Visual Evoked Potentials in Infants With Diffuse Periventricular Leukomalacia.

Periventricular leukomalacia (PVL) is characterized by necrosis of the cerebral white matter in the dorsolateral portions of the lateral ventricles. PVL causes motor, sensory, and cognitive deficits. The aim of this study was to analyze the conduction characteristics of the visual pathway in infants with diffuse PVL using visual evoked potentials (VEPs). We studied 11 healthy infants (mean age 3.3 ± 1.3 months) and 17 with diffuse PVL (mean age 2.9 ± 0.8 months and mean gestational age 31.9 ± 3.1 weeks). The N75, P100, and N135 wave latencies; the interwave N75-P100 and P100-N135 latencies; and the N75-P100 and P100-N135 amplitudes were measured in the occipital leads. VEPs were recorded during binocular stimulation at an angle of 120' from the Fz-Oz lead. Healthy children had mean N75, P100, and N135 wave latencies of 84.4 ± 5.8, 143.4 ± 30.6 and 222.9 ± 40.4 ms, respectively. The mean interwave N75-P100 and P100-N135 latencies were 59.0 ± 28.6 and 79.5 ± 13.6 ms, respectively. Compared with the healthy group, infants with PVL had longer N75 and N135 latencies at 92.3 ± 15.3 (P = .05) and 265.0 ms ± 60.3 (P = .05), respectively. The interwave latency P100-N135 (105.5 ± 29.1 ms; P = .017) was longer in children with PVL than in healthy infants. Infants with diffuse PVL had mild alterations in their N75, P100 and, particularly, their N135 latencies. These increases in P100-N135 interwave latencies could be because of damage to the geniculocortical pathways and V2-V3 networks.

Epileptic encephalopathy in children with risk factors for brain damage.

In the study of 887 new born infants with prenatal and perinatal risk factors for brain damage, 11 children with West syndrome that progressed into Lennox-Gastaut syndrome and another 4 children with Lennox-Gastaut syndrome that had not been preceded by West syndrome were found. In this study we present the main findings of these 15 subjects. In all infants multifactor antecedents were detected. The most frequent risk factors were prematurity and severe asphyxia; however placenta disorders, sepsis, and hyperbilirubinemia were also frequent. In all infants MRI direct or secondary features of periventricular leukomalacia were observed. Followup of all infants showed moderate to severe neurodevelopmental delay as well as cerebral palsy. It is concluded that prenatal and perinatal risk factors for brain damage are very important antecedents that should be taken into account to follow up those infants from an early age in order to detect and treat as early as possible an epileptic encephalopathy.

Effect of hearing aids on auditory function in infants with perinatal brain injury and severe hearing loss.

BACKGROUND: Approximately 2-4% of newborns with perinatal risk factors present with hearing loss. Our aim was to analyze the effect of hearing aid use on auditory function evaluated based on otoacoustic emissions (OAEs), auditory brain responses (ABRs) and auditory steady state responses (ASSRs) in infants with perinatal brain injury and profound hearing loss. METHODOLOGY/PRINCIPAL FINDINGS: A prospective, longitudinal study of auditory function in infants with profound hearing loss. Right side hearing before and after hearing aid use was compared with left side hearing (not stimulated and used as control). All infants were subjected to OAE, ABR and ASSR evaluations before and after hearing aid use. The average ABR threshold decreased from 90.0 to 80.0 dB (p = 0.003) after six months of hearing aid use. In the left ear, which was used as a control, the ABR threshold decreased from 94.6 to 87.6 dB, which was not significant (p>0.05). In addition, the ASSR threshold in the 4000-Hz frequency decreased from 89 dB to 72 dB (p = 0.013) after six months of right ear hearing aid use; the other frequencies in the right ear and all frequencies in the left ear did not show significant differences in any of the measured parameters (p>0.05). OAEs were absent in the baseline test and showed no changes after hearing aid use in the right ear (p>0.05). CONCLUSIONS/SIGNIFICANCE: This study provides evidence that early hearing aid use decreases the hearing threshold in ABR and ASSR assessments with no functional modifications in the auditory receptor, as evaluated by OAEs.

3D statistical parametric mapping of quiet sleep EEG in the first year of life.

This paper extends previously developed 3D SPM for Electrophysiological Source Imaging (Bosch et al., 2001) for neonate EEG. It builds on a prior paper by our group that established age dependent means and standard deviations for the scalp EEG Broad Band Spectral Parameters of children in the first year of life. We now present developmental equations for the narrow band log spectral power of EEG sources, obtained from a sample of 93 normal neonates from age 1 to 10 months in quiet sleep. The main finding from these regressions is that EEG power from 0.78 to 7.5 Hz decreases with age and also for 45-50 Hz. By contrast, there is an increase with age in the frequency band of 19-32 Hz localized to parietal, temporal and occipital areas. Deviations from the norm were analyzed for normal neonates and 17 with brain damage. The diagnostic accuracy (measured by the area under the ROC curve) of EEG source SPM is 0.80, 0.69 for average reference scalp EEG SPM, and 0.48 for Laplacian EEG SPM. This superior performance of 3D SPM over scalp qEEG suggests that it might be a promising approach for the evaluation of brain damage in the first year of life.

Reduced right frontal cortical thickness in children, adolescents and adults with ADHD and its correlation to clinical variables: a cross-sectional study.

OBJECTIVE: Some longitudinal magnetic resonance imaging (MRI) studies have shown reduced volume or cortical thickness (CT) in the frontal cortices of individuals with attention-deficit/hyperactivity disorder (ADHD). These studies indicated that the aforementioned anatomical abnormalities disappear during adolescence. In contrast, cross-sectional studies on adults with ADHD have shown anatomical abnormalities in the frontal lobe region. It is not known whether the anatomical abnormalities in ADHD are a delay or a deviation in the encephalic maturation. The aim of this study was to compare CT in the frontal lobe of children, adolescents and adults of both genders presenting ADHD with that in corresponding healthy controls and to explore its relationship with the severity of the illness. METHOD: An MRI scan study was performed on never-medicated ADHD patients. Twenty-one children (6-10 year-olds), twenty adolescents (14-17 year-olds) and twenty adults (25-35 year-olds) were matched with healthy controls according to age and sex. CT measurements were performed using the Freesurfer image analysis suite. RESULTS: The data showed regions in the right superior frontal gyrus where CT was reduced in children, adolescents and adults with ADHD in contrast to their respective healthy controls. The CT of these regions correlated with the severity of the illness. CONCLUSIONS: In subjects with ADHD, there is a thinning of the cortical surface in the right frontal lobe, which is present in the children, adolescents and in adults.

Clinical correlations of grey matter reductions in the caudate nucleus of adults with attention deficit hyperactivity disorder.

BACKGROUND: Magnetic resonance imaging (MRI) studies have shown decreased caudate volumes in individuals with attention deficit hyperactivity disorder (ADHD). However, most of these studies have been carried out in male children. Very little research has been done in adults, and the results obtained in children are difficult to extrapolate to adults. We sought to compare the volume of the caudate of adults with ADHD with that of healthy controls; we also compared these volumes between men and women. METHODS: We performed an MRI scan on 20 adults with ADHD (10 men and 10 women) aged 25-35 years and 20 healthy controls matched by age and sex. We used voxel-based morphometry with the DARTEL algorithm for image analyses. We used the specifically designed Friederichsen, Almeida, Serrano, Cortes Test (FASCT) to measure the severity of ADHD; both the self-reported (FASCT-SR) and the observer (FASCT-O) versions were used. RESULTS: The statistical parametric map showed a smaller region with low grey matter volume and a smaller concentration of grey matter in this region of the right caudate in ADHD patients than in health controls, both in the entire sample and within each sex. There was a significant correlation between the volume of this region of the caudate with the number of DSM IV-TR criteria, as well as with the total scores and most of the factors of the FASCT-SR and FASCT-O scales. A separate correlation analysis by sex gave similar results. LIMITATIONS: The study design was cross-sectional. CONCLUSION: The region of the right caudate with low grey matter volume was smaller in adults with ADHD in both sexes and was correlated with ADHD severity.

Analysis of auditory function using brainstem auditory evoked potentials and auditory steady state responses in infants with perinatal brain injury.

Approximately 2-4 % of newborns with perinatal risk factors present hearing loss. The aim of this study was to analyse the auditory function in infants with perinatal brain injury (PBI). Brainstem auditory evoked potentials (BAEPs), auditory steady state responses (ASSRs), and tympanometry studies were carried out in 294 infants with PBI (586 ears, two infants had unilateral microtia-atresia). BAEPs were abnormal in 158 (27%) ears, ASSRs in 227 (39%), and tympanometry anomalies were present in 131 (22%) ears. When ASSR thresholds were compared with BAEPs, the assessment yielded 92% sensitivity and 68% specificity. When ASSR thresholds were compared with tympanometry results as an indicator of middle-ear pathology, the assessment gave 96% sensitivity and 77% specificity. When BAEP thresholds were compared with tympanometry results, sensitivity was 35% and specificity 95%. In conclusion, BAEPs are useful test for neonatal auditory screening; they identify with more accuracy sensorineural hearing losses. ASSRs are more pertinent for identifying conductive hearing loss associated with middle-ear pathology. The consistency and accuracy of these results could be considered in additional studies.

Motor potentials by magnetic stimulation in periventricular leukomalacia.

Periventricular leukomalacia is characterized by damage to the brain's white matter and impairments in motor function. Motor-evoked potentials by transcranial magnetic stimulation evaluate corticospinal tract function. We analyzed alterations in motor-evoked potentials in newborns with periventricular leukomalacia. Thirty infants (aged 4.37 +/- 1.1 months mean +/- S.D.) were divided into three groups: 10 healthy, and 10 with focal and 10 with diffuse periventricular leukomalacia. Potentials recorded in the right abductor pollicis brevis of healthy infants indicated a total motor conduction time of 26.3 +/- 2.4 ms, central motor conduction time of 17.0 +/- 2.6 ms, and central motor conduction velocity of 12.3 +/- 2.2 m/s. In the tibialis anterior, total motor conduction time was 27.4 +/- 2.6 ms; central motor conduction time was 16.7 +/- 2.8 ms, and central motor conduction velocity was 25.2 +/- 3.4 m/s. In the focal periventricular leukomalacia and diffuse periventricular leukomalacia groups, an increase in central motor conduction time and a decrease in central motor conduction velocity (P < 0.05) were evident, without differences between the two groups. Motor-evoked potentials in periventricular leukomalacia revealed an increase in central motor conduction time and a decrease in central motor conduction velocity, without differences between diffuse and focal types.

EEG sources in a group of patients with major depressive disorders.

EEG sources were assessed in a group of patients with major moderate-severe depressive disorder (MDD) as classified by trained clinicians according to DSM-IV criteria. Frequency Domain Variable Resolution Electromagnetic Tomography (FD-VARETA) was used to calculate EEG sources. The Z-values indicated that EEG sources were abnormal (increase in current density) in all patients, with most demonstrating abnormal EEG sources in both hemispheres but with maximal inverse solution located primarily in the right. Twenty-nine patients had a predominant topography of the abnormal EEG maximal inverse solution in the frontal lobes. The remaining seven patients had a bilateral abnormal increase in current density in the superior parietal lobe. The EEG maximal abnormal inverse solution frequency was observed in both hemispheres such that the increases in current density were prevalent in alpha and theta bands. The results suggest that any of the two hemispheres could be affected by MDD, but abnormal EEG sources can be found more frequently in the right one, with the maximal abnormal inverse solution at the alpha and theta bands in frontal and parietal cortices.

Habituation of visual evoked potentials in healthy infants and in infants with periventricular leukomalacia.

OBJECTIVE: To investigate whether habituation of flash visual evoked potentials is already present during the first 3 months of life, and to explore differences between healthy infants, term infants with periventricular leukomalacia (PVL), and preterm infants with PVL. METHODS: Referential recordings to stimuli consisting of photic stimulation presented in blocks were obtained. A total of 25 blocks, 15-stimuli each, were presented. Intrablock and interblock habituation effects were analyzed. RESULTS: In healthy infants of 42-50 and 51-58 weeks of post-conceptional age (PCA), a negative central component (NCC) showed a significant decrease in amplitude due to stimulus repetition. NCC habituation was also observed in term infants with PVL at 51-58 weeks of PCA, but not in term infants with PVL at 42-50 weeks of PCA. NCC habituation was not apparent in preterm infants with PVL. CONCLUSIONS: These results suggest that the neural mechanisms of visual habituation are normally present during the first month of life, but the presence of PVL delays the emergence of these mechanisms, particularly in preterm infants. SIGNIFICANCE: The habituation of flash visual evoked potentials may be developed into a reliable tool to examine normal and abnormal development of early neural processes.

Repetitive transcranial magnetic stimulation decreases the number of seizures in patients with focal neocortical epilepsy.

PURPOSE: To evaluate the number of seizures and interictal epileptiform discharges (IEDs) in patients with focal neocortical epilepsy before, during and after rTMS. METHODS: Twelve patients (seven men and five women, mean age 29.3+/-15.8 years) were studied. An open-label study with baseline (4 weeks), intervention (2 weeks) and follow-up (8 weeks) periods was carried out. Repetitive transcranial magnetic stimulation (rTMS) with 900 pulses, intensity of 120% motor resting threshold and 0.5Hz frequency was used. A 120 channel EEG was recorded; an electrical source analysis of IEDs with Variable Resolution Electromagnetic Tomography (VARETA) was performed. The number of seizures per week and IEDs per minute were measured and compared in the three periods. RESULTS: During the basal period the mean seizure frequency was 2.25 per week; in the intervention period it decreased to 0.66 per week (F=2.825; p=0.0036) which corresponds to a 71% reduction. In the follow-up period the mean frequency was 1.14 seizures per week, that is, a 50% reduction in the number of seizures. In the visual EEG analysis, the baseline IED frequency was 11.9+/-8.3events/min; it decreased to 9.3+/-7.9 during 2 weeks of rTMS with a further reduction to 8.2+/-6.6 in the follow-up period. These differences however were not significant (p=0.190). CONCLUSION: We conclude that 2 weeks of rTMS at 0.5Hz with a figure-of-eight coil placed over the epileptic focus, determined with VARETA, decreases the number of seizures in patients with focal epilepsy, without reduction in IEDs.

Analysis of background EEG activity in patients with juvenile myoclonic epilepsy.

PURPOSE: To analyze background EEG activity of patients with juvenile myoclonic epilepsy (JME) with and without antiepileptic drugs. METHODS: We studied the background EEG activity in 18 patients with JME. The qEEG analysis included absolute power (AP), relative power (RP) and mean frequency (MF) of delta, theta, alpha and beta bands. The Z scores were calculated by comparison with population parameters based on the age-dependent regression function. Seven patients were unmedicated (UM) and eleven medicated (M). RESULTS: The UM group presented 69 (4.32%) abnormal Z scores and 227 (9.05%) in the M group (P<0.001). In the UM group, AP delta abnormal Z scores were identified in frontotemporal and occipital leads. In AP alpha and beta bands an increase in Z scores was encountered in frontoparietal leads in three patients. In addition, in three patients, the AP theta Z scores were below -1.96 and distributed in all regions. In the M group, AP beta Z scores were above 1.96 in frontoparietal leads in 7 of 11 patients. The AP delta increased above 1.96 in frontotemporal and occipital leads in 6 patients of 11. The AP alpha showed an abnormal decrease in Z scores in 5 of 11 patients, whereas other 5 patients presented normal scores. The AP theta presented 7 normal Z scores out of 11; this band exhibited the lowest number of abnormalities of the 4. CONCLUSION: Patients with JME have an increase in AP delta, alpha and beta bands, which is more evident in frontoparietal regions.

Changes in EEG current sources induced by neurofeedback in learning disabled children. An exploratory study.

The objective of this work was to explore Neurofeedback (NFB) effects on EEG current sources in Learning Disabled (LD) children, and to corroborate its beneficial consequences on behavioral and cognitive performance. NFB was given in twenty 30-min sessions to 11 LD children to reduce their abnormally high theta/alpha ratios (Experimental Group). Another five LD children with the same characteristics received a placebo treatment (Control Group). In the Control Group no changes in behavior or EEG current source were observed. In the Experimental Group, immediately after treatment children showed behavioral and cognitive improvements, but current source analysis showed few modifications; however, 2 months after treatment many changes occurred: a decrease in current of frequencies within the theta band, mainly in left frontal and cingulate regions, and enhancement in current of frequencies within the alpha band, principally in the right temporal lobe and right frontal regions, and of frequencies within the beta band, mainly in left temporal, right frontal and cingulate cortex regions. In conclusion, NFB is a possibly efficacious treatment for LD children with an abnormally high theta/alpha ratio in any lead. The changes observed in EEG current sources may reflect the neurophysiological bases of the improvement that children experienced in their behavioral and cognitive activities.

Effects of two different cycles of vagus nerve stimulation on interictal epileptiform discharges.

PURPOSE: To evaluate the effects of two cycles of vagus nerve stimulation (VNS), 30 s/5 min and 7 s/18 s on the interictal epileptiform discharges (IEDs). METHODS: Twenty patients were studied, 12 with generalized and 8 with partial seizures. An EEG of 120 channels was performed during 3 different conditions, each one lasting 30 min: basal state (BS), 30 s/5 min and 7 s/18 s VNS cycles. The number and duration of IEDs, time of IEDs in 1 min (TIEDM), IEDs/NIEDs index and the spike-free period (SFP) were determined. RESULTS: In 16 patients (80%), IED decreased during 30 s/5 min cycle (Group 1) and increased in 4 (Group 2). In Group 1, during the 30 s/5 min cycle the following variables showed a decrease: TIEDM, from 12.64 s to 9.62 s (p=0.001); IED/NIED index, from 0.53 to 0.31 (p=0.021), and IED duration, from 1.57 s to 1.05 s (p=0.015); whereas SFP duration increased from 20.06 s to 37.73 s (p=0.008). The decrease in IED was 41% and the increase in SFP 88%. In the 7s/18s cycle, only SFP had an increase, 72% (p<0.043). In Group 2, an increase in IED during both cycles was found. In the 30 s/5 min cycle, TIEDM increased 56% (p=0.042) and IED/NIED index 259% (p=0.040). CONCLUSION: VNS modifies IED in an acute form, in 80% of patients the 30 s/5 min cycle decreases the epileptiform activity and it is not modified by 7 s/18 s cycle. In 20% of patients, both cycles increase the epileptiform activity.

Interictal regional cerebral blood flow and electrical source analysis in patients with complex partial seizures.

BACKGROUND: Recently, new methods of EEG source analysis have been developed. Dipole modeling and brain distributed source analysis localize in three-dimensions the origin of the electrical source of spikes registered in EEG. With single photon emission computed tomography (SPECT), it is possible to detect hypo- and hyperperfusion zones. Our aim in this study is to compare the regions where the electrical sources are detected, with the hypoperfusion regions in patients with complex partial seizures (CPS). METHODS: The concordance of localization with dipole analysis, brain distributed source analysis and regional cerebral flow blood in patients with CPS was studied. The hypoperfusion zones detected with interictal SPECT were compared with electrical sources localized with brain electric source analysis (BESA) and brain distributed source analysis with variable resolution electromagnetic tomography (VARETA). RESULTS: Hypoperfusion zones were found to localize with the origin of dipoles in 18 cases (90%), between lobes in 17 (85%) and between mesial or lateral regions in the temporal lobe in 12 cases (60%). With VARETA, agreement between side of hypoperfusion and electrical current source localization was found in 18/20 cases (90%), with lobes in 17 (85%) and with mesial or lateral regions of the temporal lobe only in 2 cases (10%). CONCLUSIONS: Hypoperfusion zones in interictal SPECT of patients with CPS are in agreement with the origin of dipoles in 85% of the cases, but in specifics zones of temporal lobe the agreement falls to 60%. The concordance of hypoperfusion zones was better with dipole analysis than with VARETA.

Auditory steady-state responses in infants with perinatal brain injury.

Infants with perinatal brain injury present impairments in motor, visual, auditory, and cognitive functions. The most useful methods for detecting auditory alterations are auditory brainstem responses and otoacoustic emissions. Auditory steady-state responses have been reported as a reliable and objective technique for evaluating the hearing threshold. Auditory brainstem responses and auditory steady-state responses were carried out in 53 infants with perinatal brain injury and abnormal neurologic findings. With auditory brainstem responses, 33 (62.26%) infants presented normal and 20 abnormal results; 8 (15.09%) exhibited mild alterations, 8 (15.09%) moderate, and 4 (7.54%) severe alterations. With auditory steady-state responses, 17 (32.0%) infants were normal and 36 (67.9%) had abnormal results. When auditory steady-state responses were compared with auditory brainstem responses gold standard, the assessment gave 100% sensitivity, 51.51% specificity, 55.55% positive predictive value, and 100% negative predictive value. Abnormalities were mild in 21 (39.6%) infants, moderate in 10 (18.9%), and 5 (9.4%) exhibited severe hearing loss. We conclude that hearing loss is a frequent abnormality in infants with perinatal brain injury, and auditory steady-state responses have a high sensitivity for detecting hearing impairment, which is more evident in mild hearing loss for specific frequencies.

Specific EEG frequencies signal general common cognitive processes as well as specific task processes in man.

The EEG of 10 normal male young adults was recorded during the performance of three different tasks: mental calculation, verbal working memory (VWM) and spatial working memory (SWM). The stimuli used in the three tasks were the same, only the instructions to the subjects were different. Narrow band analysis of the EEG and distributed sources for each EEG frequency were calculated using variable resolution electromagnetic tomography (VARETA). At some frequencies (1.56, 4.68, 7.80 to 10.92 Hz) at least two tasks produced similar EEG patterns that were interpreted as the reflex of common cognitive processes, such as attention, inhibition of irrelevant stimuli, etc. Specific changes were also observed at 2.34, 3.12, 3.90, 5.46 and 6.24 Hz. The first three of these frequencies showed similar changes during VWM and calculus at the left frontal cortex, suggesting the activation of working memory (WM) processes. The interaction effect at these frequencies was mainly observed at the anterior cingulate cortex and frontal cortex. At 5.46 and 6.24 Hz, changes were only observed during mental calculation.

Variable resolution electromagnetic tomography (VARETA) in evaluation of compression of cerebral arteries due to deep midline brain lesions.

BACKGROUND: Hemispheric tumors produced electroencephalographic (EEG) delta activity mainly due to deafferentation of cerebral cortex. In small, deep midline lesions that compressed cerebral arteries, the most important abnormality should have been in EEG theta band that selectively responded to brain ischemia. Frequency domain-variable resolution electromagnetic tomography (FD-VARETA) has been applied satisfactorily to the study of brain tumors, cerebral infarcts, and brain hemorrhages and was shown to localize areas of hypoperfusion. METHODS: Twelve patients with deep midline lesions compressing different cerebral arteries were studied. Computer tomography (CT) and/or magnetic resonance imaging (MRI) as well as quantitative EEG with source calculation in frequency domain were obtained. Brain electromagnetic tomographies (BETs) were calculated to evaluate localization and extension of functional abnormalities. RESULTS: Ten of twelve cases presented abnormal sources in theta band as main abnormal source. In only two cases was the main source in delta band, but these cases also had abnormal Z values in theta band. In four patients there were only abnormal values in theta range. Sources of abnormal theta activity were observed in regions irrigated by the arteries compressed. CONCLUSIONS: In deep midline lesions, compression of cerebral arteries producing relative ischemia may explain abnormal EEG sources in theta band. Patients with main source in theta band showed vascular compression and some patients exhibited vasogenic edema. Thus, theta might be due to relative ischemia produced by both hypoperfusion and edema. Once again, VARETA has found to be very useful in evaluation of functional abnormalities.

Are poor readers semantically challenged? An event-related brain potential assessment.

This study explores visual event-related potentials components in a group of poor readers (PRs) and control children who carried out figure and word categorization tasks. In both tasks, every child had to categorize between animal and non-animal stimuli in an odd-ball GO-GO paradigm. During the word categorization task, PRs presented longer reaction times, a poorer performance, longer and larger P2 amplitudes, and smaller amplitudes and longer P300 latencies than controls. There were no differences in the N400 component between groups. These results suggest that semantic processing underachievement in PRs may not be a semantic deficit per se, but the late reflection of an early word codification problem, deficient use of attentional resources and lack of target identification during reading.

Sources of EEG activity in learning disabled children.

The sources of different EEG frequencies were studied in 25 normal children and 46 learning disabled (not otherwise specified) children between 7 and 11 years old. The EEG sources were computed using Frequency-domain Variable Resolution Electromagnetic Tomography which produces a three dimensional picture of the currents at each EEG frequency. Significant differences between groups were observed. LD children showed more theta activity (3.5 to 7.02 Hz) in the frontal lobes and control children more alpha (9.75 to 12.87 Hz) in occipital areas. These results may support the maturational lag hypothesis, as the neurobiological cause of learning deficiencies not otherwise specified.