Factors that influence the development of cultured neurons from the brain of the mothManduca sexta

Summary During metamorphic adult development, neurons and glial cells in the developing olfactory (antennal) lobes of the moth undergo characteristic and extensive changes in shape. These changes depend on an interplay among these two cell types and ingrowing sensory axons. All of the direct cellular interactions occur against a background of changing steroid hormone titers. Antennal-lobe (AL) neurons dissociated from stage-5 (of 18 stages) metamorphosing animals survive at least 3 wk in primary cell culture. We describe here the morphological influences on AL neurons of (1) exposure to the steroid hormone 20-hydroxyecdysone, (2) exposure to sensory axons, and (3) interactions among the AL neurons. Cultured AL neurons respond only weakly, if at all, to 20-hydroxyecdysone. They do, however, show greater total outgrowth and branching when they had been exposed in vivo to sensory axons. Because there is no direct contact between some of the neuronal types and the sensory axons at the time of dissociation, the increase in outgrowth must have been mediated via a diffusible factor(s). When AL cells (neurons and glia) are plated at high density in low volumes of medium, or when the cells are plated at low density but in the presence of medium conditioned by high-density cultures, neurite outgrowth and cell survival are increased. Nerve growth factor (NGF), epidermal growth factor (EGF), fibroblast growth factor-basic (bFGF), transforming growth factor-β (TGF _β ) and insulin-like growth factor (ILGF) had no obvious effect on neuronal morphology and thus are unlikely to underlie these effects. Our results suggest that the mature shape of AL neurons depends on developmental interactions among a number of diffusible factors.     

Axons of olfactory receptor cells of transsexually grafted antennae induce development of sexually dimorphic glomeruli in Manduca sexta

The influence of olfactory receptor cell (ORC) axons from transsexually grafted antennae on the development of glomeruli in the antennal lobes (ALs), the primary olfactory centers, was studied in the moth Manduca sexta. Normally during metamorphic adult development, the pheromone-specific macroglomerular complex (MGC) forms only in the ALs of males, whereas two lateral female-specific glomeruli (LFGs) develop exclusively in females. A female AL innervated by ORC axons from a grafted male antenna developed an MGC with three glomeruli, like the MGC of a normal male AL. Conversely, a male AL innervated by ORC axons from a grafted female antenna lacked the MGC but exhibited LFGs. ORC axons from grafted male antenna terminated in the MGC-specific target area, even in cases when the antennal nerve (AN) entered the AL via an abnormal route. Within ectopic neuromas formed by ANs that had become misrouted and failed to enter the brain, male-specific axons were not organized and formed terminal branches in many areas. The results suggest the presence of guidance cues within the AL for male-specific ORC axons. Depending on the sex of the antennal innervation, glial borders formed in a pattern characteristic of the MGC or LFGs. The sex-specific number of projection neurons (PNs) in the medial group of AL neurons remained unaffected by the antennal graft, but significant changes occurred in the organization of PN arborizations. In gynandromorphic females, LFG-specific PNs extended processes into the induced MGC, whereas in gynandromorphic males, PNs became restricted to the LFGs. The results indicate that male-and female-specific ORC axons play important roles in determining the position, anatomical features, and innervation of sexually dimorphic glomeruli.     

Temporally staggered glomerulus development in the moth Manduca sexta

Glomeruli, neuropilar structures composed of olfactory receptor neuron (ORN) axon terminals and central neuron dendrites, are a common feature of olfactory systems. Typically, ORN axons segregate into glomeruli based on odor specificity, making glomeruli the basic unit for initial processing of odorant information. Developmentally, glomeruli arise from protoglomeruli, loose clusters of ORN axons that gradually synapse onto dendrites. Previous work in the moth Manduca sexta demonstrated that protoglomeruli develop in a wave across the antennal lobe (AL) during stage 5 of the 18 stages of metamorphic adult development. However, ORN axons from the distal segments of the antenna arrive at the AL for several more days. We report that protoglomeruli present at stage 5 account for only approximately two or three of adult glomeruli with the number of structures increasing over subsequent stages. How do these later arriving axons incorporate into glomeruli? Examining the dendritic projections of a unique serotonin-containing neuron into glomeruli at later stages revealed glomeruli with immature dendritic arbors intermingled among more mature glomeruli. Labeling ORN axons that originate in proximal segments of the antenna suggested that early-arriving axons target a limited number of glomeruli. We conclude that AL glomeruli form over an extended time period, possibly as a result of ORNs expressing new odorant receptors arriving from distal antennal segments.     

Site on inhibition of the spino-bulbo-spinal reflex on stimulation of the anterior lobe of the cerebellum.

The effect of stimulation of the anterior lobe (AL) of the cerebellum on spinobulbo-spinal (SBS) reflex activity evoked by somatic and splanchnic afferentation was studied in chloralose-anaesthetized cats. Unit activity synchronous with the SBS component of the efferent discharge was observed at the level of motoneurons, descending axons of the dorsolateral and ventral funiculi and neurones of the reticular formation (RF). Conditioning stimulation of the AL inhibited this unit activity. Reticular formation units influenced from the AL had direct contact with segmental structures; these results showed that disappearance of the SBS reflex during AL conditioning is associated with the depressant effect of the cerebellar cortex on the reticular formation.     

Study of the effects of amyotrophic leukospongiosis causative agent on neuroglia cells in vitro

The influence of amyotrophic leukospongiosis (AL) causative agent on the ultrastructure of different types of cells of dissociated rat embryo brain and spinal cord cultures was studied. The AL agent belongs to the unconventional viruses (prions) and causes degenerative changes in the CNS. Large neurons and fibrous astrocytes were shown to be most sensitive. It was noted that the time of development and the degree of the dystrophic changes depend on the agent concentration. The destruction of cell membranes resulted in the pair neuron confluence. The formation of giant mitochondria with intramitochondrial inclusions was detected. It is supposed that the energetic apparatus of sensitive cells is primarily damaged by the infectious agent.     

The L1-CAM, Neuroglian, functions in glial cells for Drosophila antennal lobe development

Although considerable progress has been made in understanding the roles of olfactory receptor neurons (ORNs) and projection neurons (PNs) in Drosophila antennal lobe (AL) development, the roles of glia have remained largely mysterious. Here, we show that during Drosophila metamorphosis, a population of midline glial cells in the brain undergoes extensive cellular remodeling and is closely associated with the collateral branches of ORN axons. These glial cells are required for ORN axons to project across the midline and establish the contralateral wiring in the ALs. We find that Neuroglian (Nrg), the Drosophila homolog of the vertebrate cell adhesion molecule, L1, is expressed and functions in the midline glial cells to regulate their proper development. Loss of Nrg causes the disruption in glial morphology and the agenesis of the antennal commissural tract. Our genetic analysis further demonstrates that the functions of Nrg in the midline glia require its ankyrin-binding motif. We propose that Nrg is an important regulator of glial morphogenesis and axon guidance in AL development.     

Late EEG after-effects in humans following hyperventilation. II. A comparison between subjects with different level of adaptation to hypoxia

The late EEG after-effects following application of a short-lasting ventilatory interoceptive influence (3 min hyperventilation-HV) were studied in humans with three degrees of adaptation: students (ST) with a lower degree of training, professional alpine climbers with a high level of training (AL1) and the same subjects (AL2) in a middle position of adaptation i.e. 6 months after an expedition. ST developed late EEG after-effects, consisting mainly in an increase of the beta-2 EEG activity; AL1 showed very slight changes, while in AL2 the EEG after-effects were intermediate. It is suggested, that a lower level of adaptation facilitates the triggering through HV of processes in the cortical EEG which accompany an improvement of the brain tone.     

Dock and Pak regulate olfactory axon pathfinding in Drosophila

The convergence of olfactory axons expressing particular odorant receptor (Or) genes on spatially invariant glomeruli in the brain is one of the most dramatic examples of precise axon targeting in developmental neurobiology. The cellular and molecular mechanisms by which olfactory axons pathfind to their targets are poorly understood. We report here that the SH2/SH3 adapter Dock and the serine/threonine kinase Pak are necessary for the precise guidance of olfactory axons. Using antibody localization, mosaic analyses and cell-type specific rescue, we observed that Dock and Pak are expressed in olfactory axons and function autonomously in olfactory neurons to regulate the precise wiring of the olfactory map. Detailed analyses of the mutant phenotypes in whole mutants and in small multicellular clones indicate that Dock and Pak do not control olfactory neuron (ON) differentiation, but specifically regulate multiple aspects of axon trajectories to guide them to their cognate glomeruli. Structure/function studies show that Dock and Pak form a signaling pathway that mediates the response of olfactory axons to guidance cues in the developing antennal lobe (AL). Our findings therefore identify a central signaling module that is used by ONs to project to their cognate glomeruli.     

Innervation of the intestine in the bivalve mollusc Chione stutchburyi

Summary The innervation of the gut of the venerid bivalve mollusc, Chione stutchburyi, has been examined by fluorescence histochemistry, electron microscopy and autoradiography. Specific green and yellow varicose fluorescent fibres indicate the presence of dopaminergic and serotonergic axons, respectively. Three different types of axons can be distinguished by the morphological characteristics of their vesicles. Type I axons contain predominantly small granular vesicles (average diameter 65 nm), Type II axons possess large granular vesicles (average diameter 100 nm) and Type III axons contain large opaque vesicles (average diameter 150 nm). The granular vesicles in both Types I and II axons react positively to dichromate, and their granularity is reduced by reserpine indicating that they are monoaminergic. Only Type I axons accumulate tritiated dopamine and are selectively damaged by 6-hydroxydopamine. It is concluded that Type I axons are dopaminergic. Type II axons are serotonergic: they alone take up tritiated 5-hydroxytryptamine, and 5,7-dihydroxytryptamine selectively causes degenerative changes in these axons. Type III axons contain an unidentified neurotransmitter substance. The large opaque vesicles of these axons do not react to dichromate and are unaffected by reserpine, 6-hydroxydopamine or 5,7-dihydroxytryptamine.     

Osmometrically determined characteristics of the cell membrane of squid and lobster giant axons

Lobster and squid giant nerve fibers respond differently when subjected to osmotic challenges. The axons proper, as distinct from the total (fiber) complex formed by the axon and connective sheath, both behave as "fast" osmometers for changes in the concentration of NaCl, but the maximum degree of swelling in hyposmotic media is by about 60% in lobster and only by 20% in squid. The relative volume intercepts of the van't Hoff relation are about 0.2 for lobster and 0.4 for squid. The sheaths of both axons undergo only small, apparently passive changes in volume. Lobster axons are permeable to Cl, but squid axons are impermeable to this anion. Lobster axons are also permeable to glycerol. The implications of the data as to the nature of volume regulation of cells are discussed.     

In vitro and in vivo anti-allergic effects of Arctium lappa L

The discovery of drugs that can be used for the treatment of allergic disease is important in human health. Arctium lappa Linne (Compositae) (AL) has been used as a traditional medicine in Brazil and throughout Asia and is known to have an anti-inflammatory effect. In this study, the inhibitory effects of AL on degranulation and the release of mediators as well as on inhibition of cys-leukotriene biosynthesis by basophils were investigated. AL was selected out of 10,000 herbal extracts in a set-up for high throughput screening in which the degree of degranulation was monitored by the release of beta-hexosaminidase from rat basophil leukemia (RBL-2H3) cells. The AL extract significantly reduced degranulation and biosynthesis of cys-leukotrienes of human basophils in peripheral blood mono-nuclear cells (PBMCs) (50% inhibitory concentration [IC(50)] = 8.3 and 11.4 microg/ml, respectively). Viability and metabolic activity of the PBMCs were not affected. Although arctiin, the active component of AL that has been described in the literature, was not able to reduce degranulation in RBL-2H3 cells, a single high-performance liquid chromatography (HPLC) fraction from the AL extract inhibited beta-hexosaminidase release (IC(50) = 22.2 microg/ml). Topical administration of an aqueous extract of AL (5 mg/ear) on the ear of whey-sensitized mice 4 hrs before challenge with whey in the ear inhibited acute ear swelling by 50% in an in vivo cow's milk allergic model. The extract had no effect in this model when administered orally. In conclusion, the active component present in the active HPLC fraction of the AL extract was able to significantly reduce the release of inflammatory mediators through inhibition of degranulation and cys-leukotriene release in vitro. In addition, this active component was able to inhibit acute skin response in mice in vivo, indicating that AL is a very promising natural component for use in anti-allergic treatment.     

Central Mechanisms of Pheromone Information Processing

An advantage of using pheromones in olfactory studies is thatthey are chemical signals for which receptor neurons are evolvedand thus elicite biologically relevant odour-information tobe processed in the brain. In many vertebrate and insect species,the olfactory system is separated into a ‘main’and an ‘accessory’ division, the latter mediatingpheromone information. In moths, the pheromone information isfirst processed in the brain in a large and sexually dimorphicstructure, the macroglomerular complex (MGC) of the antennallobe (AL). Also in vertebrates the pheromone information isprocessed in specific or modified glomerular complexes. Oneprinciple question is whether individual olfactory glomeruliare functional units, processing specific information concerningboth the chemical quality and spatiotemporal features of thestimulus, like the pheromone plume. Indeed it has been shownthat the axons of different pheromone-selective receptor neuronsproject into different MGC-glomeruli. Intracellular recordingsfrom the AL projection (output) neurons also show that informationabout single components of the pheromone blend is preservedin some output pathways, whereas other output neurons respondin a unique fashion to the blend. The information about interspecificsignals, which interrupts pheromone attraction, is processedin a specific MGC-glomerulus and is to a large extent kept separatedfrom the pheromone information throughout the AL. Many of theoutput neurons accurately encode changes in the temporal characteristicsof the stimulus. Chem. Senses 21: 269–275, 1996.     

Proteins of fast axonal transport in the regenerating hypoglossal nerve of the rat

The composition of proteins conveyed by fast axonal transport in growing or regenerating axons is different from that of intact, mature axons. Consistent alterations have been observed in several different types of neurons, but adult peripheral axons (rabbit hypoglossal motoneurons) seemed to be exceptions because during their regeneration there was no increased labelling of a 23 kilodalton (kD) protein associated with the growth state. We examined the composition of fast-transported proteins, labelled by application of [35S]methionine to the hypoglossal nuclei, in intact and regenerating hypoglossal nerves of the rat. Using one- and two-dimensional electrophoresis we detected both increases and decreases in the labelling of specific polypeptides during regeneration. In particular, there was increased labelling of a 23 kD polypeptide. Changes were maximal 7 days after axotomy and subsided thereafter, coincident with reinnervation of the tongue. We conclude that hypoglossal axons show the same changes in transported protein composition which are characteristic of the growth state in other axons. Thus, we have strengthened the correlation between the growth state and changes in synthesis of a set of polypeptides of unknown function.     

Pulse-chase studies of the POMC/beta-endorphin system in the pituitary of acutely and chronically stressed rats

Experiments were carried out to determine whether stress induces biochemical changes in the pro-opiomelanocortin (POMC) system in anterior (AL) and intermediate-posterior lobe (IPL) of rat. In a series of pulse-chase experiments, acute stress led to an increase in POMC biosynthesis and shorter half-life in AL. However, when the animals were chronically stressed, the AL no longer exhibited increased POMC synthesis. On the other hand, in the IPL, acute stress did not produce any biochemical changes, but chronic stress led to an increase in POMC synthesis and shorter half-life. These data suggest that AL and IPL are affected by acute and/or chronic exposure to stress in opposite directions and that the POMC system in AL may play an important role in stress-induced analgesia.     

Interaction between geminivirus replication protein and the SUMO-conjugating enzyme is required for viral infection

Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells by using plant DNA polymerases. These viruses encode a protein designated AL1, Rep, or AC1 that is essential for viral replication. AL1 is an oligomeric protein that binds to double-stranded DNA, catalyzes the cleavage and ligation of single-stranded DNA, and induces the accumulation of host replication machinery. It also interacts with several host proteins, including the cell cycle regulator retinoblastoma-related protein (RBR), the DNA replication protein PCNA (proliferating cellular nuclear antigen), and the sumoylation enzyme that conjugates SUMO to target proteins (SUMO-conjugating enzyme [SCE1]). The SCE1-binding motif was mapped by deletion to a region encompassing AL1 amino acids 85 to 114. Alanine mutagenesis of lysine residues in the binding region either reduced or eliminated the interaction with SCE1, but no defects were observed for other AL1 functions, such as oligomerization, DNA binding, DNA cleavage, and interaction with AL3 or RBR. The lysine mutations reduced or abolished virus infectivity in plants and viral DNA accumulation in transient-replication assays, suggesting that the AL1-SCE1 interaction is required for viral DNA replication. Ectopic AL1 expression did not result in broad changes in the sumoylation pattern of plant cells, but specific changes were detected, indicating that AL1 modifies the sumoylation state of selected host proteins. These results established the importance of AL1-SCE1 interactions during geminivirus infection of plants and suggested that AL1 alters the sumoylation of selected host factors to create an environment suitable for viral infection.     

Factorization of the Michaelis functions.

Each Michaelis function that expresses the concentration of one of the species AL2, AL and A in terms of the concentration of free ligand (or its logarithm) is the product of two functions each of which represents the degree of ligation or de-ligation of a single site. These hypothetical sites have pK values of pK (SEE ARTICLE) where pK and alpha are defined by writing the two molecular pK values as pK +/- log2alpha. The factors are thus real if alpha larger than or equal to 1, i.e. if the binding of L by A is not positively co-operative. The dependence of [AL] on 1n[L] is compared with relations that represent other ligand-dependent equilibria.     

Objective Analyses of Tessellated Fundi and Significant Correlation between Degree of Tessellation and Choroidal Thickness in Healthy Eyes

A tessellated fundus is a common characteristic of myopic eyes and is an important clinical marker for the development of retinochoroidal changes. However, the exact cause and significance of tessellated fundi have not been definitively determined. We determined the degree of tessellation in fundi objectively in normal, non-pathological myopic eyes, and correlated the degree of tessellation and the choroidal thickness (CT) and axial length (AL). This was a prospective observational cross sectional study. The eyes were classified subjectively into three groups based on the degree of tessellation observed ophthalmoscopically. Digital color fundus photographs were assessed for the degree of tessellation by ImageJ, an image processing program. Three tessellated fundus indices (TFIs) were calculated and were compared to the three subjectively-determined groups. The subfoveal and nasal CTs were measured in the optical coherence tomographic images. The correlations between the TFIs and the CT were calculated. Additionally, the correlation between the TFIs and the AL was calculated. One hundred right eyes of 100 healthy volunteers (mean age 25.8±3.9 years) were studied. Ophthalmoscopically, 57 eyes were placed in the non-tessellated group, 27 eyes into the weakly tessellated group, and 16 eyes into the strongly tessellated group. There was a significant correlation between the subjective classifications and the TFI values (P<0.05, Kruskal-Wallis test). All of the TFIs were significantly associated with the subfoveal and nasal CT (R = −0.20 to −0.24, P<0.05). The TFIs were not significantly correlated with the ALs. In conclusion, the significant correlation between the subjective and objective classifications of the degree of tessellation indicates that TFIs can be used to classify the degree of tessellation. The results indicate that the differences in the CT account for the degree of tessellation.     

Multiple mitral leaflet contractile systems in the beating heart

Mitral valve closure may be aided by contraction of anterior leaflet (AL) cardiac myocytes located in the annular third of the leaflet. This contraction, observed as a stiffening of the annular region of the AL during isovolumic contraction (IVC), is abolished by beta-blockade (βB). Sub-threshold rapid pacing in the region of aorto-mitral continuity (STIM) also causes AL stiffening, although this increases the stiffness of the entire leaflet during both IVC and isovolumic relaxation (IVR). We investigated whether these contractile events share a common pathway or whether multiple AL contractile mechanisms may be present. Ten sheep had radiopaque-markers implanted: 13 silhouetting the LV, 16 on the mitral annulus, an array of 16 on the AL, and one on each papillary muscle tip. 4-D marker coordinates were obtained from biplane videofluoroscopy during control (C), βB (esmolol) and during βB+STIM. Circumferential and radial stiffness values for three AL regions (Annular, Belly, and free-Edge), were obtained from inverse finite element analysis of AL displacements in response to trans-leaflet pressure changes during IVC and IVR. βB+STIM increased stiffness values in all regions at both IVC and IVR by 35 ± 7% relative to βB (p<0.001). Thus, even when AL myocyte contraction was blocked by βB, STIM stiffened all regions of the AL during both IVC and IVR. This demonstrates the presence of at least two contractile systems in the AL; one being the AL annular cardiac muscle, involving a β-dependent pathway, others via a β-independent pathway, likely involving valvular interstitial cells and/or AL smooth muscle cells.     

GABA-mediated synaptic inhibition of projection neurons in the antennal lobes of the sphinx moth,Manduca sexta

Responses of neurons in the antennal lobe (AL) of the moth Manduca sexta to stimulation of the ipsilateral antenna by odors consist of excitatory and inhibitory synaptic potentials. Stimulation of primary afferent fibers by electrical shock of the antennal nerve causes a characteristic IPSP-EPSP synaptic response in AL projection neurons. The IPSP in projection neurons reverses below the resting potential, is sensitive to changes in external and internal chloride concentration, and thus is apparently mediated by an increase in chloride conductance. The IPSP is reversibly blocked by 100 microM picrotoxin or bicuculline. Many AL neurons respond to application of GABA with a strong hyperpolarization and an inhibition of spontaneous spiking activity. GABA responses are associated with an increase in neuronal input conductance and a reversal potential below the resting potential. Application of GABA blocks inhibitory synaptic inputs and reduces or blocks excitatory inputs. EPSPs can be protected from depression by application of GABA. Muscimol, a GABA analog that mimics GABA responses at GABAA receptors but not at GABAB receptors in the vertebrate CNS, inhibits many AL neurons in the moth.     

The role of visual experience in the formation of binocular projections in frogs

Many parts of the visual system contain topographic maps of the visual field. In such structures, the binocular portion of the visual field is generally represented by overlapping, matching projections relayed from the two eyes. One of the developmental factors which helps to bring the maps from the two eyes into register is visual input. The role of visual input is especially dramatic in the frog, Xenopus laevis. In tadpoles of this species, the eyes initially face laterally and have essentially no binocular overlap. At metamorphosis, the eyes begin to move rostrodorsally; eventually, their visual fields have a 170 degree region of binocular overlap. Despite this major change in binocular overlap, the maps from the ipsilateral and contralateral eyes to the optic tectum normally remain in register throughout development. This coordination of the two projections is disrupted by visual deprivation. In dark-reared Xenopus, the contralateral projection is nearly normal but the ipsilateral map is highly disorganized. The impact of visual input on the ipsilateral map also is shown by the effect of early rotation of one eye. Examination of the tectal lobe contralateral to the rotated eye reveals that both the contralateral and the ipsilateral maps to that tectum are rotated, even though the ipsilateral map originates from the normal eye. Thus, the ipsilateral map has changed orientation to remain in register with the contralateral map. Similarly, the two maps on the other tectal lobe are in register; in this case, both projections are normally oriented even though the ipsilateral map is from the rotated eye. The discovery that the ipsilateral eye's map reaches the tectum indirectly, via a relay in the nucleus isthmi, has made it possible to study the anatomical changes underlying visually dependent plasticity. Retrograde and anterograde tracing with horseradish peroxidase have shown that eye rotation causes isthmotectal axons to follow abnormal trajectories. An axon's route first goes toward the tectal site where it normally would arborize but then changes direction to reach a new tectal site. Such rearrangements bring the isthmotectal axons into proximity with retinotectal axons which have the same receptive fields. Anterograde horseradish peroxidase filling has also been used to study the trajectories and arborizations of developing isthmotectal axons. The results show that the axons enter the tectum before the onset of eye migration but do not begin to branch profusely until eye movement begins to create a zone of binocular space.(ABSTRACT TRUNCATED AT 400 WORDS)