Biomedical subjects
Publications and source records attributed to E G Gray.
The amyloid-related cell (ARC) of the neuritic plaques of Alzheimer's disease revealed numerous cytoplasmic projections surrounding extracellular amyloid material. It is proposed that ARC-coated vesicles fuse with the cell membrane, forming coated pits, which may empty their secretory material into the extracellular space where polymerization of amyloid filaments could occur.
This study offers a new concept on the origin and function of the hitherto enigmatic presynaptic dense projections (dps) of neurons and motor endplates. After a deuterium oxide-albumin pretreatment (da), brain tissue and motor endplate of rat and frog reveal an intricate association of smooth endoplasmic reticulum (ser), microtubules (mts) and synaptic vesicles (sv) at the presynaptic grid-active zone of synapses. The ser entwines the mts, which are clothed in svs, and impinges directly onto the presynaptic membrane as sacs or 'tubular-fibrillar' extensions. Since no dps are seen in these sections, whereas they do occur in conventionally processed material (i.e. without da pretreatment), it is suggested that the dps of conventional material may, in part, originate from improperly fixed ser at these points. Thus for the first time we demonstrate an in vivo system of ser which, because its 'finger' processes come into intimate contact with the presynaptic membrane, may be implicated in Ca2+ ion translocation, presumably out of the presynaptic bulb. Since no such tubular ser has been demonstrated in what are claimed to be sophisticated techniques (i.e. high-speed slam-freezing-freeze substitution) the actual sophistication of such methods is questioned.
The effects of prolonged alcohol consumption on the structure of prelimbic cortical synapses of the adult rat were studied. 'Perforated' synapses in alcohol-treated animals were abundant, with small spinules displaying different aspects according to the plane of section. We postulate that the central parts of the synaptic disc are more vulnerable to chronic alcohol consumption.
The influence that thyroid hormones have on the development of the cerebellar cortex is well known. Their absence in adults leads to important functional changes probably related to abnormalities in nerve terminal activity, although no morphological alterations have hitherto been described. Using ultrastructural morphometric methods, we have studied the effects of thyroidectomy on the cerebellar cortex mossy fiber terminals of adult rats with different survival periods. No significant changes in the volume and surface area of these terminals were found. The numbers of synaptic vesicles in groups 7, 15 and 30 days after thyroidectomy were significantly greater than in controls. A significant increase in coated pit formation was found when thyroidectomized groups were compared with controls. This increase is due to the lack of thyroxin since it can be counteracted by thyroxin administration. Whether this increased coated pit formation is a membrane retrieval mechanism or is related to the uptake of extracellular molecules remains to be determined.
When sections from the cerebral cortex of 5 normal rats and 2 normal monkeys were stained with saturated aqueous uranyl acetate (without block staining) and examined with the electron microscope, all 7 animal brains showed abundant membrane lamellation between apposed neuronal and neuroglial processes. Such lamellations match those described by Beck et al. (1982) in the brains of kuru-inoculated monkeys in their early stages before spongiform encephalopathy has become established. The present observations question their contention that membrane lamellation is a real phenomenon and a prelude to spongiform change in kuru encephalopathy in the monkey.
Past work on the C.N.S. of nutritionally deprived immature rats shows widespread structural and functional alterations that were not found in nutritionally deprived adults. In the present work we studied cerebellar mossy fibre terminals of adult rats, given a 8% casein diet for periods of 6, 12 and 18 months. After 12 months of the diet, mossy fibre terminals presented a large number of spherical or disc-shaped membrane-free inclusions herein referred to as presynaptic inclusions, with diameters up to 1.3 micron and formed by fine-textured subunits, compacted into 3 nm strands. This material is an extension of the cytoplasmic network of the terminal. No special relationship was observed with the active zones; synaptic vesicles found in the proximity or embedded in these inclusions were often of the flat type, whereas those close to the active zones always appeared spherical. These results show that even in adult rats synaptic terminals are areas sensitive to protein deprivation.
Alcohol consumption in rats leads to degenerative changes in the cerebellar cortex (described elsewhere). Purkinje cells show a loss of dendritic spines, but some of the intact spines elongate to 3-4 times their normal length. This is postulated as a plastic change resulting from the degeneration of the original axon terminal contact with a consequent elongation of the spine 'in search of' and finally synapsing with still viable parallel fibre terminals in the vicinity. Under these conditions the circuitry is radically altered.
Ultrastructural studies of intact tissue rarely show presynaptic microtubules, and immunocytochemical studies on tissue sections have previously been unable to demonstrate tubulin in the nerve terminal. In contrast, a microtubular coil can be readily detected in the presynaptic nerve terminal of synaptosomes. We have developed an immunocytochemical procedure on the synaptosome preparation and demonstrated, using monoclonal antibodies, that in the presynaptic terminal alpha and beta tubulin subunits are specifically restricted to the equatorial microtubular coil.
The morphological mechanisms involved in translocation of the synaptic vesicle to the presynaptic membrane, release of transmitter from the vesicle and recycling of the vesicle membrane are still far from understood. However, there is strong evidence that vesicles move along the surfaces of a specific set of highly labile presynaptic microtubules that direct the vesicles to the active zones. These microtubules are focused in a precise geometrical array, which is in register with and in contact with presynaptic dense projections of the central nervous system synapse or presynaptic dense bars of the motor endplate. These dense complexes constitute the presynaptic grid or active zones. The regular arrays of dense projections or bars are in turn coincident with rings or chains of synaptic vesicles mobilized at release sites on the presynaptic membrane (having arrived at these precise points by microtubule translocation). Thus it is suggested that the presynaptic microtubules not only translocate synaptic vesicles, but because of their ordered arrays determine, in ontogeny, the ordered structure of the presynaptic grid.
Using techniques for enhanced microtubular preservation (albumin pretreatment of Gray) occipital and pyriform cortices of rats were studied by electron microscopy at various stages of development. A close structural relationship was seen between microtubules (mts), focal membrane densities, synaptic vesicles, and presynaptic dense projections (pre-dps) during maturation. Mts were seen in the neonates to be focused onto the inner surface of immature axonic profiles merging or sometimes fragmenting there. Focal densities occur at these attachment sites and thereafter synaptic vesicles clothe the mts and abut onto these presumed primordial pre-dps. Thus, mts may contribute to the initial formation of pre-dps as well as their maintenance in the adult and may act to channel the first synaptic vesicles to the site of eventual synaptic contact and transmitter release.
Applying a morphometric analysis, the effects of chronic alcohol consumption on Golgi-impregnated Purkinje cells from the rat cerebellar cortex were evaluated. Alcohol-fed animals received ethanol up to 1, 3, 6, 12 and 18 months and controls were given the same amount of food and an aqueous solution with sucrose replacing the ethanol isocalorifically. The following parameters of Purkinje cell dendritic trees were studied: dendritic field area, branching density, total branch length, spine density and total number of spines. Results showed a progressive decrease of all parameters in alcohol-fed animals. Significant differences were found in all rats after 12 months of alcohol intake. The reduction of the dendritic network was found to be related to a drop in mean total number of dendritic branches after 6 months of alcohol consumption. It is concluded that long-term alcohol consumption induces a decrease of the synaptic receptive area of Purkinje cells which may well lead to functional changes in the cerebellum.
A research neuroscientist in his fifties, who has recovered from endogenous depression after several years, describes the experience and makes some comments on treatment and management. He found electroconvulsive therapy, mianserin, lithium carbonate and diazepam acceptable. He considers that occupational therapy and conventional psychotherapy were counter-therapeutic. He stresses how physically ill he felt when depressed, and compares this with prolonged influenza. He hypothesizes that the underlying psychological disorder in endogenous depression is the learned extinction of optimistic thoughts, and suggests that any psychotherapy used should be aimed at restoring these.
When synaptosomes are prepared from rat brain and incubated in Krebs solution, the presynaptic bulb develops a coil of microtubules (mts). Various considerations indicate that the coil does not have a cytoskeletal supportive function. Synaptosome coil mts show certain peculiarities, e.g. they thrive during incubation in Krebs solution (dendritic mts are depolymerized in Krebs solution) and they show no protofilament molecular substructure with tannic acid. Dendritic mts show clearly a 13 protofilament substructure when processed in the same way. Synaptosomal coil mts are sensitive to micromolar calcium and are depolymerized by treatment of the synaptosomes with veratridine or A23187. Our evidence indicates that coil mts of synaptosome and synaptic vesicle clothed mts of 'intact' albumin-treated synapses are different morphological and functional entities. As mentioned above, the function of coil mts remains enigmatic, while the mts seen in albumin-treated synapses could well have a role in synaptic vesicle translocation.
Electron microscopical examination indicated that incubation of slices of rat cerebral cortex in Krebs buffer at room temperature of 37 degrees C led to a rapid and more or less complete depolymerization of dendritic microtubules. The loss of dendritic microtubules did not appear to be a consequence of anoxia. Myelinated axons showed only a partial loss of microtubules and the microtubules of preterminal axons were unaffected by incubation. These results indicate differential labilities of axonal and dendritic microtubules under these conditions of incubation. Such an effect of the incubation of slices in Krebs buffer indicates a need for caution in the interpretation of experiments on slice preparations.
The microtubules in different parts of the neuron and synaptosomes were examined with respect to their stability, structure and orientation. On the basis of distribution, different labilities and differences in protofilament substructure seen by tannic acid staining, we have classified microtubules into eight major categories. Functional involvements in vesicle translocation, cytoskeletal support and the regulation of assembly/disassembly are considered.
With the electron microscope we have examined the organisation of microtubules in synapses and varicosities of central and peripheral axons and their assembly/disassembly in synaptosomes. Following fixation in a tri-aldehyde fixative containing a calcium ion chelating agent, central synapses and autonomic varicosities show up to 10 microtubule profiles. In synapses, these microtubules are found at one or both poles of the terminal. In autonomic varicosities, microtubules occupy a central zone or are located to one side. Synaptosomes also contain microtubules, probably arranged in the form of a coil. The coil lies in an equatorial plane parallel to the active zone and is closely apposed to the plasma membrane. The microtubules in intact synapses, autonomic varicosities and synaptosomes are frequently associated with mitochondria. Serial section analysis shows that the mitochondrion in synaptosomes is horse-shoe shaped and that presynaptic microtubules are continuous with those in the axon. In synaptosomes, presynaptic microtubules disassemble when exposed to colchicine (IC50 = 3 x 10(-4) M) and low temperatures (1 degree C) and reassemble following warming to 37 degrees C. Reassembly occurs with no other apparent effects on the organisation of the presynaptic terminal. The synaptosomes thus retain the capacity to assemble ordered coils of microtubules. The function of presynaptic microtubules is discussed in the light of the finding that there may be a relation between the number of turns in the microtubular coil and the size of the presynaptic terminal. An explanation is proposed for the relative scarcity of these structures at conventionally prepared synapses.
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Using techniques for enhanced microtubular preservation, including albumin pretreatment (Gray, 1975), occipital cortex of rats was studied electron microscopically at various ages of development. A close structural relationship was seen between microtubules, sacs of SER and the postsynaptic "thickening" in primordial spines and with the dense "plate" material of spine apparatuses. Stereoscopic preparations in addition show a more complicated substructure than previously described for the "plate". Microtubules may contribute to the formation of the "plate" of the spine apparatus which in turn is associated with the postsynaptic "thickening" of the mature spine. Possible functional correlates are discussed.