• Embed Doc
  • Copy Link
  • Readcast
  • Collections
  • CommentGo Back
Download
 
ARTICLE
doi:10.1038/nature10567
Melanopsin signalling in mammalian irisand retina 
T. Xue
1,2
, M. T. H. Do
1,2
{
, A. Riccio
3
, Z. Jiang
1,2
, J. Hsieh
1,4
, H. C. Wang
1,5
, S. L. Merbs
6
, D. S. Welsbie
6
, T. Yoshioka
1,7
,P. Weissgerber
8
, S. Stolz
8
, V. Flockerzi
8
, M. Freichel
8
{
, M. I. Simon
9
, D. E. Clapham
3
& K.-W. Yau
1,2,6
Non-mammalian vertebrates have an intrinsically photosensitive iris and thus a local pupillary light reflex (PLR). Incontrast, it isthought that the PLRin mammals generally requires neuronalcircuitry connecting the eye andthe brain.Here we report that an intrinsic componentof the PLR is in fact widespread innocturnal and crepuscular mammals. Inmouse,thisintrinsicPLRrequiresthevisualpigmentmelanopsin;italsorequiresPLC
b
4,avertebratehomologueofthe
Drosophila 
NorpAphospholipaseCwhichmediatesrhabdomericphototransduction.The
Plcb4
2
/
2
genotype,inadditionto removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (M1-ipRGCs), which are by far the mostphotosensitive ipRGC subtype and also have the largest response to light. Ablating in mouse the expression of bothTRPC6 and TRPC7, members of the TRP channel superfamily, also essentially eliminated the M1-ipRGC light responsebut the intrinsic PLR was not affected. Thus, melanopsin signalling exists in both iris and retina, involving a PLC
b
4-mediated pathway that nonetheless diverges in the two locations.
The discovery of ipRGCs has overturned the century-old belief thatrods and cones are the only mammalian retinal photoreceptors
1–6
.These ganglion-cell photoreceptors serve primarily non-image visualfunctions,oneofwhichisthePLR.Inanimalssuchasfish,amphibiansandbird,inadditiontotheneurallydrivenPLR,theirisitselfiscapableof autonomous, light-induced constriction
7–11
. For mammals, it isthought that the PLR generally requires neuronal circuitry throughthe brain, although sporadic reports
7,12,13
and some controversy existof an intrinsic iridic photosensitivity in occasional species, including human. Even in non-mammalian vertebrates, the photopigment driv-ingtheintrinsicPLRremainsunidentified.Inamphibiansandfishthisphotopigment has been proposed to be rhodopsin
8,9
, whereas inchicken it is suggested to be the non-opsin-based cryptochrome
11
.Wehaveexaminedtheabovequestionsmoreclosely,andfoundthattheintrinsicPLRissurprisinglywidespreadinmammals.Moreover,itbears a close relationship to the ipRGCs in phototransduction.
Intrinsic PLR in mouse and other mammals
We found that bright light triggered a pupillary constriction in anintact eye freshly isolated from a dark-adapted pigmented mouse(Fig. 1a;
n
5
3 eyes; Supplementary Methods). This photosensitivity disappeared within
,
1min after eye isolation, perhaps due to anoxia,with the pupil remaining constricted thereafter. This PLR persisted ina reduced preparation with just the eye’s anterior chamber andiris
8,11,12
under L-15 medium (Fig. 1b;
n
5
3 eyes; Supplementary Methods), even after blocking any potential parasympathetic activity totheiriswith0.5%atropine;however,thePLRagainfadedafterafew light trials.Themammalianirishasthreemaintissuelayers,allpigmentedwithmelanin: an anterior fibrovascular stroma; a middle smooth-musclelayer consisting of the circumferential sphincter muscle at the pupilperimeter and the radial dilator muscle across the iris; and a posteriorepithelium
7,14
. In non-mammalian vertebrates, the sphincter muscleitself is thought to be the light sensor
7,14
. Accordingly, we reduced themouse preparation further to the small ring of sphincter muscle, andconnected it to a strain gauge for measuring isometric tension underoxygenated Ames solution
9,10
(35–37
u
C; Supplementary Methods).The isolated sphincter muscle reproducibly gave a light-induced con-tractile force for hours. A relatively dim flash elicited a transientincrease in force that grew linearly with increasing flash intensity; thatis,proportionaltoflashintensityandwithaconstantwaveform(Fig.1cand inset, bottom; see also Supplementary Fig. 1). This flash-inducedcontraction reached a transient peak in
,
1s but decayed much moreslowly, notdissimilartothe
in situ
ipRGC-driven PLR 
15
. The responsetoanintenseflashoftenshowedahumpduringitsdecay(Fig.1cinset,top). Although the force elicited by a dim flash decayed to baselinein
#
1min, asecondidenticalflashtypicallyelicited asmallerresponseunless given in
$
8min after the first. Likewise, a near-saturated res-ponsetoanintenseflashdecayedin
,
1min,buta
,
15-mindelaywasrequired for a second identical flash to elicit a comparable response.This light adaptation was also obvious during steady light as a relaxa-tion of the force from a transient peak to a lower plateau level (Fig. 1d;
n
5
3 muscles). In contrast, acetylcholine-elicited contraction did notshow this adaptation (Supplementary Fig. 2), suggesting that thisadaptation resided in the photosignalling pathway upstream of thecontractile mechanism.The intrinsic PLR turns out to be widespread across nocturnalmammals (Supplementary Methods). Albino mice showed the samephenomenon but their sphincter muscle was more photosensitive(not shown), presumably due to higher light transmission through
1
Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
2
Center for Sensory Biology, Johns Hopkins University School ofMedicine,Baltimore,Maryland 21205,USA.
3
Departmentof Cardiology, Children’sHospitalBoston,HarvardMedical School, andHoward HughesMedical Institute,Boston,Massachusetts02115,USA.
4
BA/MS Concurrent Program in Neuroscience, Johns Hopkins University, Baltimore, Maryland 21238, USA.
5
Neuroscience Graduate Program, Johns Hopkins University School of Medicine, Baltimore,Maryland21205,USA.
6
DepartmentofOphthalmology,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,Maryland21205,USA.
7
TheZanvylKriegerMind/BrainInstitute,JohnsHopkinsUniversity,Baltimore,Maryland21238,USA.
8
Institutfu¨rExperimentelleundKlinischePharmakologie undToxikologie,Universita¨tdesSaarlandes,66421Homburg/Saar,Germany.
9
DivisionofBiology,CaliforniaInstituteofTechnology,Pasadena,California91125,USA.
{
Presentaddresses:F.M.KirbyNeurobiologyCenter,DepartmentofNeurology,Children’sHospitalBostonandHarvardMedicalSchool,Boston,Massachusetts 02115, USA (M.T.H.D.); Pharmakologisches Institut Im Neuenheimer Feld 366, 69120 Heidelberg, Germany (M.F.).
3 N O V E M B E R 2 0 1 1 | V O L 4 7 9 | N AT U R E | 6 7
Macmillan Publishers Limited. All rights reserved
 ©2011
 
the non-pigmented iris. Albino rat (Fig. 2a, left;
n
5
2 muscles;pigmented species not tested) and hamster (Fig. 2a, right;
n
5
3muscles; pigmented, but with little iridic melanin in the pupillary margin) also tested positively, with sensitivities similar to the albinomouse but with a much larger muscle force. Dog, cat and rabbit (allpigmented) also showed an intrinsic PLR (
n
5
2, 1 and 3 muscles,respectively),butwithdistinctlylowerphotosensitivityandforcepro-duction; cat and rabbit required a light step instead of flash to elicit adetectable response (Fig. 2b). Although these three species are notstrictly nocturnal, they are crepuscular (that is, active at dawn anddusk). The Nile grass rat, which has both diurnal and nocturnaltendencies
16
, also tested positively (
n
5
3 muscles, data not shown).On the other hand, the response was absent for guinea-pig (
n
5
2),ground squirrel (
n
5
4) and pig (
n
5
2) (data not shown). Groundsquirrel is strongly diurnal, whereas guinea-pig and pig are variably describedascrepuscularorevendiurnal.Noneoffourprimatespeciestested showed this phenomenon, including rhesus monkey (Fig. 2b;diurnal,
n
5
7 muscles), marmoset (diurnal,
n
5
2), owl monkey (nocturnal,
n
5
4) and bush baby (nocturnal,
n
5
2) (the latter threenot shown). Thus, nocturnal/crepuscular non-primate mammalstend to have an intrinsic PLR.
Melanopsin involvement in mammalian intrinsic PLR
The action spectrum for the isolated mouse sphincter muscle (Sup-plementary Methods) fit an A
1
-pigment spectral template
17
with
l
max 
at 480nm (Fig. 3a), indicative of the involvement of melanopsin
3–6
.Indeed, the muscle from melanopsin knockout
18
(
Opn4
2
 / 
2
) micegavenolightresponse(Fig.3b).Usingpolymerasechainreactionwithreverse transcription (RT–PCR), we detected melanopsin messengerRNA in the isolated wild-type mouse iris (Fig. 3c and Supplementary Methods). Immunostaining (in an albino background for viewing immunofluorescence) also indicated melanopsin’s presence in thesphincter muscle of wild-type, but not
Opn4
2
 / 
2
, iris (Fig. 3d andSupplementary Methods). A BAC-transgenic mouse expressing thefluorescent protein tdTomato under the melanopsin promoter
15
(
Opn4:tdTomato
, bred into an albino background) also showed fluor-escence at least in the sphincter muscle (Fig. 3e). For rat and hamster,their particularly robust sphincter-muscle photoresponses (see above)allowedcharacterizationoftheassociatedactionspectra,whichalsofita480-nm spectral template (Supplementary Fig. 3). Strictly speaking,because melanopsin seems to be more widespread than in just thesphincter muscle (Fig. 3d), contraction is not necessarily initiated by light absorbed just in the muscle itself, but this is likely.Formouse, we were ableto ruleout anysignificantroleof rhodopsinor cryptochromes in the sphincter-muscle photosensitivity (see above);namely,besidesanullcontractilephenotypeofthe
Opn4
2
/
2
muscle,theresponses from
Rho
2
/
2
(rhodopsin knockout
19
) and
Cry1
2
/
2
Cry2
2
/
2
(cryptochrome 1 and 2 double knockout
20
) mice appeared normal(Supplementary Fig. 4a). RT–PCR detected rhodopsin mRNA in the
   D  a  r   k   L   i  g   h   t
Isolated eyeball
a bcd
010203040500.00.20.40.60.81.0321Light
   N  o  r  m  a   l   i  z  e   d  p  u  p   i   l  a  r  e  a
Time (s)32160Isolated anterior chamber050100150200050100
 
Time (s)
   F  o  r  c  e   (
     μ
   N   )
Light
     μ
   N
Time (s)0180
 
0302040
     μ
   N
050100150
   F  o  r  c  e   (
     μ
   N   )
10
8
10
9
10
10
Flash intensity(photons
μ
m
–2
 )(equiv. 480 nm)(436 nm)Isolated iris sphincter muscle10
8
10
9
10
10
Figure 1
|
Intrinsic PLR of mouse. a 
, Constriction of pupil (red circle) infreshlyisolatedeye(yellowcircle),elicitedby1.2
3
10
2
3
m
W
m
m
2
2
ofwhite Xelight for
,
3s over iris (23
u
C).
b
, Intrinsic PLR in isolated anterior chamber,plotted as pupil area normalized to dark state (23
u
C). The inset shows pupil attimes indicated by arrows; white Xe light (30s of 4
3
10
2
4
m
W
m
m
2
2
) overentire iris.
c
, Flash-intensity–response relation for sphincter muscle force attransient peak of response (mean
6
s.e.m., 7 muscles) (35–37
u
C). Fit is
R
max 
1
{
e
{
I 
=
I 
0
À Á
with
R
max 
5
116
m
N,
I 
0
5
2.3
3
10
9
photons
m
m
2
2
(436-nmHg light except for brightest two intensities, which were white; 3-mm spotcovering entire muscle). Flashes were 12–400ms in duration. The inset showssampleresponsestoadimandabrightflashdelivering(attime0)7.2
3
10
7
and6.5
3
10
9
photons
m
m
2
2
(436nm), respectively.
d
, Muscle-force response to alightstep(6.1
3
10
9
photons
m
m
2
2
s
2
1
at436nm)toindicateadaptation.Flashintensities are also expressed in equivalent 480-nm photons, given thatmelanopsin is probably the signalling pigment (see Fig. 3). All wild-type micehere and in subsequent figures were C57BL/6J, unless indicated otherwise.
0200400600 
   F  o  r  c  e   (
     μ
   N   )
Flash intensity(photons
μ
m
–2
 )10
6
10
7
10
8
10
6
10
7
10
8
(equiv. 480 nm)(436 nm)Flash intensity(photons
μ
m
–2
 )10
6
10
7
10
8
05001,0001,5002,000
   F  o  r  c  e   (
     μ
   N   )
10
6
10
7
10
8
(equiv. 480 nm)(436 nm) Albino ratHamster
a
02,00002002040Time (s)
     μ
   N
0400015Time (s)
     μ
   N
02040
02004006000204060
    F  o  r  c  e   (
     μ
   N   )
Time (s)Time (s)04080120Time (s)
   F  o  r  c  e   (
     μ
   N   )
0204060
 
DogRabbit04008001,200050100Time (s)Rhesus monkey AChACh
   F  o  r  c  e   (
     μ
   N   )
b
020040002040
   F  o  r  c  e   (
     μ
   N   )
Cat
Figure 2
|
Intrinsic photosensitivity of iris sphincter muscle of other mammals.
Temperaturerange35–37
u
C.
a 
,Flash-intensity–responserelationsfrom robust light responses of albino rat and pigmented hamster muscles (twofor rat and three for hamster). Same fits as in Fig. 1, with
I 
0
(averaged acrossindividual muscles) being 4.3
3
10
7
photons
m
m
2
2
and4.6
3
10
7
photons
m
m
2
2
(436nm) for rat and hamster, respectively. Flasheswere12–400ms.Insetsshowsampleresponsesfromamuscleofeachspeciestodim and saturating flashes: 3.4
3
10
6
and 2.1
3
10
8
photons
m
m
2
2
for rat and1.6
3
10
6
and 3.6
3
10
8
photons
m
m
2
2
for hamster, respectively (436-nm Hg light).
b
, Similar experiments on dog (representative of 2 muscles), rabbit (3muscles),cat(1muscle)andrhesusmonkey(7muscles).Allstepsandtheflash(600ms) delivered 6.1
3
10
9
photons
m
m
2
2
s
2
1
(436nm). Monkey muscle(pre-incubatedwith30
m
M9-
cis
-retinalfor1h)gavenoobviouslightresponse,but responded to 50
m
M acetylcholine (ACh) in bath (black bars). Fastdeflections in the rabbit and cat experiments reflected spontaneous musclecontractions/relaxations of unknown cause. A 5-mm-diameter light spot wasused, covering the entire muscle of rat, hamster, cat and rabbit, but only partially the larger muscles of other species.
RESEARCHARTICLE
6 8 | N AT U R E | V O L 4 7 9 | 3 N O V E M B E R 2 0 1 1
Macmillan Publishers Limited. All rights reserved
 ©2011
 
dissected mouse iris (not shown; see alsoref. 21),but itssignificance isunclear.By RT–PCR, we found melanopsin mRNA in the iris of rhesusmonkey (not shown) and baboon (also diurnal; Supplementary Fig. 5),but the melanin pigmentation confounded confirmation by immuno-histochemistry. Melanopsin’s function in the primate iris is similarly unclear because there is no intrinsic PLR.
Phototransduction mechanism underlying intrinsic PLR
Melanopsin has a phylogenetic association with invertebrate rhabdo-mericvisualpigments
22
,thuspossiblysharingacommonphospholipaseC (PLC)-mediated phototransduction pathway (see ref. 23 for review).Moreover, PLC typically mediates membrane-receptor signalling insmooth muscles
24,25
. Indeed, the sphincter muscle from
Plcb4
2
/
2
mice
26
waspracticallyunresponsivetolight(Fig.4a;
n
5
5muscles).Sometimes,we observed a tiny response that disappeared after several stimuli (redtrace in Fig. 4b, top; dim flash; 3 out of 5 muscles), unlike the muchlargerandpersistentwild-typeresponse(blacktraceinFig.4b,top;dimflash). This small response could be mediated by a different PLC-
b
isoform or another minor pathway. The
Plcb4
2
 / 
2
phenotype was notdue to a defective contractile apparatus, because acetylcholine stillelicited strong contraction via muscarinic receptors on the muscle
14
(Fig. 4b, bottom). PLC
b
4 is the closest vertebrate homologue
26
of the
Drosophila
PLC (NorpA), which mediates phototransduction inrhabdomericphotoreceptors
23
.Bath-appliedU71322,aPLCinhibitor,did not block the light-induced contraction of the wild-type sphinctermuscle(notshown),butthismayreflectpoordrugpenetrationintothetissue (see below).Smooth-muscle contraction often involves intracellular Ca
2
1
release (triggered by PLC via inositol-1,4,5-trisphosphate generation)in tandem with extracellular Ca
2
1
influx 
24,25
. Indeed, blocking intra-cellular Ca
2
1
uptake with 1
m
M thapsigargin to deplete the Ca
2
1
-release pool gradually eliminated the muscle’s light response(Fig. 4c, top;
n
5
3 muscles). The muscle’s resting tension increasedand oscillated during thapsigargin application, suggesting poor intra-cellular Ca
2
1
handling.Removing extracellular Ca
2
1
likewise reducedthelightresponseby 
,
80%(Fig.4c,bottom;seealsorefs9,10,12)andpartiallyrelaxedtherestingtension.Becausemembranedepolarizationis reportedly unnecessary for the intrinsic PLR in non-mammalian vertebrates
9,10
, Ca
2
1
-permeable ion channels other than voltage-gatedCa channels are probably involved, with TRP channels—especially 
Melanopsin
Pnr 
WT
Opn4
 –/–
 
DICtdTomato
   W   T
   O  p  n   4  :   t   d   T  o  m  a   t  o
a
10
8
10
9
10
10
050100150
WT
Opn4
 –/–
03060050100
 
Time (s)
     μ
   N
   F  o  r  c  e   (
     μ
   N   )
Flash intensity(photons
μ
m
–2
in equiv. 480 nm)
bcde
Opn4
 –/–
WT
DICMelanopsinSmooth-musclemarkerDAPICombined
4004505005506000.010.11
   N  o  r  m  a   l   i  z  e   d  s  e  n  s   i   t   i  v   i   t  y
Wavelength (nm)
anterior
nterior
posterior
osterior
irispupilanterior
nterior
posterior
osterior
   I  r   i  s   R  e   t   i  n  a    R  e   t   i  n  a    I  r   i  s
β
-actin
Figure 3
|
Dependence of intrinsic PLR on melanopsin. a 
, Action spectrumof mouse muscle (wild type, pigmented) (6 muscles), with sensitivity normalizedtovalueat480nmineachexperiment.Redcirclesareaverages,andcurve is an A
1
-pigment spectral template
17
with
l
max 
5
480nm. Hg light withinterference filters used. Error bars are s.e.m.
b
, Average flash-intensity–response relation for
Opn4
2
/
2
muscle (3 muscles). Wild-type relation fromFig.1calsoshownforcomparison.Errorbarsares.e.m.Theinsetshowssampleresponses of wild-type and
Opn4
2
/
2
muscles to a saturating flash of 4.0
3
10
9
photons
m
m
2
2
(equivalent 480nm).
c
, Melanopsin (
Opn4
) mRNA detected by RT–PCR in iris and retina from wild type but not
Opn4
2
/
2
.
Pnr 
(photoreceptor-specificnuclearreceptor;alsocalled
Nr2e3
)mRNAwasusedasacontroltoruleoutcontaminationfromretinatoiris.
b
-ActinmRNAwasusedas positive control. Difference in melanopsin mRNA signal between iris andretina presumably reflects different fractional total-tissue mRNA coding formelanopsin.
d
, Immunostaining of wild-type and
Opn4
2
/
2
iris cross-sectionsfor melanopsin (green), smooth-muscle
a
-actin (red, as muscle marker) andDAPI (blue). Anterior side (stroma) up and posterior side (posteriorepithelium) down. The black arrowhead marks the pupillary edge. TheintensityofmelanopsinimmunofluorescenceappearedlowerintheiristhaninipRGCs (not shown). Additionally, although the
Opn4
2
/
2
mouse contains the
tau-lacZ 
marker gene replacing 
Opn4
(ref. 18),
b
-galactosidase activity (by X-gal labelling) was not evident in the iris (not shown), presumably due to thelowmelanopsin-promoteractivity.Scalebar:20
m
m.
e
,TdTomatofluorescencesignal detected in the iris of 
Opn4:tdTomato
but not wild-type mice. Scale bar:100
m
m. Stimuli in
b
were 436-nm Hg light, and white for the two brightestintensities, although expressed in equivalent 480-nm photons. A 3-mm-diameter spot covered the entire muscle. All force measurements at 35–37
u
C.Mice in
d
and
e
were albino (C57BL/6J-Tyr
c-2J
/J).
050100150
WT
Plcb4
 –/–
   F  o  r  c  e   (
     μ
   N   )
060
0100
Time (s)
     μ
   N
30
50
10
8
10
9
10
10
Flash intensity(Photons
μ
m
–2
in equiv. 480 nm)0204060051015Time (s)
   F  o  r  c  e   (
     μ
   N   )
0100200300050100150
Time (s)
 ACh
   F  o  r  c  e   (
     μ
   N   )
a bc
0.00.51.0
  0   C  a
  2 +
   N  o  r  m  a   l   i  z  e   d     o  r  c  e
  C  o  n   t  r  o   l   W  a  s   h  o  u   t
012
Before 0 Ca
2+
012
During 0 Ca
2+
   F  o  r  c  e   (
     μ
   N   )
0204060012
Time (s)
Washout
050100051015
0200400
   F  o  r  c  e   (
     μ
   N   )
R  e s t  i  n gt   en s i   on ch  an g e (  
   μ
N )  
1
μ
M thapsigargin
Time (min)
020
Before thapsi.
020
Early thapsi.
03060020Time (s)
1.5 h thapsi.
   F  o  r  c  e   (
     μ
   N   )
0306090–5051015–5051015
Time (min)
   F  o  r  c  e   (
     μ
   N   )
R  e s t  i  n gt   en s i   on ch  an g e (  
   μ
N )  
0 Ca
2+
0.00.51.0
   N  o  r  m  a   l   i  z  e   d     o  r  c  e
  C  o  n   t  r  o   l   T   h  a  p  s   i .
Figure 4
|
Phototransduction mechanism underlying intrinsic PLR.a 
, Average flash-intensity–response relation for
Plcb4
2
/
2
muscle (5experiments).Wild-typerelationfromFig.1cshownforcomparison.Theinsetshows sample responses of wild-type and
Plcb4
2
/
2
to a saturating flash, at4.0
3
10
9
photons
m
m
2
2
(equivalent 480nm).
b
, Top: example of a
Plcb4
2
/
2
muscle (red) showing a tiny response (
,
3
m
N) to the first few dim flashes(7.3
3
10
7
photons
m
m
2
2
) before becoming unresponsive. The wild-typeresponse (black) to the same dim stimulus is also shown for comparison.Bottom: the same
Plcb4
2
/
2
muscle responded substantially to 10
m
Macetylcholine.
c
, Thapsigargin and removal of extracellular Ca
2
1
, respectively,greatly diminished the light response. Left: time course of effect on peak force(black) generated by dim flashes (1.1
3
10
8
photons
m
m
2
2
). Resting muscletension (red) arbitrarily set as 0 before thapsigargin or 0Ca
2
1
application.Middle: sample responses. Right: collected data (4 muscles each). White Hg light for two brightest flashes in intensity-response relations of 
a 
; all otherstimuli were 436-nm Hg light. All intensities are expressed in equivalent480nmphotons.Flashesdeliveredattime0,asa3-mm-diameterspotcovering the entire muscle; 35–37
u
C. All error bars are s.e.m.
ARTICLE
 
RESEARCH
3 N O V E M B E R 2 0 1 1 | V O L 4 7 9 | N AT U R E | 6 9
Macmillan Publishers Limited. All rights reserved
 ©2011
of 00

Leave a Comment

You must be to leave a comment.
Submit
Characters: ...
You must be to leave a comment.
Submit
Characters: ...