Brain Research, 99 (1975) 49-58
49
(~ ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
EVIDENCE T H A T M O N O A M I N E S I N F L U E N C E H U M A N EVOKED POTENTIALS
E D W A R D W. P. SCHAFER AND CHARLES M. M c K E A N
Brain-Behavior Research Center, Langley Porter Neuropsychiatric Institute, University of California, Sonoma State Hospital, Eldridge, Calif. 95431 (U.S.A.) (Accepted May 20th, 1975)
SUMMARY
We have measured latency and flash/pattern differentials of the visual evoked potential (VEP) from phenylketonuric humans, while systematically manipulating rates of amine synthesis in the central nervous system using two techniques. We have observed that stimulation of monoaminergic activity in the visual processing system, either by lowering inhibitory levels of phenylalanine through dietary restriction or by a properly balanced administration of indole and catecholamine precursors, shortened VEP latencies and permitted the development of a discriminative brain response to patterned stimuli. The close temporal relationship between these electrophysiological changes and the neurocbemical manipulations following treatment initiation or discontinuation argue that monoamines play a significant role in the mediation of human sensory evoked potentials.
1NTRODUCTION
In an earlier report 24 concerning the effects of high phenylalanine levels on brain function, we noted that the excessive phenylalanine which characterizes phenylketonuria markedly inhibits central nervous system synthesis of the catecholamines and serotonin with associated reductions in cerebral concentrations of these monoamines and their amino acid precursors. Conversely, lowering plasma phenylalanine levels by dietary restriction to below 15 mg~o reduces this inhibition of cerebral amine metabolism. In the present studies, conducted concurrently to the above, we have attempted to determine the functional significance of the metabolic alterations noted previously. Specifically, does manipulation of phenylalanine levels result in observable changes of electrophysiological function as indicated by sensory evoked potentials? And further, if such electrophysiological changes occur, can one link them causally to the
50 associated alterations in the metabolism of biogenic amines thereby supporting their presumed role as neuromodulators? Two parameters of the visual evoked potential (VEP), component latency (poststimulus delay time) and flash/pattern differentiation, seemed appropriate for this investigation. Component latency provides a particularly useful measure because of its high reliability and long-term stability. Further, component latency varies predictably with pharmacological manipulation z4, decreases systematically with advancing maturation 4,7,8 and remains abnormally prolonged in humans suffering developmental retardationZ,3,9,1z, 36. The appearance of differential evoked responses to flash and pattern stimuli represents additional evidence of the brain's developing capacity for more precise edge detection. Mature humans with normal visual acuity produce differential occipital evoked potentials to flash and checkerboard pattern stimuli 1°,2s,35. Infants manifest no differential response. Production of this flash/pattern differential occurs only with increasing maturation of electrophysiological function ls,22. Thus, we measured VEP latency and flash/pattern differentials in phenylketonuric (PKU) humans while systematically manipulating the rate of amine synthesis in the central nervous system. In order to separate aminergic effects from the other metabolic consequences of excessive phenylalanine, which might also alter electrophysiological function, we employed two techniques to increase the rate of amine synthesis: (1) reduction of inhibitory concentrations of phenylalanine through dietary restriction, and (2) administration of the amine precursors L-tyrosine or L-3,4dihydroxyphenylalanine (L-DOPA), and L-5-hydroxytryptophan (L-5-HTP) without reducing the high phenylalanine levels. Consistent and significant alteration in our evoked potential parameters associated with altered cerebral amine synthesis would provide persuasive evidence for monoaminergic mediation of sensory information processing in the human brain. Previous work with animals 5,6,a°,31 has suggested such mediation. METHODS
Subjects and treatment Four profoundly retaxded, previously untreated adolescent (ages 17-18 yr) phenylketonuric patients (3 male, 1 female) served as experimental subjects. We precisely controlled and monitored blood phenylalanine concentrations in the 3 male patients by means of a totally synthetic diet of known phenylalanine composition as described previously2a. More recently, we demonstrated that by controlling the phenylalanine concentrations in blood we could also control the rate of cerebral monoamine metabolism in PKU subjects 24. We undertook the metabolic manipulations concurrently to the evoked potential measures described below. Minimal cerebral monoamine turnover occurred when blood phenylalanine exceeded 19 m g ~ while maximal turnover occurred when phenylalanine concentrations fell below 6 rag %. We compared the etectrophysiologieal responses under these two metabolic conditions. In a second series of experiments, we gave all 4 subjects the monoamine pre-
51 cursors L-tyrosine or L-DOPA and/or L-5-HTP while maintaining their blood phenylalanine concentrations above 19 rag%. We individualized the dosage of the precursor amino acids for each subject in order to obtain optimal shortening of visual evoked potential latencies. In each case precursor administration achieved demonstrable increase in metabolism of the respective amine as described previously 24. The administration of tyrosine or L-DOPA without L-5-HTP produced increased cerebral synthesis of dopamine but decreased serotonin turnover. This imbalanced condition actually produced prolongation of VEP latencies (see Table 1V). Consequently, we carefully controlled the catecholamine precursors (3.7 g tyrosine/day or 1200-1500 mg L-DOPA/day) while maintaining L-5-HTP between 300 and 600 mg/day in order to achieve optimal monoaminergic balance as indicated by the VEP response. Evoked potential measures To record his visual evoked potential, each subject sat or reclined in a darkened, shielded enclosure watching a frosted window through which an accompanying observer presented photic stimuli whenever the patient showed quiet attention to the window. We placed before this window either a checkerboard pattern transparency with checks subtending a visual angle of 20 rain at a viewing distance of 60 cm or for flash stimuli, a neutral density filter, which equated the total luminous flux of flash stimuli with that passed by the pattern transparency. Checks subtending a visual angle of from 15 to 30 min produce pattern VEPs of greatest amplitude la,12. An occipital electrode on Oz referred to A2 (10-20 electrode convention)provided EEG in a bandwidth 3dB down at 3 and 20 Hz. A PDP/8 computer averaged 200 taperecorded EEG responses to flash and 200 responses to pattern in split-half response sets of 100 each using a 500 Hz digitization rate over a post-stimulus epoch of 500 msec. Since these profoundly retarded subjects could not always cooperate fully, we encountered some difficulty in obtaining consistently reliable VEPs. Consequently, we discarded all experiments in which the latencies in milliseconds and amplitude in microvolts of VEP components FP1 and FN1 in each split-half of 100 responses varied more than ± 5 %. In all subjects, the latency measurements of the FP1 and FN1 components to flash showed sufficient reliability by this criterion to permit statistical analysis. However, within-trial amplitudes of P1 and N 1 and the latency of P2 showed less than desired stability especially in the less cooperative subjects so that we could get the necessary data for flash/pattern differentials on only 2 of the 4 subjects despite many hours of effort. After making a reliability determination, we summated all 200 responses to flash and computer scored the latencies of the FP1 and FN 1 components. These components, occurring at approximately 100 msec (FP1) and 150 msec (FN1) constitute the most prominent potentials of the occipital evoked response to flash 2v. On an X-Y plot we superimposed the VEPs to pattern and flash, characterizing as 'differential responses' only those pattern VEPs displaying the waveform configuration described consistently by many workersl°,19,26,2s; i.e., an occipital VEP to the appearance of patterned stimuli which shows a triphasic waveform with a small, surface-positive deflection at 70-90 msec followed by a surface-negative component at
52 TABLE I VEP LATENCYDIFFERENCESBETWEENHiGH (:- 19 MG~ ) AND LOW ('4~ 6 MG ~ ) SERUMPHENVLALANINE CONDITIONS
Subject
A A B B C C
V E P component
FP1 FN1 FP1 FN1 FP1 FN1
High Phe
L o w Phe
N
mean
S.D.
N
mean
S.D.
20 20 17 17 13 13
117.2 149.9 113.5 153.6 129.3 210.4
5.9 10.7 3.5 5.0 5.0 10.5
11 11 32 32 10 10
104.3 135.0 108.3 149.7 114.2 182.0
5.3 5.4 3.9 3.7 14.0 19:5
t
P
5.96 5.14 4.73 2.84 3.25 4.17
0.001 0,001 0.001 0.01 0.005 0.001
110-130 msec and a surface-positive wave at 180-200 msec (components PP1, PN1 and PP2 in Fig. 1B). To clarify any evoked potential changes contingent upon manipulation of monoamine metabolism, we also recorded the electroretinogram (ERG) to flash and pattern stimulation from the 3 male patients in the untreated, high phenylalanine condition. Further, an optometrist performed a retinoscopic examination on these 3 patients to determine their non-accommodated refractive error in the high phenylalanine condition. This examination revealed minimal refractive error for 2 of the patients while the third wore optical correction. This evaluation indicated that all 3 subjects had sufficient visual acuity to focus sharply the checkerboard pattern. We also recorded auditory evoked potentials from the vertex (Cz-A1) to loudspeaker-presented clicks at 70 dB sensation level from the 3 male patients under each condition of high or low plasma phenylatanine and scored these records for the latency of the classical Pz component20. We statistically analyzed mean VEP latency values for each subject and for grouped data under conditions of high and low serum phenylalanine and amine precursor administration using the t-test for both independent and correlated measures: RESULTS
Electroretinographic recordings taken from the 3 male patients under the high phenylalanine condition revealed normal latency and waveform configurations for the a- and b-waves of the ERG in response to both flash and pattern stimulation suggesting that the high phenylalanine concentrations left the catecholaminergic and serotonergic receptors in the retina relatively unaffected. The latency of the principal positive and negative occipital flash VEP compo. nents (FP1 and FN1)decreased significantly in all 3 male PKU patients when their plasma phenylalanine concentrations felt from untreated levels to 5.9 mg 9/0and below as a result of dietary therapy (Table I). The magnitude of this latency decrease be-
53 PPll'~ I | I I I |
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Fig. 1. Occipital visual evoked potentials to flash and checkerboard pattern stimuli from subject A under conditions of high and low serum phenylalanine. Note differential, triphasic (PP1, PN1, PP2) VEP to pattern under low phenylalanine condition not observed with high phenylalanine. tween conditions of high (above 19 mg ~ ) and low (below 6 mg ~ ) plasma phenylalanine ranged from 2 ~o to 13 ~o, and averaged a difference of 9 ~ . In parallel, the latency of the major positive (P2) component of the auditory vertex evoked potential also decreased by a significant mean 7 ~ between conditions of high and low plasma phenylalanine, as noted in data from the 3 male patients combined (t -- 4.41, df = 5, P < 0.01). The data provide clear evidence of a direct relationship between plasma phenylalanine levels and the latency of sensory evoked potential components in two modalities and from two cortical locations.
54 TABLE II EFFECT OF BALANCED AMINE PRECURSOR ADMINISTRATION ON V E P COMPONENT LATENCY IN PRESENCE OF HIGH PHENYLALANINE
Subject
A A B B C C D
VEP component
FP1 FN1 FP1 FN1 FP1 FN1 FP1
High Phe
Precursors
N
mean
S.D.
N
mean
S.D.
20 20 17 17 13 13 10
117.2 149.9 113.5 153.6 129.3 210.4 117.7
5.9 10.7 3.5 5.0 5.0 10.5 1.6
6 6 9 9 14 14 4
108.2 131.2 107.4 146.2 109.8 163.2 114.2
3.7 3.3 3.1 2.8 7.9 8.9 0.9
t
P
4.49 6.80 4.54 4.83 7.67 12.54 4.91
0.001 0.001 0.001 0.001 0.001 0.001 0.001
Analysis of reliable flash and pattern VEPs from 2 of the 3 diet-restricted subjects also indicated a relationship between plasma phenylatanine concentrations and the incidence of differential evoked potentials to flash and pattern stimuli. Fig. 1A and B illustrates undifferentiated and differentiated VEPs to flash and pattern. In the high phenylalanine conditions prior to dietary therapy, subject A failed to produce a single differentiated flash/pattern VEP (0 of 10). On the other hand, when we maintained his plasma phenylalanine below 16 mg ~ as a result of dietary restriction, he produced 100 ~o (61 of 61) differential evoked potentials over a 6-month period as illustrated in Fig. 1B. When his phenylalanine concentration exceeded 18 mg ~o he again reverted to the production of undifferentiated flash/pattern VEPs. Corroborating these findings, subject B gave only 33 ~o (3 of 9) differential VEPs under the high phenylalanine condition but 100 ~ (3 of 3) differential VEPs to flash and pattern with low plasma phenylalanine. Thus, without exception, reduction of phenylalanine levels resulted in an increased production of differential brain responses to flash and pattern. Having demonstrated an association between plasma phenylalanine level and our electrophysiological parameters, we then sought to establish whether the altered amine metabolism rather than some other metabolic consequence of excessive phenylalanine, had produced the observed changes in sensory evoked potentials. We examined the results of those experiments in which we stimulated monoamine metabolism by orally administering the precursor amino acids in the presence of high plasma phenylalanine (Tables II, III and IV). The latency of FP1 and FN1 decreased promptly in all 4 P K U patients when TABLE III INCIDENCE OF
VEP FLASH/PATTERN DIFFERENTIAL RESPONSK$ UNDER VARIOUS METABOLIC CONDITIONS
Subject
High phenylalanine
Low phenylalanine
High phenylalanine and precursors
A B
0% (0 of 10) 33 700(3 of 9)
t00% (61 of 61) 10070 (3 of 3)
100% (6 of 6) 75 70 (6 of 8)
55 TABLE IV VEP FLASH/PATTERN DIFFERENTIALS AND MEAN LATENCIES WITH VARIOUS AMINE PRECURSOR COMBINATIONS FOR SUBJECT A
% F/P differential
High Phe + 5-HTP (400-600 mg) -~ 5-HTP (300 mg) tyrosine (7.4 g) + 5-HTP (500 mg) -~ tyrosine (3.7 g)
Mean FP1- latency
0~ (0 of 10) 0 ~ (0 of 6)
117 msec 121 msec
0 ~ (0 of 2)
123 msec
100 ~ (6 of 6)
108 msec
they received a properly balanced administration of the amine precursors I.-tyrosine (3.7 g/day) or L-DOPA (1200-1500 mg/day) and L-5-HTP (300-6130 rag/day) in the presence of high plasma phenylalanine concentrations. These latency decreases, associated with amine precursor administration, ranged from 3 ~o to 22 ~o and averaged a difference of 9 ~ , which paralleled decreases observed when we increased monoamine synthesis by restriction of phenylalanine. In the two more cooperative subjects we also observed an increased incidence of differential flash/pattern VEPs upon administration of the amine precursors under the high phenylalanine condition. Subject A, who produced no differential VEPs under conditions of high plasma phenylalanine alone (0 of 10), generated 100~o (6 of 6) differential VEPs when given a properly balanced administration of amine precursors (L-tyrosine, 3.7 g; I.-5-HTP, 500 mg) in the presence of high phenylalanine concentrations. Subject B, who gave 33 ~ differential responses under the high phenylalanine condition, produced 75 ~ (6 of 8) differential VEPs to flash and pattern when given L-DOPA (1500 mg) and L-5-HTP (600 rag) in the presence of high plasma phenylalanine. The relative amounts of indole and catecholamine precursors administered critically affected both VEP latency and the incidence of flash/pattern differentials as indicated by results presented in Table IV. When subject A received either of the amine precursors alone or in excess, he manifested no differential flash/pattern responses and his VEP latencies showed values even longer than under the high phenylalanine condition alone. However, within 24 h of receiving an appropriate combination of L-tyrosine (3.7 g) and L-5-HTP (500 rag), subject A produced a clearcut flash/ pattern differential response and the latency of his FP1 and FN1 components decreased significantly. In summary, the balanced stimulation of monoaminergic activity in the visual processing system, either by lowering inhibitory levels of phenylalanine or by administration of amine precursors, shortened VEP latencies and permitted production of a discriminative brain response to patterned stimuli. Finally, in spite of these subjects' extremely limited behavioral repertoire, we
56 could record in 2 of the 4 subjects a few discrete changes closely associated with dietary alterations. When we initially restricted the diet of these PKU subjects and their plasma phenylalanine levels fell below t5 m g ~ , we observed an increase in speech production, a decrease in self-injurious behavior and a change from whining petulance to smiling cooperation. These positive changes reverted when the patients' plasma phenylalanine concentrations again rose above 20 mg ~,iDISCUSSION
We have found that stimulation of cerebral amine synthesis in PKU subjects, either by lowering plasma phenylalanine or by administering amine precursors in the presence of high phenylalanine concentrations, results in significant changes in their electrophysiological function: specifically, a decrease in the latency of evoked potential components in two different sensory modalities (visual and auditory) and from two cortical locations, as well as an increased incidence of maturationally significant flash/ pattern differential VEPs over occipital cortex. Apparently, these electrophysiological functions depend on the balanced mediation of both serotonergic and catecholaminergic neural mechanisms since neither of the amine precursors by themselves resulted in a latency decrease or the more frequent appearance of a differential response to flash/pattern. These data strongly suggest that the electrophysiologicat malfunctions in phenylketonuria do not result primarily from the high concentration of phenylalanine or its metabolites, because, despite this pathogenic condition, the amino acid precursors which singly have no ameliorative effect can, in combination, reverse the VEP abnormalities. Further, the close temporal relationship between the electrophysiological changes and the neurochemical manipulations as well as their prompt reversibility upon withdrawal of therapy argue persuasively for a definitive role by monoamines in the mediation of sensory evoked potentials. The latency differences resulting from lowered phenylalanine concentrations or amine precursor administration parallel the latency differences reported for both visual2,9 and auditory13 evoked potentials between non-PKU retardates and normal subjects. One might speculate that by increasing the rate of cerebral monoamine synthesis through dietary restriction of phenylalanine or administration of balanced amounts of amino acid precursors, electrophysiological functioning in these PKU subjects proceeded more efficiently as indicated by a latency decrease comparable to the latency differences reported between normals and retarded humans. We also noted associated behavioral improvement following phenylalanine restriction which appeared to revert when phenylalanine concentrations rose above 20 mg ~. Certain psychotropic agents which alter aminergic activity and have weltdocumented behavioral effects also produce changes in evoked potential latency. The latency changes we have observed associated with alteration in cerebral amine synthesis show comparable magnitude to the small but significant latency decreases observed in somatosensory evoked potentials upon administration of dextroamphetamine32 and to the latency increases noted with chlorpromazinea~. Dextroamphetamine presumably increases synaptic catecholamine concentrations by blocking
57 neuronal reuptakO while chlorpromazine decreases catecholaminergic activity by blocking post-synaptic receptors za. Besides the generalized latency changes observed in two sensory modalities and from two cortical locations, we also noted a more specific electrophysiological effect, the increased incidence of flash/pattern differential VEPs, contingent upon an increase in cerebral amine metabolism. The finding of normal retinograms in the presence of potentially inhibitory concentrations of phenylalanine suggests that the related disturbance in monoamine metabolism involves neural elements in the visual system central to the retina. Several workers1°, 21,29 have implicated lateral inhibition by higher-order, feature-sensitive neurons in the production of pattern-specific occipital evoked potentials. The source generators for these pattern-specific responses lie in the striate and extrastriate posterior association cortex 14-t7. The increased production of patternspecific VEPs in our subjects, contingent upon increased cerebral amine synthesis, could find explanation in an increase in the activity of feature-sensitive inhibitory neurons in striate and extrastriate cortex due to increased synaptic availability of critical monoamines present in all cortical structures. But we cannot say with certainty that our observations could not find explanation in terms of altered monoamine activity in such subcortical structures as the superior colliculus or the lateral geniculate nucleus vital to the central representation of visual inputs. Nevertheless, regardless of the mechanism by which they produce their effects, monoamines appear to influence the visual information processing reflected by the evoked potential. ACKNOWLEDGEMENTS We thank James Elliott, Sandra Udby, Mitzi Speer and David Cervantes for research assistance. Grant No. HDO1823 from the National Institute of Child Health and Human Development supported this study.
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58 9 GALBRAtTH,G. C., GLIDDEN,J. B., AND BUSK,J., Visual evoked responses in mentally retarded and nonretarded subjects, Amer. J. merit. Defic., 75 (1970) 341-348. 10 HARTER,M. R., ANDWHITE,C. T., Effects of contour sharpness and check-size on visually evoked cortical potentials, Vision Res, 8 (1968) 701-711. 11 HARTER, M, R., Evoked cortical responses to checkerboard patterns: effect of check-size as a function of retinal eccentricity, Vision Res., 10 (1970) 1365-1376. 12 HARTER,M. R., AND WHITE,C. T., Evoked cortical responses to checkerboard patterns: effect of check-size as a function of visual acuity, Electroenceph. clin. NeurophysioL, 28 (1970) 48-54. 13 HOGAN,D. D., Cortical response of retardates for AER audiometry, Amer. J. ment. Defic., 75 (1971) 474-477. 14 JEFrREYS,D. A., Striate and extra-striate origins of pattern-related visual evoked potential (VEP) components, J. Physiol. (Lend.), 211 (1970) 29-30. 15 JErFREYS, D. A., Cortical source locations of pattern-related visual evoked potentials recorded from the human scalp, Nature (Lend.), 229 (1971) 502-504. 16 JEFFREYS,D. A., AND AXFORD,J. G., Source locations of pattern-specific components of human visualevoked potentials. I. Component of striate cortical origin, Exp. Brain Res., 16 (1972) 1-21. 17 JEFFREYS,D. A., AND AXFORD,J. G., Source locations of pattern-specific components of human visual evoked potentials. II. Component of extrastriate cortical origin, Exp. Brain. Res., 16 (1972) 22--40. 18 KARMEL,B. Z., HOFFMAN,R. F., ANDFEGY, M. J., Processing of countour information by human infants evidenced by pattern-dependent evoked potentials, Child Develop., 45 (1974)39-48. 19 LESEVRE,N., ET REMOND,A., Potentials evoqu6s par l'apparition de patterns: effets de la dimension du pattern et de la densit6 des contrastes, Electroenceph. clin. Neurophysiol., 32 (1972) 593604. 20 L1NDSEY,J. W., The auditory evoked potential in man: a review, T.LT. J. Life Sci., 1 (1971) 91-1 t0. 21 MACKAY, D. M., Evoked brain potentials as indicators of sensory information processing, Neurosc. Res. Progr. Bull., 7 (1969) 18t-276. 22 MARCUS,M. M., Developmentalchange and age-related waveforms in the visual evoked response, Psychophysiology, 8 (1971) 271. 23 McKEAN,C. M., Effects of totally synthetic, low phenylalaninediet on adolescent phenylketonuric patients, Arch. Dis. Child, 46 (1971) 608-615. 24 McKEAN,C. M., The effects of high phenylalanineconcentrations on serotonin and catecholamine metabolism in the human brain, Brain Research, 47 (1972) 469-476. 25 NVBACK,H., BORZECKI,Z., ANDSEDVALL,G., Accumulation and d i s a p ~ a n c e of catecholamines formed from tyrosine-Cl4 in mouse brain: effect of some psychotropic drugS, Europ. J. PharmacoL, 5 (1968) 395-402. 26 PADMOS,P., HAAIJMAN,J. J., ANDSPEK_RIEIJSE,H., Visually evoked potentials to patterned stimuli in monkey and man, Electroenceph. clin. NeurophysioL, 35 (1973) 153-163. 27 PERRY, N. W., ANO CHILDERS,D. G., The Human Visual Evoked Response: Method and Theory, Thomas, Springfield, 111.1969, p. 10. 28 RIETVELD,W. J., TORDOIR,W. E. M., HAGENOUW,J. R. B., LUBBERS,J. A., ANDSPOOR,Tt-LA.C., Visual evoked responses to blank and to checkerboard patterned flashes, Acta physioL pharmacoL need., 14 (1967) 259-285. 29 RIETVELD,W. J., AND HAGENOUW,J. R. B., Influence on cortical responses to patterned flashes of contrast-depth and of the ratio between the combined light and dark areas, Pfliigers Arch. ges. PhysioL, 322 (1971) 235-249. 30 SABELLI,H. C., G!ARDINA, W. J., ANDBARTIZAL,F., Photic evoked cortical potentials: interaction of reserpine, monoamine oxidase inhibition and DOPA, Biol. Psychiat., 3 (1971) 273-280. 31 SABELLI,n . C., BARTIZAL,F., GIARDINA,W. J., AND MYLES,S. B, Effects of at_pha-adreaergic blockers on visual evoked responses in rabbits, Electroenceph. clin. Neurophysiol., 33 (I972) 321324. 32 SALETU,B., SALETU,M., ITIL, T. M., AND COFFIN,C., Effect of stimulatory drugs on the somatosensory evoked potential in man, Pharmakopsychiatry, 5 (1972) t29-136. 33 SALETU,B., SALETU,M., ANDITIL,T. M., Effect of minor and major tranquilizerson somatosensory evoked potentials, Psychopharmacologia (8erl.), 24 (1972) 347-358. 34 SHAGASS,C., Evoked Brain Potentials inPsyehiatry, Plenum, New York, 1972, pp. 223-240. 35 SPEHLMANN,R., The averaged electrical response to diffuse and to patterned tight in the human, Electroenceph. clin. NeurophysioL, 19 (1965)560-569. 36 STRAUMAN~S,J. J., SHAGASS,C ' ANDOWRrON, D. A., Somatosensory evoked responses in Down Syndrome, Arch. gen. Psychiat., 29 (1973) 544-549.
49
(~ ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
EVIDENCE T H A T M O N O A M I N E S I N F L U E N C E H U M A N EVOKED POTENTIALS
E D W A R D W. P. SCHAFER AND CHARLES M. M c K E A N
Brain-Behavior Research Center, Langley Porter Neuropsychiatric Institute, University of California, Sonoma State Hospital, Eldridge, Calif. 95431 (U.S.A.) (Accepted May 20th, 1975)
SUMMARY
We have measured latency and flash/pattern differentials of the visual evoked potential (VEP) from phenylketonuric humans, while systematically manipulating rates of amine synthesis in the central nervous system using two techniques. We have observed that stimulation of monoaminergic activity in the visual processing system, either by lowering inhibitory levels of phenylalanine through dietary restriction or by a properly balanced administration of indole and catecholamine precursors, shortened VEP latencies and permitted the development of a discriminative brain response to patterned stimuli. The close temporal relationship between these electrophysiological changes and the neurocbemical manipulations following treatment initiation or discontinuation argue that monoamines play a significant role in the mediation of human sensory evoked potentials.
1NTRODUCTION
In an earlier report 24 concerning the effects of high phenylalanine levels on brain function, we noted that the excessive phenylalanine which characterizes phenylketonuria markedly inhibits central nervous system synthesis of the catecholamines and serotonin with associated reductions in cerebral concentrations of these monoamines and their amino acid precursors. Conversely, lowering plasma phenylalanine levels by dietary restriction to below 15 mg~o reduces this inhibition of cerebral amine metabolism. In the present studies, conducted concurrently to the above, we have attempted to determine the functional significance of the metabolic alterations noted previously. Specifically, does manipulation of phenylalanine levels result in observable changes of electrophysiological function as indicated by sensory evoked potentials? And further, if such electrophysiological changes occur, can one link them causally to the
50 associated alterations in the metabolism of biogenic amines thereby supporting their presumed role as neuromodulators? Two parameters of the visual evoked potential (VEP), component latency (poststimulus delay time) and flash/pattern differentiation, seemed appropriate for this investigation. Component latency provides a particularly useful measure because of its high reliability and long-term stability. Further, component latency varies predictably with pharmacological manipulation z4, decreases systematically with advancing maturation 4,7,8 and remains abnormally prolonged in humans suffering developmental retardationZ,3,9,1z, 36. The appearance of differential evoked responses to flash and pattern stimuli represents additional evidence of the brain's developing capacity for more precise edge detection. Mature humans with normal visual acuity produce differential occipital evoked potentials to flash and checkerboard pattern stimuli 1°,2s,35. Infants manifest no differential response. Production of this flash/pattern differential occurs only with increasing maturation of electrophysiological function ls,22. Thus, we measured VEP latency and flash/pattern differentials in phenylketonuric (PKU) humans while systematically manipulating the rate of amine synthesis in the central nervous system. In order to separate aminergic effects from the other metabolic consequences of excessive phenylalanine, which might also alter electrophysiological function, we employed two techniques to increase the rate of amine synthesis: (1) reduction of inhibitory concentrations of phenylalanine through dietary restriction, and (2) administration of the amine precursors L-tyrosine or L-3,4dihydroxyphenylalanine (L-DOPA), and L-5-hydroxytryptophan (L-5-HTP) without reducing the high phenylalanine levels. Consistent and significant alteration in our evoked potential parameters associated with altered cerebral amine synthesis would provide persuasive evidence for monoaminergic mediation of sensory information processing in the human brain. Previous work with animals 5,6,a°,31 has suggested such mediation. METHODS
Subjects and treatment Four profoundly retaxded, previously untreated adolescent (ages 17-18 yr) phenylketonuric patients (3 male, 1 female) served as experimental subjects. We precisely controlled and monitored blood phenylalanine concentrations in the 3 male patients by means of a totally synthetic diet of known phenylalanine composition as described previously2a. More recently, we demonstrated that by controlling the phenylalanine concentrations in blood we could also control the rate of cerebral monoamine metabolism in PKU subjects 24. We undertook the metabolic manipulations concurrently to the evoked potential measures described below. Minimal cerebral monoamine turnover occurred when blood phenylalanine exceeded 19 m g ~ while maximal turnover occurred when phenylalanine concentrations fell below 6 rag %. We compared the etectrophysiologieal responses under these two metabolic conditions. In a second series of experiments, we gave all 4 subjects the monoamine pre-
51 cursors L-tyrosine or L-DOPA and/or L-5-HTP while maintaining their blood phenylalanine concentrations above 19 rag%. We individualized the dosage of the precursor amino acids for each subject in order to obtain optimal shortening of visual evoked potential latencies. In each case precursor administration achieved demonstrable increase in metabolism of the respective amine as described previously 24. The administration of tyrosine or L-DOPA without L-5-HTP produced increased cerebral synthesis of dopamine but decreased serotonin turnover. This imbalanced condition actually produced prolongation of VEP latencies (see Table 1V). Consequently, we carefully controlled the catecholamine precursors (3.7 g tyrosine/day or 1200-1500 mg L-DOPA/day) while maintaining L-5-HTP between 300 and 600 mg/day in order to achieve optimal monoaminergic balance as indicated by the VEP response. Evoked potential measures To record his visual evoked potential, each subject sat or reclined in a darkened, shielded enclosure watching a frosted window through which an accompanying observer presented photic stimuli whenever the patient showed quiet attention to the window. We placed before this window either a checkerboard pattern transparency with checks subtending a visual angle of 20 rain at a viewing distance of 60 cm or for flash stimuli, a neutral density filter, which equated the total luminous flux of flash stimuli with that passed by the pattern transparency. Checks subtending a visual angle of from 15 to 30 min produce pattern VEPs of greatest amplitude la,12. An occipital electrode on Oz referred to A2 (10-20 electrode convention)provided EEG in a bandwidth 3dB down at 3 and 20 Hz. A PDP/8 computer averaged 200 taperecorded EEG responses to flash and 200 responses to pattern in split-half response sets of 100 each using a 500 Hz digitization rate over a post-stimulus epoch of 500 msec. Since these profoundly retarded subjects could not always cooperate fully, we encountered some difficulty in obtaining consistently reliable VEPs. Consequently, we discarded all experiments in which the latencies in milliseconds and amplitude in microvolts of VEP components FP1 and FN1 in each split-half of 100 responses varied more than ± 5 %. In all subjects, the latency measurements of the FP1 and FN1 components to flash showed sufficient reliability by this criterion to permit statistical analysis. However, within-trial amplitudes of P1 and N 1 and the latency of P2 showed less than desired stability especially in the less cooperative subjects so that we could get the necessary data for flash/pattern differentials on only 2 of the 4 subjects despite many hours of effort. After making a reliability determination, we summated all 200 responses to flash and computer scored the latencies of the FP1 and FN 1 components. These components, occurring at approximately 100 msec (FP1) and 150 msec (FN1) constitute the most prominent potentials of the occipital evoked response to flash 2v. On an X-Y plot we superimposed the VEPs to pattern and flash, characterizing as 'differential responses' only those pattern VEPs displaying the waveform configuration described consistently by many workersl°,19,26,2s; i.e., an occipital VEP to the appearance of patterned stimuli which shows a triphasic waveform with a small, surface-positive deflection at 70-90 msec followed by a surface-negative component at
52 TABLE I VEP LATENCYDIFFERENCESBETWEENHiGH (:- 19 MG~ ) AND LOW ('4~ 6 MG ~ ) SERUMPHENVLALANINE CONDITIONS
Subject
A A B B C C
V E P component
FP1 FN1 FP1 FN1 FP1 FN1
High Phe
L o w Phe
N
mean
S.D.
N
mean
S.D.
20 20 17 17 13 13
117.2 149.9 113.5 153.6 129.3 210.4
5.9 10.7 3.5 5.0 5.0 10.5
11 11 32 32 10 10
104.3 135.0 108.3 149.7 114.2 182.0
5.3 5.4 3.9 3.7 14.0 19:5
t
P
5.96 5.14 4.73 2.84 3.25 4.17
0.001 0,001 0.001 0.01 0.005 0.001
110-130 msec and a surface-positive wave at 180-200 msec (components PP1, PN1 and PP2 in Fig. 1B). To clarify any evoked potential changes contingent upon manipulation of monoamine metabolism, we also recorded the electroretinogram (ERG) to flash and pattern stimulation from the 3 male patients in the untreated, high phenylalanine condition. Further, an optometrist performed a retinoscopic examination on these 3 patients to determine their non-accommodated refractive error in the high phenylalanine condition. This examination revealed minimal refractive error for 2 of the patients while the third wore optical correction. This evaluation indicated that all 3 subjects had sufficient visual acuity to focus sharply the checkerboard pattern. We also recorded auditory evoked potentials from the vertex (Cz-A1) to loudspeaker-presented clicks at 70 dB sensation level from the 3 male patients under each condition of high or low plasma phenylatanine and scored these records for the latency of the classical Pz component20. We statistically analyzed mean VEP latency values for each subject and for grouped data under conditions of high and low serum phenylalanine and amine precursor administration using the t-test for both independent and correlated measures: RESULTS
Electroretinographic recordings taken from the 3 male patients under the high phenylalanine condition revealed normal latency and waveform configurations for the a- and b-waves of the ERG in response to both flash and pattern stimulation suggesting that the high phenylalanine concentrations left the catecholaminergic and serotonergic receptors in the retina relatively unaffected. The latency of the principal positive and negative occipital flash VEP compo. nents (FP1 and FN1)decreased significantly in all 3 male PKU patients when their plasma phenylalanine concentrations felt from untreated levels to 5.9 mg 9/0and below as a result of dietary therapy (Table I). The magnitude of this latency decrease be-
53 PPll'~ I | I I I |
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t
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1 I
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200
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300 Time (mlec)
1
400
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500
Fig. 1. Occipital visual evoked potentials to flash and checkerboard pattern stimuli from subject A under conditions of high and low serum phenylalanine. Note differential, triphasic (PP1, PN1, PP2) VEP to pattern under low phenylalanine condition not observed with high phenylalanine. tween conditions of high (above 19 mg ~ ) and low (below 6 mg ~ ) plasma phenylalanine ranged from 2 ~o to 13 ~o, and averaged a difference of 9 ~ . In parallel, the latency of the major positive (P2) component of the auditory vertex evoked potential also decreased by a significant mean 7 ~ between conditions of high and low plasma phenylalanine, as noted in data from the 3 male patients combined (t -- 4.41, df = 5, P < 0.01). The data provide clear evidence of a direct relationship between plasma phenylalanine levels and the latency of sensory evoked potential components in two modalities and from two cortical locations.
54 TABLE II EFFECT OF BALANCED AMINE PRECURSOR ADMINISTRATION ON V E P COMPONENT LATENCY IN PRESENCE OF HIGH PHENYLALANINE
Subject
A A B B C C D
VEP component
FP1 FN1 FP1 FN1 FP1 FN1 FP1
High Phe
Precursors
N
mean
S.D.
N
mean
S.D.
20 20 17 17 13 13 10
117.2 149.9 113.5 153.6 129.3 210.4 117.7
5.9 10.7 3.5 5.0 5.0 10.5 1.6
6 6 9 9 14 14 4
108.2 131.2 107.4 146.2 109.8 163.2 114.2
3.7 3.3 3.1 2.8 7.9 8.9 0.9
t
P
4.49 6.80 4.54 4.83 7.67 12.54 4.91
0.001 0.001 0.001 0.001 0.001 0.001 0.001
Analysis of reliable flash and pattern VEPs from 2 of the 3 diet-restricted subjects also indicated a relationship between plasma phenylatanine concentrations and the incidence of differential evoked potentials to flash and pattern stimuli. Fig. 1A and B illustrates undifferentiated and differentiated VEPs to flash and pattern. In the high phenylalanine conditions prior to dietary therapy, subject A failed to produce a single differentiated flash/pattern VEP (0 of 10). On the other hand, when we maintained his plasma phenylalanine below 16 mg ~ as a result of dietary restriction, he produced 100 ~o (61 of 61) differential evoked potentials over a 6-month period as illustrated in Fig. 1B. When his phenylalanine concentration exceeded 18 mg ~o he again reverted to the production of undifferentiated flash/pattern VEPs. Corroborating these findings, subject B gave only 33 ~o (3 of 9) differential VEPs under the high phenylalanine condition but 100 ~ (3 of 3) differential VEPs to flash and pattern with low plasma phenylalanine. Thus, without exception, reduction of phenylalanine levels resulted in an increased production of differential brain responses to flash and pattern. Having demonstrated an association between plasma phenylalanine level and our electrophysiological parameters, we then sought to establish whether the altered amine metabolism rather than some other metabolic consequence of excessive phenylalanine, had produced the observed changes in sensory evoked potentials. We examined the results of those experiments in which we stimulated monoamine metabolism by orally administering the precursor amino acids in the presence of high plasma phenylalanine (Tables II, III and IV). The latency of FP1 and FN1 decreased promptly in all 4 P K U patients when TABLE III INCIDENCE OF
VEP FLASH/PATTERN DIFFERENTIAL RESPONSK$ UNDER VARIOUS METABOLIC CONDITIONS
Subject
High phenylalanine
Low phenylalanine
High phenylalanine and precursors
A B
0% (0 of 10) 33 700(3 of 9)
t00% (61 of 61) 10070 (3 of 3)
100% (6 of 6) 75 70 (6 of 8)
55 TABLE IV VEP FLASH/PATTERN DIFFERENTIALS AND MEAN LATENCIES WITH VARIOUS AMINE PRECURSOR COMBINATIONS FOR SUBJECT A
% F/P differential
High Phe + 5-HTP (400-600 mg) -~ 5-HTP (300 mg) tyrosine (7.4 g) + 5-HTP (500 mg) -~ tyrosine (3.7 g)
Mean FP1- latency
0~ (0 of 10) 0 ~ (0 of 6)
117 msec 121 msec
0 ~ (0 of 2)
123 msec
100 ~ (6 of 6)
108 msec
they received a properly balanced administration of the amine precursors I.-tyrosine (3.7 g/day) or L-DOPA (1200-1500 mg/day) and L-5-HTP (300-6130 rag/day) in the presence of high plasma phenylalanine concentrations. These latency decreases, associated with amine precursor administration, ranged from 3 ~o to 22 ~o and averaged a difference of 9 ~ , which paralleled decreases observed when we increased monoamine synthesis by restriction of phenylalanine. In the two more cooperative subjects we also observed an increased incidence of differential flash/pattern VEPs upon administration of the amine precursors under the high phenylalanine condition. Subject A, who produced no differential VEPs under conditions of high plasma phenylalanine alone (0 of 10), generated 100~o (6 of 6) differential VEPs when given a properly balanced administration of amine precursors (L-tyrosine, 3.7 g; I.-5-HTP, 500 mg) in the presence of high phenylalanine concentrations. Subject B, who gave 33 ~ differential responses under the high phenylalanine condition, produced 75 ~ (6 of 8) differential VEPs to flash and pattern when given L-DOPA (1500 mg) and L-5-HTP (600 rag) in the presence of high plasma phenylalanine. The relative amounts of indole and catecholamine precursors administered critically affected both VEP latency and the incidence of flash/pattern differentials as indicated by results presented in Table IV. When subject A received either of the amine precursors alone or in excess, he manifested no differential flash/pattern responses and his VEP latencies showed values even longer than under the high phenylalanine condition alone. However, within 24 h of receiving an appropriate combination of L-tyrosine (3.7 g) and L-5-HTP (500 rag), subject A produced a clearcut flash/ pattern differential response and the latency of his FP1 and FN1 components decreased significantly. In summary, the balanced stimulation of monoaminergic activity in the visual processing system, either by lowering inhibitory levels of phenylalanine or by administration of amine precursors, shortened VEP latencies and permitted production of a discriminative brain response to patterned stimuli. Finally, in spite of these subjects' extremely limited behavioral repertoire, we
56 could record in 2 of the 4 subjects a few discrete changes closely associated with dietary alterations. When we initially restricted the diet of these PKU subjects and their plasma phenylalanine levels fell below t5 m g ~ , we observed an increase in speech production, a decrease in self-injurious behavior and a change from whining petulance to smiling cooperation. These positive changes reverted when the patients' plasma phenylalanine concentrations again rose above 20 mg ~,iDISCUSSION
We have found that stimulation of cerebral amine synthesis in PKU subjects, either by lowering plasma phenylalanine or by administering amine precursors in the presence of high phenylalanine concentrations, results in significant changes in their electrophysiological function: specifically, a decrease in the latency of evoked potential components in two different sensory modalities (visual and auditory) and from two cortical locations, as well as an increased incidence of maturationally significant flash/ pattern differential VEPs over occipital cortex. Apparently, these electrophysiological functions depend on the balanced mediation of both serotonergic and catecholaminergic neural mechanisms since neither of the amine precursors by themselves resulted in a latency decrease or the more frequent appearance of a differential response to flash/pattern. These data strongly suggest that the electrophysiologicat malfunctions in phenylketonuria do not result primarily from the high concentration of phenylalanine or its metabolites, because, despite this pathogenic condition, the amino acid precursors which singly have no ameliorative effect can, in combination, reverse the VEP abnormalities. Further, the close temporal relationship between the electrophysiological changes and the neurochemical manipulations as well as their prompt reversibility upon withdrawal of therapy argue persuasively for a definitive role by monoamines in the mediation of sensory evoked potentials. The latency differences resulting from lowered phenylalanine concentrations or amine precursor administration parallel the latency differences reported for both visual2,9 and auditory13 evoked potentials between non-PKU retardates and normal subjects. One might speculate that by increasing the rate of cerebral monoamine synthesis through dietary restriction of phenylalanine or administration of balanced amounts of amino acid precursors, electrophysiological functioning in these PKU subjects proceeded more efficiently as indicated by a latency decrease comparable to the latency differences reported between normals and retarded humans. We also noted associated behavioral improvement following phenylalanine restriction which appeared to revert when phenylalanine concentrations rose above 20 mg ~. Certain psychotropic agents which alter aminergic activity and have weltdocumented behavioral effects also produce changes in evoked potential latency. The latency changes we have observed associated with alteration in cerebral amine synthesis show comparable magnitude to the small but significant latency decreases observed in somatosensory evoked potentials upon administration of dextroamphetamine32 and to the latency increases noted with chlorpromazinea~. Dextroamphetamine presumably increases synaptic catecholamine concentrations by blocking
57 neuronal reuptakO while chlorpromazine decreases catecholaminergic activity by blocking post-synaptic receptors za. Besides the generalized latency changes observed in two sensory modalities and from two cortical locations, we also noted a more specific electrophysiological effect, the increased incidence of flash/pattern differential VEPs, contingent upon an increase in cerebral amine metabolism. The finding of normal retinograms in the presence of potentially inhibitory concentrations of phenylalanine suggests that the related disturbance in monoamine metabolism involves neural elements in the visual system central to the retina. Several workers1°, 21,29 have implicated lateral inhibition by higher-order, feature-sensitive neurons in the production of pattern-specific occipital evoked potentials. The source generators for these pattern-specific responses lie in the striate and extrastriate posterior association cortex 14-t7. The increased production of patternspecific VEPs in our subjects, contingent upon increased cerebral amine synthesis, could find explanation in an increase in the activity of feature-sensitive inhibitory neurons in striate and extrastriate cortex due to increased synaptic availability of critical monoamines present in all cortical structures. But we cannot say with certainty that our observations could not find explanation in terms of altered monoamine activity in such subcortical structures as the superior colliculus or the lateral geniculate nucleus vital to the central representation of visual inputs. Nevertheless, regardless of the mechanism by which they produce their effects, monoamines appear to influence the visual information processing reflected by the evoked potential. ACKNOWLEDGEMENTS We thank James Elliott, Sandra Udby, Mitzi Speer and David Cervantes for research assistance. Grant No. HDO1823 from the National Institute of Child Health and Human Development supported this study.
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