- Email: [email protected]
Levels of dopamine and noradrenaline in the developing retina — effect of light deprivation
\
Pergamon
Int[ J[ Devl Neuroscience\ Vol[ 04\ No[ 0\ pp[ 028Ð032\ 0886 Copyright Þ 0886 ISDN[ Published by Elsevier Science Ltd Printed in Great Britain[ All rights reserved 9625Ð4637:86 ,06[99¦9[99
PII] S9625Ð4637"85#99979Ð8
LEVELS OF DOPAMINE AND NORADRENALINE IN THE DEVELOPING RETINA * EFFECT OF LIGHT DEPRIVATION RAJANI R[ J[ SHELKE\ MADEPALLI K[ LAKSHMANA\ Y[ RAMAMOHAN and TRICHUR R[ RAJU$ Departments of Neurophysiology and Neuropathology\ National Institute of Mental Health and Neuro Sciences\ Bangalore 459 918\ India "Received 16 November 0884^ revised 18 July 0885^ accepted 0 Au`ust 0885# Abstract*The e}ect of light deprivation on the levels of dopamine and noradrenaline was studied in the developing rat retina[ These transmitters were estimated in three groups of rats] "i# cycling light reared^ "ii# dark reared since birth^ and "iii# dark reared since birth\ but exposed to cycling light for 0 day prior to the estimation of catecholamines[ Our results show that "0# there is a progressive decrease in the levels of dopamine and noradrenaline in the cycling light and dark reared rats during postnatal development^ "1# dark rearing further reduces the content of dopamine and noradrenaline^ and "2# restoration of physiological "light# stimulus in the dark!reared rats during the early postnatal period results in the recovery of nor! adrenaline to a greater extent than that of dopamine[ This study demonstrates a progressive decrease in the plasticity of dopaminergic system during retinal development\ while such a decrease is not apparent in the noradrenergic system[ Þ 0886 ISDN Key words] retina\ ontogeny\ dark rearing\ dopamine\ noradrenaline[
The retina\ the photosensitive layer of the eye\ is a prototype of the central nervous system "CNS#[ Several CNS classical neurotransmitters and neuropeptides are present in the retina[ Dopamine "DA# plays an important functional role in the initial processing of visual information in the vertebrate retina[ It is known to modulate "i# the release of gamma!aminobutyric acid^18 "ii# the sensitivity of horizontal cells to photoreceptor transmitter^04 and "iii# the shedding of discs through a D1 receptor mediated mechanism[2 Dopamine!containing neurons have been demonstrated immunocytochemically in the retinae of various animals[0 Interest in this system has been on the rise since a physiological stimulus\ viz[ light\ has been shown to activate retinal DA neurons\ as demonstrated by an increase in the tyrosine hydroxylase activity in amacrine cells[02 Exposure of dark adapted retinae to constant light for 0 hr enhances DA turnover in vitro[3 Melamed et al[\19 who examined the pattern of retinal DA turnover during 13 hr\ have reported higher levels of DA and DOPAC during the light hours and lower levels during the dark hours in an adult rat retina[ Thus\ the role of light in the regulation of synthesis\ storage and release of DA in adult dopaminergic neurons has been studied extensively[3\7\19\14 However\ most of these experiments were carried out in vitro[ There are only a few reports suggesting that noradrenaline also can be a neurotransmitter in the retina[10\11 There are no quantitative studies on the NA content in the developing postnatal rat retina\ and its responsiveness to light stimulation has not been determined[ The retinal cells express a robust dopamine!stimulated adenylate cyclase activity\ several days before the appearance of synapses12\21 and then show down!regulation as development proceeds[16 The transient nature of the response\ which is mediated in part by an embryonic subtype of the D0 receptor\ may have a role in neuronal di}erentiation^ thus\ dopamine could be serving as a growth regulator in the retina[ In fact\ Lankford et al[07 provided direct evidence that dopamine\ through D0 receptor stimulation\ can modulate the growth cone motility in cultured retinal neurons[ A more recent study also suggested that DA may modulate light adaptive plasticity of horizontal cell dendrites in the gold_sh retina[29 Evidence also exists for the neurotrophic role of NA in the postnatal development of several neuronal systems[6\8\08 Since these transmitters regulate growth
$To whom correspondence should be addressed[ Tel[] 80!79!553!1010:196^ Fax] 80!79!552!0729^ E!mail] trrajuÝ nimhans[ren[nic[in[ 028
039
R[ R[ J[ Shelke et al[
and di}erentiation of neurons\20 a study on the expression of these transmitters as well as their responsiveness to physiological stimulus "light# during development is warranted[ The present study also was aimed at understanding the possible plasticity of the catecholaminergic system in the developing retina in terms of recovery of levels of catecholamines altered by light deprivation[ Accordingly\ levels of DA and NA were determined at postnatal days 5\ 00 and 10 in rats reared in "i# cycling light^ "ii# darkness since birth^ and "iii# darkness since birth but exposed to cycling light for 0 day prior to the estimation of catecholamines[
EXPERIMENTAL PROCEDURES Animals Wistar rats\ obtained from the Central Animal Research Facility\ NIMHANS\ Bangalore\ and maintained in standard laboratory conditions\ were used[ Rats were divided into three treatment groups] "i# cycling light!reared group "CLR#] rats maintained in the standard laboratory conditions with a 01 hr light]dark cycle "lights were switched on at 6]29 a[m[ and turned o} at 6]29 p[m[#^ "ii# dark!reared group "DR#] rats maintained in dark rooms from birth until the day of sacri_ce[ A dim red light was used for a brief period to feed and change the bedding^ "iii# the {experimental| group "E#] rats were maintained in dark rooms\ as for DR rats\ and exposed to cycling light for 0 day "01 hr light]dark cycle#\ prior to the measurement of catecholamines[ Each treatment group consisted of six pups belonging to three di}erent age groups] "i# postnatal day 5 "P5#^ "ii# postnatal day 00 "P00#^ and "iii# postnatal day 10 "P10#[ The rats were sacri_ced by decapitation and the eyes were removed and kept in an ice!cold Petri dish[ The HPLC measurement of neurotransmitter content The dopamine and noradrenaline contents in the retinae of di}erent treatment and age groups were measured by high performance liquid chromatography with ~uorescence detection as described by Lakshmana and Raju[06 Using the dissection microscope\ retinae were removed from between six and eight eyes and pooled for catecholamine estimation[ From the dark reared rats\ the retinae were dissected under dim red light[ Retinal tissue was homogenized in 0 ml of ice!cold perchloric acid "9[0 M# with isoproterenol as an internal standard and centrifuged at 0199g for 09 min at 3>C[ The supernatant was _ltered through a membrane of 9[34 mm pore size "Sartorius\ Gottingen\ West Germany# and an aliquot of 099 ml was injected into the equilibrated column[ The modi_ed mobile phase "pH 2[81# consisted of sodium acetate "9[91 M#\ methanol "05) v:v#\ heptane sulphonic acid "9[90264) w:v# and EDTA "9[0 mM#[ All separations were isocratic at room temperature and the ~ow rate was set at 9[8 ml:min[ The levels of DA and NA after separation were detected at the excitation wavelength of 179 nm and an emission wavelength of 204 nm[ Tissue monoamine levels were calculated by comparing peak heights with those of standards and with a correction factor related to the recovery of internal standards[ The catecholamines estimated were expressed as pmol:g wet tissue[ Data from the control and experimental groups were analysed statistically by one!way analysis of variance "ANOVA# followed by a post!hoc Duncan|s multiple!range test[
RESULTS Dopamine There was a signi_cant reduction in the DA content from p00 to p10 in CLR "F1\04 2[38^ P³9[94\ N 5#\ DR "F1\04 3[60^ P³9[94\ N 5# and E rats "F1\04 06[1^ P³9[90\ N 5#[ Comparison of DA content between di}erent treatment groups in the same age also revealed signi_cant di}erences[ There was a decrease in the DA content in the DR rats compared to CLR rats at P5 "16)\ P³9[90#\ p00 "29)\ P³9[90# and p10 "17)\ P³9[90#[ In the {experimental| rats\ DA content was signi_cantly higher than those of dark reared rats at p5 "P³9[90# and p00 "P³9[94# but not at p10 "Fig[ 0#[
E}ect of dark rearing on retinal DA and NA levels
030
Fig[ 0[ Levels of dopamine "pmol:g wet tissue# in retinae of di}erent groups of rats at postnatal days 5\ 00 and 10[ Data represent mean¦SEM of six retinae in each group[ One!way ANOVA\ followed by post!hoc Duncan|s multiple range test\ revealed signi_cant di}erences] p5 vs p00\ R R\ P³9[90^ p5 vs p10\ ¦\ P³9[94\ ¦¦\ P³9[90^ p00 vs p10\ \ P³9[94\ \ P³9[90[
Noradrenaline There was a signi_cant decrease in the NA content from p00 to p10 in CLR "F1\04 8[5^ P³9[90\ N 5#\ DR "F1\04 02[7^ P³9[94\ N 5# and E "F1\04 19[17^ P³9[90\ N 5# groups[ Comparison of NA content between di}erent treatment groups in the same age also revealed signi_cant di}erences[ The NA content in the DR rats was lower than CLR rats at p5 "00)#\ p00 "15)\ P³9[94# and at p10 "23)\ P³9[94#[ In experimental rats\ NA content was signi_cantly higher than that of DR group at p5 "P³9[94#\ p00 "P³9[94# and even at p10 "P³9[90# "Fig[ 1#[ DISCUSSION Our _ndings suggest that there appears to be a progressive decline in DA\ but not NA\ plasticity with age\ since exposure of rats for 0 day|s cycling light after dark rearing resulted in the restoration of DA level at p5 and p00 but not at p10\ whereas NA levels recovered at all time points studied[ The temporal di}erence in the optimal expression of these transmitters09 may be responsible for this di}erence in the plasticity[ Endogenous levels of DA have been shown to be stimulated by light in the dark adapted retinae[3\4\5 Exposure to 0 day|s cycling light after chronic dark rearing was su.cient for a rapid increase in the levels of catecholamines in the present study[ This may be due to activation of tyrosine hydroxylase by light\ as has been demonstrated by a number of studies[3\02\19\13 Studies in guinea!pig\ a precocious animal in which the eyes open at birth\ have shown a decreased DA content on chronic dark rearing\ from birth to 2 or 5 weeks of age[15 Although\ in rats\ the eyes open only after p09\ light di}using through the closed transparent eyelids may also be critical for the normal neurotransmitter pro_le to emerge[17
031
R[ R[ J[ Shelke et al[
Fig[ 1[ Levels of noradrenaline "pmol:g wet tissue# in retinae of di}erent groups of rats at postnatal days 5\ 00 and 10[ The rest is as in Fig[ 0[
An earlier study has speculated that catecholamine synthesis does not proceed to NA in the retina since dopamine beta hydroxylase\ the enzyme that converts DA to NA\ could not be detected by immunostaining[1 However\ Kramer05 showed NA to be released from retina by chemical stimu! lation[ Our results show the presence of NA in the rat retina already at p5\ which is consistent with previous reports from bovine retina[05\11 It has been suggested that NA found in retina could originate from sympathetic neurons innervating the retinal blood vessels[11 However\ sympathetic denervation of the retinal blood vessels had no e}ect on the histo~uorescence of NA in the retina03[ Accordingly\ the presence of NA observed in this study must be intrinsic to the retina[ The non!transmitter roles of DA and NA as growth regulators have been studied[6\8\00\08\18 In cultured retinal neurons\ DA has been shown to inhibit neurite outgrowth through the activation of D0 receptors[07 Depletion of DA in the neonates by reserpine or destruction of dopaminergic a}erents using 5!hydroxydopamine has been shown to alter the expression of peptidergic genes\ neurotrophic activity of striatal cells\ hyperinnervation by serotonergic a}erents and retarded growth of striatal neurons suggesting that DA exerts multiple in~uences on neuronal development[08 Most of the amacrine cells di}erentiate and form synaptic contacts in the inner plexiform layer during the second week of postnatal development[01 Increased levels of catecholamines\ especially the DA\ observed in the present study during this period may be critical for the above events to occur[ REFERENCES 0[ Ballesta J[\ Terenghi G[\ Thibault J[ and Polak J[ M[ "0873# Putative dopamine containing cells in the retina of seven species demonstrated by tyrosine hydroxylase immunocytochemistry[ Neuroscience 01\ 0036Ð0045[ 1[ Beazley L[ D[\ Perry V[ H[\ Baker B[ and Darby J[ E[ "0876# An investigation into the role of ganglion cells in the regulation of division and death of other retinal cells[ Dev[ Brain Res[ 22\ 058Ð072[ 2[ Besharse J[ C[\ Iuvone P[ M[ and Pierce M[ E[ "0877# Regulation of rhythmic photoreceptor metabolism] a role for post receptoral neurons[ Pro`ress in Retinal research "ed[ Osborne N[#\ pp[ 10Ð50\ Pergamon\ London[
E}ect of dark rearing on retinal DA and NA levels
032
3[ Boatright J[ H[\ Hoel M[ J[ and Iuvone P[ M[ "0878# Stimulation of endogenous dopamine release and metabolism in amphibian retina by light! and K¦!evoked depolarization[ Brain Res[ 371\ 053Ð057[ 4[ Brainard G[ C[ and Morgan W[ W[ "0876# Light!induced stimulation of retinal dopamine] a doseÐresponse relationship[ Brain Res[ 313\ 088Ð192[ 5[ Chow K[ L[\ Riesen A[ H[ and Newell R[ W[ "0846# Degeneration of retinal ganglion cells in infant chimpanzees reared in darkness[ J[ Comp[ Neurol[ 096\ 16Ð31[ 6[ DeVries S[ H[ and Baylor D[ A[ "0882# Synaptic circuitry of the retina and the olfactory bulb[ Cell 61\ 028Ð038[ 7[ Dong C[ J[\ Mc Reynolds J[ S[ "0881# Comparison of the e}ects of ~ickering and steady light in dopamine release and horizontal cell coupling in the mudpuppy retina[ J[ Neurophysiol[ 56\ 253Ð261[ 8[ Felten D[ L[\ Hallman H[ and Johnsson G[ "0871# Evidence for a neurotrophic role of noradrenaline neurons in postnatal development of the rat cortex[ J[ Neurocytol[ 00\ 008Ð024[ 09[ Foster G[ A[ "0874# Di}erential ontogeny of three putative catecholamine cell types in the postnatal rat retina[ Brain Res[ 243\ 076Ð085[ 00[ Franquinet R[ and Martelly V[ "0870# E}ects of serotonin and catecholamines on RNA synthesis in planarians] in vitro and in vivo studies[ Cell Diff[ 09\ 190Ð198[ 01[ Horsburgh G[ M[ and Safton A[ J[ "0876# Cellular degeneration and synaptogenesis in the developing retina of the rat[ J[ Comp[ Neurol[ 152\ 442Ð455[ 02[ Iuvone P[ M[\ Galli C[ L[\ Garrison!Gund C[ K[ and Ne} N[ H[ "0867# Light stimulates tyrosine hydroxylase activity and dopamine synthesis in the retinal amacrine cells[ Science 191\ 890Ð891[ 03[ Iuvone P[ M[ and Ne} N[ H[ "0870# Dopamine neurons of the retina] a simple model system for studying synaptic regulatory mechanism[ In Essays in Neurochemistry and Neuropharmacolo`y "eds Youdim M[ B[ H[\ Lovenberg W[ and Sharman D[ E[#\ Vol[ 4\ pp[ 64Ð83[ John Wiley and Sons Ltd\ New York[[ 04[ Knapp A[ G[ and Dowling J[ E[ "0876# Dopamine enhances excitatory amino acid gated conductances in cultured retinal horizontal cells[ Nature 214\ 326Ð328[ 05[ Kramer S[ G[ "0860# Dopamine] a retinal neurotransmitter * I[ Retinal uptake\ storage and light stimulated release of 2 H!dopamine in vivo[ Invest[ Opthalmol[ Vis[ Sci[ 09\ 327Ð341[ 06[ Lakshmana M[ K[ and Raju T[ R[ "0883# Endosulfan induces small but signi_cant changes in the levels of noradrenaline\ dopamine and serotonin in the developing rat brain and de_cits in the operant learning performance[ Toxicolo`y 80\ 028Ð049[ 07[ Lankford K[ L[\ Demello F[ G[\ Klein W[ L[ "0877# D0 type dopamine receptors inhibit growth cone motility in cultured retina neurons] evidence that neurotransmitters act as morphogenic growth regulators in the developing central nervous system[ Proc[ Natl Acad[ Sci[ U[S[A[ 74\ 1720Ð1723[ 08[ Lauder J[ M[ "0882# Neurotransmitters as growth regulatory signals] role of receptors and second messengers[ Trends Neurosci[ 05\ 122Ð139[ 19[ Melamed E[\ Frucht Y[\ Lemor M[\ Uzzan A[ and Resenthal Y[ "0873# Dopamine turnover in rat retina] a 13!hour light! dependent rhythm[ Brain Res[ 294\ 037Ð040[ 10[ Nesselhut T[ and Osborne N[ N[ "0871# Is noradrenaline a major catecholamine in the bovine retina<[ Neurosci[ Lett[ 17\ 30Ð34[ 11[ Osborne N[ N[ "0870# Noradrenaline\ a transmitter candidate in retina[ J[ Neurochem[ 25\ 06Ð16[ 12[ Paes de Carvalho R[ and de Mello F[ G[ "0874# Expression of A0 adenosine receptors modulating dopamine!dependent cyclic AMP accumulation in the chick embryo retina[ J[ Neurochem[ 33\ 734Ð740[ 13[ Parkinson D[ and Rando R[ R[ "0872# E}ect of light on dopamine metabolism on chick retina[ J[ Neurochem[ 39\ 28Ð 35[ 14[ Rodrigues P[ S[ and Dowling J[ E[ "0889# Dopamine induces neurite retraction in retinal horizontal cells via diacylglycerol and PKC[ Proc[ natn[ Acad[ Sci[ U[S[A[ 76\ 8582Ð8586[ 15[ Spira A[ W[ and Parkinson D[ "0880# E}ect of dark rearing on the retinal dopaminergic system in the neonatal and postnatal guinea pig[ Dev[ Brain Res[ 51\ 031Ð034[ 16[ Ventura A[ M[\ Klein W[ L[ and De Mello F[ G[ "0873# Di}erential ontogenesis of D0 and D1 dopaminergic receptors in the chick embryo retina[ Dev[ Brain Res[ 01\ 106Ð112[ 17[ Wu D[ K[ and Cepko C[ L[ "0882# Development of dopaminergic neurons is insensitive to optic nerve section in the neonatal rat retina[ Dev[ Brain Res[ 63\ 142Ð159[ 18[ Yazulla S[ and Kleinschmidt J[ "0871# Dopamine blocks carrier mediated release of GABA from retinal horizontal cells[ Brain Res[ 122\ 100Ð104[ 29[ Yazulla S[ and Studholme K[ M[ "0884# Volume transmission of dopamine may modulate light adaptive plasticity of horizontal cell dendrites in the recovery phase following dopamine depletion in gold _sh retina[ Vis[ Neurosci[ 01\ 716Ð 725[ 20[ Yeh H[ H[\ Battelle B[ A[ and Puro D[ G[ "0873# Dopamine regulates synaptic transmission mediated by cholinergic neurons of the rat retina[ J[ Neurosci[ 02\ 890Ð898[ 21[ Young L[ H[ Y[ and Dowling J[ E[ "0878# Localization of cyclic adenosine monophosphate in the teleost retina] e}ects of dopamine and prolonged darkness[ Brain Res[ 493\ 46Ð52[
Pergamon
Int[ J[ Devl Neuroscience\ Vol[ 04\ No[ 0\ pp[ 028Ð032\ 0886 Copyright Þ 0886 ISDN[ Published by Elsevier Science Ltd Printed in Great Britain[ All rights reserved 9625Ð4637:86 ,06[99¦9[99
PII] S9625Ð4637"85#99979Ð8
LEVELS OF DOPAMINE AND NORADRENALINE IN THE DEVELOPING RETINA * EFFECT OF LIGHT DEPRIVATION RAJANI R[ J[ SHELKE\ MADEPALLI K[ LAKSHMANA\ Y[ RAMAMOHAN and TRICHUR R[ RAJU$ Departments of Neurophysiology and Neuropathology\ National Institute of Mental Health and Neuro Sciences\ Bangalore 459 918\ India "Received 16 November 0884^ revised 18 July 0885^ accepted 0 Au`ust 0885# Abstract*The e}ect of light deprivation on the levels of dopamine and noradrenaline was studied in the developing rat retina[ These transmitters were estimated in three groups of rats] "i# cycling light reared^ "ii# dark reared since birth^ and "iii# dark reared since birth\ but exposed to cycling light for 0 day prior to the estimation of catecholamines[ Our results show that "0# there is a progressive decrease in the levels of dopamine and noradrenaline in the cycling light and dark reared rats during postnatal development^ "1# dark rearing further reduces the content of dopamine and noradrenaline^ and "2# restoration of physiological "light# stimulus in the dark!reared rats during the early postnatal period results in the recovery of nor! adrenaline to a greater extent than that of dopamine[ This study demonstrates a progressive decrease in the plasticity of dopaminergic system during retinal development\ while such a decrease is not apparent in the noradrenergic system[ Þ 0886 ISDN Key words] retina\ ontogeny\ dark rearing\ dopamine\ noradrenaline[
The retina\ the photosensitive layer of the eye\ is a prototype of the central nervous system "CNS#[ Several CNS classical neurotransmitters and neuropeptides are present in the retina[ Dopamine "DA# plays an important functional role in the initial processing of visual information in the vertebrate retina[ It is known to modulate "i# the release of gamma!aminobutyric acid^18 "ii# the sensitivity of horizontal cells to photoreceptor transmitter^04 and "iii# the shedding of discs through a D1 receptor mediated mechanism[2 Dopamine!containing neurons have been demonstrated immunocytochemically in the retinae of various animals[0 Interest in this system has been on the rise since a physiological stimulus\ viz[ light\ has been shown to activate retinal DA neurons\ as demonstrated by an increase in the tyrosine hydroxylase activity in amacrine cells[02 Exposure of dark adapted retinae to constant light for 0 hr enhances DA turnover in vitro[3 Melamed et al[\19 who examined the pattern of retinal DA turnover during 13 hr\ have reported higher levels of DA and DOPAC during the light hours and lower levels during the dark hours in an adult rat retina[ Thus\ the role of light in the regulation of synthesis\ storage and release of DA in adult dopaminergic neurons has been studied extensively[3\7\19\14 However\ most of these experiments were carried out in vitro[ There are only a few reports suggesting that noradrenaline also can be a neurotransmitter in the retina[10\11 There are no quantitative studies on the NA content in the developing postnatal rat retina\ and its responsiveness to light stimulation has not been determined[ The retinal cells express a robust dopamine!stimulated adenylate cyclase activity\ several days before the appearance of synapses12\21 and then show down!regulation as development proceeds[16 The transient nature of the response\ which is mediated in part by an embryonic subtype of the D0 receptor\ may have a role in neuronal di}erentiation^ thus\ dopamine could be serving as a growth regulator in the retina[ In fact\ Lankford et al[07 provided direct evidence that dopamine\ through D0 receptor stimulation\ can modulate the growth cone motility in cultured retinal neurons[ A more recent study also suggested that DA may modulate light adaptive plasticity of horizontal cell dendrites in the gold_sh retina[29 Evidence also exists for the neurotrophic role of NA in the postnatal development of several neuronal systems[6\8\08 Since these transmitters regulate growth
$To whom correspondence should be addressed[ Tel[] 80!79!553!1010:196^ Fax] 80!79!552!0729^ E!mail] trrajuÝ nimhans[ren[nic[in[ 028
039
R[ R[ J[ Shelke et al[
and di}erentiation of neurons\20 a study on the expression of these transmitters as well as their responsiveness to physiological stimulus "light# during development is warranted[ The present study also was aimed at understanding the possible plasticity of the catecholaminergic system in the developing retina in terms of recovery of levels of catecholamines altered by light deprivation[ Accordingly\ levels of DA and NA were determined at postnatal days 5\ 00 and 10 in rats reared in "i# cycling light^ "ii# darkness since birth^ and "iii# darkness since birth but exposed to cycling light for 0 day prior to the estimation of catecholamines[
EXPERIMENTAL PROCEDURES Animals Wistar rats\ obtained from the Central Animal Research Facility\ NIMHANS\ Bangalore\ and maintained in standard laboratory conditions\ were used[ Rats were divided into three treatment groups] "i# cycling light!reared group "CLR#] rats maintained in the standard laboratory conditions with a 01 hr light]dark cycle "lights were switched on at 6]29 a[m[ and turned o} at 6]29 p[m[#^ "ii# dark!reared group "DR#] rats maintained in dark rooms from birth until the day of sacri_ce[ A dim red light was used for a brief period to feed and change the bedding^ "iii# the {experimental| group "E#] rats were maintained in dark rooms\ as for DR rats\ and exposed to cycling light for 0 day "01 hr light]dark cycle#\ prior to the measurement of catecholamines[ Each treatment group consisted of six pups belonging to three di}erent age groups] "i# postnatal day 5 "P5#^ "ii# postnatal day 00 "P00#^ and "iii# postnatal day 10 "P10#[ The rats were sacri_ced by decapitation and the eyes were removed and kept in an ice!cold Petri dish[ The HPLC measurement of neurotransmitter content The dopamine and noradrenaline contents in the retinae of di}erent treatment and age groups were measured by high performance liquid chromatography with ~uorescence detection as described by Lakshmana and Raju[06 Using the dissection microscope\ retinae were removed from between six and eight eyes and pooled for catecholamine estimation[ From the dark reared rats\ the retinae were dissected under dim red light[ Retinal tissue was homogenized in 0 ml of ice!cold perchloric acid "9[0 M# with isoproterenol as an internal standard and centrifuged at 0199g for 09 min at 3>C[ The supernatant was _ltered through a membrane of 9[34 mm pore size "Sartorius\ Gottingen\ West Germany# and an aliquot of 099 ml was injected into the equilibrated column[ The modi_ed mobile phase "pH 2[81# consisted of sodium acetate "9[91 M#\ methanol "05) v:v#\ heptane sulphonic acid "9[90264) w:v# and EDTA "9[0 mM#[ All separations were isocratic at room temperature and the ~ow rate was set at 9[8 ml:min[ The levels of DA and NA after separation were detected at the excitation wavelength of 179 nm and an emission wavelength of 204 nm[ Tissue monoamine levels were calculated by comparing peak heights with those of standards and with a correction factor related to the recovery of internal standards[ The catecholamines estimated were expressed as pmol:g wet tissue[ Data from the control and experimental groups were analysed statistically by one!way analysis of variance "ANOVA# followed by a post!hoc Duncan|s multiple!range test[
RESULTS Dopamine There was a signi_cant reduction in the DA content from p00 to p10 in CLR "F1\04 2[38^ P³9[94\ N 5#\ DR "F1\04 3[60^ P³9[94\ N 5# and E rats "F1\04 06[1^ P³9[90\ N 5#[ Comparison of DA content between di}erent treatment groups in the same age also revealed signi_cant di}erences[ There was a decrease in the DA content in the DR rats compared to CLR rats at P5 "16)\ P³9[90#\ p00 "29)\ P³9[90# and p10 "17)\ P³9[90#[ In the {experimental| rats\ DA content was signi_cantly higher than those of dark reared rats at p5 "P³9[90# and p00 "P³9[94# but not at p10 "Fig[ 0#[
E}ect of dark rearing on retinal DA and NA levels
030
Fig[ 0[ Levels of dopamine "pmol:g wet tissue# in retinae of di}erent groups of rats at postnatal days 5\ 00 and 10[ Data represent mean¦SEM of six retinae in each group[ One!way ANOVA\ followed by post!hoc Duncan|s multiple range test\ revealed signi_cant di}erences] p5 vs p00\ R R\ P³9[90^ p5 vs p10\ ¦\ P³9[94\ ¦¦\ P³9[90^ p00 vs p10\ \ P³9[94\ \ P³9[90[
Noradrenaline There was a signi_cant decrease in the NA content from p00 to p10 in CLR "F1\04 8[5^ P³9[90\ N 5#\ DR "F1\04 02[7^ P³9[94\ N 5# and E "F1\04 19[17^ P³9[90\ N 5# groups[ Comparison of NA content between di}erent treatment groups in the same age also revealed signi_cant di}erences[ The NA content in the DR rats was lower than CLR rats at p5 "00)#\ p00 "15)\ P³9[94# and at p10 "23)\ P³9[94#[ In experimental rats\ NA content was signi_cantly higher than that of DR group at p5 "P³9[94#\ p00 "P³9[94# and even at p10 "P³9[90# "Fig[ 1#[ DISCUSSION Our _ndings suggest that there appears to be a progressive decline in DA\ but not NA\ plasticity with age\ since exposure of rats for 0 day|s cycling light after dark rearing resulted in the restoration of DA level at p5 and p00 but not at p10\ whereas NA levels recovered at all time points studied[ The temporal di}erence in the optimal expression of these transmitters09 may be responsible for this di}erence in the plasticity[ Endogenous levels of DA have been shown to be stimulated by light in the dark adapted retinae[3\4\5 Exposure to 0 day|s cycling light after chronic dark rearing was su.cient for a rapid increase in the levels of catecholamines in the present study[ This may be due to activation of tyrosine hydroxylase by light\ as has been demonstrated by a number of studies[3\02\19\13 Studies in guinea!pig\ a precocious animal in which the eyes open at birth\ have shown a decreased DA content on chronic dark rearing\ from birth to 2 or 5 weeks of age[15 Although\ in rats\ the eyes open only after p09\ light di}using through the closed transparent eyelids may also be critical for the normal neurotransmitter pro_le to emerge[17
031
R[ R[ J[ Shelke et al[
Fig[ 1[ Levels of noradrenaline "pmol:g wet tissue# in retinae of di}erent groups of rats at postnatal days 5\ 00 and 10[ The rest is as in Fig[ 0[
An earlier study has speculated that catecholamine synthesis does not proceed to NA in the retina since dopamine beta hydroxylase\ the enzyme that converts DA to NA\ could not be detected by immunostaining[1 However\ Kramer05 showed NA to be released from retina by chemical stimu! lation[ Our results show the presence of NA in the rat retina already at p5\ which is consistent with previous reports from bovine retina[05\11 It has been suggested that NA found in retina could originate from sympathetic neurons innervating the retinal blood vessels[11 However\ sympathetic denervation of the retinal blood vessels had no e}ect on the histo~uorescence of NA in the retina03[ Accordingly\ the presence of NA observed in this study must be intrinsic to the retina[ The non!transmitter roles of DA and NA as growth regulators have been studied[6\8\00\08\18 In cultured retinal neurons\ DA has been shown to inhibit neurite outgrowth through the activation of D0 receptors[07 Depletion of DA in the neonates by reserpine or destruction of dopaminergic a}erents using 5!hydroxydopamine has been shown to alter the expression of peptidergic genes\ neurotrophic activity of striatal cells\ hyperinnervation by serotonergic a}erents and retarded growth of striatal neurons suggesting that DA exerts multiple in~uences on neuronal development[08 Most of the amacrine cells di}erentiate and form synaptic contacts in the inner plexiform layer during the second week of postnatal development[01 Increased levels of catecholamines\ especially the DA\ observed in the present study during this period may be critical for the above events to occur[ REFERENCES 0[ Ballesta J[\ Terenghi G[\ Thibault J[ and Polak J[ M[ "0873# Putative dopamine containing cells in the retina of seven species demonstrated by tyrosine hydroxylase immunocytochemistry[ Neuroscience 01\ 0036Ð0045[ 1[ Beazley L[ D[\ Perry V[ H[\ Baker B[ and Darby J[ E[ "0876# An investigation into the role of ganglion cells in the regulation of division and death of other retinal cells[ Dev[ Brain Res[ 22\ 058Ð072[ 2[ Besharse J[ C[\ Iuvone P[ M[ and Pierce M[ E[ "0877# Regulation of rhythmic photoreceptor metabolism] a role for post receptoral neurons[ Pro`ress in Retinal research "ed[ Osborne N[#\ pp[ 10Ð50\ Pergamon\ London[
E}ect of dark rearing on retinal DA and NA levels
032
3[ Boatright J[ H[\ Hoel M[ J[ and Iuvone P[ M[ "0878# Stimulation of endogenous dopamine release and metabolism in amphibian retina by light! and K¦!evoked depolarization[ Brain Res[ 371\ 053Ð057[ 4[ Brainard G[ C[ and Morgan W[ W[ "0876# Light!induced stimulation of retinal dopamine] a doseÐresponse relationship[ Brain Res[ 313\ 088Ð192[ 5[ Chow K[ L[\ Riesen A[ H[ and Newell R[ W[ "0846# Degeneration of retinal ganglion cells in infant chimpanzees reared in darkness[ J[ Comp[ Neurol[ 096\ 16Ð31[ 6[ DeVries S[ H[ and Baylor D[ A[ "0882# Synaptic circuitry of the retina and the olfactory bulb[ Cell 61\ 028Ð038[ 7[ Dong C[ J[\ Mc Reynolds J[ S[ "0881# Comparison of the e}ects of ~ickering and steady light in dopamine release and horizontal cell coupling in the mudpuppy retina[ J[ Neurophysiol[ 56\ 253Ð261[ 8[ Felten D[ L[\ Hallman H[ and Johnsson G[ "0871# Evidence for a neurotrophic role of noradrenaline neurons in postnatal development of the rat cortex[ J[ Neurocytol[ 00\ 008Ð024[ 09[ Foster G[ A[ "0874# Di}erential ontogeny of three putative catecholamine cell types in the postnatal rat retina[ Brain Res[ 243\ 076Ð085[ 00[ Franquinet R[ and Martelly V[ "0870# E}ects of serotonin and catecholamines on RNA synthesis in planarians] in vitro and in vivo studies[ Cell Diff[ 09\ 190Ð198[ 01[ Horsburgh G[ M[ and Safton A[ J[ "0876# Cellular degeneration and synaptogenesis in the developing retina of the rat[ J[ Comp[ Neurol[ 152\ 442Ð455[ 02[ Iuvone P[ M[\ Galli C[ L[\ Garrison!Gund C[ K[ and Ne} N[ H[ "0867# Light stimulates tyrosine hydroxylase activity and dopamine synthesis in the retinal amacrine cells[ Science 191\ 890Ð891[ 03[ Iuvone P[ M[ and Ne} N[ H[ "0870# Dopamine neurons of the retina] a simple model system for studying synaptic regulatory mechanism[ In Essays in Neurochemistry and Neuropharmacolo`y "eds Youdim M[ B[ H[\ Lovenberg W[ and Sharman D[ E[#\ Vol[ 4\ pp[ 64Ð83[ John Wiley and Sons Ltd\ New York[[ 04[ Knapp A[ G[ and Dowling J[ E[ "0876# Dopamine enhances excitatory amino acid gated conductances in cultured retinal horizontal cells[ Nature 214\ 326Ð328[ 05[ Kramer S[ G[ "0860# Dopamine] a retinal neurotransmitter * I[ Retinal uptake\ storage and light stimulated release of 2 H!dopamine in vivo[ Invest[ Opthalmol[ Vis[ Sci[ 09\ 327Ð341[ 06[ Lakshmana M[ K[ and Raju T[ R[ "0883# Endosulfan induces small but signi_cant changes in the levels of noradrenaline\ dopamine and serotonin in the developing rat brain and de_cits in the operant learning performance[ Toxicolo`y 80\ 028Ð049[ 07[ Lankford K[ L[\ Demello F[ G[\ Klein W[ L[ "0877# D0 type dopamine receptors inhibit growth cone motility in cultured retina neurons] evidence that neurotransmitters act as morphogenic growth regulators in the developing central nervous system[ Proc[ Natl Acad[ Sci[ U[S[A[ 74\ 1720Ð1723[ 08[ Lauder J[ M[ "0882# Neurotransmitters as growth regulatory signals] role of receptors and second messengers[ Trends Neurosci[ 05\ 122Ð139[ 19[ Melamed E[\ Frucht Y[\ Lemor M[\ Uzzan A[ and Resenthal Y[ "0873# Dopamine turnover in rat retina] a 13!hour light! dependent rhythm[ Brain Res[ 294\ 037Ð040[ 10[ Nesselhut T[ and Osborne N[ N[ "0871# Is noradrenaline a major catecholamine in the bovine retina<[ Neurosci[ Lett[ 17\ 30Ð34[ 11[ Osborne N[ N[ "0870# Noradrenaline\ a transmitter candidate in retina[ J[ Neurochem[ 25\ 06Ð16[ 12[ Paes de Carvalho R[ and de Mello F[ G[ "0874# Expression of A0 adenosine receptors modulating dopamine!dependent cyclic AMP accumulation in the chick embryo retina[ J[ Neurochem[ 33\ 734Ð740[ 13[ Parkinson D[ and Rando R[ R[ "0872# E}ect of light on dopamine metabolism on chick retina[ J[ Neurochem[ 39\ 28Ð 35[ 14[ Rodrigues P[ S[ and Dowling J[ E[ "0889# Dopamine induces neurite retraction in retinal horizontal cells via diacylglycerol and PKC[ Proc[ natn[ Acad[ Sci[ U[S[A[ 76\ 8582Ð8586[ 15[ Spira A[ W[ and Parkinson D[ "0880# E}ect of dark rearing on the retinal dopaminergic system in the neonatal and postnatal guinea pig[ Dev[ Brain Res[ 51\ 031Ð034[ 16[ Ventura A[ M[\ Klein W[ L[ and De Mello F[ G[ "0873# Di}erential ontogenesis of D0 and D1 dopaminergic receptors in the chick embryo retina[ Dev[ Brain Res[ 01\ 106Ð112[ 17[ Wu D[ K[ and Cepko C[ L[ "0882# Development of dopaminergic neurons is insensitive to optic nerve section in the neonatal rat retina[ Dev[ Brain Res[ 63\ 142Ð159[ 18[ Yazulla S[ and Kleinschmidt J[ "0871# Dopamine blocks carrier mediated release of GABA from retinal horizontal cells[ Brain Res[ 122\ 100Ð104[ 29[ Yazulla S[ and Studholme K[ M[ "0884# Volume transmission of dopamine may modulate light adaptive plasticity of horizontal cell dendrites in the recovery phase following dopamine depletion in gold _sh retina[ Vis[ Neurosci[ 01\ 716Ð 725[ 20[ Yeh H[ H[\ Battelle B[ A[ and Puro D[ G[ "0873# Dopamine regulates synaptic transmission mediated by cholinergic neurons of the rat retina[ J[ Neurosci[ 02\ 890Ð898[ 21[ Young L[ H[ Y[ and Dowling J[ E[ "0878# Localization of cyclic adenosine monophosphate in the teleost retina] e}ects of dopamine and prolonged darkness[ Brain Res[ 493\ 46Ð52[