the vertebrate retina receives efferent inputs from different parts of the central nervous system but we still do not understand how these regulate visual processing ( ramon y cajal , 1894 ; reprant et  al . , 1989 ) . in teleosts ,
the main source of retinopetal fibers is the terminal nerve ( tn ) , which receives dense afferents from the olfactory bulb and in turn projects gnrh- and fmrfamide - containing fibers to the retina ( springer , 1983 ; zucker and dowling , 1987 ; demski , 1993 ; yamamoto and ito , 2000 ; reprant et  al . , 2007 ) .
the tn is tonically active , with a firing frequency that changes according to the physiological conditions of the animal , including arousal , motivational state , hormonal milieu , and glutamatergic inputs from various sensory systems ( abe and oka , 2006 ; wang et  al . , 2011 ) .
together , the pathways linking the olfactory bulb to the retina through the tn are known as the olfacto - retinal circuit ( orc ) .
behavioral assays examining visual threshold have shown that stimulation of the olfactory bulb by food - related amino acids induces an increase in luminance sensitivity through activation of the orc ( maaswinkel and li , 2003 ; behrens and wagner , 2004 ; li and maaswinkel , 2007 ) .
it has been suggested that an olfactory stimulus alters the processing of visual signals by decreasing the concentration of  dopamine in the retina ( huang et  al . , 2005 ) .
the sole source of dopamine in the retina of teleosts is a specialized class of amacrine cell , the interplexiform cells ( ipcs ) , which are the target of the tn ( umino and dowling , 1991 ) .
li and dowling ( 2000a ) have shown that zebrafish affected by the night blindness b mutation ( nbb ) , which provokes a progressive reduction in the number of ipcs , exhibit a 23 log unit decrease in luminance sensitivity and a profound loss of signals derived from rods .
dopamine ( da ) released from ipcs has a number of actions on the retinal circuit , which together act to enhance cone - mediated signals under bright conditions . in the outer retina ,
dopamine decreases electrical coupling between rods and cones ( ribelayga et  al . , 2008 ) , while inhibiting voltage - gated calcium currents in rods and boosting calcium currents in cones ( stella and thoreson , 2000 ) .
dopamine also inhibits electrical coupling between horizontal cells and increases their sensitivity to glutamate , resulting in less powerful negative feedback to cones ( knapp and dowling , 1987 ; devries and schwartz , 1989 ; mcmahon , 1994 ) . in the inner retina ,
actions on bipolar cells and retinal ganglion cells ( rgcs ) have also been reported , but their roles in altering retinal processing under different lighting conditions are not clearly established ( jensen and daw , 1984 ; jensen , 1992 ; heidelberger and matthews , 1994 ; li and dowling , 2000b ; ribelayga et  al . , 2002 ) .
how might the actions of dopamine underlie the modulation of retinal processing by an olfactory stimulus ?
one of the difficulties in studying a multisensory circuit is the need to conduct experiments in  vivo in order to maintain the link between the different sensory systems . in this study , we take advantage of zebrafish expressing genetically encoded calcium reporters in the synaptic terminals of bipolar cells or dendrites of rgcs ( dreosti et  al . , 2009 ; odermatt et  al . ,
these fish allow the visual signal to be monitored as it is transmitted to the inner retina and rgcs providing the output from this circuit . by imaging signals through all layers  of the inner retina , we have observed activity at the origins of the on and off channels that encode a change in light intensity with signals of opposite polarity ( schiller et  al . , 1986 ) .
here , we demonstrate that an olfactory stimulus reduces the gain but increases the sensitivity with which off bipolar cells transmit signals encoding luminance and contrast .
pharmacological manipulations in  vivo demonstrated that olfactory stimuli regulate the presynaptic calcium signal of bipolar cells by reducing the activity of d1 dopamine receptors , and electrophysiology in isolated bipolar cells demonstrated that activation of endogenous dopamine receptors potentiates voltage - dependent calcium channels that control synaptic transmission .
together , these results indicate that the orc acts through the neuromodulator dopamine to regulate synaptic transmission through the off channel in the retina .
transmission of the visual signal through the on and off pathways in the retina was assessed in transgenic zebrafish expressing sygcamp2 under the ribeye promoter ( dreosti et  al . , 2009 ) , allowing synaptic activity to be monitored across the population of bipolar cells projecting to all layers of the inner plexiform layer ( ipl ) ( figure  1a ) .
first , we investigated the effects of olfactory stimulation on the response to changes in the luminance of full - field stimuli .
all measurements were carried out between 9:00 and 11:00 a.m. , when behavioral experiments have demonstrated that the orc is most effective in modulating visual sensitivity ( maaswinkel and li , 2003 ) .
figure  1b shows responses of an individual off terminal to light steps of different intensity , before ( dark red ) and after ( light red ) the addition of 1  mm methionine to the medium surrounding the fish . in off terminals
, there was a decrease in the maximum amplitude of the response , which we term a decrease in gain .
additionally , off terminals began to respond at lower intensities , which we term an increase in sensitivity .
in contrast , the large majority of on bipolar cells were unaffected by olfactory stimulation , and an individual example is shown in figure  1c .
collected results from 84 off terminals from seven fish are shown in figures 1d1 g .
methionine reduced the response to  the brightest step of light by an average of 36.6%   8.2% ( p  <
0.0001 ) , and this effect was evident across the population of off terminals ( figure  1e ) . in parallel ( figure  1f ) ,
methionine increased the luminance sensitivity of off terminals by 0.5 log units , assessed by the lowest light level eliciting responses > 3  sd of the baseline noise . a similar shift
could be assessed by fitting the intensity - response measurements with a hill function ( figure  1 g ) and estimating i1/2 , the intensity generating a half - maximal response ( experimental procedures ) .
the increase in luminance sensitivity observed at the synaptic output of bipolar cells was quantitatively similar to the increase observed previously in ganglion cell recordings and behaviorally following olfactory stimulation ( maaswinkel and li , 2003 ; huang et  al . , 2005 ) .
the actions of methionine were almost exclusively on the off pathway . the large majority of on synapses in a population of 116 were not affected , either in terms of response amplitude or sensitivity ( figures 1h1k ) .
the exception was a small but distinct fraction of terminals ( 9% ) , in which the amplitude of the presynaptic calcium signal was increased by a factor of at least three ( arrow in figure  1i and described further in figures s1a and s1b available online ) .
this subset of on terminals was not distinguishable from others in terms of size or position in the ipl and is therefore unlikely to reflect a difference between cone - driven and mixed rod - cone bipolar cells .
no significant changes in on or off responses were observed over the same time window in the absence of methionine .
although behavioral experiments showed olfactory stimulation to be ineffective in the afternoon ( maaswinkel and li , 2003 ) , we found that methionine administration produced a qualitatively similar but  significantly smaller modulation of responses through the off pathway tested between 12:00 and 3:30 p.m. ( figures s1c  s1f ) .
many retinal neurons signal fluctuations in light intensity at frequencies up to 20  hz . to test the effects of an olfactory stimulus on the signaling of temporal contrast , we modulated full - field stimuli at 5  hz and measured the sygcamp2 signal ( 5 fish , n  = 122 terminals ) .
figure  2 shows that the most obvious effect of an increase in contrast was a steady offset in the sygcamp2 signal , reflecting a net accumulation of calcium . a change in the sygcamp2 signal in the absence of a change in the mean
the relation between the amplitude of the steady sygcamp2 signal ( a ) and contrast ( c ) is shown in figure  2b : it could be described by  a simple power function of the form a  = k  c , with   = 1.8   0.1 under control conditions . stimulation with
methionine reduced the amplitude of the rectifying response at all contrasts above 20% ( figure  2a ) . furthermore , methionine increased the exponent  of the power function to 2.9   0.2 ( p  < 0.0001 ) .
this renders off terminals more sensitive to higher contrasts at the expense of lower contrasts , e.g. , a change in temporal contrast from 80% to 90% caused a change of 23.5% f / f0 in control versus 40% f / f0 in methionine . olfactory stimulation did not , however , affect the responses of on bipolar cells ( figures 2c and 2d ) .
we also observed a small subset of on bipolar cells in which the dc presynaptic calcium levels were reduced by stimulation at 5  hz , and these were also unaffected by application of methionine ( figures s2a and s2b ) .
the inhibition of presynaptic calcium signals by olfactory stimulation was also a function of stimulus frequency .
we tested the responses of off terminals to frequencies between 0.2 and 25  hz at 90% contrast ( 6 fish , n  = 96 terminals ) and found that methionine reduced the amplitude of the sygcamp2 signal elicited by stimuli below 10  hz . again
together , the results in figures 1 , 2 , and s2 indicate that olfactory stimulation alters the processing of visual signals in the retina by two distinct actions on off bipolar cells : a suppression of the presynaptic calcium signal , resulting in a reduction in gain and an increase in luminance sensitivity .
the large majority of on bipolar cells were unaffected by this form of cross - modal regulation .
having observed an action of olfactory stimulation on the visual signal as it is transmitted by bipolar cells , we investigated how far the responses of postsynaptic ganglion cells were also affected . to monitor signals across large populations of neurons in  vivo
, we made a line of zebrafish expressing the calcium reporter gcamp3.5 under the eno2 promoter , which drives expression in rgcs ( bai et  al .
step changes in luminance were a relatively ineffective stimulus for rgcs , so we examined the effects of an olfactory stimulus on full - field stimuli modulated at 5  hz . the advantage of this in  vivo imaging approach over electrophysiology is that it allows stimulation of the olfactory system while observing activity across a large population of rgcs .
responses from rgc dendrites were classified as off ( figures 3b and 3c ) , on ( figures 3d and 3e ) , or on - off ( figures 3f and 3 g ) , according to the responses to steps of light ( figure  s3 ) .
methionine induced a reduction in gain of off and on - off rgcs at contrasts of 50% and above , without an appreciable effect on on rgcs .
these results are consistent with the reduced gain of responses to contrast observed in off bipolar cell terminals , but not on , following application of methionine ( figure  2 ) .
they also confirm that the actions of the orc are evident in the retinal output , as previously demonstrated by maaswinkel and li ( 2003 ) and huang et  al .
existing evidence suggests that a key signal is dopamine released by ipcs ( umino and dowling , 1991 ; huang et  al . , 2005 ) .
to investigate how dopaminergic signaling might be involved in modulating synaptic activity of  bipolar cells , we injected agonists or antagonists of dopamine receptors into the anterior chamber of one eye of a fish , with a parallel sham injection into the other eye acting as a control .
the first manipulation was to activate dopamine receptors by injecting the agonist [ 3h ] 2-amino-6,7-dihydroxy 1,2,3,4-tetrahydronapthalene ( adtn ) at an estimated concentration of 0.2  m ( see experimental procedures ) . in off terminals , adtn increased the amplitude of sygcamp2 responses to all but the brightest lights and luminance sensitivity ( i1/2 ) increased by a factor of 420 ( figures 4a and 4b ; n  = 92 terminals ) . in on terminals , adtn increased the amplitude of the sygcamp2 response to bright lights by 108% and increased luminance sensitivity by a factor of 15 ( figures 4c and 4d ) .
strong activation of dopamine receptors therefore potentiated presynaptic calcium signals in both on and off bipolar cells , an effect opposite to an olfactory stimulus .
further , application of methionine in the presence of  0.2  m adtn no longer depressed signals through off bipolar cells ( figures 4a and 4b ) .
both these observations are consistent with the idea that an olfactory stimulus modulates retinal function by decreasing dopamine release .
the second manipulation was to antagonize the action of endogenous dopamine by injection of 100  nm sch 23390 ( 7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol ) , a selective antagonist of dopamine d1 receptors ( mora - ferrer and neumeyer , 1996 ; bourne , 2001 ) .
sch 23390 injection resulted in a complete impairment of luminance signaling through off bipolar cells ( figures 5a and 5b ) .
in contrast , the maximum amplitude of the response in on bipolar cells was not significantly affected , although the light sensitivity ( i1/2 ) was increased by a factor of 3.8 ( figures 5c and 5d ) .
antagonizing d1 receptors therefore caused effects qualitatively similar to an olfactory stimulus : a selective decrease in the gain of signaling through the off pathway ( cf .
, we used the antagonist sulpiride at a concentration of 2  m ( lin and yazulla , 1994 ; mora - ferrer and gangluff , 2000 ) .
first , the sensitivity increased sufficiently to reduce threshold by 2 log units , likely reflecting the potentiation of rod inputs ( ribelayga et  al . , 2008 ) .
second , the luminance - response relation did not simply rise monotonically , but instead passed through a maximum ( figures 5e and 5f ) . despite these changes in circuit function , the maximum response to luminance
was reduced by 29%   5.6% when methionine was applied after injection of sulpiride , an effect similar to that of olfactory stimulation under control conditions ( figures 5e and 5 g ) .
sulpiride also increased sensitivity of signals through on bipolar cells ( 0.85 log units ) , but methionine had no effect on the amplitude of these responses ( figures 5h5j ) .
an olfactory stimulus therefore continued to cause a selective reduction of responses through the off pathway when activation of d2 receptors was blocked .
together , the results in figures 4 and 5 indicate that cross - modal regulation of retinal function depends primarily on the activity of d1 receptors . to test further the idea that activation of the orc acts by decreasing dopamine level in the retina
, we attempted to prevent these changes without interfering with the activity of dopamine receptors .
our strategy was to inject vanoxerine ( gbr 12909 ; 2  m ) , a potent and specific blocker of the transporters involved in dopamine reuptake from extracellular space and into secretory vesicles ( reith et  al .
vanoxerine administration has been reported to result in a small but steady increase in extracellular dopamine concentration , followed by a persistent  clamp  once both uptake and release are blocked ( rothman et  al . , 1991 ; lima et  al . , 1994 ; reith et  al . , 1994 ; schlicker et  al .
first , the luminance sensitivity of off terminals was increased by a factor of 26 ( figures 6a and 6b ) and of on terminals by a factor of 2 ( figures 6d and 6e ) .
notably , these increases in sensitivity were much smaller than those caused by the dopamine receptor agonist adtn ( figures 6b and 6e ) , indicating that increases in dopamine levels were relatively small and not sufficient to saturate dopamine receptors .
the second action of vanoxerine was to prevent the application of methionine from modulating luminance signaling through off bipolar cells ( figures 6a and 6c ) , consistent with the idea that this modulation occurs through changes in dopamine levels .
the manipulations of dopamine receptors and transporters shown in figures 4 , 5 , and 6 support the idea that olfactory stimulation modulates synaptic transmission from off bipolar cells by reducing dopamine levels and d1 dopamine receptor activity .
what are the cellular mechanisms by which dopamine modulates the visual signal transmitted to the inner retina ? in the outer retina of fish and mammals , dopamine acts through d1 receptors to uncouple horizontal cells providing negative feedback to the synaptic terminals of photoreceptors ( dowling , 1991 ) , but this seems an unlikely mechanism for the selective modulation of transmission through off bipolar cells given that these diverge from the on pathway downstream of photoreceptor output ( schiller et  al . , 1986 ) .
we therefore investigated the possibility that dopamine might also act directly on bipolar cells to modulate synaptic calcium signals .
mixed rod - cone ( mb1 ) bipolar cells from the retina of goldfish were isolated for electrophysiological recording ( burrone and lagnado , 1997 ) . in these neurons ,
voltage - dependent calcium channels are l - type and localized to the synaptic terminal ( burrone and lagnado , 1997 ) . in current - clamp configuration , using a standard intracellular solution , addition of 10  m dopamine depolarized bipolar cells by an average of 30.7   1.5  mv , indicating activation of a net inward current ( n  = 9 ; figures 7a and 7b ) .
the depolarization was completely reversed by blocking voltage - dependent ca channels with 100  m cadmium ( catterall et  al . ,
2003 ) , indicating that dopamine potentiates the calcium conductance ( n  = 6 ) .
the mb1 bipolar cell stands out in a preparation of dissociated retinal neurons because of its large terminal . of the bipolar cells with small terminals , off outnumber on by 3:1 ( odermatt et  al .
we also made recordings from the cell bodies of bipolar cell with small terminals , and in all three cases dopamine caused a depolarization of 15  mv .
it therefore seems very likely that dopamine also acts to enhance calcium currents in off bipolar cells .
heidelberger and matthews ( 1994 ) also observed that dopamine potentiated calcium influx in all morphological types of bipolar cell that they tested . to investigate the actions of dopamine on the calcium conductance more directly , we made voltage - clamp recordings using an intracellular solution designed to block potassium channels .
figure  7c shows the calcium currents elicited by voltage steps from 70  mv to 40  mv and 20  mv in a single cell , and figure  7d shows an example of the current - voltage relation around the threshold for activation of the calcium current , approximately 43  mv ( burrone and lagnado , 1997 ) . to quantify changes in the calcium conductance over a number of cells , we measured the amplitude of the tail current 0.5  ms after a voltage step returning to 70  mv ( dashed red line in figure  7e ) .
averaged conductance - voltage ( g - v ) relations before and after addition of 10  m dopamine are shown in figure  7f , with conductance values normalized to the maximum in the absence of dopamine ( n  = 6 cells ) .
the g - v relation could be described by a boltzmann function ( see experimental procedures ) .
addition of 10  m dopamine increased gmax by 44%   11% and shifted v1/2 from 14.2   0.4  mv to 16.5   0.4  mv ( figure  7f , p  = 0.002 ) .
the 2.3  mv shift in v1/2 to lower membrane potentials is significant in the context of the voltage signals that bipolar cells generate in response to light ( baden et  al . , 2011 ) , which are just a few millivolts in amplitude and span the voltage range at which l - type calcium channels begin to activate .
around this threshold , dopamine potentiated presynaptic calcium currents by a factor averaging 1.9 ( figure  7f ) .
these results demonstrate that dopamine can act directly on bipolar cells to increase the magnitude of the presynaptic ca current that controls transmission of the visual signal .
it seems likely that this action makes a significant contribution to the profound increase in the gain of luminance signals observed in  vivo in the presence of the dopamine receptor agonist adtn ( figure  4 ) , as well as the decrease in gain in the presence of the antagonist sch 23390 ( figure  5 ) .
if an olfactory stimulus acts to lower dopamine levels and therefore inhibits activation of presynaptic calcium channels , one might expect to observe a decrease in the basal calcium concentration in bipolar cells in darkness , with this effect being most obvious in off cells resting at more depolarized potentials .
we therefore compared resting sygcamp2 signals in bc terminals before and after the bath application of methionine ( 233 on  and 211 off from nine fish ; figure  s4 ) .
methionine induced  a statistically significant reduction in  sygcamp2 fluorescence in off terminals ( median  = 10.9% , p  < 0.01 ) but not on ( median  = 0.2% , not significant ) , providing further support for the idea that inhibition of presynaptic calcium channels is one of the mechanisms by which an olfactory stimulus reduces the gain of signaling through off bipolar cells .
the vertebrate retina receives centrifugal input from a variety of brain regions , depending on the species ( behrens and wagner , 2004 ) .
the olfacto - retinal circuit in fish is a good example of such cross - modal interactions and provides the opportunity to investigate the cellular mechanisms that regulate processing in the early visual system . by monitoring calcium signals in  vivo , we find that an olfactory stimulus reduces the gain with which changes in luminance or temporal contrast are transmitted through the off pathway , while also increasing sensitivity at lower light levels ( figures 1 , 2 , and 3 ) .
the results demonstrate that the calcium signal controlling neurotransmission from bipolar cells is a key site for regulating the flow of the visual information .
the observed modulation of presynaptic calcium responses is likely to contribute to the increase in luminance sensitivity observed behaviorally when the orc circuit is activated ( maaswinkel and li , 2003 ; huang et  al . , 2005 ) .
the chemical signal coordinating these changes in retinal performance has been suggested to be a reduction in dopamine release .
strong evidence for this idea is provided by the demonstration that a blocker of dopamine release and reuptake suppresses the change in synaptic gain and sensitivity normally caused by an olfactory stimulus ( figure  6 ) .
manipulations of dopamine receptor activity in  vivo are also consistent with this mechanism ( figures 4 , 5 , and 6 ) and , in particular , for a key role of d1 receptors ( figures 5b and 5d ) .
finally , we demonstrate that dopamine regulates the activity of voltage - dependent calcium channels in the synaptic terminals of bipolar cells , providing a direct mechanism for regulating the gain of the visual signal ( figure  7 ) .
of course , these results do not rule out the possibility that there are other sites at which orc also regulates the retinal circuit .
an overview of changes in the amplitude of the calcium signal through on and off bipolar cell terminals is shown in figure  8 .
the response is quantified as the relative change in sygcamp2 fluorescence caused by a bright step of light applied from darkness , and the various experimental conditions are ordered according to the expected level of dopamine activity , with the measurement in 100  nm of the d1 dopamine receptor antagonist sch 23390 at one extreme and in 200  nm of the agonist adtn at the other .
this comparison reveals a fundamental difference in the sensitivity of the on and off pathways to changes in retinal dopamine levels . under control conditions ,
luminance signaling through the off pathway is operating at its maximum gain ( i.e. , similar to that measured in adtn ) , whereas signaling through the on pathway is operating at its minimum gain ( measured in sch 23390 ) .
thus , although an olfactory stimulus that results in decreased dopamine levels may be expected to decrease the gain of signals through the off pathway ( figures 1 and 8a ) , it is not expected to suppress synaptic calcium signals in on bipolar cells ( figures 1 and 8b ) .
the mechanism for the differential effects of dopamine on the on and off pathways is still not clear . on and off bipolar cells
both express d1 receptors but not d2 ( fan and yazulla , 2005 ; yu and li , 2005 ) . d1 receptors act through gs proteins which couple to adenylyl cyclase to increase camp and direct activation of adenylyl cyclase by forskolin also increases bipolar ca responses ( heidelberger and matthews , 1994 )
. a possible explanation for the contrasting effect in on versus off could be differential sensitivity of the cav channels to camp that may reflect which cav channels underlie the response ( pan et  al . , 2001 ; logiudice et  al . ,
an alternative possibility is that the on and off channels are regulated by a second neuromodulator , which interacts with dopamine pathways .
for instance , iuvone and gan ( 1995 ) have demonstrated that activation of mt2 melatonin receptors antagonizes signaling through d1 dopamine receptors in bipolar cells by inhibiting camp synthesis through a gi protein , and wiechmann and sherry ( 2012 ) have found that mt2 melatonin receptors are localized to off but not on bipolar cells in xenopus laevis .
the fast decrease in melatonin concentration that occurs after dawn might therefore act to enhance selectively the sensitivity of off bipolar cells to variations in dopamine levels .
we did see a small population of on bipolar cell terminals ( 9% ) strongly potentiated by olfactory stimulation ( figures 1i , s1a , and s1b ) .
might this reflect differences in the mechanism by which glutamate released from photoreceptors act on different types of on bipolar cells ?
in zebrafish , some on bipolar cells respond through metabotropic glutamate receptors and others through a glutamate transporter with a large chloride conductance ( connaughton and nelson , 2000 ; nelson and connaughton , 2004 ) .
although the former mechanism predominates in mixed rod - cone bipolar cells with large terminals , the latter occurs in cone bipolar cells with smaller terminals .
we tested , therefore , if there was any relationship between the size of on terminals and their response to methionine , but did not find any ; i.e. , the size distribution of on terminals responding to methionine was very similar to those that did not , both varying between 0.6  m and 5  m in radius .
we also investigated whether there might be any relation between the location of on terminals within the ipl and their response to methionine and again there was not . as a consequence , at present we do not have elements to consider this as a separate subpopulation of on bipolar cells .
our results are consistent with the hypothesis that odor stimulation reduces the conductance and shifts the v1/2 of cav channels in bipolar terminals , with dopamine being the key mediator .
this mechanism is able to explain several of the observed effects of olfactory stimulation on the transmission of visual information through bipolar cells .
for example , the decreased conductance and shift in the activation of the calcium channels will lower ca influx for small depolarizations such as low contrasts , nevertheless larger depolarizations , such as high contrasts , will still be effective .
this manifests in a more nonlinear contrast response function ( figure  2b ) with greater sensitivity for higher contrasts .
the decrease in the ca channel maximum conductance also explains the lower gain seen at maximum luminance ( figure  1d ) .
this highlights the presynaptic terminal of bipolar cells as a key site for  regulating the transmission of visual signals through the retina . as well as the dramatic gain reduction
as the expected effects of reduced dopamine will shift the cav activation to more depolarized potentials , it is unlikely to explain the increased luminance sensitivity .
however , d1 receptors do enhance glutamate - gated ionic channels in off bipolar cells ( maguire and werblin , 1994 ) .
when d1 receptors are activated , ionotropic glutamate receptors generate enhanced current that will result in off bipolar cells being less sensitive to small decreases in glutamate concentration ; a similar phenomenon has been described in horizontal cells ( knapp and dowling , 1987 ) . the olfacto - retinal circuit endows the vertebrate visual system with the ability to quickly reduce the gain and increase the sensitivity of the retina in the presence of food , independently of changes in mean luminance .
a behavior that is likely to be related to this process has recently been described by stephenson et  al .
( 2011 ) , who found that zebrafish show a preference for darker areas in their environment when background levels of light are low , and brighter areas when background light levels are high . an olfactory stimulus applied in low background
would then mimick the effects of light adaptation by encouraging fish to explore brighter areas .
the reduction in gain of bipolar cell synapses transmitting the visual signal to the inner retina ( figure  1 ) , as well as the increase in sensitivity to high contrast ( figure  2 ) , is likely to be one of the mechanisms by which an olfactory stimulus allows the visual system of the zebrafish to operate in brighter areas . in the future
, it will be interesting to investigate the behavioral consequences of a selective decrease in gain of the off pathway .
certainly it would be expected to help the retina avoid saturation under bright conditions , but then so would a decrease in gain through the on pathway . a possible explanation for the selective control of the off pathway might lie in the recent study of ratliff et  al . ( 2010 ) who asked why off rgcs are so much more numerous than ons in most retinas ( including zebrafish ) .
they found that natural scenes contain an excess of negative spatial contrasts over positive , leading to the suggestion that the excess of off rgcs is a structural adaptation of the retina to the excess of darkness in natural scenes .
in zebrafish , off bipolar cells outnumber ons by a ratio of 3:1 ( odermatt et  al .
, 2012 ) , so it may be that a decrease in the gain of the off pathway is the most important adaptation required to process negative contrasts at higher mean light levels .
another possible explanation could be linked to the differential role proposed by burgess et  al .
( 2010 ) for the two systems , being the on pathway mainly involved in appetitive behaviors and the off pathway more implicated in escape responses . in this perspective
, the observed food odor - induced inhibition of the off would suppress escape responses , thus favoring appetitive behaviors .
the  re - tuning  of retinal processing by a food - related olfactory stimulus is likely to be relevant to different aspects of zebrafish behavior , but especially hunting and prey - capture .
the observed increase in the gain of the on channel relative to the off is expected to make the retina more sensitive to regions of positive contrast , such as bright spots appearing when sunlight reflects off small prey .
bright spots are an effective stimulus for eliciting prey - capture behavior in a  virtual reality  assay ( bianco et  al . , 2011 ) , and this may provide an experimental context in which to study the behavioral consequences of olfactory - visual integration .
all procedures were carried out according to the uk animals ( scientific procedures ) act 1986 and approved by the uk home office .
we made transgenic zebrafish ( danio rerio ) expressing the synaptically localized fluorescent calcium reporter sygcamp2.0 under the ribeye - a promoter , as in dreosti et  al .
( 2012 ) , or the calcium reporter gcamp3.5 under the eno2 promoter , as in bai et  al .
sygcamp2 and gcamp3.5 zebrafish were kept at a 14:10  hr light : dark cycle and bred naturally .
larvae were grown in 200  m 1-phenyl-2-thiourea ( sigma ) from 28  hr postfertilization to inhibit melanin formation ( karlsson et  al . , 2001 ) .
whole zebrafish larvae ( 811  days postfertilization [ dpf ] ) were immobilized in 2.5% low melting point agarose ( biogene ) on a glass coverslip and submersed in e2 embryo medium ( nusslein - volhard and dahm , 2001 ) .
bipolar cell terminals were imaged in  vivo using a custom - built two - photon microscope equipped with a mode - locked chameleon titanium - sapphire laser tuned to 915  nm ( coherent ) with an olympus lumplanfi 40 water immersion objective ( n.a .
emitted fluorescence was captured through both the objective and a substage oil condenser , filtered through a hq 520/60 m-2p gfp emission filter ( chroma technology ) and detected by a set of photo - multiplier tubes ( hamamatsu ) .
scanning and image acquisition were controlled under scanimage v.3.6 software ( pologruto et  al . ,
full - field light stimuli were delivered by amber leds ( luxeon ) , 590  nm band - passed  10  nm , and  controlled in igor pro 4.01 ( wavemetrics ) and time locked to image acquisition .
bipolar cell terminal responses were analyzed in terms of light sensitivity ( irradiance ) , contrast sensitivity , and frequency sensitivity .
luminance ( irradiance ) sensitivity was assessed by stimulating the dark - adapted fish with a series of flashes ( 4   3  s flashes at 6 s intervals ) at  nine different light intensities , ranging between 11 pw / mm and 110  nw / mm with 0.5 log unit steps .
maximum light intensity , 110 nw / mm , is equivalent to 3.3   10 photons / mm  s. contrast sensitivity was assessed by stimulating the dark - adapted fish with a series of 10  s light oscillations at 5  hz around a constant light level ( 55 nw / mm ) at 10 different levels of contrast , ranging from 10% to 100% of the constant light level .
finally , frequency sensitivity was assessed by stimulating the dark - adapted fish with a series of 10  s light oscillations around a constant light level ( 55 nw / mm ) at 90% contrast at 14 different frequencies , ranging from 0.2 to 25  hz .
image sequences were acquired at 10  hz ( 256   100 pixels per frame , 1  ms per line ) for the irradiance and contrast experiments and at 40  hz ( 256   25 pixels per frame , 1  ms per line ) for frequency experiments .
the stimulation of the olfactory bulb was obtained by bath application of the amino acid methionine ( sigma ) 1  mm , as in maaswinkel and li ( 2003 ) . to manipulate dopamine signaling in the retina we injected neuroactive drugs into the eye .
final concentrations of the drugs were calculated by diluting the injected concentration into the free volume of the eye .
the volume of a typical 9 dpf old zebrafish eye was assessed by three - dimensional reconstruction of the eye chamber and the lens through two - photon microscopy scanning .
the final volume was estimated as the difference between the total eye volume and the volume of the lens core .
we calculated a total free volume of 500  m . given a typical injected volume of 10  l , the final dilution factor can be approximated to 1:50 .
dopamine receptors were activated by injection of the long - lasting dopamine receptor ligand [ 3h ] 2-amino-6,7-dihydroxy 1,2,3,4-tetrahydronapthalene ( adtn ) ( sigma ) 10  m , as in li and dowling ( 2000b ) .
dopamine action on postsynaptic targets was prevented by injection of the strong dopamine d1 receptor antagonist sch 23390 ( sigma ) 2  nm , as in huang et  al .
( 2005 ) or the selective dopamine d2 receptor antagonist sulpiride ( sigma ) , as in lin and yazulla ( 1994 ) and mora - ferrer and gangluff ( 2000 ) .
finally , the level of dopamine in the circuit was frozen by injection of the dopamine release and reuptake inhibitor vanoxerine ( santa cruz biotechnology ) 2  m , as in schlicker et  al .
preprocessing was carried out in image j ( national institutes of health ) and  consisted of stack registration and kalman stack filter denoising ( filter gain  = 0.6 ) .
regions of interest ( roi ) extraction , background subtraction , and brightness normalization ( f / f0 ) were performed in igor pro 6.2 and facilitated by sarfia analysis routines ( dorostkar et  al . , 2010 ) .
the detection of active roi in the ipl was based on the thresholding of the laplacian transform of the two - photon recordings . in this way , responding bipolar cell terminals and active areas of the ganglion cell dendrites were identified in ribeye::sygcamp2 and eno2::gcamp3.5 fish , respectively .
the responses to light of bipolar cell terminals and retinal ganglion cell dendrites were characterized according to their response amplitude , i.e. , the variation in fluorescence during stimulation in comparison to baseline ( f / f0 ) .
responses to light were plotted in full , as in figure  1b , left , or in stimulus versus amplitude plots ( e.g. , figure  1b , right ) . in the case of traces representing single terminals ( e.g. , figure  1b ) , the error curve ( gray shadow in figure  1b ) represents the sem of the four trials employed to assess the terminal responsiveness ( see stimulation protocols ) . in the case of traces representing whole populations of terminals ( e.g. , figure  1d )
, the error curve represents the standard error of all the responses employed to generate the final average .
as described in the stimulation protocols section , a stimulus could be light intensity , contrast , or frequency .
intensity versus amplitude plots were obtained by averaging amplitude values over 300  ms long time windows around the maximum response occurring during the stimulation time ( e.g. , figure  1b , right ) .
contrast versus amplitude and frequency versus amplitude plots were obtained by averaging amplitude values over the whole stimulation period ( e.g. , figures 2b and s2d , respectively ) .
the intensity versus amplitude plots were fitted with hill curves , in the form a  = i 
 /i 
 + i1/2 , a being the response amplitude , i the stimulation intensity , h the hill coefficient , and i1/2 the sensitivity at half maximum , i.e. , the stimulation intensity that elicits half of the maximum response .
i1/2 has been used as a metric for the sensitivity of each intensity versus amplitude curve .
contrast versus amplitude plots were fitted with power functions , in the form a  = k  c being a the response amplitude , k a constant , c the stimulation contrast , and  the power exponent . the sensitivity shift induced by olfactory stimulation for each individual terminal ( e.g. , figure  1f )
was measured by comparing the values of the lowest light intensity eliciting a statistically significant response before and after methionine administration .
the statistical significance of a response was assessed by comparing ( t test ) the average calcium level during light stimulation with a threshold defined as three times the sd of a baseline epoch .
the effect of drugs or of olfactory stimulation has been also described in terms of percentage variation in the amplitude of response to the maximum irradiance stimulus ( e.g. , figure  1e ) .
goldfish ( carassius auratus ) were dark - adapted for 1  hr and killed by decapitation followed immediately by destruction of the brain and spinal cord under schedule 1 of the uk animals ( scientific procedures ) act 1986 .
depolarizing bipolar cells were isolated from the retina of goldfish by enzymatic digestion , using methods described by burrone and lagnado ( 1997 ) .
the standard ringer solution contained the following : 110  mm nacl , 2.5  mm cacl2 , 2.5  mm kcl , 1  mm mgcl2 , 10  mm glucose , and 10  mm hepes ( 260 mosmol l-1 , ph 7.3 ) .
the solution in the patch pipette to record voltage membrane in current - clamp experiments contained : 110  mm k - gluconate , 4  mm mgcl2 , 3  mm na2atp , 1  mm na2gtp , 0.5  mm egta , 20  mm hepes , and 10  mm na - phosphocreatine ( 260 mosmol l-1 , ph 7.2 ) . to isolate ca channel currents , the intracellular solution contained 110  mm cs - gluconate , 4  mm mgcl2 , 3  mm na2atp , 1  mm na2gtp , 10  mm tetraethylammonium chloride , 20  mm hepes , 0.5  mm egta , and 10  mm na - phosphocreatine ( 260 mosmol l-1 , ph 7.2 ) .
room temperature solutions were superfused via a fast perfusion system ( vc8-s ; ala scientific ) .
patch electrodes with 57 m tip resistance were pulled from fire - polished borosilicate glass capillary tubes using a micropipette puller ( sutter instrument ) .
voltage - clamp and current - clamp recordings were made in synaptic terminals . in voltage - clamp experiments ,
the membrane potential was held at 60  mv , and stimuli were delivered by stepping the membrane potential to 10  mv . to construct g
/ v plots the tail current amplitude measured 0.5  ms after returning to 70  mv was plotted against the preceding voltage step .
the voltage dependence of activation was determined from normalized conductance versus voltage curves , which were fitted according to the boltzmann function : g=gmax1+exp(vv1/2k),where g is the normalized conductance , v1/2 is the membrane potential at which activation is half - maximal , and k is the slope factor .
signals were recorded using an axopatch 200a amplifier ( molecular devices ) , interfaced with an itc-16 ( heka ) and controlled with pulse control 4.3 running under igor pro 5 ( wavemetrics ) .
 summarycross - modal regulation of visual performance by olfactory stimuli begins in the retina , where dopaminergic interneurons receive projections from the olfactory bulb . 
 however , we do not understand how olfactory stimuli alter the processing of visual signals within the retina . 
 we investigated this question by in  vivo imaging activity in transgenic zebrafish expressing sygcamp2 in bipolar cell terminals and gcamp3.5 in ganglion cells . 
 the food - related amino acid methionine reduced the gain and increased sensitivity of responses to luminance and contrast transmitted through off bipolar cells but not on . 
 the effects of olfactory stimulus were blocked by inhibiting dopamine uptake and release . 
 activation of dopamine receptors increased the gain of synaptic transmission in  vivo and potentiated synaptic calcium currents in isolated bipolar cells . 
 these results indicate that olfactory stimuli alter the sensitivity of the retina through the dopaminergic regulation of presynaptic calcium channels that control the gain of synaptic transmission through off bipolar cells . 
Introduction
Results
Discussion
Experimental Procedures