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    P e r g a m o nNeuroscienceand BiobehavioralReviews,Vo l. 18, No. 3, p p. 305-312. 1994Copyright1994 ElsevierScienceLtdPrinted in the USA . All rights reserved0149-7634/94$6.00 + .00

    0149 - 7634 ( 93 ) E0005- 7

    G ene Ex pression in Suprachiasmatic N ucleusand Circadian R hythms

    O G N I A N C H R I S T O V I K O N O M O V .1 A N D A L E X A N D E R G U E O R G I E V S T O Y N EV I "*Worcester Foundation for Experimental Biology, Shrewsbury, MA, 1545, USA,and tLaboratory of Physiology, Transport Medical Institute, Sofia 1233, Bulgaria

    R e c e i v e d 1 4 S e p t e m b e r 1 99 3

    I KONOMOV, O . C . AND A. G . STOYNEV. Geneexpression in suprachiasmatic nucleus and circadian rhythms. N E U -ROS CI BIOBE HAV REV 18(3) 305-312, 1 994 . -Th e suprachiasmat ic nucle i (SCN) conta in a c i rcadian sys tem consis ting ofcircad ian oscil la tor (clock) that is norm ally synchronized by the l ig ht/d ark cycle (input) a nd drives circadian rhythm s (output)tha t a re in t r ins ic to the SCN. Gene express ion of imm edia te-ear lygenes, such as c-fos andjun-B, in the vent ro la tera i SCN isassocia ted wi th c i rcadian synchroniza t ion by f ight pulses and subjec ted to c i rcadian cont ro l . Vasopress in and somatos ta t ingene expression show distinc t circad ian rhythms intrinsic to the do rsom ediai SCN with higher peptid e levels occurring duringthe day . In ad di t ion , embryonic SCN graf ted in to the bra in o f an SCN-les ioned ar rhythmic hos t def ine the per iod of theres tored c i rcadian locomo tor rhythm. Tak en together , these a nd other f indings sup por t the not ion tha t the express ion ofgenes under ly ing c i rcadian synchroniza t ion , osc i l la t ion and output takes p lace wi th in individual SCN neurons . However, noinforma t ion regarding the na ture a nd number of those neurons as wel l as the molecular mechanisms of the s ingle ce l l-c ircadianosci l la tor and output i s cur rent ly avai lable . Therefore , we prop ose a s imple two-neuron model as a f ramework for c r i t ica l lydiscussing the m olecu lar genetic strategies to analyz e the circa dian system in SC N.Circa dian system Sup rachiasm atic nucleus Gene expression

    T H E r e c u r r e n t v a r i a t io n s in m a m m a l i a n b e h a v i o r , p h y s i o l o g ya n d b i o c h e m i s t r y a r e c h a r a c t e r i z e d b y i n f e r e n t i a l s t a t i s t i c a la n a l y s is a s a s u m t o t a l o f a b r o a d s p e c t r u m o f r h y t h m s w i t hd i f f e r e n t f r e q u e n c i e s ( 2 2 ) . T h e r h y t h m s w i t h a p e r i o d l e n g t ho f a b o u t a d a y ( f r o m 1 8 t o 2 8 h ) k n o w n a s c i rc a d i a n h a v eb e e n m o s t e x t e n s i v e l y s t u d i e d . T h r e e i m p o r t a n t c h a r a c t e r i s t i c se m e r g e f r o m t h e s e s tu d i e s : (a ) t h e r h y t h m s a r e g e n e r a t e d a n dm a i n t a i n e d e n d o g e n o u s l y ; ( b ) c i rc a d i a n r h y t h m s c a n b e s y n -c h r o n i z e d b y e x t e r n a l cu e s ; a n d ( c) t h e r e is a n i n t e r n a l t e m p o -r a l o r d e r a m o n g t h e c i r c a d i a n r h y t h m s w i t h i n t h e o r g a n i s m( 3 8 ) . F o r d i d a c t i c a n d e x p e r i m e n t a l p u r p o s e s t h e c i r c a d i a ns y s te m i s p r e s e n te d a s a n i n p u t - o s c i l l a t o r ( c l o c k ) - o u t p u tm o d e l w h e r e t h e c i r c a d i a n o s c i l l a t o r i s sy n c h r o n i z e d b y i n p u ts i g n a l s a n d d r i v e s o u t p u t c i r c a d i a n r h y t h m s ( 6 5 ).I t i s a c c e p t e d t h a t t h e s u p r a c h i a s m a t i c n u c l e i (S C N ) c o n -t a i n a c i r c a d i a n o s c i l l a t o r ( c l o c k ) , r e c e i v e p h o t i c a n d n o n p h o -t i c i n p u t s f o r c l o c k e n t r a i n m e n t ( s y n c h r o n i z a t i o n ) , d e m o n -s t r a t e d i s t i n c t i n t r i n s i c c i r c a d i a n r h y t h m s , a n d p l a y a ni m p o r t a n t r o l e i n g e n e r a t i o n a n d c o o r d i n a t i o n o f b e h a v i o r a la n d e n d o c r i n e c i r c a d i a n r h y t h m s ( f o r r e c e n t r e vi e w s se e R e f .3 4 ,3 5 ,4 9 ,5 4 ). A c c o r d i n g t o th e i n p u t - c l o c k - o u t p n t m o d e l

    S C N c o n t a i n a c i r c a d i a n s y s te m a n d s e r v e c u r r e n t l y a s t h e o n l ya v a i l a b l e m a m m a l i a n b r a i n l o c u s t h a t c a n b e u s e d t o s t u d y t h en a t u r e o f t h e c i r c a d i a n c l o c k (6 5 ).T h e c e n t r a l f u n d a m e n t a l q u e s t i o n i n t h e s t u d y o f t h e c i r ca -d i a n s y s t e m i n S C N i s w h e t h e r t h e c i r c a d i a n c l o c k i s a n i n t r a -c e l l u l a r p r o p e r t y o r i s th e r e s u l t o f i n t e r c e l l u l a r i n t e r a c t i o n s .T w o g e n e r a l a p p r o a c h e s h a v e b e e n a p p l i e d to a n s w e r t h isq u e s t i o n . T h e f i r s t i s u s i n g t h e t o o l s o f c e l l u l a r n e u r o b i o l o g ya n d a t t e m p t s e i t h e r t o i d e n t i f y th e c e l l t y p e r e s p o n s i b l e f o r t h ec l o c k f u n c t io n o r t o u n d e r s t a n d t h e s p e c i fi c n e u r o n - n e u r o n o rn e u r o n - g l i a i c e ll c o m m u n i c a t i o n s t h a t s y n c h r o n i z e a n d s t a b i -l i z e t h e c i r c a d i a n o s c i l l a t o r i n S C N . O n e s t r a t e g y i s t o m a n i p u -l a t e S C N c u l t u r e s i n v i t r o t o e n s u r e t h e p r e v a l e n c e o f p a r t i c u -l a r n e u r o n s o r g l i a l c e l ls a n d s u b s e q u e n t l y t r a n s p l a n t t h e c e l l si n t o t h e b r a i n o f a n S C N - l e si o n e d a r r h y t h m i c h o s t . A n o t h e rs t r a te g y i s a c o m b i n a t i o n o f e l e c t r o p h y s i o lo g y o f b r a i n s li ce sc o n t a i n i n g S C N w i t h p h a r m a c o l o g i c a l m a n i p u l a t i o n o f i o nc h a n n e l s, s p e c if i c m e m b r a n e r e c e p t o rs , o r n e u r o n - g l i a l c o m -m u n i c a t i o n s ( 3 5 , 4 4 ) . T h e s e c o n d a p p r o a c h i s t h e m o l e c u l a rg e n e t ic a p p r o a c h . I t is b a s e d o n o b s e r v a t i o n s i n n o n m a m -m a l i a n c i r c a d i a n m o d e l s a n d a s s u m e s t h a t t h e c i r c a d i a n

    Requests for repr in ts should be addressed to O . C . Iko nom ov, Wo rces ter Foun dat ion for Exper imenta l B iology, 222 Maple Avenue,Shrewsbury, MA 1545.3 0 5

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    306 IKONOMOV AND STOYNEVclock mechanism is an intracellular property and relies onthe circadian variations in the expression of specific genes(12,18,53,66). This approach consists of initial reductive andsubsequent integrative steps. The reductive step should resultin isolation of the circadian system-related gene(s) and charac-terization of its protein product. In the integrative step theimportance of this gene should be tested in a suitable circadianmodel. For example, a mammalian circadian mutant could be"rescued" by introducing in its genome the intact gene of thecontrol (wild type) animal, thus proving the role of this partic-ular gene in the regulation of circadian rhythms.Several recent observations suggest that gene expression inSCN is associated with circadian synchronization and output.The aim of this review is to discuss the molecular geneticstrategies to the circadian function of SCN. So far, this topicis only partially addressed (4,67). In addition, the availableevidence is considered in the context of the extensive neurobi-ology of SCN that potentially offers an advantage over theother known circadian models. Subsequently, a two-neuronmodel of the circadian system in SCN is proposed. The modelsuggests feasible experiments for further understanding thecircadian system in SCN. It also reflects the gaps in our cur-rent knowledge-theoretically, we assume that single neuronspossess a molecular circadian system, a term for the expecta-tion that molecular mechanisms of gene expression underlyingcircadian synchronization, oscillation and output should becoordinated within a single cell . Finally, the model predictssome limitations in SCN-oriented research and suggests alter-native molecular genetic strategies for studies of the circadiansystem in mammals.Neurobiology of SCN-Located Circadian System andEvidence for the Role o f Gene Expression inCircadian Time-Keeping

    In all mammals, SCN are located just above the optic chi-asm and laterally from the ventral part of the third ventricle(38). In the adult rat, SCN comprise of 16-20000 neurons,associated glia and nerve terminals (71,72). SCN neurons andpresynaptic terminals are positive for more than 25 neuro-transmitters and peptides (72,73). Pract ically all neurons pro-duce gamma-aminobutyric ac id - a ma jor inhibitory transmit-ter. Approximately 4000 neurons also express vasoactiveintestinal peptide (VIP) whereas 6000 express vasopressin (VP)(37). In addition to the chemical synapses, the adult SCNshow diverse cell-to-cell interactions with multiple contactsbetween neuronal somata without gap junctions as well be-tween neurons and glial cells. The relation of this elaboratecellular network to the circadian function is sti ll unclear(5,35,72).Morphologically and electrophysiologically SCN can be di-vided into ventrolateral and dorsomedial portions (15,71,74).These two parts originate from different regions of the germi-nal epithelium (11). The ventrolateral portion of SCN receivesphotic and nonphotic afferents, displays light-induced expres-sion of several immediate-early genes and is rich in VIP-producing neurons (2,37,54,72). The neurons in this area dem-onstrate a persistent circadian rhythm in f'lring rate in vitroeven after dissociation from the dorsomedial part o f SCN(15). The dorsomedial SCN do not receive visual afferentsand contain somatostatinergic and vasopressinergic neurons(5,61,72). The latter are responsible for the circadian rhythmof VP in cerebrospinal fluid (58,60).SCN receive photic input from the retina and intergenicu-late leaflet of the lateral geniculate body. Glutamate and neu-

    ropeptide Y are among the major transmitter candidates formediating synchronizat ion to the light /dark cycle, respectively(2,54). The major nonphotic projections come from the nu-cleus raphe dorsalis and use serotonin as a neurotransmitter(2,35).It should be noted that the circadian pacemaker in SCNoperates in embryonic life before the establishment of retino-hypothalamic and local synaptic contacts and long before themanifestation of postnatal behavioral circadian rhythms (ratSCN appear as a morphological entity on ED 18) (36,48). Thedevelopment of the oscillator is predominantly under geneticcontrol whereas the role of the environment seems negligiblebecause embryonic SCN transplanted in the brain of adultarrhythmic host generates a circadian rhythm with the sameperiod as the normal nuclei in situ (11,44).Many conclusions regarding the oscillator in SCN are madepossible by the discovery of several circadian rhythms intrinsicto both embryonic and adult SCN but considered distal to theoscillator: (a) the rhythm in metabolic activity as studied bylabeled 2-deoxyglucose utilization (57); Co) in mean action po-tential rate (15); and (c) in vasopressin gene expression (31).In the rat, the rhythm of metabolic activity can be detected onembryonic day 19 whereas the VP mRNA circadian rhythmappears on embryonic day 21 when the circadian rhythm ofelectric activity is barely measurable (36,48). In addition anintrinsic circadian rhythm for somatostatin mRNA and pep-tide level is documented in adult rats (61,69). Important ly,the circadian variations in all these variables are a t maximumduring the subjective day irrespective of the overt behavioralcircadian rhythmicity, an observation that links them firmlyto the circadian pacemaker.Chronic infusion of tetrodotoxin (blocker of sodium-dependent action potentials) reversibly inactivates the inputand output pathways without altering the oscillator of SCN-located circadian system. Therefore, action potentials seemnecessary for coupling of the oscillator but not for its time-keeping function (59). The discovery of the tau mutant ham-ster, where a single autosomal gene mutation is associatedwith shorter circadian period of wheel-running (45), demon-strates that gene expression affects the mammalian circadianperiod similar to observations described in Drosophila andNeurospora (12,53). Destruction of SCN in mutant and con-trol hamsters eliminates the circadian rhythm of wheel-running. Transplantation of embryonic brain tissue contain-ing SCN into the third ventricle of arrhythmic adult hostsrestores the rhythm. Importantly, the restored circadianrhythm exhibits the period of the donor genotype (43). Thesefindings unequivocally implicate gene expression in SCN areain the control of circadian period length.Vasoactive Intestinal Peptide, Immediate-Early Genes andCircadian Synchronization in SCN

    The majority of afferent projections reaching the ventro-lateral SCN make synaptic contacts with VIP-producing neu-rons (2,72). The latter are therefore expected to play an impor-tant role in the mechanisms of entrainment. VIP-producingneurons participate in local SCN circuitry and also projectoutside the SCN (74). Other peptides, such as gastrin-releasingpeptide and peptide-histidine-isoleucine colocalize with VIP.An equimolar mixture of the three peptides produces largephase delays in rat circadian locomotor rhythm only whenapplied in SCN and around locomotor activity onset. Thelevel of VIP is lower under conditions of constant light andincreases under constant darkness (2). There is a diurnal varia-

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    G E N E E X P R E S S I O N I N S C N 307t i o n o f V I P m R N A ( 2 ) a n d p e p t i d e l e v e l ( 6 8 ) w i t h h i g h e rv a lu e s d u r in g th e n ig h t . N o c i r c a d ia n o s c i l l a t io n s in V IPm R N A a r e f o u n d u n d e r c o n s t a n t d a r k n e s s o r a f t e r e n u c i e -a t i o n ( 6 9 ) . V I P r e c e p t o r s a r e s t a b l e e v e n i n p r e se n c e o f l i g h t /d a r k c y c l e ( 5 2 ) . H o w e v e r , t h e r o l e o f V I P n e u r o n s i n t h em e c h a n i s m o f e n t r a i n m e n t r e m a i n s c o n t r o v e r s i a l . T h e r e l a -t i v e l y s l o w c h a n g e i n V I P e x p r e s s i o n a f t e r i l l u m i n a t i o n s u g -g e st s t h a t V I P n e u r o n s m a y c o n v e y i n f o r m a t i o n f o r t h e l e n gt ho f t h e l i g ht p h a s e b u t m a y n o t p a r t i c ip a t e i n t h e m e c h a n i s m so f s y n c h r o n i z a t i o n b y l i g h t p u l se s , t h a t a r e c o n s i d e r e d t o b ea l m o s t i m m e d i a t e ( 3 3, 3 8) .L i g h t - i n d u c e d S C N e x p r e s si o n o f s e v e ra l i m m e d i a t e - e a r l yg e n e s ( IE G s ) h a s r e c e n t ly a t t r a c t e d g re a t a t t e n t io n (3 , 1 4 a ,2 8 , 4 7, 5 5 ,6 4 ) . T r a n s c r i p t i o n a l a c t i v a t i o n o f I E G s i n c l u d i n g t h e. los and ju n f a m i l y i s a c o m m o n c e l lu l a r r e s p o n se t o e x tr a ce U u -l a r s ti m u l i s u c h a s h o r m o n e s , g r o w t h f a c t o r s a n d n e u r o t r a n s -m i t t e r s. T h e s t i m u l u s - t r a n sc r i p t i o n c o u p l i n g o f t h e s e g e n e s ism e d i a t e d b y s e c o n d m e s s e n g er s ( c A M P , C a 2 +, d i a c yl g l y ce r o l ).T r a n s c r i p t i o n a l a c t i v a t i o n i s f o l l o w e d b y c y t o p l a s m i c t r a n s l a -t i o n o f F o s , J u n a n d o t h e r p r o t e i n s . T h e p r o t e in s a r e t h e nt r a n s l o c a t e d t o t h e n u c l e u s w h e r e t h e y a p p e a r t o f o r m a v a r i-e t y o f p r o t e i n c o m p l e x e s . T h e h e t e r o d i m e r c o m p l e x e s o f F o sa n d J u n e x h i b i t s p e c i f i c D N A b i n d i n g t o s o - c a l l e d A P - 1 s e -q u e n c e . T h e f in a l r e s u l t o f t h i s m o le c u la r c a s c a d e i s a l t e ra t io ni n t r a n s c r i p t i o n o f t a r g e t g e n e s b e a r i n g t h e s p e c i f i c D N A -b in d in g s i te o n th e i r r e g u la to ry r e g io n s (3 9 ) .W h a t i s sp e c if i c t o S C N i n d u c t i o n o f I E G s b y l i g h t pu l s es ?T h e i n d u c t i o n o f c-fos , jun-B, N G F I - A a n d N G F I - B m R N A sa n d c - F o s a n d J u n - B p r o t e i n s t a k e s p l a c e i n t h e v e n t r o l at e r a lS C N o f a d u l t r a t s a n d h a m s t e r s o n l y w h e n l i g h t p u ls e s a rea p p l i e d d u r in g th e s u b je c t iv e n ig h t (2 8 , 2 9 , 5 5 ,5 6 , 6 4 ) . T h e c-losa n d j u n - B i n d u c t io n o c c u r s a f t e r l i g h t p u l s e s a s s h o r t a s 5m i n a n d r e a c h es m a x i m a l m R N A l e v e l o n l y 3 0 ra i n a f t er l i g h te x p o s u re (2 8 , 2 9 ) . T h e s a m e l ig h t p u l s e s s h i f t c i r c a d ia n lo c o -m o t o r r h y t h m s w i t h d i f f e re n t b e h a v i o r a l o u t c o m e i n e a r l y a n dla t e s u b je c t iv e n ig h t r e s u l t in g in p h a s e d e la y o r a d v a n c e in th eo n s e t o f l o c o m o t o r a c t i v it y (2 9 ,3 8 ). T r e a t m e n t w i t h n o n c o m -p e t i t i v e b l o c k e r o f N M D A r e c e p t o r s t h a t p r e v e n t s t h e b e h a v -io ra l p h a s e s h i f t (1 0 ) a l s o b lu n t s t h e l i g h t - in d u c t io n o f c-fos int h e v e n t r o l at e r a l S C N ( 1 ). I n t h i s c o n t e x t I E G s a p p e a r t o f o r ma n i n p u t t o t h e o s c i l l a t o r f o r i t s s y n c h r o n i z a t i o n t o t h e l i g h tp u l se s . T h e r e i s n o e v i d e n c e f o r c e l l u la r c o l o c a l i z a t io n o f I E G sa n d V I P g e n e e x p r e s s i o n ( 6 7 ) . P r o t e i n e x t r a c t s o f S C N o b -t a in e d 2 h a f t e r l i g h t p u l se d u r in g th e s u b je c t iv e n ig h t ( s u p p o s -e d l y c o n t a i n i n g c - F o s - J u n - B p r o t e i n h e t e r o d i m e r s ) p o s s e s sm a x i m a l b i n d i n g a c t i v i t y t o c o n s e n s u s A P - I s e q u e nc e s i n ge lr e t a rd a t io n a s s a y (2 9 ) . T a k e n to g e th e r th e s e o b s e rv a t io n s s u p -p o r t t h e f o l l o w i n g s e q u e n c e o f e v e n t s: l ig h t - , r e t i n o h y p o t h a -l a m ic t r a c t -- , e x c i t a to ry t r a n s m i t t e r (g lu t a m a te ) - -, N M D Ar e c e p t o rs - , i n d u c t i o n o f I E G s -- , c l o c k g e n e / s / e x p r e s s i o n .T h e p o s s i b l e i n t e r a c t i o n b e t w e e n t h e I E G s p r o d u c t s a n d t h ec l o c k g e n e / s / r e g u l a t o r y r e g i o n a s t h e i r p o t e n t i a l t a r g e t i sc o m p l i c a t e d b y t h e c o n s t i tu t i v e a n d a b u n d a n t p r e s e n c e o fo t h e r m e m b e r s o f J u n p r o t e i n f a m i l y i n S C N . T h e l a t t e r m a yf o r m h o m o - o r d i ve r se h e t e ro d i m e r s a n d o c c u p y th e s a m eD N A b i n d i n g d o m a i n s a s th e h e t e r o d i m e r s o f l i g h t- i n d u c e dc - F o s a n d J u n - B ( 4) .U n d e r c o n d i t i o n s o f c o n s t a n t i l l u m i n a t i o n c-los p r o t e i nd e m o n s t r a t e s c i r c a d i an r h y t h m w i t h 2 - t im e s h i g h e r l e ve ls d u r -i n g t h e s u b j ec t i v e n i g h t ( 1 4b ) . U n d e r c o n s t a n t d a r k n e s s c-losi s r e p o r t e d to b e s l ig h t ly h ig h e r o n ly a t c i r c a d ia n t im e 2 2 ( l a t es u b j e c t i v e n i g h t ) ( 6 4 ) . T h e r h y t h m m a y r e f l e c t t h e c i r c a d i a nc o n t r o l o f c-fos i n d u c t i o n b y l i g h t o r th e i n c r e a s e d l o c o m o t o ra c t i v i t y o f t h e r a t s d u r i n g t h e s u b j e c t i v e n i g h t . I t s h o u l d b em e n t i o n e d t h a t t h e c i r c a d i a n p r o f i l e o f th e c - los e x p re s s io n i s

    o p p o s i t e t o t h e o t h e r S C N - i n t r i ns i c c i r c a d ia n r h y t h m s . C - f o si n d u c ib i l i t y in r a t S C N i s r e p o r t e d a l s o a s e a r ly a s o n e m b ry -o n i c d a y 1 8. T h e i n d u c t i o n o f c - f o s i n t h e f e t u s is m e d i a t e dt h r o u g h D i - d o p a m i n e r e c e p to r s a n d d o e s n o t d e m o n s t r a t e c i r -c a d ia n v a r i a t io n s (7 5 ) .N o n - p h o t i c s t i m u l i a l s o s h i f t t h e c i r c a d i a n l o c o m o t o rr h y t h m b u t w i t h o u t c-fos i n d u c t i o n i n S C N ( 3 3) . A s o p p o s e dto th i s , m c la to n in in j e c t io n s c a n in d u c e c-fos e x p re s s io n inS C N a t c i r c a d i a n t i m e s w h e n m e l a t o n i n i s n o t e f f e c t iv e b e h a v -io ra l ly (2 7 ). F in a l ly , i n t r a c e re b ro v c n t r i c u la r i n j e c t io n s o f c a r -b a c h o l m im ic l i g h t - in d u c e d p h a s e s h i f t s i n f r e e - ru n n in g lo c o -m o t o r r h y t h m i c it y w i t h o u t c-fos i n d u c t io n (9 ) . T h e s e s tu d ie ss u g g e s t t h a t c-fos e x p re s s io n i s n o t a n in d i s p e n s a b le p a r t o ft h e s y n c h r o n i z a t i o n m e c h a n i s m i n a d u l t a n i m a l s a n d m a y b ei m p o r t a n t o n l y i n s y n c h r o n i z a t i o n b y l ig h t p u l se . I t s e em sd i f f ic u l t t o d e t e r m i n e w h i c h i f a n y o f t h e I E G s r e a ll y l ea d s t oth e o s c i l l a to r . O n e p o s s ib i l i ty s e em s to t e s t k n o w n t a rg e t g e n e sf r o m o t h e r s y s t e m s i n t h e c o n t e x t o f S C N . A n o t h e r s t r a t e g ym a y b e t o c l o n e g e n e s i n d u c e d b y l i g h t i n S C N a f t e r I E G s( s e c o n d w a v e g e n e s ) a n d a m o n g t h e m i d e n t i f y t h e p o t e n t i a lt a rg e t g e n e s .I t i s n o t e w o r t h y t h a t a n o t h e r t r a n s c r i p t i o n f a c t o r i s e x -p re s s e d s e l e c t iv e ly in S C N b u t b e h a v e s d i f f e re n t ly f ro m th eI E G s m e n t i o n e d a b o v e . T h i s g e n t i s c a l l e d O c t - 2 a n d i s am e m b e r o f t h e P O U f a m i l y o f o c t a m e r b i n d i n g p r o t e i n s ( 19 ).I n t h e f e t al S C N O c t - 2 m R N A i s p r e s en t a s e a r l y a s o n e m b r y -o n ic d a y 1 8 a n d i s a l s o d e te c t e d in s u p ra o p t i c n u c le i . I n a d u l tr a t h y p o t h a l a m u s O c t - 2 i s e x p r e s s e d p r e d o m i n a n t l y i n S C Na n d d o e s n o t d e m o n s t r a t e c i r c a d i a n v a ri a t i o n s u n d e r c o n s t a n td a rk n e s s . L ig h t p u l s e s in d u c in g c-fos t r a n s c r ip t i o n d o n o t a f -f e c t m a r k e d l y O c t - 2 m R N A l ev el ( 50 ). A n o t h e r g e n e t h a t i se x p re s s e d s e le c t iv e ly in S C N i s V G F , a n a m e d e s ig n a te d fo r ag e n e e n c o d in g a 7 1 2 a m in o a c id p ro te in r e g u la t e d b y n e rv eg r o w t h f a c t o r i n a d r e n a l c h r o m a f f i n P C I 2 c el ls . I t i s p r e s e n ts t r o n g l y i n S C N a t p o s t n a t a l d a y 4 a n d i n t h e d o r s o m e d i a lS C N a n d in t e rg e n ic u la t e l e a f l et o f a d u l t r a t b ra in (7 2 ). B o thg e n e s a re s e l e c t iv e ly e x p re s s e d in S C N b u t d o n o t f i t t o th ec i r c a d i a n i n p u t - c l o c k - o u t p u t m o d e l b e c a u s e n o l i g h t i n d u c -ib i l i t y o r c i r c a d ia n v a r i a t io n s a re r e p o r t e d . H o w e v e r , t h e g e n ee x p re s s io n r e l a t e d to th e c i r c a d ia n fu n c t io n m a y n o t n e c e s s a r -i l y e x h ib i t s u c h c h a ra c te r i s t ic s . T h e re fo re , fu r th e r e x p e r im e n -t a l e v id e n c e is n e e d e d r e g a rd in g th e ro l e o f t h e s e tw o g e n e s inS C N f u n c t i o n . I t s h o u l d b e n o t e d , h o w e v e r , t h a t i n a d d i t i o nt o i t s c ir c a d i a n f u n c t i o n S C N h a v e a l s o o t h e r r e g u l a t o r y f u n c -t io n s (4 1 ) th a t m a y b e b a s e d o n s p e c i f i c g e n e e x p re s s io n .A s a l r e a d y m e n t i o n e d , t h e l ig h t i n d u c t i o n o f c-los , jun-B,N G F I - A a n d N G F I - B i s u n d e r c i r c a d i a n c o n t r o l b e c a u s e t h es a m e l ig h t p u l s e s a re in e f fe c t iv e d u r in g th e s u b je c t iv e d a y(2 8 , 5 5 , 5 6 ) . T h i s o b s e rv a t io n o f fe r s a n o th e r s t r a t e g y fo r s tu d -i e s a im e d a t t h e o s c i l l a to r , n a m e ly to d i s s e c t t h e s t im u lu s -t r a n s c r i p t io n c a s c a d e f o r a p a r t i cu l a r I E G a n d l o c a t e t h e s t e pu n d e r c i r c a d ia n c l o c k c o n t r o l . R e c e n t ly t h is w a s a t t e m p t e df o r t h e m e c h a n is m o f c-los t r a n s c r ip t io n (17 ) . T h e r e g u la to ryr e g i o n o f c-fos g e n e c o n ta in s a s e q u e n c e c a l l e d c A MP -re s p o n s iv e e l e m e n t . In c re a s e in in t r a c e l lu l a r c A MP c o n te n t o rC a + a c t i v it y tr i g g er s C R E B ( c A M P r e s p o n s e e l em e n t b i n d i n gp r o t e i n ) p h o s p h o r y l a t i o n . T h e p h o s p h o r y l a t e d C R E B b i n d st h e c A M P - r e s p o n s i v e e l e m e n t a n d t u r n s o n c-fos t r a n s c r ip -t i on . I m m u n o h i s t oc h e m i s t r y o f t h e p h o s p h o r y l a te d a n d n o n -p h o s p h o r y l a te d f o r m s o f C R E B r e v ea ls t h a t C R E B p h o s p h o r -y la t io n t a k e s p l a c e o n ly d u r in g th e s u b je c t iv e n ig h t w h e n l ig h tp u l s e s in d u c e c-los t r a n s c r i p ti o n . C R E B i s n o t p h o s p h o r y l a t e dd u r i n g t h e s u b j e c t i v e d a y . T h i s o b s e r v a t i o n s h o w s t h a t t h ec i r c a d i a n c o n t r o l o f t h e c-los s t im u lu s - t r a n s c r ip t io n c a s c a d el ie s u p s t r e a m t o C R E B p h o s p h o r y l a t i o n .

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    308 IKONOMOV AND STOYNEVFinally, transgenic mice with null mutation in c-fos proto-oncogene (25) may offer another useful model for decipheringthe genetic basis of circadian synchronization. To look forvariations in the behavioral rhythms o f currently availabletransgenic mice vs. their controls seems unique combinationof advanced molecular genetics and biostatistics that mayform a potentially fruitful area of research.

    Direct Molecular Strategies to the Genetic Basis ofCircadian Oscillator in SCNThree different strategies aim at genes whose expressionis directly related to the circadian oscillator: (a) isolation o fSCN-specific proteins in mutant hamsters; (b) cloning of ratSCN-specific genes; and (c) SCN-expression of Drosophila pergene homologs.Two sets of SCN proteins that markedly differ betweenthe control (with free running per iod of wheel-running 24 h),heterozygous (22 h) and homozygous mutant hamsters (20 h)are distinguished by two-dimensional gel electrophoresis. A

    protein with molecular mass of 33 KDa (P33tau) is missingonly in SCN extracts from homozygous mutants. Similar insize, protein P33b appears as a doublet in heterozygous ham-sters. Finally, another protein, P32tau, is a chain of spots witha striking difference in pattern among the groups. The samedifferences are observed however in protein extracts from ce-rebral cortex. Therefore, these putative clock proteins seemto be present throughout the brain (26). Their sequence andpossible circadian variations are not yet known.The unique role of SCN in the regulation of circadianrhythms allows the assumption that there are SCN-specificgene(s). Differential screening of a subtracted SCN eDNAlibrary with SCN and hypothaiamic population probes haveso far failed to detect genes expressed selectively in SCN(8,20). The same technology was successfully applied in isola-tion of several eDNA clones strongly induced in rat dentategyrus after kainic acid treatment (8,42).Another approach is based on the assumption that the mo-lecular mechanisms of circadian time-keeping share genes con-served during evolution. Three recent reports offer evidencethat mammalian homologs of Drosophila period (per) geneare present in SCN. Ishida et al. (23) use a 2.5 kbp restrictionDNA fragment homologous to the Drosophila per gene GlyThr repeat (a stretch containing 20 Gly Thr pairs). The 2.1kbp open reading frame of this DNA presumably codes forprotein containing the Gly Thr repeat. The fragment gives ahybridization signal in various brain nuclei, pineal body, cor-pus striatum, etc. and shows light/dark variations in SCNneurons with higher intensity occurring during the day. Diur-nal variations are absent for the signal in the dentate gyrus.The per gene repeat, however, is not indispensable in Dro-sophila since its deletion does not seem to alter the circadianrhythm of the transformed flies (32). A restriction fragmentof the per gene missing the Gly Thr repeat (1.9 kbp) givesdistinct hybridization bands with genomic DNA from differ-ent mammalian species. Using specific primers and polymer-ase chain reaction Maier et al. (32) report amplification of perhomologs from several eDNA libraries and polyadenylatedRNA from mouse and hamster SCN, hamster heart, andAplysia and Bulla eyes. The results suggest that per homologsare transcribed in all studied circadian oscillators, includingSCN. The per homologs are probably not abundant becausefull-length clones are not isolated f rom the eDNA librariesused in spite of the successful homolog amplification from thesame source. Finally, anti-per polyclonal antibody against a

    peptide from the gene region that is tightly conserved amongDrosophila species, recognizes in immunoblots of SCN ex-tracts a protein with molecular mass of 115 kDa and a hetero-geneous antigen /s/ rangin g from 160-170 kDa. In situ immu-nohistochemistry gives a prominent signal in the dorsomedialpart of SCN. Strong staining is also found in other parts ofthe brain, for example in the hypothalamic supraoptic andparaventricular neurons and fibers. No reliable daily cyclingin the intensity and the number of labeled cells in the SCN isobserved. The same antibody also stains cells of Aplysia andBulla eyes that are known models of circadian oscillators (62).In summary, in spite of the many studies the ultimate stepof isolating gene/ s/ and/o r proteins participating in SCN-located circadian oscillator remains to be achieved.Circadian Rhythms in Vasopressin and SomatostatinGene Expression in Rat SC N

    Rat VP gene consists of 3 exons and 2 introns with totallength of 2.2 kbp. The VP mRNA contains approximately 750nueleotides (around 250 bases form the polyadenylated tail atthe 3' end). The translation of this message results in synthesisof a precursor peptide, pro-vasopressin-neurophysin, that has3 peptide components-neurophysin, glycopeptide and VP(31). Vasopressinergic somata are located mostly in thedorsomedial part of SCN. The vasopressinergic projectionsoutside the SCN reach mainly neurons in the hypothalamicparaventricular nucleus, dorsomedial nucleus and organumvasculosum laminae terminalis (21). Within the adult murineSCN there is a complex network where vasopressinergic axonsmake synaptic contacts predominantly with dendrites of othervasopressinergic neurons. These neurons are also engaged inextensive membrane to membrane appositions and multiplepuncta adherentia (5,72). Pharmacological studies combinedwith electrophysiology of SCN slices in vitro reveal that SCNneurons contain VI of the two VP membrane receptor sub-types (30) . Interestingly, the circadian sensitivity to VP ishigher during the subjective night (i.e., opposite to the normalpattern of VP synthesis and secretion).The vasopressinergic neurons of the SCN form a separatephysiological system, different from the well-known magno-cellular hypothaiamic system that is implicated in the regula-tion of water-salt balance and blood pressure. The SCN-located neurons are responsible for the circadian rhythm ofvasopressin level in cerebrospinal fluid (58). The rhythm re-sembles the circadian variations in mean firing activity withhigher values seen during the subjective day, although severaldifferences are found on closer inspection o f the experimentaldata (15,16). The circadian variations in VP level in cerebro-spinal fluid disappear after complete lesions of SCN (60) andare manifested after transplantation of embryonic SCN to thebrain o f VP-deficient Brattleboro rats (14). In SCN explants,tetrodotoxin treatment blocks the secretion of VP, suggestingthat the release is triggered by sodium-dependent action poten-tiais (13) . The rhythm of vasopressin secretion has been re-cently documented in chronic SCN cultures where it shows aperiod o f 27 h (40).The VP content in SCN oscillates in a similar manner to itscerebrospinal level and shows a free-running period of 24 1/2h (76). On this basis, one may expect that the circadian regula-tion of VP gene expression is at the level of mRNA transcrip-tion and processing. Indeed, in situ hybridization studies ofcontrol and VP-deficient Bratfleboro rats show that VPmRNA level in SCN is markedly higher during the day than atnight (70). The VP mRNA also demonstrates different sizes

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    G E N E E X P R E S S I O N I N S C N 3 09b e i n g l o n g e r i n t h e d a y - t i m e t h a n a t n i g h t b e c a u s e o f v a r i a -t i o n s i n p o l y a d e n y l a t e d t a i l le n g t h ( 5 1 ). T h e c i r c a d i a n v a r i a -t i o n s i n b o t h s t u d i e s a r e s p e c i f i c c h a r a c t e r i s t i c s o f S C N a n da r e n o t f o u n d i n t h e o t h e r V P - c o n t a i n i n g h y p o t h a l a m i c n u cl e i.T h e v a r i a t i o n i n V P m R N A t a i l l e n g t h i s n o t s i g n i f i c a n t l ya f f e c t e d b y a d r e n a l e c t o m y , c a s t r a ti o n , o v a r i e c t o m y , p h a r m a -c o l o g i c a l c e n t r a l s e r o t o n i n d e p l e t i o n b y p a r a - c h l o r o p h e n y l -a l a n i n e , a n d m e l a t o n i n o r b e n z o d i a z e p a m t r e a t m e n t . T h et r a n s i t io n f r o m t h e d a y t o t h e n ig h t m R N A f o r m c o n t i n u e s inv i t r o w h e n s t u d i e d i n e x p l a n t s ( 6 ). F i n a l l y , i n v i t r o r u n o na n a l y s is a n d s e p a r a t i o n o f n u c l e a r f r o m c y t o p l a s m i c R N As u g g es t t h a t S C N - s p e c i fi c c i r c a d i a n v a r i a ti o n s i n V P m R N Ap r o c e s s i n g o c c u r i n t h e n u c l e u s a t t h e l e v e l o f V P g e n e t r a n -s c r i p t i o n ( 7 ).S o m a t o s t a t i n g e n e e x p r es s i o n in d o r s o m e d i a l S C N e x h i b it sd i s t in c t c i r c a d i a n r h y t h m u n d e r c o n d i t i o n s o f c o n s t a n t d a r k -n e s s o r a f t e r e n u c l e a ti o n . T h e h i g h e s t m R N A l e ve l i s o b s e r v e da t t h e t r a n s i t i o n f r o m s u b j e c ti v e n ig h t t o s u b j e c t i v e d a y a n dp r e c e d e s t h e m a x i m a l p e p t i d e l e ve l w h i c h i s f o u n d i n t h e m i d -d l e o f t h e s u b j e c t i v e d a y ( 6 1 , 6 9 ) .

    B o t h c i r c a d i a n r h y t h m s o f V P a n d s o m a t o s t a t i n g en e ex -p r e s s i o n a r e c o n s i d e r e d o u t p u t r h y t h m s ( i . e . , a r e d i s t a l t o t h ec i r c a d i a n o s c i l l a t o r in t h e S C N ) . O n e r e a s o n f o r t h i s i s t h el o c a l i z a ti o n o f t h e r e s p e c t iv e n e u r o n s i n t h e d o r s o m e d i a l S C Nt h a t d o n o t r e c e i v e d ir e c t v is u a l a f f e r e n t a ti o n . I n a d d i t i o n ,V P - d e f i c i e n t B r a t t l e b o r o r a t s d e m o n s t r a t e d i s t i n c t b e h a v i o r a lc i r c a d i a n r h y t h m s ( 46 ). T h e c i r c a d i a n r h y t h m o f f o o d i n -t a k e i n t h o s e r a t s i s p r a c t ic a l l y n o t a f f e c t e d b y c o n t i n u o u s I Vi n f u s i o n o f v a s o p r e s s i n ( 6 3 ) . T h u s , t h e r o l e o f V P a n ds o m a t o s t a t i n i n t h e c i r c a d i a n f u n c t i o n o f S C N i s c u r r e n t l yu n k n o w n .I n p r i n c i p l e t h e s t u d y o f S C N - l o c a t e d c i rc a d i a n o s c i l l a to rm a y s t a r t f r o m t h e o u t p u t ( i . e . , t h e r e g u l a t i o n o f V P o r s o -

    m a t o s t a t i n g e n e e x p r e s s i o n ) . I t s e e m s h o w e v e r , t h a t t h e d o r -s o m e d i a l S C N n e u r o n s r e c e i v e s y n a p t i c i n p u t a s a m e a n s f o rc l o c k i n f o r m a t i o n a n d t h e r e f o r e o n e m a y e x p e c t t h a t s u c ha n i n p u t i n i t i a t e s a s e p a r a t e c a s c a d e o f s t i m u l u s - t r a n s c r i p t i o nc o u p l i n g . I t r e m a i n s t o b e e l u c i d a t e d w h e t h e r t h i s c a s c a d es h a r e s s i m i l a r i t i e s w i t h l i g h t - i n d u c e d I E G s e x p r e s s i o n a n dw h e t h e r t h e c i r c a d ia n r h y t h m o f V P a n d s o m a t o s t a t i n g e n ee x p r e s s i o n i s a f f e c t e d b y l i g h t - p u l s e s k n o w n t o i n d u c e I E G sa n d s h i ft b e h a v i o r a l r h y t h m s .Two-Neuron Model, Predictions and Prospective

    W e p r o p o s e a s i m p l e m o d e l c o m b i n i n g g e n e e x p r e ss i o nw i t h t h e c e l lu l a r n e u r o b i o l o g y o f S C N . T h e m o d e l i s b a s e do n t w o g e n e r a l a s s u m p t i o n s : ( a ) t h e c i r c a d i a n t i m e - k e e p i n gm e c h a n i s m i s a n i n t r a c e l lu l a r p r o p e r t y ; a n d ( b ) v a l i d i t y o ft h e c i r c a d i a n i n p u t - c l o c k - o u t p u t m o d e l a t t h e l e v el o f g e n ee x p r e s si o n in S C N . T h e f i r s t a s s u m p t i o n s t e m s f r o m o b s e r v a -t i o n s i n o t h e r c i r c a d i a n s y s t e m s w h e r e g e n e x p r e s s i o n h a sb e e n s h o w n t o p l a y a n i m p o r t a n t r o l e as w e l l a s f r o m t h ed i s c o v e ry o f m a m m a l i a n c i r c a d i a n m u t a n t s ( 1 8, 45 ) . T h e s e c-o n d a s s u m p t i o n i s b a s e d o n t h e e x t en s i ve s tu d y o f S C N a s ac i r c a d i a n p a c e m a k e r t o g e t h e r w i t h t h e o b s e r v a t i o n s f o r r h y t h -m i c i t y i n th e e x p r e s s i o n o f g e n e s a s s o c i a t e d w i t h c i r c a d i a ns y n c h r o n i z a t i o n a n d o u t p u t i n S C N .W h i c h S C N c e l l s a r e t h e n t h e c a n d i d a t e c l o c k c e l l s ? W ef a v o r t h e i d e a t h a t t h e c e l ls a r e n e u r o n s , l o c a t e d i n t h e v e n t r o -l a t e r a l S C N a n d e x p r e s s i n g I E G s a f t e r l i g h t p u l s e s t h a t a r ee f f e c t i v e i n s h i f t i n g b e h a v i o r a l c i r c a d i a n r h y t h m s . S e v e r a l in -d i r e c t l in e s o f e v i d e n c e s u p p o r t t h i s n o t i o n : ( a ) p e r s is t e n c e o fc i r c a d i a n r h y t h m o f n e u r o n a l m e a n f i ri n g r at e i n v e n t r o l a t e ra lS C N i n v i t r o a f t e r d i s s o c i a t i o n f r o m t h e r e s t o f t h e n u c l e i (1 5 );( b ) p r a c t i c a l l y a l l i n p u t p r o j e c t i o n s m a k e s y n a p t i c c o n t a c t s

    INPUT v~ CIRCADIAN v~ OUTPUTOSCILLATOR

    l i g h t ~ r e t i n o -h y p o t h a l a m i c t r a c t

    A

    f i m p u l s e r a t e

    lE G s ~ c l o c k g e n e sm e t a b o l i s m

    V P m R N A "~I t r a n s c r ip t i o n lL ~ , . p r o c e s s i n g , )o m a t o s t a t i n

    J L , e v e

    V Ps e c r e t i o nA

    ventro la tera l SCN d o r s o m e d l a l S C NFIG . 1 . Two-neuron m odel of the c i rcadian sys tem in SCN. The f i r s t neuron i s assumed to be located in vent ro la tera lSCN. Its t ime-keeping mechanism depends on gene expression. The neuron receives photic input from the retina forc lock synchroniza t ion to the external l ight /dar k cycle . Light pulses phase-shi ft ing behaviora l c i rcadian rhythms induceimm ediate-e arly genes (IEGs) expression in th e first neuron . IE Gs produ cts possibly affect circadian clock gene expres-s ion . On the o the r hand lEG s induct ion i s under c i rcadian cont ro l and l imi ted only to the subjec tive n ight . In pr inc ip le ,the f i r s t neuron can in corpora te a comple te c i rcadian sys tem in case tha t some informat ion for the poss ib le influence o fthe osc i l la tor on the c i rcadian rhythms of f i r ing ra te and metabol i sm is available . The input an d ou tput pa thwaysemploy ac t ion p otent ia ls for adjus t ing the c lock o r pro pagat ing the t ime inform at ion, respectively . The second neuronlies in the dorso me dial part o f SCN a nd is vasopressinergic or somatostatinergic. Unknown sequence of events l inks thesynapt ic input to the c i rcadian rhythm in VP mR NA t ranscr ip t ion and polyadenyla ted a l l format ion and som atos ta t inmR NA l eve l.

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    310 IKONOMOV AND STOYNEVwith neurons in the same area (2,72); (c) fast and selectiveinductio n of IEGs in neurons of ventrolateral SCN by shortlight pulses that shift behavioral circadian rhythms togetherwith abolishment of IEG induction by treatments that blockthe behavio ral effect of light pulses (1,28); (d) almost immedi-ate clock resetting mechanism (33,38); and (e) intracellularcircadian control on IEGs expression in the same neuro n (29,55,56). It is expected that th e closest circadian out put rhyth mis the rhythm in electrical discharge or neuronal metabolicactivity. Unfortunately, we are missing experimental evidencesupporting this possibility. Therefore, in the model, we in-troduce the second neuron located in the dorsomedial SCNwhere circadian rhythms in the expression of vasopressin andsomatostatin genes are already documented. The timing infor-mation from the first neuron is propagated as action poten-tials. Unkn own are the transmitter, its postsynaptic receptors,and stimulus-transcription mechanisms coupling the timingsignal with the VP an d som atost atin gene expression (Fig. l) .Some aspects of the present model are amenable to experi-mental test. The effect of light pulses inducing IEGs o n thecircadian rhythms of VP a nd somatostatin gene expression inSCN may answer the questionable validity of the circadianinp ut-c lock -ou tput model. It will be more difficult to testthe hypothesis that circad ian function- related gene expressiontakes place in individualSCN neurons. The pr oblem stems fromthe absence of suitable molecular markers for the possible inter-actions between the presumable clock gene products and the in-trinsic circadian variations in glucose uptake and mean firingfrequency. Such markers could assist in defining the inp ut-o ut-put time course in models of phase shift and ind icate the propertime for hypothetical clock gene isolation. Unfortunate ly, incontrast to behavioral rhythms, the SCN-intrinsic circadianvariations are low in amplitude and do not show sharp onsetor offset. In addition, the results are inevitably from groups ofanimals with all subsequent difficulties for frequent time-sampling and statistical interpretation of the results.

    All molecular strategies reviewed above are strictly SCN-oriented. However, the two-neuron model predicts fimitationsin regard to their potential success. If the assumption that theneuron s expressing c-fos upo n light pulse are the "clock" cellsis true, then they will constitute only a few percent of theSCN neurons and associated glial cells. Combined with thepossibility that the gene products of interest may be no t abu n-dant, this could lead to a situation where the presumablecircadian genes are below the curre nt level of detection. There-fore, the success in SCN-orien ted strategies depends on treat-ments that will marked ly and specifically affect the expressionof the c ircadian func tion-r elated genes together with the devel-opment of a very sensitive system for their differential isola-tion.An alternative molecular genetic strategy is the genetic link-age analysis of crosses between mam mal ian circadian mutantsand control animals. Such analysis may result in identificationof the DNA locus responsible for the phenotypic difference,as has been pr eviously useful in studies of experim ental hyper-tension (24). Th e advantag e of this strategy is the use of geno-mic DNA (easy to obtain in large amounts) combined with aclear phenotypic difference.In summary, the molecular genetic approach to the circa-dian system in SCN offers initial views suggesting that geneexpression is associated with circadian synchro nizatio n andoutput. Obviously, the limited available knowledge do notprovide information on the nature of the mammalian circa-dian clock which still remain s a mystery.

    ACKNOWLEDGEMENTThanks are due to Drs. P. J. Morgane, W. J. Schwartz, A. C.Shisheva, M. H. Jacob, and J. D. Levine for constructive criticismand help in preparation of this review. The stimulating discussions

    with Dr. Y. Citri and his collaborators during the stay of O.C.I . at theWeizmann Institute of Science are gratefully acknowledged.REFERENCES

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