Cell-to-cell transfer of glial proteins to the squid giant axon: The glia- neuron protein transfer hypothesis

The hypothesis that glial cells synthesize proteins which are transferred to adjacent neurons was evaluated in the giant fiber of the squid (Loligo pealei). When giant fibers are separated from their neuron cell bodies and incubated in the presence of radioactive amino acids, labeled proteins appear in the glial cells and axoplasm. Labeled axonal proteins were detected by three methods: extrusion of the axoplasm from the giant fiber, autoradiography, and perfusion of the giant fiber. This protein synthesis is completely inhibited by puromycin but is not affected by chloramphenicol. The following evidence indicates that the labeled axonal proteins are not synthesized within the axon itself. (a) The axon does not contain a significant amount of ribosomes or ribosomal RNA. (b) Isolated axoplasm did not incorporate [(3)H]leucine into proteins. (c) Injection of Rnase into the giant axon did not reduce the appearance of newly synthesized proteins in the axoplasm of the giant fiber. These findings, coupled with other evidence, have led us to conclude that the adaxonal glial cells synthesize a class of proteins which are transferred to the giant axon. Analysis of the kinetics of this phenomenon indicates that some proteins are transferred to the axon within minutes of their synthesis in the glial cells. One or more of the steps in the transfer process appear to involve Ca++, since replacement of extracellular Ca++ by either Mg++ or Co++ significantly reduces the appearance of labeled proteins in the axon. A substantial fraction of newly synthesized glial proteins, possibly as much as 40 percent, are transferred to the giant axon. These proteins are heterogeneous and range in size from 12,000 to greater than 200,000 daltons. Comparisons of the amount of amino acid incorporation in glia cells and neuron cell bodies raise the possibility that the adaxonal glial cells may provide an important source of axonal proteins which is supplemental to that provided by axonal transport from the cell body. These findings are discussed with reference to a possible trophic effect of glia on neurons and metabolic cooperation between adaxonal glia and the axon.     

The fine structure of the nerve cord of Myxicola infundibulum (annelida,polychaeta)

Summary Ultrastructural observations of the giant axon of Myxicola infundibulum reveal that the axoplasm contains neurofilaments, a few neurotubules and mitochondria. Finger-like projections issuing from the glial cells of the sheath encircle the giant axon at various angles. The space between the axolemma and sheath is 125 Å. Branches of the giant axon are also surrounded by a glial sheath as they course through the neuropil. Some branches of the giant axon seem to fuse with certain neurons, creating a syncytial arrangement between the giant axon and these neurons.Many small nerve fibers course longitudinally in the neuropil of the nerve cord. Most of these axons are separated from each other by a space of 200 Å without intervening glial processes. Synapses in the neuropil have both clear 600 Å vesicles and larger dense core vesicles suggesting chemical transmission. Some, but not all, of the synaptic areas show thickened membranes and dense material in the synaptic cleft.This study was supported in part by PHS NS-07740 to R.L.P., J.A.B. is a NDEA Predoctoral Fellow in the Department of Physiology.     

Distribution of Acid Protease Activity in the Squid Nervous System

Abstract: Acid protease activity was measured in homogenized stellate ganglion, axoplasm extruded from the squid giant axon, homogenized fin nerves, and in lysed synaptosomes prepared from the optic lobe of the squid. At least two different acid protease classes were distinguished on the bases of their inhibitor profiles. Acid protease activity was present in each of the above tissues except extruded axoplasm. This result suggests that the acid protease activity found in our homogenized finnerves might be located not within the axons but rather in glial cells or extracellular tissue. The absence of acid protease activity in extruded axoplasm indicates that acid proteases are unlikely to play a significant role in the catabolism of intracellular proteins along the length of the axon.     

Transfer of molecules from glia to axon in the squid may be mediated by glial vesicles

Although the transfer of glial proteins into the squid giant axon is well documented, the mechanism of the transfer remains unknown. We examined the possibility that the transfer involved membrane-bound vesicles, by taking advantage of the fact that the fluorescent compound, 3,6-acridinediamine, N,N,N,',N'-tetramethylmonohydride [acridine orange (AO)], rapidly and selectively stains vesicular structures in glial cells surrounding the giant axon. We labeled cleaned axons (1–3 cm long) by incubation for 1 min in filtered seawater (FSW) containing AO. Because the AO was concentrated in glial vesicular organelles, these fluoresced bright orange when the axon was examined by epifluorescence microscopy. To look for vesicle transfer, axoplasm was extruded from such AO-treated axons at various times after labeling. During the initial 15 min, an increasing number of fluorescent vesicles were observed. No further increases were observed between 15 and 60 min post AO. The transfer of the fluorescent vesicles into the axoplasm seemed to be energy dependent, as it was inhibited in axons treated with 2 mM KCN. These results suggest that a special mode of exchange exists between the adaxonal glia and the axon, perhaps involving phagocytosis by the axon of small portions of the glial cells.     

A light and electron microscopic study of betaine/GABA transporter distribution in the monkey cerebral neocortex and hippocampus

The present study aimed to elucidate the distribution of betaine/γ-aminobutyric acid (GABA) transporter-1 (BGT-1) in the normal monkey cerebral neocortex and hippocampus by immunoperoxidase and Immunogold labelling. BGT-1 was observed in pyramidal neurons in the cerebral neocortex and the CA fields of the hippocampus. Large numbers of small diameter dendrites or dendritic spines were observed in the neuropil. These made asymmetrical synaptic contacts with unlabelled axon terminals containing small round vesicles, characteristic of glutamatergic terminals. BGT-1 label was observed in an extra-perisynaptic region, away from the post-synaptic density. Immunoreactivity was not observed in portions of dendrites that formed symmetrical synapses, axon terminals, or glial cells. The distribution of BGT-1 on dendritic spines, rather than at GABAergic axon terminals, suggests that the transporter is unlikely to play a major role in terminating the action of GABA at a synapse. Instead, the osmolyte betaine is more likely to be the physiological substrate of BGT-1 in the brain, and the presence of the transporter in pyramidal neurons suggests that these neurons utilize betaine to maintain osmolarity.     

Ultrastructural changes in the gracile nucleus of the spontaneously diabetic BB rat.

The present study describes the structural changes in the gracile nucleus of the spontaneously diabetic BB rat. At 3-7 days post-diabetes, axons, axon terminals and dendrites showed electron-dense degeneration. Degenerating axons were characterized by swollen mitochondria, vacuolation, accumulation of glycogen granules, tubulovesicular elements, neurofilaments and dense lamellar bodies. Degenerating axon terminals consisted of an electron-dense cytoplasm containing swollen mitochondria, vacuoles and clustering of synaptic vesicles. These axon terminals made synaptic contacts with cell somata, dendrites and other axon terminals. Degenerating dendrites were postsynaptic to normal as well as degenerating axon terminals. At 1-3 months post-diabetes, degenerating electron-dense axons, axon terminals and dendrites were widely scattered in the neuropil. Macrophages containing degenerating electron-dense debris were also present. At 6 months post-diabetes, the freshly degenerating neuronal elements encountered were similar to those observed at 3-7 days. However, there were more degenerating profiles at 6 months post-diabetes compared to the earlier time intervals. Terminally degenerating axons were vacuolated and their axoplasm appeared amorphous. It is concluded that degenerative changes occur in the gracile nucleus of the spontaneously diabetic BB rat.     

Neurofilamentous network and filamentous matrix preserved and isolated by different techniques from squid giant axon

The contribution of the neurofilamentous network to the structure of the squid giant axon was analyzed electron-microscopically. Axial 10-nm filaments cross-linked by radial 5-nm bridges form a network that is present in preparations prepared by a variety of techniques. The axoplasm is differentiated into dense and less dense regions. In the presence of Co(II) ions, the neurofilamentous network was remarkably well preserved and appeared to be associated with a dense web of fine filament matrix, which also was identified in extracted axoplasm and in fractions enriched with neurofilament protein complex. In the presence of La(III) ions, the neurofilamentous network had a coarse and open appearance. The stereo images of extracted and critical-point dried axoplasm suggested that the neurofilamentous network contains ordered lattice-like regions. Extracted preparations of extruded axoplasm and fractions enriched with neurofilament protein complex suggested that the properties of the network are determined by the neurofilament protein complex. It is proposed that the neurofilamentous network is the essential determinant of the form of the axon, and that the order within the network is determined by the radial components of the network. The structures observed in the different preparations are not artifacts, but rather are related closely to their native state in the axon.     

THE SYNTHESIS OF CHOLINE PHOSPHOGLYCERIDES IN THE GIANT FIBRE SYSTEM OF THE SQUID

The mechanisms and pathways of synthesis of phosphatidylcholine in the giant fibre system of the squid (Loligo vulgaris) have been examined by incubating the stellate ganglion-nerve preparation or its separated compartments in an artificial bathing solution with labelled choline. Other experiments were done by dissecting the whole stellate ganglion into axoplasm, axon sheath, giant fibre lobe, small fibres and ganglion residue, after incubation. The initial rate of choline incorporation into choline phosphoglycerides was severalfold higher in the lobe than in the axon. Higher lipid radioactivity was recovered in the axon sheath as compared to the axoplasm, and in the small fibres as compared to the ganglion residue which contains its cell bodies. The production of phosphorylcholine and CDP-choline in the intact ganglion-nerve preparation during incubation with choline points to the occurrence of the net synthesis pathway for phosphatidylcholine in this material. Base-exchange activity was also observed in the axon and giant fibre lobe preparations in vitro, but no indication can yet be given whether it also takes place in intact preparations. Electrical stimulation and‘depolarizing’conditions enhance choline phosphorylation in the squid axon and lobe, but decrease phosphatidylcholine labelling.     

Ultrastructural studies on neuromuscular contacts and the formation of junctions in the flight muscle of Antheraea polyphemus (Lep.)

In the moth Antheraea polyphemed at the onset of adult development. The subsequent breakdown of the isolated motor stulongated vesicles similar in structure to channels of smooth ER, appear in large numbers in the axoplasm. Their nature as well as the functional aspects of early axonal changes are discussed. From the 7th day onward two types of axonal breakdown become prominent. The first is characterized 0y swelling axon profiles, distorted vesicles and strongly shrunken mitochondria, uhile shrinking axon profiles containing tightly packed mitochondria and unaltered vesicles are typical of the second. Both types presumably take place independently of each other in different axon terminals. Axons and the contents of at least the first type are finally removed by transformation into lamellar bodies. Glial processes obviously behave independently of degenerating terminals; they loose any contact with them and never act as phagocytes for axon remnants. During the whole period of breakdown undifferentiated contacts between nerve fibers and muscle anlagen are present but synaptic structures as in normal developing dlm have never been observed. This fact, in comparison with earlier studies, suggests a lack of trophic nervous activity on the muscle anlagen tissue. A short time after removal of the isolated stumps new nerve tracts appear between dlm-fibers (which are, of course, strongly retarded in development). They are presumably sensory wing nerves which lack a guide structure to the central target, due to axotomy. Neuromuscular contacts or even junctions formed by axons of these nerves have occasionally been detected on the dlm. Their nature is discussed. Wallerian axon degeneration is compared to the normal, metamorphic breakdown of the innervation of the larval dlm-precursor. In contrast to the former, glial processes here remain in contact with the terminals. Glia and axons first swell. Then most glial processes are transformed into lamellar bodies whereas neurites shrink and become electron-dense. Axonal organelles remain intact for a long period.     

Effects of stimulation and rest on the ultrastructure of the excitatory neuromuscular junctions of Locusta migratoria L.

Summary The terminals of the fast axon on extensor tibiae muscle fibres of Locusta were examined in untreated nerve-muscle preparations and in preparations stimulated electrically at frequencies varying from 0.5 to 100 Hz. The ultrastructure of the terminals in preparations stimulated at the lower range of these frequencies, which induce twitch contractions of the muscles, is similar to that of the controls. Stimulation at the higher frequencies induced tetanic muscle responses and rapid fatigue of the muscles after which they would not respond again to high frequency stimulation for about 1 h. This loss and recovery of the responses of the muscles is correlated with changes in the ultrastructural appearance of the terminals, in particular in the number and shape of the synaptic vesicles. The ultrastructure of these recovering axon terminals closely resembles that of the controls.R.P. Botham gratefully acknowledges the SRC for financial assistance     

Axonal and presynaptic RNAs are locally transcribed in glial cells.

In the last few years, the long-standing opinion that axonal and presynaptic proteins are exclusively derived from the neuron cell body has been substantially modified by the demonstration that active systems of protein synthesis are present in axons and nerve terminals. These observations have raised the issue of the cellular origin of the involved RNAs, which has been generally attributed to the neuron soma. However, data gathered in a number of model systems indicated that axonal RNAs are synthesized in the surrounding glial cells. More recent experiments on the perfused squid giant axon have definitively proved that axoplasmic RNAs are transcribed in periaxonal glia. Their delivery to the axon occurs by a modulatory mechanism based on the release of neurotransmitters from the stimulated axon and on their binding to glial receptors. In additional experiments on squid optic lobe synaptosomes, presynaptic RNA has been also shown to be synthesized locally, presumably in nearby glia. Together with a wealth of literature data, these observations indicate that axons and nerve terminals are endowed with a local system of gene expression that supports the maintenance and plasticity of these neuronal domains.     

Kainic Acid Differentially Affects the Synaptosomal Release of Endogenous and Exogenous Amino Acidic Neurotransmitters

Presynaptic actions of kainic acid have been tested on uptake and release mechanisms in synaptosome-enriched preparations from rat hippocampus and goldfish brain. Kainic acid increased in a Ca2+-dependent way the basal release of endogenous glutamate and aspartate from both synaptosomal preparations, with the maximum effect (40-80%) being reached at the highest concentration tested (1 mM). In addition, kainic acid potentiated, in an additive or synergic way, the release of excitatory amino acids stimulated by high K+ concentrations. Kainic acid at 1 mM showed a completely opposite effect on the release of exogenously accumulated D-[3H]aspartate. The drug, in fact, caused a marked inhibition of both the basal and the high K+-stimulated release. Kainic acid at 0.1 mM had no clear-cut effect, whereas at 0.01 mM it caused a small stimulation of the basal release. The present results suggest that kainic acid differentially affects two neurotransmitter pools that are not readily miscible in the synaptic terminals. The release from an endogenous, possibly vesiculate, pool of excitatory amino acids is stimulated, whereas the release from an exogenously accumulated, possibly cytoplasmic and carrier-mediated, pool is inhibited or slightly stimulated, depending on the external concentration of kainic acid. Kainic acid, in addition, strongly inhibits the high-affinity uptake of L-glutamate and D-aspartate in synaptic terminals. All these effects appear specific for excitatory amino acids, making it likely that they are mediated through specific recognition sites present on the membranes of glutamatergic and aspartatergic terminals. The relevance of the present findings to the mechanism of excitotoxicity of kainic acid is discussed.     

Transfer of molecules from glia to axon in the squid may be mediated by glial vesicles.

Although the transfer of glial proteins into the squid giant axon is well documented, the mechanism of the transfer remains unknown. We examined the possibility that the transfer involved membrane-bound vesicles, by taking advantage of the fact that the fluorescent compound, 3,6-acridinediamine, N,N,N,',N'-tetramethylmonohydride [acridine orange (AO)], rapidly and selectively stains vesicular structures in glial cells surrounding the giant axon. We labeled cleaned axons (1-3 cm long) by incubation for 1 min in filtered seawater (FSW) containing AO. Because the AO was concentrated in glial vesicular organelles, these fluoresced bright orange when the axon was examined by epifluorescence microscopy. To look for vesicle transfer, axoplasm was extruded from such AO-treated axons at various times after labeling. During the initial 15 min, an increasing number of fluorescent vesicles were observed. No further increases were observed between 15 and 60 min post AO. The transfer of the fluorescent vesicles into the axoplasm seemed to be energy dependent, as it was inhibited in axons treated with 2 mM KCN. These results suggest that a special mode of exchange exists between the adaxonal glia and the axon, perhaps involving phagocytosis by the axon of small portions of the glial cells.     

Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis

BACKGROUND: Axon pruning is involved in establishment and maintenance of functional neural circuits. During metamorphosis of Drosophila, selective pruning of larval axons is developmentally regulated by ecdysone and caused by local axon degeneration. Previous studies have revealed intrinsic molecular and cellular mechanisms that trigger this pruning process, but how pruning is accomplished remains essentially unknown. RESULTS: Detailed analysis of morphological changes in the axon branches of Drosophila mushroom body (MB) neurons revealed that during early pupal stages, clusters of neighboring varicosities, each of which belongs to different axons, disappear simultaneously shortly before the onset of local axon degeneration. At this stage, bundles of axon branches are infiltrated by the processes of surrounding glia. These processes engulf clusters of varicosities and accumulate intracellular degradative compartments. Selective inhibition of cellular functions, including endocytosis, in glial cells via the temperature-sensitive allele of shibire both suppresses glial infiltration and varicosity elimination and induces a severe delay in axon pruning. Selective inhibition of ecdysone receptors in the MB neurons severely suppressed not only axon pruning but also the infiltration and engulfing action of the surrounding glia. CONCLUSIONS: These findings strongly suggest that glial cells are extrinsically activated by ecdysone-stimulated MB neurons. These glial cells infiltrate the mass of axon branches to eliminate varicosities and break down axon branches actively rather than just scavenging already-degraded debris. We therefore propose that neuron-glia interaction is essential for the precisely coordinated axon-pruning process during Drosophila metamorphosis.     

Microtubules and actin in giant nerve fibers of the spiny lobster, Panulirus argus

Giant axons of the spiny lobster, Panulirus argus, are filled with microtubules that are decorated with fine, irregular filaments. Mitochondria and membrane-limited clear vesicles are the only other distinguishable elements in the axoplasm and are located around the periphery of the axon near the axolemma. Neither 100 A neurofilaments nor 70 A microfilaments are evident in fixed, intact axons or in negatively stained axoplasm. Actin-like microfilaments are a prominent constituent of the glial cells that closely ensheathe the axons, and gel electrophoresis studies suggest that most of the actin in the nerve fibers is located in the glia rather than in the axons. Studies of isolated axoplasm indicate that microtubules are the primary elements stabilizing the axoplasm. The microtubules in the isolated axoplasm are disrupted by Ca2+ in the medium in the presence of protease inhibitors.     

Electron microscopic histochemistry of acetylcholinesterase of rat brain by Karnovsky's method

Summary Karnovsky's electron microscopic acetylcholinesterase method was successfully applied to rat brain fixed by vascular perfusion with either 2% glutataldehyde or 4% formaldehyde. 2% glutaraldehyde showed better fine structure but worse preservation of the enzyme than 4% formaldehyde.In the neuropil of the caudate nucleus, locus coeruleus and dorsal nucleus of the vagus, AChE activity was most intensely demonstrated on the plasma membranes of preterminal axons and somewhat less strongly on those of axon terminals and contacting dendritic branches. The axoplasm and synaptic vesicles were usually negative, while the cytoplasm and neurotubules of the dendritic branches showed some activity. In the nodule and uvula of the cerebellum moderate activity was exhibited on the synaptic contacts between the mossy fiber endings and granule cell dendrites. In the hypothalamus and other autonomic regions the characteristic coexistence of AChE and granulated vesicles of axon terminals could be demonstrated.In the perikaryon of positive nerve cells, AChE was observed strongly in the cytoplasm, disseminated irregularly or attached to the endoplasmic reticulum, while it was absent in the mitochondria and lysosomal dense bodies.     

FORMATION OF MYELIN IN THE CENTRAL NERVOUS SYSTEM OF MICE AND RATS, AS STUDIED WITH THE ELECTRON MICROSCOPE

Sections of brain and spinal cord of mice and rats at 1, 3, 5, and 17 days after birth were examined with the electron microscope. In the early stages of myelinization only two to four lamellae surround the axon. These laminae are formed from the plasma membraces of glial processes, and in particular of oligodendroglial processes. In a later stage of myelinization a larger number of flattened glial processes surround the axon with enclosed cytoplasm trapped within some of the membranes. Multiple extensions of the membranes of the flattened glial processes to the lamellae of the myelinated sheath are evident at this stage, with a variable number of membranes within the sheath at various positions along the fiber. Newly formed myelinated sheaths are sometimes larger than their enclosed fibers, extending as a projection of sheath which does not surround axoplasm. Loci are present in which the myelinated sheath is incomplete or interrupted.     

The Fine Structure of Photoreceptor Terminals in the Compound Eye of Pandalus borealis (Crustacea)

Seven of the photoreceptor axons of each ommatidium in the compound eye of the prawn Pandalus borealis end in two layers in the optic lamina. They have expanded terminals in the optic cartridges; four distally and three proximally in each cartridge. All seven receptor terminals are presynaptic to one lamina monopolar neuron (M₂) of the cartridge. This monopolar neuron is situated centrally in the cartridge and has a thick axis fibre with radially arranged branches, and its axon has a terminal in medulla externa. At the synapses, an arrowlike presynaptic bar is found facing three postsynaptic profiles. The receptor terminals have several characteristics. Their cytoplasm is filled with empty and coated vesicles, and contains numeorus large mitochondria and clusters of tubular elements. There is a longitudinally arranged fascicle of filaments partly surrounded by electron-dense amorphous material in the terminals. Centrally towards M₂, numerous neural spines invaginate into the terminal. Along the entire terminal periphery, there are invaginations from the glial cells. The terminals also form small knoblike protrusions extending into the surrounding glial cells.     

Stimulation-induced changes at crayfish (Procambarus clarkii) neuromuscular terminals

Summary The fine structure of neuromuscular terminals of the single excitor axon was examined in the limb stretcher muscle of the crayfish Procambarus clarkii. A morphometric comparsion of the neuromuscular terminals of the left and right limbs of a control crayfish showed them to be similiar in qualitative as well as quantitative features. The excitor axon to the stretcher muscle of the right side was stimulated, by backfiring its branches in the adjacent opener muscle, at 20 Hz for 4–5 h per day over 4–5 days. The stretcher muscle on the left side was not stimulated and served as a control. Morphometric analysis of stimulated terminals revealed an increase in the number of dense bars and synaptic vesicles compared to their non-stimulated, contralateral counterparts. Since dense bars are regarded as active sites of transmitter release, changes in their number provide a morphological basis for synaptic plasticity.     

GABA immunoreactivity in the human cerebellar cortex: a light and electron microscopical study

The distribution of gamma-aminobutyric acid (GABA) in surgical samples of human cerebellar cortex was studied by light and electron microscope immunocytochemistry using a polyclonal antibody generated in rabbit against GABA coupled to bovine serum albumin with glutaraldehyde. Observations by light microscopy revealed immunostained neuronal bodies and processes as well as axon terminals in all layers of the cerebellar cortex. Perikarya of stellate, basket and Golgi neurons showed evident GABA immunoreactivity. In contrast, perikarya of Purkinje neurons appeared to be negative or weakly positive. Immunoreactive tracts of longitudinally- or obliquely-sectioned neuronal processes and punctate elements, corresponding to axon terminals or cross-sectioned neuronal processes, showed a layer-specific pattern of distribution and were seen on the surface of neuronal bodies, in the neuropil and at microvessel walls. Electron microscope observations mainly focussed on the analysis of GABA-labelled axon terminals and of their relationships with neurons and microvessels. GABA-labelled terminals contained gold particles associated with pleomorphic vesicles and mitochondria and established symmetric synapses with neuronal bodies and dendrites in all cortex layers. GABA-labelled terminals associated with capillaries were seen to contact the perivascular glial processes, basal lamina and endothelial cells and to establish synapses with subendothelial unlabelled axons.