Multifocal ERG Responses in Infants

Abstract

purpose. To assess function of the central retina in 10-week-old infants, multifocal electroretinograms (mfERGs) were recorded. mfERG responses represent postreceptor retinal activity.

methods. In infants (n = 23) and adults (n = 10), mfERG responses to both unscaled and scaled 61 hexagon arrays were recorded. Amplitude and implicit time of negative (N₁, N₂) and positive (P₁) peaks of the first-order kernel were examined. The response from the entire area stimulated and responses to concentric rings were analyzed separately. The overall averaged response of the first slice of the second-order kernel was also evaluated. Results from infants and adults were compared.

results. Amplitudes of the infants’ responses (N₁, P₁, N₂) were significantly smaller and implicit times were significantly longer than those of adults. In infants, amplitude and implicit time varied little with eccentricity. In adults, amplitude decreased with eccentricity, whereas implicit time varied little. In infants, the second-order kernel was relatively more attenuated than the first-order kernel.

conclusions. The infants’ mfERG responses indicated immaturities of processing in the central retina. Infant-adult differences in the distribution of cones and bipolar cells may account for the results.

Cone-mediated electroretinographic (ERG) responses to full-field stimuli are relatively mature early in infancy. Specifically, the sensitivity and saturated amplitude of the cone photoresponse are 60% to 70% of the adult values by age 10 weeks. ¹ Peripheral cones are the main contributors to these ERG responses, whereas cones in the macula, which constitutes less than 5% of the total retinal area, contribute little. Thus, little is known about cone function in the immature central retina. It is well recognized that foveal cone structure does not reach maturity until well into childhood ^{2 3} and that visual acuity has a protracted course of development. ⁴  

Although the maximum amplitude of the cone-driven postreceptor component, the b-wave, is nearly the same in infants as in adults, the shape of the b-wave stimulus/response function differs. In infants, stimulus/response function does not show a falloff with increasing intensity. ¹ This absence of the photopic hill ^{5 6 7 8 9} in infants has been attributed to immaturity of the ON and OFF bipolar cell responses. ¹  

The multifocal ERG (mfERG) provides information about the functional topography of the central retina by recording responses from a large number of small, discrete regions. ^{10 11} Cone-initiated activity in the bipolar cells is the main contributor to the mfERG response. ^{11 12} The goal of the present study was to use the mfERG to investigate postreceptor processes in the central retina of healthy young infants. 

Methods

Subjects

Term-born 10-week-old infants (median, 70 days; range, 61–77 days; n = 23) were recruited by mail. All had been born within 10 days of their due dates and were in good general health. Healthy adult control subjects (median, 24 years; range, 22–51 years, n = 10) were also studied. Ophthalmic examination disclosed no abnormality in any subject. All infants had grating acuity ⁴ within the 95% prediction limits of normal for age. The infants, whose spherical equivalents (median, +2.00 diopters [D]; range, +1.00 to +4.00 D) were within the 95% prediction limits of normal, ¹³ did not wear optical correction during testing. Optical defocus has little effect on the amplitude or implicit time of the first-order mfERG response. ^{14 15} The median ETDRS acuity of adults was 20/16 (range, 20/13 to 20/24). Adults wore their optical correction, if any, during the test; median spherical equivalent was −0.88 D (range, −7.75 D to Plano). Written, informed consent was obtained from control subjects and parents of the infants after explanation of the nature and possible consequences of the study. This study conformed to the tenets of the Declaration of Helsinki and was approved by the Children’s Hospital Committee on Clinical Investigation. 

Procedure

The left pupil was dilated with cyclopentolate 1%. After 30 minutes, proparacaine 0.5% was instilled and a bipolar Burian-Allen electrode was placed on the left cornea. A ground electrode was placed on the skin over the left mastoid. Responses were differentially amplified (bandpass, 0.3–100 Hz; gain, 100,000), digitized, and displayed (VERIS 4.1 system; EDI, San Mateo, CA). The real-time input signal from the electrode was monitored, and segments contaminated by noise were rejected and recorded again. 

An array of 61 hexagons around a central fixation cross was presented on a high resolution monitor (Nortech Imaging Technologies, Mount Prospect, IL) at a 75-Hz frame rate. At the 45-cm viewing distance, the horizontal extent of this array was 43.2°. Average luminance of the stimulus was approximately 100 cd/m², and the contrast between white and black hexagons was greater than 90%. Each hexagon alternated between white and black using a pseudo-random m-sequence with exponent 14. There were 2¹⁴ − 1 m-sequence steps during the 3-minute 38-second recording period, which was divided into eight 27-second segments. 

Subjects were tested with unscaled and scaled stimuli. In the unscaled condition, all hexagons were of equal size; this stimulus makes no assumptions about the underlying distribution of retinal cells. In the scaled condition, hexagon size was scaled with eccentricity such that, in adults, all hexagons produced approximately equal amplitude responses. ¹⁰ A flip of a coin determined which condition was recorded first. Sixteen of the 23 infants completed testing in both conditions, five completed it with scaled stimuli only, and two completed it with unscaled stimuli only. Six of the 10 adults were tested in both conditions, and four were tested with scaled stimuli only. 

One of the investigators (ABF) held the infant on her lap and supported the chin during the test. To reduce the demands on the infant’s fixation, the recording was broken into eight segments. At the start of each segment, the infant’s gaze was attracted to the center of the display by a small yarn doll (33 × 8 mm) that was jiggled and then removed when recording began. An observer continuously monitored the infant’s fixation during recording. Responses were recorded only when the observer reported that the infant was alert and looking at the center of the monitor. If the infant looked away from the center of the display, the segment was discarded and re-recorded. A control experiment showed that an observer could reliably report when adult subjects looked outside the central 2.8° hexagon. A second control experiment with an adult (RMH) showed that changes of fixation within the central hexagon produced by scanning along the border of that hexagon throughout the trial did not appreciably change the amplitude or implicit time of any component. Menz et al. ¹⁶ also found that small changes of fixation within the central hexagon have no significant effect on the overall topography of the mfERG response. 

Analyses

Responses to unscaled and scaled stimuli (Fig. 1)were processed using the VERIS (EDI) software, with one iteration of artifact removal and spatial averaging with one-sixth of the surrounding responses. ¹⁷ We refer to response density (nV/deg²) as amplitude. Amplitude of the first-order kernel was measured from the baseline to the trough (N₁, N₂) or peak (P₁) of the waveform. Implicit time was measured from the start of the trace to the trough or peak. First, the overall response of the entire stimulated retinal area was assessed by averaging the responses to all 61 hexagons. The amplitude and implicit time of each component of this overall averaged response of infants were compared with those of adults using t-tests. 

Next, responses for each subject were combined in five concentric rings using the VERIS (EDI) software. Ring 1 was the central hexagon only. The amplitude and implicit time of the three components (N₁, P₁, N₂) of the first-order kernel were evaluated as a function of group (infant, adult) and eccentricity (ring 1, 2, 3, 4, 5) using a two-factor, repeated-measures analysis of variance. ¹⁸ Post hoc comparisons were made using the Scheffé test. 

The first slice of the second-order kernel represented in the averaged response to all 61 hexagons was also examined. Peak-to-trough amplitude of the second-order response was measured. The ratio of this amplitude to P₁ amplitude of the first-order response was calculated. ^{19 20 21} The ratio does not depend on absolute amplitude. The ratio in infants and adults was compared using a t-test. For all statistical tests, the level of significance was P ≤ 0.01. 

Results

The waveform of the averaged first-order kernel of the mfERG was similar in infants and adults for both unscaled and scaled stimuli (Fig. 2) . For unscaled and scaled stimuli (Fig. 3) , the amplitude of P₁ and N₂ and the implicit time of N₁, P₁, and N₂ differed significantly between infants and adults; N₁ amplitude did not differ significantly. These results illustrate the principal features of the infant response: reduced amplitude and increased implicit time. 

Ring averages for first-order kernel responses in three infants and one adult are shown in Figure 4for unscaled and scaled stimuli. In Figure 5 , mean infant and adult N₁, P₁, and N₂ amplitudes (± SEM) are plotted for each ring (rings 1 to 5). The difference between infants and adults was greatest for ring 1, the central hexagon, and was least for ring 5, the most peripheral ring. In adults, amplitude decreased markedly with eccentricity. In infants, amplitude changed little with eccentricity for unscaled stimuli; for scaled stimuli, amplitude in ring 1 was slightly larger than in rings 2 to 5. 

For the ring averages, analysis of variance indicated that for both unscaled and scaled stimuli, the mean amplitude of N₁, P₁, and N₂ differed significantly between infants and adults (Table 1) . There was a significant interaction between group and eccentricity. For the central hexagon and ring 2, N₁, P₁, and N₂ amplitudes were significantly smaller in infants than in adults (Scheffé test). For the peripheral rings (4 and 5), infant and adult amplitudes did not differ significantly. 

Implicit time of all components for both unscaled and scaled conditions was significantly longer in infants than in adults (Fig. 6 ; Table 1 ). The infant-adult difference did not vary with ring number and was larger for N₂ than for the earlier components, N₁ and P₁. 

The first slice of the second-order kernel in three infants and an adult is shown in Figure 7 . The mean amplitude of the second-order response (Fig. 8 , upper) was significantly smaller in infants than in adults for both unscaled and scaled stimuli. In infants, second-order responses were relatively more attenuated than first-order responses. The average ratio of second-order to first-order response was significantly lower in infants than in adults (Fig. 8 , lower). 

Discussion

The present results show that the photopic responses of the central retina are significantly smaller and slower in infants than in adults (Figs. 3 5 6)and that in infants they vary little with eccentricity (Figs. 5 6) . The amplitude difference between infants and adults decreases with eccentricity (Fig. 5) . The large central peak observed in adults is not found in infants. The similarity of infant and adult amplitudes in the peripheral rings is consistent with the relative maturity of the infants’ full-field cone ERG, which is dominated by peripheral retinal activity. ¹  

In infants, the distribution of cones in the central retina is nearly flat. ^{2 3 22} Specifically, results of analysis by Candy et al. ²³ showed that cone density varies from 15,000 cones/mm² at the fovea to 12,500 cones/mm² at 10° in the infant retina compared with approximately 200,000 cones/mm² at the fovea and 11,300 cones/mm² at 10° in the adult retina. If the relative density of bipolar cells is like that of cones, as it is in simian retina, ^{24 25 26 27} the amplitude of the infants’ mfERG response would vary little with eccentricity. This would account for the results displayed in Figure 5 . 

Small changes in fixation within the central hexagon are unlikely to account for the differences between infant and adult mfERG responses. As previous results from adults ¹⁶ and our control experiment show, such eye movements have little effect. Furthermore, the shallow gradient of receptors in the infant eye ²³ would be likely to reduce the impact of unstable fixation in infant mfERG. 

The first slice of the second-order kernel is thought to reflect nonlinear adaptive processes. ^{11 28 29} The amplitude of the second-order response is very small in infants (Figs. 7 8) , indicating that these adaptive processes are not well developed in them. The present results add to previous knowledge obtained through the study of full-field ERG oscillatory potentials, ³⁰ which also document immature neural processes in postreceptor retina of young infants. These retinal immaturities may explain, in part, the protracted course of visual development. 

 Supported by National Eye Institute Grant EY 10597.

 Submitted for publication June 12, 2008; revised July 30, 2008; accepted October 20, 2008.

 Disclosure: R.M. Hansen, None; A. Moskowitz, None; A.B. Fulton, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Sample mfERG records of infant 15. The 61 first-order responses to unscaled (upper trace array) and scaled (lower trace array) stimuli are shown. This infant had amplitudes that were near the median in both conditions. In each panel, a schematic drawing of the stimulus is shown.

Figure 2.

 

First-order kernel responses averaged across all 61 hexagons in the unscaled (upper) and scaled (lower) conditions. Responses are shown for infants who had P₁ amplitudes near the minimum (infant 19), median (infant 15), and maximum (infant 14) values in both stimulus conditions. Responses from an adult (adult 10) with P₁ amplitude near the median are also shown (lowest trace in each). For each subject, waveforms in the unscaled and scaled conditions are similar. In each panel, the calibration bar pertains to all subjects.

Figure 3.

 

Amplitude (upper graphs) and implicit time (lower graphs) of overall averaged responses for unscaled (left) and scaled (right) stimuli in infants (filled bars) and adults (open bars). Means (± SEM) for N₁, P₁, and N₂ of the first-order kernel are shown.

Figure 4.

 

Ring averages to unscaled (upper) and scaled (lower) stimuli. The response to the central hexagon (ring 1) and the average response to all hexagons in each concentric ring (rings 2–5) are shown. Responses from infants with P₁ amplitude at the minimum (infant 19), median (infant 15), and maximum (infant 14) are shown. For comparison, ring averages for an adult (adult 10) are also plotted. The calibration bar (lower right) pertains to all.

Figure 5.

 

Mean amplitude (± SEM) for infants (filled circles) and adults (open circles) for N₁, P₁, and N₂ of the first-order kernel plotted for rings 1 to 5. Responses to unscaled stimuli are shown on the left, and responses to scaled stimuli are shown on the right.

Figure 6.

 

Mean implicit time (± SEM) for infants (filled circles) and adults (open circles) for N₁, P_1, and N₂ of the first-order kernel plotted for rings 1 to 5. Responses to unscaled stimuli are shown on the left, and responses to scaled stimuli are shown on the right.

Table 1.

 

Analysis of Variance for Ring Averages

	Amplitude											Implicit Time
		Unscaled					Scaled					Unscaled					Scaled
		F	df	P	F	df	P	F	df	P	F	df	P
N1
Group	9.27	1	<0.001	18.80	1	<0.001	25.60	1	<0.001	23.93	1	<0.001
Ring	57.47	4	<0.001	66.05	4	<0.001	1.72	4	0.152	0.72	4	0.578
Group × ring	31.19	4	<0.001	24.53	4	<0.001	0.19	4	0.939	0.49	4	0.742
P1
Group	24.39	1	<0.001	46.14	1	<0.001	28.08	1	<0.001	58.70	1	<0.001
Ring	81.10	4	<0.001	154.51	4	<0.001	1.54	4	0.197	0.02	4	0.999
Group × ring	94.86	4	<0.001	58.78	4	<0.001	0.43	4	0.789	0.91	4	0.463
N2
Group	39.36	1	<0.001	38.40	1	<0.001	20.97	1	<0.001	42.71	1	<0.001
Ring	73.75	4	<0.001	94.61	4	<0.001	0.42	4	0.795	1.12	4	0.349
Group × ring	79.70	4	<0.001	26.77	4	<0.001	1.51	4	0.206	1.44	4	0.226

Figure 7.

 

The second-order response averaged across all 61 hexagons in the unscaled (upper) and scaled (lower) conditions. The peak and trough used to specify amplitude are indicated. Responses are shown for the same subjects as in Figures 2 and 4 . The calibration bar pertains to all subjects.

Figure 8.

 

The amplitude of the second-order kernel plotted (upper graphs) for unscaled (left) and scaled (right) stimuli for infants (filled bars) and adults (open bars). The ratio of the amplitude of the second- to first-order response (lower graphs) is plotted for infants and adults. Means (± SEM) are shown.