Decreased serotonergic receptor binding in rhombic lip-derived regions of the medulla oblongata in the sudden infant death syndrome.

The sudden infant death syndrome (SIDS) is postulated to result from a failure of homeostatic responses to life-threatening challenges (e.g. asphyxia, hypercapnia) during sleep. The ventral medulla participates in sleep-related homeostatic responses, including chemoreception, arousal, airway reflex control, thermoregulation, respiratory drive, and blood pressure regulation, in part via serotonin and its receptors. The ventral medulla in humans contains the arcuate nucleus, in which we have shown isolated defects in muscarinic and kainate receptor binding in SIDS victims. We also have demonstrated that the arcuate nucleus is anatomically linked to the nucleus raphé obscurus, a medullary region with serotonergic neurons. We tested the hypothesis that serotonergic receptor binding is decreased in both the arcuate nucleus and nucleus raphé obscurus in SIDS victims. Using quantitative autoradiography, 3H-lysergic acid diethylamide (3H-LSD binding) to serotonergic receptors (5-HT1A-D and 5-HT2 subtypes) was measured blinded in 19 brainstem nuclei. Cases were classified as SIDS (n = 52), acute controls (infants who died suddenly and in whom a complete autopsy established a cause of death) (n = 15), or chronic cases with oxygenation disorders (n = 17). Serotonergic binding was significantly lowered in the SIDS victims compared with controls in the arcuate nucleus (SIDS, 6 +/- 1 fmol/mg tissue; acutes, 19 +/- 1; and chronics, 16 +/- 1; p = 0.0001) and n. raphé obscurus (SIDS, 28 +/- 3 fmol/mg tissue; acutes, 66 +/- 6; and chronics, 59 +/- 1; p = 0.0001). Binding, however, was also significantly lower (p < 0.05) in 4 other regions that are integral parts of the medullary raphé/serotonergic system, and/or are derived, like the arcuate nucleus and nucleus raphé obscurus, from the same embryonic anlage (rhombic lip). These data suggest that a larger neuronal network than the arcuate nucleus alone is involved in the pathogenesis of SIDS, that is, a network composed of inter-related serotonergic nuclei of the ventral medulla that are involved in homeostatic mechanisms, and/or are derived from a common embryonic anlage.

Tritiated-naloxone binding to brainstem opioid receptors in the sudden infant death syndrome.

The sudden infant death syndrome (SIDS) is defined as the sudden death of an infant under 1 year of age that remains unexplained after a thorough case investigation, including a complete autopsy. We hypothesized that SIDS is associated with altered 3H - naloxone binding to opioid receptors in brainstem nuclei related to respiratory and autonomic control. We analyzed 3H - naloxone binding in 21 regions in SIDS and control brainstems using quantitative tissue receptor autoradiography. Three groups were analyzed: SIDS (n = 45); acute controls (n = 14); and a chronic group with oxygenation disorders (n = 15). Opioid binding was heavily concentrated in the caudal nucleus of the solitary tract, nucleus parabrachialis medialis, spinal trigeminal nucleus, inferior olive, and interpeduncular nucleus in all cases analyzed (n = 74). The arcuate nucleus on the ventral medullary surface contained negligible binding in all cases (n = 74), and therefore binding was not measurable at this site. We found no significant differences among the three groups in the age-adjusted mean 3H - naloxone binding in 21 brainstem sites analyzed. The only differences we have found to date between SIDS and acute controls are decreases in 3H - quinuclidinyl benzilate binding to muscarinic cholinergic receptors and in 3H - kainate binding to kainate receptors in the arcuate nucleus in alternate sections of this same data set. The present study suggests that there is not a defect in opioid receptor binding in cardiorespiratory nuclei in SIDS brainstems.

Developmental changes in heterogeneous patterns of neurotransmitter receptor binding in the human interpeduncular nucleus.

The interpeduncular nucleus (IPN) exhibits many complex features, including multiple subnuclei, widespread projections with the forebrain and brainstem, and neurotransmitter heterogeneity. Despite the putative importance of this nucleus, very little is known about its neurochemical development in the human. The human IPN is cytoarchitectonically simple, unlike the rat IPN, which displays considerable heterogeneity. In the following study, we hypothesized that the developing human IPN is neurochemically heterogeneous despite its cytological simplicity. The chemoarchitecture in this study was defined by neurotransmitter receptor binding patterns by using quantitative tissue autoradiography for the muscarinic, nicotinic, serotoninergic, opioid, and kainate receptors. We examined neurotransmitter receptor binding in the developing human IPN in a total of 15 cases. The midbrains of five midgestational fetuses (19-26 gestational weeks) and six infants (38-74 postconceptional weeks) were examined. The midbrain of one child (4 years) and three adults (20-68 years) were analyzed as indices of maturity. At all ages examined, high muscarinic binding was localized to the lateral subdivision of the IPN, high serotoninergic binding was localized to the dorsal IPN, and high opioid receptor binding was localized to the medial IPN. The developmental profile was unique for each radioligand. We report a heterogenous distribution of neurotransmitter receptor binding in the developing human IPN, which supports a complex role for it in human brain function.

Decreased kainate receptor binding in the arcuate nucleus of the sudden infant death syndrome.

The human arcuate nucleus is postulated to be homologous to ventral medullary surface cells in animals that participate in ventilatory and blood pressure responses to hypercarbia and asphyxia. Recently, we reported a significant decrease in muscarinic cholinergic receptor binding in the arcuate nucleus in victims of the sudden infant death syndrome compared with control patients that died of acute causes. To test the specificity of the deficit to muscarinic cholinergic binding, we examined kainate binding in the arcuate nucleus in the same database. We assessed 3H-kainate binding to kainate receptors with tissue receptor autoradiography in 17 brainstem nuclei. Analysis of covariance was used to examine differences in binding by diagnosis, adjusted for postconceptional age (the covariate). Cases were classified as SIDS, 47; acute control, 15; and chronic group with oxygenation disorder, 17. (Acute controls are infants who died suddenly and unexpectedly and in whom a complete autopsy established a cause of death). The arcuate nucleus was the only region in which there was a significant difference in the age-adjusted mean kainate binding between the SIDS group (37+/-2 fmol/mg tissue) and both the acute controls (77+/-4 fmol/mg tissue) (p < 0.0001) and the chronic group (69+/-4 fmol/mg tissue) (p < 0.0001). There was a positive correlation between the density of muscarinic cholinergic and kainate binding in the SIDS cases only (R = 0.460; p = 0.003). The neurotransmitter deficit in the arcuate nucleus in SIDS victims involves more than one receptor type relevant to carbon dioxide and blood pressure responses at the ventral medullary surface.

Three-dimensional distribution of [3H]quinuclidinyl benzilate binding to muscarinic cholinergic receptors in the developing human brainstem.

Acetylcholine has been implicated in brainstem mechanisms of cardiac and ventilatory control, arousal, rapid eye movement (REM) sleep, and cranial nerve motor activity. Virtually nothing is known about the developmental profiles of cholinergic perikarya, fibers, terminals, and/or receptors in the brainstems of human fetuses and infants. This study provides baseline information about the quantitative distribution of muscarinic cholinergic receptors in fetal and infant brainstems. Brainstem sections were analyzed from 6 fetuses (median age: 21.5 postconceptional weeks), 4 premature infants (median age: 26 postconceptional weeks), and 11 infants (median age: 53 postconceptional weeks). One child and three adult brainstems were examined as indices of maturity for comparison. The postmortem interval in all cases was less than or equal to 24 hours (median: 10 hours). Muscarinic receptors were localized by autoradiographic methods with the radiolabeled antagonist [3H]quinuclidinyl benzilate ([3H]QNB). Computer-based methods permitted quantitation of [3H]QNB binding in specific nuclei and three-dimensional reconstructions of binding patterns. By midgestation, muscarinic cholinergic receptor binding is already present and regionally distributed, with the highest binding levels in the interpeduncular nucleus, inferior colliculus, griseum pontis, nucleus of the solitary tract, motor cranial nerve nuclei, and reticular formation. During the last half of gestation, [3H]QNB binding decreases in most, but not all of the nuclei sampled. The most substantial decline occurs in the reticular formation of the medulla and pons, a change that is not fully explained by progressive myelination and lipid quenching. Binding levels remain essentially constant in the inferior olive and griseum pontis. Around the time of birth or shortly thereafter, the relative distribution of binding becomes similar to that in the adult, with the highest levels in the interpeduncular nucleus and griseum pontis, although binding levels are higher overall in the infant. In the rostral pontine reticular formation, paramedian bands of high muscarinic binding are present which do not correspond to a cytoarchitectonically defined nucleus. By analogy to animal studies, these bands may comprise a major cholinoreceptive region of the human rostral pontine reticular formation involved in REM sleep. In the human interpeduncular nucleus in all age periods examined, muscarinic binding localizes to the lateral portions bilaterally, indicative of a heterogeneous chemoarchitecture. Muscarinic binding is high in the arcuate nucleus, a component of the putative respiratory chemosensitive fields along the ventral surface of the infant medulla. This observation is consistent with the known effects of muscarinic agents on chemosensitivity and ventilatory responses applied to the ventral medullary surface in animal models. The nonuniform distribution of muscarinic binding in the caudorostral plane in individual brainstem nuclei, as illustrated by three-dimensional reconstructions, underscores the need for rigorous sampling at precisely matched levels in quantitative studies. This study provides basic information toward understanding the neurochemical basis of brainstem disorders involving dysfunction of autonomic and ventilatory control, arousal, and REM sleep in preterm and full-term newborns and infants and for developing cholinergic drugs for such disorders in the pediatric population.

Developmental changes in [3H]kainate binding in human brainstem sites vulnerable to perinatal hypoxia-ischemia.

The human brainstem is especially susceptible to hypoxia-ischemia in early life. To test the hypothesis that the period of vulnerability of the developing human brainstem to hypoxia-ischemia correlates with a transient elevation in kainate receptor binding, we compared the quantitative distribution of [3H]kainate binding in brainstem nuclei between four fetuses (19-26 gestational weeks), four infants (one to nine months), and three "mature" individuals (one child and two adults) without neurological disease. Quantitative tissues autoradiography was used. [3H]Kainate binding decreased in all brainstem regions from early life to maturity with the most significant decreases occurring in nuclei thought to be especially vulnerable to perinatal hypoxia-ischemia (e.g. principal inferior olive, griseum pontis, inferior colliculus and reticular core). The highest binding in the fetal and infant period was found primarily in the major cerebellar-relay nuclei. In the inferior olive and arcuate nucleus, binding increased from the fetal to the infant period, and then fell 50-61% to low mature levels. In the griseum pontis, binding decreased 60% between the fetal and mature periods. In the reticular formation, binding fell 67-78% from the fetal to mature period. These data support a correlation between the period of brainstem vulnerability to hypoxia-ischemia in early life to transient elevation in kainate binding, and are particularly relevant to the topographic brainstem patterns in perinatal hypoxia-ischemia of infantile olivary gliosis, pontosubicular necrosis and reticular core damage. Striking localization of [3H]kainate binding to rhombic lip derivatives further suggests that kainate receptors may be involved in the development and function of human brainstem-cerebellar circuitry.

Anatomic distribution of the growth-associated protein GAP-43 in the developing human brainstem.

GAP-43 is a membrane phosphoprotein whose expression is high in neurons undergoing development or remodeling of axonal connections. This study used a monospecific antibody to GAP-43 to investigate the sequences of fiber tract elongation and synaptic development in the human brainstem. Immunocytochemistry was performed in 14 fetal and infant brainstems; two child and adult cases were also examined for comparison. At midgestation, GAP-43 immunostaining was moderately intense across nuclei and fiber tracts, except for the corticospinal tract, where levels were higher, and cranial nerve nucleus VII, superior olive, inferior colliculus, inferior olivary hilum, inferior cerebellar peduncle, medial lemniscus, and medial longitudinal fasciculus, where staining was nearly absent. By the end of the neonatal period, the relative distribution of GAP-43 immunostaining appeared well-established and similar, although not identical, to that in the child and adult brainstem. Immunostaining was absent or negligible in almost all the cranial nerve somato- and branchiomotor nuclei, auditory-relay nuclei, and vestibular nuclei, while remaining intense in visceral-related nuclei, reticular formation, cochlear nucleus, and periaqueductal gray. Staining was also virtually absent in all fiber tracts at birth, except for the corticospinal tract and central tegmental tract. Persistence of GAP-43 staining in the corticospinal tract past the fetal period suggests that this tract remains in a plastic state beyond initial axonal elongation. Intense immunostaining in visceral-related nuclei into adulthood suggests that these regions may continue to undergo synaptic reorganization. This study provides baseline information relevant to understanding developmental brainstem disorders in early human life.