Nervous System Of Xenopus Laevis Research Articles

Morpholino Studies in Xenopus Brain Development.

Antisense morpholino oligonucleotides (MOs) have become a valuable method to knockdown protein levels, to block with mRNA splicing and to interfere with miRNA function. MOs are widely used to alter gene expression in development of Xenopus and Zebrafish, where they are typically injected into the fertilized egg or blastomeres. Here we present methods to use electroporation to target delivery of MOs to the central nervous system of Xenopus laevis or Xenopus tropicalis tadpoles. Briefly, MO electroporation is accomplished by injecting MO solution into the brain ventricle and driving the MOs into cells of the brain with current passing between 2 platinum plate electrodes, positioned on either side of the target brain area. The method is relatively straightforward and uses standard equipment found in many neuroscience labs. A major advantage of electroporation is that it allows spatial and temporal control of MO delivery and therefore knockdown. Co-electroporation of MOs with cell type-specific fluorescent protein expression plasmids allows morphological analysis of cellular phenotypes. Furthermore, co-electroporation of MOs with rescuing plasmids allows assessment of specificity of the knockdown and phenotypic outcome. By combining MO-mediated manipulations with sophisticated assays of neuronal function, such as electrophysiological recording, behavioral assays, or in vivo time-lapse imaging of neuronal development, the functions of specific proteins and miRNAs within the developing nervous system can be elucidated. These methods can be adapted to apply antisense morpholinos to study protein and RNA function in a variety of complex tissues.

Bulk Electroporation of Retinal Ganglion Cells in Live Xenopus Tadpoles

Individual neurons in the developing nervous system of Xenopus laevis can be visualized by the targeted delivery of a fluorophore. The fluorophore can be delivered as a fluorescent dye or DNA that encodes a fluorescent protein. Local iontophoresis is a method that works well for transfer of fluorescent dye to retinal ganglion cells (RGCs) in the eye, but it does not give a high yield for delivery of DNA. This is largely because the degree of pigmentation of the eyes, even in albino strains, makes it difficult to visualize RGC somata during pipette positioning. Bulk retinal electroporation is a better approach for delivery of plasmid DNA to RGC. The method described here works best in tadpoles older than stage 42.

Spatiotemporal sequence of appearance of NPFF-immunoreactive structures in the developing central nervous system of Xenopus laevis

Neuropeptide FF-like immunoreactive (NPFFir) cells and fibers were analyzed through development of Xenopus laevis. The first NPFFir cells appeared in the embryonic hypothalamus, which projected to the intermediate lobe of the hypophysis, the brainstem and spinal cord. Slightly later, scattered NPFFir cells were present in the olfactory bulbs and ventral telencephalon. In the caudal medulla, NPFFir cells were observed in the nucleus of the solitary tract only at embryonic and early larval stages. Abundant NPFFir cells and fibers were demonstrated in the spinal cord. The sequence of appearance observed in Xenopus shares many developmental features with mammals although notable differences were observed in the telencephalon and hypothalamus. In general, NPFF immunoreactivity developed earlier in amphibians than in mammals.

Distribution and effects of PACAP, VIP, nitric oxide and GABA in the gut of the African clawed frog Xenopus laevis.

The distribution and possible effects on gastrointestinal motility of pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide and gamma-amino-butyric acid (GABA) were investigated in the African clawed frog (Xenopus laevis) using immunohistochemistry and in vitro strip preparations. PACAP- and VIP-immunoreactive nerve fibres were common in the myenteric plexus as well as in the longitudinal and circular muscle layers all along the gastrointestinal tract. Double labelling demonstrated a close correlation between PACAP and VIP immunoreactivities, indicating that the two neurotransmitters are colocalised within the enteric nervous system. Occasionally, PACAP- and VIP-positive nerve cell bodies were seen in the myenteric or submucous plexa. In addition, VIP immunoreactivity coexisted with helospectin immunoreactivity. Nitric oxide synthase (NOS)-immunoreactive nerve cells were found in the myenteric plexus at an average density for the whole gastrointestinal tract of 4584 +/- 540 cells cm(-2). The NOS-immunoreactive nerve cells were usually multipolar with an average size of 11.3 +/- 3.7 x 23.2 +/- 6.6 microm. Some NOS-immunoreactive nerve fibres were VIP-immunoreactive but not all VIP-positive fibres showed NOS immunoreactivity. GABA immunoreactivity was found in nerve fibres and nerve cells in the myenteric plexus of all regions of the gut. Few GABA-immunoreactive nerve fibres were VIP-immunoreactive. PACAP 27, VIP, sodium nitroprusside (a nitric oxide donor; NaNP) and GABA caused similar responses on spontaneously contracting circular preparations of the cardiac stomach of X. laevis. The mean force developed was decreased, mainly by a reduction in resting tension, while the amplitude of contractions was not necessarily affected. The NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) increased the mean force developed, indicating a nitrergic tone in the preparations. In contrast, PACAP 27, VIP, NaNP, GABA and L-NAME had no significant effect on longitudinal strip preparations from the duodenum. These results indicate that PACAP, VIP, nitric oxide and GABA, which are known to be important inhibitory neurotransmitters in other vertebrates, are widely spread in the enteric nervous system of Xenopus laevis and may be involved in the inhibitory control of gastric motility. Although no effect of PACAP, VIP, nitric oxide or GABA on the longitudinal strips of the duodenum was seen in this study, this does not rule out the possibility that they might play an important role in controlling intestinal motility as well.

A retinoid-binding lipocalin, Xlcpl1, relevant for embryonic pattern formation is expressed in the nervous system of Xenopus laevis.

The importance of retinoids in early development has been increasingly recognized during the past decade. Their transport and action are mediated by extracellular, intracellular and nuclear proteins. Here we describe the isolation and characterization of a cDNA, Xlcpl1, coding for a lipocalin that is most likely involved in the transport of retinoids in the embryonic nervous system of Xenopus laevis. Lipocalins generally are carriers for small hydrophobic molecules. The expression of Xlcpl1 shows a distinct spatial and temporal pattern: transcription was observed in the anterior-most part of the neural plate at embryonic stages 13-14 whereas in adult brain, Xlcpl1 is expressed exclusively in the choroid plexus. In addition, expression was also found at the dorsal borders of the eye anlagen and in the otic vesicle of mid-neurula stage embryos. In order to gain insight into the molecular function of Xlcpl1, we caused ectopic overexpression by injecting fertilized eggs with either an excess of sense mRNA or DNA constructs under the control of the EF1α promotor. The resulting embryos developed a dysmorphogenised anterior neural system, in particular malformed heads and eyes. Phenotypic defects were therefore similar to those obtained by retinoic acid treatment at the gastrula stage or overexpression of other proteins involved in retinoic acid signalling in the early embryo. Retinoic acid treatment disrupts the endogenous gradient of retinoids that supposedly controls antero-posterior differentiation. By analogy, we propose that local overexpression of Xlcpl1 reflects precisely the situation of exogenous application of retinoic acid. In fact, we have shown biochemically that Xlcpl1 is suited to mediate the cellular retinoid signal.

Effects of Intermediate Filament Disruption on the Early Development of the Peripheral Nervous System ofXenopus laevis

The principal function of intermediate filaments is to strengthen cells. Their developmentally regulated, tissue-specific patterns of expression further suggest that they modulate cellular structural properties during development. To explore the role of intermediate filaments in development, we injected RNA encoding a truncated form of theXenopus laevismiddle-molecular-weight neurofilament protein (NF-M) into embryonic frog blastomeres at the 2-cell stage. A similar truncated form of mammalian NF-M disrupts neurofilaments (Type IV) and vimentin (Type III) intermediate filaments in transfected fibroblasts. In cultures made from dissociated neural tubes and their adjacent myotomes, the resultant protein disrupted both desmin filaments in muscle cells and neurofilaments in neurons during the first day of culture, which corresponds to stage 35/36 in the intact embryo. We next examined the effects of this truncated neurofilament protein on development of the nervous system. The greatest effects were seen on development of cranial and primary motor nerves, which were severely stunted as late as stage 37/38. In addition to these effects, ectopic neurons also appeared immediately beneath the epidermis along the flank of tadpoles expressing the truncated neurofilament protein. Whereas the former effects on peripheral nerve development were nearly identical to effects obtained with injected neurofilament antibodies, the ectopic neurons were novel, suggesting they resulted from the disruption of intermediate filaments other than the neurofilaments. These experiments thus implicate intermediate filaments in several functions important for normal neural development.

Immunohistochemical investigation of ?-aminobutyric acid ontogeny and transient expression in the central nervous system ofXenopus laevis tadpoles

Immunohistochemical investigation of ?-aminobutyric acid ontogeny and transient expression in the central nervous system ofXenopus laevis tadpoles

Isolation and embryonic expression of Xel-1, a nervous system-specific Xenopus gene related to the elav gene family

We have identified a new member of the elav gene family in Xenopus laevis. This gene, Xel-1, like the other elav-related genes, encodes a putative RNA-binding protein that contains three RNA Recognition Motifs and is solely expressed in the nervous system. Xel-1 is most likely the Xenopus homologue of Hel-N1, one of the three known human genes related to elav. Xel-1 is not expressed in early neural precursors but rather in differentiating neurons of the central nervous system, as well as in the cranial and the spinal ganglion cells. Xel-1 thus appears to be an early differentiation marker for both the central and the peripheral nervous system of Xenopus laevis.

Spatial and temporal expression of phosphorylated and non-phosphorylated forms of neurofilament proteins in the developing nervous system of Xenopus laevis

Immunocytochemical studies of developing Xenopus laevis embryos and tadpoles (stages 12 1 2 to 46 ) were performed using a panel of 11 monoclonal antibodies to phosphorylated and non-phosphorylated forms of the neurofilament proteins. These included nine antibodies to the middle molecular weight neurofilament protein (XNF-M, 175 kDa), and two additional antibodies to non-phosphorylated forms of the other two neurofilament proteins (XNF-L, 73 kDa; XNF-H, 205 kDa). The developmental expression of XNF-M, XNF-L and XNF-H, and the progressive phosphorylation of XNF-M in the rhombencephalon, spinal cord, and optic nerve were studied using these antibodies. In the spinal cord and rhombencephalon, non-phosphorylated forms of XNF-M were initially detected during neural tube stages (stages 22–26), one day before XNF-L and XNF-H at early tadpole stages (stage 35/36). In the eye, XNF-M was observed initially during tailbud stages (stage 29/30), but neither XNF-L nor XNF-H was seen even by stage 46 (swimming tadpole). The phosphorylation of XNF-M occurred over a protracted period of several days, both in the neural tube and visual system, and could be divided into four phases. (1) When initially expressed, XNF-M was hypophosphorylated. This was indicated by the early immunostaining of axons and cell bodies with antibodies to dephosphorylated epitopes on XNF-M and by the absence of staining with antibodies to phosphorylated epitopes. (2) After a short timelag (3–9 h) axons were stained by some, but not all antibodies to phosphorylated epitopes. (3) Approximately one day later, all antibodies to phosphorylated epitopes stained the relevant axons. However, XNF-M was not yet fully phosphorylated, as indicated by the continued staining of these axons with antibodies to dephosphorylated epitopes of XNF-M. (4) Two to 3 days after the initial expression of XNF-M, dephosphorylated epitopes disappeared from the axons, establishing the adult pattern. During development, the most heavily phosphorylated neurofilament proteins present at a given stage were found first in distal regions of the axons and progressed gradually toward the neuronal perikarya as development proceeded. This gradient of phosphorylation, established early within the axon, suggests that neurofilaments in the axons mature from their distal ends toward the cell body, a process which may be regulated by local factors within the axons themselves. The similarity of the basic features of NF-M phosphorylation in mammalian, avian, and amphibian axons underscores the importance of this phenomenon for the development of a mature axon.

Gene expression in the embryonic nervous system of Xenopus laevis.

Development of the nervous system in the amphibian embryo is initiated during gastrulation by an inductive interaction between chordamesoderm and dorsal ectoderm. The induced ectoderm forms the neural plate while uninduced ectoderm generates epidermis. We screened for genes activated during gastrulation and expressed specifically in the nervous system of Xenopus laevis in the expectation that clones representing such genes will constitute useful markers for the study of early neurogenesis. Probes were prepared from adult brain RNA by subtraction with RNA from ovary and from different combinations of adult kidney, muscle, and skin; cDNA libraries prepared from early to late neurula embryo RNA were screened with these probes. Six clones were chosen for further study. Three of these clones are not represented in the maternal RNA population but are activated at the late gastrula stage; the other three increase from a maternal base. Expression of five of the genes is restricted to the neural plate during embryogenesis, and all six are restricted to the central nervous system in premetamorphic tadpoles and adults. One of the clones encodes an apparently neurospecific isoform of beta-tubulin; the identity of the other clones is unknown. Expression of all six genes is suppressed in axis-deficient embryos that lack dorsal structures including the brain.

Monoclonal antibodies against larval nervous system of Xenopus laevis: Their specificities and application to analysis of neural development

Monoclonal antibodies were raised against larval tissues from Xenopus laevis. Two of them which bound to neural tissue were examined for their cytological specificities by indirect immuno-fluorescence on larval frozen sections. One of the monoclonal antibodies (NMI) had affinity for both neural and muscular tissues, while the other (NI) bound exclusively to neural tissue. NMI was shown in the neural tissue to possess a high affinity for the axons from a large population of neurons and possibly the radial fibers from ependymal cells as well. On the other hand, Nl showed a high specificity for some cellular elements other than axons in the white matter, most likely dendrites. For both antibodies, binding to cell bodies could not be detected.The spatial and temporal distribution of the NMI and Nl antigens was investigated. In the mid-trunk region the NMI antigen in axons was first detected at stage 29/30, nearly 10 h earlier than the Nl antigen (stage 33/34). In a larva at stage 37/38, the axonal NMI antigen was distributed throughout the nervous system, whereas the distribution of Nl antigen was restricted to the brainstem and the trunk spinal cord. The Nl antigen-positive area continued expanding rostrocaudally with increasing developmental age. The distribution of NMI and Nl antigens, thus, follows patterns which are consistent with the previously shown general patterns of neuronal process development; the earlier outgrowth of axons than dendrites. and the rostrocaudal gradient in process development.We also examined the two monoclonal antibodies in a cell culture system derived from hatching larvae and found that cytological specificity was substantially conserved; NMI possessed affinity for both neuronal and muscular cells and NI bound exclusively to neuronal cells, though Nl bound to cell bodies as well as processes in culture.

Mapping the auditory central nervous system of Xenopus laevis with 2-deoxyglucose autoradiography

We report here on our use of 2-deoxyglucose autoradiography to identify acoustically-sensitive nuclei in the brain of the South African clawed frog, Xenopus laevis. The superior olive, lateral lemniscus, torus semicircularis, and posterocentral thalamus all selectively accumulated radioactively labeled deoxyglucose during acoustic stimulation. In frogs with middle ear bones removed on one side, the laminar nucleus of the torus was more heavily labeled on the side opposite the intact ear. Some cells within this area are known to concentrate sex hormones. This finding is, to our knowledge, the only example of a hormone-sensitive auditory nucleus in vertebrates.

A biochemical comparison of Xenopus laevis and mammalian myelin from the central and peripheral nervous systems

Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P0 protein and a basic protein of slightly larger mol wt. than the P1 protein of rat myelin.

DDT-like action of allethrin in the sensory nervous system of Xenopus laevis

DDT and allethrin induced repetitive activity in the lateral-line organ of the clawed toad, Xenopus laevis. As measured by the number of spikes per train, both insecticides showed a definite negative temperature coefficient of activity in this sense organ. In isolated peripheral nerves of Xenopus allethrin produced effects similar to those of DDT, i.e., a large negative afterpotential and repetitive activity. As in the case of DDT, the allethrin-induced repetitive firing in these nerves occured only in sensory fibres. However, the origin of the repetitive activity recorded from the allethrin- or DDT-treated lateral-line organ was not located in the afferent nerve fibres as such and this repetitive activity required an intact sense organ. It is concluded that the action of DDT and allethrin in the lateral-line organ is not primarily on the hair cells, but on the impulse-generating process in the afferent nerve terminal, probably by an effect on the sodium conductance of the membrane. The results demonstrate a striking similarity in action between DDT and allethrin, despite the marked difference in their molecular structure. It is suggested that the dimethylcyclopropane group of allethrin is responsible for the DDT-like action of this insecticide.

Patterns of acoustically evoked discharges of neurons in the mesencephalon of the bullfrog.

ArticlesPATTERNS OF ACOUSTICALLY EVOKED DISCHARGES OF NEURONS IN THE MESENCEPHALON OF THE BULLFROGH. David PotterH. David PotterPublished Online:01 Nov 1965https://doi.org/10.1152/jn.1965.28.6.1155MoreSectionsPDF (3 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByEffect of sound pressure level on contralateral inhibition underlying duration-tuned neurons in the mammalian inferior colliculusRoberto Valdizón-Rodríguez, Dominika Kovaleva, and Paul A. Faure28 June 2019 | Journal of Neurophysiology, Vol. 122, No. 1Tuning for rate and duration of frequency-modulated sweeps in the mammalian inferior colliculusJames A. Morrison, Roberto Valdizón-Rodríguez, Daniel Goldreich, and Paul A. 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Casseday, and Ellen Covey1 May 1997 | Journal of Neurophysiology, Vol. 77, No. 5Modelling the response of auditory midbrain neurons in the grassfrog to temporally structured monaural stimuliHearing Research, Vol. 43, No. 2-3Influence of envelope rise time on neural responses in the auditory system of anuransHearing Research, Vol. 36, No. 2-3Coincidence detection in auditory neurons: A possible mechanism to enhance stimulus specificity in the grassfrogHearing Research, Vol. 30, No. 2-3Single-unit characteristics in the auditory midbrain of the immobilized grassfrogHearing Research, Vol. 18, No. 3Detection of amplitude-modulated tones by frogs: Implications for temporal processing mechanismsHearing Research, Vol. 14, No. 2The auditory system of anuran amphibiansProgress in Neurobiology, Vol. 22, No. 1Tonotopic organisation of the auditory midbrain nuclei of the midwife toad (Alytes obstetricans)Hearing Research, Vol. 9, No. 1Mapping the auditory central nervous system ofXenopus laevis with 2-deoxyglucose autoradiographyBrain Research, Vol. 249, No. 1Spectro-temporal characteristics of single units in the auditory midbrain of the lightly anaesthetised grass frog (Rana temporaria L.) investigated with tonal stimuliHearing Research, Vol. 6, No. 1Spectro-temporal characteristics of single units in the auditory midbrain of the lightly anaesthetised grass frog (Rana temporaria L.) Investigated with noise stimuliHearing Research, Vol. 5, No. 2-3Binaural interaction in the inferior colliculus of the frogExperimental Neurology, Vol. 37, No. 3Single unit studies in the midbrain auditory area of CaimanZeitschrift f�r Vergleichende Physiologie, Vol. 71, No. 3Elektro-physiologische Untersuchungen an der H�rbahn der Schleie (Tinca tinca [L.])Zeitschrift f�r Vergleichende Physiologie, Vol. 57, No. 1 More from this issue > Volume 28Issue 6November 1965Pages 1155-1184 https://doi.org/10.1152/jn.1965.28.6.1155PubMed5883734History Published online 1 November 1965 Published in print 1 November 1965 Metrics

The structure of myelin sheaths in the central nervous system of Xenopus laevis (Daudin).

The structure of myelinated nerve fibres has been studied in the spinal cord and optic nerve of the tadpoles of Xenopus laevis. Potassium permanganate-fixed material was examined with the electron microscope. The myelin sheath itself is made up of spirally arranged lamellae in which the intraperiod and dense lines alternate. Inside the myelin sheath an inner cytoplasmic process surrounds the axon and where the external surfaces of its bounding membrane come together an internal mesaxon is formed. The intraperiod line begins within the mesaxon and the dense line usually begins in the same region by apposition of the cytoplasmic surfaces of the membrane. The width of each lamella is 140 A. The outer line in the sheath is the dense line and this terminates in a tongue where the cytoplasmic surfaces of the myelin-forming glial cell separate. Thus, central myelin in Xenopus tadpoles is arranged in the same way as peripheral myelin, the only difference being that in central fibres, cytoplasm on the outside of the sheath is confined to that present in the tongue. For this reason adjacent central sheaths come into apposition without any intervening material being present. When this occurs an intraperiod line is formed between them.