Modulation of neuronal migration by NMDA receptors.

The N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor is essential for neuronal differentiation and establishment or elimination of synapses in a developing brain. The activity of the NMDA receptor has now been shown to also regulate the migration of granule cells in slice preparations of the developing mouse cerebellum. First, blockade of NMDA receptors by specific antagonists resulted in the curtailment of cell migration. Second, enhancement of NMDA receptor activity by the removal of magnesium or by the application of glycine increased the rate of cell movement. Third, increase of endogenous extracellular glutamate by inhibition of its uptake accelerated the rate of cell migration. These results suggest that NMDA receptors may play an early role in the regulation of calcium-dependent cell migration before neurons reach their targets and form synaptic contacts.

Selective role of N-type calcium channels in neuronal migration.

Analysis of neuronal migration in mouse cerebellar slice preparations by a laser scanning confocal microscope revealed that postmitotic granule cells initiate their migration only after the expression of N-type calcium channels on their plasmalemmal surface. Furthermore, selective blockade of these channels by addition of omega-conotoxin to the incubation medium curtailed cell movement. In contrast, inhibitors of L- and T-type calcium channels, as well as those of sodium and potassium channels, had no effect on the rate of granule cell migration. These results suggest that N-type calcium channels, which have been predominantly associated with neurotransmitter release in adult brain, also play a transient but specific developmental role in directed migration of immature neurons before the establishment of their synaptic circuits.

Intracellular Ca2+ fluctuations modulate the rate of neuronal migration.

Transient elevations of intracellular Ca2+ levels play critical roles in neuronal development, but such elevations have not been demonstrated in migrating neurons. Here, we show that the amplitude and frequency components of Ca2+ fluctuations are correlated positively with the rate of granule cell movement in cerebellar microexplant cultures. Moreover, depression of the amplitude and frequency components of Ca2+ fluctuations by blockade of Ca2+ influx across the plasma membrane results in a reversible retardation of cell movement. These results indicate that the combination of amplitude and frequency components of intracellular Ca2+ fluctuations may provide an intracellular signal controlling the rate of neuronal cell migration.

Cerebellar cortical-layer-specific control of neuronal migration by pituitary adenylate cyclase-activating polypeptide.

Migration of immature neurons is essential for forming the cortical layers and nuclei. Impairment of migration results in aberrant neuronal cytoarchitecture, which leads to various neurological disorders. Neurons alter the mode, tempo and rate of migration when they translocate through different cortical layers, but little is known about the mechanisms underlying this process. Here we show that endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) has short-term and cortical-layer-specific effects on granule cell migration in the early postnatal mouse cerebellum. Application of exogenous PACAP significantly slowed the migration of isolated granule cells and shortened the leading process in the microexplant cultures of the postnatal day (P)0-3 cerebella. Interestingly, in the cerebellar slices of P10 mice, application of exogenous PACAP significantly inhibited granule cell migration in the external granular layer (EGL) and molecular layer (ML), but failed to alter the movement in the Purkinje cell layer (PCL) and internal granular layer (IGL). In contrast, application of PACAP antagonist accelerated granule cell migration in the PCL, but did not change the movement in the EGL, ML and IGL. Inhibition of the cAMP signaling and the activity of phospholipase C significantly reduced the effects of exogenous PACAP on granule cell migration. The PACAP action on granule cell migration was transient, and lasted for approximately 2 h. The duration of PACAP action on granule cell migration was determined by the desensitization of its receptors and prolonged by inhibiting the protein kinase C. Endogenous PACAP was present sporadically in the bottom of the ML, intensively in the PCL, and throughout the IGL. Collectively, these results indicated that PACAP acts on granule cell migration as "a brake (stop signal) for cell movement." Furthermore, these results suggest that endogenous PACAP slows granule cell migration when the cells enter the PACAP-rich PCL, and 2 h later the desensitization of PACAP receptors allows the cells to accelerate the rate of migration and to actively move within the PACAP-rich IGL. Therefore, endogenous PACAP may provide a cue that regulates granule cell migration in a cerebellar cortical-layer-specific manner.

Recognition, adhesion, transmembrane signaling and cell motility in guided neuronal migration.

Recent studies indicate that migration of neurons from their place of origin to their final destination requires the orchestration of multiple molecular events, including the selection of a pathway by cell recognition receptors, the formation of adhesive interactions with cellular and extracellular substrates through multiple adhesion molecules and the activation of specific ion channels and receptors that provide second messenger mediated signals for the diverse cellular mechanisms involved in cell motility. New approaches allow for the examination of the role of individual molecular components that mediate these processes.

Lack of correlation of N-myc gene amplification with prognosis in localized neuroblastoma: a Pediatric Oncology Group study.

Multiple copies of N-myc proto-oncogene are only rarely detected in localized neuroblastomas (NBs), and the prognostic relevance of amplification in this subset of patients is not clear. We analyzed a series of 850 children with NB admitted to a Pediatric Oncology Group NB Biology Study and identified six patients with localized NBs harboring N-myc gene amplification. Three patients whose tumors showed favorable histology by Shimada classification and low-risk histological features according to the Joshi classification have remained disease-free, whereas two of three patients with unfavorable histology tumors have developed recurrent disease. Although earlier studies have indicated that N-myc amplification is associated with diploid DNA content, flow cytometric analysis revealed that only two of the localized tumors contained stem lines with diploid DNA content. Loss of chromosome 1p was not detected by fluorescence in situ hybridization in the two tumors examined. N-myc protein was detected by immunohistochemical studies in four of the five NBs analyzed. However, N-myc protein was not visualized in one of the tumors with stroma-rich histology, and Western blot analysis revealed only low levels of N-myc protein expression in another NB with favorable histology. These studies indicate that the presence of N-myc amplification in localized NBs does not necessarily portend an adverse outcome. Furthermore, the biological features of this subset of N-myc-amplified NBs appear to differ from those of more advanced N-myc-amplified tumors.

Mutations of the p53 gene are involved in Ewing's sarcomas but not in neuroblastomas.

We have investigated the frequency of p53 gene mutations in Ewing's sarcoma (ES) and neuroblastoma (NB) by using polymerase chain reaction-single strand conformation polymorphism analysis for genomic DNA or complementary DNA generated from total RNA. Mutations of the p53 gene were found in six of seven ES cell lines: a missense mutation of TGC (Cys)-->TAC (Try) at codon 141 in one, a missense mutation of CGT (Arg)-->TGT (Cys) at codon 273 in one, a missense mutation of TGC (Cys)-->TTC (Phe) at codon 176 in three, and one base deletion of CGC-->CG at codon 283 in one. Further analysis of 14 ES and related primary tumors showed mutations of the p53 gene in only two: one base insertion of CCG-->CCCG at codon 152 in one and a missense mutation of GGC (Gly)-->GTC (Val) at codon 154 in the other. Both of the two tumors were obtained from patients with an advanced stage disease. Three of the eight ESs with mutations of the p53 gene showed the same missense mutation at codon 176, suggesting the mutational hot spot of the p53 gene in ESs. In contrast to ES, none of 6 NB cell lines or 48 NB tumors including advanced-stage ones with or without N-myc amplification showed any aberration of the p53 gene. Our findings suggest that mutations of the p53 gene in ES might represent late genetic events related to tumor progression, and that aberrations of the p53 gene might not be involved in the development or the progression of NB.

Mode and tempo of tangential cell migration in the cerebellar external granular layer.

After their final mitosis, cerebellar granule cells remain in the external granular layer (EGL) for 20-48 hr before initiating their radial migration across the molecular layer (ML), but the significance of this latent period is not well understood. In the present study, we used a confocal microscope to examine morphogenetic changes and behavior of postmitotic granule cells restricted to the EGL in slice preparations of the postnatal mouse cerebellum. We found that, coincident with the extension of two uneven horizontal processes oriented parallel to the longitudinal axis of the folium, postmitotic granule cells start to migrate tangentially in the direction of the larger process. Interestingly, their morphology and the speed of cell movement change systematically with their position within the EGL. The rate of tangential cell movement is fastest (approximately 14.8 micrometer/hr) in the middle of the EGL, when cells have two short horizontal processes. As granule cells elongate their somata and extend longer horizontal processes at the bottom of the EGL, they move at a reduced rate (approximately 12.6 micrometer/hr). At the interface of the EGL and ML where cells migrate tangentially at the slowest rate (approximately 4.1 micrometer/hr), their somata round and then begin to extend couples of the descending processes into the ML. After the stationary period, granule cells abruptly extend a single vertical process and initiate the transition from tangential to radial migration, reshaping their rounded somata into a vertically elongated spindle. These observations suggest that tangential migration of granule cells within the EGL may provide the developmental mechanisms for their appropriate allocation across parasagittal compartments of the expanding cerebellar cortex.

Functional pacemaking area in the early embryonic chick heart assessed by simultaneous multiple-site optical recording of spontaneous action potentials.

Pacemaking areas in the early embryonic chick hearts were quantitatively assessed using simultaneous multiple-site optical recordings of spontaneous action potentials. The measuring system with a 10- X 10- or a 12 X 12-element photodiode array had a spatial resolution of 15-30 microns. Spontaneous action potential-related optical signals were recorded simultaneously from multiple contiguous regions in the area in which the pacemaker site was located in seven- to nine-somite embryonic hearts stained with a voltage-sensitive merocyanine-rhodanine dye (NK 2761). In the seven- to early eight-somite embryonic hearts, the location of the pacemaking area is not uniquely determined, and as development proceeds to the nine-somite stage, the pacemaking area becomes confined to the left pre-atrial tissue. Analysis of the simultaneous multiple-site optical recordings showed that the pacemaking area was basically circular in shape in the later eight- to nine-somite embryonic hearts. An elliptical shape also was observed at the seven- to early eight-somite stages of development. The size of the pacemaking area was estimated to be approximately 1,200-3,000 micron2. We suggest that the pacemaking area is composed of approximately 60-150 cells, and that the pacemaking area remains at a relatively constant size throughout the seven- to nine-somite stages. It is thus proposed that a population of pacemaking cells, rather than a single cell, serves as a rhythm generator in the embryonic chick heart.

Effects of calcium on electrical propagation in early embryonic precontractile heart as revealed by multiple-site optical recording of action potentials.

The effects of Ca2+ on electrical propagation in early embryonic precontractile chick hearts were studied optically using a voltage-sensitive merocyanine-rhodanine dye. Spontaneous optical signals, corresponding to action potentials, were recorded simultaneously from 25 separate regions of the eight-to-nine-somite embryonic primitive heart, using a square photodiode array. Electrical propagation was assessed by analyzing the timing of the signals obtained from different regions. Electrical propagation in the heart was suppressed by either lowering or raising extracellular Ca2+. Similar effects were produced by a Ca2+ ionophore (A23187). We have also found that electrical propagation across the primordial fusion line at the midline of the heart was enhanced by increasing, and depressed by lowering, external Ca2+. One possible interpretation is that intercellular communication in the embryonic precontractile heart is regulated by the level of the intracellular Ca2+ concentration, and it is suggested that intercellular communication across the primordial fusion line strongly depends on external Ca2+.

p27KIP1 deletions in childhood acute lymphoblastic leukemia.

The p27KIP1 gene, which encodes a cyclin-dependent kinase (CDK) inhibitor, has been assigned to chromosome band 12p12, a region often affected by cytogenetically apparent deletions or translocations in childhood acute lymphoblastic leukemia (ALL). As described here, fluorescence in situ hybridization (FISH) analysis of 35 primary ALL samples with cytogenetic evidence of 12p abnormalities revealed hemizygous deletions of p27KIP1 in 29 cases. Further analysis of 19 of these cases with two additional gene-specific probes from the 12p region (hematopoietic cell phosphatase, HCP and cyclin D2, CCND2) showed that p27KIP1 is located more proximally on the short arm of chromosome 12 and is deleted more frequently than either HCP or CCND2. Of 16 of these cases with hemizygous deletion of p27KIP1, only eight showed loss of HCP or CCND2, whereas loss of either of the latter two loci was uniformly associated with loss of p27KIP1. Missense mutations or mutations leading to premature termination codons were not detected in the coding sequences of the retained p27KIP1 alleles in any of the 16 ALL cases examined, indicating a lack of homozygous inactivation. By Southern blot analysis, one case of primary T-cell ALL had hemizygous loss of a single p27KIP1 allele and a 34.5-kb deletion, including the second coding exon of the other allele. Despite homozygous inactivation of p27KP1 in this case, our data suggest that haploinsufficiency for p27KIP1 is the primary consequence of 12p chromosomal deletions in childhood ALL. The oncogenic role of reduced, but not absent, levels of p27KIP1 is supported by recent studies in murine models and evidence that this protein not only inhibits the activity of complexes containing CDK2 and cyclin E, but also promotes the assembly and catalytic activity of CDK4 or CDK6 in complexes with cyclin D.

Optical monitoring of early appearance of spontaneous membrane potential changes in the embryonic chick medulla oblongata using a voltage-sensitive dye.

Using a voltage-sensitive merocyanine-rhodamine dye (NK2761) and a 12 x 12-element photodiode matrix array, we recorded optically spontaneous membrane potential changes in a slice preparation from the embryonic chick brain stem during early development. The spontaneous optical signals, related to membrane potential changes, showed a simple monophasic shape with a relatively long duration, and they were synchronized among the different regions in the medulla oblongata. The spontaneous signals were first detected from seven-day-old embryos, and were not present in six-day-old embryos. The spontaneous signals appeared sporadically, and their frequency was very low. Three modes of optical signals termed "singlet-mode", "doublet-mode", and "triplet-mode" were observed. In the doublet- and triplet-modes, the spatial pattern of the first signal was primarily similar to that of the singlet-mode signal, whereas the signal size and spatial extent of the second and third signals appeared to decay.

Optical imaging of the spatiotemporal patterning of neural responses in the embryonic chick superior cervical ganglion.

Multiple-site optical recording of transmembrane potential changes with a voltage-sensitive dye was used to reveal the functional expression and developmental changes of the postsynaptic potentials in the early embryonic chick superior cervical ganglion. The ganglia were isolated from five- to 12-day-old chick embryos with preganglionic nerve fibres (vertebral and/or cervical carotic nerves) attached. The preparations were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). Voltage-related optical (absorbance) changes were recorded simultaneously from 127 contiguous loci in the preparation, using a 12 x 12-element photodiode array. Optical changes having two components were evoked by preganglionic nerve stimulation. One component was the fast spike-like signal and another the delayed slow signal. The amplitude of the slow signal was decreased by repetitive stimulation, reduced by low external calcium ion concentrations and eliminated in the presence of manganese or cadmium ions. The slow signals were also eliminated in the presence of D-tubocurarine. Accordingly, we concluded that the slow signal corresponds to cholinergic excitatory postsynaptic potentials. In the five- and six-day-old superior cervical ganglia, only the fast optical signals (referred to as the action potentials) were recorded. Slow optical signals (referred to as the excitatory postsynaptic potentials) were detected from preparations older than seven days. The amplitude of the slow optical signal gradually increased, together with an expansion of the response area, as the developmental stage proceeded from seven to 10 days. To compare the distribution patterns of the neural responses evoked by stimuli applied to the cervical carotic and vertebral nerves, we have mapped and imaged the spatial patterning of the synaptic responses. In the maps, the positions of the peak size regions of the slow signals were assessed, and we found that there were differences in the location of these areas for the cervical carotic vs vertebral nerves. From these experimental results, we conclude that synaptic function within the chick superior cervical ganglion is initiated at the seven-day-old embryonic stage, and reaches a maximum level at 10 days. Synaptic transmission at these stages is mediated solely by nicotinic acetylcholine receptors. The spatial mapping of the synaptic responses reveals that the neural populations related synaptically to the cervical carotic and vertebral nerves are located separately within the ganglion, even at an early developmental stage.

Expression of angiogenic factors and tumor progression in human neuroblastoma.

PURPOSE: The growth and metastasis of malignant tumors is largely dependent on angiogenesis. Angiogenic factors produced by tumor cells are known to promote tumor angiogenesis. The aim of this study was to investigate which angiogenic factor is the most important in the progression of neuroblastoma (NB). PROCEDURE: The relative expression levels of vascular endothelial growth factor-A (VEGF-A), VEGF-C, basic fibroblast growth factor (bFGF), and platelet-derived endothelial growth factor (PD-ECGF/TP) were studied in 28 NB tumor specimens by real-time quantitative reverse transcriptase/polymerase chain reaction (RT-PCR). The relationships between the expression of these four angiogenic factors and stage, patient age, primary site, MYCN copy number, and lymph node metastasis were analyzed. RESULTS: High VEGF-A expression was correlated with stage 4 disease (blood-borne metastasis). No relationship between VEGF-A expression and age, primary site, MYCN copy number, or lymph node metastasis was found. The expression of VEGF-C, bFGF, or PD-ECGF/TP showed no correlation with stage, age, primary site, MYCN copy number, or lymph node metastasis. CONCLUSIONS: Our findings suggest that VEGF-A, but not VEGF-C, bFGF, or PD-ECGF/TP, may be associated with progression of NB. VEGF-A could be a target for antiangiogenic therapy for disseminated NB.

Optical assessment of spatially ordered patterns of neural response to vagal stimulation in the early embryonic chick brainstem.

Spatial dynamic patternings of electrical responses to vagal nerve stimulations in the embryonic chick brainstem preparation were assessed by means of simultaneous multiple-site optical recordings of electrical activity. The vagus/brainstem preparations were dissected from early 7-day-old chick embryos (165-172 h after incubation), and they were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). Application of depolarizing square current pulses to the vagus nerve fibers using a suction electrode evoked voltage-related optical (absorbance) signals that were recorded simultaneously from 127 contiguous sites in the whole brainstem preparation using a 12 x 12-element photodiode array. The optical signals evoked by the vagus nerve stimulation appeared to be concentrated longitudinally in the central region and in the lateral region ipsilateral to the site of brainstem stimulation. These response areas were orderly changed according to changes in the strength and in the duration of the stimulating current: the response area expanded as the strength or the duration of the stimulating current was increased. The size of the evoked optical signals also depended on the strength and on the duration of the stimulating current. We also measured critical (threshold) values of the strength and duration of the stimulating current to produce the optical responses, and we found that there was a regionally ordered distribution in the brainstem. In the experiments on transverse slices of the brainstem, the evoked optical responses were detected from limited areas near the dorsal surface of the stimulated side, and the response pattern depended on the strength and duration of the stimulating current in a manner similar to that in the whole brainstem preparations. On the basis of the data obtained from these experiments, we have constructed 3 kinds of response maps for the embryonic brainstem, and the results suggest the functionally ordered arrangement of the neurons in the vagus-related area in the embryonic brainstem.

Hemizygous deletions of chromosome band 16q24 in Wilms tumor: detection by fluorescence in situ hybridization.

Loss of heterozygosity (LOH) for markers on chromosome arm 16q in Wilms tumor has been linked to an increased risk of treatment failure. We therefore postulated that fluorescence in situ hybridization (FISH) with probes from this region might enhance current strategies for identifying high-risk patients at diagnosis. In a blinded comparative pilot study of 19 Wilms tumor samples from 18 patients with favorable histology, FISH and DNA polymorphism analysis yielded concordant results in 14 cases, either retention (n = 6) or loss (n = 8) of chromosome arm 16q markers. Discordant findings in 4 of the 5 remaining cases resulted from detection of LOH, but no loss by FISH. Two of these cases, directly comparable at marker D16S422, appeared to have tumor-specific uniparental disomy, in that 2 copies of D16S422 and the 16 centromere were evident, despite LOH. In 2 other cases, the discrepancies could be explained by LOH confined to loci distal to the D16S422 locus. In the fifth case, FISH detected 2 distinct populations of tumor cells, one characterized by normal diploidy and the other by monosomy 16, whereas DNA polymorphism analysis failed to indicate LOH altogether. Thus, FISH confirmed the presence of allelic loss (hence, the possible location of biologically important tumor suppressor genes) on the distal long arm of chromosome 16 in cases of favorable-histology Wilms tumor, with the advantages of technical simplicity, successful analysis of samples that were otherwise uninformative by analysis of DNA polymorphisms, and the addition of internal controls for chromosomal aneusomy. We suggest that combined analysis of the chromosome 16q region in Wilms tumor by FISH and DNA polymorphism analysis would improve evaluations to identify high-risk patients who might benefit from alternative therapy.

Optical recording of membrane potential responses from early embryonic chick ganglia using voltage-sensitive dyes.

Changes in absorbance of voltage-sensitive merocyanine-rhodanine dyes were used to monitor electrical responses in the semilunar ganglion of 4-10-day-old developing chick embryos. The electrical responses were simultaneously recorded from many positions in the ganglion. Stimulation of the afferent nerve fibers (the ophthalmic division of cranial nerve V) with a suction electrode led to changes in light absorption of the stained ganglia. With both the depolarizing and hyperpolarizing pulses, the change was largest at 700 nm and was eliminated at a wavelength of 620 nm where the voltage-dependent absorption change of the dyes disappears. In the 4-10-day-old embryonic ganglia, two types of optical membrane potential responses, 'non-conducted' and 'conducted' responses, were identified. The non-conducted response varied with the intensity of the stimulus and had the nature of an electrotonic spread. Furthermore, this non-conducted response exhibited an 'initial upstroke-response' followed by the steady-state plateau evoked by larger depolarizing pulses. The conducted responses were blocked by tetrodotoxin (TTX) or by high external potassium concentration. The incidence of the conducted responses increased as development proceeded from the 5th to the 10th day of age. Thus, the TTX-sensitive action potential activity is probably generated initially in the semilunar ganglion during the 5-10-day-stage of development. These data represent the first demonstration of membrane potential responses in early embryonic intact nervous system. Furthermore, these studies demonstrate the usefulness of voltage-sensitive dyes in the analysis of the organizing process of embryonic neuronal functions during these early stages of development.

Laparoscopic resection of an adrenal neuroblastoma detected by mass screening that grew in size during the observation period.

Neuroblastomas (NB) identified by mass screening tests are characterized by benign features. Recently, laparoscopic resection has been applied to the treatment of patients with small adrenal NB (<2-3 cm). However, an increasing number of cases of small NB are followed without any treatment in Japan because many cases regress spontaneously. We describe a case of right adrenal NB detected by mass screening that increased in size during an observation period of 8 months. In this case, laparoscopic resection was performed successfully. The size of the tumor was 27 x 20 x 18 mm at diagnosis and 51 x 42 x 35 mm when it was excised. Small adrenal NB that do not regress during the observation period may require laparoscopic resection before they reach 5 cm in maximum diameter.

Computerized tomography in Moyamoya syndrome.

Twelve patients with Moyamoya syndrome were examined by computerized tomography (CT); nine children and three adults. In one patient, CT was normal. Significant abnormalities on the precontrast CT in the remaining 11 patients were: cortical atrophy with or without varying degree of ventricular dilatation, and irregularly shaped, various sized, often multiple or bilateral lucent foci in the cortex, subcortical white matter and the central gray matter. After contrast enhancement, no significant increase in the attenuation number was observed in and around the lucent foci except in one patient. In three patients, there was a slight increase in density in the central gray matter, however the increase in density was slight when the dense angiographic vascular networks in the basal ganglia were considered.

Conduction pattern of excitation in the amphibian atrium assessed by multiple-site optical recording of action potentials.

Spontaneous action potentials were monitored from multiple sites in the bullfrog atrium using a voltage-sensitive merocyanine-rhodanine dye together with a 100-element photodiode matrix array, and we have assessed the spread of the excitation from the pacemaker. Isochrone curves of conduction were obtained by timing the initiation of the action potential-related optical signals: we constructed maps of the spread. Excitatory waves appeared to conduct radially from the pacemaking area over the atrium, and the conduction velocity in the left atrium exceeded that in the right atrium.