A survey of genetic diversity and reproductive biology of Puya raimondii (Bromeliaceae), the endangered queen of the Andes.

Puya raimondii Harms is an outstanding giant rosette bromeliad found solely around 4000 m above sea level in the Andes. It flowers at the end of an 80 - 100-year or even longer life cycle and yields an enormous (4 - 6 m tall) spike composed of from 15,000 to 20,000 flowers. It is endemic and currently endangered, with populations distributed from Peru to the north of Bolivia. A genomic DNA marker-based analysis of the genetic structure of eight populations representative of the whole distribution of P. raimondii in Peru is reported in this paper. As few as 14 genotypes out of 160 plants were detected. Only 5 and 18 of the 217 AFLP marker loci screened were polymorphic within and among these populations, respectively. Four populations were completely monomorphic, each of the others displayed only one to three polymorphic loci. Less than 4 % of the total genomic variation was within populations and genetic similarity among populations was as high as 98.3 %. Results for seven cpSSR marker loci were in agreement with the existence of a single progenitor. Flow cytometry of seed nuclear DNA content and RAPD marker segregation analysis of progeny plantlets demonstrated that the extremely uniform genome of P. raimondii populations is not compatible with agamospermy (apomixis), but consistent with an inbreeding reproductive strategy. There is an urgent need for a protection programme to save not only this precious, isolated species, but also the unique ecosystem depending on it.

Association of DNAse sensitive chromatin domains with the nuclear periphery in 3T3 cells in vitro.

DNAse sensitive chromatin, putative transcriptionally competent sequences, exists either as pan-nuclear speckles in cells with nuclei which exhibit a flat geometry, or as a shell apposed to the nuclear envelope in cells with spheroidal nuclei. To test the hypothesis that DNAse sensitive chromatin is similarly associated with the nuclear periphery in cell types with a very flat geometry such as 3T3 fibroblasts, cells were subjected to hypotonic expansion to change their nuclei from a flat ellipsoid to a spheriod. This was based on the assumption that such a spatial association is not resolvable due to the interdigitation at the nuclear midplane of DNAse sensitive chromatin associated with the upper and lower nuclear surfaces. In situ nick translation was used to visualize the distribution of DNAse sensitive chromatin as a function of nuclear geometry. Both unexpanded and expanded cells exhibit DNAse sensitive chromatin as a dome at the apical side of the nucleus, i.e., that aspect of the cell facing the culture medium. The results argue for a polarized association of DNAse sensitive chromatin with the nuclear envelope and indicate that the nuclear periphery may function as a compartment for the spatial coupling of transcription and nucleo-cytoplasmic transport.

Nuclear topology of murine, cerebellar Purkinje neurons: changes as a function of development.

The interphase nucleus is a structurally ordered, three-dimensional structure, in which specific chromatin domains occupy distinct spatial positions that can, in turn, be modified with changes in cell function. A fundamental goal in developmental neurobiology is the identification of mechanisms that dictate the orderly expression of genes in a cell-specific manner. Given that different neuronal populations feature a characteristic spatial topology of centromeric sequences, the positioning of specific DNA sequences may constitute such a mechanism. We tested the hypothesis that the cell-specific nuclear topology in fully differentiated neurons is acquired before or during that stage at which neuron-specific sequences are first expressed. For this, we assessed the number and spatial distribution of centromeric domains in the murine, cerebellar Purkinje neuron as a function of postnatal development. Centromeric domains were localized by immunofluorescence of centromere-associated kinetochore proteins and visualized by confocal microscopy. Kinetochores are known to cluster in Purkinje neurons. Thus, the number of signals discerned is always less than the chromosome complement of the species. The number of signals observed in adults (10.8 +/- 0.46) (mean +/- SEM) is established by postnatal day 15 (P15), after a transient decrease from 11.44 +/- 0.44 at P0 to 8.78 +/- 0.24 at P3. The distribution of signals characteristic of the adult, with the majority located at the nucleolus, is established by P5 and is associated with a decrease in the fraction of signals at the nuclear periphery. These changes are temporally associated with the onset of processes such as dendritic differentiation and synaptic maturation and might serve the process of differentiation by placing specific sequences into transcriptionally competent, nuclear sites.

Dynamics of structure-function relationships in interphase nuclei.

The interphase nucleus is a topologically ordered, three-dimensional structure. While it remains unclear whether this structural organization also represents compartmentalization of function, the presence of the latter would likely be reflected in the spatial coupling of molecular factors involved in related events. This review summarizes morphological evidence, derived from in situ experiments, which indicates the existence of compartmentalization of both chromatin and non-chromatin components in the interphase nucleus. Moreover, the review addresses the spatial relationships of these components relative to each other and correlates these spatial relationships with such nuclear functions as transcription, splicing and nucleo-cytoplasmic transport of pre-mRNA. Given that it is increasingly recognized that such spatial relationships are dynamic, the review also addresses the emerging concept that the spatial intranuclear organization changes with changes in cell function, a concept which supports the hypothesis that the spatial organization of the interphase nucleus may represent one of the fundamental control mechanisms in gene expression.

Kindling-induced neurogenesis in the dentate gyrus of the rat.

Kindling, a form of neuronal plasticity produced by repeated low intensity electrical brain stimulation, leads to epileptic seizures. To address possible causes of this phenomenon, we have prepared amygdala-kindled animals and measured neurogenesis, by bromodeoxyuridine incorporation. Early, when focal seizures were present, there was no evidence of a change in the rate of hippocampal neurogenesis. In contrast, during the later phases of kindling, when secondary generalization was well established and motor seizures were present, neurogenesis was enhanced by 75-140%, depending on the hippocampal region. Double labelling with the neuron-specific marker TOAD-64 demonstrated the presence of numerous new-born granule neurons in the kindled animals. We propose that the newly-born neurons contribute to the cellular changes and behavioral symptoms associated with this type of epileptiform brain plasticity.

A specific conformation of the territory of chromosome 17 locates ERBB-2 sequences to a DNase-hypersensitive domain at the nuclear periphery.

Chromatin in interphase nuclei exhibits a topology that is associated with the transcriptional state of cells. We examined the spatial, intranuclear distribution of chromosome 17 and the ERBB-2 (HER2/neu) sequence thereon, relative to that of DNase-hypersensitive chromatin (DHC), in breast tumour cells exhibiting different levels of expression of ERBB-2. These sequences were specifically associated with the nuclear periphery, within a band of DHC. The remainder of the chromosome 17 mass showed no preferential position within the nucleus. The peripheral placement of ERBB-2 sequences is associated with a specific conformation of chromosome 17. We propose that the conformational organization of chromosome territories might represent a fundamental control mechanism in gene expression.

Changes in morphology and spatial position of coiled bodies during NGF-induced neuronal differentiation of PC12 cells.

Interphase nuclei are organized into structural and functional domains. The coiled body, a nuclear organelle of unknown function, exhibits cell type-specific changes in number and morphology. Its association with nucleoli and with small nuclear ribonucleo-proteins (snRNPs) indicates that it functions in RNA processing. In cycling cells, coiled bodies are round structures not associated with nucleoli. In contrast, in neurons, they frequently present as nucleolar "caps." To test the hypothesis that neuronal differentiation is accompanied by changes in the spatial association of coiled bodies with nucleoli and in their morphology, PC12 cells were differentiated into a neuronal phenotype with nerve growth factor (NGF) and coiled bodies detected by immunocytochemical localization of p80-coilin and snRNPs. The fraction of cells that showed coiled bodies as nucleolar caps increased from 1.6 +/- 0.9% (mean +/- SEM) in controls to 16.5 +/- 1.6% in NGF-differentiated cultures. The fraction of cells with ring-like coiled bodies increased from 17.2 +/- 5.0% in controls to 57.8 +/- 4.4% in differentiated cells. This was accompanied by a decrease, from 81.2 +/- 5.7% to 25.7 +/- 3.1%, in the fraction of cells with small, round coiled bodies. SnRNPs remained associated with typical coiled bodies and with ring-like coiled bodies during NGF-induced recruitment of snRNPs to the nuclear periphery. Together with the observation that coiled bodies are also present as nucleolar caps in sensory neurons, the results indicate that coiled bodies alter their morphology and increase their association with nucleoli during NGF-induced neuronal differentiation.

Spatial repositioning of centromeric domains during regrowth of axons in nuclei of murine dorsal root ganglion neurons in vitro.

Specific chromatin domains within interphase nuclei are organized in cell type specific distributions and are rearranged in association with changes in cell function. Axotomy leads to changes in gene expression. Dorsal root ganglion (DRG) neurons in vitro are a model for axotomy because they are detached from their axons in preparation for the culturing procedure. In a test of the hypothesis that neurons regrowing in vitro undergo rearrangement of specific chromatin domains, changes in the distribution of centromere-associated kinetochores proteins within DRG neurons were assessed as a function of time in vitro. Camparison of the kinetochore distributions in neurons in situ to those 24 h after placement into culture showed that the mean proportion (+/-S.E.M.) of kinetochore signals in the karyoplasm decreased from 41.0 +/- 1.8% to 28.6 +/- 3.3%, while the proportion at the nucleolus increased from 35.2 +/- 2.0% to 48.4 +/- 2.9%. This indicated redistribution of centromeric domains to the nucleolus. Between 1 day and 16 days in vitro, signals were redistributed to the nuclear periphery, indicated by an increase in the proportion of signals in this nuclear compartment from 23.0 +/- 4.3% to 37.6 +/- 3.4% and a decrease in the proportion of signals from 48.4 +/- 2.9% to 23.0 +/- 2.3% at the nucleolus. The results indicate that neurite regrowth following axotomy is associated with changes in nuclear topology. The reorganization that occurs within 24 h is speculated to be associated with a recapitulation of a cytoskeletal development program, while later changes in centromeric distributions may be related to cues elicited by in vitro conditions.

Transposition of DNase hypersensitive chromatin to the nuclear periphery coincides temporally with nerve growth factor-induced up-regulation of gene expression in PC12 cells.

To test the hypothesis that the nonrandom organization of the contents of interphase nuclei represents a compartmentalization of function, we examined the relative, spatial relationship of small nuclear ribonucleoproteins (snRNPs) and of DNase I hypersensitive chromatin (DHC) in rat pheochromocytoma cells. In controls, DHC and snRNPs colocalized as pan-nuclear speckles. During nerve growth factor-induced differentiation, both snRNPs and DHC migrated to the nuclear periphery with the migration of DHC preceding that of snRNPs, resulting in their transient separation. The formation of DHC shells temporally coincided with an up-regulation of neurofilament light chain mRNA. This indicates that the expression of this sequence may be associated with its spatial transposition to the nuclear periphery.

Dopamine secretion by PC12 cells microencapsulated in a hydroxyethyl methacrylate--methyl methacrylate copolymer.

A rat pheochromocytoma cell line (PC12) was encapsulated in a water-insoluble hydroxyethyl methacrylate-methyl methacrylate copolymer by interfacial precipitation from a polyethylene glycol 200 solution into phosphate-buffered saline. The resulting capsules (660 +/- 44 microns in diameter; 84 +/- 27 microns wall thickness) contained viable PC12 cells in a spheroidal arrangement, much like tumour spheroids, the latter grown on surfaces unsuitable for cell attachment. In these spheroids, the viable cells formed a band approximately 100 microns thick, surrounding an inner core of necrotic cells. A similar arrangement was seen 14, 28 and 42 days after encapsulation, with capsules maintained in an in vitro tissue culture environment; the annular ring was roughly constant in size, although the packing density appeared to increase over the 6 week observation period. During the first 4 weeks, when measurements were made the encapsulated cells converted a tetrazolium dye (MTT) into an insoluble formazan product, in a time-after-encapsulation-dependent manner. This indicated that PC12 cells retained viability despite encapsulation and an ability to increase (at least in part) their metabolic capacity, presumably by a combination of proliferation and altered cellular activity. The encapsulated PC12 cells also secreted dopamine when incubated in a high potassium release medium but not in a low potassium, conventional tissue culture medium (RPMI 1640). Consistent with the MTT results, the amount of dopamine released was also dependent on the time after encapsulation, as well as the cell density at the time of encapsulation.

Organization of centromeric domains in hepatocyte nuclei: rearrangement associated with de novo activation of the vitellogenin gene family in Xenopus laevis.

The existence of a function-dependent, nonrandom organization of chromatin domains within interphase nuclei is supported by evidence which suggests that specific chromatin domains undergo spatial rearrangement under conditions which alter gene expression. Exposure to estrogen of male Xenopus laevis hepatocytes in vitro results in de novo activation of vitellogenin mRNA production and vitellogenin protein synthesis and provides an ideal model to study the association between chromatin organization and changes in gene expression. In a test of the hypothesis that the de novo induction of vitellogenesis in male X. laevis is associated with a spatial rearrangement of specific chromatin domains, centromeric regions were localized by immunofluorescent labeling of associated kinetochore proteins in naive and in estrogen-treated, vitellogenic cells. Analyses by confocal scanning laser microscopy of the three-dimensional spatial distribution of kinetochores in estrogen-treated male hepatocytes showed that a significantly greater proportion of signals was associated with the nuclear periphery than in non-estrogen-treated, naive male cells. In hepatocyte nuclei, quantification of kinetochore signal sizes using image analysis showed that these signals were fewer in number and showed greater variation in size than those of cells in metaphase, with larger signals exhibiting total normalized fluorescence intensities of two, three, four, and five times that associated with kinetochore signals of metaphase cells. These observations are taken to reflect the existence of clustering of kinetochores and, by extension, of centromeres in these cells. In summary, the results show that centromeric domains within interphase nuclei of Xenopus hepatocytes occur as clusters and that these domains undergo spatial rearrangement under conditions which alter the transcriptional state of the cell.

Ultrastructural localization of filamentous actin within neuronal interphase nuclei in situ.

Previous biochemical studies utilizing isolated nuclei and nuclear matrices have shown actin to be a constituent of the interphase nucleus. In addition, recent ultrastructural work has shown the presence of actin and myosin within nuclei of interphase cells in situ. It was unclear, however, whether this intranuclear actin is present in the unpolymerized globular actin or the filamentous (F)-actin form. The present work, using confocal microscopy and ultrastructural cytochemical techniques, demonstrates the presence of F-actin within interphase nuclei of intact, uncompromised, dorsal root ganglion neurons in vitro and in vivo. Labeling by FITC-phalloidin detected the presence of intranuclear F-actin adjacent to the nucleolar periphery in a small fraction of cells in vitro, an observation confirmed by three-dimensional reconstruction. Ultrastructural analyses of cells exposed to heavy meromyosin (HMM), showed the presence of typical "arrowhead" complexes. The observation that these complexes were associated with nucleoli confirms that the intranuclear ligand detected by FITC-phalloidin indeed represents F-actin. Postembedding labeling with HMM conjugated to 20-nm colloidal gold (HMM-Au20) resulted in labeling similar to that obtained with HMM. However, HMM-Au20 was found to label a much larger fraction of cells, both in vitro and in vivo, than did FITC-phalloidin or HMM. This finding indicates that labeling with HMM-Au20 more accurately reflects the extent of actin polymerization in nuclei. Results from double labeling with HMM-Au20 and an antibody to alpha-sarcomeric actin confirmed that only a small amount of nuclear actin is in the F-form. Together, these results represent a first ultrastructural demonstration of the presence of F-actin in nuclei of neurons. While the role of nuclear F-actin has yet to be defined, the results suggest that F-actin may represent a component of the molecular motor responsible for the dynamic positioning of specific chromatin domains into the tissue-specific, nonrandom patterns observed in many cell types.

Cytochemical localization of actin and myosin aggregates in interphase nuclei in situ.

Biochemical and ultrastructural studies on isolated nuclear compartments have previously shown actin and myosin to be constituents of interphase nuclei. In the present work, immunocytochemistry, in conjunction with confocal microscopy and ultrastructural immunogold techniques, shows that interphase nuclei of intact dorsal root ganglion neurons and of PC12 cells contain actin and myosin. Nuclear actin was observed to be distributed throughout the nucleoplasm occurring as distinct aggregates. Frequently, prominent actin aggregates were associated with the nucleolar periphery, often near nucleolar satellites. Ultrastructurally, actin was observed to be associated with linear, electron-dense structures, putatively identified as chromatin fibers, extending from nucleoli. Use of three antibodies against subclasses of alpha-actin isoforms revealed that nuclear actin is more closely related to alpha-sarcomeric actin than to alpha-smooth muscle actin. Those aggregates associated with the nucleolus were found to be in the polymerized F-actin form, in a small fraction of neurons, as assessed by FITC-phalloidin. A myosin-like antigen was also observed to occur as intranuclear aggregates. Quantitative assays of the distribution of actin and myosin aggregates by nearest neighbour analysis indicated a distribution characterized as uniform and failed to reveal statistically significant associations between any set of aggregates. The evidence presented herein indicates that actin and myosin are constituent proteins of interphase nuclei in situ of both normal mammalian and transformed mammalian cells.

Distribution of snRNPs, splicing factor SC-35 and actin in interphase nuclei: immunocytochemical evidence for differential distribution during changes in functional states.

Small nuclear ribonucleoproteins (snRNPs) play an integral role in the processing of pre-mRNA in eukaryotic nuclei. snRNPs often occur in a speckled intranuclear distribution, together with the non-snRNP splicing factor SC-35. snRNPs have also been shown to be associated with actin in the nuclear matrix, suggesting that both actin and snRNPs may be involved in the processing and transport of transcripts. The work reported here was undertaken to compare the spatial relationship of snRNPs, SC-35, and intranuclear actin in neuronal and non-neuronal cell types. In undifferentiated PC12 cells and in non-neuronal cells growing in association with dorsal root ganglion neurons, confocal immunocytochemistry revealed a typical, speckled distribution of snRNP aggregates, which colocalized with the SC-35 splicing factor. In contrast, a unique snRNP distribution was observed in dorsal root ganglion neurons in vitro and in PC12 cells differentiated by nerve growth factor. In nuclei of these cells, snRNPs were predominantly located at the periphery where they formed a spherical shell apposed to the nuclear envelope. Ultrastructural immunogold labelling of snRNPs in dorsal root ganglion neurons in vitro confirmed this distribution. In contrast, SC-35 remained distributed in a speckled pattern throughout nuclei of dorsal root ganglion neurons and PC12 cells, even in cases where snRNPs were almost exclusively positioned at the nuclear periphery. In non-neuronal cells in dorsal root ganglion cultures and in undifferentiated PC12 cells, snRNP aggregates were frequently associated with actin aggregates, as determined by Nearest Neighbor Analyses. In PC12 cells, this spatial relationship was altered during nerve growth factor-induced differentiation, prior to the time at which these cells showed morphological evidence of differentiation. Specifically, Nearest Neighbor Analyses between snRNP and actin aggregates in PC12 cells exposed to nerve growth factor for 4 hours revealed that snRNP and actin aggregates exhibited a closer association than in undifferentiated cells. These results suggest that sites of pre-mRNA processing and transcription may differ between cell types, and that the functions of snRNPs and actin within interphase nuclei may be related. The results also indicate that the distribution of snRNPs is dynamic and that it may depend upon the functional state of the cell as well as upon its state of differentiation.

Diethylstilbestrol alters the morphology and calcium levels of growth cones of PC12 cells in vitro.

Diethylstilbestrol (DES) is a synthetic estrogen with carcinogenic properties. DES is known to alter cytoskeletal components, including the organization of actin stress fibres in C6 rat glioma cells. In a test of the hypothesis that DES disrupts actin filaments of growth cones in neuron-like cells, DES-induced changes in filopodial lengths were quantified in rat pheochromocytoma (PC12) cells in vitro. DES significantly altered growth cone morphology, with collapse of growth cone filopodia and neurite retraction invariably occurring at a concentration of 10 microM. At 5 microM DES, transient reductions in total filopodial lengths occurred. At DES concentrations of 0.1 nM and 1 nM, reductions in total filopodial lengths occurred in a fraction of growth cones. Evidence exists which shows that growth cone activity and morphology are intimately linked to levels of intracellular, free calcium and that DES increases such levels. Measurements of free intracellular calcium levels by fluorescence microscopy, at times concurrent with the DES-induced reduction in total filopodial lengths, showed that calcium levels were indeed significantly increased by 10 microM DES. Labelling of filamentous actin (f-actin) with FITC-phalloidin showed that the f-actin distribution in growth cones exposed to DES could not be differentiated from the distribution found in spontaneously retracting growth cones. Together with evidence which showed that growth cone motility was not affected, the results are taken to indicate that DES, rather than acting directly on the cytoskeleton, exerts its effects indirectly, by a calcium-induced destabilization of actin filaments in the growth cone.

Spatial rearrangement and enhanced clustering of kinetochores in interphase nuclei of dorsal root ganglion neurons in vitro: association with nucleolar fusion.

Interphase nuclei of several cell types display distinct, nonrandom arrangements of specific chromatin domains. It has been suggested that this arrangement is associated with the functional commitment of the cell and results from compartmentalization of specific DNA sequences to transcriptionally competent sites. In a test of the hypothesis that such topological organization is established during cellular differentiation, the spatial distribution of centromeres was determined, in dorsal root ganglion neurons in vitro, using immunocytochemistry in conjunction with fluorescence microscopy, confocal laser microscopy, and ultrastructural immunogold techniques. Kinetochores occurred as clusters, in association with nucleoli and with the nuclear envelope. Neurons at different stages of differentiation, as determined by nucleolar distribution, exhibited a distinct, stage-specific, spatial organization of kinetochores. Morphometric analyses, together with serial reconstruction, indicated that progressive clustering of kinetochores accompanies differentiation and that such clustering occurs in association with nucleolar fusion. The data presented indicate that the chromatin organization observed in the fully differentiated state may be the result of controlled rearrangements of specific chromatin domains during differentiation and that the mechanism governing such rearrangement and the process of cellular differentiation may be linked.

Morphological assessment of hepatoma cells (HepG2) microencapsulated in a HEMA-MMA copolymer with and without Matrigel.

Hepatoma cells (HepG2), an anchorage-dependent cell line, were microencapsulated in a HEMA-MMA polyacrylate membrane to which the cells do not adhere. This environment was altered by the coencapsulation of Matrigel, a reconstituted extracellular matrix derived from the Engelbreth-Holm-Swarm (EHS) mouse tumor basement membrane, to provide sites for cell attachment. The effect on the cells of these two capsule microenvironments during a 2-week in vitro culture period was assessed by examining the spatial arrangement, morphology, and viability of the cells using light microscopy and scanning electron microscopy (SEM). In preparation for microscopy, dissolution of the polymer was prevented by the use of frozen sections embedded in a water-soluble compound. Similarly, freeze cleavage of conductively stained capsules permitted SEM observation of the capsule interior along with ultrastructural detail of the cells. In the absence of Matrigel, cells in HEMA-MMA capsules were found to form aggregates in intracapsular pockets with central necrosis occurring at day 7 in large aggregates. The coencapsulation of HepG2 cells with Matrigel, resulted in an initially uniform distribution of essentially individual cells with aggregates appearing later within the Matrigel. Many cells within these capsules had remained viable when examined up to day 14 with only limited cellular necrosis, implying a favorable environment for microencapsulated HepG2 cells.

Nuclear membrane modifications in polytene nuclei of Drosophila melanogaster: serial reconstruction and cytochemistry.

The nuclear envelope of polytene nuclei of salivary glands of Drosophila melanogaster displays modifications consisting of nuclear envelope invaginations (NEI) and evaginations (NEE). Ultrastructural analyses combined with three-dimensional reconstruction and cytochemistry show that NEI are bounded by a single membrane and that they may arise as invaginations of the inner nuclear membrane. NEI extend deeply into the nucleus. The lumens of NEI may collapse resulting in membranous sheets which may combine with those arising from adjacent NEI to form intranuclear structures resembling annulate lamellae. All NEI are associated with NEE. In contrast to NEI, NEE are enclosed in a double membrane morphologically identical to the nuclear envelope. While NEI and NEE share wheat germ agglutinin binding properties with the nuclear envelope, they differ in their ability to localize lanthanum. Pore annuli of NEI display complete lack of lanthanum binding, while those of NEE exhibit minor deposition of this cation. In contrast, pore annuli of the nuclear envelope are specifically and significantly decorated by lanthanum. A conceptual model based on the results obtained suggests that NEI are formed by invaginations of the inner nuclear membrane, together with accompanying modifications of pore complexes.

Rearrangement of centromeric satellite DNA in hippocampal neurons exhibiting long-term potentiation.

In situ hybridization in conjunction with three-dimensional reconstruction was used to examine the topology of satellite DNA (sDNA) sequences in hippocampal CA1 neurons. In slices fixed immediately after preparation, 4-5 signals/nucleus were detected in CA1, CA3 and dentate neurons. 70-80% of 154 neurons examined in these 3 areas displayed all signals at the nuclear periphery. In the remaining fraction of neurons, sDNA signals were divided between the nucleolus and the nuclear periphery. sDNA signals were consistently localized to the nuclear midplane. Slices left to equilibrate in artificial cerebral spinal fluid for 1 h, in the absence of potentiation, exhibited a significant increase in the total number of signals/nucleus in CA1 and dentate neurons. This increase in the number of signals occurred in both nucleolar and peripheral compartments, with the number of the nucleolar compartment nearly doubling. The total number of signals/nucleus was found to be consistently reduced in tetanized CA1 neurons (4.89 +/- 0.09 signals/nucleus, n = 195, P less than 0.05) as compared to neurons from unpotentiated slices (5.27 +/- 0.10 signals/nucleus, n = 81). A similar decrease in the total number of signals/nucleus was also observed in CA1 neurons exposed to N-methyl-D-aspartate (NMDA), from 5.27 +/- 0.10 signals/nucleus (n = 81) to 5.00 +/- 0.08 signals/nucleus (n = 215, P less than 0.05). In contrast, dentate neurons, employed as internal controls, did not exhibit any change in number and compartmentalization of sDNA signals.(ABSTRACT TRUNCATED AT 250 WORDS)