Behavior of nucleolus in the tobacco male meiocytes involved in cytomixis.

Behavior of nucleolus during the nuclear migration between plant cells (cytomixis) is studied for the first time in the tobacco male meiosis. As is shown, the nucleolus is located in a nonrandom manner in the migrating nuclei. In the majority of cases, the nucleolus resides on the nuclear pole strictly opposite to the cytomictic channel. Owing to this localization, the nucleolus extremely rare enters the recipient cell, so that the nucleolar material is in most cases undetectable in the micronuclei formed after cytomixis. When a whole nucleus migrates from a donor cell to recipient, the nucleolus can leave the nucleus and remain in the donor cells either alone or with a small amount of chromatin. The causes underlying a nonrandom location of the nucleolus in cytomictic cells are discussed. It is assumed that the nucleolar material contacts the cytoplasmic cytoskeleton, which prevents migration of the nucleolus into another cell within the nucleus. The potential use of cytomixis as a model for studying the nuclear motion is discussed.

Localization of rDNA at nucleolar structural components by immunoelectron microscopy.

Fluorescence in situ hybridization (FISH) shows that DNA encoding ribosomal RNA cistron (rDNA) is localized to small speckles scattered in the nucleolus and the nucleolus-associated chromatin (NAC). This technique cannot however precisely locate rDNA in the nucleolar ultrastructural components such as the fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC). In situ hybridization at the electron microscopic level is suitable for localization of rDNA at the ultrastructural level. We have tried to determine the precise localization of rDNA in the nucleolus of a higher plant by electron microscopic (EM) in situ hybridization using biotin-labeled 18S rDNA and demonstrated that it is exclusively localized in the fibrillar centers (FCs) and the nucleolus-associated chromatin (NAC). A secondary antibody coupled to the smallest (5 nm) colloidal gold particles was used in this technique to increase the label. Another important factor to increase the label was pretreatment with proteinase. Convincing results are obtained when the samples are pretreated with 1 microg/mL proteinase K for 45 min at 37 degrees C before immunogold labeling.

Elucidating chromatin and nuclear domain architecture with electron spectroscopic imaging.

Electron microscopy has been the 'gold standard' of spatial resolution for studying the structure of the cell nucleus. Electron spectroscopic imaging (ESI) offers advantages over conventional transmission electron microscopy by eliminating the need for heavy-atom contrast agents. ESI also provides mass-dependent and element-specific information at high resolution, permitting the distinguishing of structures that are primarily composed of protein, DNA, or RNA. The technique can be applied to understand the structural consequences of epigenetic modifications, such as modified histones, on chromatin fiber morphology. ESI can also be applied to elucidate the multifunctional behavior of subnuclear 'organelles' such as the nucleolus and promyelocytic leukemia nuclear bodies.

In situ comparative studies on subnucleolar distribution and configuration of plant rDNA.

The distribution and configurations of nucleolar DNA in Pisum sativum L., Allium sativum L., Triticum aestivum L. were analyzed by specific cytochemical staining using NAMA-Ur. It has been observed that in the nucleoli of different plant species, the DNA occupied different positions in different areas, which may imply a different status and strategy of rDNA transcription. Our results showed irregular clumps of rDNA surrounding FCs in semi-circular formations in P. sativum and T. aestivum, indicating a regular pattern of rDNA distribution and supporting the helix model of rDNA configuration. The rDNA was condensed in some regions and uncondensed in others. Nucleolus-associated chromatin extended from outside the nucleolus to the periphery of the FCs via nucleolar channels, which suggests a possible origin for nucleolar DNA.

The nucleolar structure and the activity of NopA100, a nucleolin-like protein, during the cell cycle in proliferating plant cells.

For the purpose of gaining knowledge of the relationships between cell proliferation and ribosome biogenesis, as two fundamental mutually interconnected cellular processes, studies were performed on cell populations synchronized in their cell-cycle progression by treatment with hydroxyurea, followed by sampling at different times after its removal. A structural rearrangement of the nucleolus was observed throughout the interphase, along with changes in the relative amounts of different nucleolar subcomponents. A structural model of nucleolar organization was associated with each interphase period. Throughout interphase, the nucleolin-like protein, NopA100, was immunodetected in the dense fibrillar component of the nucleolus, preferentially near fibrillar centers and its levels were shown to increase from G1 to G2. A western blotting analysis of soluble nuclear protein extracts with anti-NopA100 antibody resulted in the intense labeling of a 100-kDa band, but also of a series of proteins related to it, suggesting that NopA100 undergoes a physiological process of proteolytic maturation, similar to that described for mammalian nucleolin, but not reported in other biological model systems. Physiological proteolysis of NopA100, related to cell-cycle progression, was confirmed after the nuclei extracted from synchronized cells were treated with the protease inhibitor, leupeptin, which resulted in an increase of the 100-kDa band at the expenses of the decrease of some other bands, according to the cell-cycle stages. We therefore conclude that there is a relationship between the increase in nucleolar activity, cell-cycle progression, nucleolar structure, the activity of NopA100, and the proteolysis of this nucleolin-like protein.

Subcellular localization of cadmium in the root cells of Allium cepa by electron energy loss spectroscopy and cytochemistry.

The ultrastructural investigation of the root cells of Allium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver

Identification of specific plant nucleolar phosphoproteins in a functional proteomic analysis.

The soluble fraction of nuclear proteins is a functionally significant fraction, since it has been shown that it contains ribonucleoproteins active in nuclear RNA metabolism. The aim of this work was to detect variations associated with cell proliferation, by comparing two-dimensional proteomes obtained from the soluble fractions of onion nuclei isolated from actively proliferating root meristematic cells versus nonmeristematic root cells. In particular, we have studied the physicochemical features of the major nucleolar protein NopA100, a highly phosphorylated, nucleolin-like protein. A total of 384 spots were quantified in meristematic nuclei, while only 209 were detected in nonmeristematic nuclei. The comparison of both proteomes resulted in the determination of specific spots for each proliferative state and those which were common to both cases. Furthermore, among these latter, we could discriminate quantitative differences. Interestingly, well-known nucleolar proteins, such as RNA polymerase I, B23 and the nucleolin-like protein NopA100, were significantly increased in proliferating cells. Western blots with anti-NopA100 antibody demonstrated 26 spots in the meristematic sample. All the spots detected were clustered at 100 kDa and were distributed through an isoelectric point (pI) range of 4.3-6.6. In contrast, only seven spots were found in the extract from nonmeristematic nuclei, and the pI range was shortened to 4.8-6.1. These results indicate that the state of NopA100 phosphorylation correlates with the degree of nucleolar activity, i.e. the protein is more highly phosphorylated in cycling cells. We have also analyzed the bidimensional silver staining of the nucleolar organizing region (Ag-NOR) pattern of the soluble nuclear fraction in order to identify plant cell phosphoproteins that are considered to be markers of proliferation. These experiments demonstrated that NopA100, the onion, nucleolin-like protein, is an Ag-NOR protein. In addition we found that the plant homologue of the vertebrate nucleolar phosphoprotein B23 migrated as two clusters of acidic spots, 43 and 42 kDa respectively in molecular mass. The differences between these features and those described for mammalian cells is discussed. Our results demonstrate that the use of protein fractionation procedures with functional significance and the location of candidate spots by indirect techniques are advantageous, complementary methods to random selection procedures for proteomic studies involving further mass spectrometry analysis.

Deletion and site-specific mutagenesis of nucleolin's carboxy GAR domain.

Vertebrate nucleolin is an abundant RNA-binding protein in the dense fibrillar component of active nucleoli. Nucleolin is modular in composition. Its amino-terminal third contains alternating acidic and basic domains, its middle section contains four consensus RNA-binding domains (cRBDs), and its carboxy-terminus contains a distinctive glycine/arginine-rich (GAR) domain with several RGG motifs. The arginines within these motifs are asymmetrically dimethylated. Several laboratories have shown that the GAR domain is necessary but not sufficient for the efficient localization of nucleolin to nucleoli. We examined the distribution of endogenous fibrillarin, Nopp140, and B23 when full-length and DeltaGAR nucleolin were expressed exogenously as enhanced green fluorescent protein (EGFP)-tagged fusions. Only B23 redistributed when DeltaGAR-EGFP was expressed at moderate to high levels, suggesting an in vivo interaction between nucleolin and B23. Next we substituted all ten arginines within the GAR domain of Chinese hamster ovary (CHO) nucleolin with lysines to test the hypothesis that methylation of the carboxy GAR domain is necessary for the nucleolar association of nucleolin. The lysine-substituted mutant was not an in vitro substrate for the yeast protein methyltransferase, Hmt1p/Rmt1. It was, however, able to associate properly with interphase nucleoli and with interphase pre-nucleolar bodies upon recovery from hypotonic shock. We conclude, therefore, that although the GAR domain is necessary for the efficient localization of nucleolin to nucleoli, methylation of this domain is not required for proper nucleolar localization.

In situ visualization of rDNA arrangement and its relationship with subnucleolar structural regions in Allium sativum cell nucleolus.

We used a DNA-specific staining technique to show the two states of DNA component distributed in the nucleolar region of Allium sativum cells. One state is the extended DNA fiber, and the other is the condensed DNA clump. In situ hybridization demonstrated that the extended DNA fiber was an rRNA gene. Anti-fibrillarin antibody immunolabeling revealed that these rRNA genes were located in the dense fibrillar component near the fibrillar center, including at the periphery of the fibrillar center. None was in the dense fibrillar component far away from the fibrillar center. The condensed DNA clump was located in the fibrillar center. Further observations showed that the rRNA genes in the nucleolus were all arranged around the fibrillar center and associated with the DNA clumps in the fibrillar center. Results of statistical analysis showed that the distribution region of rRNA genes occupied about one-third of the total dense fibrillar component region. Ag-NOR protein showed a similar distribution pattern to that of rDNA. Immunolabeling of an anti-RNA/DNA hybrid antibody demonstrated that the transcription sites of rRNA were located at the periphery of the fibrillar center and in the dense fibrillar component near the fibrillar center, and these sites were consistent with the location and arrangement of rDNA shown in situ. These results demonstrated that transcription of rRNA takes place around the fibrillar center and at the periphery, whereas the dense fibrillar component that was far away from fibrillar center was the non-transcription region. The DNA clumps within the fibrillar center were probably the anchoring sites for rDNA arrangement.

Effects of hyperthermia and differentiation on cultured Dunn osteosarcoma cells.

We investigated the characteristics of morphology, DNA synthesis and argyrophilic nucleolar organizer regions (AgNORs) on murine Dunn osteosarcoma cells in response to heat (42 degrees C, 1 h) or dibutyryl cyclic adenosine 3',5'-monophosphate (Bt(2)cAMP). The cell morphology changes to a fibroblast-like appearance with long and thin protoplastic processes with the reduction of DNA synthesis by heat. It is closely similar to a response by Bt(2)cAMP. In the presence of 3 mM Bt(2)cAMP, the mean number of AgNORs was significantly decreased in 48 h compared with the untreated group. It was increased conversely by heat. Among these responsive cells, we can also find many cells stained without discrimination by the use of AgNORs staining. The present study provides a new clue to support differentiation of osteosarcoma cells from the viewpoint of hyperthermia in vitro.

Combination of electron microscopic in situ hybridization and anti-DNA antibody labelling reveals a peculiar arrangement of ribosomal DNA in the fibrillar centres of the plant cell nucleolus.

The fibrillar centres (FCs) in the nucleoli of Allium cepa usually contained compact dense chromatin, which was always surrounded with light fibrous material (LFM). Distribution of 18S ribosomal DNA (rDNA) in the FCs was examined by in situ hybridization at the light and electron microscopic levels and the results were compared with those obtained by immunogold labelling with anti-DNA antibodies. Anti-DNA antibodies heavily labelled the dense chromatin of the FCs but scarcely labelled the LFM. However, electron microscopic in situ hybridization using the 18S rDNA probe showed that the label in the dense chromatin was extremely weak compared with that obtained by the anti-DNA antibody labelling: the specific label with anti-DNA antibodies of the dense chromatin was about 15 times as much as that of the LFM, whereas the specific label with in situ hybridization in the dense chromatin was only about 1.7 times higher than in the LFM. These results suggest that the rDNA encoding rRNA is preferentially released from the dense chromatin and that non-transcribed intergenic spacers remain in the dense chromatin as the anchoring sites of rDNA.

Effect of cadmium on the nucleoli of meristematic cells of onion Allium cepa L: an ultrastructural study.

This study of the effect of cadmium on nucleolar ultrastructure was carried out with meristematic cell populations of Allium cepa L. Meristems, grown at 25 degrees C, were treated with 10 ppm cadmium chloride. Conventional and silver staining techniques were carried out, and the ultrastructure was analyzed using electron microscopy. Observation showed alterations in the nucleoli of the cells that had been treated with cadmium and this effect varied according to the time of exposure to the metal. After 4h of treatment, nucleolar segregation was observed in interphase, probably because of the effect of cadmium on the synthesis of ribosomal RNA precursors. A decrease in the fibrillar to granular component ratio also occurred in the cells exposed to Cd2+ for 8 h. Some changes were observed in the G1 cells; their chromatin still remained very condensed, and prenucleolus bodies remained scattered within the nucleus. At the same time, there was a large amount of interchromatin granules. These changes produced by cadmium resembled those produced during inhibition of RNA synthesis. The fibrillar bodies, another morphologic feature, resulting from a blocked transcription, were also evidenced. All these observations suggest that one of the ways that cadmium exercises its toxicity is by altering the biosynthesis of the preribosomal RNA precursor.

Jun, Fos and Krox in the thalamus after C-fiber stimulation: coincident-input-dependent expression, expression across somatotopic boundaries, and nucleolar translocation.

Expression of the inducible transcription factors Jun, Fos and Krox is commonly used to map neurons in the brain that are activated by sensory inputs. However, some neurons known to be electrically excited by such inputs do not always express these factors. In particular, stimulation of hindlimb sensory nerve C-fibers induces expression of c-Fos in the medial thalamus (the mediodorsal, intermediodorsal, centrolateral and centromedial), but not in the lateral thalamus (the ventroposterolateral, ventroposteromedial and posterior group). We hypothesized that c-Fos expression might only occur in these lateral areas after more complex stimulation patterns, or that only other transcription factors can be induced in these areas by such stimuli. Thus we examined the effects of single, repeated and coincident C-fiber inputs on expression of six inducible transcription factors in the medial, lateral and reticular thalamus of the rat. A weak C-fiber input caused by noxious mechanical stimulation of the skin of one hindpaw did not induce expression of c-Fos, FosB, Krox-20 or Krox-24; but it did reduce the basal expressions of c-Jun and JunD in both the medial and lateral areas. An intense input produced by electrical stimulation of all the C-fibers in one sciatic nerve also failed to induce expression of c-Fos, FosB, Krox-20 or Krox-24 in the medial or lateral areas. However, in the medial thalamus it increased c-Jun and reduced the basal expression of JunD, whereas in the lateral thalamus it had no effect on c-Jun but again reduced the basal expression of JunD. With repeated stimulation, i.e. when the noxious stimulus was applied to the contralateral hindpaw 6 h after the sciatic stimulation, there was again no induction of c-Fos, FosB or Krox-20 in the medial thalamus; but there was an increase in c-Jun and Krox-24, and a decrease in JunD levels. In the lateral thalamus the repeated stimulation again failed to induce c-Fos, but the expressions of FosB, c-Jun and Krox-24 were increased, and that of JunD was again reduced. With coincident stimulation, i.e. when a stimulus was applied to each hindpaw simultaneously, c-Fos and Krox-24 remained absent; but there was a marked induction of FosB and Krox-20, a strong repression of c-Jun, and no effect or a reduction of the basal levels of JunD. This coincident stimulation also caused FosB to appear in the nucleolus of many thalamic neurons. MK-801, but not L-NAME, blocked all these changes. In summary, noxious stimulation affects the expression of all transcription factors in the medial, lateral and reticular thalamus in a complex manner depending upon the inducible transcription factor considered, the thalamic nucleus, and the stimulation paradigm. The expression of some transcription factors uniquely after simultaneous inputs suggests they act as coincidence detectors at the gene level.

Structural components of the nuclear body in nuclei of Allium cepa cells.

Nuclear bodies have long been noted in interphase nuclei of plant cells, but their structural component, origin and function are still unclear by now. The present work showed in onion cells the nuclear bodies appeared as a spherical structure about 0.3 to 0.8 microm in diameter. They possibly were formed in nucleolus and subsequently released, and entered into nucleoplasm. Observation through cytochemical staining method at the ultrastructural level confirmed that nuclear bodies consisted of ribonucleoproteins (RNPs) and silver-stainable proteins. Immunocytochemical results revealed that nuclear bodies contained no DNA and ribosomal gene transcription factor (UBF). Based on these data, we suggested that nuclear bodies are not related to the ribosome or other gene transcription activities, instead they may act as subnuclear structures for RNPs transport from nucleolus to cytoplasm, and may also be involved in splicing of pre-mRNAs.

Distribution and transcription activity of nucleolar DNA in higher plant cells.

By using the NAMA-Ur DNA selective staining method, we have observed in situ the location of nucleolar DNA in onion cells and found it at the boundary between fibrillar centres (FC) and dense fibrillar component (DFC) in transcriptionally active nucleolus. We have also used anti-NOR serum, which is identified as the RNA Polymerase I transcription factor (UBF) antibody, to study its reactivity with higher plant cells and demonstrated this factor associated to the DFC but not present at the interior of FC. Finally, by employing anti-DNA/RNA hybrid antibodies, we labeled the transcriptionally active rRNA genes in active nucleolus and testified that at the boundary between FC and DFC. The results provide the evidence that the boundary between FC and DFC is the genuine transcription site of rRNA genes in nucleolus.

High resolution detection of rRNA and rDNA in plant nucleoli with different activities by in situ hybridization.

In the present work we perform in situ hybridization with probes to different stretches of rDNA and electron microscopy of nucleoli with different activities, to gain insight into the ultrastructural organization of transcription and processing in the plant nucleolus. The main ultrastructural nucleolar components: fibrillar centers (FC), dense fibrillar component (DFC), and granular component (GC), are arranged in different ways depending on nucleolar activity. Heterogeneous FCs containing RNP fibrils and nucleolar perichromatin granules are frequently seen in nucleoli in the process of activation. DNA-RNA in situ hybridization with biotinylated probes spanning different sequences of the rDNA unit followed by immunogold detection of biotin, demonstrated the localization of the ribosomal transcripts in DFC, mainly in the zones around the FCs, in GC, and in the periphery of pale FC. The internal region of the heterogeneous FCs is labeled only in cells in the process of activation of transcription after dormancy. The distribution of the U3 probe indicates that the processing of the rRNA takes place in the DFC and inside the heterogeneous FCs, in which transcription occurs. DNA-DNA hybridization demonstrates the presence of rDNA in the compact and extended chromatin located in the interior and at the periphery of FCs and in nucleolar associated chromatin. Our results support the view that the plant nucleolus has a highly dynamic morphological and functional organization composed of a bipartite domain formed by FCs surrounded by DFC, which is associated with rRNA transcription and processing, and the GC representing a store of preribosomal particles.

Simultaneous localization of transcription and early processing markers allows dissection of functional domains in the plant cell nucleolus.

Nucleolar transcription in isolated onion cell nuclei was visualized, after Br-UTP incorporation, under the conventional fluorescence microscope, the confocal microscope, and the transmission electron microscope. The confocal microscopy study of transcription was combined with immunodetection of fibrillarin, a component of the RNP complex involved in the early processing of pre-rRNA. Superposition of transcription and fibrillarin images from the same optical section showed some small "black holes" in the nucleolus, around which a lateral and radial differentiation of labeling was observed: laterally, zones corresponding to transcription labeling alternated with zones of fibrillarin labeling; radially, areas of transcription gradually became areas of colocalization of transcription and fibrillarin, and, further outward, of fibrillarin alone, which occupied the major part of the labeled nucleolar area. Three-dimensional reconstruction of the nucleolar transcription labeling, from confocal optical sections, showed clusters of foci arranged around an area of low or no labeling. Thin labeled extensions, connecting single foci, were observed. Visualization of transcription at the ultrastructural level identified the black holes as fibrillar centers, in view of their size and the absence of labeling in them. In fact, most of the labeling was observed in discrete areas of the dense fibrillar component, near fibrillar centers, including the transition area between these two components. This observation was supported by a quantitative study. Otherwise, the outline of fibrillar centers did not appear entirely surrounded by particles, and a minor proportion of particles was detected dispersed throughout the dense fibrillar component. As a complementary study, the transcription factor upstream binding factor (UBF) and the protein NopA64, a plant nucleolin homologue, were immunolocalized. Small foci of UBF localization alone and other foci in which the two protein markers overlapped were observed. The outer areas of the nucleolus showed the exclusive presence of NopA64. Under the electron microscope, UBF labeling, quantitatively assessed, appeared as clusters of particles, most of them surrounding fibrillar centers. A graphic model is presented to give a molecular interpretation of these data.

[Nucleolar apparatus of neurosecretory cells in the hypothalamus of rats of different age during acute immobilization stress]. / Kharakteristika iadryshkovogo apparata neirosekretornykh kletok gipotalamusa u krys raznogo vosrasta pri ostroi immobilizatsii zhivotnykh.

To determine condition and adaptive capacity of cell nucleoli at ageing, the effect of acute immobilization on the hypothalamic neurosecretory cells was investigated in young and old male Wistar rats. Using immunohistochemical methods and nucleolometry we have shown that: 1) the nuclear volume in all neurosecretory cells is increased; 2) the share of cells, containing nucleoli with marginal position or multiple nucleoli in the nuclei, displays opposite changes in young and in old rats under stress condition. We suppose that adaptive mechanisms are different in young and old animals. Although, both kinds of morphological reorganization result in the increase in functional activity.

Cofactors in in vitro induction of apoptosis in HL60 cells by all-trans retinoic acid (ATRA).

The aim of this study was to determine the culture conditions that could modulate the induction of apoptosis by all-trans retinoic acid (ATRA). Cell viability was evaluated by trypan blue test, differentiation by nitro blue tetrazolium test, and apoptosis by morphological analysis. ATRA induced apoptosis in HL60 cells only when more than 100,000 cells/mL were seeded, while differentiation was induced regardless of the seeded cell concentration. Reduction in the concentration of foetal calf serum or glutamine in the medium led to a weak increase in apoptosis. In contrast, a dramatic enhancement of apoptosis occurred when the culture medium was supplemented with glucose or when the culture pH was decreased. These characteristics were independent of the mechanism of action of ATRA, but the action of glucose could be of significance in diabetic patients. An exchange of supernatants after 3 days of culture showed that supernatants from control cultures seeded at high cell density were better apoptosis inducers than supernatants from cultures treated with ATRA, but seeded at low cell density. Factor(s) in this supernatant which induced apoptosis was (were) removed by ultrafiltration. In conclusion, our results showed that ATRA alone cannot induce apoptosis, but can do so in conjunction with cofactors. The depletion of some components of the medium and the appearance of secreted macromolecule(s) could be cofactor(s) in the induction of apoptosis.

[Changes in the oxytocinergic neurosecretory cells in the accessory magnocellular neuroendocrine nuclei of the rat hypothalamus during aging]. / Izmeneniia oksitotsinergicheskikh neirosekretornykh kletok v dobavochnykh krupnokletochnykh neiroéndokrinnykh iadrakh gipotalamusa u krys pri starenii.

Histophysiology of accessory magnocellular nuclei of hypothalamus was studied in young (3-6 months) and old (14-29 months) male Wistar rats. Using non-labeled antibodies to oxytocin and karyolometry activation of protein synthesis combined with inhibition of oxytocin transport along the fibres was shown to occur in 4 (circular, fornical, dorso- and ventrolateral) of 5 accessory nuclei in old rats. This was not observed in anterior commissural nucleus. A hypothesis on compensatory role of accessory magnocellular nuclei of hypothalamus in regulation of neuroendocrine homeostasis in old rats is discussed.