Teratogenic potential of two neurotropic drugs, haloperidol and dextromoramide, tested on mouse embryos

Potential teratogenic activity of two neurotropic drugs, haloperidol and dextromoramide tartrate, was tested by using the mouse embryo experimental model. Like numerous other drugs of this class these two are also embryotoxic. After treatment 1 hour into the 9th gestation day they induce the neurotropic syndrome of malformations comprising exencephaly, craniorachischisis, kinking of the spinal cord, brachyury, and dilation of the fourth brain ventricle. In addition, dextromoramide tartrate was found to induce one more, so far unknown, neural tube defect, namely the ectopia of the neural tube. Delay of treatment by 1 or 2 hours tends to displace the location of the neural tube defects along the length of the neural axis towards the anterior and posterior directions. Even after accumulating these results at relatively high doses, it is difficult to estimate human reproductive risks from this animal data for human therapeutic doses.     

All-or-none craniorachischisis in Loop-tail mutant mouse chimeras

Mouse embryos homozygous for the mutant gene Loop-tail (Lp) are characterized by craniorachischisis, an open neural tube extending from the midbrain to the tail. In the present study, experimental chimeric mice containing mixtures of genetically mutant (from Lp/+ x Lp/+ matings) and genetically normal cells were produced. Our aim was to determine whether a 'rescue,' phenotypic gradient, or intermediate expression (i.e. alternating areas of open and closed neural tube) would be observed in these chimeras. We report our analyses of Loop-tail mutant chimeras (n = 82) by gross examination, progeny testing and quantitative analysis of glucose phosphate isomerase (GPI) isozyme levels. An all-or-none craniorachischisis in Loop-tail mutant chimeras was observed. Two multicolored adult chimeras, without any gross evidence of a neural tube defect, were shown to be homozygous Loop-tail chimeras (Lp/Lp in equilibrium +/+) by progeny testing. These results indicate that the normal phenotype can be expressed in the presence of mutant cells. Conversely, six neonates with craniorachischisis were shown to be chimeras by GPI analyses. These results show that the full mutant phenotype can be expressed even when one-third to one-half of the cells are genotypically wild-type. This study did not determine which tissue is primarily responsible for the defective neurulation in this mutant, but suggests that a 'threshold' mechanism underlies the Loop-tail mutant phenotype. In some chimeras that threshold is not reached and the neural tube remains open, whereas in other chimeras the threshold is reached and the neural tube closes completely.     

Circletail, a new mouse mutant with severe neural tube defects: chromosomal localization and interaction with the loop-tail mutation.

Circletail (Crc) is a new mouse mutant that exhibits a severe form of neural tube defect, craniorachischisis, in which almost the entire neural tube fails to close. This phenotype is seen in very few other mutants, the best characterized of which is loop-tail (Ltap(Lp), referred to hereafter as Lp). We tested the possibility of allelism between Lp and Crc by intercrossing Lp/+ and Crc/+mice. A proportion of double heterozygotes (Lp/+,Crc/+) exhibit craniorachischisis, revealing failure of complementation. However, genetic analysis shows that Crc is not linked to the markers that flank the Lp locus and cannot, therefore, be an allele of Lp. A genome-wide scan has localized the Crc gene to a region of 8.8 cM on central chromosome 15. Partial penetrance of the craniorachischisis phenotype in Crc/+,Lp/+double heterozygotes suggests the existence of a third, unlinked genetic locus that influences the interaction between Crc and Lp.     

Cell polarity pathways converge and extend to regulate neural tube closure

Neural tube defects, such as spinabifida, craniorachischisis and anencephaly, are some of the most common birth defects in humans. Recent studies in mouse model systems suggest that craniorachischisis is associated with mutations in genes that regulate cell polarity. Using Xenopus as a model system, Wallingford and Harland have now shed light on the mechanism by which these pathways affect neural tube closure.     

Oral-Facial Clefts and Associated Malformations in the Squirrel Monkey (Saimiri sciureus)

Of the 414 squirrel monkey pregnancies recorded at this institution since 1977, seven (1.7%) have resulted in offspring with clefts of the lip and/or palate. Associated malformations include a ventricular septal defect, renal agenesis, anal atresia, axial skeletal anomalies, and craniorachischisis (anencephaly and spina bifida). Three of these infants are the result of consanguineous matings.     

The novel mouse mutant, chuzhoi, has disruption of Ptk7 protein and exhibits defects in neural tube, heart and lung development and abnormal planar cell polarity in the ear

Background

The planar cell polarity (PCP) signalling pathway is fundamental to a number of key developmental events, including initiation of neural tube closure. Disruption of the PCP pathway causes the severe neural tube defect of craniorachischisis, in which almost the entire brain and spinal cord fails to close. Identification of mouse mutants with craniorachischisis has proven a powerful way of identifying molecules that are components or regulators of the PCP pathway. In addition, identification of an allelic series of mutants, including hypomorphs and neomorphs in addition to complete nulls, can provide novel genetic tools to help elucidate the function of the PCP proteins.

Results

We report the identification of a new N-ethyl-N-nitrosourea (ENU)-induced mutant with craniorachischisis, which we have named chuzhoi (chz). We demonstrate that chuzhoi mutant embryos fail to undergo initiation of neural tube closure, and have characteristics consistent with defective convergent extension. These characteristics include a broadened midline and reduced rate of increase of their length-to-width ratio. In addition, we demonstrate disruption in the orientation of outer hair cells in the inner ear, and defects in heart and lung development in chuzhoi mutants. We demonstrate a genetic interaction between chuzhoi mutants and both Vangl2 ^Lp and Celsr1 ^Crsh mutants, strengthening the hypothesis that chuzhoi is involved in regulating the PCP pathway. We demonstrate that chuzhoi maps to Chromosome 17 and carries a splice site mutation in Ptk7. This mutation results in the insertion of three amino acids into the Ptk7 protein and causes disruption of Ptk7 protein expression in chuzhoi mutants.

Conclusions

The chuzhoi mutant provides an additional genetic resource to help investigate the developmental basis of several congenital abnormalities including neural tube, heart and lung defects and their relationship to disruption of PCP. The chuzhoi mutation differentially affects the expression levels of the two Ptk7 protein isoforms and, while some Ptk7 protein can still be detected at the membrane, chuzhoi mutants demonstrate a significant reduction in membrane localization of Ptk7 protein. This mutant provides a useful tool to allow future studies aimed at understanding the molecular function of Ptk7.     

Recurrent neural tube defects associated with partial trisomy 2p22-pter: report of two siblings and review of the literature

We report on two male siblings with partial trisomy 2p22-pter and partial monosomy 15q26-qter resulting from a maternally derived translocation t(2;15)(p22;q26). Both fetuses had different neural tube defects (craniorachischisis in the first fetus and anencephaly in the second fetus) which were detected by sonographic examination at the end of the first trimester of pregnancy. This report demonstrates the importance of chromosomal analysis in the etiologic exploration of neural tube defects and supports the importance of 2p24 triplication in neural tube development.     

The interference of naloxone hydrochloride in the teratogenic activity of opiates

Diamorphine hydrochloride, methadone hydrochloride, and the synthetic enkephalin analogue FK 33-824 are potent teratogens for the central nervous system in mouse embryos. They induce the "neurotropic syndrome of malformations," which is restricted to the central nervous system if administered during the critical period of neural tube closure. Pretreatment with corresponding equimolecular doses of the antagonist naloxone hydrochloride applied 30 minutes before treatment with the opiate agonists abolishes the major severe malformations, i.e., exencephaly, craniorachischisis, and brachyury, and reduces the number of cases of kinking of the spinal cord. Dilation of the fourth brain ventricle remains unaffected. It is suggested that the mechanism of interference in the teratogenicity of the opiates by naloxone hydrochloride reported here is based on competition for opiate receptors. In general, these observations are regarded as evidence that the pharmacological affinity of opiate agonists to receptors in the central nervous system is responsible for the malformations caused by them in this system.     

Clinicoanatomical study of thalamic stimulation for pain relief

Our small experiences with electrical stimulation in the VPL and VPM for dysesthetic pain show that it provoked only paresthesia and induced some relief of pain. It does not increase the beta-endorphin level in CSF. To clarify the anatomical substrata in VPL stimulation, neuroanatomical studies were done about the inputs to VPL in man, monkey and cat by the Fink-Heimer method. The spinothalamic tract terminates in VPL in a patchy fashion in the monkey. The corticothalamic fibers from SI and SII cortex project to VPL and VPM in somatotopical organization in the cat. SI and SII cortices have reciprocal connections, in addition to projections to area 5 or SIII cortex. The corticofugal fibers to the magnocellular and gigantocellular tegmental fields are suggested in addition to the dorsal column nuclei, spinal trigeminal nuclei and spinal posterior horn in cat. The medial lemniscus input to VPL and the above neural circuits are thought to be associated with VPL stimulation.     

Rescue of the neural tube defect of loop-tail mice by a BAC clone containing the Ltap gene

The mouse mutant loop-tail (Lp) is an accepted model for the study of neural tube defects (NTDs) in humans. Whereas Lp/+ heterozygotes show a mild tail defect (looped), homozygous Lp/Lp embryos show a very severe form of NTD, with a completely open neural tube from the hindbrain region to the caudal portion of the spinal cord (craniorachischisis). We have recently identified a positional candidate for Lp on chromosome 1, designated as Ltap. Here, we have used an in vivo complementation approach in transgenic mice to attempt to correct the looped-tail phenotype with a bacterial artificial chromosome clone (BAC280A23) that harbors a full-length copy of the Ltap gene. Genotype:phenotype correlations in Lp/+ heterozygotes carrying BAC280A23 show that this clone can rescue the looped-tail phenotype in two independent founder lines (P < 0.05 and P < 0.0001). Importantly, BAC280A23 is also observed to rescue the lethal NTD of Lp/Lp homozygotes, because several viable transgenic Lp/Lp mice could be identified and appeared normal (P < 0.05). Results from these gain-of-function transgenic animals strongly suggest that the positional candidate Ltap present in this BAC is indeed the gene that is defective in loop-tail.     

Development of the notochord in normal and malformed human embryos and fetuses.

In view of its possible involvement in early embryogenesis and teratogenesis, the developmental characteristics of the human notochord were studied by light and electron microscopy and immunohistochemistry on 20 human conceptuses (5th-22nd week). At the earliest embryonic stages examined, the notochord is closely related to both the pharyngeal endoderm and the neuroectoderm of the posterior (tail) end of the neural tube. In both regions the interspace is bridged by slender cytoplasmic processes, lined with basal lamina and filled with amorphous extracellular material containing collagen types III and IV and laminin. The notochordal cells express cytokeratin brightly and vimentin weakly. As embryonic age progresses, the notochord gradually separates from the epithelia, becomes the axis of developing spinal column and undergoes progressive cell degeneration and rearrangement within the vertebral bodies. This is associated with extensive production of extracellular material and the first appearance of fibronectin. Intracellularly, the expression of vimentin gradually increases, while that of cytokeratin slightly weakens. Changes in the notochord parallel other developmental events in axial organs. In anencephalic fetuses the course of the notochord is irregular and partly interrupted with segments outside the basichondrocranium in specimens with craniorachischisis.     

猕猴神经干细胞的诱导分化、干性维持及同种脑内移植

为探索猕猴神经干细胞分化及特性维持,推进神经干细胞临床应用研究,该实验以绿色荧光蛋白(green fluorescence protein,GFP)为标记探讨猕猴胚胎干细胞向玫瑰花环(rosettes)结构神经干细胞的分化及其碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和表皮生长因子(epidermal growth factor,EGF)的扩增培养。结果表明:1)建立了稳定高效的猕猴神经干细胞分化体系,在该分化体系下,GFP标记猕猴胚胎干细胞在分化的第12天时,95%以上的细胞分化为神经干细胞;2)分化得到的Rosettes结构神经干细胞经bFGF/EGF扩增后,能够较好地维持其Rosettes结构;3)经bFGF/EGF扩增后的rosettes结构神经干细胞移植到猕猴脑内后能够较好的存活并向神经元分化,即bFGF/EGF扩增培养能较好地维持Rosettes结构的神经干细胞,且移植到猕猴脑内的该细胞亦能够较好地存活并向神经元分化,该结果为神经干细胞应用于临床提供了基础理论依据。     

Identification of the mouse Loop‐tail gene: a model for human craniorachischisis?

Neural tube defects are one of the commonest human birth defects, with more than 0.5% of some populations affected. Mouse models are being used in an attempt to identify genes that could be involved in these malformations. Only two mouse mutations are known to lead to craniorachischisis, failure of closure of almost the entire neural tube. Two recent papers report that the gene for one of these, Loop-tail, has now been identified and sequenced.1, 2 It has been given the designation Ltap/Lpp1 and appears to function in floor plate formation. It will be of great interest to investigate the role of this gene in human neural tube defects.     

Neural computations underlying the exertion of force: a model

We have developed a model that simulates possible mechanisms by which supraspinal neuronal signals coding forces could converge in the spinal cord and provide an ongoing integrated signal to the motoneuronal pools whose activation results in the exertion of force. The model consists of a three-layered neural network connected to a two-joint-six-muscle model of the arm. The network layers represent supraspinal populations, spinal cord interneurons, and motoneuronal pools. We propose an approach to train the network so that, after the synaptic connections between the layers are adjusted, the performance of the model is consistent with experimental data obtained on different organisms using different experimental paradigms: the stiffness characteristics of human arm; the structure of force fields generated by the stimulation of the frog's spinal cord; and a correlation between motor cortical activity and force exerted by monkey against an immovable object. The model predicts a specific pattern of connections between supraspinal populations coding forces and spinal cord interneurons: the weight of connection should be correlated with directional preference of interconnected units. Finally, our simulations demonstrate that the force generated by the sum of neural signals can be nearly equal to the vector sum of forces generated by each signal independently, in spite of the complex nonlinearities intervening between supraspinal commands and forces exerted by the arm in response to these commands.     

MALFORMATIONS OF THE CENTRAL NERVOUS SYSTEM INDUCED BY NEUROTROPIC DRUGS IN MOUSE EMBRYOS

Out of a sample of fifteen neurotropic drugs consisting of seven antidepressants and anti-psychotics, two antianxiety drugs, one anticonvulsant, three opiates and two synthetic analgesics, twelve were found to be teratogenic for mouse embryos, causing malformations of the central nervous system. After single injections of the teratogenic dose administered at the very beginning of the ninth day of gestation, four days later, i.e. in 13-day-old embryos, the induced defects appeared to make up a recurring syndrome of malformations which consists of several abnormalities present in various frequencies either individually or in combination in the same embryos. These malformations are: exencephaly, craniorachischisis, cervical and thoraco-lumbar myeloschisis, hydrocephalic dilatation of the fourth brain ventricle, Z-shaped kinking of the spinal cord and lumbar hydromyelia. In addition, after administration of some of the drugs, branchyury or anury with or without lumbar myeloaplasia were recorded.
In general the results reported here seem to suggest that because of their possible affinity neurotropic drugs are potentially teratogenic for the embryonic central nervous system if applied at the time of the neural tube closure although it is known that there are drugs in this group which do not cause any malformations of the central nervous system and that many non-neurotropic agents do cause such malformations. Secondly, the results seem to suggest also that the position of the malformations along the cerebro-spinal axis may be depending to some extent on the pharmacological properties of the drugs tested. These conjectures are treated here as entirely provisional pending further investigations.     

Mutations in Planar Cell Polarity Gene SCRIB Are Associated with Spina Bifida

Neural tube defects (NTDs) (OMIM #182940) including anencephaly, spina bifida and craniorachischisis, are severe congenital malformations that affect 0.5–1 in 1,000 live births in the United States, with varying prevalence around the world. Mutations in planar cell polarity (PCP) genes are believed to cause a variety of NTDs in both mice and humans. SCRIB is a PCP-associated gene. Mice that are homozygous for the Scrib p.I285K and circletail (Crc) mutations, present with the most severe form of NTDs, namely craniorachischisis. A recent study reported that mutations in SCRIB were associated with craniorachischisis in humans, but whether SCRIB mutations contribute to increased spina bifida risk is still unknown. We sequenced the SCRIB gene in 192 infants with spina bifida and 190 healthy controls. Among the spina bifida patients, we identified five novel missense mutations that were predicted-to-be-deleterious by the PolyPhen software. Of these five mutations, three of them (p.P1043L, p.P1332L, p.L1520R) significantly affected the subcellular localization of SCRIB. In addition, we demonstrated that the craniorachischisis mouse line-90 mutation I285K, also affected SCRIB subcellular localization. In contrast, only one novel missense mutation (p.A1257T) was detected in control samples, and it was predicted to be benign. This study demonstrated that rare deleterious mutations of SCRIB may contribute to the multifactorial risk for human spina bifida.     

Distribution of neurotensin-like immunoreactivity in the central nervous system of the Formosan monkey

The distribution of neurotensin-like immunoreactivity was investigated in the central nervous system of the Formosan monkey employing immunohistochemical techniques. Neurotensin-containing cells were found to be widely distributed in the forebrain. The principal densities of neurotensin-like neuronal perikarya were located in the limbic system, the basal ganglion and the cerebral cortex; particularly in the amygdala, the septum, the neostriatum, the claustrum and the insula. The stria terminalis and the preoptic area were also rich in immunostained neurotensin-like neurons. A large number of immunoreactive fibers were observed from the cerebral cortex to the spinal cord in locations such as the median eminence, the arcuate nucleus, the hippocampus, the central gray and the dorsal horn of the spinal cord. We analyzed in detail the distribution of neurotensin-like immunoreactivity in the brain of the Formosan monkey, and compared these results with those obtained in the brain of the rat, Japanese monkey and human. Some possible implications regarding differences in location of this peptide are also briefly discussed.     

Clinical, genetic, and epidemiological factors in neural tube defects.

We examined clinical, genetic, and epidemiologic factors among 512 probands with nonsyndromal neural tube defects (NTDs). Data were analyzed after grouping the probands in four different ways with respect to pathological features and putative pathogenic mechanisms. Apparently unrelated congenital anomalies occurred more frequently among probands with craniorachischisis (62%), encephalocele (30%), or multiple NTDs (25%) than among probands with anencephaly (14.7%) or spina bifida (10.1%) (P much less than .0001). Unrelated congenital anomalies occurred less often among probands with low spina bifida (6.7%) than among probands with high spina bifida (19.5%). NTDs were seen in 7.8% of the siblings of probands with high spina bifida but in only 0.7% of the siblings of probands with low spina bifida, in 2.2% of the siblings of anencephalic probands, and in none of the siblings of probands with craniorachischisis, encephalocele, or multiple NTDS (P less than .001). In all 16 families in which two siblings had NTDs, both had either defects of the type associated with abnormal primary neurulation or defects of the type associated with abnormal canalization. High spina bifida and multiple NTDs were found more frequently than expected among the Sikh probands (P less than .02). The frequency of non-NTD congenital anomalies was higher among siblings of Sikh probands (8.8%) than among siblings of other probands (2.4%) (P less than .05). This excess was due to the occurrence of hydrocephalus without spina bifida in four of 68 siblings of Sikh probands.     

Implantation of BM mesenchymal stem cells into injured spinal cord elicits de novo neurogenesis and functional recovery: evidence from a study in rhesus monkeys

BACKGROUND: Transplantation of mesenchymal stem cells (MSC) in rodent models has proved to be an effective therapeutic approach for spinal cord injury (SCI). However, further studies in primate models are still needed before clinical application of MSC to patients. METHODS: MSC were isolated from rhesus monkey BM and induced ex vivo to differentiate into neural lineage cells. Induced cells were labeled with Hoechst 33342 and injected into the injured sites of rhesus SCI models. Function of the injured spinal cord was assessed using Tarlov behavior assessment, sensory responses and electrophysiologic tests of cortical somatosensory-evoked potential (CSEP) and motor-evoked potential (MEP). In vivo differentiation of the implanted cells was demonstrated by the presence of neural cell markers in Hoechst 33342-labeled cells. The re-establishment of the axonal pathway was demonstrated using a true blue (TB) chloride retrograde tracing study. RESULTS: Monkeys achieved Tarlov grades 2-3 and nearly normal sensory responses 3 months after cell transplantation. Both CSEP and MEP showed recovery features. The presence of the neural cell markers neurofilament (NF), neuro-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) was observed in approximately 10% of Hoechst 33342-labeled cells. TB, originally injected at the caudal side of injured sites, was traceable in the rostral thoracic spinal cord, red nucleus and sensory motor cortex. DISCUSSION: Our results suggest that the implantation of MSC-derived cells elicits de novo neurogenesis and functional recovery in a non-human primate SCI model and should harness the clinical application of BM MSC in SCI patients.     

Dynamic Neural Network Models of the Premotoneuronal Circuitry Controlling Wrist Movements in Primates

Dynamic recurrent neural networks were derived to simulate neuronal populations generating bidirectional wrist movements in the monkey. The models incorporate anatomical connections of cortical and rubral neurons, muscle afferents, segmental interneurons and motoneurons; they also incorporate the response profiles of four populations of neurons observed in behaving monkeys. The networks were derived by gradient descent algorithms to generate the eight characteristic patterns of motor unit activations observed during alternating flexion-extension wrist movements. The resulting model generated the appropriate input-output transforms and developed connection strengths resembling those in physiological pathways. We found that this network could be further trained to simulate additional tasks, such as experimentally observed reflex responses to limb perturbations that stretched or shortened the active muscles, and scaling of response amplitudes in proportion to inputs. In the final comprehensive network, motor units are driven by the combined activity of cortical, rubral, spinal and afferent units during step tracking and perturbations.The model displayed many emergent properties corresponding to physiological characteristics. The resulting neural network provides a working model of premotoneuronal circuitry and elucidates the neural mechanisms controlling motoneuron activity. It also predicts several features to be experimentally tested, for example the consequences of eliminating inhibitory connections in cortex and red nucleus. It also reveals that co-contraction can be achieved by simultaneous activation of the flexor and extensor circuits without invoking features specific to co-contraction.