- Email: [email protected]
Prenatal Impairment of Brain Serotonergic Transmission in Infants
PRENATAL IMPAIRMENT OF BRAIN SEROTONERGIC TRANSMISSION IN INFANTS GABRIEL MANJARREZ, MD, PHD, IGNACIA CISNEROS, MD, ROCIO HERRERA, MD, MSC, FELIPE VAZQUEZ, MD, MSC, ALEJANDRO ROBLES, AND JORGE HERNANDEZ, MD, PHD
Objective To evaluate whether the free fraction of L-tryptophan (L-Trp) and the N1/P2 component of the auditory evoked potentials (AEPs) are associated with impaired brain serotonin neurotransmission in infants with intrauterine growth restriction (IUGR). Study design We measured free, bound, and total plasma L-Trp and recorded the N1/P2 component of AEP in a prospective, longitudinal, and comparative study comparing IUGR and control infants. Results Plasma free L-Trp was increased and the amplitude of N1/P2 component was significantly decreased in IUGR relative to control infants. The free fraction of L-Trp and N1/P2 component had a negative association. Conclusions In newborns with IUGR, the changes in measured plasma free fraction of L-Trp and in the amplitude the N1/ P2 component of the AEP suggest an inverse association between free L-Trp and components of the AEP. The changes observed in the free fraction of L-Trp and AEP may be causally associated with brain serotonergic activity in utero. In IUGR, epigenetic factors such as stress-induced disturbances in brain serotonin metabolism or serotonergic activity, identifiable by alterations in AEP, influence cerebral sensory cortex development and may be causally associated with serotonin-related disorders in adulthood. (J Pediatr 2005;147:592-6)
n the human brain, serotonergic neurons are present in the fifth week of gestation, and they increase rapidly through the 10th week of fetal life. By week 15, the typical organization of the serotonergic system into the raphe nuclei is complete.1-5 Evidence exists to support the role of serotonin in the normal process of sensory cortex formation.6-11 Serotonin depresses or facilitates cortical neuronal activity that is dependent on the type of receptor involved;12,13 hence it may control gain factors and excitability levels of cortical neurons. Decreased serotonin availability increases neuronal cortical activity in the auditory cortex, which in turn is reflected in the amplitude of the N1/P2 component of auditory evoked potentials (AEPs).14-16 An opposite effect on the auditory cortex is observed when serotonergic neuronal activity increases. N1 and P2 waveforms recorded from the scalp are AEP components generated by the supratemporal plane of the superior temporal and lateral gyri, and are considered representative of auditory cortex integrative functions.14-17 In rats, intrauterine growth restriction (IUGR) leads to increased brain serotonin synthesis accompanied by an increase in the free fraction of plasma L-tryptophan (L-Trp).18-21 The free fraction of plasma L-Trp and N1/P2 ratio and its components correlate significantly, and they appear to be reliable indicators of changes in serotonergic neurotransmission in rats with IUGR.16 The normal pattern of the N1/P2 component is disrupted in rats with IUGR.16 This change in the free fraction of plasma L-Trp has also been detected in human infants with IUGR.18,22 The purpose of the present study was to test
I
ACD AEP AgCl C Cz EEG
592
Citric acid, sodium citrate, dextrose Auditory evoked potentials Silver/silver chloride Control group Vertex Electroencephalogram
FGR GABA IUGR L-Trp MS SS
Fetal growth ratio g-amino butyric acid Intrauterine growth restriction L-tryptophan Sum of squares minus Sum of squares
From the Laboratory of Developmental Neurochemistry, Specialties Hospital and Department of Biomedical Engineering, XXI Century National Medical Center and Service of Neonatology, Gynecology-Obstetrics Hospital ‘‘4’’, Mexican Institute of Social Security, Mexico City, Mexico and Laboratory of Neurontogeny, Department of Physiology, Biophysics and Neurosciences, Center of Research and Advanced Studies, Mexico City, Mexico. Supported by grants from the Mexican Institute of Social Security and CONACYT (FP-0038/740 and 30764-M). Submitted for publication Sep 13, 2004; last revision received May 11, 2005; accepted Jun 13, 2005. Reprint requests: Gabriel Manjarrez, Laboratory of Developmental Neurochemistry, Specialties Hospital, XXI Century National Medical Center, Mexican Institute of Social Security, Av. Cuauhte´moc 330, Col. Doctores, CP 06720, Mexico City, Mexico. E-mail: [email protected]. 0022-3476/$ - see front matter Copyright ª 2005 Elsevier Inc. All rights reserved. 10.1016/j.jpeds.2005.06.025
the hypothesis that changes in the free fraction of plasma LTrp and in the N1/P2 component of AEP may be associated and are altered in infants with IUGR. Changes in L-Trp and alterations in N1/P2 waveforms may identify developmental metabolic and cerebrocortical anomalies caused by disrupted serotonergic activity during cerebral cortex differentiation.
METHODS Subjects Parents gave consent for their infants to participate in this study. The study group comprised 25 newborns from the service of Neonatology, Gynecology-Obstetrics, Hospital 4, Mexican Institute of Social Security. The group with IUGR included 13 newborns of both sexes with body weight below the 10th percentile of intrauterine growth curves,23 fetal growth ratio (FGR) < 0.90,24 and ponderal index of 2.14 ± 0.10.25 The control group included 12 newborns with body weight between the 10th and 90th percentiles of the same curves, FGR > 0.90, and ponderal index of 2.63 ± 0.06. No clinical signs of other pathologies were observed in either group. All newborns were breast-fed and also fed with a complement of protein-modified milk diluted to 16% that supplies 20 cal/30 mL, with 3.98 ± 0.81 mg L-Trp/100 mL. Maternal milk and protein-modified milk have similar L-Trp content (FAO Nutrition Studies, Rome 1970, items 375 and 383, Geigy Scientific tables, 7th ed. Basel, Switzerland, CibaGeigy, Ltd).26
Recording of the N1/P2 Component of the AEPs Recordings were obtained with one electrode referenced on the vertex (Cz), in a sound-attenuated and electrically shielded room adjacent to the recording apparatus. AgCl electrodes were used (electroencephalogram [EEG] disk electrode NE-101, 10 mm diameter). Binaural 1-KHz tones, lasting 20 ms randomized between 1500 and 2000 ms at 60 dB, were presented in pseudorandomized order through headphones. Data were digitally collected with a sampling rate of 1000 Hz. Artifact rejection was done with 0.1-Hz and 200-Hz filters, from 50 ms prestimulus to 500 ms poststimulus. Fifty sweeps were amplified with a Grass module (preamplifier and amplifier model 7P5, wide-band AC EEG; Star Med, CITY and STATE) with a gain of 10,000 and recorded on computer paper. The x-y graphs of the AEPs were examined, and prominent peaks were identified and measured using specific software (multi-lab card with programmable Cain PCL-812P6). Latencies in milliseconds (60 to 120 ms for N1 and 110 to 210 ms for P2) and amplitudes in microvolts (mV) were also calculated. The amplitude of the N1/P2 component of the AEP was considered as the sum of mV’s between the crests of the waves N1 and P2.
Biochemical Assays At 1, 30, and 60 days after birth, 2 mL of blood was collected through venipuncture in borosilicate tubes containing Prenatal Impairment Of Brain Serotonergic Transmission In Infants
Table. Clinical data of infants with intrauterine growth restriction and normal controls
Gestational age (wk) Ponderal index Fetal growth ratio Body weight (gm) (days) 1 30 60 Body length (cm) (days) 1 30 60
Intrauterine growth restriction n = 13
Controls n = 12
38.3 ± 0.2 2.14 ± 0.10* 61.8 ± 3.2*
39.5 ± 0.2 2.63 ± 0.06 104.6 ± 3.2
1845 ± 93.9* 2544 ± 142.2* 3475 ± 179.0*
3246 ± 90.5 4200 ± 174.4 5638 ± 143.3
44.2 ± 1.0* 48.3 ± 1.0* 50.9 ± 1.4*
49.5 ± 0.9 54.0 ± 0.8 58.3 ± 1.2
Each point represents the mean value ± SD. Body weight (Treatment: SS = 1340, Df = 5, MS = 268.1. Residual SS = 1340, Df = 5, MS = 5.946). Body length (Treatment: SS = 392.0, Df = 2, MS = 196. Residual SS = 3152, Df = 31, MS = 1.07). Difference were determined by Wilcoxon test and ANOVA. *p < 0.001.
450 mL of ACD solution (citric acid 9.9 mg, sodium citrate 3.6 mg, and dextrose 11 mg, buffered with Tris acetate 50 mmol, pH 7.4). Blood samples were obtained between 7 and 8 AM and 4 hours after the last feeding, were immediately cooled (to 0 to 4C) and centrifuged at 500 3 g in a Sorvall RC5C refrigerated centrifuge. Plasma aliquots were used for the various biochemical assays. An ultrafiltered plasma sample was obtained using Centriflo-Amicon CF50A membranes (Danvers, MA) for the free fraction of plasma L-Trp. The high-performance liquid chromatography fluorescent method of Peat et al26 was used to quantify the free fraction and total plasma L-Trp. The difference between these 2 was considered to be the fraction bound to albumin.
Data Analysis Mean values and standard deviations were used for normally distributed data. Differences among mean values were analyzed for significance using Wilcoxon’s signed rank test and analysis of variance, with a level significance of P < .05.
RESULTS The IUGR and control infants are described in Table. Free plasma L-Trp concentrations were increased in the IUGR group relative to the control group, confirming previous observations.18,22 Bound L-trp was decreased, and total L-trp was unchanged (Figure 1). The group with IUGR demonstrated a significantly decreased amplitude of the N1/P2 component (P < .05) (Figure 2) and decreased latencies of P1, N1, and P2 (P < .05) (Figure 2). In the control group there was an increase only in N1 latency on day 60, and no changes in P1 and N1 latencies with age were observed (Figure 3). 593
Figure 1. Plasma L-Trp concentration. Each point represents the mean value ± standard deviation from 13 infants with IUGR and 12 normal controls. All determinations were done in duplicate samples. (A) Treatment: (SS) = 496.1, degrees of freedom (df) = 5, (MS) = 99.21; residual: SS = 63.06, df = 69, MS = 0.79140. (B) Treatment: SS = 356.5, df = 5, MS = 71.30; residual: SS = 524.5, df = 60, MS = 8.7.42. (C) Treatment: SS = 148.7, df = 5, MS = 29.74; residual: SS = 499.8, df = 60, MS = 8.742. *P < .05; **P < .01; ***P < .001; Wilcoxon’s test and analysis of variance.
Figure 2. AEPs obtained from the Cz reference electrode at a stimulation intensity of 60 dB in controls (—) and infants with IUGR (- - -). Each recording represents the mean values from the infants in that group. *P < .001; Wilcoxon’s test, comparing N1/P2 component amplitude of IUGR with controls.
DISCUSSION We measured the electrical activity of the auditory cortex using the N1/P2 component of AEP to assess whether it was associated with biochemical indicators of serotonin metabolism in infants with IUGR. We believe that the alterations in the N1/P2 component reflect impaired cortical activity caused by developmental anomalies of the serotonergic signal during corticogenesis provoked by IUGR. Our results confirm previous data from rats and humans with IUGR, demonstrating that the free faction of L-Trp was significantly elevated in plasma and brain.16,18,20,22 This early hyperserotonergic neuronal activity may negatively influence brain corticogenesis, particularly the normal sensory cortex conformation and later function. In older rats, the plasma free fraction of L-Trp and its binding to albumin may play a major role in the regulation 594
Manjarrez et al
Figure 3. Latencies of P1, N1, and P2 components of AEPs in infants with IUGR (n = 13) and controls (n = 12) at age 30 and 60 days. N1: treatment, SS = 2164, df = 3, MS = 721.4; residual, SS = 1932, df = 46, MS = 42.00., P2: treatment, SS = 17400, df = 3, MS = 5801; residual, SS = 2327, df = 46, MS = 50.58. P1: treatment, SS = 1864, df = 3, MS = 621.4; residual, SS = 1532, df = 46, MS = 32.00. *P < .01; **P < .001; Wilcoxon’s test and analysis of variance.
of the amount of L-Trp available for transport to the brain through the blood-brain barrier, thereby stimulating the synthesis of 5-HT (serotonin) by serotonergic neurons and their activity.27 In rats with IUGR, there is an increase in brain serotonin during the fetal period18-20 associated with an increase in free plasma L-Trp in the mother’s plasma. This serotonin precursor stays elevated in the offspring and chronically activates tryptophan-5-hydroxylase, resulting in serotonin synthesis in various brain regions, including the sensory cortex.16,28 A similar effect may be expected from the plasma free fraction of L-Trp in human infants with IUGR, in whom L-Trp free fraction is significantly elevated.18,22 Early alteration of the serotonin precursor’s availability through the placental–fetal barrier appears to be an effective way to activate serotonin synthesis in the brain,29 increase serotonin release, and modify its early signaling function in cortical neuronal assembly. Our results indicate that the greater the L-Trp free fraction in plasma, the lower the N1/P2 component amplitude. This observation strongly suggests a functional or causal relationship between brain serotonin activity and changes in the N1/P2 ratio. These components of auditory cortex electrical activity (N1/P2) are the result of spatial and temporal integration of various neuronal processes. Electrical dipole source analyses have identified 2 main components in each hemisphere: a tangential dipole source representing activation of the primary auditory cortex and a radial dipole generated by the activity in the secondary auditory cortex.14,15 The N1/P2 component has been identified in human infants, but whether its source is the same as in the adult brain remains controversial.30 Because the N1/P2 component can be induced by The Journal of Pediatrics November 2005
auditory stimulus and is better detected on the corresponding auditory projections of the scalp, that it reflects cortical integration of auditory activity in the human infant seems reasonable. In the normal and IUGR rat brain, serotonin plays an important role in regulating auditory evoked cortical responses.16 Thus we propose, based on the current biochemical and electrophysiological results, that in humans the response of the auditory cortex to sound intensity stimulus may be a function of brain serotonergic activity. Moreover, as in infants with IUGR, this response can be developmentally disrupted by anomalous sensory cortex conformation caused by gestational metabolical stress. Serotonin, together with other molecules (eg, norepinephrine, dopamine, acetylcholine), may act as ‘‘enabling factors’’ in the process of ‘‘experience-induced plasticity’’ in the developing visual cortex.11 Serotonin may control whether inputs can be strengthened or weakened and may influence longterm potentiation or long-term depression by lowering thresholds. Kojic et al11 reported that in the developing visual cortex, the serotonin receptors 5-HT2C occur only at a critical period on layer IV and that, depending on their density, they may facilitate either long-term potentiation or long-term depression of plastic responses. Long-term potentiation or long-term depression is implicated in the synaptic remodeling and refinement of neural connections during development. These findings support the significance of serotonin’s role in early cerebral corticogenesis and the later consequences of modifying its normal availability. Increased serotonin transmission during the prenatal period may later provoke altered behavior in sensory cortices and reproducible, quantitative differences in auditory cortex responses detected using AEPs to measure decreases in the amplitude of the sound-intensity dependent N1/P2 component (present study). Because there are abundant serotonin-innervated g-aminobutyric acid circuits that inhibit the neuronal responses in the brain,12 it is possible that augmented serotonin activity may be reflected in decreased amplitude of N1/P2 and changes in the onset of response, as demonstrated by the retarded latencies observed in infants with IUGR. Latency retardation was also noted in visual evoked potentials from infants with IUGR.31 This effect of IUGR on latencies may be related to modified development of thalamocortical pathways.31 Although there is a reported tendency for latencies in brain electrical sensory responses to decrease with age in normal children, this phenomenon was not observed in the present study, presumably because of the limited duration of the developmental period studied. In conclusion, it appears that in utero epigenetic factors influencing cerebral cortex development are associated with metabolic disturbances in brain serotonin synthesis induced by nutritional stress during the fetal period. The influence of early cortical changes is demonstrated by impairments in the postnatal function of the sensory cortex and can be identified through measurable changes in AEP responses to intensity modulated auditory stimuli. It is likely that other sensory responses are altered as well;28 if so, then the capacity to react to environmental signals will be different in newborns with IUGR with disturbed sensory responses. Ultimately, these Prenatal Impairment Of Brain Serotonergic Transmission In Infants
newborns’ cognitive development may be impaired, and thus IUGR will be shown to have life-long consequences and to represent a significant cause of diverse serotonin-related brain disorders in adults. We thank Dr. Jack Segal for his help in editing this manuscript.
REFERENCES 1. Steinbusch HWM. Distribution of serotonin immunoreactivity in the central nervous system of the rat cell bodies and terminals. Neuroscience 1981;6:557-618. 2. Takahashi H, Nakashima S, Ohama E, Takeda S, Ikuta F. Distribution of serotonin-containing cell bodies in the brainstem of the human fetus determined with immunohistochemistry using antiserotonin serum. Brain Dev 1986;8:355-65. 3. Sundstrom E, Kolare S, Souverbie F, Samuelsson EB, Pschera H, Lunell NO, et al. Neurochemical differentiation of human bulbospinal monoaminergic neurons during the first trimester. Brain Res Dev Brain Res 1993;75:1-12. 4. Levallois C, Valence C, Baldet P, Privat A. Morphological and morphometric analysis of serotonin-containing neurons in primary dissociated cultures of human rhombencephalon: a study of development. Brain Res Dev Brain Res 1997;99:243-52. 5. Shen WZ, Luo ZB, Zheng DR, Yew DT. Immunohistochemical studies of the development of 5-HT (serotonin) neurons in the nuclei of the reticular formations of human fetuses. Pediatr Neurosci 1989;15:291-5. 6. Lambe EK, Krimer LS, Goldman-Rakic PS. Differential postnatal development of catecholamine and serotonin inputs to identified neurons in prefrontal cortex of rhesus monkey. J Neurosci 2000;20:8780-7. 7. D’Amato JR, Blue ME, Largent BL, Lynch DR, Ledbetter DJ. Ontogeny of the serotonergic projection to rat neocortex: transient expression of a dense innervation to primary sensory areas. Proc Natl Acad Sci USA 1987; 84:4322-6. 8. Bennett-Clarke CA, Chiaia NL, Rhoades RW. Contributions of raphecortical and thalamocortical axons to the transient somatotopic pattern of serotonin immunoreactivity in the rat cortex. Somatosens Mot Res 1997;14: 27-33. 9. Lebrand C, Cases O, Adelbrecht C, Doye A, Alvarez C, El Mestikawy S, et al. Transient uptake and storage of serotonin in developing thalamic neurons. Neuron 1996;17:823-35. 10. Lavdas AA, Blue EM, Lincoln J, Parnavelas GJ. Serotonin promotes the differentiation of glutamate neurons in organotypic slice cultures of the developing cerebral cortex. J Neurosci 1997;17:7872-80. 11. Kojic L, Dyck HR, Gu Q, Douglas MR, Matsubara J, Cynader SM. Columnar distribution of serotonin-dependent plasticity within kitten striate cortex. Proc Natl Acad Sci USA 2000;97:1841-4. 12. Zhou F-M, Hablitz JJ. Activation of serotonin receptors modulates synaptic transmission in rat cerebral cortex. J Neurophysiol 1999;82:2989-99. 13. Morales M, Bloom FE. The 5-HT3 receptor is present in different subpopulations of GABAergic neurons in rat telencephalon. J Neurosci 1997;17:3157-67. 14. Hegerl U, Juckel G. Intensity dependence of auditory evoked potentials as an indicator of central serotonergic neurotranmission: a new hypothesis. Biol Psychiatry 1993;33:173-87. 15. Juckel G, Molna´r M, Hegerl U, Cse´pe V, Karmos G. Auditory-evoked potentials as indicator of brain serotonergic activity: first evidence in behaving cats. Biol Psychiatry 1997;41:1181-95. 16. Manjarrez GG, Hernandez ZE, Robles OA, Gonzalez RM, Hernandez RJ. Development impairment of auditory evoked N1/P2 component in rats undernourished in utero: its relation to brain serotonin activity. Dev Brain Res 2001;127:149-55. 17. Semple MN, Scott BH. Cortical mechanisms in hearing. Curr Opin Neurobiol 2003;13:167-73. 18. Hernandez RJ, Manjarrez GG, Chagoya GG. Newborn humans and rats malnourished in utero: free plasma L-tryptophan, neutral amino acids and brain serotonin synthesis. Brain Res 1989;488:1-13. 19. Manjarrez GG, Chagoya GG, Hernandez RJ. Perinatal brain serotonin metabolism in rats malnourished in utero. Biol Neonate 1988;54:232-40.
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20. Manjarrez GG, Magdaleno VM, Chagoya GG, Hernandez RJ. Nutritional recovery does not reverse the activation of brain serotonin synthesis in the ontogenetically malnourished rat. Int J Dev Neurosci 1996;14: 641-8. 21. Manjarrez GG, Chagoya GG, Hernandez RJ. Early nutritional changes modify the kinetics and phosphorylation capacity of tryptophan-5-hydroxylase. Int J Dev Neurosci 1994;12:695-702. 22. Manjarrez GG, Contreras JL, Chagoya GG, Hernandez RJ. Free tryptophan as indicator of brain serotonin synthesis in infants. Pediatr Neurol 1998;18:57-62. 23. Lubchenco LO, Hansman C, Dressleer M, Boyd E. Intrauterine growth as estimated from live-born birth weight date at 24 to 42 weeks of gestation. Pediatrics 1963;32:793-800. 24. Kramer MS, Oliver M, McLean FH, Dougherty GE, Willis DM, Usher RH. Determinants of fetal growth and body proportionality. Pediatrics 1990; 86:18-26. 25. Miller HC, Hassanein K. Diagnosis of impaired fetal growth in newborn infants. Pediatrics 1971;48:511-22.
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26. Peat M, Gibb JW. High-performance liquid chromatography determination of indoleamines, dopamine, and norepinephrine in rat brain with fluorometric detection. Anal Biochem 1983;128:275-80. 27. Boadle-Biber? Regulation of serotonin synthesis. Prog Biophys Mol Biol 1993;60:1-15. 28. Gutierrez OG, Manjarrez GG, Gonzalez C, Lopez S, Herrera R, Medina AI, et al. Neither increased nor decreased availability of cortical serotonin (5HT) disturbs barrel field formation in isocaloric undernourished rat pups. Int J Dev Neurosci 2002;20:497-501. 29. Hernandez-RJ, Chagoya G. Brain serotonin synthesis and Na1, K1-ATPase activity are increased postnatally after prenatal administration of L-tryptophan. Dev Brain Res 1986;25:221-6. 30. Ohlrich SE, Barnet BA, Weiss PI, Shanks B. Auditory evoked potential development in early childhood: a longitudinal study. Electroencephalogr Clin Neurophysiol 1978;44:411-23. 31. Amiel-Tison C, Cabrol D, Denver R, Jarreau P-H, Papiernik E, Piaza P-V. Fetal adaptation to stress: acceleration of fetal maturation and earlier birth triggered by placental insufficiency in humans. Early Hum Dev 2004;78:15-27.
The Journal of Pediatrics November 2005
Objective To evaluate whether the free fraction of L-tryptophan (L-Trp) and the N1/P2 component of the auditory evoked potentials (AEPs) are associated with impaired brain serotonin neurotransmission in infants with intrauterine growth restriction (IUGR). Study design We measured free, bound, and total plasma L-Trp and recorded the N1/P2 component of AEP in a prospective, longitudinal, and comparative study comparing IUGR and control infants. Results Plasma free L-Trp was increased and the amplitude of N1/P2 component was significantly decreased in IUGR relative to control infants. The free fraction of L-Trp and N1/P2 component had a negative association. Conclusions In newborns with IUGR, the changes in measured plasma free fraction of L-Trp and in the amplitude the N1/ P2 component of the AEP suggest an inverse association between free L-Trp and components of the AEP. The changes observed in the free fraction of L-Trp and AEP may be causally associated with brain serotonergic activity in utero. In IUGR, epigenetic factors such as stress-induced disturbances in brain serotonin metabolism or serotonergic activity, identifiable by alterations in AEP, influence cerebral sensory cortex development and may be causally associated with serotonin-related disorders in adulthood. (J Pediatr 2005;147:592-6)
n the human brain, serotonergic neurons are present in the fifth week of gestation, and they increase rapidly through the 10th week of fetal life. By week 15, the typical organization of the serotonergic system into the raphe nuclei is complete.1-5 Evidence exists to support the role of serotonin in the normal process of sensory cortex formation.6-11 Serotonin depresses or facilitates cortical neuronal activity that is dependent on the type of receptor involved;12,13 hence it may control gain factors and excitability levels of cortical neurons. Decreased serotonin availability increases neuronal cortical activity in the auditory cortex, which in turn is reflected in the amplitude of the N1/P2 component of auditory evoked potentials (AEPs).14-16 An opposite effect on the auditory cortex is observed when serotonergic neuronal activity increases. N1 and P2 waveforms recorded from the scalp are AEP components generated by the supratemporal plane of the superior temporal and lateral gyri, and are considered representative of auditory cortex integrative functions.14-17 In rats, intrauterine growth restriction (IUGR) leads to increased brain serotonin synthesis accompanied by an increase in the free fraction of plasma L-tryptophan (L-Trp).18-21 The free fraction of plasma L-Trp and N1/P2 ratio and its components correlate significantly, and they appear to be reliable indicators of changes in serotonergic neurotransmission in rats with IUGR.16 The normal pattern of the N1/P2 component is disrupted in rats with IUGR.16 This change in the free fraction of plasma L-Trp has also been detected in human infants with IUGR.18,22 The purpose of the present study was to test
I
ACD AEP AgCl C Cz EEG
592
Citric acid, sodium citrate, dextrose Auditory evoked potentials Silver/silver chloride Control group Vertex Electroencephalogram
FGR GABA IUGR L-Trp MS SS
Fetal growth ratio g-amino butyric acid Intrauterine growth restriction L-tryptophan Sum of squares minus Sum of squares
From the Laboratory of Developmental Neurochemistry, Specialties Hospital and Department of Biomedical Engineering, XXI Century National Medical Center and Service of Neonatology, Gynecology-Obstetrics Hospital ‘‘4’’, Mexican Institute of Social Security, Mexico City, Mexico and Laboratory of Neurontogeny, Department of Physiology, Biophysics and Neurosciences, Center of Research and Advanced Studies, Mexico City, Mexico. Supported by grants from the Mexican Institute of Social Security and CONACYT (FP-0038/740 and 30764-M). Submitted for publication Sep 13, 2004; last revision received May 11, 2005; accepted Jun 13, 2005. Reprint requests: Gabriel Manjarrez, Laboratory of Developmental Neurochemistry, Specialties Hospital, XXI Century National Medical Center, Mexican Institute of Social Security, Av. Cuauhte´moc 330, Col. Doctores, CP 06720, Mexico City, Mexico. E-mail: [email protected]. 0022-3476/$ - see front matter Copyright ª 2005 Elsevier Inc. All rights reserved. 10.1016/j.jpeds.2005.06.025
the hypothesis that changes in the free fraction of plasma LTrp and in the N1/P2 component of AEP may be associated and are altered in infants with IUGR. Changes in L-Trp and alterations in N1/P2 waveforms may identify developmental metabolic and cerebrocortical anomalies caused by disrupted serotonergic activity during cerebral cortex differentiation.
METHODS Subjects Parents gave consent for their infants to participate in this study. The study group comprised 25 newborns from the service of Neonatology, Gynecology-Obstetrics, Hospital 4, Mexican Institute of Social Security. The group with IUGR included 13 newborns of both sexes with body weight below the 10th percentile of intrauterine growth curves,23 fetal growth ratio (FGR) < 0.90,24 and ponderal index of 2.14 ± 0.10.25 The control group included 12 newborns with body weight between the 10th and 90th percentiles of the same curves, FGR > 0.90, and ponderal index of 2.63 ± 0.06. No clinical signs of other pathologies were observed in either group. All newborns were breast-fed and also fed with a complement of protein-modified milk diluted to 16% that supplies 20 cal/30 mL, with 3.98 ± 0.81 mg L-Trp/100 mL. Maternal milk and protein-modified milk have similar L-Trp content (FAO Nutrition Studies, Rome 1970, items 375 and 383, Geigy Scientific tables, 7th ed. Basel, Switzerland, CibaGeigy, Ltd).26
Recording of the N1/P2 Component of the AEPs Recordings were obtained with one electrode referenced on the vertex (Cz), in a sound-attenuated and electrically shielded room adjacent to the recording apparatus. AgCl electrodes were used (electroencephalogram [EEG] disk electrode NE-101, 10 mm diameter). Binaural 1-KHz tones, lasting 20 ms randomized between 1500 and 2000 ms at 60 dB, were presented in pseudorandomized order through headphones. Data were digitally collected with a sampling rate of 1000 Hz. Artifact rejection was done with 0.1-Hz and 200-Hz filters, from 50 ms prestimulus to 500 ms poststimulus. Fifty sweeps were amplified with a Grass module (preamplifier and amplifier model 7P5, wide-band AC EEG; Star Med, CITY and STATE) with a gain of 10,000 and recorded on computer paper. The x-y graphs of the AEPs were examined, and prominent peaks were identified and measured using specific software (multi-lab card with programmable Cain PCL-812P6). Latencies in milliseconds (60 to 120 ms for N1 and 110 to 210 ms for P2) and amplitudes in microvolts (mV) were also calculated. The amplitude of the N1/P2 component of the AEP was considered as the sum of mV’s between the crests of the waves N1 and P2.
Biochemical Assays At 1, 30, and 60 days after birth, 2 mL of blood was collected through venipuncture in borosilicate tubes containing Prenatal Impairment Of Brain Serotonergic Transmission In Infants
Table. Clinical data of infants with intrauterine growth restriction and normal controls
Gestational age (wk) Ponderal index Fetal growth ratio Body weight (gm) (days) 1 30 60 Body length (cm) (days) 1 30 60
Intrauterine growth restriction n = 13
Controls n = 12
38.3 ± 0.2 2.14 ± 0.10* 61.8 ± 3.2*
39.5 ± 0.2 2.63 ± 0.06 104.6 ± 3.2
1845 ± 93.9* 2544 ± 142.2* 3475 ± 179.0*
3246 ± 90.5 4200 ± 174.4 5638 ± 143.3
44.2 ± 1.0* 48.3 ± 1.0* 50.9 ± 1.4*
49.5 ± 0.9 54.0 ± 0.8 58.3 ± 1.2
Each point represents the mean value ± SD. Body weight (Treatment: SS = 1340, Df = 5, MS = 268.1. Residual SS = 1340, Df = 5, MS = 5.946). Body length (Treatment: SS = 392.0, Df = 2, MS = 196. Residual SS = 3152, Df = 31, MS = 1.07). Difference were determined by Wilcoxon test and ANOVA. *p < 0.001.
450 mL of ACD solution (citric acid 9.9 mg, sodium citrate 3.6 mg, and dextrose 11 mg, buffered with Tris acetate 50 mmol, pH 7.4). Blood samples were obtained between 7 and 8 AM and 4 hours after the last feeding, were immediately cooled (to 0 to 4C) and centrifuged at 500 3 g in a Sorvall RC5C refrigerated centrifuge. Plasma aliquots were used for the various biochemical assays. An ultrafiltered plasma sample was obtained using Centriflo-Amicon CF50A membranes (Danvers, MA) for the free fraction of plasma L-Trp. The high-performance liquid chromatography fluorescent method of Peat et al26 was used to quantify the free fraction and total plasma L-Trp. The difference between these 2 was considered to be the fraction bound to albumin.
Data Analysis Mean values and standard deviations were used for normally distributed data. Differences among mean values were analyzed for significance using Wilcoxon’s signed rank test and analysis of variance, with a level significance of P < .05.
RESULTS The IUGR and control infants are described in Table. Free plasma L-Trp concentrations were increased in the IUGR group relative to the control group, confirming previous observations.18,22 Bound L-trp was decreased, and total L-trp was unchanged (Figure 1). The group with IUGR demonstrated a significantly decreased amplitude of the N1/P2 component (P < .05) (Figure 2) and decreased latencies of P1, N1, and P2 (P < .05) (Figure 2). In the control group there was an increase only in N1 latency on day 60, and no changes in P1 and N1 latencies with age were observed (Figure 3). 593
Figure 1. Plasma L-Trp concentration. Each point represents the mean value ± standard deviation from 13 infants with IUGR and 12 normal controls. All determinations were done in duplicate samples. (A) Treatment: (SS) = 496.1, degrees of freedom (df) = 5, (MS) = 99.21; residual: SS = 63.06, df = 69, MS = 0.79140. (B) Treatment: SS = 356.5, df = 5, MS = 71.30; residual: SS = 524.5, df = 60, MS = 8.7.42. (C) Treatment: SS = 148.7, df = 5, MS = 29.74; residual: SS = 499.8, df = 60, MS = 8.742. *P < .05; **P < .01; ***P < .001; Wilcoxon’s test and analysis of variance.
Figure 2. AEPs obtained from the Cz reference electrode at a stimulation intensity of 60 dB in controls (—) and infants with IUGR (- - -). Each recording represents the mean values from the infants in that group. *P < .001; Wilcoxon’s test, comparing N1/P2 component amplitude of IUGR with controls.
DISCUSSION We measured the electrical activity of the auditory cortex using the N1/P2 component of AEP to assess whether it was associated with biochemical indicators of serotonin metabolism in infants with IUGR. We believe that the alterations in the N1/P2 component reflect impaired cortical activity caused by developmental anomalies of the serotonergic signal during corticogenesis provoked by IUGR. Our results confirm previous data from rats and humans with IUGR, demonstrating that the free faction of L-Trp was significantly elevated in plasma and brain.16,18,20,22 This early hyperserotonergic neuronal activity may negatively influence brain corticogenesis, particularly the normal sensory cortex conformation and later function. In older rats, the plasma free fraction of L-Trp and its binding to albumin may play a major role in the regulation 594
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Figure 3. Latencies of P1, N1, and P2 components of AEPs in infants with IUGR (n = 13) and controls (n = 12) at age 30 and 60 days. N1: treatment, SS = 2164, df = 3, MS = 721.4; residual, SS = 1932, df = 46, MS = 42.00., P2: treatment, SS = 17400, df = 3, MS = 5801; residual, SS = 2327, df = 46, MS = 50.58. P1: treatment, SS = 1864, df = 3, MS = 621.4; residual, SS = 1532, df = 46, MS = 32.00. *P < .01; **P < .001; Wilcoxon’s test and analysis of variance.
of the amount of L-Trp available for transport to the brain through the blood-brain barrier, thereby stimulating the synthesis of 5-HT (serotonin) by serotonergic neurons and their activity.27 In rats with IUGR, there is an increase in brain serotonin during the fetal period18-20 associated with an increase in free plasma L-Trp in the mother’s plasma. This serotonin precursor stays elevated in the offspring and chronically activates tryptophan-5-hydroxylase, resulting in serotonin synthesis in various brain regions, including the sensory cortex.16,28 A similar effect may be expected from the plasma free fraction of L-Trp in human infants with IUGR, in whom L-Trp free fraction is significantly elevated.18,22 Early alteration of the serotonin precursor’s availability through the placental–fetal barrier appears to be an effective way to activate serotonin synthesis in the brain,29 increase serotonin release, and modify its early signaling function in cortical neuronal assembly. Our results indicate that the greater the L-Trp free fraction in plasma, the lower the N1/P2 component amplitude. This observation strongly suggests a functional or causal relationship between brain serotonin activity and changes in the N1/P2 ratio. These components of auditory cortex electrical activity (N1/P2) are the result of spatial and temporal integration of various neuronal processes. Electrical dipole source analyses have identified 2 main components in each hemisphere: a tangential dipole source representing activation of the primary auditory cortex and a radial dipole generated by the activity in the secondary auditory cortex.14,15 The N1/P2 component has been identified in human infants, but whether its source is the same as in the adult brain remains controversial.30 Because the N1/P2 component can be induced by The Journal of Pediatrics November 2005
auditory stimulus and is better detected on the corresponding auditory projections of the scalp, that it reflects cortical integration of auditory activity in the human infant seems reasonable. In the normal and IUGR rat brain, serotonin plays an important role in regulating auditory evoked cortical responses.16 Thus we propose, based on the current biochemical and electrophysiological results, that in humans the response of the auditory cortex to sound intensity stimulus may be a function of brain serotonergic activity. Moreover, as in infants with IUGR, this response can be developmentally disrupted by anomalous sensory cortex conformation caused by gestational metabolical stress. Serotonin, together with other molecules (eg, norepinephrine, dopamine, acetylcholine), may act as ‘‘enabling factors’’ in the process of ‘‘experience-induced plasticity’’ in the developing visual cortex.11 Serotonin may control whether inputs can be strengthened or weakened and may influence longterm potentiation or long-term depression by lowering thresholds. Kojic et al11 reported that in the developing visual cortex, the serotonin receptors 5-HT2C occur only at a critical period on layer IV and that, depending on their density, they may facilitate either long-term potentiation or long-term depression of plastic responses. Long-term potentiation or long-term depression is implicated in the synaptic remodeling and refinement of neural connections during development. These findings support the significance of serotonin’s role in early cerebral corticogenesis and the later consequences of modifying its normal availability. Increased serotonin transmission during the prenatal period may later provoke altered behavior in sensory cortices and reproducible, quantitative differences in auditory cortex responses detected using AEPs to measure decreases in the amplitude of the sound-intensity dependent N1/P2 component (present study). Because there are abundant serotonin-innervated g-aminobutyric acid circuits that inhibit the neuronal responses in the brain,12 it is possible that augmented serotonin activity may be reflected in decreased amplitude of N1/P2 and changes in the onset of response, as demonstrated by the retarded latencies observed in infants with IUGR. Latency retardation was also noted in visual evoked potentials from infants with IUGR.31 This effect of IUGR on latencies may be related to modified development of thalamocortical pathways.31 Although there is a reported tendency for latencies in brain electrical sensory responses to decrease with age in normal children, this phenomenon was not observed in the present study, presumably because of the limited duration of the developmental period studied. In conclusion, it appears that in utero epigenetic factors influencing cerebral cortex development are associated with metabolic disturbances in brain serotonin synthesis induced by nutritional stress during the fetal period. The influence of early cortical changes is demonstrated by impairments in the postnatal function of the sensory cortex and can be identified through measurable changes in AEP responses to intensity modulated auditory stimuli. It is likely that other sensory responses are altered as well;28 if so, then the capacity to react to environmental signals will be different in newborns with IUGR with disturbed sensory responses. Ultimately, these Prenatal Impairment Of Brain Serotonergic Transmission In Infants
newborns’ cognitive development may be impaired, and thus IUGR will be shown to have life-long consequences and to represent a significant cause of diverse serotonin-related brain disorders in adults. We thank Dr. Jack Segal for his help in editing this manuscript.
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