Quantitative changes in the total proteins of the cells of the hypothalamic supraoptic nucleus after extreme exposures: acute immobilization and deprivation of the paradoxical sleep phase

It has been shown that 1-hour acute immobilization of rats resulted in a decrease of total protein content (per 1 cell) in neurones (by 17% in cytoplasm and 22% in nucleus) of the supraoptic nucleus of the hypothalamus and in their glial satellite cells (by 10%), mainly at the expense of the nuclei of the latter. These changes remained at the same level in the neuronal cytoplasm and satellite cells within 6 hours after cessation of acute immobilization, being abolished in the neuronal nuclei. Deprivation of REM sleep for 24 hours affected these parameters to the same extent as acute immobilization. However, 6-hour rest was sufficient for normalisation in satellite cells and for decrease in protein deficiency in the neuronal nuclei; yet protein deficiency still sustained in the neuronal cytoplasm. REM sleep deprivation was also accompanied by a decrease in tritium incorporation into satellite cell proteins (by 18%); after 3-hour rest, incorporation of the label into satellite cells was restored to the initial level being increased by 20% in neuronal nuclei; another 3-hour rest resulted in normalisation of labeling in the neuronal nuclei and in 27% increase of labeling in the satellite cells.     

Dark and light neurons in the cortical plate of the forebrain of the swamp turtle]

Electron microscopic study of the neuronal composition of Emys orbicularis cortical zones as compared with the light microscopy data has revealed that the distinctions in the cell cytoplasm ultrastructure with respect to the dendrite branching pattern and the peculiarities of their localization in the cortical plate are suggestive of a possible metabolic heterogeneity and can serve as a foundation of the morpho-functional characteristics of the neurons. A few neuronal types of neurons have been revealed: 1. light neurons distinguished by the cytoplasm matrix devoid of any electron density, a poorly developed endoplasmic reticulum and a few cytoplasmatic organells and inclusions; 2. light neurons having slight osmophility of the cytoplasm matrix, a more developed endoplasmic reticulum with elongated cisternae, a great number of organells including neurofilaments; 3. dark neurons having a high degree of cytoplasm matrix and the nucleus, a large number of the nuclei, cytoplasm, dendrites and axons in contrast to the above described patterns.     

Induction of Cbeta splice variants and formation of novel forms of protein kinase A type II holoenzymes during retinoic acid-induced differentiation of human NT2 cells

Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are critical regulators of neuronal differentiation. The expression, levels and activities of PKA subunits were studied prior to and during differentiation of the human neuronal precursor cell line NTera 2 (NT2). Undifferentiated NT2 cells expressed mainly cytoplasmic PKA type I, consisting of the regulatory subunit RIalpha and the catalytic subunit Calpha. Low levels of PKA type II consisting of RIIalpha or RIIbeta associated with Calpha were also detected, mainly in the cytoplasm and in the Golgi-centrosomal area. During retinoic acid-induced differentiation, the RIalpha and RIIalpha expressions remained in the cytoplasm, while we observed a strong upregulation of RIIbeta, located to the whole cytoplasm including neurite extensions. This upregulation coincided with increased PKA-specific activity accompanied by a strong induction of a number of neuronal-specific Cbeta splice variants that together with RIIbeta form novel PKAII holoenzymes. Formation of novel PKAII holoenzymes may imply specific PKA features which may have consequences for the process of neuronal differentiation and nerve cell function.     

Three-dimensional organization of the cytoplasmic neuroglobin-immunopositive structures in the rat medulla oblongata neurons

Neuroglobin is an iron-containing protein, most abundant in the vertebrate nervous system. Since neuroglobin is able to bind oxygen reversibly owing to the heme prosthetic group, it was believed that its function is an intercellular transport of oxygen in the nervous system and accumulation of oxygen for energy supply of cells in hypoxic conditions. In this work, a three-dimensional reconstruction of the neuroglobin distribution in large neurons of the rat medulla oblongata was carried out by means of immunocytochemistry and confocal laser microscopy. Positive neuroglobin immunocytochemical reaction was observed mainly in the perinuclear areas of large nerve cells exhibiting a discrete staining of the cytoplasm. Examination under the microscope at a high magnification revealed some neuroglobin-immunopositive granules, ring-like objects 1–2 μm in diameter, as well as linear and branched structures in neuronal cytoplasm and, occasionally, in the proximal segments of neuronal processes. Three-dimensional reconstruction of the neuroglobin-immunopositive structures showed that they mainly have the form of continuous lines and curves interlaced in some sites, about 1.0–1.5 μm thick, forming a complex network in the cytoplasm. The neuroglobin-immunopositive complexes found for the first time in neuronal cytoplasm are not identical to any known cytoplasmic compartments of nerve cells, but the diameter of their elements, as well as the shape and location suggest a possible link of neuroglobin with a mitochondrial network.     

Neuronal cell death in transmissible spongiform encephalopathies (prion diseases) revisited: from apoptosis to autophagy

Neuronal autophagy, like apoptosis, is one of the mechanisms of the programmed cell death (PCD). In this review, we summarize the presence of autophagic vacuoles in experimentally induced scrapie, Creutzfeldt–Jakob disease and Gerstmann–Sträussler–Scheinker (GSS) syndrome. Initially, a part of the neuronal cytoplasm was sequestrated by concentric arrays of double membranes; the enclosed cytoplasm appeared relatively normal except that its density was often increased. Next, electron density of the central area dramatically increased. The membranes then proliferated within the cytoplasm in a labyrinth-like manner and the area sequestrated by these membranes enlarged into a more complex structure consisting of vacuoles, electron-dense areas and areas of normally-looking cytoplasm connected by convoluted membranes. Of note, autophagic vacuoles form not only in neuronal perikarya but also in neurites and synapses. Finally, a large area of the cytoplasm was transformed into a collection of autophagic vacuoles of different sizes. On a basis of ultrastructural studies, we suggest that autophagy plays a major role in transmissible spongiform encephalopathies (TSEs) and may even participate in a formation of spongiform change.     

Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non-neuronal cells

Recent studies have revealed that accumulation of prion protein (PrP) in the cytoplasm results in the production of aggregates that are insoluble in non-ionic detergents and partially resistant to proteinase K. Transgenic mice expressing PrP in the cytoplasm develop severe ataxia with cerebellar degeneration and gliosis, suggesting that cytoplasmic PrP may play a role in the pathogenesis of prion diseases. The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non-neuronal cells. Transient expression of cytoplasmic PrP produced juxtanuclear aggregates reminiscent of aggresomes in human embryonic kidney 293 cells, human neuroblastoma BE2-M17 cells and mouse neuroblastoma N2a cells. Time course studies revealed that discrete aggregates form first throughout the cytoplasm, and then coalesce to form an aggresome. Aggresomes containing cytoplasmic PrP were 1-5-microm inclusion bodies and were filled with electron-dense particles. Cytoplasmic PrP aggregates induced mitochondrial clustering, reorganization of intermediate filaments, prevented the secretion of wild-type PrP molecules and diverted these molecules to the cytoplasm. Cytoplasmic PrP decreased the viability of neuronal and non-neuronal cells. We conclude that any event leading to accumulation of PrP in the cytoplasm is likely to result in cell death.     

Cytochrome c binds to inositol (1,4,5) trisphosphate and ryanodine receptors in vivo after transient brain ischemia in gerbils

Previously we have shown that the biphasic efflux of mitochondrial protein cytochrome c to cytoplasm is one of the important events of the delayed postichemic neuronal death. We concluded that early and transient appearance of cytochrome c in cytoplasm of cells recovering after ischemia was decisive for initiation of the pathological signaling cascade leading to neuronal death, but the precise mechanism remained unknown. In vitro cytochrome c was identified as a messenger that coordinates mitochondrial-endoplasmatic reticulum interactions that drive apoptosis. Here we show that in vivo cytochrome c interacts with inositol (1,4,5) trisphosphate receptor type 1 in gerbil hippocampus subjected to transient brain ischemia and short reperfusion. Moreover, cytochrome c binds also to ryanodine receptor type 2, the role of which in postischemic neuronal death is suggested. The complexes could be coimmunoprecipitated by antibodies against any of the two proteins. Our data verified that the mechanism observed in vitro applies to the pathological in vivo situation.     

Experimental protein malnutrition in squirrel monkeys: cytochemical interaction of nucleolus and cytoplasm in the anterior horn cells of the spinal cord of Saimiri sciureus.

Nuecleolo-cytoplasmic relationships have been studied using histochemical techniques in the spinal cord of healthy neonates compared with those born to mothers malnourished through most of the gestation period, and healthy and severely protein-malnourished young adult squirrel monkeys. Formaldehyde-fixed as well as fresh frozen tissues were used for histological study and for histochemical techniques to demonstrate lipids, carbohydrates, nucleic acids and enzymes of various metabolic cycles. Changes in the neuronal cytoplasm of the low protein animals appear to be related to impaired protein metabolism and energy transport. The nucleolus reacts to cytoplasmic changes by enlarging its size and by active synthesis of secretion of nucleolar material, which is then passed into the cytoplasm through an evagination of the nuclear envelope. The greater the degree of chromatolysis in the cytoplasm, the more active the nucleolus appears to be in terms of its enlarged size and transfer of secretion products into the cytoplasm. It is believed that the perinuclear accumulation of secretion products in the neuronal cytoplasm of the protein-deficient animals has its origin in the nucleolus. Such an accumulation in the severely malnourished animals appears to be a compensatory mechanism for the increased cytoplasmic catabolism and loss of ribonucleoprotein material in order to facilitate additional protein synthesis for cell survival.     

A CRM1-Mediated Nuclear Export Signal Is Essential for Cytoplasmic Localization of Neurogenin 3 in Neurons

Neurogenin 3 (Ngn3), a proneural gene, regulates dendritogenesis and synaptogenesis in mouse hippocampal neurons. Ngn3 is transiently exported from the cell nucleus to the cytoplasm when neuronal polarity is initiated, suggesting that the nucleo-cytoplasmic transport of the protein is important for its action on neuronal development. In this study, we identified for the first time a functional nuclear export sequence (NES2; ¹³¹YIWALTQTLRIA¹⁴²) in Ngn3. The green fluorescent protein (EGFP)-NES2 fusion protein was localized in the cytoplasm and its nucleo-cytoplasmic shuttling was blocked by the CRM1 specific export inhibitor leptomycin B. Mutation of a leucine residue to alanine (L135A) in the NES2 motif resulted in both cytoplasmic and nuclear localization of the EGFP-NES2 fusion protein and in the nuclear accumulation of ectopic full-length myc-Ngn3. In addition, point mutation of the leucine 135 counteracted the effects of Ngn3 on neuronal morphology and synaptic inputs indicating that the cytoplasmic localization of Ngn3 is important for neuronal development. Pharmacological perturbation of the cytoskeleton revealed that cytoplasmic Ngn3 is associated with microtubules.     

A combined Bodian-Nissl stain for improved network analysis in neuronal cell culture

Bodian and Nissl procedures were combined to stain dissociated mouse spinal cord cells cultured on coverslips. The Bodian technique stains fine neuronal processes in great detail as well as an intracellular fibrillar network concentrated around the nucleus and in proximal neurites. The Nissl stain clearly delimits neuronal cytoplasm in somata and in large dendrites. A combination of these techniques allows the simultaneous depiction of neuronal perikarya and all afferent and efferent processes. Costaining with little background staining by either procedure suggests high specificity for neurons. This procedure could be exploited for routine network analysis of cultured neurons.     

A Combined Bodian-Nissl Stain for Improved Network Analysis in Neuronal Cell Culture

Bodian and Nissl procedures were combined to stain dissociated mouse spinal cord cells cultured on coverslips. The Bodian technique stains fine neuronal processes in great detail as well as an intracellular fibrillar network concentrated around the nucleus and in proximal neurites. The Nissl stain clearly delimits neuronal cytoplasm in somata and in large dendrites. A combination of these techniques allows the simultaneous depiction of neuronal perikarya and all afferent and efferent processes. Costaining with little background staining by either procedure suggests high specificity for neurons. This procedure could be exploited for routine network analysis of cultured neurons.     

Ultrastructure of neuroglial contacts in crayfish stretch receptor

In order to explore neuroglial relationships in a simple nervous system, we have studied the ultrastructure of the crayfish stretch receptor, which consists of only two mechanoreceptor neurons enwrapped by glial cells. The glial envelope comprises 10–30 glial layers separated by collagen sheets. The intercellular space between the neuronal and glial membranes is generally less than 10–15 nm in width. This facilitates diffusion between neurons and glia but restricts neuron communication with the environment. Microtubule bundles passing from the dendrites to the axon through the neuron body limit vesicular transport between the perikaryon and the neuronal membrane. Numerous invaginations into the neuron cytoplasm strengthen glia binding to the neuron and shorten the diffusion pathway between them. Double-membrane vesicles containing fragments of glial, but not neuronal cytoplasm, represent the captured tips of invaginations. Specific triads, viz., “flat submembrane cisterns - vesicles - mitochondria”, are presumably involved in the formation of the invaginations and double-membrane vesicles and in neuroglial exchange. The tubular lattice in the glial cytoplasm might transfer ions and metabolites between the glial layers. The integrity of the neuronal and glial membranes is impaired in some places. However, free neuroglial passage might be prevented or limited by the dense diffuse material accumulated in these regions. Thus, neuroglial exchange with cellular components might be mediated by transmembrane diffusion, especially in the invaginations and submembrane cisterns, by the formation of double-walled vesicles in which large glial masses are captured and by transfer through tubular lattices. This work was supported by RFBR (grants 05-04-48440 and 08-04-01322) and Minobrnauki RF (grant 2.1.1/6185).     

Studies on the cellular localization and distribution of glucocorticoid receptor and estrogen receptor immunoreactivity in the central nervous system of the rat and their relationship to the monoaminergic and peptidergic neurons of the brain

By means of monoclonal antibodies against the rat liver glucocorticoid receptor (GR) and the human estrogen receptor (ER), in combination with an immunocytochemical analysis, it has been possible to map out GR and ER immunoreactive (IR) neurons in the rat central nervous system and GR IR glial cells in the white matter. The GR IR is located in the cytoplasm and especially in the nucleus while the ER IR is only demonstrated in the nuclei of the neurons. Upon adrenalectomy the GR IR appears to be present exclusively in the cytoplasm, while after castration the ER IR is still exclusively present in the nuclei. Upon corticosterone treatment of the adrenalectomized rat the GR IR is again predominantly found in the nuclei of the neurons. These results indicate that the occupied GR and the unoccupied and occupied ER are located in the nuclei and the unoccupied GR in the cytoplasm. Evidence has been presented that large numbers of monoamine and peptide nerve cell bodies contain GR IR. Furthermore, neuronal GR IR is found in neuronal populations all over the central nervous system, especially in the cerebral cortex, the thalamus and the hypothalamus, indicating a major role of GR in regulating the metabolic and synaptic functions of the brain. The ER IR is instead limited to certain neuronal populations, mainly those of the preoptic area, the bed nucleus of the striae terminalis and the arcuate nucleus, suggesting a specific role in control of LHRH secretion and reproductive behaviour.     

Monoclonal antibody to rat brain actin antigenically enhanced with HVJ (Sendai Virus) M protein

Summary Monoclonal antibody to rat brain actin was easily produced using HVJ (Sendai Virus) M protein to enhance the antigenicity of the actin. This monoclonal antibody was determined to be IgM with a kappa light chain. By immunoblot analysis the antibody was also shown to react with rat brain actin but not with HVJ M protein on nitrocellulose sheets. Utilizing the antibody, neuronal cytoplasm in the cerebral cortex, the anterior and posterior horns in the spinal cord, the spinal ganglion and astrocytes showed positive immunohistochemical staining by light microscopy. However, Purkinje cells showed variable staining, some staining intensely, while others were negative. All of neurons in specific anatomical locations showed always positive staining but variable intensities. Vascular walls were stained only faintly. By electron microscopy, neuronal cytoplasm showed diffuse positive staining. Other areas showed a positive reaction, including dendrites, the postsynaptic densities, and a few capillary endothelial cells and arterial smooth muscle cells. The results suggest that the HVJ M protein was effective for producing monoclonal antibody to brain actin, and that the antibody could be utilized for the immunohistochemical study of neuronal elements in both normal and pathological conditions.     

Monoclonal antibody to rat brain actin antigenically enhanced with HVJ (Sendai virus) M protein

Monoclonal antibody to rat brain actin was easily produced using HVJ (Sendai Virus) M protein to enhance the antigenicity of the actin. This monoclonal antibody was determined to be IgM with a kappa light chain. By immunoblot analysis the antibody was also shown to react with rat brain actin but not with HVJ M protein on nitrocellulose sheets. Utilizing the antibody, neuronal cytoplasm in the cerebral cortex, the anterior and posterior horns in the spinal cord, the spinal ganglion and astrocytes showed positive immunohistochemical staining by light microscopy. However, Purkinje cells showed variable staining, some staining intensely, while others were negative. All of neurons in specific anatomical locations showed always positive staining but variable intensities. Vascular walls were stained only faintly. By electron microscopy, neuronal cytoplasm showed diffuse positive staining. Other areas showed a positive reaction, including dendrites, the postsynaptic densities, and a few capillary endothelial cells and arterial smooth muscle cells. The results suggest that the HVJ M protein was effective for producing monoclonal antibody to brain actin, and that the antibody could be utilized for the immunohistochemical study of neuronal elements in both normal and pathological conditions.     

BRAIN-SPECIFIC ANTIGENS IN THE QUAKING MOUSE DURING ONTOGENY

Abstract— By means of crossed Immunoelectrophoresis the concentrations of 7 brain-specific antigens have been investigated during the ontogenic development of normal and Quaking mice. Two proteins, the glial fibrillary acidic protein and the brain-specific membrane protein D5 were found to be strongly increased in mutant brains. The synaptosomal antigen synaptin (Cl), the 14-3-2 protein of neuronal cytoplasm, and the neuronal membrane antigens D1, D2 and D3 were all present at normal levels in mutant brains.     

Ultrastructural study of the Central Nervous System of Endemic Geocentrophora(Prorhynchida,Platyhelminthes) from Lake Baikal

The ultrastructure of the central nervous system of four species of the Geocentrophoragroup (Prorhynchida, Lecithoepitheliata) has been studied with Geocentrophora waginias a model organism. This is the first ultrastructural study of the nervous system in flatworms of the endemic Geocentrophoragroup from Lake Baikal. The neurons are characterized by invaginations of the plasma membrane with extracellular material (ECM) extending deep into the neuronal cytoplasm. Two types of neuron are distinguished on the basis of the character of the cytoplasm and the content of neuronal vesicles. Several more vesicle types are observed in the nerve processes in the neuropile. The following kinds of neuronal release sites are distinguished: single synapses, shared synapses, nonsynaptic release sites characterized by omega profiles and neuromuscular contacts. Special features of the synapses in the species of Geocentrophoraare pronounced local thickenings of the presynaptic membrane connected to paramembraneous densities. The ECM-filled invaginations and the local presynaptic thickenings are features that distinguish the neurons of Geocentrophoraspp. from previously described turbellarian neurons.     

Protein–protein interactions of huntingtin in the hippocampus

Molecular Biology - Huntingtin (HTT) occurs in the neuronal cytoplasm and can interact with structural elements of synapses. Huntington’s disease (HD) results from pathological expansion of a...     

Histochemical diagnosis of the neurons of cholinergic synaptic transmission

The authors studied neurons of the medulla oblongata of 5 human fetuses (22-27 weeks of development). Cholinacetyltransferase (CAT) activity was examined by the Berth method. Three neuronal types were diagnosed in the nuclei of the medulla oblongata with regard to CAT localization in the cytoplasm and synapses: (a) cholinergic-cholinoceptive neurons having CAT in the cytoplasm and in the innervating afferent fibers; (b) cholinergic-noncholinoceptive neurons with high CAT content, innervated with noncholinergic afferent fibers; (c) noncholinergic-cholinoceptive neurons carrying cholinergic synapses.