W. Frost White

Early developmental changes in [3H]nicotine binding in the human brainstem

Little is known about the developmental profile of nicotinic cholinergic receptors in the developing human brain, despite the potential importance of such information in understanding the pathogenesis of neurological abnormalities or increased risk for the sudden infant death syndrome in offspring exposed to nicotine in utero. In this study, we determined the distribution of [3H]nicotine binding in the developing human brainstem by quantitative tissue autoradiography. In midgestational fetuses, [3H]nicotine binding sites were heavily concentrated in tegmental nuclei related to cardiopulmonary integration, arousal, attention, rapid eye movement sleep, and somatic motor control. Over the last half of gestation, [3H]nicotine binding decreased 60-70% in the tegmental nuclei, with a significant difference in binding between midgestation and early infancy. In contrast, there was essentially no change in [3H]nicotine binding in the major cerebellar-relay nuclei (principal inferior olive and griseum pontis) between the same time-points. Tritium quenching by increasing lipid (myelin) content in tissue sections did not account for the decreases in [3H]nicotine binding in tegmental nuclei. Based upon the high levels of [3H]nicotine binding at midgestation, combined with experimental data demonstrating trophic properties for acetylcholine, we postulate that nAChRs a role in the development of the brainstem tegmentum during this period, and that once this role is fulfilled, nicotinic cholinergic binding decreases and remains low thereafter. Alternatively, nicotinic cholinergic receptors may be critical for other developmentally related functions and/or neurotransmission in the brainstem tegmentum at midgestation. The high levels of [3H]nicotine binding in the brainstem tegmentum at midgestation and its rapidly changing profile over late gestation further suggest that mid-to-late gestation is a developmental period during which this region is likely to be most vulnerable to the harmful effects of nicotine in maternal cigarette smoke. The baseline information provided in this study is potentially relevant towards understanding attention deficits and risk for the sudden infant death syndrome in offspring exposed to cigarette smoke in utero.

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.

Brainstem 3H-Nicotine Receptor Binding in the Sudden Infant Death Syndrome

Maternal cigarette smoking during pregnancy has been shown to be a major risk factor for the sudden infant death syndrome (SIDS). We hypothesized that SIDS is associated with altered 3H-nicotine binding to nicotinic receptors in brainstem nuclei related to cardiorespiratory control and/or arousal. We analyzed 3H-nicotine binding in 14 regions in SIDS and control brainstems using quantitative tissue receptor autoradiography. Three groups were analyzed: SIDS (n = 42), acute controls (n = 15), and a chronic group with oxygenation disorders (n = 8). The arcuate nucleus, postulated to be important in cardiorespiratory control and abnormal in at least some SIDS victims, contained binding below the assay detection limits in all (SIDS and control) cases. We found no significant differences among the 3 groups in mean 3H-nicotine binding in the 14 brainstem sites analyzed. When a subset of the cases were stratified by the history of the presence or absence of maternal cigarette smoking during pregnancy, however, we found that there was no expected increase (upregulation) of nicotinic receptor binding in SIDS cases exposed to cigarette smoke in utero in 3 nuclei related to arousal or cardiorespiratory control. This finding raises the possibility that altered development of nicotinic receptors in brainstem cardiorespiratory and/or arousal circuits put at least some infants, i.e. those exposed to cigarette smoke in utero, at risk for SIDS, and underscores the need for further research into brainstem nicotinic receptors in SIDS in which detailed correlations with smoking history can be made.

CP-101,606, an NR2B subunit selective NMDA receptor antagonist, inhibits NMDA and injury induced c-fos expression and cortical spreading depression in rodents.

(1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606) is a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors containing the NR2B subunit. This compound was used to investigate the role of NR2B containing receptors in three responses to NMDA receptor activation in vivo. In mouse, CP-101,606 completely inhibited increases in fos-like immunoreactivity in dentate gyrus caused by a subconvulsant intraperitoneal dose of NMDA. In rat, the compound completely blocked cortical c-fos mRNA induction following focal injury in parietal cortex and the initiation and propagation of electrically induced cortical spreading depression. Inhibition of these responses by CP-101,606 indicates that c-fos induction and cortical spreading depression are dependent on activation of NMDA receptors containing the NR2B subunit. Since NMDA receptor dependent c-fos induction and cortical spreading depression may contribute to neuron loss after focal CNS injury, inhibition of these responses by CP-101,606 may contribute to the neuroprotective efficacy of the compound.

Effects of the NMDA antagonist CP-98,113 on regional cerebral edema and cardiovascular, cognitive, and neurobehavioral function following experimental brain injury in the rat

The present study examined the effects of CP-98,113, an N-methyl-d-aspartate (NMDA) receptor blocker, on cardiovascular variables, neurobehavioral motor function, spatial memory deficits, and cerebral edema formation following lateral (parasagittal) fluid-percussion (FP) brain injury in the rat. In Study 1, we compared the cardiovascular effects of i.p. administration of CP-98, 113 at 15 min postinjury at doses of 1 mg/kg, 2 mg/kg, 5 mg/kg, or 20 mg/kg (n=8/dose). Animals receiving 1 mg/kg to 5 mg/kg CP-98,113 showed slight but nonsignificant decreases in blood pressure, while those receiving the highest dose (20 mg/kg) showed significant hypotension. Based upon those observations, the 5 mg/kg dose was chosen as the optimal dose for subsequent behavioral studies. In Study 2, 15 min following lateral FP brain injury of moderate severity (2.5 atm), animals randomly received either CP-98,113 (5 mg/kg, i.p., n=23) followed by a 24-h subcutaneous infusion (1.5 mg kg-1 h-1) by means of a miniature osmotic pump, or identical volume of vehicle (n=24), and were evaluated for neurologic motor function (n=11/drug vs. 11/vehicle), memory function, and cerebral edema (n=12/drug vs. 13/vehicle). CP-98,113 (5 mg/kg) significantly attenuated neurologic motor dysfunction at 24 h (p<0.01) and 2 weeks (p<0.05) postinjury, reduced posttraumatic impairment in spatial memory observed at 48 h postinjury (p<0.001), and significantly reduced focal brain edema in the cortex adjacent to the site of maximal injury at 48 h postinjury (injury penumbra) (p<0.001). These results suggest that blockade of the NMDA receptor may attenuate the deleterious sequelae of traumatic brain injury.

Developmental Changes in [3H]Lysergic Acid Diethylamide ([3H]LSD) Binding to Serotonin Receptors in the Human Brainstem

The ontogeny of serotonin receptors in the human brainstem is largely unknown, despite the putative roles of serotonin in neural development, synaptic transmission, brainstem modulation of vegetative functions, and clinical disorders of serotonergic function. This study provides baseline information about the quantitative distribution of [3H]LSD binding to serotonergic receptors (5-HT1A-D, 5-HT2) in the human brainstem, from midgestation through maturity, with a focus upon early infancy. Brainstems were analyzed from 5 fetuses (19–25.5 weeks postconception), 5 infants (42–55.5 weeks postconception), and 3 mature individuals (4, 20, and 52 years). Tissue autoradiography was used with [3H]LSD for total serotonergic receptor binding and [3H]LSD and serotonin for nonspecific binding; computer-based quantitation was applied. The highest levels of [3H]LSD binding occurred prenatally throughout the brainstem. At all ages, the highest relative binding localized to the rostral raphe. A marked decline in [3H]LSD binding occurred between the midgestation and infancy in brainstem regions involved in control of cardiovascular function, respiration, and pain. The fetal peak in [3H]LSD binding to 5-HT receptors is consistent with a trophic role of serotonin in immature human brainstem, and a decrease, between midgestation and infancy, in serotonergic modulation of vegetative functions controlled by the brainstem

A 3,4-dihydroxyphenylalanine oxidation product is a non-N-methyl-D-aspartate glutamatergic agonist in rat cortical neurons.

Applications of solutions of 2,4,5-trihydroxyphenylalanine (TOPA or 6-hydroxyDOPA) to rat cortical neurons in culture monitored under whole-cell voltage clamp with patch electrodes resulted in currents which could be nearly completely blocked by the non-N-methyl-D-aspartate (non-NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but only weakly antagonized by the NMDA antagonist D.L-2-amino-5-phosphonovalerate (APV). Thus, TOPA can generate glutamatergic responses by interacting preferentially with non-NMDA receptors in cortical neurons. As these results show that a product closely related to the catecholamine precursor 3,4-dihydroxyphenylalanine (DOPA) has glutamatergic agonist properties, it is conceivable that catecholamine-containing brain areas may be at special risk for excitotoxic damage under certain conditions.

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.

CP-101,606: An NR2B-Selective NMDA Receptor Antagonist

Glutamate and aspartate play dual roles in the central nervous system as essential amino acids and the principal excitatory neurotransmitters. The theory of excitotoxicity presents the paradoxical view that these excitatory amino acids may also become endogenous neurotoxins any time the brain’s energy homeostasis is compromised (16,23,42,59). Cerebral ischemia and traumatic brain injury result in acute energy depletion and cellular depolarization. This triggers a dramatic increase in extracellular glutamate levels (10,11) due to presynaptic glutamate release and/or reversal of neuronal and glial glutamate transporters (63,64). The result is a prolonged overactivation of glutamate receptors which, through an incompletely understood cascade of events, leads to neuron death. Glutamate receptor activity is also hypothesized to play a role in the neuron death associated with chronic neurodegenerative conditions such as Alzheimer’s disease and Parkinson’s disease. In these latter conditions, subtle but chronic deregulation in neuronal energy metabolism renders neurons susceptible to excitotoxicity from physiological glutamate receptor activity (1,23,36). Given the premise of excitotoxicity as a central event in neuron loss associated with both acute and chronic neurodegenerative conditions, glutamate-receptor inhibition has been an aggressively pursued therapeutic strategy to treat these conditions. There are four major classes of glutamate receptors: N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainate, and metabotropic (2). Of these, NMDA receptors appears to be especially important to the excitotoxic process. The NMDA receptor is an ion channel gated by synaptically released glutamate in the presence of the coagonist glycine (29,32) and concomitant depolarization (38). The NMDA receptor is permeable to Na and Ca; it is Ca influx through the receptor that

Developmental Changes in Neurotransmitter Receptor Binding in the Human Periaqueductal Gray

The periaqueductal gray (PAG) plays a central role in the integration of defense responses to threatening or stressful stimuli. Little is known about the neurochemical development of the human PAG around the time of birth, when the fetus makes the transition to extrauterine life and independent defense responses are needed. We analyzed receptor binding to selected neurotransmitters implicated in PAG function in 7 fetuses (19 to 26 gestational weeks), 9 infants (38 to 74 postconceptional weeks), 1 child (4 years), and 3 adults (20 to 68 years). Tissue autoradiography was used with radioligands for opioid, nicotinic, muscarinic, kainate, and serotoninergic receptors. By midgestation, binding to nicotinic, muscarinic, serotoninergic, opioid, and kainate receptors is already localized to the human PAG. The subsequent developmental profiles are unique for each radioligand. Binding to nicotinic and serotoninergic receptors decreases significantly from the fetal to mature periods, but at different tempos. In contrast, there is no significant change from midgestation to infancy for muscarinic, kainate, and opioid binding: between infancy and the mature period there is a downward trend in binding for muscarinic and kainate receptors and an upward trend for opioid receptors. This study provides baseline information about the neurochemical development of the human PAG in early life. This information is of value in considering the neurochemical substrate of the maturation of defense responses in human infancy, and in evaluating potential neurochemical disorders of the developing human PAG