Dynamic properties of axonal transport of proteins and glycoproteins: A study based on the effects of metaphase blocking drugs in the developing optic pathway of chick embryos

Some properties of the axonal transport of proteins and glycoproteins along the optic pathway of chick embryos and newly hatched chicks were studied by labelling retinal ganglion cells with 3H-proline or 3H-fucose. A study of the effects of colchicine (COL) and vinblastine (VLB) on embryonic axonal transport was also carried out. Marked changes in the efficiency of axonal transport were found throughout development. In particular, the fraction of retinal ganglion cell proteins which is rapidly exported toward tectal terminals increases during embryonic life but steadily decreases after hatching. Glycoprotein transport behaves similarly except that its efficiency is relatively higher at stages when critical events of synaptic maturation in the tectum are reported to occur. Embryonic axonal transport is blocked by COL and VLB at very low intravitreal concentrations. Retinal protein synthesis and the morphology of ganglion cells are profoundly altered by the drugs: in general, COL and VLB effects were much more marked in embryonic than in mature neurons. An analysis of the time course of rapid transport along embryonic optic axons was carried out by reducing the efflux of labelled proteins from the eye by giving VLB intravitreally 2 h after the pulse. It revealed some peculiar features in the retino-tectal migration of glycoproteins and confirmed their progressive accumulation within terminals as previously described by radioautography. These results suggest that axonal transport of proteins during embryonic life undergoes changes in parallel with synaptic maturation. It may thus be considered as one of the factors controlling the genesis of neuronal networks.     

Essential role of somatic and synaptic protein synthesis and axonal transport in long-term synapse-specific facilitation at distal sensorimotor connections in Aplysia

To investigate further the cellular mechanisms underlying long-term facilitation (LTF) and long-term synapse-specific facilitation (LTSSF), we studied the role of axonal transport and somatic and synaptic protein synthesis at proximal and distal synapses of Aplysia siphon sensory neurons (SNs). The long soma-synapse distances (2.5 to 3 cm) of the SN distal synapses impose important temporal and mechanistic constraints on long-term facilitation and on intracellular signaling. Excitatory postsynaptic potentials (EPSPs) evoked by SNs in central and peripheral siphon motor neurons were used to assay LTF 24-30 h after various pharmacological treatments. Inhibition of protein synthesis via anisomycin application at either the SN soma or distal synapses blocked the induction of LTF and LTSSF normally produced by synaptic application of the facilitating transmitter serotonin (5-hydroxytryptamine). Further, disruption of axonal transport by application of nocodazole to the isolated siphon nerve completely blocked LTF at distal synapses. These results indicate an essential role for somatic and synaptic protein synthesis and active axonal transport in LTSSF at distal synapses, and raise intriguing questions for current synaptic marking/capture models of synapse specificity and LTF.     

Effects of Colchicine Application to Preganglionic Axons on Choline Acetyltransferase Activity and Acetylcholine Content and Release in the Superior Cervical Ganglion

Abstract: These experiments investigate the effect of block, by colchicine, of fast axonal transport in the cat's cervical sympathetic trunk (CST) on the superior cervical ganglion's choline acetyltransferase (ChAT) enzyme activity, acetylcholine (ACh) content, and ACh release. Electron microscopy on the segment of the CST exposed to colchicine 1 or 4 days earlier showed disappearance of microtubules and accumulation of vesicles and smooth membrane tubules but no disruption of the axonal cytomatrix. At 4 days following colchicine treatment, the number and size of synaptic boutons per grid square in the ganglion ipsilateral to the colchicine-treated CST were similar to those in the control ganglion. At 2 and 4 days following exposure of the CST to colchicine, ChAT activity in the ipsilateral ganglion was reduced to 76 ± 8 and 54 ± 6% of control values, respectively. ACh stores in the ganglia were also reduced (to 81 ± 6% of control values at 2 days and to 51 ± 5% of control values at 4 days). Ganglionic transmission and its sensitivity to blockade by hexamethonium during 2-Hz CST stimulation were not impaired at day 4 postcolchicine. ACh release evoked by 2-Hz stimulation of colchicine-treated axons was similar to release from untreated axons, despite the decrease in the ganglionic ACh content. In contrast, ACh release evoked by 20-Hz stimulation was depressed. The amount of ACh released during 5-Hz stimulation in the presence of vesamicol by the terminals of colchicine-treated axons was similar to that released by the terminals of untreated axons. These results suggest the following conclusions: (a) Colchicine-sensitive fast axonal transport contributes significantly to maintaining ChAT stores in preganglionic axon terminals. (b) The half-life of ChAT in sympathetic preganglionic terminals is ～4 days. (c) One consequence of colchicine-induced block of axonal transport is a reduced ACh content of preganglionic nerve terminals. (d) This decrease in ACh content appears to be the result of a loss in a reserve transmitter pool, whereas the size of the readily releasable compartment is maintained.     

Patterned synaptic drive to locust flight motoneurons after hemisection of thoracic ganglia

Intracellular recordings were carried out on locust flight motoneurons after hemisection of individual thoracic ganglia. With the exception of minimal surgical manipulations, the animals were intact and able to perform tethered flight. Analysis of the synaptic drive recorded in the motoneurons during flight motor activity revealed the extent to which ganglion hemisection influenced the premotor rhythm generating network.

Effect of colchicine on axonal transport and morphology of retinal ganglion cells

Summary Intraocular injection of colchicine in doses which do not affect the protein synthesis in the retina has profound effects on the axonal transport of protein in the retinal ganglion cells of the rabbit. Rapid axonal transport in these cells is completely inhibited after treatment with relatively low amounts of colchicine. In contrast to this, a certain fraction of the slow axonal transport is resistant to colchicine treatment. Colchicine in doses which completely inhibits fast axonal transport caused discrete morphological changes in the perikaryon and in the axon of the retinal ganglion cell. No disappearance of microtubules and no general proliferation of neurofilaments was observed in the perikaryon of the retinal ganglion cells. There was a slight or moderate increase in the number of filaments in the intra-retinal part of the axons of the retinal ganglion cells.This work has been supported by grants from the Swedish Medical Research Council (B71-12X-2543-03, B71-13X-2226-05A) and the Swedish National Cancer Society (265-B70-02X).     

Parasitoid wasp sting: a cocktail of GABA, taurine, and beta-alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host

The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal-giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI-TOF-MS revealed high levels of GABA (25 mM), and its receptor agonists beta-alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal-giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin-sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host.     

Embryology of the thoracic and abdominal ganglia of the large milkweed bug, Oncopeltus fasciatus (Dallas), (Hemiptera, Lygaeidae)

In this study, the condensation of the three thoracic and 11 abdominal segmental ganglia to form a prothoracic and central nerve mass during embryogenesis is described. During katatrepsis, many changes occur in the organization of these ganglia; this study suggests that some of these changes are caused by mechanical forces acting on the ventral nerve cord at this time. The ventral nerve cord begins its anterior migration and coalescence ten hours after katatrepsis and is completed 63 hours later. The central ganglion is made up of the meso- and metathoracic ganglia and seven abdominal ganglia. Intrasegmental median cord nuclei are shown to form glial elements in the median sagittal plane of the neuropile and in the longitudinal connectives. Intersegmental median cord neuroblasts migrate into the posterior gangliomeres but, apparently, degenerate soon after katatrepsis. Lateral cord cells bordering on the neuropile form a glial investment that surrounds this fiber tract region. Peripheral lateral cord cells are shown to form the cells of the outer ganglionic sheath, the perineurium.     

Formation of postural asymmetry in rat hindlimbs during colchicine-induced unilateral blockade of axonal transport in cortico-lumbar projections

The possibility of the formation of spinal cord functional asymmetry by the blockade of axonal transport in corticolumbar projections with colchicine was investigated. To identify the blockade of axonal transport, the method of retrograde transport of horseradish peroxidase was used. The blockade of axonal transport led to the formation of the asymmetric functional status of the spinal cord, manifesting in postural asymmetry of the hind limbs and characteristic changes in the pattern of bioelectrical activity of the flexor muscles. An endogenous factor inducing postural asymmetry in intact recipients was detected in the cerebrospinal fluid of colchicine treated animals. Based on the experimental data the conclusion is drawn that interruption of normal axonal transport attests to the destruction of central neurons.     

Projection areas and branching patterns of the tympanal receptor cells in migratory locusts,Locusta migratoria and Schistocerca gregaria

Summary In Locusta migratoria and Schistocerca gregaria, the projection areas and branching patterns of the tympanal receptor cells in the thoracic ganglia were revealed. Four auditory neuropiles can be distinguished on each side of the ventral cord, always located in the anterior part of the ring tract in each neuromere (two in the meta-, one in the meso-, and one in the prothoracic ganglion). Some of the receptor fibres ascend to the suboesophageal ganglion. There are distinct subdivisions within the auditory, frontal metathoracic and mesothoracic neuropiles. The arrangement of the terminal arborisations of the four types of tympanal receptor cells according to their different frequency-intensity responses is somatotopic and similar in the two ganglia. Here the receptor cells of type-1 form a restricted lateroventral arborisation. Cells of type-4 occupy the caudal part with a dorsorostral extension. Cells of type-2 and -3 arborise in a subdivision between both. Most of the stained low-frequency receptors (type-1, -2, and -3) terminate either in the metathoracic or, predominantly, in the mesothoracic ganglion. In contrast, the high-frequency cells (type-4) ascend to the prothoracic ganglion. The receptor fibres of the different types of receptor cells differ in diameter.Abbreviations aRT anterior part of the ring tract - cf characteristic frequency - MVT median ventral tract - SEG suboesophageal ganglion - SMC supramedian commissure - VMT ventral median tract - VIT ventral intermediate tract Supported by the Deutsche Forschungsgemeinschaft; part of program A7 in Sonderforschungsbereich 305 (Ecophysiology)     

Synaptic plasticity in a regenerated crayfish phasic motoneuron.

Crustacean neuromuscular systems provide many advantages for the study of synaptic transmission and plasticity. The present study examines aspects of synaptic transmission in the phasic, fast closer excitor (FCE) motoneuron of regenerated crayfish claws. Excitatory postsynaptic potentials (EPSPs) fatigued rapidly and showed poor long-term facilitation (LTF) in the smallest of regenerating claws. EPSPs in larger regenerating claws fatigued less and showed pronounced facilitation. These observations were not the same as those previously made during primary development of this motoneuron (Lnenicka and Atwood, 1985a, J. Neuroscience 5:459-467). Hence, regeneration is not the recapitulation of primary development. In situ stimulation of the FCE is known to lead to long-lasting adaptation of synaptic performance. This adaptation is age dependent; it is expressed in young but not old animals. In the regenerated FCE of old animals, we observed a novel form of long-lasting adaptation to imposed activity: EPSPs showed large initial EPSPs and did not exhibit resistance to fatigue during maintained stimulation. This indicates that aged motoneurons can express adaptive changes to increased activity following axonal regeneration, but that the adaptive changes are the opposite to what is observed in nonregenerated motoneurons.     

Synaptic plasticity in a regenerated crayfish phasic motoneuron

Crustacean neuromuscular systems provide many advantages for the study of synaptic transmission and plasticity. The present study examines aspects of synaptic transmission in the phasic, fast closer excitor (FCE) motoneuron of regenerated crayfish claws. Excitatory postsynaptic potentials (EPSPs) fatigued rapidly and showed poor long-term facilitation (LTF) in the smallest of regenerating claws. EPSPs in larger regenerating claws fatigued less and showed pronounced facilitation. These observations were not the same as those previously made during primary development of this motoneuron (Lnenicka and Atwood, 1985a, J. Neuroscience 5:459–467). Hence, regeneration is not the recapitulation of primary development. In situ stimulation of the FCE is known to lead to long-lasting adaptation of synaptic performance. This adaptation is age dependent; it is expressed in young but not old animals. In the regenerated FCE of old animals, we observed a novel form of long-lasting adaptation to imposed activity: EPSPs showed large initial EPSPs and did not exhibit resistance to fatigue during maintained stimulation. This indicates that aged motoneurons can express adaptive changes to increased activity following axonal regeneration, but that the adaptive changes are the opposite to what is observed in nonregenerated motoneurons. © 1992 John Wiley & Sons, Inc.     

Parasitoid wasp sting: A cocktail of GABA,taurine, and β‐alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host

The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal‐giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI‐TOF‐MS revealed high levels of GABA (25 mM), and its receptor agonists β‐alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal‐giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin‐sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host. © 2006 Wiley Periodicals, Inc. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Modulation of syllable period‐selective phonotaxis by prothoracic neurones in crickets (Acheta domesticus): juvenile hormone,picrotoxin and photoinactivation of the ON1 neurones

Abstract In response to model calling songs (CSs), the phonotaxis of female Acheta domesticus ranges from being very selective to unselective. Within 15 min of nanoinjecting juvenile hormone III (JHIII) or picrotoxin (PTX) into the prothoracic ganglion, females become more selective for syllable period (SP) than in pre‐tests. Controls for JHIII experiments, including nanoinjection of acetone into the prothoracic ganglion or nanoinjection of JHIII into the metathoracic ganglion, do not influence selectivity. Similarly, nanoinjection of saline into the prothoracic ganglion and nanoinjection of PTX outside of the prothoracic ganglion does not change the overall selectivity of the female’s phonotaxis. These results indicate that circuits in the prothoracic ganglion modulate the SP‐selectivity of phonotaxis. Photoinactivating both of the ON1 prothoracic auditory interneurones in old females that were previously unselective for SP also results in greater SP‐selectivity during phonotaxis. Evidence suggesting that ON1 has this effect via its inhibitory input to L3 (another prothoracic auditory neurone) includes: photoinactivation of one ON1 neurone causes angular errors in the female’s orientation to CSs at 85 dB (above the threshold of the L3), stimulation with 60 dB CSs (above the threshold of ON1 but below the threshold of L3) does not induce errors in angular orientation, inactivation of ON1 in old crickets results in greater angular errors (85 dB stimulus) than it does when ON1 is inactivated in young females, and photoinactivation of ON1 increases the firing rate of the L3 neurone.     

Rapid introduction of long-lasting synaptic changes at crustacean neuromuscular junctions

In this review we present recent evidence implicating second-messenger systems in two forms of long-lasting synaptic change seen at crustacean neuromuscular junctions. Crustacean motor axons are endowed with numerous terminals, each possessing many individual synapses. Some synapses appear to be quiescent or impotent, but can be recruited in response to imposed functional demands. Supernormal impulse activity leads to long-term facilitation (LTF) which persists for many hours. During the persistent phase, additional synapses are physiologically effective, and morphological changes in synapses are seen at the ultrastructural level. Pulsatile application of serotonin, a neuromodulator, also enhances synaptic transmission, but this enhancement declines more rapidly than LTF. Elevation of intraterminal Ca2+ is neither necessary nor sufficient for long-lasting enhancement of transmission, but activation of A-kinase is necessary. LTF is set in motion by an unknown depolarization-dependent mechanism leading to A-kinase activation, whereas serotonin facilitation depends for its initiation on the phosphatidylinositol system. The initial phase of serotonin facilitation may be accounted for by production of inositol triphosphate, whereas the secondary long-lasting phase appears to require participation of both C kinase and A kinase. Neither LTF nor serotonin facilitation requires an intact neuron; both are presynaptic phenomena expressed by the nerve terminals. Brief comparison is made with long-lasting synaptic changes in other systems.     

The actions of phenol and pentachlorophenol (PCP) on axonal conduction, ganglionic synaptic transmission, and the effect of pH changes

1. A comparison of phenol, pentachlorophenol (PCP) and procaine effects on axonal conduction were studied in vitro in the sciatic nerves of toad. PCP and procaine were respectively 6.3 and 3.15 times more potent than phenol in blocking axonal conduction. 2. Effects of PCP on synaptic transmission were studied in vitro in the eighth sympathetic ganglion of toad. 3. Axonal conduction block and synaptic transmission block by phenol was reversible, but not that by PCP. 4. When the PCP ionization was increased, a lesser per cent reached the site of action, reducing its capacity to block the axonal conduction and ganglionic transmission. 5. PCP plus, 3,4-Diaminopyridine (3,4-DAP) decreased synaptic transmission block from post-ganglionic compound action potential (CAP) responses to supramaximal preganglionic stimulation.     

The effects of injecting Juvenile Hormone III into the prothoracic ganglion on phonotaxis by female crickets Gryllus bimaculatus

Nanoinjection of Juvenile Hormone III (JH III) into the prothoracic ganglion causes virgin female crickets Gryllus bimaculatus De Geer to become more phonotactically selective for the syllable periods (SPs) of model calling songs. Females responding to all, or almost all, of the SPs presented before JH III injection significantly narrow their responses to a range of SPs that is usually centered on the SPs included in the conspecific males' calling song. Control injections of acetone (i.e. the solvent for JH III) into the prothoracic ganglion do not significantly change the recipient females' phonotactic responses. Injection of JH III into the metathoracic ganglion also has no effect the females' phonotactic choices.     

Axonal transport: a quantitative study of retained and transported protein fraction in the cat

The fast axonal transport of proteins was studied in the cat sciatic nerve after injection of [3H]leucine into the spinal ganglion or the ventral horn of the seventh lumbar segment. The amount of transported proteins after ganglion injection was linearly related to the amount of label present at the ganglion. At variable intervals after ganglion or spinal cord injection, the sciatic nerves were sectioned in some experiments. The transport of proteins continued in the peripheral nerve stump in a wavelike manner, but the advancing wave leaves a labeled trail behind. A fraction of this trail corresponds to proteins moving at slower velocities than the velocity of proteins in the wave front. Another fraction of the trail corresponds to molecules retained by the axons. Each nerve segment of 5 mm in length retains 1.5% of the transported proteins, and the profile of retained proteins along the sciatic nerves follows a single exponential function. From the proportion of retained proteins, the concentration of transported proteins at the terminals of branching axons as a function of the branching ratio was estimated. In the case of motor axons innervating the soleus muscle of the cat, the concentration of recently transported proteins at the nerve terminals would be approximately 0.83% of the proteins leaving the spinal cord. This low concentration of transported proteins at the nerve terminals may explain the lability of neuromuscular synapses when axonal transport is decreased or interrupted.     

Mass spectrometric analysis of FMRFamide-like immunoreactive neurons in the prothoracic and subesophageal ganglion of Periplaneta americana

MALDI-TOF mass spectrometry combined with immunocytochemistry and retrograde labeling, was used to study the expression pattern and morphology of Pea-FMRFamide-related peptides in single neurons of the prothoracic ganglion and the subesophageal ganglion (SEG) of the American cockroach Periplaneta americana. In contrast to the postero-lateral cells (PLCs) of the meta- and mesothoracic ganglion, the prothoracic FMRFamide-related peptides expressing neurons not only extend in the posterior median nerve but also in an anterior median nerve, which is described herein. The peptidome of the prothoracic PLCs is identical with that of the meso- and metathoracic neurons, respectively. In this study, we identified a truncated form of Pea-FMRFa-24 which was found to be more abundant than the peptide originally designated as Pea-FMRF-24. FMRFamide-related peptides expressing postero-lateral cells were also detected in the labial neuromere of the SEG. Although their projection could not be solved, mass spectrometric analyses revealed the same peptide complement in these neurons as found in the thoracic postero-lateral cells. In all neurons which we studied no co-localized peptides of other peptide families were observed.     

Embryonic development of the central projection of auditory afferents (Schistocerca gregaria, Orthoptera, Insecta)

The auditory system of Schistocerca gregaria is a well investigated sensory network in the adult grasshopper. Here we present a first study on the embryonic development of this neuronal network. Focussing on the auditory receptor cells we show that they differentiate axonal processes at around 45% of embryonic development. These axons fasciculate with the intersegmental nerve and enter the central nervous system by 45-50% of development. First collaterals sprout into the major arborization area, the frontal auditory projection area of the metathoracic ganglion by 60%. This projection increases in density until an adult-like morphology is established by 90% of development. Furthermore, by the end of embryogenesis all three types of receptor fiber projections can be distinguished. This development is independent of a hearing ability, which develops much later during postembryonic life. The auditory projection co-develops with the fusion of neuromeres to the metathoracic ganglion, the formation of the target neuropile areas and the expression of the synapse associated molecule synapsin. Fasciclin I and Lachesin, both potential axon-guidance molecules, are expressed strongly on both, peripheral and central auditory pathways and, although much weaker, within the synaptic target area.     

The effect of colchicine on the transport of axonal protein in the chicken

1. Small doses (1-10mug) of colchicine injected into the ventral horn of the spinal cord of the chicken caused paralysis in the legs. 2. Colchicine had no effect on the incorporation of leucine into proteins of the spinal cord but markedly decreased the total amount of protein flowing into the axons of the sciatic nerve. 3. This axonal flow of protein proceeded at two rates: a high rate (300mm/day) and a low rate (2mm/day). Although both groups of proteins were affected, the slow transport of protein was more profoundly blocked by colchicine. 4. The results suggest that axonal flow is dependent on the neurotubular system in the axon.