Amanda Pellegrino de Iraldi

The neurotubular system of the axon and the origin of granulated and non-granulated vesicles in regenerating nerves

SummaryElectronmicroscope observations have been made on compressed sciatic nerves and preganglionic afferents to the superior cervical ganglia of rats. After 6 hours, the proximal regenerating stumps of both myelinated and unmyelinated axons become filled with enlarged neurotubules and vesicles. Granulated vesicles of 750–900 Å, having a dense core become abundant in all types of regenerating axons and they increase in number after 24 hours. The vesicular material is formed by dilatation and pinching off from neurotubules. The existence of a neurotubular system within the axon, connected with the Golgi complex at the perikaryon and involved in the formation of vesicles, is postulated. The presence of granulated vesicles in all types of regenerating axons and nerve terminals is discussed in relation with their possible functional significance. The distal stumps of compressed sciatic nerves show, after 6 hours, a considerable increase in membranous material within the axoplasm mainly represented by multivesicular and lamellar bodies. This reaction, which is interpreted as being autolytic, is compared with the regenerative reaction of the proximal stump.

Isolation of synaptic vesicles from nerve endings of the rat brain.

SINCE the discovery of ‘synaptic vesicles’ as a specific component of synapses1,2 it was suggested that they could be associated with the storage and possibly with the synthesis of transmitter substances. This concept was supported by degeneration experiments3 and specially by the stimulation of some cholinergic terminals4, which suggested that within the ending a balance exists between the formation and release of synaptic vesicles. However, the isolation of such a sub-cellular component remained as the most direct approach to the elucidation of the physiological significance and chemical nature of these vesicles.

Cyclic AMP-Responsive Element Binding Protein in Brain Mitochondria

Abstract: Cyclic AMP‐responsive element binding protein (CREB) is critically involved in many important brain functions, including the formation of long‐term memory. CREB is the best characterized member of a family of transcription factors (CREB/ATF family) recognized to be important nuclear targets for intracellular signal transduction systems. Here we show, by using different approaches, that CREB is unexpectedly localized to mitochondria of the rat brain. Controlled subcellular fractionation of hippocampus and cerebral cortex showed that both synaptic and nonsynaptic mitochondria exhibited immunoreactivity to the phosphorylated form of CREB (pCREB). Moreover, CREB extracted from synaptic mitochondria is able to be phosphorylated by the catalytic subunit of protein kinase A and dephosphorylated by protein phosphatase 1 or 2B. DNA mobility shift assays showed the presence of binding activity to the calcium—cyclic AMP‐responsive element in mitochondrial extracts from hippocampus; this binding complex was specifically supershifted by an anti‐CREB antibody. Immunoelectron microscopic analysis of hippocampal subcellular fractions revealed that pCREB immunoreactivity is localized in close association with the inner mitochondrial membrane. These results, together with recent findings describing the presence and phosphorylation of CREB in developing dendrites, suggest that CREB may participate in different mechanisms involved in the communication between extracellular signals and the expression of genes.

Action of reserpine on the osmium tetroxide zinc iodide reactive site of synaptic vesicles in the pineal nerves of the rat

SummaryThe technique of Champy-Maillet was used to stain pineal nerves in an electron microscopic study. Synaptic vesicles appear as a negative image of that revealed with osmium tetroxide. They are totally stained and 34% of the total vesicles show a paler core, with a mean diameter of 280 Å. Reactive granules with a mean diameter of 255 Å, were observed with the histochemical technique of Wood for catechol and indoleamines. These reactive granules may correspond to the clear cores revealed with the osmium tetroxide-zinc iodide technique and the dense cores visualized with osmium tetroxide alone. These results seem to indicate that the osmium tetroxide-zinc iodide technique stains a component present in the synaptic vesicles which is not catechol or indoleamine.Under reserpine the osmium tetroxide-zinc iodide reactive material disappears and the vesicles show a smaller size and a more tubular shape. The assumption is made that the long lasting blocking action of reserpine could be related to the release of some component present into the matrix of the granule.

Action of reserpine, iproniazid and pyrogallol on nerve endings of the pineal gland

Summary Drugs that may change the metabolism of catecholamines and indolamines have been used in order to investigate the nature of the granulated vesicles found in adrenergic nerve endings min the pineal gland of the rat. In normal endings, 30–40 per cent of the vesicles are granulated and the rest are homogeneous. The proportion of granulated vesicles diminish considerably between 2–48 hr after a single injection of reserpine and this is slowly restored to normal levels in a period of 8 days. A single injection or chronic treatment with iproniazid (a MAO inhibitor) increases the proportion of granulated vesicles, and also the size of the vesicles and granules. Iproniazid prior to reserpine protects the nerve endings from the depletion of granulated vesicles. Pyrogallol (a COMT inhibitor) does not change the concentration of granulated vesicles and does not protect the nerve ending from the action of reserpine. These results are interpreted as supporting the concept that the granulated vesicles contain the adrenergic transmitter. The possible mechanism of action of these drugs on the submicroscopic structure of adrenergic nerve endings is discussed.

Granulated vesicles in the pineal gland of the mouse

SummaryPineal glands of normal adult mice, 7 to 42 days after bilateral superior cervical gangliectomy and 5 and 16 hours after one dose of reserpine (10 mg/kg) were studied under the electron microscope. The architecture of the gland is basically similar to that of other mammalian pineal glands previously studied. Mouse pinealocytes are polymorphic cells with perivascular and intercellular processes. Its most prominent feature is the presence of abundant granulated vesicles with a mean diameter of 1100 Å and a dense core of about 800 Å intermingled with clear vesicles of similar size distributed throughout the cytoplasm and more concentrated in perivascular and intercellular processes. These processes were seen in continuity with the perykaryon and remained after bilateral superior cervical gangliectomy. Processes containing the plurivesicular component of adrenergic nerves situated in perivascular and intercellular spaces disappeared after bilateral superior cervical gangliectomy. Reserpine depleted small granulated vesicles of nerves but the larger ones of perikaryon and polar processes remained almost unchanged.The histochemical technique of Wood was positive for catechol- and indolamines in the nerves. The reaction was negative in the perikaryon and polar processes.The significance of these findings is discussed.

Granulated vesicles in retinal synapses and neurons.

SummaryThe electron microscope study of the retina of rats revealed the presence of granulated vesicles in synapses and neurons. Of the three different types of nerve endings observed in the inner plexiform layer, two were found to contain granulated vesicles. Type 1 nerve ending, belonging to bipolar cells, is devoid of granulated vesicles and together with type 2 nerve endings and ganglion cell dendrites forms glomerular-like synapses. Type 2 nerve endings, belonging to amacrine cells, have granulated vesicles of 640 Å, and type 3 nerve endings, probably representing centrifugal fibers and located at the outermost zone of the inner plexiform layer, have granulated vesicles of 1140 Å.Granulated vesicles were also found in perikarya of the inner nuclear layer in close association with the Golgi complex of these cells. Of the two types of neurons observed in the ganglion cell layer, the clear type neuron exhibits granulated vesicles in its cytoplasm.The correspondence of these findings with those obtained with the histochemical fluorescence technique for catecholamines is discussed. The suggestion is made that the granulated vesicles could represent the site of storage of retinal catecholamines.The electron microscope study of the retina of rats revealed the presence of granulated vesicles in synapses and neurons. Of the three different types of nerve endings observed in the inner plexiform layer, two were found to contain granulated vesicles. Type 1 nerve ending, belonging to bipolar cells, is devoid of granulated vesicles and together with type 2 nerve endings and ganglion cell dendrites forms glomerular-like synapses. Type 2 nerve endings, belonging to amacrine cells, have granulated vesicles of 640 Å, and type 3 nerve endings, probably representing centrifugal fibers and located at the outermost zone of the inner plexiform layer, have granulated vesicles of 1140 Å. Granulated vesicles were also found in perikarya of the inner nuclear layer in close association with the Golgi complex of these cells. Of the two types of neurons observed in the ganglion cell layer, the clear type neuron exhibits granulated vesicles in its cytoplasm. The correspondence of these findings with those obtained with the histochemical fluorescence technique for catecholamines is discussed. The suggestion is made that the granulated vesicles could represent the site of storage of retinal catecholamines.

Isolation of oligodendroglial cells from young and adult whole rat brains using an in situ generated percoll density gradient

A method for the isolation of oligodendroglial cells from young and adult whole rat brains, using a Percoll density gradient is presented. The minced tissue, incubated in a balanced salt solution containing 0.1% trypsin is further dissociated by forcing it through nylon screens to 145 and 74 ?m pore size. The crude suspension is then mixed with an isosmotic Percoll solution and centrifuged for 15 min. An in situ generated density gradient allows the separation of five bands, only one of which (Band C) lying between ?1.050 and ?1.062 contains cellular elements. The isolated cells show the typical morphological characteristics of oligodendroglia. A detailed morphological study of the cells isolated from whole brains of 10-, 30- and 120-day old rats is presented for the first time in the literature and immunocytochemical characterization is carried out using specific (antigalactocerebroside) and non specific (anti-glial fibrillary acidic protein) anti-sera. The method is simple and rapid and isosmotic conditions are maintained throughout, resulting in a better preservation of cell integrity. It represents an improvement over the two previous methods described in the literature and will be useful for studying different developmental events (biochemical and morphological) occurring in oligodendroglial cells at early stages of myelin formation.

Catecholamine and serotonin in granulated vesicles of nerve endings in the pineal gland of the rat

p-Chloropherrylalaninc, a drug that selectively inhibits the synthesis of serotooin, has been used in order to invcstigate the localization of this amine in nerves of the pineal gland of rats. Glands of normal and treated rats were studied under the electron microscope, after fixing with osmium tetroxide and glutaraldehyde, and applying specific histochemistry for catechol and indolamines. Fluorometric determinations of scrotonin, noradrenalinc and dopaminc were also made in normal and treated glands. In normal glands about 35 % of granulated vesicles were observed in nerve endings fixed with osmium tetroxide or glutaraldehyde. After treatment with p-chlorophenylalanine the dense core of the granulated vesicles disappeared in glands fixed with glutaraldehyde but remained unchanged or increased in number in those fixed in osmium tetroxide. p-Chlorophcnylalanine produced an 87% decrease in serotonin content of the pineal gland while noradrenalinc and dopamine content did not diminish, The correlation between morphological and biochemical data suggests that both catechol and indolamines are stored in granulated vesicles in the pineal nerves of the rat. This assumption was confirmed with the specific histochemical technique of wood to differentiate catechol and indolamines.

Noradrenaline and dopamine content of normal, decentralized and denervated pineal gland of the rat☆

Abstract Noradrenaline and dopamine were determined with flourimetric methods in normal, decentralized and denervated pineal glands of the rat. A high content in both amines was found in the normal gland. While noradrenaline decreases 44.5 per cent with decentralization and almost disappears with bilateral superior cervical gangliectomy, the dopamine content is not significantly affected with either procedure. These results give support to the concept that the granulated vesicles of adrenergic nerves contain noradrenaline 3 . They suggest the existence of a central regulation for the content of noradrenaline in the peripheral sympathetic neurons. They also prove that there is a rich extraneuronal pool of dopamine in the pineal gland of the rat.