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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Alterations of visual and auditory evoked potentials in fragile X syndrome
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Inga Sophia Knoth a,b,∗ , Phetsamone Vannasing a , Philippe Major a , Jacques L. Michaud a , Sarah Lippé a,b a b
Research Center of the CHU Ste-Justine Mother and Child University Hospital Center, University of Montreal, Quebec, Canada Centre de Recherche en Neuropsychologie et Cognition, University of Montreal, Quebec, Canada
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Article history: Received 10 February 2014 Received in revised form 30 April 2014 Accepted 14 May 2014
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Keywords: Fragile X syndrome Intellectual disability Autism Sensory information processing Auditory evoked potential Visual evoked potential
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1. Background
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Background: Fragile X Syndrome (FXS) is the most common monogenic form of intellectual disability and one of the few known monogenic causes of autism. It is caused by a trinucleotide repeat expansion in the FMR1 (‘Fragile X Mental Retardation 1’) gene, which prevents expression of the ‘Fragile X Mental Retardation Protein’ (FMRP). In FXS, the absence of FMRP leads to altered structural and functional development of the synapse, while preventing activity-based synapse maturation and synaptic pruning, which are essential for normal brain development and cognitive development. Possible impairments in information processing can be non-invasively investigated using electrophysiology. Methods: We compared auditory (AEP) and visual (VEP) evoked potentials in twelve adolescents and young adults (10–22 years) affected by FXS to healthy controls matched by chronological age (N = 12) and developmental age of cognitive functioning (N = 9; 5–7 years), using analysis of variance. Results: In the visual modality, the N70 and N2 amplitude have been found increased in FXS in comparison to the chronological, but not the developmental control group at occipital sites, whereas in the auditory modality N1, P2 and N2 amplitude as well as N2 latency have been found increased in FXS, relative to both chronological and developmental control groups at mid-central sites. Conclusions: The AEP/VEP profile suggests disruptions in sensory processing specific to FXS that exceed immaturity of physiological activity. In addition, the auditory modality seems to be more affected than the visual modality. Results are discussed in light of possible underlying neuronal mechanisms, including deficits in synaptic pruning and neuronal inhibition that might account for a hyperreactive nervous system in FXS. © 2014 Published by Elsevier Ltd. on behalf of ISDN.
Fragile X Syndrome (FXS) is the most common monogenic form of intellectual disability (ID) and affects about 2% of male patients with ID (Ropers and Hamel, 2005). It is caused by a trinucleotide repeat expansion in the FMR1 (‘Fragile X Mental Retardation 1’) gene, which is located on the X-chromosome. Women can also be affected but the penetrance of the mutation is reduced and its expressivity more variable in them (Bennetto et al., 2001). The FMR1 mutation prevents expression of the ‘Fragile X Mental
∗ Corresponding author at: Research Center of the CHU Ste-Justine Mother and Child University Hospital Center, University of Montreal, 3175 Chemin de la CôteSainte-Catherine, Montreal, QC H3T 1C5, Canada. Tel.: +1 514 345 4931x4590. E-mail addresses: [email protected], [email protected] (I.S. Knoth).
Retardation Protein’ (FMRP), which is known to repress the translation of specific mRNAs in response to the activation of metabotropic Glutamate Receptors (mGluRs) (Bear et al., 2004). FMRP directly targets approximately 5% of all mRNAs (Darnell and Klann, 2013). However, changes in protein synthesis observed in the absence of FMRP affect about 20% of pre-synaptic protein synthesis (Darnell and Klann, 2013). Thus, secondary alterations are believed to additionally account for the changes in protein synthesis observed in the absence of FMRP (Darnell and Klann, 2013). Alterations in protein synthesis result in a loss of synaptic plasticity in FXS (Bassell and Warren, 2008). Structurally, dendritic spines are increased in number and appear elongated whereas synapses appear immature in FXS patients and fragile X knockout mice (Comery et al., 1997). Thus, the absence of FMRP is likely to prevent activity-based synapse maturation and synaptic pruning, which are essential for normal brain development (Weiler and Greenough, 1999) and cognitive development (Schneider et al., 2009).
http://dx.doi.org/10.1016/j.ijdevneu.2014.05.003 0736-5748/© 2014 Published by Elsevier Ltd. on behalf of ISDN.
Please cite this article in press as: Knoth, I.S., et al., Alterations of visual and auditory evoked potentials in fragile X syndrome. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.05.003
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2 Table 1 Demographics of the study population.
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Variable
FXS patients
Chronological age matched controls
Developmental age matched controls
N Age range Mean age (SD) IQ range Mean IQ (SD)
12, 4 ♀ 10–22 years 14.7 (3.75) 32–93 51 (±16.57)
12, 3 ♀ 11–32 years 16.9 (±6.02) 87–129 113 (±14.05)
9, 3 ♀ 5–7 years 5.8 (±0.83) 97–118 108 (±7.25)
Patients affected by FXS frequently show deficits in language, executive functions, visuo-spatial and social cognition. Further, they tend to show aberrant behavior, emotional instability and hyperarousal to sensory stimulation (Schneider et al., 2009). Most of the symptoms found in FXS are typical of the autistic spectrum; about 30% of male individuals with FXS meet the full diagnostic criteria for autism. FXS is thus considered one of the few known monogenic causes of autism (Rogers et al., 2001). Further, reduction of FMRP levels have been found in the cerebrellar vermis of adult subjects with autism who were not diagnosed with FXS, suggesting that common neurobiological mechanisms might account for the shared symptoms between non-syndromic autism and FXS (Fatemi et al., 2011). However, autistic symptoms vary considerably in their intensity between patients affected by FXS (Schneider et al., 2009). Disrupted pathways in synaptic plasticity, the potential link between the FMR1 mutation and the learning disability often found in FXS, are likely to be associated with impairments in mechanisms of information processing (Belmonte and Bourgeron, 2006). Early sensory processing can be non-invasively investigated using the AEP/VEP technique that records local field potentials, which are summarized postsynaptic potentials from large groups of neurons (Luck, 2005). Studies investigating AEP/VEPs in FXS so far exclusively used oddball paradigms and mostly studied AEPs (St Clair et al., 1987; Castrèn et al., 2003; Van Der Molen et al., 2012a,b). The most consistent findings were enhanced N1 and decreased P3 amplitudes, as well as prolonged N2 latencies in FXS compared to healthy age-matched controls, whereas findings concerning other components tended to be more variable. Thus, some AEPs appeared to be specifically altered in FXS, revealing disruptions in early sensory processing. In this study we aim to investigate to which extent the altered AEP/VEPs in FXS can be explained by immature as opposed to otherwise disrupted sensory processing. Since parameters of AEP/VEPs specifically change with brain development (Lippé et al., 2007, 2009), the altered AEP/VEPs in FXS might reflect immature physiological activity due to deficits in synaptic pruning. In this case, the AEP/VEPs in FXS would resemble those of individuals on the same level of cognitive functioning. However, given that the absence of FMRP has been found to interfere with functional and structural brain development, we hypothesize that the disruptions in sensory processing reflected by AEP/VEPs exceed immaturity. In order to distinguish between immaturity and specific alterations of the AEP/VEPs, we compared the FXS patients to an additional control group matched to the developmental age of cognitive functioning, assessed by Intelligence Quotient (IQ). Further, we aimed to investigate differences in the extent of impairments in sensory processing between auditory and visual modality in FXS. The only previous study investigating VEPs in FXS found the auditory modality to be more affected than the visual modality (Van Der Molen et al., 2012a), which matches modality differences in performance found in FXS (Schneider et al., 2009; Van Der Molen et al., 2010). This indicates that FMRP absence might affect sensory processing differently depending on modality. We hypothesize that the VEPs appear less altered in FXS than the AEPs. We thereby examine whether the extent to which the AEP/VEP
alterations can be explained by physiological immaturity varies between modalities.
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2. Methods
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2.1. Participants
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Twelve FXS patients aged from 10 to 22 years diagnosed with full mutation of the FMR1 gene were compared to 21 healthy controls matched by chronological age or developmental age and gender (Table 1). The developmental control group contains children whose chronologic age matches the developmental age of patients with intellectual disability (IQ < 70). Note that not all patients meet the criteria for intellectual disability. A total of 18 FXS patients had been tested; six patients were excluded from data analysis due to epileptic activity, difficulties in testing and extensive movement artifacts. Patients were recruited on the basis of DNA analysis previously conducted by geneticists at the CHU Sainte-Justine Mother and Child University Hospital Center in Montreal. Healthy controls were recruited at the Ste-Justine Hospital, the University of Montreal, kindergartens and summer day camps. Four of the twelve FXS patients had also been diagnosed with autistic disorder; eight FXS patients showed language delay and nine FXS patients were also diagnosed with Attention Deficit Hyperactivity Disorder (ADHD). Five of the tested patients did not take any medication, while seven patients were medicated with psychostimulant (5× methylphenidate, 2× atomoxetine, 1× amphetamine mixed salts) and/or antidepressant (1× citalopram) drugs to treat symptoms of autism, attention deficit hyperactivity disorder, depression and anxiety. All patients underwent detailed physical examinations in the developmental clinic of the hospital following their diagnosis. None of the patients has been diagnosed with hearing deficits within the scope of these evaluations. Parents reported normal hearing and normal or corrected-to-normal vision in all patients and control participants upon specific request. Healthy controls had no history of brain injuries, psychiatric or neurological illnesses and did not take any medication. All participants were born at term and right-handed. Intelligence in patients and controls was examined using the completely non-verbal Leiter-R International Performance Scale (Roid and Miller, 1997) for children and adolescents and the Wechsler Abbreviated Scale of Intelligence (Wechsler, 1999) for adults. The nonverbal scale was chosen in order to reduce the impact of language deficits in patients on the global IQ result. Developmental age of patients was calculated on the basis of IQ in order to match them with healthy controls. Autistic behavior was quantified using the repetitive behavior questionnaire (Lam and Aman, 2007) and the aberrant behavior checklist (Aman et al., 1985), which were completed by parents of patients and minor control participants. The study protocol was reviewed and approved by the ethics, administrative, and scientific committees at the Ste-Justine’s Hospital Research Center. Informed consent was obtained before the experiment from participants and parents or legal caregivers following a full explanation of the procedures undertaken.
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2.2. Apparatus and stimuli
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Auditory and visual stimuli were generated by a Dell GX150 PC using E-Prime 1.0 (Psychology Software Tools Inc., Pittsburgh, PA, USA). The EEG recording took place in a dark soundproof experimental chamber. Auditory stimulation consisted of 50 ms broadband noise presented 150 times in a randomly distributed interstimulus interval varying from 1200 to 1400 ms at 79 dB SPL intensity and 16-bit resolution as in a developmental study previously conducted in our laboratory (Lippé et al., 2009). The two speakers (Optimus XTS 24, Boston, MA, USA) were located laterally at 30 cm distance from the subject’s ears. During auditory stimulation all subjects watched a silent movie. Following this, visual stimulation consisted of a black and white checkerboard stimulus presented at a reversal rate of 1 Hz, meaning that the checkerboard changed every 500 ms, and subtending a visual angle of 2◦ . The original and reversed checkerboard stimulus were presented 200 times each. Stimuli had a luminance of 40 cd/m2 and were displayed on a 40.5 cm × 30.5 cm ViewSonic monitor (ViewSonic, Canada) at 114 cm distance from the participant’s eyes. This visual paradigm has been created for a developmental study previously conducted in our laboratory (Lippé et al., 2007). An assistant observed whether the participant looked at the screen at all times and gave a signal whenever the participant looked elsewhere, in order to exclude these EEG segments from analyses. The assistant likewise directed the attention of participants to the screen by holding small objects in the lower middle part of the screen and talking to them if necessary.
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Fig. 1. t-Test brain topographies comparing auditory N1, P2 and N2 amplitude between FXS patients and chronological/developmental controls. t-Values 3.1 were considered for defining ROIs.
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A dense array EEG system containing 128 electrodes was used for recording (Electrical Geodesics System Inc., Eugene, OR, USA). The vertex was used as the reference electrode during recording and impedances were maintained below 40 k (Tucker, 1993). Signals were acquired and processed by a G4 Macintosh computer using NetStation EEG Software (Version 2.0). Digitalization of EEG data was carried out at a sampling rate of 250 Hz in 1024 ms epochs and an analog 0.01–100 Hz bandpass filter was applied. Off-line analyses were carried out with BrainVision Analyser software, version 2.0 (Brain Products, Munich, Germany). Data were digitally filtered with a 1–50 Hz filter for the visual and a 1–30 Hz filter for the auditory experiment and re-referenced to an average reference. Eye movement artifacts were corrected using semi-automatic Ocular Correction ICA as implemented in BrainVision Analyser. Algorithmic artifact rejection of voltage exceeding ±100 V was followed by visual data inspection of segmented data in which segments with artifacts were manually rejected.
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2.3. Auditory and visual evoked potential analysis
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Artifact-free segments were averaged and baseline corrected. In the auditory paradigm, an average of 146 artifact-free segments were available for the patient group and an average of 149 segments for the chronological and developmental control group. In the visual paradigm, an average of 191 segments were available for patients, 198 for chronological controls and 191 for developmental controls.
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Number of presented segments did not differ between the three groups (auditory condition: (F(2,30) = 1.75, p = 0.19), visual condition: (F(2,30) = 7.7, p = 0.08)). In order to define regions of interest (ROI) for statistical comparison between groups, BrainVision-assisted t-tests were computed on amplitude topographies during the three time windows showing maximal differences between the responses evoked in FXS compared to the two control groups (Figs. 1 and 2). Note that the potentials with opposing polarity to the AEPs/VEPs observed temporally in the auditory condition and frontally in the visual condition reflect opposing charges of the same dipole source generated by AEPs/VEPs primary ROI (central for the auditory condition and occipital for the visual condition) (Scherg, 1990; Di Russo et al., 2002). Results from dipolar ROIs are reported, but interpreted as the same activity as the primary ROIs. Electrodes within the selected ROI were averaged and thereupon components were individually selected for each subject in each associated ROI. For lateral regions, a region on the opposing hemisphere was defined as well. Parameters of all defined ROI are given in Table 2. Amplitudes were defined from baseline (200 ms prestimulus) to the highest amplitude of each component and latencies were defined from stimulus onset to the highest point of each component.
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2.4. Statistical analysis
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Statistical analyses were performed using SPSS statistics, version 20 (IBM Corp., Armonk, NY, USA). Firstly, we compared the following variables between male and female FXS patients: IQ; N1, N2 and P2 amplitudes and latencies at the corresponding
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Table 2 Parameters of defined ROI for every component. Auditory condition Component
N1
P2
N2
ROI (number of electrodes included)
Mid-central (6) Centro-parietal left (7) Centro-parietal right (7) Temporal left (6) Temporal right (6)
Mid-central (6) Temporal left (6) Temporal right (6)
Mid-central (6)
Visual condition Component ROI (number of electrodes included)
N70
P100
N2
Centro-occipital (7) Parietal left (5) Parietal right (5) Frontal left (4) Frontal right (4)
Temporal left (4) Temporal right (4) Frontal left (4) Frontal right (4)
Centro-occipital (6) Parietal left (7) Parietal right (7) Fronto-central (8) Frontal left (4) Frontal right (4)
Please cite this article in press as: Knoth, I.S., et al., Alterations of visual and auditory evoked potentials in fragile X syndrome. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.05.003
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Fig. 2. t-Test brain topographies comparing visual N70, P1 and N2 amplitude between FXS patients and chronological/developmental controls. t-Values 3.1 were considered for defining ROIs.
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ROI for AEPs and N70, P100 and N2 amplitudes and latencies at the corresponding ROI for VEPs. Since female patients are known to be less affected, due to their intact X-chromosome, which mitigates the outcome of the FMR1 mutation, we verified whether male and female patients differed from each other in respect of ERPs or can be considered as a single group. Student’s t-test has been used and log transformations were applied to non-normally distributed data. Analysis was then carried out between FXS patients, chronological and developmental age matched controls for the variables listed above using analysis of variance (ANOVA). For the evoked potentials, a mixed-design ANOVA (group × ROI) using Greenhouse–Geisser correction has been carried out. If significant interactions between group and ROI were found, one-way ANOVA was carried out for every ROI separately. If significant differences between groups were detected, Tukey’s test was carried out for post hoc analysis. Effect sizes are provided for significant ANOVA results. Data obtained from the abnormal behavior questionnaire and the repetitive behavior scale were only compared between FXS patients and the chronological control group, since most of the items cover behavior that is perceived as normal in pre-school children. Significance level for all statistical tests was set to 5% (˛ = 0.05).
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3. Results
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3.1. Gender, medication and autism
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developmental age matched control group (p = 0.001), while both control groups do not significantly differ from each other. Table 3 shows mean values of aberrant and repetitive behavior in FXS patients and chronological controls as reported by parents/care givers in the aberrant behavior questionnaire and the repetitive behavior scale. 3.3. AEPs Group averages of the AEP responses in mid-central sites are presented in Fig. 3. Table 4 shows mean amplitudes and latencies at electrode FCz and Cz for every group.
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With a mean of 68.25 (±17.11), female patients had higher IQs than male patients (M = 43.38, ±8.38) (p = 0.006). However, no significant differences between male and female FXS patients were found in amplitude or latency of VEP components N70, P100 and N2, as well as in the AEP components N1, P2 and N2 in all ROI (p > 0.05). Further, IQ, as well as amplitude and latency of AEP/VEP components did neither differ significantly between medicated and non-medicated patients, nor between patients that were and were not diagnosed with autism (p > 0.05). Thus, all FXS patients were combined into one group for analysis.
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3.2. IQ, aberrant and repetitive behavior
3.3.1. Auditory N1 Even though a significant group × ROI interaction has been found in N1 latency (F(5,76) = 2.29, p = 0.054, R2 = 0.15), subsequent one-way ANOVAs did not detect a significant difference between the three groups in any ROI. A group × ROI interaction has been found for N1 amplitude (F(4,56) = 10, p = 0.001, R2 = 0.67). In subsequent one-way ANOVAs, a significant difference between groups could be found at centro-parietal left (F(2,30) = 14.32, p = 0.001, R2 = 0.96), centro-parietal right (F(2,30) = 12.37, p = 0.001, R2 = 0.82), mid-central (F(2,30) = 12.77, p = 0.001, R2 = 0.85) and temporal left (F(2,30) = 12.22, p = 0.001, R2 = 0.82) sites. Post hoc analysis revealed that FXS patients had a higher N1 amplitude than both control groups at centro-parietal left (p = 0.001 for both control groups), centro-parietal right (p = 0.004 for chronological and p = 0.001 for developmental controls) and mid-central (p = 0.015 for chronological and p = 0.001 for developmental controls) sites. At the temporal left site, developmental controls showed a smaller amplitude than FXS patients (p = 0.001) and chronological controls (p = 0.019).
One-way ANOVA shows a difference in IQ between FXS patients, chronological and developmental age matched controls (F(2,28) = 67.12, p = 0.00, R2 = 0.83). FXS patients differ from the chronologic age matched control group (p = 0.001) and the
3.3.2. Auditory P2 No significant group × ROI interaction has been found for P2 latency. Concerning P2 amplitude, a significant interaction was found (F(3,46) = 14.28, p = 0.001, R2 = 0.95). Subsequent one-way
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Please cite this article in press as: Knoth, I.S., et al., Alterations of visual and auditory evoked potentials in fragile X syndrome. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.05.003
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Table 3 Mean values (SD) of abnormal and repetitive behavior in participants as reported by their parents/caregivers. Scale
FXS patients
Chronological controls
p-Value
Irritability Lethargy Stereotypical Behavior Hyperactivity Inappropriate Speech Self Mutilation Compulsive Behavior Ritualized Behavior Immutable Behavior Restrictive Behavior
4 (4.54)* 4.13 (5.11)* 1.75 (3.24) 3.35 (4.34)* 3.25 (3.45)* 1.78 (2.22)* 1.78 (2.49) 1.89 (1.96)* 4.22 (4.92)* 1.22 (1.64)*
0.78 (1.09) 0.33 (0.52) 0 (0.00) 1.52 (0.51) 0.33 (0.52) 0.17 (0.41) 0.33 (0.82) 0 (0.00) 0.17 (0.41) 0 (0.00)
0.04 0.04 0.08 0.02 0.02 0.03 0.06 0.01 0.02 0.03
*
Significant difference between FXS and chronological control group (p < 0.05).
Fig. 3. Group averages of AEPs at mid-central ROI for the FXS group and the two control groups. 0 ms marks stimulus onset.
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ANOVAs showed difference between groups at mid-central (F(2,30) = 18.73, p = 0.001, R2 = 1.25), temporal left (F(2,30) = 6.8, p = 0.004, R2 = 0.45) and temporal right (F(2,30) = 10.17, p = 0.001, R2 = 0.68) sites. According to post hoc analysis, P2 amplitude was higher in FXS compared to both control groups at mid-central sites (p = 0.001 for both control groups) and higher in FXS compared to the chronological control group at temporal left (p = 0.003) and temporal right (p = 0.001) sites.
3.3.3. Auditory N2 Since only one ROI of interest was defined for auditory N2, one-way ANOVA was immediately performed for amplitude and latency. A difference between groups could be found for latency (F(2,30) = 8.32, p = 0.001, R2 = 0.55) and amplitude (F(2,30) = 13.23, p = 0.001, R2 = 0.88). Post hoc analysis revealed that latency was longer in FXS compared to both control groups (p = 0.019 for chronological and p = 0.001 for developmental controls). Amplitude was found to be higher in FXS compared to both control
groups (p = 0.001 for chronological and p = 0.002 for developmental controls). 3.4. VEPs
Parameter
FXS patients
N1
Amplitude Latency
−1.79 (1.16)* , T 97.33 (15.3)
P2
Amplitude Latency
4.9 (1.8)* , T 181.33 (28.5)
N2
Amplitude Latency
−2.3 (0.84)* , T – 321.67 (35.92)* , T
*
–
T
–
–
–
294
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Mean amplitudes and latencies at occipito-central sites for every subgroup are presented in Table 5. Fig. 4 shows the group averages of the VEP response at electrode Oz. 3.4.1. Visual N70 No significant group × ROI interaction was found for N70 latency. As for amplitude, a group × ROI interaction was found (F(4,55) = 3.77, p = 0.001, R2 = 0.25). Subsequent one-way ANOVAs revealed difference between groups at occipito-central (F(2,30) = 3.79, p = 0.034, R2 = 0.25), left parietal (F(2,30) = 7.36, p = 0.033, R2 = 0.49) and frontal right (F(2,30) = 7.08, p = 0.003, R2 = 0.47) sites. Amplitude was found higher in FXS compared to chronological controls in occipito-central (p = 0.026) and left
Table 4 Mean amplitudes in V and latencies in ms (SD) for AEP components in FXS patients, chronological control group and developmental control group at mid-central sites. Component
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Chronological control group
Developmental control group
−0.62 (0.73) 90 (12.8)
0.68 (1.08) 90 (34)
1.67 (0.97) 170 (22.79)
2.49 (0.97) 158.22 45.92)
−0.89 (0.60) 277.67 (26.88)
−1.08 (0.69) 257.33 (49.92)
Significant difference between FXS and chronological control group (p < 0.05). Significant difference between FXS and developmental control group (p < 0.05).
Please cite this article in press as: Knoth, I.S., et al., Alterations of visual and auditory evoked potentials in fragile X syndrome. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.05.003
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Table 5 Mean amplitudes in V and latencies in ms (SD) at occipito-central sites for VEP components in FXS patients, chronological control group and developmental control group. Component
Parameter
FXS patients
Chronological control group
Developmental control group
N70
Amplitude Latency
−2.11 (1.33)* 83 (21.47)
−0.81 (0.58) 80 (7.03)
−1.54 (1.44) 76.89 (7.15)
P100
Amplitude Latency
4.08 (3.11) 122 (31.19)
3.29 (2.01) 118.67 (16.12)
3.88 (1.7) 114.22 (4.06)
N2
Amplitude Latency
−1.29 (0.83)* 319.33 (35.44)
−0.37 (0.48) 289 (39.67)
−1.04 (0.99) 288.44 (54.24)
*
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Significant difference between FXS and chronological control group (p < 0.05).
parietal (p = 0.002) sites. At the right frontal ROI, amplitude was found higher in FXs compared to both control groups (p = 0.003 for chronological and p = 0.033 for developmental controls). 3.4.2. Visual P100 No significant group × ROI interaction was found for P100 latency. P100 amplitude showed a significant group × interaction (F(3,50) = 3.97, p = 0.001, R2 = 0.27). Subsequent one-way ANOVAs showed group differences at right temporal (F(2,30) = 4.54, p = 0.019, R2 = 0.3) and left frontal (F(2,30) = 5.3, p = 0.011, R2 = 0.36) sites. P100 amplitude was found to be higher in developmental controls than in FXS patients at right temporal (p = 0.018) and left frontal (p = 0.013) sites. 3.4.3. Visual N2 No significant group × ROI interaction was found for visual N2 latency. As for amplitude, the interaction was found to be significant (F(3,49) = 5.94, p = 0.001, R2 = 0.4). Subsequent one-way ANOVAs showed group differences in left parietal (F(2,30) = 3.52, p = 0.042, R2 = 0.23), right parietal (F(2,30) = 4.8, p = 0.016, R2 = 0.32), occipito-central (F(2,30) = 4.47, p = 0.020, R2 = 0.3), fronto-central (F(2,30) = 7.6, p = 0.002, R2 = 0.5), left frontal (F(2,30) = 8.9, p = 0.001, R2 = 0.59) and right frontal (F(2,30) = 4.25, p = 0.024, R2 = 0.28) sites. A higher amplitude in FXS compared to the chronological control group has been found in left parietal (p = 0.035), right parietal (p = 0.012), occipito-central (p = 0.018), left frontal (p = 0.001) and right frontal (p = 0.018) sites. N2 amplitude at fronto-central sites was found to be higher in FXS compared to both control groups (p = 0.002 for chronological and p = 0.032 for developmental controls).
4. Discussion FXS patients showed alterations in AEP/VEPs compared to healthy aged-matched controls. However, we found that auditory processing is more impaired than visual processing in FXS patients. Furthermore, AEPs in FXS do not only alter in comparison to age-matched healthy peers, but also compared to children with the same developmental age of intellectual functioning. Auditory information processing in FXS patients does thus not resemble immature information processing, but has its own particularities. Visual information processing however appears immature in FXS. A summary of ERP alterations in FXS found in our study is given in Tables 6 and 7. Specifically, an increased auditory N1 amplitude and no difference in N1 latency have been found in our patient population, replicating the findings of relevant AEP studies conducted with FXS patients so far (St Clair et al., 1987; Castrèn et al., 2003; Van Der Molen et al., 2012a,b). We also found an increased visual N70 amplitude in FXS, which differs at right frontal sites from both control groups, whereas it differs in centro-occipital and right temporal sites only from the chronological control group, while no difference in latency was found. Increased N1 amplitude seems to be somewhat specific to FXS when compared to other forms of ID and autism that often present a difference in N1 latency and no difference (or even a reduction) in N1 amplitude (Knoth and Lippé, 2012). A higher N1 amplitude in FXS suggests that more neurons are synchronously active in response to the sensory stimulation in FXS than in healthy controls (Rojas et al., 2001). This could mean that FXS patients have a surplus of neurons in brain regions that are involved in N70/N1 generation. Alternatively, neuronal activation could be less inhibited in FXS than in healthy controls.
Fig. 4. Group averages of VEPs at occipito-central ROI for the FXS group and the two control groups. 0 ms marks stimulus onset.
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Amplitude
Latency
N1
Increased in FXS compared to both control groups at left, right and mid-central sites
No difference
P2
N2
Increased in FXS compared to both control groups at mid-central sites Increased in FXS compared to chronological controls at right and left temporal sites Increased in FXS compared to both control groups at mid-central sites
No difference
Increased in FXS compared to both control groups at mid-central sites
Table 7 Alterations of VEPs in FXS obtained in this study. Component N70
P100
N2
366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396
Amplitude Increased in FXS compared to chronological controls in occipito-central and left parietal sites Increased in FXS compared to both control groups at opposing dipolar potential (right frontal ROI) Decreased in FXS compared to developmental controls at right temporal and left frontal sites Increased in FXS compared to chronological controls at left frontal, right frontal, left parietal, right parietal and occipito-central sites Increased in FXS compared to both control groups at opposing dipolar potential (centro-frontal ROI)
Latency No difference
No difference
No difference
The first theory, a surplus of neurons, is supported by neuroanatomical aberrations in FXS. Among other alterations, greater gray matter volume in the occipital cortex has been found in infants with FXS compared to normally developed children and children with non-syndromic delay (Hoeft et al., 2008). The occipital cortex contains most of the visual cortex, which is believed to be involved in visual N70 generation (Shigeto et al., 1998). In contrast, the superior temporal gyrus, which contains the primary auditory cortex and is believed to be involved in auditory N1 generation (Näätänen and Picton, 1987), is found to be smaller in FXS, relative to healthy controls (Gothelf et al., 2008). However, FXS children between the age of 1 and 3 years showed a greater gray matter increase relative to chronologically and developmentally age matched controls over the course of 2 years, until the age of 3–5 years, in several brain structures, including not only temporaloccipital regions, but also the superior temporal gyrus. This could indicate deficient synaptic pruning (Hoeft et al., 2010) in areas that are believed to be involved in generation of auditory N1 and visual N70. Synaptic pruning, a neuroregulatory process in which unnecessary neurons and synapses are reduced in order to strengthen more efficient neuronal configurations, might be impaired in FXS, according to animal model studies (Weiler and Greenough, 1999; Pfeiffer and Huber, 2007, 2009). In consequence, more redundant neurons, which have not been eliminated through synaptic pruning, would respond to sensory stimulation in a sort of non-specific arousal response, accounting for the increased auditory N1 and visual N70 amplitude. This finds additional support in studies that investigated auditory N1 habituation in FXS (Castrèn et al., 2003; Van Der Molen et al., 2012b). In both studies, controls showed a reduction of N1 amplitude after several presentations of the same stimulus, while
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this N1 habituation did not occur in FXS. The reduction of N1 amplitude in controls suggests that less neurons synchronously respond to a stimulus after several repetitions, which could reflect a reduction of the non-specific arousal or novelty response that generally occurs after the first appearance of a sensory stimulus (Karhu et al., 1997). Thus, deficient synaptic pruning might lead to an excess of less adapted synaptic configurations that non-specifically respond to sensory stimulation, while reducing the capacity for more efficient synaptic connections. This mechanism might be reflected in increased auditory N1 amplitude and a lack of N1 habituation. The second theory, impaired neuronal inhibition as an explanation for increased N70/N1 amplitudes, finds support in a study that investigated resting state EEG in FXS (Van Der Molen and Van Der Molen, 2013). Van der Molen et al. found an increased relative theta power and a decreased relative upper alpha in FXS, compared to healthy controls. Alpha oscillations in EEG are believed to be involved in neural inhibitory regulation mechanisms that gate information by reducing the processing capabilities of a given area (Jensen and Mazaheri, 2010). A reduced alpha rhythm in FXS could indicate impaired sensory gating mechanisms, which fail to inhibit redundant neural activity. In consequence, there would be more neuronal activation, which might be reflected in increased N1 amplitude. Lastly, we investigated auditory and visual basic N2 in FXS. In the visual modality, no difference in N2 latency was found, which differs from the results reported by Van der Molen and colleagues, who found an increased visual N2 latency in FXS, compared to chronological controls (Van Der Molen et al., 2012a). However, since the mean latency of our developmental group lies between FXS patients and chronological controls, the difference between FXS patients and chronological controls would have been significant in a direct comparison. Further, visual N2 amplitude was only found to be larger in FXS compared to the chronological control group in most ROI and only differed from the developmental control group at fronto-central sites. Since N2 amplitude is known to decrease with age (Lippé et al., 2007), it can be argued that the visual N2 is rather immature in FXS than specifically altered. Moreover, prolonged N2 latencies seem to be a general phenomenon in ID (Knoth and Lippé, 2012). Even though our study provides exciting evidence for information processing deficits in FXS, there are two limitations that should be mentioned. Firstly, the sample size can be considered as rather small. In addition to the general difficulty of finding patients with a rare disease, such as FXS, hyperactive and impulsive behavior, as well as social anxiety in many patients posed another problem, since EEG recordings require calmness and AEP/VEP cannot be studied in sedated patients. Secondly, it is an issue that more than half of our patient population was medicated during the EEG recording with psychostimulant and/or other drugs. As psychoactive drugs are known to have an influence on the parameters of AEP/VEPs, medication might have been a confounding variable in our study. However, statistical testing did not reveal significant differences in amplitude or latency of the investigated ERP components between medicated and non-medicated patients. Further, psychoactive drugs are more likely to reverse the effects of the treated pathology on the evoked potential profile so that it no longer differs from that of controls (Broyd et al., 2005). Thus, it seems unlikely that the altered ERP profile, which we detected in our patients, reflects drug effects rather than the FXS pathology.
5. Conclusions The present study presents a profile of altered AEP/VEPs in FXS, which likely reflects impairments in basic neural sensory processing. The additional comparison to a control group matched by
Please cite this article in press as: Knoth, I.S., et al., Alterations of visual and auditory evoked potentials in fragile X syndrome. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.05.003
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developmental age of cognitive functioning of patients with ID leads to the conclusion that AEP components in FXS are not simply immature, but appear specifically altered. Conversely, the visual N70 and N2 amplitudes do not differ between FXS patients and developmental controls at occipito-central sites. Notably, information processing seems to be more severely impaired in the auditory than in the visual modality. The knowledge that visual processing is less affected, as well as indications of a hyperreactive nervous system, should be considered in the design of behavioral treatments for FXS patients.
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Competing interests
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The authors declare that they have no competing interests. Authors’ contributions
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ISK participated in the study design, coordinated the study, carried out recruitment of patients and controls, EEG recording, AEP/VEP and statistical analysis and drafted the manuscript. PV participated in EEG recordings and AEP/VEP analysis. JM participated in the DNA analysis and in the study design. PM examined EEGs for epileptic activity. SL conceived the study, participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.
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Acknowledgements
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This research was supported by a Scottish Rite Charitable Foundation of Canada grant to Sarah Lippé. We thank Domitille Malfait for carrying out the neuropsychological evaluation of most of the patients and Patricia Laniel and Maude Joannette for their help in the acquisition of EEG data. We thank language editor J. Arthur White for significantly revising the manuscript.
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Alterations of visual and auditory evoked potentials in fragile X syndrome
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Inga Sophia Knoth a,b,∗ , Phetsamone Vannasing a , Philippe Major a , Jacques L. Michaud a , Sarah Lippé a,b a b
Research Center of the CHU Ste-Justine Mother and Child University Hospital Center, University of Montreal, Quebec, Canada Centre de Recherche en Neuropsychologie et Cognition, University of Montreal, Quebec, Canada
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Article history: Received 10 February 2014 Received in revised form 30 April 2014 Accepted 14 May 2014
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Keywords: Fragile X syndrome Intellectual disability Autism Sensory information processing Auditory evoked potential Visual evoked potential
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1. Background
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Background: Fragile X Syndrome (FXS) is the most common monogenic form of intellectual disability and one of the few known monogenic causes of autism. It is caused by a trinucleotide repeat expansion in the FMR1 (‘Fragile X Mental Retardation 1’) gene, which prevents expression of the ‘Fragile X Mental Retardation Protein’ (FMRP). In FXS, the absence of FMRP leads to altered structural and functional development of the synapse, while preventing activity-based synapse maturation and synaptic pruning, which are essential for normal brain development and cognitive development. Possible impairments in information processing can be non-invasively investigated using electrophysiology. Methods: We compared auditory (AEP) and visual (VEP) evoked potentials in twelve adolescents and young adults (10–22 years) affected by FXS to healthy controls matched by chronological age (N = 12) and developmental age of cognitive functioning (N = 9; 5–7 years), using analysis of variance. Results: In the visual modality, the N70 and N2 amplitude have been found increased in FXS in comparison to the chronological, but not the developmental control group at occipital sites, whereas in the auditory modality N1, P2 and N2 amplitude as well as N2 latency have been found increased in FXS, relative to both chronological and developmental control groups at mid-central sites. Conclusions: The AEP/VEP profile suggests disruptions in sensory processing specific to FXS that exceed immaturity of physiological activity. In addition, the auditory modality seems to be more affected than the visual modality. Results are discussed in light of possible underlying neuronal mechanisms, including deficits in synaptic pruning and neuronal inhibition that might account for a hyperreactive nervous system in FXS. © 2014 Published by Elsevier Ltd. on behalf of ISDN.
Fragile X Syndrome (FXS) is the most common monogenic form of intellectual disability (ID) and affects about 2% of male patients with ID (Ropers and Hamel, 2005). It is caused by a trinucleotide repeat expansion in the FMR1 (‘Fragile X Mental Retardation 1’) gene, which is located on the X-chromosome. Women can also be affected but the penetrance of the mutation is reduced and its expressivity more variable in them (Bennetto et al., 2001). The FMR1 mutation prevents expression of the ‘Fragile X Mental
∗ Corresponding author at: Research Center of the CHU Ste-Justine Mother and Child University Hospital Center, University of Montreal, 3175 Chemin de la CôteSainte-Catherine, Montreal, QC H3T 1C5, Canada. Tel.: +1 514 345 4931x4590. E-mail addresses: [email protected], [email protected] (I.S. Knoth).
Retardation Protein’ (FMRP), which is known to repress the translation of specific mRNAs in response to the activation of metabotropic Glutamate Receptors (mGluRs) (Bear et al., 2004). FMRP directly targets approximately 5% of all mRNAs (Darnell and Klann, 2013). However, changes in protein synthesis observed in the absence of FMRP affect about 20% of pre-synaptic protein synthesis (Darnell and Klann, 2013). Thus, secondary alterations are believed to additionally account for the changes in protein synthesis observed in the absence of FMRP (Darnell and Klann, 2013). Alterations in protein synthesis result in a loss of synaptic plasticity in FXS (Bassell and Warren, 2008). Structurally, dendritic spines are increased in number and appear elongated whereas synapses appear immature in FXS patients and fragile X knockout mice (Comery et al., 1997). Thus, the absence of FMRP is likely to prevent activity-based synapse maturation and synaptic pruning, which are essential for normal brain development (Weiler and Greenough, 1999) and cognitive development (Schneider et al., 2009).
http://dx.doi.org/10.1016/j.ijdevneu.2014.05.003 0736-5748/© 2014 Published by Elsevier Ltd. on behalf of ISDN.
Please cite this article in press as: Knoth, I.S., et al., Alterations of visual and auditory evoked potentials in fragile X syndrome. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.05.003
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2 Table 1 Demographics of the study population.
49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104
Variable
FXS patients
Chronological age matched controls
Developmental age matched controls
N Age range Mean age (SD) IQ range Mean IQ (SD)
12, 4 ♀ 10–22 years 14.7 (3.75) 32–93 51 (±16.57)
12, 3 ♀ 11–32 years 16.9 (±6.02) 87–129 113 (±14.05)
9, 3 ♀ 5–7 years 5.8 (±0.83) 97–118 108 (±7.25)
Patients affected by FXS frequently show deficits in language, executive functions, visuo-spatial and social cognition. Further, they tend to show aberrant behavior, emotional instability and hyperarousal to sensory stimulation (Schneider et al., 2009). Most of the symptoms found in FXS are typical of the autistic spectrum; about 30% of male individuals with FXS meet the full diagnostic criteria for autism. FXS is thus considered one of the few known monogenic causes of autism (Rogers et al., 2001). Further, reduction of FMRP levels have been found in the cerebrellar vermis of adult subjects with autism who were not diagnosed with FXS, suggesting that common neurobiological mechanisms might account for the shared symptoms between non-syndromic autism and FXS (Fatemi et al., 2011). However, autistic symptoms vary considerably in their intensity between patients affected by FXS (Schneider et al., 2009). Disrupted pathways in synaptic plasticity, the potential link between the FMR1 mutation and the learning disability often found in FXS, are likely to be associated with impairments in mechanisms of information processing (Belmonte and Bourgeron, 2006). Early sensory processing can be non-invasively investigated using the AEP/VEP technique that records local field potentials, which are summarized postsynaptic potentials from large groups of neurons (Luck, 2005). Studies investigating AEP/VEPs in FXS so far exclusively used oddball paradigms and mostly studied AEPs (St Clair et al., 1987; Castrèn et al., 2003; Van Der Molen et al., 2012a,b). The most consistent findings were enhanced N1 and decreased P3 amplitudes, as well as prolonged N2 latencies in FXS compared to healthy age-matched controls, whereas findings concerning other components tended to be more variable. Thus, some AEPs appeared to be specifically altered in FXS, revealing disruptions in early sensory processing. In this study we aim to investigate to which extent the altered AEP/VEPs in FXS can be explained by immature as opposed to otherwise disrupted sensory processing. Since parameters of AEP/VEPs specifically change with brain development (Lippé et al., 2007, 2009), the altered AEP/VEPs in FXS might reflect immature physiological activity due to deficits in synaptic pruning. In this case, the AEP/VEPs in FXS would resemble those of individuals on the same level of cognitive functioning. However, given that the absence of FMRP has been found to interfere with functional and structural brain development, we hypothesize that the disruptions in sensory processing reflected by AEP/VEPs exceed immaturity. In order to distinguish between immaturity and specific alterations of the AEP/VEPs, we compared the FXS patients to an additional control group matched to the developmental age of cognitive functioning, assessed by Intelligence Quotient (IQ). Further, we aimed to investigate differences in the extent of impairments in sensory processing between auditory and visual modality in FXS. The only previous study investigating VEPs in FXS found the auditory modality to be more affected than the visual modality (Van Der Molen et al., 2012a), which matches modality differences in performance found in FXS (Schneider et al., 2009; Van Der Molen et al., 2010). This indicates that FMRP absence might affect sensory processing differently depending on modality. We hypothesize that the VEPs appear less altered in FXS than the AEPs. We thereby examine whether the extent to which the AEP/VEP
alterations can be explained by physiological immaturity varies between modalities.
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2. Methods
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2.1. Participants
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Twelve FXS patients aged from 10 to 22 years diagnosed with full mutation of the FMR1 gene were compared to 21 healthy controls matched by chronological age or developmental age and gender (Table 1). The developmental control group contains children whose chronologic age matches the developmental age of patients with intellectual disability (IQ < 70). Note that not all patients meet the criteria for intellectual disability. A total of 18 FXS patients had been tested; six patients were excluded from data analysis due to epileptic activity, difficulties in testing and extensive movement artifacts. Patients were recruited on the basis of DNA analysis previously conducted by geneticists at the CHU Sainte-Justine Mother and Child University Hospital Center in Montreal. Healthy controls were recruited at the Ste-Justine Hospital, the University of Montreal, kindergartens and summer day camps. Four of the twelve FXS patients had also been diagnosed with autistic disorder; eight FXS patients showed language delay and nine FXS patients were also diagnosed with Attention Deficit Hyperactivity Disorder (ADHD). Five of the tested patients did not take any medication, while seven patients were medicated with psychostimulant (5× methylphenidate, 2× atomoxetine, 1× amphetamine mixed salts) and/or antidepressant (1× citalopram) drugs to treat symptoms of autism, attention deficit hyperactivity disorder, depression and anxiety. All patients underwent detailed physical examinations in the developmental clinic of the hospital following their diagnosis. None of the patients has been diagnosed with hearing deficits within the scope of these evaluations. Parents reported normal hearing and normal or corrected-to-normal vision in all patients and control participants upon specific request. Healthy controls had no history of brain injuries, psychiatric or neurological illnesses and did not take any medication. All participants were born at term and right-handed. Intelligence in patients and controls was examined using the completely non-verbal Leiter-R International Performance Scale (Roid and Miller, 1997) for children and adolescents and the Wechsler Abbreviated Scale of Intelligence (Wechsler, 1999) for adults. The nonverbal scale was chosen in order to reduce the impact of language deficits in patients on the global IQ result. Developmental age of patients was calculated on the basis of IQ in order to match them with healthy controls. Autistic behavior was quantified using the repetitive behavior questionnaire (Lam and Aman, 2007) and the aberrant behavior checklist (Aman et al., 1985), which were completed by parents of patients and minor control participants. The study protocol was reviewed and approved by the ethics, administrative, and scientific committees at the Ste-Justine’s Hospital Research Center. Informed consent was obtained before the experiment from participants and parents or legal caregivers following a full explanation of the procedures undertaken.
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2.2. Apparatus and stimuli
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Auditory and visual stimuli were generated by a Dell GX150 PC using E-Prime 1.0 (Psychology Software Tools Inc., Pittsburgh, PA, USA). The EEG recording took place in a dark soundproof experimental chamber. Auditory stimulation consisted of 50 ms broadband noise presented 150 times in a randomly distributed interstimulus interval varying from 1200 to 1400 ms at 79 dB SPL intensity and 16-bit resolution as in a developmental study previously conducted in our laboratory (Lippé et al., 2009). The two speakers (Optimus XTS 24, Boston, MA, USA) were located laterally at 30 cm distance from the subject’s ears. During auditory stimulation all subjects watched a silent movie. Following this, visual stimulation consisted of a black and white checkerboard stimulus presented at a reversal rate of 1 Hz, meaning that the checkerboard changed every 500 ms, and subtending a visual angle of 2◦ . The original and reversed checkerboard stimulus were presented 200 times each. Stimuli had a luminance of 40 cd/m2 and were displayed on a 40.5 cm × 30.5 cm ViewSonic monitor (ViewSonic, Canada) at 114 cm distance from the participant’s eyes. This visual paradigm has been created for a developmental study previously conducted in our laboratory (Lippé et al., 2007). An assistant observed whether the participant looked at the screen at all times and gave a signal whenever the participant looked elsewhere, in order to exclude these EEG segments from analyses. The assistant likewise directed the attention of participants to the screen by holding small objects in the lower middle part of the screen and talking to them if necessary.
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Fig. 1. t-Test brain topographies comparing auditory N1, P2 and N2 amplitude between FXS patients and chronological/developmental controls. t-Values 3.1 were considered for defining ROIs.
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A dense array EEG system containing 128 electrodes was used for recording (Electrical Geodesics System Inc., Eugene, OR, USA). The vertex was used as the reference electrode during recording and impedances were maintained below 40 k (Tucker, 1993). Signals were acquired and processed by a G4 Macintosh computer using NetStation EEG Software (Version 2.0). Digitalization of EEG data was carried out at a sampling rate of 250 Hz in 1024 ms epochs and an analog 0.01–100 Hz bandpass filter was applied. Off-line analyses were carried out with BrainVision Analyser software, version 2.0 (Brain Products, Munich, Germany). Data were digitally filtered with a 1–50 Hz filter for the visual and a 1–30 Hz filter for the auditory experiment and re-referenced to an average reference. Eye movement artifacts were corrected using semi-automatic Ocular Correction ICA as implemented in BrainVision Analyser. Algorithmic artifact rejection of voltage exceeding ±100 V was followed by visual data inspection of segmented data in which segments with artifacts were manually rejected.
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2.3. Auditory and visual evoked potential analysis
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Artifact-free segments were averaged and baseline corrected. In the auditory paradigm, an average of 146 artifact-free segments were available for the patient group and an average of 149 segments for the chronological and developmental control group. In the visual paradigm, an average of 191 segments were available for patients, 198 for chronological controls and 191 for developmental controls.
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Number of presented segments did not differ between the three groups (auditory condition: (F(2,30) = 1.75, p = 0.19), visual condition: (F(2,30) = 7.7, p = 0.08)). In order to define regions of interest (ROI) for statistical comparison between groups, BrainVision-assisted t-tests were computed on amplitude topographies during the three time windows showing maximal differences between the responses evoked in FXS compared to the two control groups (Figs. 1 and 2). Note that the potentials with opposing polarity to the AEPs/VEPs observed temporally in the auditory condition and frontally in the visual condition reflect opposing charges of the same dipole source generated by AEPs/VEPs primary ROI (central for the auditory condition and occipital for the visual condition) (Scherg, 1990; Di Russo et al., 2002). Results from dipolar ROIs are reported, but interpreted as the same activity as the primary ROIs. Electrodes within the selected ROI were averaged and thereupon components were individually selected for each subject in each associated ROI. For lateral regions, a region on the opposing hemisphere was defined as well. Parameters of all defined ROI are given in Table 2. Amplitudes were defined from baseline (200 ms prestimulus) to the highest amplitude of each component and latencies were defined from stimulus onset to the highest point of each component.
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2.4. Statistical analysis
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Statistical analyses were performed using SPSS statistics, version 20 (IBM Corp., Armonk, NY, USA). Firstly, we compared the following variables between male and female FXS patients: IQ; N1, N2 and P2 amplitudes and latencies at the corresponding
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Table 2 Parameters of defined ROI for every component. Auditory condition Component
N1
P2
N2
ROI (number of electrodes included)
Mid-central (6) Centro-parietal left (7) Centro-parietal right (7) Temporal left (6) Temporal right (6)
Mid-central (6) Temporal left (6) Temporal right (6)
Mid-central (6)
Visual condition Component ROI (number of electrodes included)
N70
P100
N2
Centro-occipital (7) Parietal left (5) Parietal right (5) Frontal left (4) Frontal right (4)
Temporal left (4) Temporal right (4) Frontal left (4) Frontal right (4)
Centro-occipital (6) Parietal left (7) Parietal right (7) Fronto-central (8) Frontal left (4) Frontal right (4)
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Fig. 2. t-Test brain topographies comparing visual N70, P1 and N2 amplitude between FXS patients and chronological/developmental controls. t-Values 3.1 were considered for defining ROIs.
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ROI for AEPs and N70, P100 and N2 amplitudes and latencies at the corresponding ROI for VEPs. Since female patients are known to be less affected, due to their intact X-chromosome, which mitigates the outcome of the FMR1 mutation, we verified whether male and female patients differed from each other in respect of ERPs or can be considered as a single group. Student’s t-test has been used and log transformations were applied to non-normally distributed data. Analysis was then carried out between FXS patients, chronological and developmental age matched controls for the variables listed above using analysis of variance (ANOVA). For the evoked potentials, a mixed-design ANOVA (group × ROI) using Greenhouse–Geisser correction has been carried out. If significant interactions between group and ROI were found, one-way ANOVA was carried out for every ROI separately. If significant differences between groups were detected, Tukey’s test was carried out for post hoc analysis. Effect sizes are provided for significant ANOVA results. Data obtained from the abnormal behavior questionnaire and the repetitive behavior scale were only compared between FXS patients and the chronological control group, since most of the items cover behavior that is perceived as normal in pre-school children. Significance level for all statistical tests was set to 5% (˛ = 0.05).
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3. Results
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3.1. Gender, medication and autism
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developmental age matched control group (p = 0.001), while both control groups do not significantly differ from each other. Table 3 shows mean values of aberrant and repetitive behavior in FXS patients and chronological controls as reported by parents/care givers in the aberrant behavior questionnaire and the repetitive behavior scale. 3.3. AEPs Group averages of the AEP responses in mid-central sites are presented in Fig. 3. Table 4 shows mean amplitudes and latencies at electrode FCz and Cz for every group.
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With a mean of 68.25 (±17.11), female patients had higher IQs than male patients (M = 43.38, ±8.38) (p = 0.006). However, no significant differences between male and female FXS patients were found in amplitude or latency of VEP components N70, P100 and N2, as well as in the AEP components N1, P2 and N2 in all ROI (p > 0.05). Further, IQ, as well as amplitude and latency of AEP/VEP components did neither differ significantly between medicated and non-medicated patients, nor between patients that were and were not diagnosed with autism (p > 0.05). Thus, all FXS patients were combined into one group for analysis.
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3.2. IQ, aberrant and repetitive behavior
3.3.1. Auditory N1 Even though a significant group × ROI interaction has been found in N1 latency (F(5,76) = 2.29, p = 0.054, R2 = 0.15), subsequent one-way ANOVAs did not detect a significant difference between the three groups in any ROI. A group × ROI interaction has been found for N1 amplitude (F(4,56) = 10, p = 0.001, R2 = 0.67). In subsequent one-way ANOVAs, a significant difference between groups could be found at centro-parietal left (F(2,30) = 14.32, p = 0.001, R2 = 0.96), centro-parietal right (F(2,30) = 12.37, p = 0.001, R2 = 0.82), mid-central (F(2,30) = 12.77, p = 0.001, R2 = 0.85) and temporal left (F(2,30) = 12.22, p = 0.001, R2 = 0.82) sites. Post hoc analysis revealed that FXS patients had a higher N1 amplitude than both control groups at centro-parietal left (p = 0.001 for both control groups), centro-parietal right (p = 0.004 for chronological and p = 0.001 for developmental controls) and mid-central (p = 0.015 for chronological and p = 0.001 for developmental controls) sites. At the temporal left site, developmental controls showed a smaller amplitude than FXS patients (p = 0.001) and chronological controls (p = 0.019).
One-way ANOVA shows a difference in IQ between FXS patients, chronological and developmental age matched controls (F(2,28) = 67.12, p = 0.00, R2 = 0.83). FXS patients differ from the chronologic age matched control group (p = 0.001) and the
3.3.2. Auditory P2 No significant group × ROI interaction has been found for P2 latency. Concerning P2 amplitude, a significant interaction was found (F(3,46) = 14.28, p = 0.001, R2 = 0.95). Subsequent one-way
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Table 3 Mean values (SD) of abnormal and repetitive behavior in participants as reported by their parents/caregivers. Scale
FXS patients
Chronological controls
p-Value
Irritability Lethargy Stereotypical Behavior Hyperactivity Inappropriate Speech Self Mutilation Compulsive Behavior Ritualized Behavior Immutable Behavior Restrictive Behavior
4 (4.54)* 4.13 (5.11)* 1.75 (3.24) 3.35 (4.34)* 3.25 (3.45)* 1.78 (2.22)* 1.78 (2.49) 1.89 (1.96)* 4.22 (4.92)* 1.22 (1.64)*
0.78 (1.09) 0.33 (0.52) 0 (0.00) 1.52 (0.51) 0.33 (0.52) 0.17 (0.41) 0.33 (0.82) 0 (0.00) 0.17 (0.41) 0 (0.00)
0.04 0.04 0.08 0.02 0.02 0.03 0.06 0.01 0.02 0.03
*
Significant difference between FXS and chronological control group (p < 0.05).
Fig. 3. Group averages of AEPs at mid-central ROI for the FXS group and the two control groups. 0 ms marks stimulus onset.
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ANOVAs showed difference between groups at mid-central (F(2,30) = 18.73, p = 0.001, R2 = 1.25), temporal left (F(2,30) = 6.8, p = 0.004, R2 = 0.45) and temporal right (F(2,30) = 10.17, p = 0.001, R2 = 0.68) sites. According to post hoc analysis, P2 amplitude was higher in FXS compared to both control groups at mid-central sites (p = 0.001 for both control groups) and higher in FXS compared to the chronological control group at temporal left (p = 0.003) and temporal right (p = 0.001) sites.
3.3.3. Auditory N2 Since only one ROI of interest was defined for auditory N2, one-way ANOVA was immediately performed for amplitude and latency. A difference between groups could be found for latency (F(2,30) = 8.32, p = 0.001, R2 = 0.55) and amplitude (F(2,30) = 13.23, p = 0.001, R2 = 0.88). Post hoc analysis revealed that latency was longer in FXS compared to both control groups (p = 0.019 for chronological and p = 0.001 for developmental controls). Amplitude was found to be higher in FXS compared to both control
groups (p = 0.001 for chronological and p = 0.002 for developmental controls). 3.4. VEPs
Parameter
FXS patients
N1
Amplitude Latency
−1.79 (1.16)* , T 97.33 (15.3)
P2
Amplitude Latency
4.9 (1.8)* , T 181.33 (28.5)
N2
Amplitude Latency
−2.3 (0.84)* , T – 321.67 (35.92)* , T
*
–
T
–
–
–
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Mean amplitudes and latencies at occipito-central sites for every subgroup are presented in Table 5. Fig. 4 shows the group averages of the VEP response at electrode Oz. 3.4.1. Visual N70 No significant group × ROI interaction was found for N70 latency. As for amplitude, a group × ROI interaction was found (F(4,55) = 3.77, p = 0.001, R2 = 0.25). Subsequent one-way ANOVAs revealed difference between groups at occipito-central (F(2,30) = 3.79, p = 0.034, R2 = 0.25), left parietal (F(2,30) = 7.36, p = 0.033, R2 = 0.49) and frontal right (F(2,30) = 7.08, p = 0.003, R2 = 0.47) sites. Amplitude was found higher in FXS compared to chronological controls in occipito-central (p = 0.026) and left
Table 4 Mean amplitudes in V and latencies in ms (SD) for AEP components in FXS patients, chronological control group and developmental control group at mid-central sites. Component
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Chronological control group
Developmental control group
−0.62 (0.73) 90 (12.8)
0.68 (1.08) 90 (34)
1.67 (0.97) 170 (22.79)
2.49 (0.97) 158.22 45.92)
−0.89 (0.60) 277.67 (26.88)
−1.08 (0.69) 257.33 (49.92)
Significant difference between FXS and chronological control group (p < 0.05). Significant difference between FXS and developmental control group (p < 0.05).
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Table 5 Mean amplitudes in V and latencies in ms (SD) at occipito-central sites for VEP components in FXS patients, chronological control group and developmental control group. Component
Parameter
FXS patients
Chronological control group
Developmental control group
N70
Amplitude Latency
−2.11 (1.33)* 83 (21.47)
−0.81 (0.58) 80 (7.03)
−1.54 (1.44) 76.89 (7.15)
P100
Amplitude Latency
4.08 (3.11) 122 (31.19)
3.29 (2.01) 118.67 (16.12)
3.88 (1.7) 114.22 (4.06)
N2
Amplitude Latency
−1.29 (0.83)* 319.33 (35.44)
−0.37 (0.48) 289 (39.67)
−1.04 (0.99) 288.44 (54.24)
*
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Significant difference between FXS and chronological control group (p < 0.05).
parietal (p = 0.002) sites. At the right frontal ROI, amplitude was found higher in FXs compared to both control groups (p = 0.003 for chronological and p = 0.033 for developmental controls). 3.4.2. Visual P100 No significant group × ROI interaction was found for P100 latency. P100 amplitude showed a significant group × interaction (F(3,50) = 3.97, p = 0.001, R2 = 0.27). Subsequent one-way ANOVAs showed group differences at right temporal (F(2,30) = 4.54, p = 0.019, R2 = 0.3) and left frontal (F(2,30) = 5.3, p = 0.011, R2 = 0.36) sites. P100 amplitude was found to be higher in developmental controls than in FXS patients at right temporal (p = 0.018) and left frontal (p = 0.013) sites. 3.4.3. Visual N2 No significant group × ROI interaction was found for visual N2 latency. As for amplitude, the interaction was found to be significant (F(3,49) = 5.94, p = 0.001, R2 = 0.4). Subsequent one-way ANOVAs showed group differences in left parietal (F(2,30) = 3.52, p = 0.042, R2 = 0.23), right parietal (F(2,30) = 4.8, p = 0.016, R2 = 0.32), occipito-central (F(2,30) = 4.47, p = 0.020, R2 = 0.3), fronto-central (F(2,30) = 7.6, p = 0.002, R2 = 0.5), left frontal (F(2,30) = 8.9, p = 0.001, R2 = 0.59) and right frontal (F(2,30) = 4.25, p = 0.024, R2 = 0.28) sites. A higher amplitude in FXS compared to the chronological control group has been found in left parietal (p = 0.035), right parietal (p = 0.012), occipito-central (p = 0.018), left frontal (p = 0.001) and right frontal (p = 0.018) sites. N2 amplitude at fronto-central sites was found to be higher in FXS compared to both control groups (p = 0.002 for chronological and p = 0.032 for developmental controls).
4. Discussion FXS patients showed alterations in AEP/VEPs compared to healthy aged-matched controls. However, we found that auditory processing is more impaired than visual processing in FXS patients. Furthermore, AEPs in FXS do not only alter in comparison to age-matched healthy peers, but also compared to children with the same developmental age of intellectual functioning. Auditory information processing in FXS patients does thus not resemble immature information processing, but has its own particularities. Visual information processing however appears immature in FXS. A summary of ERP alterations in FXS found in our study is given in Tables 6 and 7. Specifically, an increased auditory N1 amplitude and no difference in N1 latency have been found in our patient population, replicating the findings of relevant AEP studies conducted with FXS patients so far (St Clair et al., 1987; Castrèn et al., 2003; Van Der Molen et al., 2012a,b). We also found an increased visual N70 amplitude in FXS, which differs at right frontal sites from both control groups, whereas it differs in centro-occipital and right temporal sites only from the chronological control group, while no difference in latency was found. Increased N1 amplitude seems to be somewhat specific to FXS when compared to other forms of ID and autism that often present a difference in N1 latency and no difference (or even a reduction) in N1 amplitude (Knoth and Lippé, 2012). A higher N1 amplitude in FXS suggests that more neurons are synchronously active in response to the sensory stimulation in FXS than in healthy controls (Rojas et al., 2001). This could mean that FXS patients have a surplus of neurons in brain regions that are involved in N70/N1 generation. Alternatively, neuronal activation could be less inhibited in FXS than in healthy controls.
Fig. 4. Group averages of VEPs at occipito-central ROI for the FXS group and the two control groups. 0 ms marks stimulus onset.
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Amplitude
Latency
N1
Increased in FXS compared to both control groups at left, right and mid-central sites
No difference
P2
N2
Increased in FXS compared to both control groups at mid-central sites Increased in FXS compared to chronological controls at right and left temporal sites Increased in FXS compared to both control groups at mid-central sites
No difference
Increased in FXS compared to both control groups at mid-central sites
Table 7 Alterations of VEPs in FXS obtained in this study. Component N70
P100
N2
366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396
Amplitude Increased in FXS compared to chronological controls in occipito-central and left parietal sites Increased in FXS compared to both control groups at opposing dipolar potential (right frontal ROI) Decreased in FXS compared to developmental controls at right temporal and left frontal sites Increased in FXS compared to chronological controls at left frontal, right frontal, left parietal, right parietal and occipito-central sites Increased in FXS compared to both control groups at opposing dipolar potential (centro-frontal ROI)
Latency No difference
No difference
No difference
The first theory, a surplus of neurons, is supported by neuroanatomical aberrations in FXS. Among other alterations, greater gray matter volume in the occipital cortex has been found in infants with FXS compared to normally developed children and children with non-syndromic delay (Hoeft et al., 2008). The occipital cortex contains most of the visual cortex, which is believed to be involved in visual N70 generation (Shigeto et al., 1998). In contrast, the superior temporal gyrus, which contains the primary auditory cortex and is believed to be involved in auditory N1 generation (Näätänen and Picton, 1987), is found to be smaller in FXS, relative to healthy controls (Gothelf et al., 2008). However, FXS children between the age of 1 and 3 years showed a greater gray matter increase relative to chronologically and developmentally age matched controls over the course of 2 years, until the age of 3–5 years, in several brain structures, including not only temporaloccipital regions, but also the superior temporal gyrus. This could indicate deficient synaptic pruning (Hoeft et al., 2010) in areas that are believed to be involved in generation of auditory N1 and visual N70. Synaptic pruning, a neuroregulatory process in which unnecessary neurons and synapses are reduced in order to strengthen more efficient neuronal configurations, might be impaired in FXS, according to animal model studies (Weiler and Greenough, 1999; Pfeiffer and Huber, 2007, 2009). In consequence, more redundant neurons, which have not been eliminated through synaptic pruning, would respond to sensory stimulation in a sort of non-specific arousal response, accounting for the increased auditory N1 and visual N70 amplitude. This finds additional support in studies that investigated auditory N1 habituation in FXS (Castrèn et al., 2003; Van Der Molen et al., 2012b). In both studies, controls showed a reduction of N1 amplitude after several presentations of the same stimulus, while
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this N1 habituation did not occur in FXS. The reduction of N1 amplitude in controls suggests that less neurons synchronously respond to a stimulus after several repetitions, which could reflect a reduction of the non-specific arousal or novelty response that generally occurs after the first appearance of a sensory stimulus (Karhu et al., 1997). Thus, deficient synaptic pruning might lead to an excess of less adapted synaptic configurations that non-specifically respond to sensory stimulation, while reducing the capacity for more efficient synaptic connections. This mechanism might be reflected in increased auditory N1 amplitude and a lack of N1 habituation. The second theory, impaired neuronal inhibition as an explanation for increased N70/N1 amplitudes, finds support in a study that investigated resting state EEG in FXS (Van Der Molen and Van Der Molen, 2013). Van der Molen et al. found an increased relative theta power and a decreased relative upper alpha in FXS, compared to healthy controls. Alpha oscillations in EEG are believed to be involved in neural inhibitory regulation mechanisms that gate information by reducing the processing capabilities of a given area (Jensen and Mazaheri, 2010). A reduced alpha rhythm in FXS could indicate impaired sensory gating mechanisms, which fail to inhibit redundant neural activity. In consequence, there would be more neuronal activation, which might be reflected in increased N1 amplitude. Lastly, we investigated auditory and visual basic N2 in FXS. In the visual modality, no difference in N2 latency was found, which differs from the results reported by Van der Molen and colleagues, who found an increased visual N2 latency in FXS, compared to chronological controls (Van Der Molen et al., 2012a). However, since the mean latency of our developmental group lies between FXS patients and chronological controls, the difference between FXS patients and chronological controls would have been significant in a direct comparison. Further, visual N2 amplitude was only found to be larger in FXS compared to the chronological control group in most ROI and only differed from the developmental control group at fronto-central sites. Since N2 amplitude is known to decrease with age (Lippé et al., 2007), it can be argued that the visual N2 is rather immature in FXS than specifically altered. Moreover, prolonged N2 latencies seem to be a general phenomenon in ID (Knoth and Lippé, 2012). Even though our study provides exciting evidence for information processing deficits in FXS, there are two limitations that should be mentioned. Firstly, the sample size can be considered as rather small. In addition to the general difficulty of finding patients with a rare disease, such as FXS, hyperactive and impulsive behavior, as well as social anxiety in many patients posed another problem, since EEG recordings require calmness and AEP/VEP cannot be studied in sedated patients. Secondly, it is an issue that more than half of our patient population was medicated during the EEG recording with psychostimulant and/or other drugs. As psychoactive drugs are known to have an influence on the parameters of AEP/VEPs, medication might have been a confounding variable in our study. However, statistical testing did not reveal significant differences in amplitude or latency of the investigated ERP components between medicated and non-medicated patients. Further, psychoactive drugs are more likely to reverse the effects of the treated pathology on the evoked potential profile so that it no longer differs from that of controls (Broyd et al., 2005). Thus, it seems unlikely that the altered ERP profile, which we detected in our patients, reflects drug effects rather than the FXS pathology.
5. Conclusions The present study presents a profile of altered AEP/VEPs in FXS, which likely reflects impairments in basic neural sensory processing. The additional comparison to a control group matched by
Please cite this article in press as: Knoth, I.S., et al., Alterations of visual and auditory evoked potentials in fragile X syndrome. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.05.003
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developmental age of cognitive functioning of patients with ID leads to the conclusion that AEP components in FXS are not simply immature, but appear specifically altered. Conversely, the visual N70 and N2 amplitudes do not differ between FXS patients and developmental controls at occipito-central sites. Notably, information processing seems to be more severely impaired in the auditory than in the visual modality. The knowledge that visual processing is less affected, as well as indications of a hyperreactive nervous system, should be considered in the design of behavioral treatments for FXS patients.
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Competing interests
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The authors declare that they have no competing interests. Authors’ contributions
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ISK participated in the study design, coordinated the study, carried out recruitment of patients and controls, EEG recording, AEP/VEP and statistical analysis and drafted the manuscript. PV participated in EEG recordings and AEP/VEP analysis. JM participated in the DNA analysis and in the study design. PM examined EEGs for epileptic activity. SL conceived the study, participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.
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Acknowledgements
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This research was supported by a Scottish Rite Charitable Foundation of Canada grant to Sarah Lippé. We thank Domitille Malfait for carrying out the neuropsychological evaluation of most of the patients and Patricia Laniel and Maude Joannette for their help in the acquisition of EEG data. We thank language editor J. Arthur White for significantly revising the manuscript.
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References
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Please cite this article in press as: Knoth, I.S., et al., Alterations of visual and auditory evoked potentials in fragile X syndrome. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.05.003
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