Outgrowth of nerve fibres from Drosophila central nervous system cultured in vitro

Explants of the central nervous system of Drosophila have been shown to produce nerve fibres in vitro. The effects of various culture conditions on fibre outgrowth have been examined. Nervous tissue could form nerve fibres in vitro when the explants were obtained from mid-embryonic or early- and mid-pupal stages, but not when they were obtained from larvae or late-pupae. The effect of the temperature-sensitive mutation shibire^ts has been investigated by placing mutant explants into culture at permissive (17°C) or restrictive (28°C) temperatures. No differences in the extent of fibre outgrowth between wild-type and shibire^ts were observed, regardless of the temperature of cultivation.     

Effects of Substantia Nigra Lesions on the Locomotor and Stereotypy Responses to Amphetamine

IT is usually supposed that amphetamine produces behavioural effects which include an increase of spontaneous motor activity and the elicitation of stereotyped behaviours¹, by causing a release of endogenous catecholamines in the central nervous system². This view is, for example, supported by the observation that amphetamine can release the catecholamines noradrenaline (NA) and dopamine (DA) from the central nervous system in vitro² and in vivo^{3, 4} and that inhibition of catecholamine biosynthesis blocks the amphetamine effect⁵. Anatomical studies of the distribution of neurones containing catecholamine however, raise, questions about the general applicability of this hypothesis⁶.     

Central neurophysiological processing of joint pain on the basis of studies performed in normal animals and in models of experimental arthritis.

On the basis of anatomical and electrophysiological studies, this review summarizes first, the data dealing with the transmission of joint inputs in the central nervous system of normal animals at the spinal and supraspinal levels. It appears that in these conditions neuronal responses to mechanical noxious stimuli of the joints are relatively few and (or) weak. Second, in sharp contrast, the studies performed in polyarthritic rats have emphasized the profound changes in the activities (spontaneous firing and responsiveness) of the somatosensory neurones at various levels of the central nervous system (CNS), including the thalamus and primary somatosensory cortex; many were spontaneously active and a majority of them could be maximally activated by gentle mechanical stimuli applied to the inflamed joints. Although the change in the sensitivity of the peripheral mechanoreceptors has a major role in the modifications described in the CNS, additional observations have suggested a complex interaction between peripheral and central processes. On the basis of the recent data obtained in poly- and mono-arthritic animals; the following phenomena have been successively considered: the segmental and hetero-segmental "cross-talk" and their possible relationship with referred pain; the involvement of "new" neuronal populations as a possible basis of a selective system for joint pain; and the possible involvement of changes in the various control systems that normally modulate the nociceptive inputs at different levels of the CNS.     

ELECTRICAL RESPONSES FROM THE LATERAL-LINE NERVES OF FISHES : V. RESPONSES IN THE CENTRAL NERVOUS SYSTEM

Records of spontaneous discharge of nerve impulses, similar to that previously described in catfish and in trout, have been obtained from lateral-line nerves of goldfish and perch, by the use of concentric micro electrodes slipped under the nerve in situ. These impulses have been followed into the central nervous system. They enter the tuberculum acusticum and thence apparently spread diffusely through the cerebellum. Cutting the lateral-line nerve on one side silences the ipsilateral tuberculum acusticum, but only reduces the intensity of ipsilateral cerebellar activity. Cutting the remaining lateral-line nerve silences activity throughout the tuberculum acusticum and the cerebellum. The maintenance of tonic activity in the tuberculum acusticum by way of lateral-line discharge may account for the inhibitory effects of the lateral-line system on auditory responses.     

Ultrastructure and Function of Cephalopod Chromatophores

SYNOPSIS. Each chromatophore organ consists of a pigment celland of several radial muscle fibers that represent separatecells. The pigment granules are contained within an elasticsacculus within the pigment cell. The sacculus is attached aroundthe equator of the chromatophore to the cell membrane by zonalhaptosomes. In turn, the cell membrane is attached to the radialmuscle fibers by a dense basal lamina. The cell membrane ofthe retracted chromatophore is highly folded. Contraction ofthe radial muscle fibers is initiated by (a) excitatory junctionpotentials, (b) miniature potentials, or (c) spike potentials.The latter arise spontaneously in the muscle fibers when thesehave undergone some internal (metabolic?) change. The contractionof the muscle fibers causes expansion of the pigment-containingsacculus. Relaxation of the muscle fibers permits the sacculusto assume its original lenticular or near-spherical shape; theenergy for this is stored within theexpanded elastic componentsof the sacculus. In normal skin the chromatophore organs areentirely under the control of the central nervous system, themuscle fibers being activated only by local, excitatory postsynapticpotentials initiated by motor nerve impulses. That postsynapticpotentials are non-propagating insures that individual motorfibers can be activated individually, thus permitting a delicatecontrol of skin color by recruitment as well as by frequency.Tonic contractions and pulsations, involving spontaneous releaseof transmitter from nerve terminals and spike generation withinthe muscle fibers, respectively, are the result of altered,abnormal conditions within the skin.     

Pacemaker Potentials for the Periodic Burst Discharge in the Heart Ganglion of a Stomatopod, Squilla oratoria

From somata of the pacemaker neurons in the Squilla heart ganglion, pacemaker potentials for the spontaneous periodic burst discharge are recorded with intracellular electrodes. The electrical activity is composed of slow potentials and superimposed spikes, and is divided into four types, which are: (a) "mammalian heart" type, (b) "slow generator" type, (c) "slow grower" type, and (d) "slow deficient" type. Since axons which are far from the somata do not produce slow potentials, the soma and dendrites must be where the slow potentials are generated. Hyperpolarization impedes generation of the slow potential, showing that it is an electrically excitable response. Membrane impedance increases on depolarization. Brief hyperpolarizing current can abolish the plateau but brief tetanic inhibitory fiber stimulation is more effective for the abolition. A single stimulus to the axon evokes the slow potential when the stimulus is applied some time after a previous burst. Repetitive stimuli to the axon are more effective in eliciting the slow potential, but the depolarization is not maintained on continuous stimulation. Synchronization of the slow potential among neurons is achieved by: (a) the electrotonic connections, with periodic change in resistance of the soma membrane, (b) active spread of the slow potential, and (c) synchronization through spikes.     

Bioelectric activity is required for regional specificity of sensory ganglion projections to spinal cord explants cultured in vitro

Summary Chronic blockade of spontaneous nerve impulses by means of tetrodotoxin leads to abnormally diffuse afferent projections into spinal cord cross-sections cultured for two to six weeks in vitro. In addition, even untreated explants which show a low level of spontaneous cord discharges failed to develop the normal degree of dorsal pathway selectivity. It is therefore concluded that centrally generated neuronal activity may play an important role in eliminating exuberant connections which, during early development, are transiently present in this part of the nervous system.     

Physiological control of the chromatophores of Austrolestes annulosus (Odonata)

The chromatophore-containing integument of Austrolestes annulosus always changes from a dark to a blue colour phase in vitro and therefore functions independently of a central control mechanism. In isolated abdominal segments, the reverse colour change occurs independently of the presence of central nervous system. The amount of change decreases with the size of the piece of integument; it varies over different parts of the body of the same intact insect, and it varies both in vitro and in vivo with time of day.A variety of experiments, involving ligation of abdominal segments and ablation of their ganglia, show that the terminal ganglion is primarily responsible for the release of a darkening factor which migrates in an anterior direction. Other ventral ganglia have a similar function but are of less importance.Transplants of terminal ganglia and injection of their extracts fail to restore capacity for colour change in ligature-isolated segments which have had their own ganglia removed. These findings, supported by histological observations, suggest that the darkening factor is a neurosecretion.     

Spontaneous electrical activity recorded from the aphid central nervous system

Whilst many classes of insecticides target the insect central nervous system (CNS), their effects in the CNS of pest aphids have not been demonstrated. In this report, we describe an electrophysiological method for recording spontaneous neuronal activity from the giant willow aphid (Tuberolachnus salignus). Using extracellular recording electrodes and two analysis methods (threshold and template search), spontaneous spike activity was shown to exhibit sensitivity to the neuroexcitatory insecticide imidacloprid. This method allows changes in the frequency of action-potentials to be monitored during direct bath exposure to chemical agents, enabling a means of assessing and comparing neurotoxic effects of insecticides in a previously inaccessible superfamily of pest insects.     

Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo.     

Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat,chick and human)

Summary Explants of embryonic and fetal spinal cords (rat, chick, and human) develop and maintain in vitro many cytologic and bioelectric properties characteristic of central nervous tissues in situ. Despite the thickness of the cord explants, a condition which appears to be necessary for differentiation, considerable neuronal development becomes visible to light microscopic examination.The mature expiant consists of large concentrations of small neurons interspersed with large neurons and neuroglia, bounded by a broad tract of nerve fibers and capped by a neuropil. Myelination in rat cultures usually begins 2–3 weeks after explantation in both the explant and outgrowth zone. Myelin is of the central glial type unless the cord explants are grown with their meningeal covering. In the latter case the myelination pattern abruptly changes to the peripheral Schwannian type as the axons penetrate the meninges.Bouton-like endings are observed in the neuropil of rat cord explants (whole-mounts) impregnated with silver; but most neurons are only partially blackened. In 20 sections, neuronal somas and dendrites are identified in negative image with blackened bouton-like endings suggesting synapses.Chick spinal cord, when grown in Rose chambers, becomes more thinly spread so that more detailed interrelationships can be visualized in the living neuronal somas, neuritic processes and termini. Bouton-like endings on neuronal somas have been selectively stained, vitally, with methylene blue.Complex bioelectric activity can be evoked in these long-term spinal cord explants by electric stimuli localized to various regions of the cord tissues as well as to attached dorsal-root ganglia. The long-lasting after-discharge patterns and the neuropharmacologic sensitivity of the responses show remarkable similarity to the activity of synaptic networks of the central nervous system in situ. These functions develop gradually during the first week after explantation of fetal rat cord tissues — more slowly in cultures explanted before the establishment of reflex arcs in utero, and more rapidly in cord explants from older fetuses. Reference is made to the companion electron microscopy study of older fetuses, which shows that characteristic synaptic structures, although extremely rare at the time of explantation, are abundant in later stages of the culture's development. This confirms the functional evidence that synapses are able to develop in organized culture conditions.This study was supported by grants NB-00858 and NB-03814 from the National Institute of Neurological Diseases and Blindness, United States Public Health Service.Research Career Development Fellow (Award NB-K3-2904 from the NINDB, USPHS).Research Career Award 5K6-GM-15,372 from U.S. National Institutes of Health.     

Micropropagation of Laburnum anagyroides Medic. through axillary shoot regeneration

An in vitro propagation system based on the proliferation of axillary buds has been developed for Laburnum anagyroides. Culture initiation was influenced by explanting season, with the maximum response obtained from explants harvested in spring and autumn while the lowest response was noted in summer explants. The best shoot induction was observed on Murashige and Skoog medium (MS) supplemented with 2.22 μM 6-benzylaminopurine (BAP). The basal media, type of cytokinin, and explant type were the most important factors affecting shoot multiplication of L. anagyroides. Medium comprised of ½MS salts was found to be more efficient for axillary shoot multiplication compared to full-strength MS or Wood Plant Medium (WPM) when using identical growth regulators. Shoot tip explants were more responsive than nodal segments of microshoots for micropropagation. In vitro-derived shoots, >10 mm in length, were successfully rooted in medium containing ¼MS salts and 2.68 μM α-naphtalene acetic acid (NAA). In vitro-regenerated plantlets were adapted to ex vitro conditions and transferred to a greenhouse. This is the first report for successful in vitro propagation of L. anagyroides from bud explants of mature trees.     

"Action potentials" in Neurospora crassa, a mycelial fungus.

Occasional spontaneous "action potentials" are found in mature hyphae of the fungus Neurospora crassa. They can arise either from low-level sinusoidal oscillations of the membrane potential or from a linear slow depolarization which accelerates into a rapid upstroke at a voltage 5-20 mV depolarized from the normal resting potential (near-180 mV). The "action potentials" are long-lasting, 1-2 min and at the peak reach a membrane potential near-40 mV. A 2-to 8-fold increase of membrane conductance accompanies the main depolarization, but a slight decrease of membrane conductance occurs during the slow depolarization. Two plausible mechanisms for the phenomenon are (a) periodic increases of membrane permeability to inorganic ions, particularly H+ or Cl- and (b) periodic decreases in activity of the major electrogenic pump (H+) or the Neurospora membrane, coupled with a nonlinear (inverse signoid) current-boltage relationship. Identification of action potential-like disturbances in fungi means that such behavior has now been found in all major biologic taxa which have been probed with suitable electrodes. As yet there is no obvious function for the events in fungi.     

Mutation of pescadillo disrupts oligodendrocyte formation in zebrafish

Background

In vertebrates, the myelin sheath is essential for efficient propagation of action potentials along the axon shaft. Oligodendrocytes are the cells of the central nervous system that create myelin sheaths. During embryogenesis, ventral neural tube precursors give rise to oligodendrocyte progenitor cells, which divide and migrate throughout the central nervous system. This study aimed to investigate mechanisms that regulate oligodendrocyte progenitor cell formation.

Methodology/Principal Findings

By conducting a mutagenesis screen in transgenic zebrafish, we identified a mutation, designated vu166, by an apparent reduction in the number of oligodendrocyte progenitor cells in the dorsal spinal cord. We subsequently determined that vu166 is an allele of pescadillo, a gene known to play a role in ribosome biogenesis and cell proliferation. We found that pescadillo function is required for both the proper number of oligodendrocyte progenitors to form, by regulating cell cycle progression, and for normal levels of myelin gene expression.

Conclusions/Significance

Our data provide evidence that neural precursors require pes function to progress through the cell cycle and produce oligodendrocyte progenitor cells and for oligodendrocyte differentiation.     

Refining the Ciona intestinalis Model of Central Nervous System Regeneration

Background

New, practical models of central nervous system regeneration are required and should provide molecular tools and resources. We focus here on the tunicate Ciona intestinalis, which has the capacity to regenerate nerves and a complete adult central nervous system, a capacity unusual in the chordate phylum. We investigated the timing and sequence of events during nervous system regeneration in this organism.

Methodology/Principal Findings

We developed techniques for reproducible ablations and for imaging live cellular events in tissue explants. Based on live observations of more than 100 regenerating animals, we subdivided the regeneration process into four stages. Regeneration was functional, as shown by the sequential recovery of reflexes that established new criteria for defining regeneration rates. We used transgenic animals and labeled nucleotide analogs to describe in detail the early cellular events at the tip of the regenerating nerves and the first appearance of the new adult ganglion anlage.

Conclusions/Significance

The rate of regeneration was found to be negatively correlated with adult size. New neural structures were derived from the anterior and posterior nerve endings. A blastemal structure was implicated in the formation of new neural cells. This work demonstrates that Ciona intestinalis is as a useful system for studies on regeneration of the brain, brain-associated organs and nerves.     

Electrophysiologic and morphologic properties of neurons in dissociated chick spinal cord cell cultures

Neurons dissociated from embryonic chick spinal cords mature in relatively sparse cell culture and survive in vitro for several weeks. They generate action potentials and form both excitatory and inhibitory chemical synapses with one another. By electrophysiologic and morphologic criteria, it appears that the neuronal population (after 2–3 weeks) is made up of a variety of different cell types; few, if any, are motoneurons. Neuron cell bodies are not covered by glia or satellite cells and nerve processes are not myelinated. Thus, the cultures should permit more direct microelectrode and pharmacologic analysis of differentiation of cell specific properties and of synapse formation than is possible in the intact central nervous system.     

Incorporation and Rate of Loss of S35-Methionine by Adult Rat Trachea in Organ Cultures and in Living Animals

Full-thickness pieces of adult rat trachea were supported on rayon on the surface of clotted medium in watch glasses. Differentiated epithelium was reduced in height during 25 days of cultivation because basal cells and some columnar cells migrated to cover exposed parts of the explants and because some differentiated cells died and were shed. S³⁵-methionine was (a) placed on explants in vitro and (b) injected intraperitoneally in living rats. Cultured tissues and tissues of living rats were examined by autoradiography at 4 and 24 hours and 4, 7, and 11 days after labeling. Although migratory undifferentiated epithelial cells appeared in cultured trachea, all living epithelial cells in vitro incorporated and subsequently lost S³⁵-methionine to the same extent as did epithelium of intact rats. The biologic half-life of methionine in rat tracheal epithelium in vivo and in vitro was about 5 days.     

The mode of regulation of the corpus allatum activity during starvation in adult females of the Colorado potato beetle, Leptinotarsa decemlineata (Say)

When two-day-old female Leptinotarsa decemlineata were starved, their corpus allatum activity, as measured by the radiochemical in vitro assay, was significantly reduced after 24 hr. Such a reduction was not observed when the nerve connections between the central nervous system and the retrocerebral complex were severed and the beetles starved up to 5 days. In some experiments, the rate of juvenile hormone biosynthesis in vitro, was substantiated by measurement of the juvenile hormone titre in the haemolymph by physico-chemical methods. It is concluded that intact nervous connections between the central nervous system and the corpora allata are essential for restraining the juvenile hormone biosynthesis during the initial stages of starvation.Corpora allata from 1-day starved insects were considerably stimulated in vitro by farnesenic acid indicating that juvenile hormone synthesis is controlled enzymatically at a stage prior to the final two steps in the pathway. However, on day 5 of starvation, rate-limitation may occur after formation of this intermediate, since farnesenic acid stimulation was much less at this time.Corpora allata of adult females newly emerged from the soil were activated within 4 hr regardless of feeding.     

Afferent innervation shapes the dendritic branching pattern of the Medial Giant Interneuron in grasshopper embryos raised in culture

In the adult grasshopper the Medial Giant Interneuron (MGI) receives synaptic input from the peripheral sensory neurons of the cercus. We prevented this innervation in grasshopper embryos by cutting off one or both cerci at a stage when the first sensory axons are just beginning to reach the central nervous system (CNS), and the MGI has not yet formed its mature branching pattern. Following this operation the embryos were raised in vitro for 3–9 days, and the MGI injected with the fluorescent dye Lucifer Yellow to determine its morphology. The development of the deprived cells was then compared to that of the normal MGI (described in M. Shankland and C. S. Goodman, 1982, Develop. Biol., 92, 483–500) and of cultured, but unoperated, controls to ascertain whether these presynaptic axons influence the embryonic growth and branching of the MGI's dendrites. The results of these experiments show that dendrite formation is enhanced in regions of the neuropil containing sensory axon terminals and that the afferents exert their influence locally on restricted portions of the branching structure. The enhanced growth of innervated dendrites appears to occur at the expense of dendritic outgrowth elsewhere, suggesting that the growing dendrites may be competing for a limited supply of some cellular component necessary for continued growth. Thus, the MGI's final branching pattern is at least partially dictated by the spatial distribution of presynaptic axons within the embryonic nervous system.     

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, ''99). The cerebral cortex consists of two basic neuronal types: excitatory and inhibitory. These cells arise in distinct areas and migrate into the cortex along different routes (Pearlman et al., ''98). Inhibitory interneurons migrate tangentially from subcortical sources, mostly from different regions of the ganglionic eminences (Gelman et al., ''09; Xu et al., ''04). Their movement requires precise spatiotemporal control imposed by environmental cues, to allow for the establishment of proper cytoarchitecture and connectivity in the cerebral cortex (Caviness & Rakic, ''78; Hatten, ''90; Rakic, ''90). To study the migratory behavior of cells generated in proliferative zones of the ganglionic eminences (GE) in newborn ferrets in vitro we used a 3 dimensional culture arrangement in a BD Matrigel Matrix. The culture setup consisted of two GE explants and a source of tested proteins extracted from the cerebral cortex and adsorbed on fluorescent latex Retrobeads IX positioned between the explants (Hasling et al., ''03; Riddle et al., ''97). After 2-3 days of culture, the cells start to appear at the edge of the explant showing a propensity to leave the tissue in a radial direction. Live imaging allowed observation of migratory patterns without the necessity of labeling or marking the cells. When exposed to fractions of the protein extract obtained from isochronic ferret cortex, the GE cells displayed different behaviors as judged by quantitative kinetic analysis of individual moving cells.