Journal of Neuropathology and Experimental Neurology
Copyright q 2003 by the American Association of Neuropathologists
Vol. 62, No. 11
November, 2003
pp. 1178 1191
Serotonergic Brainstem Abnormalities in Northern Plains Indians with the
Sudden Infant Death Syndrome
HANNAH C. KINNEY, MD, LESLIE L. RANDALL, RN, MPH, LYNN A. SLEEPER, ED SC, MARIAN WILLINGER, PHD,
RICHARD A. BELLIVEAU, AB, NATASA ZEC, MD, PHD, LUCIANA A. RAVA, BA, LAURA DOMINICI, BA,
SOLOMON IYASU, MB, BS, BRADLEY RANDALL, MD, DONALD HABBE, MD, HARRY WILSON, MD,
FREDERICK MANDELL, MD, MARY MCCLAIN, RN, MS, AND THOMAS K. WELTY, MD
Abstract. The rate of the sudden infant death syndrome (SIDS) among American Indian infants in the Northern Plains is
almost 6 times higher than in U.S. white infants. In a study of infant mortality among Northern Plains Indians, we tested the
hypothesis that receptor binding abnormalities to the neurotransmitter serotonin (5-HT) in SIDS cases, compared with autopsied controls, occur in regions of the medulla oblongata that contain 5-HT neurons and that are critical for the regulation of
cardiorespiration and central chemosensitivity during sleep, i.e. the medullary 5-HT system. Tritiated-lysergic acid diethylamide binding to 5-HT1A-D and 5-HT2 receptors was measured in 19 brainstem nuclei in 23 SIDS and 6 control infants using
tissue receptor autoradiography. Binding in the arcuate nucleus, a part of the medullary 5-HT system along the ventral surface,
in the SIDS infants (mean age-adjusted binding 7.1 6 0.8 fmol/mg tissue, n 5 23) was significantly lower than in controls
(mean age-adjusted binding 13.1 6 1.6 fmol/mg tissue, n 5 5) (p 5 0.003). Binding also demonstrated significant diagnosis
3 age interactions (p , 0.04) in 4 other nuclei that are components of the 5-HT system. These data suggest that medullary
5-HT dysfunction can lead to sleep-related, sudden death in affected SIDS infants, and confirm the same binding abnormalities
reported by us in a larger dataset of non-American Indian SIDS and control infants. This study also links 5-HT abnormalities
in the arcuate nucleus with exposure to adverse prenatal exposures, i.e. cigarette smoking (p 5 0.011) and alcohol (p 5
0.075), during the periconceptional period or throughout pregnancy. Prenatal exposure to cigarette smoke and/or alcohol may
contribute to abnormal fetal medullary 5-HT development in SIDS infants.
Key Words:
Alcohol; American Indian/Alaska Native; Arcuate nucleus; Cigarette smoking; Raphé; Sudden infant death.
INTRODUCTION
The sudden infant death syndrome (SIDS) is defined
as the sudden death of an infant under 1 year of age that
remains unexplained by a complete autopsy, death scene
From the Departments of Pathology (HCK) Neurology (HCK, RAB,
NZ, LAR, LB), and Pediatrics (FM), Children’s Hospital and Harvard
Medical School, Boston, Massachusetts; Division of Reproductive
Health (LLR, SI), National Center for Chronic Disease Prevention and
Health Promotion, U.S. Centers for Disease Control and Prevention,
Atlanta, Georgia; New England Research Institutes (LAS), Watertown,
Massachusetts; National Institute of Child Health and Human Development (MW), National Institutes of Health, Bethesda, Maryland; Department of Pathology (BR), University of South Dakota Medical
School, Sioux Falls, South Dakota; Department of Pathology (DH),
Rapid City Regional Hospital, Rapid City, South Dakota; Department
of Pathology (HW), Providence Memorial Hospital, El Paso, Texas;
Department of Pediatrics (MM), Boston Medical Center, Boston, Massachusetts; Aberdeen Area Indian Health Service (TKW), Rapid City,
South Dakota.
Correspondence to: Hannah C. Kinney, MD, Department of Pathology, Children’s Hospital (Enders Building 206), 300 Longwood Avenue, Boston, MA 02115. E-mail: [email protected]
The National Institute for Child Health and Human Development,
U.S. Centers for Disease Control and Prevention, and Indian Health
Service funded the Aberdeen Area Indian Health Service (AAIMS)
through interagency agreements. The neuropathologic analysis in the
AAIMS was supported by subcontract NICHD-CRMC-92-05 (HCK).
The content of this publication does not necessarily reflect the views
or policies of the U.S. Department of Health and Human Services or
the Indian Health Service, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
investigation, and review of the clinical record (1). Typically, a seemingly healthy infant is found dead after a
sleep period, indicating that the death occurred during
sleep or a transition between sleep and waking. Despite
a national risk reduction campaign leading to an almost
50% decline in the overall SIDS rate in the United States,
SIDS remains the leading cause of postneonatal infant
mortality, with an incidence of 0.6/1000 live births. A
wide disparity exists in SIDS rates among different racial
and ethnic groups in the United States, particularly between certain American Indian tribes and all other races
combined, with an overall SIDS rate of 1.5/1,000 in
American Indian infants (2). The reason(s) for the high
SIDS rate in American Indian infants of particular tribes
is unknown, although genetic and/or environmental factors likely play a role. Especially prevalent in certain
American Indian populations is maternal cigarette smoking, a factor that increases the risk for SIDS 2- to 4-fold
or higher in American Indian (3, 4), as well as non-American Indian, populations (5, 6).
There are no reports of intrinsic brainstem abnormalities in American Indian infants dying of SIDS that may
put them at risk for sleep-related sudden death. In contrast, we found that many non-American Indian SIDS
cases have abnormal serotonergic receptor binding compared to autopsy controls in regions of the medulla that
contain serotonergic neurons and are involved in homeostatic control, the so-called medullary serotonergic system (7, 8). The affected components of the medullary
1178
1179
SEROTONERGIC ABNORMALITIES IN SIDS
serotonergic system in SIDS cases included the raphé obscurus (key component of the caudal raphé complex), arcuate nucleus along the surface of the ventral medulla
(which contains serotonergic receptors, terminals, and a
neuronal subpopulation), nucleus paragigantocellularis
lateralis (PGCL) (which likely contains the human homologue of the rodent preBötzinger complex important
in respiratory rhythm patterning (9), nucleus gigantocellularis (GC), and intermediate reticular zone. These regions of the medulla are involved in autonomic responses, thermoregulation, respiratory drive, upper airway
reflexes, chemosensitivity to carbon dioxide, and protective arousal mechanisms (7, 8). Indeed, the overall function of the medullary serotonergic raphé and extra-raphé
neurons may be to modulate and integrate autonomic and
somatomotor control according to the individual’s level
of arousal and/or brain pH and carbon dioxide (8–10).
The Aberdeen Area Infant Mortality Study (AAIMS)
provided the extraordinary opportunity to correlate medullary serotonergic system abnormalities with extensive
clinical material and infant information in the same cases.
The Aberdeen Area, serving Indian communities in North
Dakota, South Dakota, Nebraska, and Iowa, has the highest rate of infant mortality (15.6/1000 live births, 1992–
1994) in the Indian Health Service (2). The AAIMS
found that the SIDS rate for the Aberdeen Area was 4.5/
1,000 live births between 1993 and 1996, a rate that was
5.7 times the national rate for white infants during this
same period. The AAIMS established that SIDS accounted for 52% of the American Indian infant deaths during
this period (2). In the epidemiological limb of the
AAIMS, in which data from the autopsied SIDS infants
were compared to those from 2 living controls, a maternal
history of alcohol use, particularly binge drinking during
the first trimester, and greater than 2 layers of clothing at
night were significant risk factors for SIDS. Maternal cigarette smoking during pregnancy was not a statistically
significant risk factor, likely due to a combination of the
small sample size of SIDS cases and the high prevalence
of prenatal smoking in the mothers of SIDS infants and
the living controls in the Aberdeen Area (2). Nevertheless, the direction of the odds ratio was consistent with
the expected higher risk among infants of smokers, and
thus the results of the epidemiological study did not contradict the importance of maternal smoking as a potential
risk factor for SIDS (2).
In this study, we tested the hypotheses that 1) serotonergic receptor binding is on average lower in the arcuate
nucleus in American Indian SIDS infants compared to
autopsy control infants dying of known causes, as found
in non-American Indian SIDS cases (7); 2) serotonergic
receptor binding decreases with increasing age in SIDS
cases, but no correlation exists between binding and age
in controls (i.e. diagnosis 3 age interaction), in components extra-raphé of the medullary serotonergic system
as found in non-American Indian SIDS cases (7); and 3)
a serotonergic receptor binding deficiency in the arcuate
nucleus in American Indian infants correlates with maternal cigarette smoking and/or alcohol use during pregnancy, suggesting that the teratogens of cigarette smoke,
nicotine, and/or alcohol play key roles in the pathogenesis of postnatal medullary serotonergic dysfunction.
MATERIALS AND METHODS
Clinical Information
The AAIMS involved the analysis of 72 infant deaths, 56
(78%) of which were autopsied. Twenty-nine brains (52% of
the autopsied cases) were available for detailed anatomic and
neurochemical studies. SIDS cases were classified based upon
the standard definition above (n 5 23) (1); autopsy control cases
were infants who died suddenly, but in whom a complete autopsy established an anatomic cause of death (n 5 6). Information for all SIDS and autopsied control cases was available
from maternal interviews, standardized autopsies, death scene
investigations, and medical chart reviews (2).
Receptor Binding Studies and Generation of
Brainstem Autoradiograms
We selected 3H-lysergic acid diethylamide (LSD), which
binds to the 5-HT1A-D and 5-HT2 receptor subtypes, as the radioligand in order to compare the results of the AAIMS to those
of the non-American Indian population that we studied previously (7). Horizontally sectioned blocks of unfixed were stored
frozen at 2708C, and subsequently cryostat sectioned at 20 mm.
Unfixed, slide-mounted 20-mm sections of the brainstem were
preincubated in 300 mM Tris-maleate buffer (pH 7.4) for 30
min at room temperature. For determination of total binding,
sections were incubated with 5 nM 3H-LSD (New England Nuclear, Boston, MA) in 300 mM Tris-maleate buffer (pH 7.4)
with 0.1% ascorbic acid for 60 min at room temperature. For
determination of nonspecific binding, adjacent sections were incubated with 100 mM serotonin (Sigma, St. Louis, MO) added
to the buffer, for 60 min at room temperature. After washing
and drying, the sections were placed in cassettes and exposed
to 3H-sensitive film (LKB Ultrofilm-3H) for 8 weeks. Each cassette also contained a set of 3H-standards (Amersham, Piscataway, NJ) for conversion of optical density of silver grains in
autoradiograms to specific activities of tissue-bound in femtomoles/milligram of tissue (fmole/mg tissue).
Receptor binding density (expressed as the specific activity
of tissue-bound ligand) was analyzed in 19 brainstem nuclei of
each specimen (all nuclei were not available in all cases). For
each specimen, the selected nuclei were analyzed at a defined
level of the brainstem (2 autoradiograms for each nucleus). The
arcuate nucleus was analyzed at 6 defined levels of the medulla.
To ascertain anatomic boundaries of brainstem nuclei, selected
tissue sections that generated the autoradiograms were stained
with cresyl violet or hematoxylin and eosin, and compared with
corresponding autoradiogram. The brainstem nuclei were classified according to the atlas of Olszewski and Baxter (11), with
the exception of the intermediate reticular nucleus and raphé
nuclei, which were classified according to the atlas of Paxinos
and Huang (12).
J Neuropathol Exp Neurol, Vol 62, November, 2003
1180
KINNEY ET AL
TABLE 1
Comparison of Serotonergic Receptor Binding (fmol/mg tissue) in 19 Brainstem Nuclei between SIDS Cases
and Control Infants*
Site
ARC
HG
PIO
CENT
NTS
DMX
GC
PGCL
ROB
IRZ
PBL
LC
PONOR
GRPO
MEDR
PAG
CUN
RD
ICOL
SIDS age-adjusted
mean 6 SE (n)
7.1
37.4
18.4
38.5
46.5
51.1
6
6
6
6
6
6
0.8
3.12
1.5
2.6
3.8
3.3
57.8 6 5.8
38.9
42.5
47.3
13.0
66.8
57.4
45.5
6
6
6
6
6
6
6
4.0
5.2
7.1
2.8
31.8
6.3
4.0
35.0 6 3.6
(23)
(17)
(23)
(22)
(20)
(20)
(22)
(23)
(8)
(22)
(16)
(16)
(16)
(16)
(6)
(15)
(15)
(15)
(15)
Controls
age-adjusted
mean 6 SE (n)
13.1
40.2
15.2
36.3
41.3
50.3
6
6
6
6
6
6
1.6
5.8
2.9
5.0
6.9
6.0
40.8 6 11.7
42.8
45.4
42.5
9.1
54.9
55.6
39.7
6
6
6
6
6
6
6
7.1
9.3
12.7
1.6
13.0
10.9
7.0
37.4 6 6.2
(5)
(5)
(6)
(6)
(6)
(6)
(6)
(6)
(2)
(6)
(5)
(5)
(5)
(5)
(1)
(5)
(5)
(4)
(5)
Diagnosis
p value
Age
p value
0.003
0.669
0.337
0.698
0.517
0.903
0.460
0.079
0.675
0.028
0.937
0.752
Diagnosis 3
age interaction
p value
0.010
0.043
0.232
0.147
0.639
0.790
0.744
0.243
0.746
0.888
0.475
0.018
0.244
0.402
0.234
0.208
0.081
0.005
0.736
0.018
0.026
0.031
* p values are from analysis of covariance model without interaction. The diagnosis 3 postconceptional age interaction p value
is reported only when p , 0.05. Age-adjusted means are not present when a significant interaction is present. In this setting, ageadjusted means are not presented because the mean difference varies with age. Abbreviations: ARC, arcuate nucleus; HG,
hypoglossal nucleus; PIO, principal inferior olive; CENT, nucleus centralis; NTS, nucleus of the solitary tract; DMX, dorsal
motor nucleus of the vagus; GC, nucleus gigantocellularis; PGCL, nucleus paragigantocellularis lateralis; ROB, nucleus raphé
obscurus; IRZ, intermediate reticular zone; PBL, nucleus parabrachialis lateralis; LC, locus coeruleus; PONOR, nucleus pontis
oralis; GRPO, griseum pontis; MEDR, median raphé; PAG, periaqueductal gray; CUN, nucleus cuneiformis; RD, nucleus raphé
dorsalis; ICOL, inferior colliculus.
The defined brainstem levels at which the analysis was performed (with the atlas plate number of each level, included the
following: (a) caudal medulla, level of nucleus gracilis (Plate
VIII), for analysis of the arcuate nucleus; (b) caudal medulla,
level of area postrema (Plate X), for analysis of the arcuate
nucleus; (c) mid-medulla, level of nucleus of Roller (Plate XII),
for analysis of the hypoglossal nucleus, dorsal motor nucleus
of the vagus, nucleus of the solitary tract, raphé obscurus, nucleus centralis medullae, principal inferior olive, intermediate
reticular zone, and arcuate nucleus; (d) rostral medulla, level of
nucleus prepositus hypoglossi (Plate XIV), for analysis of the
nucleus gigantocellularis, nucleus paragigantocellularis lateralis, raphé obscurus, intermediate reticular zone, and arcuate nucleus; (e) rostral medulla, level of the rostral limit of the nucleus
of the solitary tract (Plate XVI), for analysis of the nucleus
gigantocellularis, nucleus paragigantocellularis lateralis, raphé
obscurus, and arcuate nucleus; (f) pontomedullary junction
(Plate XVIII), for analysis of the arcuate nucleus; (g) rostral
pons, level of nucleus parabrachialis lateralis (Plate XXVIII)
for analysis of the locus coeruleus, nucleus parabrachialis lateralis, median raphé nucleus, nucleus pontis oralis, and
griseum pontis; (h) caudal midbrain, level of the decussation of
the superior cerebellar peduncles (Plate XXXII) for analysis of
the inferior colliculus, periaqueductal gray (PAG), raphé dorsalis, nucleus cuneiformis, and interpeduncular nucleus.
J Neuropathol Exp Neurol, Vol 62, November, 2003
Optical density measured in the autoradiograms was converted to specific activities of tissue-bound ligand (in fmol/mg
tissue). Receptor binding density was determined in each brainstem nucleus by digitizing the boundaries of the nucleus upon
the color image of the autoradiogram displayed on the computer
monitor. All neurochemical analysis was performed blinded,
without knowledge of the diagnosis or age of the case. Specific
activity data were depicted as computer-generated images, with
a linear color scale of specific activity divided into 15 colorcoded intervals of quantitative values.
Statistical Analysis
We compared mean serotonergic receptor binding by case
diagnosis (SIDS versus control) adjusted for postconceptional
age using analysis of covariance (Table 1). For brainstem nuclei, where the mean difference by case diagnosis was not independent of age, the diagnosis 3 age interaction p value was
reported; age-adjusted means were omitted because in this setting the mean difference varies with age (Table 1). Because the
experimental conditions and data analysis for the 3H-LSD binding studies in the AAIMS were identical to those used for the
original, non-American Indian dataset (7), we combined the
binding values from the 2 datasets for each nucleus to increase
statistical power. We were also able to combine the binding
SEROTONERGIC ABNORMALITIES IN SIDS
values from the 2 datasets because we used the same 3H-standards for the exposure of the autoradiographic films. Moreover,
the absolute binding values for each nucleus were comparable
between the 2 datasets (data not shown), and the same patterns
of binding (e.g. diagnosis 3 age interaction) were present in
the same nucleus.
For the AAIMS data, we compared maternal and infant characteristics by case diagnosis using the Fisher exact test for categorical factors, the Student t-test for normally distributed variables, and the Wilcoxon rank sum test for non-normally
distributed continuous and ordinal variables. Association between serotonergic receptor binding in the arcuate nucleus and
maternal and infant characteristics was evaluated using linear
regression. Multivariate linear regression modeling was also
conducted to determine independent correlates of serotonergic
receptor binding. Variables that had a univariate p value of less
than 0.20 were considered candidate predictors in the multivariate model. All findings with a p value of 0.05 or less were
considered statistically significant. No adjustments were made
to p values to control for the number of variables examined.
All analyses were conducted using SAS (Statistical Analysis
System version 8.1, Cary, NC) and S-Plus (33 S-Plus 2000,
Insightful Corp., Seattle, WA).
RESULTS
Clinicopathologic Information
The causes of death in the autopsy control population
were acute respiratory infection, n 5 2; meningococcal
sepsis, n 5 1; chronic encephalopathy consistent with
perinatal hypoxia-ischemia, n 5 1; and congenital heart
disease presenting as sudden death, n 5 2. The age- and
body length-adjusted mean brain weight was not significantly different between the SIDS (adjusted mean 615
6 17 grams, n 5 20) and control cases (adjusted mean
652 6 34 grams, n 5 5) (p 5 0.343). Moreover, age- and
body length-adjusted brain weight was not significantly
different between infants whose mothers smoked (adjusted mean 632 6 19 grams, n 5 17) or did not smoke
(adjusted mean 619 6 34 grams, n 5 6) during pregnancy
(p 5 0.753), nor between infants whose mothers drank
alcohol (adjusted mean 624 6 18 grams, n 5 18) or did
not drink (adjusted mean 644 6 34 grams, n 5 5) during
pregnancy (p 5 0.609), whether or not they were SIDS
cases. We found no difference in the postmortem interval
between SIDS and control cases and there was no effect
of postmortem interval upon serotonergic receptor binding in the nuclei analyzed (data not shown).
Serotonergic Receptor Binding Analysis
Tritiated-lysergic acid diethylamide binding to serotonergic receptors (5-HT1A-D and 5-HT2 subtypes) was measured in 19 brainstem nuclei in a sample of serial sections
in SIDS (n 5 23) and autopsy control cases (n 5 6) using
the technique of quantitative tissue receptor autoradiography (Table 1) (7). Of note, binding levels were not
1181
available for all nuclei in all cases (Table 1). TritiatedLSD binding in the arcuate nucleus in the SIDS cases
(mean age-adjusted binding, 7.1 6 0.8 fmol/mg tissue, n
5 23) was significantly lower than that of autopsy controls (mean age-adjusted binding, 13.1 6 1.6 fmol/mg
tissue, n 5 5) (p 5 0.003), after adjusting for postconceptional age (Table 1; Fig. 1A). When the arcuate data
from the AAIMS and our original, non-American Indian
dataset (7) were combined, there was a total of 90 cases,
with a highly significant difference between SIDS (n 5
73, mean age-adjusted binding, 6.2 6 0.5 fmol/mg tissue)
and controls (n 5 17, mean age-adjusted binding, 17.3 6
0.9 fmol/mg tissue)(p , 0.0001) (Fig. 1B). Lowered 3HLSD binding was not present in all SIDS cases in the
arcuate nucleus in the AAIMS, as illustrated by the presence of binding levels in individual SIDS cases in the
control range (Fig. 1A). Measurements in the raphé obscurus were limited, with small numbers precluding valid
statistical analysis due to the fact that brainstems were
hemisected through the midline at autopsy for simultaneous analysis of neurochemistry and (formalin-fixed)
neurohistology.
Four nuclei demonstrated decreasing binding with increasing age in the SIDS group, but no correlation between binding and age in the control group (diagnosis 3
age interaction). These nuclei were the GC (interaction p
5 0.010), PGCL (interaction p 5 0.043), and intermediate reticular zone (interaction p 5 0.026) in the medulla
and the raphé dorsalis in the midbrain (interaction p 5
0.031) (Table 1). This diagnosis 3 age interaction is
graphically illustrated in the GC to demonstrate visually
that the SIDS infants have elevated 3H-LSD binding levels in early infancy that progressively decline to significantly lower levels in late infancy (Fig. 2). In comparison, the binding levels in the control cases are essentially
unchanged across the same time period. As postulated,
the GC, PGCL, and intermediate reticular zone demonstrated the same binding patterns (diagnosis 3 age interaction) between SIDS and control cases as in the original,
non-American Indian population (7).
Severe Arcuate Nucleus Hypoplasia in 2 SIDS Cases
In 2 SIDS cases (9% of the SIDS cases analyzed), the
arcuate nucleus was almost completely absent, i.e. severely hypoplastic, as well as deficient in 3H-LSD binding (Fig. 3). While these 2 cases demonstrated almost a
total absence of the arcuate nucleus upon visual inspection in a sample of serial sections, all other SIDS cases
with lowered 3H-LSD binding in the study had an arcuate
nucleus anatomically, albeit of variable volumes. The
clinical histories of the 2 cases with severe arcuate nucleus hypoplasia were typical of other SIDS cases in the
AAIMS. One infant boy died suddenly and unexpectedly
during a sleep period at 2.5 postnatal weeks. The mother
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1182
KINNEY ET AL
Fig. 1. A: Scatter plot of 3H-LSD binding levels (fmol/mg tissue) in the arcuate nucleus in SIDS (solid triangles) and controls
(open circles) according to postconceptional age, with added regression lines for comparison (SIDS, solid line; controls, dotted
line). B: Scatter plot of 3H-LSD binding levels (fmol/mg tissue) in the arcuate nucleus according to postconceptional age in
SIDS and control cases combined from the AAIMS dataset and the previously reported, non-American Indian dataset (8). The
symbols are used as follows: solid black triangles, non-American Indian SIDS cases (n 5 50); open black circles, non-American
Indian controls (n 5 12); solid blue triangles, Aberdeen (American Indian) SIDS cases (n 5 23), and open blue circles, Aberdeen
(American Indian) controls (n 5 5).
J Neuropathol Exp Neurol, Vol 62, November, 2003
SEROTONERGIC ABNORMALITIES IN SIDS
1183
Fig. 2. The pattern of 3H-LSD binding [fmol/mg tissue] with a significant diagnosis 3 age interaction (p 5 0.005) is illustrated
for the combined dataset of the AAIMS and original, non-American Indian cases (8) in the nucleus gigantocellularis. Each symbol
represents a single case, with added regression lines for comparison. The symbols are used as follows: solid black triangles, nonAmerican Indian SIDS (n 5 48); open black circles, non-American Indian controls (n 5 10); solid blue triangles, Aberdeen
(American Indian) SIDS cases (n 5 22); and open blue circles, Aberdeen (American Indian) controls (n 5 6).
reported smoking 3 cigarettes/day 3 months prior to pregnancy, 2 cigarettes/day in the second trimester, and no
cigarettes during the first and third trimesters. She also
reported drinking 6 drinks/day on 4 to 5 days/month 3
months before and during the first trimester, with no alcohol use during the second and third trimesters. She admitted to binge drinking (.4 drinks in a single day) 12
to 15 times in the 3 months before and during the first
trimester. The second infant, a girl, died suddenly and
unexpectedly during a sleep period at 8 postnatal weeks
(Fig. 3). The mother reported smoking 10 cigarettes/day
3 months prior to pregnancy and in the first and third
trimesters. She also reported drinking 10 drinks/day on 5
days/month during the 3 months prior to pregnancy, 4
drinks/day on 1 day/month in the first trimester, and 8
drinks/day on 10 days/month during the third trimester,
with binge drinking in the 3 months prior to pregnancy
and during the third trimester. The autopsy in both cases
was remarkable for intrathoracic petechiae involving the
heart and lungs, a typical finding in SIDS postmortem
examinations.
Clinical Correlations
We compared multiple sociodemographic and healthrelated variables between the SIDS victims and autopsy
controls (Table 2). All variables were not present for each
case. The autopsy controls were more likely to be low
birth weight, premature, never breast-fed, and born with
lower 1-minute and 5-minute Apgar scores than the SIDS
cases in this autopsy study (Table 2). Compared with the
living controls in the AAIMS study, the autopsy controls
also tended to be premature, low birth weight, less often
breast-fed, and put to bed at night with total clothing and
covers .5 (2); however, the autopsy and living control
groups had similar rates of exposure to maternal cigarette
smoke and binge drinking 3 months before or during
pregnancy (2).
Using all SIDS and control autopsy data combined in
the AAIMS, we examined the association between 3HLSD binding in the arcuate nucleus and clinical variables
obtained in the epidemiologic component of the AAIMS
(Tables 3, 4). Mean binding in the arcuate nucleus was
similar (p . 0.05) in groups defined by multiple socioeconomic, infant, and maternal variables (Table 3). Mean
binding differed significantly, however, in the infants of
smoking and nonsmoking mothers (p 5 0.011), with
higher mean binding observed in infants of mothers who
did not smoke before or during pregnancy (Table 4).
Mean binding was marginally lower in the group of infants whose mothers drank prior to or during pregnancy
(p 5 0.075), and in those whose mothers engaged in
binge drinking 3 months prior to or during pregnancy (p
5 0.080) (Table 4).
We used multivariate analysis of the association of serotonergic receptor binding in the arcuate nucleus and
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KINNEY ET AL
Fig. 3. The differential binding patterns for the radioligand 3H-LSD to the 5-HT1A-D and 5-HT2 receptors are compared between
a SIDS and control case at comparable caudal and rostral levels of the medulla. The receptor binding density mosaics are
standardized to the same color-code for visual comparison of binding levels between the 2 cases. Low serotonergic receptor
binding characterizes the arcuate nucleus along the pyramids (ventral surface) in the control case (white arrowheads); the mean
value averaged over multiple levels in the caudorostral plane was 10.0 fmol/mg tissue in this control case. In contrast, there is
essentially no arcuate nucleus in the SIDS case along the pyramidal surface based upon examination of the same sections stained
for cresyl violet (data not shown), and, consequently, there is no serotonergic receptor binding at this site. The mean value of
binding in the arcuate nucleus averaged over multiple levels of the medulla in the caudorostral plane was 3.3 fmol/mg in this
SIDS case. Note that the highest binding levels were in the raphé obscurus (ROb) in both the SIDS and control cases, and that
binding levels were comparable in the nucleus of the solitary tract (nTS) and principle inferior olive (PIO). In contrast, binding
levels are slightly higher in the in the GC (nucleus gigantocellularis) and nucleus paragigantocellularis (PGL) in the SIDS case
compared to the control case. Abbreviation: SpV, spinal subdivision of the trigeminal nucleus.
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SEROTONERGIC ABNORMALITIES IN SIDS
TABLE 2
Comparison of Selected Sociodemographic and Health-Related Variables between SIDS Cases and Control Infants
in the AAIMS*
Variable
n
SIDS
Infant male sex
Infant birth weight (grams)
Infant birth length (cm)
Infant gestational age at birth (weeks)
Gravidity‡
1-minute Apgar‡
5-minute Apgar‡
Ever breast-fed
Age of primary caretaker (yr)
Father’s age (yr)
Education of primary caretaker (yr)‡
Education of father (yr)‡
Married
Phone in home
Household income #15,000/year
Maternal smoking, 3 months before or during pregnancy
Maternal alcohol use, 3 months before or during pregnancy
Maternal binge drinking, 3 months before or during pregnancy
Bed sharing with parent
22
22
22
20
22
22
22
21
20
21
21
22
22
20
20
20
20
22
Usual placement of infant
Prone
Lateral
Supine
22
3
10
9
13.6%
45.5%
40.9%
Usually found during the night
Prone
Lateral
Supine
22
5
7
10
22.7%
31.8%
45.5%
5
0
2
3
0%
40.0%
60.0%
Total clothing 1 covers .5
Number of well baby visits‡
Number of prenatal care visits‡
Crowding index†‡
19
22
21
22.7%
1.0
5.5
1.0
5
4
5
5
40.0%
2.0
7.0
1.0
54.5%
3,302 6 413
48.3 6 7.0
39.1 6 1.8
3.5
8.0
9.0
63.6%
26.2 6 6.5
28.2 6 6.3
11.0
12.0
72.7%
36.4%
85.0%
80.0%
80.0%
65.0%
63.6%
N
Controls
p value
5
5
5
5
5
5
5
20.0%
2,372 6 866
46.8 6 7.5
35.2 6 4.1
4.0
7.0
8.0
0%
32.1 6 10.1
28.6 6 11.0
12.0
12.0
80.0%
60.0%
80.0%
60.0%
60.0%
60.0%
40.0%
0.326
0.073
0.679
0.103
0.735
0.057
0.005
0.033
0.115
0.913
0.283
0.428
1.000
0.371
1.000
0.562
0.562
1.000
0.371
5
4
5
3
5
5
5
5
5
5
5
5
0
3
1.000
0%
60.0%
40.0%
0.667
0.580
0.946
0.912
0.965
* Mean values 61 standard deviation and percentages are shown.
† Crowding index, number of total people living in the household/number of the total rooms in the household.
‡ Median.
selected clinical variables to identify independent correlates of binding. Multivariate analyses revealed that the
most significant predictor of lowered arcuate binding was
case status, i.e. SIDS (p , 0.01), and that cigarette smoking 3 months before or during pregnancy was likewise
an independent predictor, but not as significant as case
status (Table 5). These results indicate that binding is
higher in infants whose mothers do not smoke, but that
other, possibly unmeasured factors, account for more than
half of the observed variability in serotonergic receptor
binding. The multivariate analysis also suggests that maternal drinking in the 3 months before or during pregnancy is not an independent predictor of serotonergic
binding in the arcuate nucleus, and that smoking and
drinking are somewhat correlated with each other. The
mean difference in binding for SIDS versus autopsy controls (5 fmol/mg tissue) was not altered by adjustment for
drinking as long as smoking was already in the model,
but the mean difference in binding associated with smoking is slightly smaller when drinking is present in the
model.
DISCUSSION
The neuropathologic analysis in the AAIMS indicate
that many American Indian infants in the Northern Plains
share with many non-American Indian infants a common
intrinsic defect in the medullary serotonergic system that
may put them at risk for sudden death. Serotonergic receptor binding abnormalities in SIDS infants likely reflect
serotonergic dysfunction in critical medullary pathways
that prevents the infants from properly detecting hypoxic,
hypercarbic, asphyxial, and/or hypotensive stimuli during
sleep, particularly in the prone (face-down) or face-covered position; consequently, these infants can not mount
J Neuropathol Exp Neurol, Vol 62, November, 2003
1186
KINNEY ET AL
TABLE 3
Mean (6SD) Serotonergic Receptor Binding (fmol/mg tissue) in the Arcuate Nucleus for Selected Maternal
Sociodemographic and Health Characteristics and Infant Care Characteristics in the AAIMS (n 5 27)*
Variables
Maternal characteristics
Married
Education ,13 years
Gravida .2
Transport problem for prenatal care
Home visit by PHN
Maternal pre-pregnancy BMI ,25
History of gestational diabetes
History of other STDs
History of urinary tract infection
History of bleeding $20 wks
gestation
Newborn characteristics
Infant male sex
Birth weight ,3,000 grams
Gestation ,37 weeks
Hyperbilirubinemia
Hypoglycemia
Meconium staining
Mean 6 SD
Comparison group
8.5
20
22
8.6
21
8.3
5
5.5
10
7.1
16
7.3
2
5.9
5
8.0
10
7.8
2 10.9
6
6
6
6
6
6
6
6
6
6
4.7
3.8
4.1
3.6
2.5
3.1
6.5
1.6
4.7
0.6
Unmarried
Education $ 13 years
Gravida #2
No transport problem for prenatal care
No home visit by PHN
Maternal Pre-pregnancy BMI $25
No history of gestational diabetes
No history of other STDs
No history of urinary tract infection
No history of bleeding $20 wks gestation
8.1 6 1.9
7
4
7.1 6 6.6
6
8.7 6 4.8
22
9.0 6 4.0
16
9.4 6 4.8
10 10.0 6 5.3
22
9.2 6 3.7
19
8.4 6 4.2
16
8.8 6 4.0
19
8.0 6 3.7
8.2
9.7
8.7
8.1
4.9
5.2
6
6
6
6
6
6
4.2
2.5
3.7
3.6
4.6
3.0
Infant female sex
Birth weight $3000 grams
Gestation $37 weeks
No hyperbilirubinemia
No hypoglycemia
No meconium staining
14
21
19
11
21
23
n
13
6
6
14
3
3
n
Mean 6 SD p value
8.5
8.0
8.4
7.6
8.8
8.7
6
6
6
6
6
6
0.778
0.525
0.846
0.086
0.176
0.112
0.253
0.844
0.554
0.300
4.2
4.5
4.5
4.1
4.2
4.2
0.859
0.388
0.905
0.740
0.144
0.171
6.2 6 6.0
0.533
Other sociodemographic characteristics
Paternal education ,13 years
22
8.0 6 3.8
Paternal education $13 years
Variables
Phone in home
Household income #15,000/yr
11
21
7.7 6 4.4
8.1 6 4.4
No phone in home
Household income .15,000/year
16
8.8 6 4.0
4 10.0 6 3.5
0.504
0.425
Ever breast-fed
Well baby visits ,3
Room sharing with parent
Bed sharing with parent
14
18
26
16
7.0
7.6
8.4
7.8
Never breast-fed
Well baby visits $3
No room sharing with parent
No bed sharing with parent
12
9.8 6 4.2
5 10.4 6 4.5
1
7.2 6 *
11
9.2 6 4.1
0.090
0.188
0.771
0.406
Usually placed
Prone
Lateral
Supine
3
13
11
9.7 6 4.3
7.4 6 4.7
9.2 6 3.5
Usually found during the night
Prone
Lateral
Supine
5
9
13
9.1 6 3.6
7.9 6 4.0
8.5 6 4.7
2
6
7
6.5 6 1.1
8.0 6 6.0
7.9 6 5.5
Layers of clothing .2
Layers of covers .2
Total clothing 1 covers .5
6
6
6
6
3.8
3.9
4.2
4.2
2
0.490
0.864
Layers of clothing #2
Layers of covers #2
Total clothing 1 covers #5
25
21
20
8.5 6 4.3
8.5 6 3.6
8.6 6 3.7
0.503
0.801
0.721
* Abbreviations: Trim, trimester; PHN, public health nurse; BMI, body mass index; SD, standard deviation.
successful protective responses, such as head-turning
and/or arousal to the adverse stimuli, and they go on to
die (8). The AAIMS neuropathology data are critically
important because they confirm in an independent population the finding of medullary serotonergic abnormalities in SIDS cases originally reported by us in a completely separate dataset (7), and they suggest an intrinsic
brainstem defect in SIDS cases in American Indians, a
high-risk population. The AAIMS neuropathology data
J Neuropathol Exp Neurol, Vol 62, November, 2003
are also important because they link a specific brainstem
neurochemical abnormality to prenatal exposure to maternal cigarette smoke, a major risk factor for SIDS
throughout the world. The possibility of an underlying
brainstem vulnerability influenced by, and potentially due
to, prenatal factors has long been suspected in SIDS research (8). Evidence for this hypothesis is now provided
by the linkage of the extensive epidemiological and neuropathologic data of the AAIMS that could only be col-
1187
SEROTONERGIC ABNORMALITIES IN SIDS
TABLE 4
Mean (6SD) Serotonergic Receptor Binding (fmol/mg tissue) in Arcuate Nucleus for Variables Related to Maternal
Smoking and Alcohol Drinking 3 Months Prior to and During Pregnancy*
Number
Variables
T
Maternal smoking
Any use, 3 months before or during pregnancy
Any use 3 months before pregnancy
Trimester 1
S
Mean 6
C
SD
Number
Comparison group
T
S
C Mean 6 SD
Mean difference
p
(95% CI)
value
19 16 3 7.2 6 3.7 No use, before or during
pregnancy
6
4 2
12.1 6 4.0
4.93
(1.27, 8.59)
0.011
19 16 3 7.2 6 3.7 No use 3 months before
pregnancy
17 14 3 7.6 6 3.7
No use trimester 1
6
4 2
12.1 6 4.0
0.011
8
6 2 10.13 6 5.1
4.93
(1.27, 8.59)
2.56
(21.16, 6.27)
1.27
(22.45, 5.00)
0.69
(23.06, 4.45)
1.97
(21.32, 5.26)
0.168
Trimester 2
9
7 2 7.6 6 4.3
No use trimester 2
16 13 3
8.9 6 4.4
Trimester 3
9
7 2 8.0 6 4.0
No use trimester 3
16 13 3
8.7 6 4.5
11
8 3
9.6 6 4.7
19 16 3 7.5 6 3.8 No use before or during
pregnancy
6
4 2
11.1 6 4.9
3.56
0.075
(20.39, 7.50)
19 16 3 7.5 6 3.8 No use 3 months before
pregnancy
14 12 2 8.0 6 4.0
No use trimester 1
6
4 2
11.1 6 4.9
11
8 3
8.9 6 4.8
3.56
(20.39, 7.50)
0.89
(22.74, 4.51)
0.48
(24.45, 5.41)
2.14
(22.00, 6.27)
3.11
(20.41 6.63)
16 14 2 7.6 6 3.6 No Postnatal use
Postnatal
Maternal drinking
Any use, 3 months before or during pregnancy
Any use 3 months before pregnancy
Trimester 1
Trimester 2
4
4 0 8.0 6 2.6
No use trimester 2
21 16 5
8.5 6 4.6
Trimester 3
6
6 0 6.8 6 2.7
No use trimester 3
19 14 5
8.9 6 4.6
Binge drinking, 3
months before or
during pregnancy
Binge drinking 3
months before pregnancy
Trimester 1
16 13 3 7.3 6 3.9 No binge drinking, before
or during pregnancy
15 12 3 7.2 6 4.1 No binge drinking 3
months before pregnancy
8 8 0 8.0 6 3.9
No binge trimester 1
0.487
0.706
0.229
0.075
0.618
0.843
0.296
9
7 2
10.4 6 4.4
10
8 2
10.1 6 4.2
2.82
0.108
(20.66, 6.31)
17 12 5
8.6 6 4.5
0.753
0.60
(23.27, 4.46)
0.781
0.76
(24.80, 6.31)
0.272
2.40
(22.01, 6.80)
Trimester 2
3
3 0 7.7 6 3.1
No binge trimester 2
22 17 5
8.5 6 4.5
Trimester 3
5
5 0 6.5 6 2.9
No binge trimester 3
20 15 5
8.9 6 4.5
0.080
* Abbreviations: T 5 Total sample size; S 5 SIDS sample size; C 5 Control infant sample size.
lected by a multidisciplinary, multicultural team in a
tight-knit and supportive community with a high SIDS
rate.
Limitations of the Study
A limitation of this study is the small size of the dataset, particularly the control group. Despite the small
sample size, statistically significant differences were
found in serotonergic receptor binding in the same regions and with the same patterns of altered binding (e.g.
diagnosis 3 age interaction) between the SIDS and control groups that were found in the larger dataset originally
reported by us (7). The confirmation of the same binding
abnormalities in the small dataset as in the larger dataset
attests, in our opinion, to the strength and validity of the
findings. A second limitation is that the autopsy control
infants were not representative of living controls. This
limitation is not unique to the AAIMS, but is inherent in
all pediatric autopsy case/control studies because normal
infants do not die. Compared to the living controls in the
AAIMS, the autopsy controls tended to be premature, low
birth weight, less often breast-fed, and put to bed at night
with heavy clothing and covers. Nevertheless, the autopsy and living control infants had similar rates of exposure
J Neuropathol Exp Neurol, Vol 62, November, 2003
1188
KINNEY ET AL
TABLE 5
Multivariate Analysis
Variable
Mean group
difference
Partial squared
correlation
p value
A. Multivariate linear regression model for serotonergic receptor binding (fmol/mg tissue) in the arcuate nucleus: by diagnosis
and smoking: n 5 25 with adjusted R2 5 0.425.
Diagnosis (SIDS vs Control)
25.02
0.296
0.006
Maternal smoking 3 months prior to or during
24.05
0.238
0.016
pregnancy
B. Multivariate linear regression model for serotonergic receptor binding (fmol/mg tissue) in the arcuate nucleus: by diagnosis
and smoking & drinking: n 5 25 with adjusted R2 5 0.421.
Diagnosis (SIDS vs Control)
24.82
0.283
0.009
Maternal smoking 3 months prior to or during
23.58
0.185
0.041
pregnancy
Maternal drinking 3 months prior to or during
21.49
0.038
0.374
pregnancy
C. Multivariate linear regression model for serotonergic receptor binding (fmol/mg tissue) in the arcuate nucleus: by diagnosis
and smoking & binge drinking: n 5 25 with adjusted R2 5 0.451.
Diagnosis (SIDS vs Control)
25.16
0.326
0.004
Maternal smoking 3 months prior to or during
23.35
0.174
0.048
pregnancy
Maternal binge drinking 3 months prior to or dur21.93
0.087
0.172
ing pregnancy
to maternal cigarette smoke and binding drinking 3
months before or during pregnancy, the 2 clinical variables of most interest in the clinicopathologic correlations
in this study. Moreover, the mean brain weights adjusted
for age and body length were not significantly different
between the SIDS and control groups, nor between infants exposed to prenatal cigarette smoke or alcohol compared to infants without these exposures, whether they
were SIDS or control cases. This information suggests
that overall brain development was comparable between
the SIDS and control cases, and underscores the significance of the identification of brainstem neurochemical
differences between the 2 groups. Nevertheless, the small
sample size and the general recognition that autopsy control populations are not truly representative of living controls, caution is warranted in the interpretation of clinical
variables associated with lowered serotonergic binding in
the arcuate nucleus. The association of lowered serotonergic receptor binding with maternal cigarette smoke
and alcohol use is thus hypothesis-generating, and requires confirmation in a larger human infant autopsy dataset (a major logistical and potentially unfeasible undertaking) and in animal models.
A third limitation of this study is that the analysis concerning prenatal exposures to cigarette smoke and alcohol
was based upon maternal self-reporting. In order to obtain biochemical measurements of maternal alcohol and
cigarette use during pregnancy (e.g. serum cotinine levels), a prospective study design is necessary. In the
J Neuropathol Exp Neurol, Vol 62, November, 2003
AAIMS, however, the design was retrospective, and no
measurements of prenatal exposures were available from
mothers whose infants subsequently died of SIDS or other causes. Of note, the questions about alcohol use in the
AAIMS were formally validated in the population prior
to the study (14). It was determined that the proportion
of pregnant control women in the AAIMS who reported
drinking during the third trimester (6.6%) was similar to
the proportion of mothers in the Aberdeen Area (6.3%)
who reported drinking during pregnancy on birth certificates between 1994 and 1996.
Abnormal Medullary Serotonergic Receptor Binding in
SIDS Infants in the AAIMS
The major findings in this study are that serotonergic
receptor binding is significantly lowered in SIDS cases
compared to controls in the arcuate nucleus, and that it
is significantly altered (diagnosis 3 age interaction) in
the GC, PGCL, intermediate reticular zone, and raphé
dorsalis in the SIDS compared to control cases. Essentially these same abnormalities were found in the previous SIDS and control dataset reported by us (7). The
human arcuate nucleus along the ventral medullary surface is considered the ventral extension of the caudal raphé complex, and a part of the medullary serotonergic
system as defined by us (8). It contains a small subpopulation of serotonergic neurons, as well as serotonergic
fibers and receptors (7, 8). We postulate that the serotonergic neuronal subpopulation in the arcuate nucleus is
SEROTONERGIC ABNORMALITIES IN SIDS
homologous to known chemosensitive serotonergic neurons on the ventral surface of the rat medulla (8, 10, 14).
Anatomic and neurochemical abnormalities, including
lowered serotonergic receptor binding, occur in the arcuate nucleus in non-American Indian SIDS cases (7, 8,
15–20). The finding of hypoplasia of the arcuate nucleus
associated with lowered serotonergic binding in 2 SIDS
infants in the AAIMS is comparable to that in non-American Indian SIDS and control datasets in which aplasia
or severe hypoplasia of the arcuate nucleus has been reported in 5% to 56% of SIDS cases (15, 19, 20). The
finding of a structural underdevelopment of the arcuate
nucleus points to a prenatal origin of the abnormality
around the time of the proliferation and migration of future arcuate neurons in the first and early second trimester
from its embryonic anlage, the rhombic lip (8, 15).
We found a different pattern of serotonergic receptor
binding in the GC, PGCL, intermediate reticular zone,
and raphé dorsalis in the SIDS cases than that found in
the arcuate nucleus. In the arcuate nucleus, binding was
significantly lower in the SIDS compared to controls, but
the binding levels did not change with increasing postconceptional age in either the SIDS or control cases. In
contrast, binding in the GC, PGCL, intermediate reticular
zone, and raphé dorsalis was initially elevated in the
SIDS cases and declined to lower values with increasing
postconceptional age, whereas the binding levels did not
change with age in the control cases, i.e. there was a
significant diagnosis 3 age interaction between the SIDS
and control groups. The GC, PGCL, and intermediate reticular zone, subdivisions of the reticular formation, contain the extra-raphé populations of serotonergic neurons
in the medulla, and are the extra-raphé components of the
medullary serotonergic system that are involved in homeostatic control (8). These extra-raphé (lateral) serotonergic neurons extensively interconnect with the raphé
(midline) serotonergic neurons, particularly the raphé obscurus (8). In baseline (control) developmental studies,
we have shown that there is a significant decline in serotonergic receptor binding in these extra-raphé nuclei
from high levels at midgestation to low levels around
term birth, with a plateau at low levels across the first
year of postnatal life (21). Consequently, the decline from
high to low binding levels in these same regions in the
SIDS cases who died during the first year of life may
reflect delayed maturation of these binding levels, with a
shift to the fall in binding levels from the appropriate
period between midgestation to birth, to the inappropriate
period between birth and the end of the first postnatal
year. Alternately, this distinctive pattern of binding in the
GC, PGCL, intermediate reticular zone, and raphé dorsalis may reflect adaptive changes in the SIDS cases, with
receptor upregulation around birth followed by decompensation and receptor downregulation (loss) over the
first postnatal year.
1189
Clinical Correlations with Abnormal Serotonergic
Receptor Binding in the Arcuate Nucleus
in the SIDS Infants
Given the extensive epidemiologic data in the AAIMS,
we had the unique opportunity to explore possible clinical
correlations with abnormal serotonergic receptor binding
in the arcuate nucleus. We found no association between
3
H-LSD binding in the arcuate nucleus and multiple socioeconomic, infant, and maternal values. Despite the
lack of association, we provide all of these data above
due to their uniqueness in the SIDS literature, and their
potential value in formulating hypotheses concerning
clinical antecedents to brainstem serotonergic binding abnormalities in human infants. Mean 3H-LSD binding in
the arcuate nucleus, however, was significantly lower (p
5 0.011) in infants of mothers who smoked before or
during pregnancy, and was marginally lower in infants
who mothers who drank before or during pregnancy (p
5 0.075), and in infants whose mothers engaged in binge
drinking 3 months prior to or during pregnancy (p 5
0.080), than in infants whose mothers did not engage in
these behaviors. We used multivariate analysis of the association of serotonergic receptor binding in the arcuate
nucleus and selected clinical variables to identify independent correlates of binding. Given the small sample
size and the general recognition that the autopsy control
population does not necessarily reflect normal living infants, this analysis is considered exploratory. Multivariate
analyses revealed that the most significant predictor of
lowered arcuate binding was case status, i.e. SIDS (p ,
0.01), and that cigarette smoking 3 months before or during pregnancy was likewise an independent predictor, but
not as significant as case status. Thus, a ‘‘SIDS-specific’’
factor appears to be the most important variable associated with lowered serotonergic binding in the arcuate nucleus, and that is likely directly involved in the pathogenesis of this abnormality. Prenatal exposure to cigarette
smoking, on the other hand, may be a risk factor that
compounds the underlying intrinsic defect, or alternately,
is directly responsible for the defect in some cases. The
multivariate analysis also suggests that maternal drinking
in the 3 months before or during pregnancy is not an
independent predictor of serotonergic binding in the arcuate nucleus, and that smoking and drinking are somewhat correlated with each other.
Serotonergic Receptor Binding Abnormalities in SIDS
Infants and Adverse Prenatal Exposures
While the cause and pathogenesis of the serotonergic
receptor binding abnormalities in the medulla in the SIDS
cases of the AAIMS are unknown, the significant association of lowered binding in the arcuate nucleus with
maternal cigarette smoking in the periconceptional period
(3 months prior to pregnancy) and during pregnancy, and
J Neuropathol Exp Neurol, Vol 62, November, 2003
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KINNEY ET AL
its marginal association with maternal alcohol use, notably binge drinking, in the periconceptional period and
during pregnancy, suggest that the defect likely originates
during gestation and is influenced by toxic exposures.
One or more of the components of cigarette smoke, potentially nicotine, hypoxia-ischemia secondary to nicotine’s effects upon placental and fetal vascular perfusion,
and alcohol, alone or in combination with cigarette
smoke, may impinge upon the development of the medullary serotonergic system that is already abnormal in
certain SIDS infants, thereby increasing the risk for expression of the abnormality.
In experimental systems, inter-relationships between
nicotine or alcohol and the development and/or function
of the brainstem serotonergic neurons are well documented. Cholinergic pathways via nicotinic receptors, for
example, influence serotonergic neurotransmission; nicotine in doses relevant to human consumption targets the
brain serotonergic system; and prenatal exposure to nicotine affects postnatal serotonergic development in the
brainstem (22–25). In the human midgestational fetus,
nicotinic receptors are heavily concentrated in medullary
nuclei with serotonergic neurons (26), including the caudal raphé complex and PGCL, which are known to project to the human fetal arcuate nucleus (27); after fetal
life, nicotinic binding decreases dramatically in medullary serotonergic nuclei to negligible levels in the infant
(26). One potential outcome of prolonged stimulation to
the transiently over-expressed nicotinic receptors in fetal
serotonergic circuits by exogenous (maternal) nicotine is
desensitization, with a potentially long-lasting effect
upon the intrinsic serotonergic defect in the critical regions (26). Prenatal exposure to alcohol also adversely
affects the development of the serotonergic neurons, especially in the rostral raphé complex in rats (28). This
observation is particularly relevant to the AAIMS because serotonergic receptor binding is abnormal (diagnosis 3 age interaction) in the SIDS infants in the raphé
dorsalis, a component of the rostral raphé. The raphé dorsalis projects widely to regions throughout the forebrain,
and is involved in arousal, cognition, motor control, and
mood. Prenatal alcohol treatment in rodent models decreases the formation and migration of serotonergic neurons in the rostral raphé, and decreases their number by
20% to 30% (28). In addition, it reduces serotonin levels
in the brainstem, decreases the density of serotonin neurons in the brainstem raphé, and decreases serotonergic
innervation of target forebrain areas (29–31). Serotonergic agonists can prevent several adverse effects of alcohol
upon the development of serotonergic brainstem systems
(30). Thus, experimental data underscore the idea that
prenatal exposures to nicotine and alcohol, either separately or in combination, can disrupt the development of
serotonergic brainstem neurons in utero.
J Neuropathol Exp Neurol, Vol 62, November, 2003
Conclusions
In conclusion, SIDS infants in the AAIMS demonstrated serotonergic receptor binding abnormalities in the
medullary serotonergic system, which likely put them at
risk for sleep-related, sudden death during a vulnerable
developmental period. The AAIMS neuropathology data
point to future avenues for SIDS research, including the
study of the prenatal development of the human medullary serotonergic system, the elucidation of the specific
serotonergic receptor subtypes at fault in the SIDS cases,
and the potentially harmful effects of cigarette smoke,
nicotine, alcohol, and/or chronic hypoxia upon medullary
serotonergic development in perinatal animal models. Of
utmost importance, the AAIMS reinforces a key message
of current risk reduction campaigns for SIDS, i.e. strictly
avoid cigarette smoking and alcohol use during pregnancy. Our data underscore this message by suggesting that
prenatal exposure to cigarette smoke and alcohol may
cause or exacerbate a defect in medullary serotonergic
development, and thus they provide a biologically plausible mechanism for brainstem injury related to adverse
prenatal exposures in affected SIDS infants in high-risk
populations.
ACKNOWLEDGMENTS
We would like to extend our appreciation to the many families who
participated in this study. We also appreciate help with the statistical
analysis from Ms. Annie Zhang, MPH, MB; with the technical procedures from students in the Native American Four Directions Summer
Program at Harvard Medical School, Boston, MA; and with final manuscript preparation from Ms. Rachael J. Keefe. We appreciate the assistance provided by the Aberdeen Area Tribal Chairman’s Health
Board, the 10 participating Tribal Communities, the Office of Epidemiology, Aberdeen Area Indian Health Service, and the Perinatal Infant
Mortality Review Committee.
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syndrome (SIDS): Deliberations of an expert panel convened by
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Received March 10, 2003
Revision received July 21, 2003
Accepted August 18, 2003
J Neuropathol Exp Neurol, Vol 62, November, 2003