of light o n c-fos or jun-B m R N A was observed in
 Biological Rhythms: From Gene Expression to Behavior 11

*
V., ^ . ' ί Λ

* « · -, ·

Light

..." * . * ·. '

200 μΜ 50 μΜ

F I G . 4. /rc siiw h y b r i d i z a t i o n of c-fos m R N A in t h e S C N of h a m s t e r s 30 m i n u t e s

after t h e o n s e t of a 300 sec light pulse at C T 1 9 . T o p p a n e l s s h o w t h e S C N r e g i o n
from a d a r k c o n t r o l a n i m a l . Left side is a d a r k field p h o t o m i c r o g r a p h of e m u l s i o n
a u t o r a d i o g r a p h y . R i g h t side is a h i g h e r m a g n i f i c a t i o n b r i g h t field p h o t o m i c r o -
g r a p h . L o w e r p a n e l s s h o w t h e S C N r e g i o n from a h a m s t e r receiving light a t C T 1 9 .
( F r o m K o r n h a u s e r et al.,26 c o p y r i g h t b y Cell Press.)

other areas of the b r a i n , including retinorecipient regions such as the

intergeniculate leaflet ( I G L ) (although we have detected a m o d e s t p h o t i c
induction in the I G L with F o s i m m u n o c y t o c h e m i s t r y ) . T h e elevation of
c-fos m R N A levels following light stimulation is rapid a n d transient, with
m a x i m a l levels occurring a b o u t 30 m i n u t e s after the onset of l i g h t . 26
Similar to c-fos m R N A , the highest levels of jun-B m R N A are detected 30
minutes after the onset of the light pulse, a n d h a v e r e t u r n e d nearly to basal
levels by 120 m i n u t e s . 27 Clearly, this t e m p o r a l correlation of the
expression of c-fos a n d of jun-B, suggests t h a t F o s a n d jun-B proteins are
available to dimerize with one a n o t h e r to form the transcription factor,
A P - 1 (see below).

Photic threshold of c-fos mRNA induction

As described a b o v e , the response of the h a m s t e r circadian system to
varying levels of illumination has been characterized. T h e m a g n i t u d e of
12 Light and Biological Rhythms in Man

the phase shift in l o c o m o t o r activity exhibits a m o n o t o n i e , saturable

dependence on the irradiance of the light stimulus. Figure 5 illustrates the
a m o u n t of behavioral p h a s e shift p r o d u c e d by 5 m i n u t e light pulses (503
n m wavelength) of varying irradiance occurring at C T 1 9 . 26 T o c o m p a r e
the sensitivity to light of the c-fos m R N A induction a n d of this p h o t i c
response of the circadian system, we performed in situ hybridization
following 5 m i n u t e light stimuli of different illumination levels, a n d
quantified the a m o u n t of specific c-fos hybridization within the S C N . T h e
threshold for induction of c-fos is indistinguishable from the threshold for
light-induced phase shifts, consistent with the idea c-fos is involved in the
e n t r a i n m e n t m e c h a n i s m of the p a c e m a k e r . 26

0 12 24
Time (h)

F I G . 5 . D e p e n d e n c e of c-fos m R N A i n d u c t i o n a n d phase-shifting of t h e l o c o m o t o r
activity r h y t h m o n light i r r a d i a n c e . L i g h t pulses of i n c r e a s i n g i r r a d i a n c e were
given at C T 1 9 . Left side s h o w s in situ h y b r i d i z a t i o n of c-fos m R N A in t h e S C N
region. P a n e l 1 s h o w s n o light ( d a r k c o n t r o l ) ; p a n e l s 2 - 5 s h o w i n c r e a s i n g light
i r r a d i a n c e . R i g h t side s h o w s the l o c o m o t o r activity r h y t h m s of h a m s t e r s e x p o s e d
to light pulses at C T 1 9 at t h e s a m e i r r a d i a n c e . ( F r o m K o r n h a u s e r et al.,26
c o p y r i g h t by Cell Press.)
 Biological Rhythms: From Gene Expression to Behavior 13

Circadian gating o f c-fos and j u n - Β induction by

light

Light exposure d u r i n g the subjective night of the h a m s t e r ' s circadian cycle

causes a p h a s e shift in the activity r h y t h m of the a n i m a l . Light exposure
d u r i n g the subjective day, by c o n t r a s t , p r o d u c e s n o effect o n the p h a s e of
the r h y t h m of l o c o m o t o r activity (Figure 6A). T h e p h o t i c i n d u c t i o n of
c-fos gene expression displays a similar p h a s e - d e p e n d e n c e : the ability of

3 9 14 19 21
Circadian time (h)

F I G . 6. C i r c a d i a n p h a s e - d e p e n d e n c e of l i g h t - i n d u c e d b e h a v i o r a l p h a s e shifts (A),
c-fos m R N A i n d u c t i o n in t h e S C N (B), a n d jun-Β m R N A i n d u c t i o n in t h e S C N of
h a m s t e r s . P h o t i c i n d u c t i o n of c-fos a n d jun-Β were m e a s u r e d at five p h a s e s of t h e
cycle. P h o t i c i n d u c t i o n w a s seen o n l y d u r i n g t h e subjective n i g h t a n d w a s
21
c o r r e l a t e d w i t h l i g h t - i n d u c e d p h a s e shifts of t h e activity r h y t h m . ( F r o m
K o r n h a u s e r et al., c o p y r i g h t by Science.)

light to stimulate c-fos is restricted to the same times of the circadian cycle
when light causes a behavioral p h a s e shift (Figure 6B). In situ hybridiza-
tion studies d e m o n s t r a t e t h a t the i n d u c t i o n of jun-B m R N A following a
5 m i n u t e light pulse are likewise d e p e n d e n t on circadian p h a s e (Figure
6C). Light pulses at C T 1 4 , which p r o d u c e a phase-delay in the h a m s t e r ' s
activity r h y t h m , dramatically elevate jun-B m R N A levels; light at C T 1 9
a n d at C T 2 1 causes a p h a s e - a d v a n c e a n d also induces jun-B m R N A .
D u r i n g the subjective d a y , at C T 3 a n d C T 9 , light neither p r o d u c e s p h a s e
shifts n o r induces the expression ofjun-B. T h e expression of c-fos a n d jun-B
m R N A , then, are b o t h gated by the circadian 27 p a c e m a k e r , a n d are b o t h
induced u n d e r the same t e m p o r a l c o n d i t i o n s .
14 Light and Biological Rhythms in Man

Photic regulation of AP-1 DNA binding activity in

the SCN

T h e co-induction of jun-B a n d c-fos m R N A by light suggests t h a t levels of

the heterodimeric A P - 1 complex in the S C N are increased by light
stimulation. T o determine whether light indeed alters a m o u n t s of A P - 1
factor capable of specifically binding to a consensus D N A recognition 27 site,
we performed D N A - b i n d i n g gel mobility shift a s s a y s . Whole-cell
extracts were p r e p a r e d from microdissections of h a m s t e r brain tissue
32
containing the S C N . These S C N extracts were i n c u b a t e d with a
P - l a b e l e d oligonucleotide c o n t a i n i n g consensus AP-1 sites a n d then
electrophoresed. A protein complex of retarded mobility was detected
using extract p r e p a r e d from h a m s t e r s 60 or 120 minutes after the onset of a
5 m i n u t e light pulse, presented at C T 1 9 S C N extracts from h a m s t e r s
receiving the same handling, but n o light pulse, c o n t a i n e d low levels of
A P - 1 binding activity m e a s u r e d by this in vitro assay. T h e time course of
A P - 1 induction was elevated at 1 h o u r a n d peaked at 2 h o u r s after light
stimulation. As in the case of c-fos a n d jun-B mRNA, the induction of A P - 1
by light was also p h a s e - d e p e n d e n t . Light induced A P - 1 at C T 1 4 a n d
C T 1 9 , b u t did n o t induce A P - 1 at C T 6 . T h u s , the circadian clock also gates
the p h o t i c induction of A P - 1 activity. These results d e m o n s t r a t e t h a t light
increases levels of A P - 1 protein complex implying t h a t p h o t i c stimulation
is coupled to A P - 1 regulated changes in gene transcription in the S C N .
T a k e n together, these experiments suggest t h a t p h o t i c e n t r a i n m e n t in
m a m m a l s m a y involve transcriptionally regulated signal t r a n s d u c t i o n
processes, a n d that A P - 1 m a y play a role in conveying p h o t i c information
within the circadian system.

Light induces p h o s p h o r y l a t i o n o f t h e t r a n s c r i p t i o n
f a c t o r , C R E B , in t h e S C N

In cell culture systems, the signaling p a49 t h w a y s t h a t regulate the expression

of c-fos have been extensively s t u d i e d . In P C 1 2 cells, the c A M P response
element binding protein, C R E B , mediates the transcriptional activation of
c-fos2+in response to stimuli t h a t elevate the second messenger c A M P or
Ca signaling p a t h w a y s1.3These
3 p a t h w a y s converge at the level of C R E B
p h o s p h o r y l a t i o n on S e r 49
a n d this p h o s p h o r y l a t i o n event is necessary for
the 2transcriptional
+ activation of C R E B - r e g u l a t e d target g e n e s . Because
C a is a likely second messenger in n e u r o t r a n s m i t t e r regulated p a t h w a y s
such as the r e t i n o h y p o t h a l a m i c input to the S C N , we asked whether
C R E B m a y play a role in the p h o t i c regulation of c-fos. In a very fruitful 11
collaboration with D a v i d G i n t y , Michael G r e e n b e r g a n d c o l l e a g u e s , we
have examined the p h o s p h o r y l1a3t3i o n of C R E B using a novel a n t i b o d y t h a t
specifically recognizes the S e r site of C R E B . Light exposure of h a m s t e r s
 Biological Rhythms: From Gene Expression to Behavior 15

at C T 1 9 caused the r a p i d p h o s p h o r y l a t i o n of C R E B in the S C N w i t h o u t

any detectable changes in the a m o2u n+t of "total C R E B " . D N A mobility
shift assays for C R E B using the C a / c A M P response element ( C a - C R E ,
sequence T G A C G T T T ) of the c-fos p r o m o t e r as a p r o b e , suggests t h a t the
major C a - C R E b i n d i n g factor in the S C N is C R E B r a t h 11 er than another
m e m b e r of the C R E B family such as A T F - 1 or C R E M .
T o determine w h e t h e r the light-induced p h o s p h o r y l a t i o n of C R E B is
p h a s e 11d e p e n d e n t , we examined h a m s t e r s d u r i n g the subjective d a y t i m e at
C T 6 . At this time, light fails t o cause 27 b o t h b e h a v i o r a l p h a s e shifts a n d
immediate-early gene i n d u c t i o n . Light also failed t o induce C R E B
p h o s p h o r y l a t i o n in the S C N at C T 6 . T h e absence of C R E B p h o s p h o r y l a -
tion was n o t d u e t o a decrease in C R E B because n o changes in the level of
total C R E B could be detected in the S C N at different phases of the
circadian cycle. T h u s , light-induced C R E B p h o s p h o r y l a t i o n a n d c-fos
i n d u c t i o n are b o t h gated by the circadian clock. These experiments
strongly suggest t h a t the circadian gating m e c h a n i s m regulating i m m e d i -
ate-early genes acts u p s t r e a m of the p h o s p h o r y l a t i o n of C R E B . It will be of
great interest to e x a m i n e kinases responsibility for C R E B p h o s p h o r y l a -
tion, as well as o t h e r regulatory sites in the c-fos p r o m o t e r such as the
serum response element a n d the factors t h a t interact with it.

U n r e s o l v e d issues c o n c e r n i n g F o s / A P - 1

T h u s far the relationship between behavioral p h a s e shifting a n d F o s / A P - 1

i n d u c t i o n is correlative. Are these t w o responses causally related, with
F o s / A P - 1 acting as a step in the p h o t i c e n t r a i n m e n t p a t h w a y ? O r ,
alternatively, are these t w o responses coincidental? At the present time it
has n o t been possible t o o b t a i n a definitive answer to this issue. T h e
current state is as follows. T h e p h o t i c i n d u c t i o n of F o s is a n a t o m i c a l l y
specific a n d restricted t o the S C N a n d t h e I G L . T h e p h o t i c i n d u c t i o n of
F o s in the S C N is tightly correlated with the b e h a v i o r a l phase-shifting
effects of light u n d e r a n u m b e r of c o n d i t i o n s . These include p h a s e
dependence a n d p h o t i c threshold. T h e F o s response is m o d a l i t y specific
a n d is associated 1 298 with photically induced b u t n o t non-photically i n d u c e d
p h a s e s h i f t s . ' This indicates t h a t F o s is related to the p h o t i c i n p u t
r a t h e r t h a n t o a phase-shifting m e c h a n i s m per se. T h e r e q u i r e m e n t for F o s
i n d u c t i o n h a1s8 1, tested
6 by p h a r m a c o l o g i c a 2
l 5, b l o c k a d e by NMDA 67
antagonists, ' by n o n - N M D A a n t a g o n i s t s a n d by m e c a m y l a m i n e .
In all three cases, agents t h a t blocked b e h a v i o r a l p h a s e shifting t o light
also a t t e n u a t e d F o s i n d u c t i o n . These experiments are all consistent with
the hypothesis t h a t F o s is a c o m p o n e n t of the p h o t i c e n t r a i n m e n t p a t h w a y
in r o d e n t s . H o w e v e r , experiments t h a t selectively r e m o v e 21 F o s by o t h e r
m e a n s such as antisense oligonucleotides or gene k n o c k o u t h a v e n o t yet
been c o m p l e t e d . Clearly, experiments t h a t directly a n d specifically test t h e
16 Light and Biological Rhythms in Man

r e q u i r e m e n t for F o s / A P - 1 in p h o t i c e n t r a i n m e n t are i m p o r t a n t goals for

future w o r k .

Conclusions

A l t h o u g h we are far from a concrete u n d e r s t a n d i n g of the clock

m e c h a n i s m in m a m m a l s , substantial progress o n a n u m b e r of fronts using
diverse a p p r o a c h e s suggests the following five generalizations.

7. Vertebrate circadian systems

There is a substantial diversity in the organization of circadian systems at

the physiological level, b u t only a few discrete loci within the nervous
systems of animals act as circadian p a c e m a k e r s . T h e m a m m a l i a n S C N , the
avian pineal gland a n d the a m p h i b i a n retina define three central elements
of the circadian system in vertebrates 4in , which 22 94 5, oscillator
459
5 5 and pacemaker
functions have been d e m o n s t r a t e d . ' ' '

2. A cellular circadian clock

In spite of the diversity at the physiological level, the circadian clock

a p p e a r s to be a cellular entity even in multicellular o r g a n i s m s . T h e
strongest case for this is seen in chick pineal cells in which a circadian
oscillator55system with a p h o t i c e n t r a i n m e n t p a t h w a y can be studied in cell
c u l t u r e . P h o t o r e c e p t i o n , clock function a n d m e l a t o n i n biosynthesis all
a p p e a r to be cellular properties of a single cell type, the pinealocyte.
Recently the S C N a n d the retina have also been 3 37
1 s h o w n to be capable of
expressing circadian r h y t h m s in cell c u l t u r e . '

3. Coupling pathways
T h e coupling p a t h w a y s (inputs a n d o u t p u t s ) of the circadian clock within
the o r g a n i s m are specific t o each system, a n d these specific differences in
coupling can explain m a n y a p p a r e n t differences in "clock m e c h a n i s m "
r e p o r t e d in various systems. A m o n g vertebrates, the differences between
the coupling p a t h w a y s in the m a m m a l i a n S C N a n d the chick pineal gland
29
are self-evident. E n t r a i n m e n t p a t h w a y s of S C N 55
cells are neurally
c o u p l e d , whereas those in chick pineal cells are n o t . These differences
in input p a t h w a y s can explain, for example, why neurally coupled
circadian oscillators such as the3 S90C,N4 are phase-shifted by n e u r o t r a n s m i t -
ters a n d second m e s s e n g e r s , while non-neurally 3 65
4 coupled circadian
oscillators such as the chick pineal gland are n o t . '