Electrophysiological evidence of atypical visual change detection in adults with autism.

Although atypical change detection processes have been highlighted in the auditory modality in autism spectrum disorder (ASD), little is known about these processes in the visual modality. The aim of the present study was therefore to investigate visual change detection in adults with ASD, taking into account the salience of change, in order to determine whether this ability is affected in this disorder. Thirteen adults with ASD and 13 controls were presented with a passive visual three stimuli oddball paradigm. The findings revealed atypical visual change processing in ASD. Whereas controls displayed a vMMN in response to deviant and a novelty P3 in response to novel stimuli, patients with ASD displayed a novelty P3 in response to both deviant and novel stimuli. These results thus suggested atypical orientation of attention toward unattended minor changes in ASD that might contribute to the intolerance of change.

Minimization of cochlear implant artifact in cortical auditory evoked potentials in children.

OBJECTIVES: In congenitally deaf children fit with a cochlear implant, little is known about the maturation of the auditory cortex. Cortical auditory evoked potentials are a useful methodology to study the auditory cortical system of children with cochlear implants. Nevertheless, these recordings are contaminated by a cochlear implant artifact. The objective of this study was to use independent component analysis to minimize the artifact of the cochlear implant to study cortical auditory evoked potentials. STUDY DESIGN: Prospective study. METHOD: A total of 5 children ranging in age from 21 to 49 months who were fitted with a cochlear implant for at least 6 months were included in this study. The stimuli were pure tones (750 Hz, 200 ms duration, 70 dB SPL) presented with an irregular interstimulus interval (1000-2000 ms) via loud speakers. The cortical auditory evoked potentials were recorded from 17 Ag-AgCl electrodes referenced to the nose. The peak latency and amplitude of each deflection culminating at the fronto-central and temporal sites were analyzed. MAIN OUTCOME MEASURES: The P100-N250 peak latencies and amplitudes of the cortical auditory evoked potentials recorded from children fitted with cochlear implants. Scalp map potentials distributions were done for each child for the N250 wave. RESULTS: The use of independent component analysis permitted to minimize the cochlear implant artifact for the five children. Cortical auditory evoked potentials were recorded at fronto-central and temporal sites. Scalp map potentials distributions for the N2 wave showed activation of temporal generators contralateral at the CI for the five children. CONCLUSION: This preliminary electrophysiological study confirms the value and the limits of independent component analysis. It could allow longitudinal studies in cochlear implant users to examine the maturation of auditory cortex. It could also be used to identify objective cortical electrophysiological measures to help the fitting of CIs in children.

Vietnamese chickens: a gate towards Asian genetic diversity.

BACKGROUND: Chickens represent an important animal genetic resource and the conservation of local breeds is an issue for the preservation of this resource. The genetic diversity of a breed is mainly evaluated through its nuclear diversity. However, nuclear genetic diversity does not provide the same information as mitochondrial genetic diversity. For the species Gallus gallus, at least 8 maternal lineages have been identified. While breeds distributed westward from the Indian subcontinent usually share haplotypes from 1 to 2 haplogroups, Southeast Asian breeds exhibit all the haplogroups. The Vietnamese Ha Giang (HG) chicken has been shown to exhibit a very high nuclear diversity but also important rates of admixture with wild relatives. Its geographical position, within one of the chicken domestication centres ranging from Thailand to the Chinese Yunnan province, increases the probability of observing a very high genetic diversity for maternal lineages, and in a way, improving our understanding of the chicken domestication process. RESULTS: A total of 106 sequences from Vietnamese HG chickens were first compared to the sequences of published Chinese breeds. The 25 haplotypes observed in the Vietnamese HG population belonged to six previously published haplogroups which are: A, B, C, D, F and G. On average, breeds from the Chinese Yunnan province carried haplotypes from 4.3 haplogroups. For the HG population, haplogroup diversity is found at both the province and the village level (0.69).The AMOVA results show that genetic diversity occurred within the breeds rather than between breeds or provinces. Regarding the global structure of the mtDNA diversity per population, a characteristic of the HG population was the occurrence of similar pattern distribution as compared to G. gallus spadiceus. However, there was no geographical evidence of gene flow between wild and domestic populations as observed when microsatellites were used. CONCLUSIONS: In contrast to other chicken populations, the HG chicken population showed very high genetic diversity at both the nuclear and mitochondrial levels. Due to its past and recent history, this population accumulates a specific and rich gene pool highlighting its interest and the need for conservation.

Revealing fine scale subpopulation structure in the Vietnamese H'Mong cattle breed for conservation purposes.

BACKGROUND: During the last decades, there has been an acceleration of the loss of domestic animal biodiversity. For conservation purposes, the genetic diversity of the H'Mong cattle, an indigenous local breed was studied. Single-nucleotide polymorphisms (SNP) of the SRY gene and mtDNA D-Loop sequence were analysed to clarify the origin of the breed. The genetic diversity was assessed through genetic data with twenty-five FAO microsatellites, and morphometric data with five body measurements from 408 animals sampled from eight districts of the Ha Giang province. RESULTS: The SRY genes were all of the zebu type. Among the 27 mtDNA haplotypes, 12 haplotypes were of the taurine type and the remaining 15 of the zebu type. This indicates female taurine introgression in the zebu H'Mong. The observed and expected heterozygosity ranged from 0.616 to 0.673 and from 0.681 to 0.729 respectively according to district, with low genetic differentiation (F ST = 0.0076). Multivariate analysis on morphometric and genetic data shows a separation of districts into two groups following a south-west/north-east cline and admixture analysis confirmed the two clusters, but no differentiation of taurine introgression between clusters was observed. A possible admixture with the Yellow cattle breed from a neighbouring province was suggested through genetic data and householder interviews. CONCLUSIONS: In this study we demonstrate the interest of fine-scale sampling for the study of genetic structure of local breeds. Such a study allows avoiding erroneous conservation policies and on the contrary, proposes measures for conserving and limiting crossbreeding between the H'Mong and the Yellow cattle breeds.

Combining surveillance and expert evidence of viral hemorrhagic septicemia freedom: a decision science approach.

The ability to combine evidence streams to establish disease freedom or prioritize surveillance is important for the evaluation of emerging diseases, such as viral hemorrhagic septicemia virus (VHSV) IVb in freshwater systems of the United States and Canada. Waterways provide a relatively unconstrained pathway for the spread of VHSV; and structured surveillance for emerging disease in open systems has many challenges. We introduce a decision framework for estimating VHSV infection probability that draws from multiple evidence streams and addresses challenges associated with the assessment of emerging disease. Using this approach, historical and risk-based evidence, whether empirical or expert-derived, supplement surveillance data to estimate disease probability. Surveillance-based estimates of VHSV prevalence were described using beta distributions. Subjective likelihood ratios (LRs), representing contextual risk, were elicited by asking experts to estimate the predicted occurrence of risk factors among VHSV-affected vs. VHSV-unaffected watersheds. We used the odds form of Bayes' theorem to aggregate expert and surveillance evidence to predict the risk-adjusted posterior probability of VHSV-infection for given watersheds. We also used LRs representing contextual risk to quantify the time value of past surveillance data. This evidence aggregation model predicts disease probability from the combined assessment of multiple sources of information. The method also provides a flexible framework for iterative revision of disease freedom status as knowledge and data evolve.

Cross-modal processing of auditory-visual stimuli in a no-task paradigm: a topographic event-related potential study.

OBJECTIVE: The aim of this study was to investigate in healthy adults the electrophysiological correlates of auditory-visual interactions involved in perception of bimodal events in a no-task paradigm. METHODS: Event-related potentials were recorded in response to unimodal auditory (A), unimodal visual (V) and bimodal (AV) stimuli. Cross-modal interactions were estimated using the additive [AV-(A+V)] model. RESULTS: The spatio-temporal analysis of ERPs and scalp current densities revealed several interaction patterns occurring at both early and late stages of sensory cortical processing: (1) amplitude decrease of the unimodal auditory N1 wave as early as 55ms, (2) amplitude increase of the unimodal auditory P2 wave from 150 to 195ms concomitant with new neural activity over the right fronto-temporal region, and (3) amplitude increase of the late unimodal visual response within 245-350ms post-stimulus. CONCLUSIONS: Our study provides evidence that several patterns of cross-modal interactions can be generated even if no task is required from subjects. SIGNIFICANCE: The paradigm used here can thus be utilized for studying the maturation of the cross-modal processes in young children and in children with pathological development.

Transcytosis of pancreatic bile salt-dependent lipase through human Int407 intestinal cells.

In previous studies, we have shown that the bile-salt-dependent-lipase (BSDL), secreted by pancreatic acinar cells and secreted into the duodenal lumen, can be transcytosed through intestinal cells up to the lamina propria. In this study, we used an in vitro system to provide insights into the apical to basolateral transport of BSDL, across the intestinal barrier. The Int407 human epithelial cell line, grown under conditions that optimize polarity, was used as a tight epithelium model. We attempted to delineate uptake mechanisms and the transcytotic pathway followed by this pancreatic enzyme within the intestinal Int407 cells, which do not produce BSDL. When added to the apical reservoir of Transwell-grown Int407 cells, BSDL was shown to first interact with the apical membrane. Further, BSDL forms clusters that are internalized via clathrin-coated pits. Following endocytosis, BSDL is directed to a nocodazole- and colchicin-sensitive multivesicular compartment. Interestingly, this protein transits through the Golgi apparatus, where it was found to colocalize with the KDEL retrieval-receptor. Finally, enzymatically active intact BSDL was released at the basolateral membrane level. This is the first demonstration for an apical-to-basolateral transcytotic pathway of a secreted pancreatic digestive enzyme through polarized intestinal cells.

Effects of arachidonic and docosahexaenoic acids on secretion and degradation of bile salt-dependent lipase in AR4-2J cells.

In this study we demonstrated that two polyunsaturated fatty acids, arachidonic acid (AA, n-6) and docosahexaenoic acid (DHA, n-3), modulate the secretion of bile salt-dependent lipase (BSDL) by pancreatic AR4-2J cells. The effects of AA and DHA were also compared with that of the monounsaturated fatty acid, oleic acid (OA). Our results showed that the chronic treatment of cells with AA or DHA, that did not affect the biosynthesis rate of BSDL, similarly decreased the amount of secreted BSDL and perturbed the intracellular partitioning of the enzyme, whereas OA had no effect. Particularly, AA and DHA induced the retention of the enzyme in microsomes and lowered its content in the cell cytosol. We have further shown that AA treatment decreased the ubiquitination of the protein, and consequently diminished its export toward the cytosol, a result that might explain the retention of BSDL in microsomes and correlated with membrane phospholipids alteration. The retained protein was further degraded by a nonproteasomal pathway that likely involves ATP-dependent endoplasmic reticulum proteases. These findings concerning the regulation of the pancreatic BSDL secretion by two polyunsaturated acids, AA and DHA, might be of physiological importance in the plasmatic and cellular cholesterol homeostasis.

Preferential expression of reg I beta gene in human adult pancreas.

In human pancreas two genes, reg I alpha and reg I beta, have been characterized but only the reg I alpha protein has been isolated from human pancreatic secretion. To examine their respective physiological roles in fetal and adult pancreas we have compared the patterns of gene expression using a specific RT-PCR method. No progressive evolution in the two mRNAs levels was observed during fetal development (16--41 weeks). A discoordinate expression of the two genes was found with a higher level of reg I alpha mRNA in fetus and a higher level of regI beta in adult. In addition, if reg I alpha mRNA level was correlated with the expression of genes encoding exocrine proteins in adults, reg I beta mRNA level presented no correlation with any ductular, endocrine, or exocrine gene expression. In human pancreatic cell lines we showed the only expression of reg I beta gene and protein. All these data suggest that the two reg genes and proteins could play different roles in the pancreas.

Impairment of bile salt-dependent lipase secretion in AR4-2J rat pancreatic cells induces its degradation by the proteasome.

Bile salt-dependent lipase (BSDL, EC 3.1.1.13) is a lipolytic enzyme normally secreted by the pancreatic acinar cell. Co- and post-translational modifications, such as N- and O-linked glycosylation, regulate the secretion of this enzyme; therefore it was of first importance to determine the behaviour of BSDL under conditions that impaired its secretion. Using AR4-2J pancreatic cells as model, we showed, particularly when BSDL secretion is impaired, that proteasome inhibitors increased the amount of intracellular BSDL, suggesting that the proteasome is involved in the degradation of this protein. This was strengthened by the detection of ubiquitinated BSDL and of degradation product. Our results suggested that both ubiquitination and degradation of the enzyme occurred at the level of the cytosolic side of microsome membranes. ATP hydrolysis appears essential in ubiquitinated BSDL association with membranes and degradation. Furthermore, under normal secretory conditions, we have shown that a fraction of ubiquitinated BSDL is neither O-glycosylated nor N-glycosylated, suggesting that the N-glycosylation-deficient proteasome substrate does not reach the Golgi and could be degraded by the ER-associated degradation machinery. However, another fraction of ubiquitinated BSDL that is deficient in O-glycosylation, carries out endoglycosidase H-insensitive N-linked glycans, meaning that a second system, that detects abnormal BSDL molecules, could also operate at the level of the Golgi compartment. Consequently, it appears that impairment of BSDL secretion consecutive to secretion inhibition or to a deficient glycosylation leads to the proteasome-ubiquitin-dependent degradation of the protein. Therefore, this pathway is part of the quality control involved in BSDL secretion.

Peripheral auditory asymmetry in infantile autism.

Difficulty in filtering relevant auditory information in background noise is one of the features of autism. Auditory filtering processes can be investigated at the peripheral level as they are hypothesized to involve active cochlear mechanisms which are regulated by the efferent activity of the medial olivocochlear (MOC) system. The aim of the present work was therefore to assess these peripheral auditory processes in 22 children and adolescents with autism compared with age- and gender-matched normal controls. Active cochlear mechanisms were evaluated with transiently evoked otoacoustic emissions (TEOAEs) and MOC system efficiency was assessed via TEOAEs which are decreased when stimulating the contralateral ear with noise. The MOC system evaluation was performed on 18 of the 22 children. In both studies, results were analysed according to age (from 4 to 10 years and from 11 to 20 years). The main result concerns the asymmetry of the efferent system which differs in individuals with autism. Several neural processes might be hypothesized as involved in the results obtained as the MOC system which originates in the brainstem received regulating controls from upper brain structures including auditory cortex. Lateralization abnormalities at the auditory periphery may reflect indirectly a problem at a higher level of auditory processing. A second important result shows a decrease in TEOAE amplitude with age, in patients, that may correspond to a decrease in hearing sensitivity.

Control of pancreatic bile-salt-dependent-lipase secretion by the glucose-regulated protein of 94 kDa (Grp94).

Bile-salt-dependent lipase (BSDL; EC 3.1.1.13) is an enzyme expressed by the pancreatic acinar cell and secreted as a component of the pancreatic juice. During its route towards secretion, BSDL is associated with intracellular membranes by means of a multiprotein folding complex, which includes the glucose-regulated protein of 94 kDa (Grp94). We have postulated that the association of BSDL with membranes is required for the complete O-glycosylation of the protein, which diverts BSDL from a degradation route and consequently allows its secretion. To further characterize the role of Grp94 in BSDL secretion, we have studied the effect of a ribozyme specifically targeted to Grp94 mRNA. This ribozyme has been transfected into AR4-2J cells, and we have shown that a decrease in Grp94 expression leads to a concomitant decrease in BSDL secretion and expression. Geldanamycin (GA), which alters Grp94 functions, also affects the release of BSDL into the culture medium of AR4-2J cells. BSDL expressed in GA-treated AR4-2J cells is unstable. Furthermore, under conditions that decrease the level of BSDL secretion, no intracellular accumulation of the enzyme was observed, suggesting that BSDL that cannot associate with (or be structured by) Grp94 could be rapidly degraded. We have further shown that this degradation probably occurs via the ubiquitin-dependent pathway. Altogether, these results indicate that Grp94 has a pivotal role in BSDL folding and in the sorting of this pancreatic enzyme.

Maturation of frontal and temporal components of mismatch negativity (MMN) in children.

The mismatch negativity (MMN) response of auditory ERPs in adults appears to result from several overlapping components involving both frontal and temporal brain areas. Our aim was to test whether a similar configuration could be observed in children, and to examine the maturation rates of the different components. MMN (standard tones: 1000 Hz, deviants: 1100 Hz) was recorded from 28 scalp electrodes in 24 healthy children aged from 5 to 10 and in eight adults for comparison. Scalp current density analysis revealed both temporal and frontal components in children of all ages as well as in adults. Moreover the amplitudes of the temporal components were significantly greater in children than in adults, whereas the frontal components were similar at all ages. The results strongly suggest that MMN is mediated by at least two separate neural systems, and that the frontal system matures earlier than the sensory-specific system.

Roles of molecular chaperones in pancreatic secretion and their involvement in intestinal absorption.

This review focuses on the contribution of molecular chaperones in the secretory process of digestive enzymes and their interaction with enterocytes. By using biochemistry and immunocytochemistry, we have shown that Grp94, Cpn10, Cpn60, and protein disulfide isomerase (PDI) are present all along the rough endoplasmic reticulum-Golgi-granule secretory pathway of the pancreatic acinar cells and are secreted into the acinar lumen. Two other molecular chaperones, Grp78 and the Hsp70, appear to be restricted to the rough endoplasmic reticulum and the trans-Golgi apparatus, respectively. We have found that chaperones can be associated with pancreatic enzymes along the secretory pathway. Indeed, double immunogold and immunocoprecipitation revealed an association between Cpn60 and the colipase-dependent lipase (CDL) and between Grp94 and the bile salt-dependent lipase (BSDL). These complexes are secreted into the acinar lumen and diverted to the duodenal lumen. These findings led us to investigate these enzyme-chaperone complexes in intestinal tissue. Grp94, Cpn60, and PDI are present on microvilli and on the endosomal compartment of enterocytes. Furthermore, we have shown that the Grp94-BSDL complexes are internalized by enterocytes through classical endocytosis. Upon dissociation of the BSDL-Grp94 complex in the late endosome, BSDL is transferred to the basolateral membrane. We propose that Grp94 interacts with specific receptors and/or could force the associated protein to adopt a specific conformation that allows its binding to corresponding membrane receptors and its internalization by enterocytes. These two hypotheses need not to be exclusive. The existence of such a pancreatic secretion-intestinal absorption link speaks in favor of a coordinated functional connection between these two entities, through molecular chaperones, in order to optimize intestinal activities.

Opposite effects of GABA(A) and GABA(C) receptor antagonists on the b-wave of ERG recorded from the isolated rat retina.

The largest component in the fully dark-adapted ERG is a corneal-positive response, known as the b-wave, and believed to originate from depolarizing (ON-type) bipolar cells. The two types of GABA receptors, GABA(A) and GABA(C) have been reported to exist on bipolar cells in rat retina. The goal of these experiments was to find whether these GABA receptors participate in the generation of the b-wave of electroretinogram (ERG). ERGs were recorded from the isolated rat retinas. The P(2)(t) component, obtained by subtracting the ERGs measured before the application of 50 micrograms APB from those measured after the application of 50 micrograms APB, was used as an indicator of depolarizing bipolar cell activity. Photovoltages, the fast P(3)(t) component of ERG, were registered between the two microelectrodes across the rod outer segments. Bicuculline and 3-aminopropylphosphonic acid (3-APA) were used as selective antagonists of GABA(A) and GABA(C) receptors, respectively. It was found that the GABA(A) and GABA(C) receptors antagonists have opposite effects on the b-wave: bicuculline increased the b-wave amplitude, while 3-APA reduced the amplitude of the b-wave. Neither bicuculline nor 3-APA affect photoreceptors.

The affinity binding sites of pancreatic bile salt-dependent lipase in pancreatic and intestinal tissues.

In previous studies, we have shown that the bile salt-dependent lipase (BSDL) associates with the Grp94 molecular chaperone, an association that appears to play essential roles in the folding of BSDL. More recently, combined biochemical and immunocytochemical investigations were carried out to show that the transport of BSDL occurs via an association with the Grp94 all along the pancreatic secretory route (ER-Golgi-granules). The Grp94-BSDL complex is secreted with the pancreatic juice into the acinar lumen and reaches the duodenal lumen, where it is internalized by enterocytes. The dissociation of the complex could take place within the endosomal compartment because BSDL continues further on its way to the basolateral membrane of the enterocyte. To localize the affinity binding sites of pancreatic BSDL in pancreatic and duodenal tissues, we have used an affinity-gold ultrastructural technique. BSDL coupled to gold particles appears to interact with specific sites in tissue sections. This was confirmed by another indirect morphological approach using biotin-labeled BSDL and streptavidin-gold complexes on tissue sections. We have shown that BSDL associates with sites in the pancreatic secretory pathway compartments and in the microvilli, the endosomal compartment, and the basolateral membrane of enterocytes. By biochemical approaches, biotin-labeled BSDL displayed affinities with proteins of 180-190 kD in both pancreatic and duodenal tissues. We have also shown that the Grp94-BSDL complexes, which are insensitive to denaturing conditions, are present in pancreatic homogenate but not in duodenal lysate. Thus, BSDL is able to bind protein complexes formed by either BSDL-Grp94 or Grp94 dimers. (J Histochem Cytochem 48:267-276, 2000)

Partial rescue of the ocular retardation phenotype by genetic modifiers.

The or(J) allele of the murine ocular retardation mutation is caused by a premature stop codon in the homeodomain of the Chx10 gene. When expressed on an inbred 129/Sv strain, the or(J) phenotype is characterized by microphthalmia and a thin, poorly differentiated retina in which the peripheral portion is affected to a greater extent than the central portion. Such mutant retinae lack differentiated bipolar cells and the optic nerve typically fails to form, leading to blindness. Here, we show that progeny from an outcrossed backcross between 129/Sv-or(J) /or(J) and Mus musculus castaneus produce animals that are homozygous for the or(J) mutation and exhibit a much ameliorated eye phenotype. Although not of normal size, such modified or(J) eyes are significantly larger than those in 129/Sv-or(J) /or(J) mice, and contain a better organized retina which includes bipolar cells. Furthermore, optic nerves are frequently present, and the eyes show a degree of function as reflected by electroretinogram and pupillary response. As in 129/Sv-or(J) /or(J) mice, however, modified or(J) eyes show incomplete growth and a lack of cell differentiation in the periphery of the retina. The selective, and apparently nonmodifiable, effect of the ocular retardation phenotype on the periphery of the retina indicates that Chx10 plays an important role in the central-to-peripheral gradient of retinal development. These findings demonstrate that the ocular retardation phenotype can be greatly modified by the genetic background, and help to define a role for Chx10 in ocular development.

Auditory associative cortex dysfunction in children with autism: evidence from late auditory evoked potentials (N1 wave-T complex).

OBJECTIVES: Auditory processing at the cortical level was investigated with late auditory evoked potentials (N1 wave-T complex) in 4-8-year-old autistic children with mental retardation and compared to both age-matched normal and mentally retarded children (16 children in each group). METHODS: Two negative peaks which occurred in the 80-200 ms latency range were analyzed according to stimulus intensity level (50 to 80 dB SPL): the first culminated at fronto-central sites (N1b) and the second at bitemporal sites (N1c, equivalent to Tb of the T complex). The latter wave was the most prominent and reliable response in normal children at this age. RESULTS: Our results in autistic children indicated abnormalities of this wave with markedly smaller amplitude at bitemporal sites and pronounced peak latency delay (around 20 ms). Moreover, in both reference groups the intensity effect was found on both sides whereas in autistic children it was absent on the left side but present on the right. CONCLUSION: These findings in autistic children showing very disturbed verbal communication argue for dysfunction in brain areas involved in N1c generation i.e., the auditory associative cortex in the lateral part of the superior temporal gyrus, with more specific left side defects when auditory stimulus have to be processed.

A dissection of the electroretinogram from the isolated rat retina with microelectrodes and drugs.

The origins of the a- and b-wave of the ERG were studied using simultaneous recordings made across the receptor layer and the full thickness of a piece of isolated albino rat retina. An inwardly directed current flowing across the rod outer segments was eliminated from the recording when postsynaptic activity was blocked with cobalt or when current source density measurements were made along the length of the outer segments. Rod photovoltages were inferred by removing extraneous field potentials from the recordings made across the photoreceptor layer. The spatial properties of the photovoltage indicates the responses came from an area about 100 microm in diameter. The glutamate analog. APB, which blocks depolarizing bipolar cells, eliminated the b-wave but left the a-wave unaffected. The ERG component due to depolarizing bipolar cells was inferred by subtracting recordings obtained before and after APB. After treatment with APB a slow component remained. This component was completely blocked by barium (200 microM), which blocks potassium channels on Müller cells. Barium had virtually no effect on low-intensity photovoltages but did affect the amplitude and shape of the saturated responses. Barium increased the amplitude of the component of the ERG which underlies the b-wave. It was concluded that the depolarizing bipolar cells directly generate the b-wave of the ERG.