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Early Human Development 55 (1999) 51–62

The effect of sleep state on electroretinographic(ERG) activity during early human development

a b b ,c a ,*˜Marcela Pena , David Birch , Ricardo Uauy , Patricio Peiranoa ˜ ´Laboratorio de Sueno y Neurobiologıa Funcional, INTA, Universidad de Chile, P.O. Box 138-11,

Santiago, ChilebRetina Foundation of the Southwest, Dallas TX, USA

c ´ ´Unidad de Nutricion Clınica, INTA, Universidad de Chile, Santiago, Chile

Accepted 29 December 1998

Abstract

To assess the effects of sleep state on human retinal electric responses, full-field elec-troretinograms were obtained in a cross-sectional study from 123 preterm infants at 36, 40 and56 weeks of post-gestational age. At each age, electroretinographic recordings were assigned toone of two groups according to whether the infants were in active sleep or quiet sleep. Bothsleep states were determined behaviorally. Pure rod, maximal, 30Hz flicker and light adaptedsingle cone responses were evaluated when a sleep state was clearly established. Peak-to-peakamplitudes of most electroretinographic responses were significantly larger in active sleeprelative to quiet sleep at 36 and 40 weeks of post-gestational age. We speculate that largeramplitudes during active sleep may play a role in the maturation of the visual system. 1999Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Sleep states; Electroretinogram; Premature; Infant; Human

1. Introduction

During early human development, organization of sleep states and retinal activityare functional expressions of central nervous system (CNS) maturation. Further sleep

*Corresponding author. Tel.: 1 56-2-678-1447; fax: 1 56-2-221-4030.E-mail address: [email protected] (P. Peirano)

0378-3782/99/$ – see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved.PI I : S0378-3782( 99 )00006-7

˜52 M. Pena et al. / Early Human Development 55 (1999) 51 –62

state organization has been used as a sensitive predictor of neuro-behavioralmaturation and cognitive development [1–3].

Under normal conditions, two sleep states, active sleep (AS)- or REM sleep- andquiet sleep (QS)- or non REM sleep-alternate periodically within a sleep episode.Each sleep state represents a specific manner of CNS functional integration evidencedby the sustained concordance of characteristic patterns in different biologicalvariables. The alternation of sleep states, representing particular modes of CNSfunctional integration, is generated endogenously and recurs periodically in apredictable manner.

Preterm and full-term infants sleep most of their life throughout the first year, andAS is the prevailing state during the neonatal period and the first months of life.Wakefulness (WA), less than 15% of the total sleeping time, is expressed mainly as areflex behavioral state [4,5]. With advancing age WA increases and AS decreasesconcurrently. Thus AS and WA have been proposed as two functionally equivalentbehavioral states during early development, both representing active states of theCNS [6,7]: AS provides endogenous stimulation, while WA provides environmental(exogenous) stimulation. During this age span the quantity of QS also increases andits characteristics evolve toward adult patterns [8].

Sleep states affect a wide range of biological activities, including the activity of theauditory system [9,10], autonomic nervous system balance [11], brain energyexpenditure [12], blood flow distribution in different regions of the CNS [13],thermoregulation [14], hormonal secretion [15] and neurotransmitter activities in theCNS [16,17]. Many of these state-dependent modulations have been studied only inanimals or adult humans.

Sleep states modify the human visual system responses such as the visual-evoked-potential triggered by a flash of light [18,19]. Recently, Galambos et al. demonstratedsleep state-related changes in the electroretinogram (ERG) of adult rats [20]. To thebest of our knowledge sleep state-related modifications of human retinal functionhave not previously been reported. The purpose of this study was to determinewhether naturally occuring sleep states modulate ERG activity throughout earlyhuman development.

2. Subjects and methods

2.1. Subjects

This cross-sectional study was carried out on 123 healthy appropriate-for-gestation-al age preterm infants, grouped according to post-gestational age (pGA): 36, 40 or 56weeks. Characteristics of different groups are presented in Table 1. There was nohistory of significant pathological conditions defined as necrotizing enterocolitis,neonatal sepsis, intracraneal haemorrhage greater than grade II, retinopathy ofprematurity greater than grade III, mechanical ventilation for respiratory problemslonger than 7 days, serious congenital defects or, diseases that alter normal growth.

˜M. Pena et al. / Early Human Development 55 (1999) 51 –62 53

Table 1Clinical data

Age Sleep state P(pGAweeks) AS QS

n median inter n median interquartile quartilerange range

Birth gestational age 36 8 30.7 3.2 7 30.8 2.0 NS(pGA weeks) 40 13 30.6 3.1 18 31.9 2.5 NS

56 9 31.8 2.5 21 31.0 2.0 NSPostnatal age at ERG 36 8 5.8 1.8 7 5.5 1.1 NS(weeks) 40 13 9.2 2.9 18 8.2 2.9 NS

56 9 23.9 3.1 21 25.2 3.4 NSBirth weight (g) 36 8 1539 250 7 1605 245 NS

40 13 1538 390 18 1600 304 NS56 9 1647 253 21 1557 283 NS

NS: no significant difference between infant-groups evaluated during AS or QS, P . 0.05.AS: active sleep.QS: quiet sleep.

All infants were fed human milk or formula which met nutritional recommendationsfor low birth weight infants.

The study was approved by INTA–University of Chile’s ethical committee whichmonitors the participation of human as experimental subjects. Parental writtenconsent was obtained after providing a detailed explanation of the methods ofrecording and the aim of the study.

2.2. Full-field electroretinogram.

The ERG represents the summed retinal activity in response to light stimulation.The ERG measured at the cornea contains an early corneal-negative component(a-wave) and a slow corneal-positive component (b-wave) (Fig. 1). The a-wavecorresponds to photoreceptor activity while the b-wave reflects bipolar cell activity inthe inner nuclear layer [21]. The ERG was recorded from the left eye of each infant,following the International Standard Protocol endorsed by the International Societyfor Clinical Electrophysiology of Vision [22,23], with a widely-used variant for the30 Hz flicker response described elsewhere [21].

Four responses were obtained in the following order: (1) Rod response. Thisresponse reflects rod activity. It was obtained follow at least 30 min of dark-adaptation, with a short wavelength stimulus (l 5 440 nm; half-bandwidth 5 47max

nm), retinal illuminance 5 2 0.1 log scot.td 2 s and 0.2 Hz frequency of stimulation;(2) Maximal response. This response reflects a mixed dark-adapted rod and coneresponse. It was obtained in the dark, with a white light stimulus, retinal illuminance5 1.6 log scot.td 2 s and 0.2 Hz frequency of stimulation; (3) 30 Hz flicker coneresponse. This response reflects cone activity. It was obtained in the dark, with a


Fig. 1. Components and parameters of an ERG response (example). a-wave, b-wave, b-wave implicit time(b-it) and peak-to-peak amplitude (p-p amp) are indicated in the maximal retinal response. The curvecorresponds to the computer average of 20 isolated responses, in an infant at 56 pGA.

white light stimulus, retinal illuminance 5 1.3 log phot.td 2 s and 30 Hz frequency ofstimulation; and (4) Light-adapted cone response. This response reflects cone activity.It was obtained under light-adaptation (after 10 min of exposure to a continuous

22background luminance of 1.5 log cd*m ), with a white light stimulus, retinalilluminance 5 1.6 log phot.td 2 s and 0.2 Hz frequency of stimulation. Fig. 2 showsa typical example of these four responses, at 56 pGA weeks.

2.3. Sleep states scoring.

Sleep states were scored continuosly throughout the recording on a minute-to-minute basis. The following specific behaviors were assessed visually: (a) the eyelidcleavage of the right eye (open or close); (b) rapid eye movements (presence orabsence); (c) body movements (presence or absence), (d) facial mimicry (presence orabsence); (e) respiratory rhythm (regular or irregular); and (f) thoraco-abdominalrespiratory relationship (in-phase or out-of-phase). QS was established when infantspresented the right eye closed, absence of rapid eye movements (REM), absence ofbody movements, absence of facial mimicry, regular respiration and with in-phasethoraco-abdominal relationship. AS was established when infants presented the righteye closed, presence of REM, presence of body movements, presence of facialmimicry, irregular respiration and with out-of-phase thoraco-abdominal relationship.In order to assure that light stimuli were performed in a well established AS or QS,


Fig. 2. Typical ERG-responses. (a) Rod response, (b) Maximal retinal response, (c) 30Hz flicker coneresponse and (d) Light adapted cone response. Each curve represents the average of 20 or more responses,in an infants at 56 pGA. Amplitude (in mv) is indicated on vertical axis, and time (in ms) on horizontalaxis.

they were applied when all parameters for AS or QS were simultaneously presentduring the corresponding minute. In general the agreement between visually observedand electrophysiologically recorded patterns to define sleep states in infancy variesfrom 80 to 90% [8,24–27].

2.4. Procedure

ERG recordings were performed in an electrically insulated and dark room,between 2:00 and 4:00 PM, after infants were fed and during the occurrence of anatural sleep episode (nap). The pupil of the left eye was dilated with a combinationof cyclopentolate hydrochloride 0.2% and phenylephrine hydrochloride 1%. Theinfant was dark adapted (with a black eye patch) for 30 min in the company of aparent or guardian. During this period the infant was fed and fell asleep following itsusual routine. Under dim red illumination, the infant was placed in a cradle, the eyepatch was removed and two drops of 0.5% propacaine hydrochloride was instillated.After 5 min, a bipolar Burian–Allen contact lens electrode, filled with methyl-cellulose hydroxipropyl 2.5%, was placed on the cornea of the left eye, and a groundelectrode was placed on the forehead. Pupil diameter at the begining and the end oftesting was measured to calculate retinal illuminance. A xenon lamp within a


Ganzfeld dome was used to provide brief (20 ms duration) flash stimuli. The infant’seye was continuously checked through a small aperture in the dome to verify thecorrect positioning of the pupil with respect to the lens. Responses were recordedonly when the pupil was clearly visible through the contact lens electrode. The flashintensity and the interstimulus interval were controlled by a photostimulator (GrassPS22C). At least 20 responses for each stimulus condition were amplified (gain 5

10.000; bandwidth, 2 to 300 Hz), averaged by computer with appropriate software,and stored on hard disk for off-line analyses. The parameters analyzed in all the ERGresponses (Fig. 1) were peak-to-peak amplitude and b-wave implicit time (time fromflash to major positive component).

2.5. Statistical analysis

Mean amplitude and implicit time of each ERG response were calculated for ASand QS at 36, 40 and 56 pGA weeks. Results were compared among ages in acorresponding sleep state, and between sleep states at a corresponding age usingKruskal–Wallis ANOVA, Dunn’s test for post-hoc comparisons from STATISTICAfor Windows, 4.5 software.

3. Results

3.1. Effects of post-gestational age

During AS and QS, a significant age-related increase was observed in peak-to-peakamplitude of the maximal response, the flicker response and light-adapted coneresponse (P , 0.001, P , 0.003 and 0.04, respectively) (Table 2). B-wave implicittimes of the same responses showed a significant decrease between 40 and 56 pGAweeks (P , 0.001) (Table 3). Finally, the rod response parameters were not comparedby pGA due to the limited number of infants that had adequate recording at 36 and 56pGA weeks.

3.2. Effects of sleep state

1. Mean amplitude (Table 2).(a) Rod response. Peak-to-peak amplitude at 40 pGA weeks did not differ

statistically between sleep states (P , 0.34). At 36 and 56 pGA weeks, this measurewas not compared by sleep states due to the limited number of infants from whomrod responses were obtained.

(b) Maximal response. Peak-to-peak amplitude was significantly greater during ASthan QS at 36 and 40 pGA weeks (P , 0.027 and P , 0.0009 respectively). At 56pGA weeks, the peak-to-peak amplitude was not significantly different between sleepstates (P , 0.37).

(c) 30 Hz flicker cone response. Peak-to-peak amplitude was significantly greaterduring AS than QS at 40 pGA weeks (P , 0.037). At 36 and 56 pGA weeks,


Table 2Amplitude (amp)

ERG-response Age Sleep state P(pGAweeks) AS QS


Rod response p-p amp 40 6 12.2 5.1 6 10.0 5.9 NSMaximal retinal p-p amp 36 9 40.2 25.3 8 23.7 14 , .05response 40 12 85.4 20.8 32 37.5 32.9 , .001

56 11 162.3 88.2 26 143.5 99.3 NS30Hz flicker p-p amp 36 8 2.7 2.2 6 1.8 1.6 stcone response 40 17 3.7 2.8 17 1.7 2.0 , .05

56 7 5.1 7.1 19 3.0 1.9 stLight adapted p-p amp 36 6 17.9 13.9 7 6.3 9.4 stcone response 40 17 29.1 20.2 16 11.8 13.9 , .01

56 8 34.0 27.7 18 21.3 18.1 st

AS: active sleep.QS: quiet sleep.NS: no significant difference between infant-groups evaluated during AS or QS.st: statistical tendency (0.05 , P , 0.1).p-p: peak-to-peak.

peak-to-peak amplitude showed a suggestive tendency to be greater during AS thanQS (P , 0.06 and P , 0.06 respectively).

(d) Light adapted cone response. Peak-to-peak amplitude was significantly greater

Table 3b-Wave implicit time

ERG-response Age Sleep state P(pGAweeks) AS QS


Rod response b-it 40 6 129.7 6.6 6 128.1 20.2 NSMaximal retinal b-it 36 9 79.7 11.7 8 76.2 9.6 NSresponse 40 12 76.2 10.2 32 79.7 14.4 NS

56 11 62.5 6.2 26 65.6 10.9 NS30Hz flicker b-it 36 8 50.8 2.3 6 50.0 3.5 NScone response 40 17 49.6 2.0 17 48.4 2.0 NS

56 7 44.5 6.3 19 43.8 3.9 NSLight adapted b-it 36 6 55.1 9.4 7 52.3 1.6 NScone response 40 17 50.8 5.5 16 53.9 4.3 NS

56 8 41.0 3.1 18 41.0 2.3 NS

AS: active sleep. QS: quiet sleep. NS: no significant statistical difference between infants-groups evaluated during AS orQS (P . 0.1).


during AS than QS at 40 pGA weeks (P , 0.009). At 36 and 56 pGA weekspeak-to-peak amplitude tended to be higher during AS than QS (P , 0.062 and 0.084respectively).

2. Mean implicit time (Table 3)For all ERG responses at all ages, the b-wave implicit time was not statistically

different between sleep states.

4. Discussion

The primary finding in this study is that the sleep state of the human infantinfluences the magnitude of the retinal response to light stimulation. Specifically, ourresults suggest that sleep states modulate the ERG of healthy human preterm infantsby affecting amplitude. For each ERG at every post-natal age, amplitudes weregreater during AS than QS. The effect was evident in both cone-mediated and mixedrod and cone responses to a maximal stimulus. The effect was statistically significantfor the majority of comparisons and for most, the effect remained significant afterapplying the Bonferroni adjustment [28] for multiple comparisons. Implicit time,which primarily reflects photoreceptor sensitivity to light, did not vary with sleepstate. Thus, the ERG variation appears to reflect state-dependent modulation ofpost-receptoral activity. This relationship between sleep states and evoked retinalactivity is consistent with results of studies showing state-dependent modulation ofnumerous biological variables [9,10,12–20], and may reflect a functional synchroni-zation process between the CNS and the retina. Modifications of this modulation withadvancing age could be associated with neuronal and sleep-wake states maturationthroughout early development.

Greater ERG amplitude during AS and influence of age. AS is associated withgreater peak-to-peak amplitude and this effect is more evident at 36 and 40 pGA than56 pGA weeks. Several studies have shown that the endogenous (internal) activationthat occurs during AS is essential for CNS maturation. This is particularly relevant atthe onset of WA state, characterized by the neonate’s limited ability to profit fromexogenous (external) stimuli [6,7,29]. For instance, the threshold of different sensorysystems is high and sensitivities are low [22] reducing thus the capacity of theneonate to process information during WA. It is well accepted that the maturation ofsensorimotor systems is dependent on the activity induced by exogenous stimulation[30,31]. The activity originating from endogenous stimulation during AS may have asimilar or greater influence on CNS maturation. In this respect, Roffwarg et al. [29]noted that visual pathways were activated during periods of AS. They surmised that aprocess of intense information processing was stimulating the visual system fromwithin, suggesting that AS served to functionally stimulate immature neurons andsynapses ‘internally’ at an age when infants were in a state of partial sensorydeprivation during WA. More recently, spontaneous retinal activation has beendescribed in animals during early development. Ganglion cells of neonatal mam-malian retina produce spontaneous bursts of activity in the absence of lightstimulation [32,33]. The assessment of the relationship between retinal electrical


activity and sleep states may provide new insights in this respect. Supporting thisconcept, it has recently been shown that AS deprivation aggravates the anatomicaland functional deletereous effects of monocular deprivation on lateral geniculatenucleus in kittens [6]. Furthermore, the effect of AS deprivation in unocluded kittensresulted in an impact of higher magnitude than the one provoked by monoculardeprivation [6,53,54]. Our results suggest that greater retinal activity during ASrepresents a biological condition that favors the maturation of the retina (and probablyother structures within the visual system) in early stages of development. There isalso evidence from the present study that the early functional association betweenretinal activity and sleep states is attenuated at 56 pGA weeks, when the quantity ofWA has increased [34–36] and its quality improved [37], paralleling the decrease ofthe amount of AS especially during the day-time.

Functional Synchronization. The functional synchronization (integration) betweencenters controlling sleep-states and retinal activity could originate through eitherneural or non-neural mechanisms, which are not necessarily mutually exclusive.

1. Neural mechanisms. Retinopetal fibers have been identified in numerousmammal species including monkey [38] with cellular bodies located in differentzones of the CNS, including those involved in sleep states regulation [38,39]. To thebest of our knowledge there is no data in the literature that demonstrate the presenceof retinopetal fibers in the human. In animals the retina is known to be an importantcontroller of eye growth and refractive development. Although the mechanisms bywhich the retina controls this process remain unclear, at least two differentmechanisms have been proposed, one within the eye (feedback from retinal ganglioncells) and the other in the brain (feedback from the CNS) [40]. In humans, retinaldysfunction during early ontogenesis, has been associated with higher incidence ofrefractive errors, possibly due to an imbalance of ON/OFF activity in the retina [41].A centrifugal regulation of retinal activity during early human development couldaccount for the present results.

The retina expresses numerous neuro-modulators and neurotransmitters and bothneural and non-neural mechanisms could participate in regulating their activities.Different neuro-modulators or neurotransmitters (i.e. dopamine, adrenaline, acetyl-choline), can modify the ERG amplitude and/or implicit time in mammals [42–44].One model of sleep-wake cycle regulation proposes that a permanent competingcholinergic /monoaminergic balance in the CNS is the principal mechanism thatcontrols the expression and maintenance of different sleep-wake states [45]. DuringAS the function of the cholinergic system prevails, while that of the monoaminergicone predominates during WA; during QS both system are expressed without anevident predominance. This balance would optimize the synaptic efficiency of themonoaminergic systems (thermoregulation, sustained attention) specially activeduring WA [17]. It is possible that retinal neuromodulatory and neurotransmittingactivities could be differently modified by sleep states.

2. Non-neural mechanisms. These mechanisms could include any of severalnaturally occuring sleep states-related metabolic modifications. The retina is a highlyirrigated organ representing the highest oxygen consumption by unit of weight of allhuman tissues. Changes in blood flow, oxygen and glucose supports and/or acid-base


balance modify retinal activity, and have been shown to produce reversible modi-fications in the ERG, in different species, ages and experimental conditions [46–50].To the best of our knowledge, there is no data in the literature describingmodifications of retinal activity according to the naturally occuring changes of theseparameters under physiological conditions of the sleep-wake cycle.

Cerebral blood flow, as well as oxygen and glucose consumption, are significantlymodified by sleep states. The full-term human newborn shows higher cerebral bloodflow during AS than QS [13]. Similar results have been reported in adults [51]. Inaddition greater oxygen and glucose consumption have been demonstrated in differentareas of the CNS during AS in adults mammals [11,52]. The normal modulation ofacid /base balance during sleep states remains unknow. It seems reasonable thatretinal activity may also be modulated by state-dependent changes in blood flow,oxygen and/or glucose support or acid /base balance.

In conclusion, our results demonstrate a modulatory effect of sleep states on retinalactivity during early human development. We speculate that this modulation partici-pates in early retinal development. Further research is needed to evaluate thefunctional implications of these results.

5. Notation

CNS, central nervous systemAS, active sleepQS, quiet sleepWA, wakefulnessERG, electroretinogrampGA, post-gestational ageREM, rapid eye movements

Acknowledgements

This work was supported by grants from Fondecyt (CONICYT, Chile) 1930820,NIH HD-22380, and ECOS/CONICYT C96B03. The authors would like to expresstheir gratitude to the mothers and infants who participated in this investigation.

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