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detection of 4-hydroxynonenal modified proteins. Free Radic. Res 1996;25: 149-1 59. Oda Y, Imai S, Nakanishi I, et al. Immunohistochemical study on choline ...
detection of 4-hydroxynonenal modified proteins. Free Radic Res 1996;25:149-1 59 17. Oda Y , Imai S, Nakanishi I, et al. Immunohistochemical study on choline acetyltransferase in the spinal cord of patients with amyotrophic lateral sclerosis. Pathol Int 1995;45:933-939 18. Lovell MA, Ehmann WD, Mattson MP, et al. Elevated 4-hydroxynonenal in ventricular fluid in Alzheimer’s disease. Neurobiol Aging 1997;18:457-461 19. Mark RJ, Lovell MA, Markesbery WR, et al. A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid P-peptide. J Neurochem 1997;68:255-264 20. Keller JN, Pang Z, Geddes JW, et al. Impairment of glucose and glutamate transport and induction of mitochondrial oxidative stress and dysfunction in synaptosomes by amyloid-P peptide: role of the lipid peroxidation product 4-hydroxynonenal. J Neurochem 1997;69:273-284 21. Keller JN, Mark RJ, Bruce AJ, et al. 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes. Neuroscience 1997;80:685-696 22. Blanc EM, Keller JN, Fernandez S, et al. 4-Hydroxynonenal, a lipid peroxidation product, inhibits glutamate transport in astrocytes. Glia 1998;22:149-160 23. Kostic V, Jackson-Lewis V, de Bilbao F, et al. Bcl-2: prolonging life in a transgenic mouse model of familial amyotrophic lateral sclerosis. Science 1997;277:559-562 24. Bruce-Keller AJ, Begley JG, Fu W, et al. Bcl-2 protects isolated plasma and mitochondrial membranes against lipid peroxidation induced by hydrogen peroxide and amyloid P-peptide. J Neurochem 1997;70:31-39

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Seasonal variation of Interferon-y Production in Progressive Multiple Sclerosis Konstantin E. Balashov, M D , PhD, Michael J. Olek, D O , Derek R. Smith, MD, Samia J. Khoury, M D , and Howard L. Weiner, MD

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. There is increased interferon (1FN)-y secretion in MS patients in vitro, and IF”-y administration induces exacerbations of disease suggesting a link between IFN-y and disease activity. We observed significantly increased IFN-y production in the autumn and winter months compared with the spring and summer months in chronic progressive MS, and this increase was linked to endogenous interleukin (1L)-12 production. Increased seasonal IFN-y was not observed in normal control subjects, and there were no seasonal changes in IL-10 in progressive MS. These results suggest a potential environmental link between dysregulated IFN-y production and MS disease progression and pathogenesis. Balashov KE, Olek MJ, Smith DR, Khoury SJ, Weiner HL. Seasonal variation of interferon-y production in progressive multiple sclerosis. A n n Neurol 1998;44:824- 828

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Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system postulated to be a Thl type T-cell-mediated autoimmune disease’ that is linked to environmental factors such as viruses.2 Interferon (1FN)-y, a cytokine that is the hallmark of Thl type immune responses, appears to play an important role in disease pathogenesis, as increased production of IFN-y precedes clinical attacks3 and treatment of MS patients with recombinant IFN-y-induced exacerbations of the disease.* Furthermore, within the nervous system, the inflammatory process is characterized by increased IFN-y expre~sion.~Activated peripheral blood mononuclear cells (PBMCs) also produce significantly higher levels of IFN-y in progressive MS,6 and we have recently observed that increased endogenous interleukin (1L)- 12 production via CD40-CD40 ligand interactions between antigen-presenting cells and T cells was responsible for upregulated T-cell receptormediated IFN-y secretion in progressive MS’ and that macrophages produce increased IL- 12 in progressive M S 8 MS is generally believed to be related to environmental factors, although these factors are not well defined. Correlations, however, have been found between viral infections and exacerbations of MS.9,10It has also been reported that attacks may vary according to the season.”,l2 We have been studying the mechanism of raised IFN-y secretion for the last 2 to 3 years. This provided the opportunity to determine whether seasonal variation in an immunological measure such as IFN-7, which is potentially linked to disease pathogenesis, varied depending on the time of the year.

Materials and Methods Subjects Chronic progressive MS patients consisted of 23 men and 37 women with a n average age of 46.8 2 7.4 years (mean t SD) a n d an EDSS of 5.4 1.6 (mean 2 SD). Most patients (45/60) had secondary progressive MS, and the remainder had primary progressive MS. Patients had not received immunosuppressive therapy in the past or steroid treatment in the 6 months prior to blood drawing. T h e control group consisted of healthy subjects (26 m e n and 27 women with a n average age of 40.1 ? 10.2 years [mean 2 SD]). Some (30%) samples included those reported in previous publication~.’,’~

*

From the Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. Received Dec 18, 1997, and in revised form Jun 11, 1998. Accepted for publication Jun 11, 1998. Address correspondence to Dr Weiner, Center for Neurologic Diseases, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Boston, MA 02115.

the American Neurological Association

Cell Culture and Cytokine Enzyme-Linked Immunosorbent Assay PBMCs (1 X 106/ml)were isolated in heparinized venous blood and stimulated with soluble anti-CD3 mAb (OKT3) as described previ~usly.~ Cytokine Enzyme-Linked Immunosorbent Assay Secretion of IFN-y and IL-10 was measured in 48-hour su-

pernatants by using cytokine enzyme-linked immunosorbent assay (ELISA) protocols and reagents from PharMingen (San Diego, CA) as previously described.’

Results Seasonal Variation of Anti-CD3-Induced IFN-y Secretion i n MS During the course of our previous studies,’ we noticed that raised levels of IFN-y secretion appeared to vary depending on the time of the year. Because we had

Fig 1. Seasonal variation of intevferon (IFN)-y production in progressive multiple sclerosis (MS). Peripheral blood mononuclear cells (PBMCs) (1 X l@/ml) were isolated from progressive M S patients and tested in the spring (March-May) (n = 28), summer @me-August) (n = I2), autumn (SeptemberNovember) (n = 15), and winter (December-Februay) (n = 18) months or from healthy control subjects in the spring (n = 8), summer (n = 8), autumn (n = 12), and winter (n = I @ months. PBMCs were stimulated with anti-CD3 mAb as described in Materials and Methods section. AfZer 2 days, the levels of IFN-y production in culture supernatants were measured by enzyme-linked immunosorbent assay and are expressed as the mean ? SEM in picograms per milliliter. IFN-y production of progressive M S patients tested in the autumn (2,307 2 365 pg/ml) was significantly higher (p < 0.005)than it was in PBMC cultures from M S patients tested in the spring (737 2 157 pglml), from MS patients tested in the summer (687 2 223 pg/ml), or from healthy control subjects tested in the autumn (769 +- I53 pg/ml). IFN-y production of progressive MS patients tested in the winter (I,67G ? 373 pg/ml) was significantly higher (p < 0.05)that it was in PBMC cultures from M S patients tested in the spring (737 -+ 157 pg/ml) or from healthy control subjects tested in the winter (784 -t I05 pg/ml).

** Controls H CPMS

Spring

Summer

i

Autumn

Winter

studied this immunological measure over a 2-year period, we were able to analyze our data based on the time of the year when blood was drawn. As shown in Figure 1, maximal IFN-y production in progressive MS patients was detected during the autumn and winter months (1,962 2 264 pg/ml, n = 33) as compared to that in progressive MS patients studied during the spring and summer months (722 5 127 pg/ml, n = 40, p < 0.001) or as compared with that in healthy control subjects during the autumn and winter months (778 2 87 pg/ml, n = 28, p < 0.001). The peak of IFN-y production in progressive MS was detected in period from October through December (2,532.6 f 353.4, n = 20). We also measured IL-10 as a control cytokine in the same cell supernatants. There were no significant variations in the levels of IL-10 detected in progressive MS patients during the autumn and winter months (1,295 -t 201 pg/ml, n = 23) as compared with the spring and summer months (1,424 f 268 pg/ml, n = 21). In addition, when the ratio of IFN-y to IL-10 for the same cell supernatants in progressive MS patients was calculated in the autumn and winter months (4.1 2 1.4, n = 22) and compared with that in the spring and summer months (0.54 2 0.2, n = 21), it appeared significantly higher ( p = 0.019) in the autumn and winter period. Although these studies were not designed as serial measurements in individual patients, we were able to identify 4 patients and 4 control subjects who had blood drawn at two different time points. As shown in Table 1, for this small group of subjects, there was a significant difference in IFN-y secretion for MS patients depending on when their blood was drawn which was consistent with what we observed in the large cohort of patients studied. To determine if patients experienced relapses or clinical progressions during the months when the IFN-y levels were highest, we retrospectively analyzed the clinical records of EDSS scores. The EDSS in patients seen during the October through December period worsened by 0.55 points in the 6-month period associated with elevated IFN-y, whereas the EDSS in those patients seen during the March through August period worsened by 0.52 points. Only 1 patient reported a respiratory tract infection at the time of the visit. Furthermore, in a separate immunological study at our Center for Neurologic Diseases, 7 progressive MS patients were tested serially two to three times over 1 year using the same protocol of anti-CD3induced IFN-y secretion. For this study, measurement of cytokines was performed with a different IFN-y ELISA kit (Biosource International, Camarillo, CA) using a different recombinant IFN-y standard which gave higher IFN-y levels. For the 7 progressive MS patients, IFN-y levels were 11,282 2 1,634 pg/ml in

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Table. Serial Measurements of IFN-y Production in Individual Subject? September-January

February-August

Subject

IFN-y (pg/ml)

Time

IFN-y (pg/ml)

Time

MS MS MS MS

3,698 2,190 1,394 2,083

December 1993 October 1994 September 1994 November 1994

298 412 535

June 1995 April 1995 March 1995 February 1995

1 2

3 4

Mean 5

SEM

Control Control Control Control

1 2 3 4

338 627 445 298

Mean

SEM

427 2 74

I+_

155

2,342 2 485b

350 ? 81 October 1994 October 1994 January 1996 January 1996

April 1995

375 64 1 236 220

August 1995 August 1994 August 1994

368 2 97

"Peripheral blood mononuclear cells (PBMCs) were separated from either healthy control subjects or chronic progressive multiple sclerosis (MS) patients on 2 different days. PBMCs (1 X 106/ml) were activated with anti-CD3 monoclonal antibody (1 g/ml) for 48 hours. Cell culture supernatants were collected, and interferon-? was measured (in picograms per milliliter) by enzyme-linked immunosorbent assay as described in the Material and Methods section. ' p < 0.01 versus both chronic progressive MS patients in February through August and controls in September through January.

the October through December period and 4,537 2 1,381 pg/ml in the February through August period ( p < 0.01). Endogenous IL-12 Mediated Seasonal Upregulation of IFN-y Secretion in Progressive M S Patients We have observed that there is increased IL-12 production by anti-CD3-activated PBMCs in progressive MS.' To determine whether endogenous IL-12 was involved in the seasonal increase of IFN-y secretion by activated PBMCs, we added neutralizing anti-IL- 12 antibody to PBMC cultures of progressive MS patients tested during different seasons. As shown in Figure 2 , neutralization of endogenous IL-12 led to significant suppression of IFN-y production in the group of progressive MS patients tested during the autumn period (2,443 -t 446 pg/ml without neutralizing antibody; 843 -t- 159 pg/ml with neutralizing anti-IL-12, n = 12, p < 0.005). In contrast, no effect of neutralizing anti-IL- 12 was detected in the group of progressive MS patients tested in the spring period or in healthy control subjects tested in both the autumn and spring periods. IL- 10 secretion was not significantly changed in the presence of neutralizing anti-IL-12 antibody (data not shown).

Discussion The seasonal variations in the levels of IFN-y in progressive MS that we observed are consistent with reports describing seasonal variations in the frequency of onsets and exacerbations in relapsing-remitting MS. Interestingly, seasonal changes in MS course varied in different geographical regions. For example, the peak exacerbation rate in relapsing-remitting MS in north-

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without neutralizing Ab neutralizing anti-IL-12 Ab h

B

2 2

2000

v

940

E

1OW

Y

.

Controls in Autumn

CP MS

in Autumn

Controls in Spring

CP MS in Spring

Fig 2. Endogenous interleukin (IL)-12 mediates increased interferon (IFN)-y production in progressive multiple sclerosis (MS) patients. Peripheral blood mononuclear cells (PBMCs) (I X I&/ml) from progressive MS patients tested in the autumn (n = 12) or in the spring (n = 7) and from healthy control subjects tested in the autumn (n = 9 ) or in the spring (n = 3) were stimulated with anti-CD3 mAb ds described in Materials and Methods section. Neutralizing goat anti-human IL-12 (10 pg/ml, R d D Systems, Minneapolis, M N ) was added to the parallel cultures. AJter 2 days, the levels of IFN-y production in culture supernatants were measured by enzyme-linked immunosorbent assay and are expressed as mean ? SEM in picograms per milliliter. IFN-y production o f progressive M S patients tested in the autumn in the presence of 10 p g h l of neutralizing anti-IL-12 antibody (843 2 159 pglml) was significantly reduced (p < 0.00s) versus that of cell cultures from the same patients tested without neutralizing A6 (2,443 2 4 4 6 p g h l ) .

east Ohio was observed in the July through October period,' in the winter and spring months in Switzerland," and in the warmer months in Arizona." A correlation between viral upper respiratory tract infections and exacerbations of relapsing-remitting MS has been rep~rted,'~'"but the effect of infections on progressive MS has not been investigated. The marked changes in the levels of IFN-y in progressive MS patients that we observed in the September to December period may be related to different local environment factors in New England. The change in temperature, risk of infections, and other factors may also be involved. Many infections such as respiratory syncytial virus, parainfluenza type 3, rotavirus, rubella, and parvovirus B19 undergo cycles lasting 1 year with marked seasonal variations.'* It is unknown which defects in the immunoregulation of IFN-y (or IL-12) expression are responsible for the seasonal cytokine upregulation that we observed in progressive MS patients but not in healthy control subjects. Of note, only 1 of 60 patients we studied had a clinically evident upper respiratory tract infection at the time of the blood drawing. Another theoretical possibility is that the amount of sunlight could also play a role, as there is decreased vitamin D in the winter months and the active form of vitamin D has been reported to inhibit IFN-y ~ecretion.'~ Also, W irradiation has immunomodulatory effects and UV exposure is lowest during the October to February period.I6 Within the nervous system, the inflammatory process in MS plaques is characterized by increased IL-12 p40 messenger RNA expression" and an increased number of CD4+ T cells expressing the CD40 ligand." IL-12 is a potent cytokine which upregulates IFN-y in most systems," and IFN-y also enhances IL-12 production by means of activated antigenpresenting cells in our system.' Thus, regulation of these two cytokines (IL-12 and IFN-y) is closely related. IL-12 can upregulate both IFN-y and IL-10 in the same human T-cell cultures.'" Neutralizing antiIL-12 Ab was effective only in decreasing the increased levels of IFN-y produced by activated PBMCs, however. N o effect of neutralizing anti-IL-12 on the levels of IL-10 from the same PBMC cultures was detected. Neutralizing anti-IL-2 Ab, however, downregulated the levels of both IFN-y and IL-10 produced by activated PBMCs in both MS patients and controls (data not shown), consistent with the role of IL-2 as a generalized growth factor associated with T-cell activation. Understanding the environmental factors that are involved in the regulation and triggering of the Thl type-associated cytokines IL-I2 and IFN-y in progressive MS may help to elucidate immunopathological mechanisms in MS and ultimately provide new approaches for disease treatment.

This study was supported by NIH grant NS23132, the National Multiple Sclerosis Society, and the Nancy Davis Center without Walls.

References 1. Martin R, McFarland HF, McFarlin DE. Immunological as-

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pects of demyelinating diseases. Annu Rev Immunol 1992;10: 153-187 Johnson RT. The possible viral etiology of multiple sclerosis. Adv Neurol 1975;13:1-45 Beck J, Rondot P, Catinot L, et al. Increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger off exacerbations? Acra Neurol Scand 1988;78:3 18-323 Panitch HS, Hirsch RL, Haley AS, et al. Exacerbations of multiple sclerosis in patients treated with gamma interferon. Lancet 1987;1:893- 896 Woodroofe MN, Cuzner ML. Cytokine mRNA expression in inflammatory multiple sclerosis lesions: detection by nonradioactive in sku hybridization. Cytokme 1993;5:583-588 Noronha A, Toscas A, Jensen MA. Interferon beta decreases T cell activation and interferon gamma production in multiple sclerosis. J Neuroimmunol 199346:145-1 53 Balashov KE, Smith DR, Khoury SJ, et al. Increased IL-I2 production in progressive multiple sclerosis: induction by activated CD4+ T cells via CD40 ligand. Proc Natl Acad Sci USA 1997; 94599- 603 Comabella M, Balashov K, Issazadeh S, et al. Elevated interleukin-12 in progressive multiple sclerosis correlates with disease activity and is normalized by pulse cyclophosphamide therapy. J Clin Invest 1998; 102:671-678 Sibley WA, Bamford CR, Clark K. Clinical viral infections and multiple sclerosis. Lancet 1985;1:1313-1315 Anderson 0, Lygner R-E, Bergstrom T, et al. Viral infections trigger multiple sclerosis relapses: a prospective seroepidemiological study. J Neurol 1993;240:417-422 Wuthrich R, heder HP. The seasonal incidence of multiple sclerosis in Switzerland. Eur Neurol 1970;3: 157-164 Bamford CR, Sibley WA, Thies C. Seasonal variation of multiple sclerosis exacerbations in Arizona. Neurology 1983;33: 897-901 Smith D, Balshov K, Hafler D, et al. Immune deviation following cyclophosphamide/methylprednisolonetreatment of multiple sclerosis: increased IL-4 and associated eosinophilia. Ann Neurol 1997;42:313-318 Noah ND. Cyclical patterns and predictability in infection. Epidemiol Infect 1989;102:175-190 Amento EP. Vitamin D and the immune system. Steroids 1987;49:55-72 Araneo BA, Dowel1 T, Moon HB, Daynes RA. Regulation of murine lymphocyte production in vivo: ultraviolet radiation exposure depresses IL-12 and enhances IL-4 production by T cells through an IL-I-dependent mechanism. J Immunol 1989;143: 1737-1 744 Windhagen A, Newconibe J, Dangond F, et al. Expression of costimulatory molecules B7-1 (CD80), B7-2 (CD86), and interleukin 12 cytokine in multiple sclerosis lesions. J Exp Med 1995;182: 1985-1 996 Gerritse K, Laman JD, Noelle RJ, et al. CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Proc Natl Acad Sci USA 1996;93:2499-2501 Trinchieri G. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu Rev Immunol 1995; 13:251-276

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20. Windhagen A, Anderson D, Carrizosa A, et al. IL-12 induces human T cells secreting IL-I0 with IFN-7. J Immunol 1996; 157:1127-1131

Abnormal Somatosensory Homunculus in Dystonia of the Hand William Bara-Jimenez, M D , Maria Jose Catalan, M D , Mark Hallett, M D , and Christian Gerloff, M D

physiological mapping techniques. The monkeys were also noted to have abnormal hand control and performed poorly on motor tasks. Although it remains speculative whether or not the motor difficulties seen in these monkeys are equivalent to human dystonic movements, the investigators proposed that learninginduced dedifferentiation of the sensory cortex may contribute to the genesis of dystonia. This highly interesting proposal, which is of potential therapeutic relevance, was based on the speculative analogy that the S1 is abnormally organized in patients with dystonia. In the present study, we tested the hypothesis that the organization of finger representations in the S1 of dystonic patients is abnormal.

Patients and Methods Abnormalities of the sensory system have been proposed as causative factors for dystonia. By mapping the human cortical hand somatosensory area of 6 patients with focal dystonia of the hand, we found an abnormality of the normal homuncular organization of the finger representations in the primary somatosensory cortex (SI). Although a remote antecedent event or even a developmental anomaly cannot entirely be ruled out, our findings may support the concept that abnormal plasticity is involved in the development of dystonia. Bara-Jimenez W , Catalan MJ, Hallett M, Gerloff C. Abnormal somatosensory homunculus in dystonia of the hand. Ann Neurol 1998;44:828- 83 1

sensory system In addition to motor disturbances, abnormalities have been reported3 in patients with dystonia. Focal dystonia may occur in persons working under conditions of repetitive movements and sensory inputs (ie, occupational hand cramps) .* Repetitive peripheral sensory stimulation and movements induced plastic changes of the primary somatosensory cortex (S1) in animal studies of learning and neuroplasticit^.^,' Similarly, plasticity-mediated cortical S 1 reorganization in humans followed the alteration of afferent inputs.' In a study of the relationship between repetitive movements and cortical plasticity in monkeys subjected to repetitive hand opening and closing,' plastic reorganization of the hand area in the S1 occurred after months of training as demonstrated by invasive electro-

From the Human Motor Control Section, Medical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD. Received Oct 27, 1997, and in revised form May 26, 1998. Accepted for publication Jun 10, 1998. Address correspondence to Dr Hallett, NINDS, NIH, Building 10, Room 5N226, 10 Center Drive, MSC-1428, Bethesda, MD, 20892-1428.

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We mapped cortical S1 finger representations in 6 righthanded patients (mean age, 52 years) with dystonia (mean duration, 8.8 years) of the right hand. Motor function was assessed by the Burke-Fahn-Marsden dystonia movement scale.' Mean score on this scale was 4.6 (range, 2.0 to 12.0, indicating mild to severe degrees of dystonia). In 5 patients, symptoms were limited to the right hand. Another patient had additional evidence of mild right-sided torticollis and a history of spasmodic dysphonia. Three subjects had taskspecific dystonia (writing in 2, instrument playing in 1). In the remaining 3 patients, dystonic symptoms were associated with several different tasks or even at rest (2/6 patients). There was no evidence of left hand dystonia. All results were compared with those from 6 normal (control) subjects closely matched with the patients for the variables of age and gender. All subjects had normal neurological examinations, except for dystonia in the patients. Study protocols were approved by the Institutional Review Board; all subjects gave their written informed consent for the study.

Topographic Mapping The N20 peak of somatosensory evoked potentials (SEPs) was used for mapping as it is known to be generated in the anterior bank of the postcentral gyrus" and has been used successfully to demonstrate homuncular organization of the S1 hand area in normal SEPs were recorded while the thumb ( D l ) or little finger (D5) of the right hand was stimulated by ring electrodes applied to the middle and distal phalanges, with the anode placed proximally. The stimulus was square-wave electrical pulses delivered at a rate of 1.7 Hz for 0.2 msec via a Grass S 1 1 stimulator (Grass Intruments, Astro-Med, Inc, West Wanvick, RI). Four blocks of 250 trials were recorded and later averaged to obtain a total of 1,000 responses for each finger. Finger stimulation was performed randomly to avoid the effect of order. Data were acquired from 122 tin surface electrodes (average spacing -2 cm) mounted in a cap. Signals were sampled at a rate of 5000 Hz. The recording band pass was set at 1 to 1,000 Hz (direct current amplifiers and software by NeuroScan Inc, El Paso, TX). Linked ears served as a reference. A moving dipole model was employed for the source reconstruction, and the x, y, and z coordinates of the respective generators were obtained at the latency where the residual