Indian 10urnal of Experimental Biology Vol. 37, November 1999, pp . 1053- 1064

Review Article

Light sensitivity of the photoperiodic response system in higher vertebrates: Wavelength and intensity effects

Vinod Kumar & Sangeeta Rani

Department of Zoology, University of Lucknow, Lucknow 226 007 , India

Most species use daily li ght in one way or the other in regulation of their short and/ or long term activities. Li ght is perceived by pigment(s) present in the retinal (RP) and/ or extra-retinal photoreceptors (ERPs). ERPs may be located at various sites in the body but in non-mammalian vertebrates they are found predominantl y in the pineal body and hypoth a­lamic region of the brain ,- Li ght radiations directly penetrate brain ti ssues to reach and stimulatc the hypothalamic (dcep­brain) photoreceptors. How does li ght information fin all y reach to the clock is not fully understood in man y vertebrate groups? In mammals, however, the light informati on from the retina to the clock (the hypothalami c suprachiasmatic nu cl ei, SeN) is rel ayed throu gh the retino-hypothalamic tract (RHT) which ori ginates from the retinal gangli on cell s, and through the geniculo-hypothalamic tract (G HT) which originates from the photically responsive cells of a portion of the lat eral gen­icul ate nucleus (LGN), called the intergeni cu late leanet (lGL).

A response to light (the photoperiodic response) is the result of the interpretation of li ght information by the photoperi­odic system. Apart from the duration , the animals use the gradual shi fts in the intensity and wavelength of daily li ght to regu late thei r photoperiodic clock system. The wavelengths to which photoreceptors are maximall y sensiti ve or the wave­lengths which have greater access to the photoreceptors can induce a maximal response. There can also be differenti al ef­fects of wavelength and intensit y of light on circadian process(e~) involved in the entrainment and induction of the photo­periodic clock. This may have some adapti ve implicat ions. Entrainment to dail y light-dark (LD) cyc le may be achieved at dawn or dusk , depending whether the animal is day- or night-acti ve, when there is relatively low intensity of li ght. By CO Il ­

tras t, photoperiodic induction in many species occurs during long days of spri ng and summer when plenty of dayli ght at hi gher intensity is avail able later in the day.

The thrust of evolutionary bio logy is that the changes in nature produce biological stresses that lead to ei­ther adaptation or ex tinct ion of a gi ven species . Adaptive advantage is the key to synchroni zation of physiological and behavioural activities to environ­mental factors such that these occur at the most fa­vourable time of the year. Natural selec ti on ensures that the organi sms choose environmental cue(s) th at is(are) predictable and that help them anticipate the "appropriate season" . At a given latitude, the annual variat ion in daily light (day length , photoperiod) is extremely predictable and used by Illost species in one way or the other in regulation of their short and/ or long term activities. Apart from the durati on, other important characteri stics of dai ly li ght are its quantity (light intensity) and quali ty (colour, spectra l compo­sition). The gradual al terati ons in the intensit y and wavelength of li ght that occur in natural cond it ions can also provide reliable informati on about the time­of-day and/or the ti me-of- year. The intensity refers to the amount of photons avai lab le in the li ght source, and the co lour is the sensat ion ex peri enced as a resul t of activati on of certain classes of photoreceptors by

selected wavelengths of visible li ght spectrum (380 to 760 nm; commonly expressed as VIBGYOR where violet to blue correspond to short wave lengths , green and yell ow to mid wave lengths and red to long wavelengths) .

It is important that day length is percei ved and measui'ed correct ly before it is transduced to effect an appropriate photoperiod-induced chan ge. Thus, the photoperiodic induction (for example, gonada l growth, body fattening etc .) is the result of li ght­induced effects on the whole photoperi odic machi n­ery (the photoperi odic response system) of an ani ma l that can be very specific depend ing upon the wave­length (spectrum, colour) and intensity or li ght. In thi s articl e, we aim to summarize bri efl y th e progress of research th at has been made in studyin g the effects of wavelength and intensity of li ght on photoperi odic regu lati on of physiological fun cti ons, based on stud­ies in hi gher vertebrates with spec ial emphasis placed on birds and mammals. In particul ar, we wil l restrict oursel ves to two photoperi od ic e\'ents , the photore­ception and the photoperi odi c time measurement , in thi s communication . At the end , we gtve a brief ac-

1054 INDIAN J EXP BIOL, NOVEMBER 1999

count of light effects on other (non-reproductive) cir­cadian functions as an overview of light sensitivity of the circadian system in organisms.

Photoreception The environmental illumination useful for animal

physiology is perceived by at least two forms of photoreceptor: visual and non-visual photoreceptors. Whereas visual photoreceptors function as image de­tectors, non-visual photoreceptors detect photoperiod and synchronize physiology with respect to environ­mental time. Thus, the two divergent sensory tasks seem to have led to the evolution of specialized pho­toreceptor systems I . In contrast to visual, our under­standing of anatomy, physiology and biochemistry of the non-visual photoreceptor system is poor. Here, we briefly focus upon the non-visual photoreceptor which are important for the photoperiodic timing.

In vertebrates, light used to synchronize physiol­ogy with respect to environmental time is perceived by pigment(s) in the photoreceptors present in the eye (retinal photoreceptors, RP), pineal and/or extra­retinal (extrapineal) structures (extra-retinal photore­ceptors, ERPs). In general, photopigments can be characterized by their biochemistry and by the physiological responses they mediate. All vertebrate photopigments characterized so far are remarkably similar in consisting of an opsin protein coupled to a chromophore derived from an II-cis form of vitamin A retinaldehyde2a

.b

.c

.

In non-mammalian vertebrates , the pineal organ is an important photoreceptive system. The pineal com­plex of many fish and amphibi ans contain pinealo­cytes which have ultrastructural properti es that re­semble retinal photo receptors and show light depend­ent e lectrica l act ivity, and thus act as photoreceptors (see revi ew by Dodt and Meiss l'). In reptiles, pinea­locytes act photoreceptors, and it has been shown in the isolated pinea l of Ano/is li zards that me latonin production can be entrained to the li ght:dark (LD) cycle4

. Spec tra l sens itivity of pineal photoreceptor may be species specific but , in general, the peak sen­sitivities of most photoreceptors range in between 520-530 nm.

Photoreception with respect to light regulated phys io logical functi ons has been ex tensive ly stud ied in birds. As is true of mammals, retinal ill umination in birds reaching the putative clock site in the hypo­th alamu s is re layed through the re ti no-hypoth alamic tract (RHT)5 a [ract of nerve f ibers ori ainat ina fro m , b b

the retinal ganglion cells . The prec ise rol e of RHT­mediated photic information in avian photoperiodism is unclear given the fact that photi c e ffect s onto re­productive axis survive in the enucleated birds67

. The pineal in birds differ from visual photoreceptors and the pineal photoreceptors of the lower vertebrates in lacking the ordered cytoplasmic laminae and per­forms photoreceptive and c lock functionss.0 . Chicken pineal is the most studied photoreceptive system. It contains a photoreceptive molecule that receives en­vironmental light signal and transmit s this signal to the oscillator. This photoreceptive molecule is sug­gested to be rhodopsin like, although the action spec­trum of this molecule as obtained by inhibitory ef­fects of light on the N-acetyl transferase (NAT), the enzyme involved in the melatonin synthesis, was broader in shape (maximum at 500·520 nm) than that of the absorpt ion spectrum of rhodopsin . Immuno­histochemical studies show rhodopsin-like immu­noreactivity in bird pineal cell s (e.g . in the chicken and quail), but the identity of pineal pigment is still unclear. Recent molecular studies (for example see lO

)

have identified pinopsin (a major pigment ) and io­dopsin (a minor pigment) as the pinea l pigment , and still the existence of a third or more photorecepti ve molecule cannot be ruled out. Taken together, vari ous lines of e vidence seem to sugges t that the p ineal and retinal photoreceptors may have ph oto-transducti on (re lay of photic information) cascades similar to eac h other, although they may not have iden tical path ways .

Apart from the retina and pineal , the brain in non­mammalian vertebrates contains photorecepto rs that are involved in transfer of li ght information to the photoperiodic response syste m. ERPs are s ituated in the brain (call ed encephalic photoreceptors or dee p­brain photoreceptors, DBPs) , parti c ularl y in the hy­pothalami c regions. Avail able data ra ise the poss ib il ­ity that there arc several types of ph otorece ptor pho­topi gments (cone-like, rod-like or diffe re nt from both), and depending on species at leas t two types of photoreceptor ce ll : CSF-containing neuron s (found in larval lamprey, reptil es and birds) and c lass ica l neu­rosecretory neurons within the nuc le us magnoce llu ­lari s preopticus (NMPO; fi sh and amphibian s) (for more deta il s see l I) .

ERPs have been imp lica ted in scve ra l d iffe rcnt ar­eas of ph ysi o logy but , in a li species ex ami ned. they pl ay a criti ca l role in the regul ati on of c ircadian and reproducti ve responses to li ght. In reptiles, they ha ve been shown medi ating exc lu s ive ly th e c ircad ia n c n-

KUM AR & RANI; WAVELENGTH AND INTENSITY EFFECTS 1055

trainment since blinded, pinealectomized and parietal eye (if present) removed lizards are readily entrain­able to light:dark cycle ' 2. ERPs are qu ite sensitive since lizards can be entrained to light as dim as I lux ' 3. A more detailed study of ex traretinal photore­ception has been done on AnaUs lizards (A nalis caro­Iinensis)14 . In this spec ies, Cerebrospinal fluid (CSF)­contacting neurons in the septal area of the brain rep­resent the ERPs and bind to anti-opsin anti serum. Further, the anti-opsin antibodies recogn ized a 40 kD protein in ocular, pineal and onl y in septa l brain ar­eas, and the anterior brain of Anolis contained spe­cific retinoids ( II-cis and all-trans-retinoids) associ­ated with phototransduction. In birds, ERPs have been suggested to be located in the CSF-contacting neurons within the septal and tuberal areas of the brain 's.

The following table summarizes the important characteristics of retinal and extraretinal photorecep­tor cell s measured in terms of light sense of these re­ceptorsl 6:

Characteri sti cs Extraretinal Retinal

Image analys is poor or absent prominent Ontogeny early late Mode of transduct ion neural and humoral neural (humoral ?) Refractory periods well establi shes absent Cave dwelling survival degeneration Function orientati on in time orientation in space

It is probable that extraretinal photoreception ex­isted prior to the evolution of lateral eyes since the most primitive eyeless organisms definitely utilized the daily LD cycle as an entraining stimulus. Image­forming vision needs a specialised structure like an eye, irradiance detection for the photoentrainment does not require any such specialised structure. Rather, these irradiance detecting encephalic photore­ceptors could be closely associated with their effector system within the brain. The fact that most primitive vertebrate brain of lampreys contains di stinct popula­tion of photoreceptors which are associated with dif­ferent behavioural and physiological functions but all they are labelled by one or more of the anti-opsin an­tibodies2b (indicating the presence of opsin, V A opsin (vertebrate ancient opsin), photopigment) favour for early evolution of encephalic photoreceptors. The eyes were "wired" into the photoreceptive system when they evolved . There may be some selective ad­vantages for having extraretinal photoreception since ERPs survived even after evolution of eyes in major-

ity of vertebrates. It appears that retin al and extra photoreceptors extract different kinds of information from the light envi ronment, e.g. parametri c (conti nu­ous action occurring throughout li ght phase of an LD cycle) versus non-parametric (discontinuous action occurring at LD or DL transiti ons . Alternatively, it may be that ERPs are integral part of the timing de­vice (c lock) itself and therefore evo lutionary con­served as its cell component: These considerat ions however raise an important questi on: wh y mammals apparentl y do not possess ERPs? There is no con­vincing explanation for thi s riddl e, but there can be some speculations. One of these could be that mam­mals apparently evolved from ni ght acti ve mammal­like reptiles which natura ll y hid during the day in dark places, and thus, the developing brain of these animals probably did not ex peri ence sufficient ligh t during critical periods of its ontogeny. The absence of specific stimuli during critical peri od of ontogeny can result the loss of function that has to be induced by the stimuli . ERPs which develop early in ontogeny degenerated whereas RP cells which develop late in ontogeny survived since they probably rece ived suffi­cient light. RPs took over visual function in a re­shaped visual system and photic control of endoge­nous rhythmicity.

In mammals, photoreception occurs exc lusively through the eyes (the retina). However, one recent study by Campbell and Murph yl 7" suggests that ex ­traocular exposure (behind the knee) may cause phase-shifts of the circadian clock in human s. This important result which challenged the belief that adu lt mammals are incapable of using ERPs for the cir­cadian phototransduction , however, could not be proved by another similar study of Lockley el al. 17b

in wh ich extraocular exposure (behind the knee) of human.s to light failed to suppress the circadian rhythm of melatonin secretion , a direct function of the circadian system. So, unless replicated by othe,' labo­ratories, the extraocular circadian phototransduction in mammals is to be considered with a lot of caution. At present, retinal phototransduction remains only effective pathway for photic regulation of circadian system in mammals. The exact ocular (ret inal) photo­receptors involved are not known since ve ry recentl y Lucas et 01.1 8 found that the mice which had no rods or cones (rdlrd cl mi ce, the strain of mice that had combined the rd mutation and a transgeni c ablation of cones) showed normal suppress ion of pineal mela­tonin in response to monochromat ic li ght of wave-

1056 INDI AN J EXP BIOL, NOVEMBER 1999

length 509 nm; thi s finding clearl y suggests that mammals have additi onal ocu lar photoreceptors which they use in the regulati on of their photoperi ­od ic timing. The photic information from eyes to the suprachiasmatic nucleus (SeN), the important clock site in mammals that is located in the anterior hypo­thalamus is conveyed through RHT which is com­posed of very thinly myelinated ret inal ganglion cell axons that emerge from the chiasma to terminate in the ventral SeN in rats l9 and the entire SeN in Syrian hamster2o

. The second photic pathway reaching SeN is the geniculc-hypothalamic tract (GHT) which ori gi nates in photically responsive cell s in a portion of the lateral gen icu late nucleus (LGN) ca lled the intergenicu late leaflet (IGL)21. Treep et a/.22 clearly de monstrated that stimulati on in the IGL region anti­dromically activates a retinal projection that reaches both the IGL and the SeN via co ll ateral fibers. The relay of light is mediated chemicall y by synaptic re­lease of glutamate and other excitatory amino ac ids (EAA) which act through specific EAA receptors (see Kumar et at. 2J for details) as well as of a host of neu­rotransmitters like neuropeptide Y (NPY), enkepha­line (ENK) etc .. A simple schematic di agram of how photic information is relayed to the clock components in mammals is shown in Fig. I.

Action spectrum studies help in characterization of pineal and ERPs. All opsin and I I-cis retinaldehyde based photopigments have a characteristic shape to their absorbance and action spectra even though they may have a very different wavelength of maximum sensitivity. An action spect rur.l for ERPs in Japanese quail was similar to the acti on spectra of pineal re­sponses although the receptors in volved were clearly not in the pineal24

. It may be noted however that the studies on action spectra of light provide a basis for

findin g the pigment(s) responsible for a spec ific photoresponse, but they do not necessarily te ll how many photoreceptors are in volved. Bra in pholore­ceptors were discovered more than five decades ago by Benoit and hi s colleagues but the acti on spect ra for the photoperiodic responses have been studied on ly for a few species as yet (see espec iall /'·ls).

In non-mammalian vertebrates, the retina and pi n­eal organ may have both photorecepti ve and ti me­measuring capabilities. Whether photorecepti on by these two structures is meant for the entrainment of their own osc illator or they further re lay the photic information to the brain (hypothalamic) pacemaker is unclear. This is important in view of the fact that enucleated (both eyes surgica l I y removed) house sparrows respond fu ll y to stimulatory effects (e.g . photoinduced gonadal growth) of light , and circadian locomotor acti vity rhythms in blind sparrows can en­train to LD cyc les . Also, pinealectomy does not im­pair photoperiodic inductio n in many species (see review29.3o) and pinea lectomi zed birds show onl y gradual loss of circad ian rhythmi ci ty of locomotor activity (see review~ I ). These clear y argue for the principal ro le of hypoth alamic photoreceptors in the circad ian photoresponse system at least in birds. Furthermore, whether these different organs of photo­reception work synerges ti call y or work independen tl y remains an open questi on. A conservat i ve exp lanation at the moment could be that the photorecepti on by the pineal and eye (ret ina) playa modulating ro le on the circadian pacemaking system located e lsewhere in the brain which may include the photoreceptor, the clock or both.

How do DBPs communicate with the photoperi­odic machinery is not absolutely clear') In birds , how­ever, DBPs co-express proteins characteri st ics of

Fig. I-A schematic diagram of photic relay from the retina to the clock components in mammals. EAA - excilatory amin o acids ; E lK -enkephaline; LGN - lateral genicu late nucleus; NPY - neuropeptide Y; SeN - suprach iasmatic nucleus

KUMAR & RANI: WAVELENGTH AND INTENSITY EFFECTS 1057

retinal photoreceptors, as well as vasoactive-intestinal polypeptide (VIP)! ). These putative opsi n-express ing DBP have fibers lying in c lose appos ition with gona­dotropin releasing hormone (GnRH)-expressing cell s which mi ght mean that they communicate with GnRH neurons, and vice versa. There is also ev idence of seasonal variation in these brain peptides: ex press ion of VIP-like immunoreactivity is highest in photore­fractory an imals while GnRH-li ke immunoreactivity is highest in photosensitive birds . Also, expression of these brain peptides is correlated with changes in plasma prolact in and lutei ni zing hormone (LH). All this might be taken to suggest that putative DBP are involved somehow in med iating the photoperiod­induced seasonal cyclicity. How is the relay of light information from photoreceptors to the clock compo­nents done, which is so crucial for the measurement of day length, is not known? The issue may be more clear once the definiti ve clock sites in avian brain become known.

The direct effects of light intensity on vertebrate photoreceptors have not been monitored. A large number of studies have however investigated the in­tensity affects on reproducti ve and non-reproductive functions that are mediated by photoreceptors. The spectrum of light intensity effects includes the effects on food and feeding, reproduction , immune system to general health32.J3

. Both low and high light intensiti es can cause convulsions and death in the African weaver finch 34 . Since the demonstration of the role of light intensity in the manipUlation of starling's repro­ductive response to day length 3

), the effects of inten­sity on photosexual response has been studied in

b· d b b h' '1 '6 ] '1,7.,9 d some Ir s, 0 w Ite qual ' , apanese quat - - , 0 -. k 40 d . d k4! h 42-4'; mestlc tur ey , omestlc uc , ouse sparrow -,

white-crowned sparrow46. Of these, the studies on house sparrow are most comprehensive.

A photoperiodic response is maximally induced by the wavelength(s) at which photoreceptor(s) mediat­ing such effect is most sensitive or by wavelengths which have greater access to the photoreceptors. Photoreceptors seem to have precise spectral sensi­tivity. When blackheaded bunting were subjected to a stimulatory day length of 13L: I I D in white, green (528 nm) and red (654 nm) colours at 100 lux inten­sity, photostimulation (weight gain and testis growth) occurred in all groups but the tes tes were significantl y larger in birds that were exposed to red light (Fi g. 2). Similar responses occur in other bird species (see Oishi and Lauber37 for detail s). In mammals too, the

wave length- and intensity-dependent photoperi odic inducti on has been reported. In the hamster. I hr pulse of green, blue or near ultraviolet light. but not red or yellow li ght , presented to animals during dark period blocked the co llapse of the reproduct ive sys­tem. Also, at equal irrad iances diffe rent bandwidths of light have different effects on short photoperi od induced collapse of the hamster reproductive sys­tem47

.48. The effects of light spectrum have been re­ported in lower vertebrates as well. Joshi and Udaykumar49 found that the effects on ovari an fol­licular kinetics in the frog, Raila cY({llophlyclis , were max imum of red ligh t, foll owed by ye ll ow and green li ght.

Photoperiodic t.ime measurement (PTM , the pho­toperiodic clock)

The precision with which seasonal res ponses are temporally organized clearl y suggests that they are regu lated by a clock mechani sm that is fine-tuned by the light environment. Experimen tal ev idence con­clude that endogenous circa-rhythms , synchroni zed to time-of-day (circadian rhythm; circa - about , dies -day) and/ or time-of-year (ci rcannual rhythm; circa -

about, annum - year), help measure the length of the day. It is believed that the entrainment and induct ion of the circadian rhythm of photoperi odi c photosens i-

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Fig. 2-Testicular re~ponsc of thc photoscll siti \ 'C blackheaded bunting (ElI1beriza lIleln llOCefJhala) cxposed to a I()ng photopel iod ( 13L: lID: L: 100 Ix. D: 0 Ix) of while (w). green (g). and red (r) light. The un stimulated birds werc maintained under shan days ensuring their sensitivity to light. Note that red li ght group indi ­cates significantly larger response cOlllpared with white ( w )

and/or green (g) li ght group (red rawn from KUlllar and Rani")

1058 INDIAN J EXP BIOL, NOVEMBER 1999

tivity (CRPP) by photoperiod are key to the time­keeping process. Intensity and wavelength are the characteristics of 24 hr daily cycle of illumination that are important to the photoperiodic entrainment and induction of CRPP.

The models Pittendrigh50 has summarized in theoretical model s

how could the circadian organization be involved in PTM50

-52

. Briefly, a photoperiodic clock is circadian and operates as if there is coincidence between phases of the photoperiodic oscillator and the light:dark (LD) cycle - the external coincidence, or there is interac­tion between two or more independent oscillators as a result of exposure to LD cycle- the internal coinci­dence (Fig. 3). The central feature of the external co­incidence model, as originally conceived by Erwin Buenning53 and later widely used to explain the pho­toperiodic phenomena, is that light exerts phase­control over the circadian osci llation to enable its dual actions, of phasing the oscillation and of effect­ing the photoperiodic induction by extending (or not extending) into the photoinducible phase (<I>i), lo­cated early in the subjective night. Remarkably, so much experimental effort has gone into testing the Buenning hypothesis without making any significant achievement in unraveling the mechanism how light exerts phase control over CRPP. Furthermore, there can be species variation in the ability of <I>i to free-

External coincidence

Internal . coincidence

2 Days

run in constant condition (e.g. constant darkness. DD) and such differences raise the question as to whether the photoperi odic clocks are genuinely and qual ita­tively different between the species. Clearly, in spe­cies like AlIa/is lizards and Japanese quail the cir­cadian system driving <I>i is weakly se lf-sustaining, but in species like golden hamster, white-crowned sparrow and blackheaded bunting the c ircad ian sys­tem driving <l>i is strongly se lf-susta ining. These as­pects of the photoperiodic clock have been discussed in detail by Kumar and Follett:>".

CRPP: Phasic effects Relati ve ly less is known concern ing spectral se nsi ­

tivity of phasic components of circad ian effec ts of light. Indirect ev idence from a few spec ies nonethe­less show that light intens ity at a threshold is required for the photoperiodic entrainment and induction of the photoperi od ic clock mediating full go nadal growth and development. In the bunting (ElIlberi-:,{{ melanocephala and Emberiza brulliceps) held in a skeleton photoschedule, the rate of gonadal growth is very slow in 100 lux li gh t intensity 55.56 . Also. in Japanese quail I hr night-interrupti on at an intensity of 850 lux stimulates significan tly larger (P<0.02) testes than those exposed to at 250 lux .1,} There is strong evidence supporting the role of li ght intensity as synchronizer of clock-regulated long term seasonal responses . At equater where the change in day lengt h

Long day

1 2 Days

Fig. 3-Models of mechanism(s) involved in the photoperiodic time - measurement in long day animals. In the external coincidence

model (A), coincidence of light with the photoinduci ble phase (<I>i ), which fa il s early in the night, leads to a photoperiodic respon se un­der long days . In internal coincidence, photoperiodic stimUlation under long days occurs due to changes in the phase relationship (and thereby coincidence) between two circadian oscillators (B). Non-stimulation under short days is due to non-coincidence of li ght with the

<I>i (external coineidence- Fig. 3A), or due to a different relationship between two or more circadian oscillators (in te rnal coincidencc­Fig. 3B) (redrawn from Kumar and Follett54

).

KUM AR & RAN I: WAVELENGTH AND INTENS ITY EFFECTS 1059

over the season is so small that it cannot be used as a reliable photoperiodic index of the changing season, animals seem to rely on the change in light intensity over the season to regulate their responses. Simulat­ing high and low intensity cycle of the nature, Gwin­ner and Scheuerlein57 have demonstrated that light intensity could reliably be used by an equatorial avian species in synchronizing its annual reproductive cy­cle.

We have for the first time attempted elucidating the phasic effects on CRPP of light intensity and light spectrum in birds using a skeleton paradigm. Induc­tive effects of I hr light pulse in a skeleton photo­period (6L:6D: I L: liD) were intensity- and spectrum­dependent. At 50- and 100 lux intensities of two dif­ferent wavelengths (green - 528 nm, and red - 654 nm), 1 hr light pulses induced fattening and gonadal growth in the blackheaded bunting only under red light55

. At a given intensity, red light is more effective than white light and white light is more effective than green light. In other words, the short wavelengths of light are less effective than the long wavelengths in stimulation of av ian photoneuroendocrine (PNE)­system.

Induction VS . entrainment

Differential responses to LD cycles that contain same inductive but varying intensity and wavelength of entraining pul se, or vice-versa, indicate the wave­length- and intensity-dependent entrainment of CRPP. The threshold light intensity for entrainment seems to be lower than that for induction. In the migratory bunting, a 20 lux entraining or inducing light pul se fail to induce full testis growth . Al so, there can be differences in light intensity thres holds between en­training and inducing li ght pu lses.'i5. 56 . Cou ld it be that the entrainment and induct ion of photoperiod ic re­sponses are two separate phenomena? In any case, it may be much easier to entrain the c ircadi an system than to convert the photoinductive e ffects into a neu­roendoc rine event such as the lipogenes is (body fat­.teni ng) and the gametogenes is (testicular growth). It remains however unclear whethe r the difference is at the level of endocrine system or at photoperiodic c lock or of both. Furthermore, whether the photoperi­odi c entrainment and inducti on are mediated by dif­ferent classes of photoreceptors is not known. The phase-spec ific sensiti vity (entrainment by low and induction by hi gh light intensities) of photoperiodi c clock to li ght intensity may however have some

adaptive implicat ions. For example, entrainment to daily LD cycle may be achieved by phas ic effects of light at re lati vely low intensity during daily twilight hours (dawn and dusk) . By contrast, photoperi odic induction in a long day species like bunting occurs during long day lengths of spring and summer when plenty of day light at higher intensity is avail ab le .

There can al so be differential effects depending how light is applied. Light in wide range of wave­lengths is stimulatory when applied direc tl y on to brain areas, thus directly illuminating ERPs, but not when applied from outside. In the duck, ERPs that mediate light effects respond to both ' blue and red lights when light is introduced through a quartz rod directly into the brain41. ERPs in quail also respond to both blue (455 nm) and orange-ye ll ow (575 nm) light when radioluminescent pa int was implanted directl y in specific brain areas25

.

All observations from spectral studies are consis­tent with the idea that the difference in photoperiodic effects at a given intensity by different light wave­lengths is due to the difference in number of photons

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Fig. 4-Shows the importance of pos ition-dependent ill uminati on of the photoinducible phase (<I)i ) in the blac kheaded Imllfill g.l· (Emberiza melallocephala). The photoinductive elTec t of Shr light pul se. given at various intervals during 88hr constant da rk ness (~O) on plasma concentratio ns of LH in bun tings. Hori zonta l bar at the bottom indicate time of Sh li ght pu lses. A grou p was main­tained on short days and received an Hhr pu lsc at CT Ii - 19: thus served as contro l (C) . denoted by open symbol. left ill body ot the graph . The graph shows the change in LH as determin cd by sub­tracting . the pre- pulse pl asma Ll i pro lil e from thc post-pulse pl asma pro fil es (redrawn from Ku nwr (' / (I I. 1116)

1060 INDIAN J EXP BIOL, NOVEMBER 1999

received by the photoreceptorss8. At equal energy

levels, the ·number of photons emitted is larger in red light than in green or blue light and the penetration to brain tissues, thereby access to the photoreceptors (capture of large number of photons by the photopig­ments), of red light (long wavelengths) is far more faster and deeper than of blue or green light (short wavelengths)24,28)5)7,41. This is although inconsistent with the fact the deep brain photopigments in birds which are known to mediate the photoperiodic effects are not be maximall y sensit ive to red light, rather they are maximally sensitive to green li ght24, but they re­spond greater to red light since red light has greater access to them because of being dominant in the light environment and having greater penetrance through the brain ti ssues.

Temporal photosensitivity

Intensity and spectrum of li ght are not the onl y factors that induce seasonal responses in birds. If that was the case, regard less of the photoperiodic sched­ule, the light illumination at given spectrum and in­tens ity should evoke si milar physiological effects but it does not: Light pulses given at different times in the photosensi ti ve phase of CRPP induces differential response (Fig. 4). This supports the view that an "ap­propriate" photoperiod is a far more important facto r, of course after when the light intensity threshold is at tained , In other words, a hi gher intensity or a longer wavelength of light cannot act as a substi tute for a " long" photoperiod. The study of BurgerS') clearly shows that regardl ess of the increase or decrease in light intensity, a photoperiod of I 0.5L: 13.50 (a non­stimulatory photoperiod per se) did not st imulate spermatogenesis in male sta rli ngs. However, recentl y, Bentl ey et 01.60 have shown that the decrease in light intensity alters the perception of day length in star­lings : when groups of starlings were kept on 18L:60 at 3-, 13-,45- and 108- lu x li gh t int ensities, the photo­periodic responses observed in these groups were comparab le to those exposed to different photoperi­ods, viz. II L: 130, 13L: I: D, 16L:80 and 18L:60, respecti ve ly , Thus, there can be a fasc inating poss i­bility that to a certain extent light intensity and pho­toperiod may have synerg isti c effec ts. We are work­ing on the poss ibility if wavelengt h of li ght could also produce synergisti c effects along wi th li ght in tensity and photoperiod .

Non-reproductive circadian functions It is beyond the scope of thi s artic le to summari ze

light effects on the circadian fun ctions, other than reproduction, known so extensively but we intend to cite a . few important findings that wi ll indicate the spectrum of such studies. It is speculated that li ght information that is so unique in twi light times when there are large changes in irradiance as well as very precise changes in light spectrum is utili zed by ani ­mals to regulate their ci rcadian ti ming system. Fresh water alga Chlamydol11onos6 1 and the marine alga Gonyaulax62 are good examples of how spect ral in­format ion is used by the circadi an system in organ­isms with simple organization. A number of findin gs on vertebrates also suggest that the spectral changes of li ght affect the circadian system. Blue li ght incre­ments are more effec ti ve in advancing the start of ac­tivity than blue decrements and ye llow decrements are more effecti ve than ye ll ow increment s(" in the evening-acti ve wild rabbits. In go lden hamster, the sensitivity of the circadian system to li gh t spectrum was maxi mum near 500 nm and is sim il ar to the ab­sorption spectrum for rhodopsi n6~ . In the bat reti na , Joshi and Chandrashekaran6s suggest the ex istcnce of two classes of photoreceptors: S photoreceptors that have max imum sensiti vity at the wave length 430 nm and mediate delay phase shifts, and M photoreceptors which have maximum sensiti vity at the wavelengih 520 nm and mediate advance phase s hift ~ . However, such li ght-spectrum based regu larion of ci rcad ian system in vertebrates has not been thoroughl y studied. For ins tance, we do know that the circadian system of mouse66 is sensi ti ve to both green li ght and near-UV irradiance, but we do not know how the signal s from these different spectral channels are uti li zed by the mammalian circad ian system.

The intensity of li ght acts as dominant cxtcrnal time cue (entra ining agent, Zeilgehn - a German word, Zeit- time, geher - giver) fo r the entrainment or the clock in most organi sms, as shown in majorit y of cascs by its effects on the circadi an rhythm in loco­motor ac ti vity . Even uncleI' constant conclition:-- , li ghting can have important effec ts on rhythms that prov ide clues as to how the synchroni zation can hap­pen. The leve l of constant illuminat ion to whi ch an anima l is ex posed affects the ex pressed period (repre­sented ' by the Greek letter, tau 1) of the frccnlll ning rhythm (freerun is a chronobiol ogica l jargon that de­notes the express ion of a rhyth m with its peri od Linder constant conditions) . These effec ts were first sumlll (J-

KUMAR & RANI: WAVELENGTH AND INTENSITY EFFECTS 1061

rized more than three decades ago by J. Aschoff in what became known as "Aschoff' s Rule" (for details see67). This rule states that as constant light intensity increases, 't lengthens for nocturnal species and short­ens for diurnal species. With other regularities of the effects of lighting on the circadian system, like the effects on activity (represented by Greek letter alpha, a) and rest (represented by Greek letter rho, p) of the daily activity rhythm, added to Aschoff's Rule, they became known as "Circadian Rule". This then states that as constant light intensity increases, tau shortens, activity level increases, and the ratio of active phase to rest phase (a : p) increases for diurnal animals. The opposite effects are true of nocturnal animals. This rule holds pretty well for most groups of verte­brates that have been studied : fi sh, reptiles, amphibi­ans, birds and nocturnal mammals. The rule is less successful with most invertebrates , curiously seems not to apply to diurnal mammals, many of which re­spond as if they were, in fact, nocturnal.

The light effects are clearly seen in regulati on of rhythm in melatonin sec retion that occurs in night regardless of the nature of animals, diurnal , nocturnal or crepuscular. In starlings (Kumar et al., unpub­lished), daytime light intensity had some effects on the circadian pattern of plasma melatonin. There was also difference in the phasing of the melatonin rhythm: peak melatonin levels tended to occur earlier in LDdim ( I: 0.2 lux) than in LDbright (500 : 0.2 lux). The relationship between the li ght intensity at day and/or ni ght and the pattern of pl asma melatonin and/ or enzymes in volved in the melatonin synthes is has been studi ed also in a few mammalian spec ies6R-n .

Besides reproducti ve and circad ian functions, viz. rhythms in locomotor acti vity and melatonin secre­tion , the light of different illuminance and wavelength influence a variety of other responses in birds. Wiltschko and hi s co ll eaguesn7~ have investi gated the effects of I ight wavelength on magnetoreception in the avian mi grants (e.g. Tasmanian sil ver eyes, Zosterops I. haeralis). They have found that these migran ts were well oriented in their appropriate mi­gratory direction under 'wh ite' (full spectrum), blue and green li ght but were disoriented under 633 nm li ght in the red region of the light spectrum7ns.

Light illuminance and wavelength also affect growth of birds76-80

. When large white turkey hens at the age of 30 weeks were exposed to blue, green and red or incandescent light equalized at a photon out-

put, a colour-dependent effect of li ght was found on the cellular and humoral immune responses but not on stress status33

. The light colour has also been found to affect body temperature, kidney fun ction, sex ual function, adrenal function and pituitary fun cti on in birds32

.

Light-dependent compass ori entati on has also been reported in other vertebrates, such as fishess1

, am-h'b ' 8? 81 '1 84 K' P I lans -. " reptl es and mammals ' although

wavelength dependence seems to differ from th at or birds. In primitive chordate like ri ver lamprey, LOll/­

petra japonica, neuronal activity seems to be influ­enced by the light colour. It was inhibited by the li ght of short wavelengths and excited by middle to long wavelengths ; the maximum sensiti viti es of the in­hibitory and excitatory responses were at about 380 and 540 nm, respectively. Since environmental li ght contains both inhibitory and exc itatory components, the neurons would keep both sensitivities during day­time and could measure the variati on in the spectral composition. Tosini and Aver/ 6 ha ve reported that the spectral composition of I ight mi ght be an i mpor­tant variable in mediating the thermoregul atory proc­esses in lizards.

The circadian literature is full of references wherein a li ght pulse given at different times in the circadian cycle evokes differenti al responses, meas­ured in the context of circadian locomotor rhythms as phase shifts . A graphical representati on of such phase shift induced in the oscillator by li ght pulses is kn own as the phase response curve (PRC). PRCs obtained by the light pulse of varying intensity and/ or durati on (few seconds to few hours) as well as varying co lours are available for a number of organisms including plants. A few examples are: circad ian rh ythm or mating reacti on in Paramecillll1 hllr.\·(/ rioK7

, of CO2

output of Bryophyllum fedlschellko / K, of ec los ion

rhythm in Drosophila ~'i - 'i l , of petal movement s in Kalanchoe92 and of ac ti vity rhythm in Ra II 11.1' e. l'lI­

lans91. Three well-documented in stances are those of

the sporulati on rhythm of the fungus, Pi/obo /IIS'i~, the ec los ion rhythm of the Drosophi/(/'is and the circad ian rhythm of fli ght activity in the bat. Hipposideros speoris96 all of which respo nd with phase shi fts to ex tremely short li ght flashes of just 0.5 msec. Li ght fl ashes of different durati ons (0.063 - 3.33 msec .) phase shifts the circadian rhythm in fli ght act ivit y in the bat, Hipposideros speori.I·96 Kl ante and Stei n­lechner97 report in Djungari an hamster that a single weak red light pulse given 2 h before regular " li ghts

1062 INDIAN J EXP BIOL, NOVEMBER 1999

on" had acute and long term effects persistIng for several days following exposure; this indicates high sensitivity of circadian system to red light pulses during later part of the night.

As yet, it has been difficult to produce a PRC for the photoperi odic oscillator because it cannot be monitored directl y. Formal analyses of the photoperi­odic clock are done employing various LD cycle paradigms54 and examining their effects on the physiological and/ or behavioural markers of the clock system, assuming that these markers faithfully predict the photosensitive phase of the circadian photoperiodic c1ock'J8.'J'J. However, recent studies on few species including Japanese quail which show dis­sociation of photoperiodi c responses (LH rise) from other circadian locomotor acti vity rhythm, question if the properties of photoperiodic osc ill ator can be studied using any other marker of the circadian sys­temI OO

-I02

•

A final remark It is intriguing that CRPP responsible for photoin­

duction of reproductive responses is st imulated at such a hi gh li ght intensity when other circadi an sys­tems are known to be affec ted by very low intensities of light. A further conundrum in the photoperi odic literature is that while red light is the most effect ive agent for the stimulation of PNE-system in birds, it is considered as "safe li ght" (i .e. non-inducti ve) for the stimulation of PNE-system in mammals. Is it because of the differences in the sensiti vity of photoreceptors or because of the di ffe rences in the degree of pene­tration of red li ght through brain ti ssues overlying photoreceptors in two vertebrate c lasses? It is more puzzling when basic characteri stics of the photore­ceptors among birds and mammals have great simi­larity with each other. For example, although the anatomy of their eyes differs, the cones of human and fow l are both trichromat ic l03 and the relative spectral luminositi es of chicken , pigeon and man are the same I04,10., .

Acknowledgement Resources from CSIR research grant to YK were

utilized in preparat ion of thi s rev iew paper.

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