Fluctuations of pol I and fibrillarin contents of the nucleoli.

Nucleoli are formed on the basis of ribosomal DNA (rDNA) clusters called Nucleolus Organizer Regions (NORs). Each NOR contains multiple genes coding for RNAs of the ribosomal particles. The prominent components of the nucleolar ultrastructure, fibrillar centers (FC) and dense fibrillar components (DFC), together compose FC/DFC units. These units are centers of rDNA transcription by RNA polymerase I (pol I), as well as the early processing events, in which an essential role belongs to fibrillarin. Each FC/DFC unit probably corresponds to a single transcriptionally active gene. In this work, we transfected human-derived cells with GFP-RPA43 (subunit of pol I) and RFP-fibrillarin. Following changes of the fluorescent signals in individual FC/DFC units, we found two kinds of kinetics: 1) the rapid fluctuations with periods of 2-3 min, when the pol I and fibrillarin signals oscillated in anti-phase manner, and the intensities of pol I in the neighboring FC/DFC units did not correlate. 2) fluctuations with periods of 10 to 60 min, in which pol I and fibrillarin signals measured in the same unit did not correlate, but pol I signals in the units belonging to different nucleoli were synchronized. Our data indicate that a complex pulsing activity of transcription as well as early processing is common for ribosomal genes.

Nucleolar DNA: the host and the guests.

Nucleoli are formed on the basis of ribosomal genes coding for RNAs of ribosomal particles, but also include a great variety of other DNA regions. In this article, we discuss the characteristics of ribosomal DNA: the structure of the rDNA locus, complex organization and functions of the intergenic spacer, multiplicity of gene copies in one cell, selective silencing of genes and whole gene clusters, relation to components of nucleolar ultrastructure, specific problems associated with replication. We also review current data on the role of non-ribosomal DNA in the organization and function of nucleoli. Finally, we discuss probable causes preventing efficient visualization of DNA in nucleoli.

GSI-I (Z-LLNle-CHO) inhibits γ-secretase and the proteosome to trigger cell death in precursor-B acute lymphoblastic leukemia.

Gamma secretase inhibitors (GSIs) comprise a growing class of compounds that interfere with the membrane-bound Notch signaling protein and its downstream intra-nuclear transcriptional targets. As GSI-I (Z-LLNle-CHO) is also a derivative of a widely used proteosome inhibitor MG-132, we hypothesized that this compound might be active in precursor-B acute lymphoblastic leukemia (ALL) cell lines and patient samples. We found that GSI-I treatment of precursor-B ALL blasts induced apoptotic cell death within 18-24 h. With confirmation using RNA and protein analyses, GSI-I blocked nuclear accumulation of cleaved Notch1 and Notch2, and inhibited Notch targets Hey2 and Myc. Microarray analyses of 207 children with high-risk precursor-B ALL demonstrate that Notch pathway expression is a common feature of these neoplasms. However, microarray studies also implicated additional transcriptional targets in GSI-I-dependent cell death, including genes in the unfolded protein response, nuclear factor-κB and p53 pathways. Z-LLNle-CHO blocks both γ-secretase and proteosome activity, inducing more robust cell death in precursor-B ALL cells than either proteosome-selective or γ-secretase-selective inhibitors alone. Using Z-LLNle-CHO in a nonobese diabetes/severe combined immunodeficiency (NOD/SCID) precursor-B ALL xenograft model, we found that GSI-I alone delayed or prevented engraftment of B-lymphoblasts in 50% of the animals comprising the experimental group, suggesting that this compound is worthy of additional testing.

Changes in extracellular space volume and geometry induced by cortical spreading depression in immature and adult rats.

Changes in extracellular space (ECS) diffusion parameters, DC potentials and extracellular potassium concentration were studied during single and repeated cortical spreading depressions (SD) in 13-15 (P13-15), 21 (P21) and 90-day-old (adult) Wistar rats. The real-time iontophoretic method using tetramethylammonium (TMA+)-selective microelectrodes was employed to measure three ECS parameters in the somatosensory cortex: the ECS volume fraction alpha (alpha = ECS volume/total tissue volume), ECS tortuosity lambda (increase in diffusion path length) and the nonspecific TMA+ uptake k'. SD was elicited by needle prick. SD was significantly longer at P13-15 than at P21 and in adults. During SD, alpha in all age groups decreased from 0.21-0.23 to 0.05-0.09; lambda increased from 1.55-1.65 to 1.95-2.07. Ten minutes after SD, alpha (in adults) and lambda (all age groups) increased compared to controls. This increase persisted even 1 hour after SD. When SD was repeated at 1 hour intervals, both alpha and lambda showed a gradual cumulative increase with SD repetition. Our study also shows that cortical SD is, as early as P13, accompanied by severe ECS shrinkage and increased diffusion path length (tortuosity) with values similar to adults, followed by a long-lasting increase in ECS volume and tortuosity when compared to pre-SD values.

Diffusion parameters in the striatum of rats with 6-hydroxydopamine-induced lesions and with fetal mesencephalic grafts.

Functional recovery after transplantation of dopaminergic cells into the lesioned striatum is dependent on widespread diffusion of the transmitter released by the graft. In the present study, we investigated the diffusion parameters of the extracellular space in the striatum of control, 6-hydroxydopamine-lesioned, intrastriatally grafted, and sham-grafted rats in vivo. We used two types of grafts-single macrografts or multiple micrografts. The real-time iontophoretic tetramethylammonium method enabled us to extract three extracellular space diffusion parameters: volume fraction, alpha, tortuosity, lambda, and nonspecific uptake of tetramethylammonium, k'. Compared with controls (alpha = 0.19, lambda = 1.59), in lesioned animals both alpha and lambda were lower (alpha = 0.14, lambda = 1.50). alpha and lambda were increased inside macro-and micrografts, where alpha = 0.24 and lambda = 1.80, and in sham-grafted areas, where alpha = 0.24 and lambda = 1.72. In regions outside the grafts (alpha = 0.15, lambda = 1.51) or in sham grafts (alpha = 0.14, lambda = 1.49), the values of alpha and lambda were similar to the values observed in lesioned striatum. Nonspecific uptake (k') did not differ among the groups. Our results show that, compared with control, alpha and lambda were decreased in dopamine-depleted areas and increased in areas with grafts. Multiple but smaller graft deposits, in contrast to their enlarged capability for dopaminergic reinnervation, impair the conditions for diffusion and extrasynaptic transmission in a larger area of the striatum than do single macrografts, presumably because of more extensive tissue damage, cell loss, and astrogliosis.

Learning deficits in aged rats related to decrease in extracellular volume and loss of diffusion anisotropy in hippocampus.

The extracellular space (ECS) is the microenvironment of the nerve cells and an important communication channel, allowing for long-distance extrasynaptic communication between cells. Changes in ECS size, geometry, and composition have been reported in diverse (patho)physiological states, including aging. In the present study, real-time tetramethylammonium (TMA+) iontophoresis was used to quantify ECS diffusion parameters in different brain regions of adult and behaviorally characterized aged rats. Prior to ECS diffusion measurement, superior and inferior learners were selected from a large group of aged rats, according to their performance in the open-field water maze. The main finding was that the degree of impaired maze performance of old rats correlates, firstly, with decrease in ECS volume, loss of diffusion anisotropy in hippocampus, and degree of astrogliosis, and secondly, with disorganization of the astrocytic processes and reduction of hippocampal ECS matrix molecules. Importantly, no significant differences were found in the density of neurons in any region of the hippocampus or dentate gyrus. The alterations in hippocampal diffusion parameters evident in aged animals with severe learning deficits could account for the learning impairment, due to their effects on extrasynaptic volume transmission and/or on the "cross-talk" between synapses, which has been suggested to be involved in neural processes associated with learning and memory formation.

Effect of elevated K(+), hypotonic stress, and cortical spreading depression on astrocyte swelling in GFAP-deficient mice.

Glial fibrillary acidic protein (GFAP) is the main component of intermediate filaments in astrocytes. To assess its function in astrocyte swelling, we compared astrocyte membrane properties and swelling in spinal cord slices of 8- to 10-day-old wild-type control (GFAP(+/+)) and GFAP-knockout (GFAP(-/-)) mice. Membrane currents and K(+) accumulation around astrocytes after a depolarizing pulse were studied using the whole-cell patch-clamp technique. In vivo cell swelling was studied in the cortex during spreading depression (SD) in 3 to 6-month-old animals. Swelling-induced changes of the extracellular space (ECS) diffusion parameters, i.e., volume fraction alpha and tortuosity lambda, were studied by the real-time iontophoretic tetramethylammonium (TMA(+)) method using TMA(+)-selective microelectrodes. Morphological analysis using confocal microscopy and quantification of xy intensity profiles in a confocal plane revealed a lower density of processes in GFAP(-/-) astrocytes than in GFAP(+/+) astrocytes. K(+) accumulation evoked by membrane depolarization was lower in the vicinity of GFAP(-/-) astrocytes than GFAP(+/+) astrocytes, suggesting the presence of a larger ECS around GFAP(-/-) astrocytes. Astrocyte swelling evoked by application of 50 mM K(+) or by hypotonic solution (HS) produced a larger increase in [K(+)](e) around GFAP(+/+) astrocytes than around GFAP(-/-) astrocytes. No differences in alpha and lambda in the spinal cord or cortex of GFAP(+/+) and GFAP(-/-) mice were found; however, the application of either 50 mM K(+) or HS in spinal cord, or SD in cortex, evoked a large decrease in alpha and an increase in lambda in GFAP(+/+) mice only. Slower swelling in GFAP(-/-) astrocytes indicates that GFAP and intermediate filaments play an important role in cell swelling during pathological states.

Astrocytes, oligodendroglia, extracellular space volume and geometry in rat fetal brain grafts.

Fetal neocortex or tectum transplanted to the midbrain or cortex of newborn rats develops various degrees of gliosis, i.e. increased numbers of hypertrophied, glial fibrillary acidic protein-positive astrocytes. In addition, there were patches or bundles of myelinated fibres positive for the oligodendrocyte and central myelin marker Rip, and increased levels of extracellular matrix molecules. Three diffusion parameters--extracellular space volume fraction alpha (alpha = extracellular volume/total tissue volume), tortuosity lambda (lambda = square root(D/ADC), where D is the free and ADC is the apparent tetramethylammonium diffusion coefficient) and non-specific uptake k'--were determined in vivo from extracellular concentration-time profiles of tetramethylammonium. Grafts were subsequently processed immunohistochemically to compare diffusion measurements with graft morphology. Comparisons were made between the diffusion parameters of host cortex and corpus callosum, fetal cortical or tectal tissue transplanted to host midbrain ("C- and T-grafts") and fetal cortical tissue transplanted to host cortex ("cortex-to-cortex" or C-C-grafts). In host cortex, alpha ranged from 0.20 +/- 0.01 (layer V) to 0.21 +/- 0.01 (layers III, IV and VI) and lambda from 1.59 +/- 0.03 (layer VI) to 1.64 +/- 0.02 (layer III) (mean +/- S.E.M., n = 15). Much higher values were found in "young" C-grafts (81-150 days post-transplantation), where alpha = 0.34 +/- 0.01 and lambda = 1.78 +/- 0.03 (n = 13), as well as in T-grafts, where alpha = 0.29 +/- 0.02 and lambda = 1.85 +/- 0.04 (n = 7). Further analysis revealed that diffusion in grafts was anisotropic and more hindered than in host cortex. The heterogeneity of diffusion parameters correlated with the structural heterogeneity of the neuropil, with the highest values of alpha in gray matter and the highest values of lambda in white matter bundles. Compared to "young" C-grafts, in "old" C-grafts (one year post-transplantation) both alpha and lambda were significantly lower, and there was a clear decrease in glial fibrillary acidic protein immunoreactivity throughout the grafted tissue. In C-C-grafts, alpha and lambda varied with the degree of graft incorporation into host tissue, but on average they were significantly lower (alpha = 0.24 +/- 0.01 and lambda = 1.66 +/- 0.02, n = 8) than in young C- and T-grafts. Well-incorporated grafts revealed less astrogliosis, and alpha and lambda values were not significantly higher than those in normal host cortex. The observed changes in extracellular space diffusion parameters could affect the movement and accumulation of neuroactive substances and thus impact upon neuron-glia communication, synaptic and extrasynaptic transmission in the grafts. The potential relevance of these observations to human neuropathological conditions associated with acute or chronic astrogliosis is considered.

Effects of nitric oxide inhibition on the spread of biotinylated dextran and on extracellular space parameters in the neostriatum of the male rat.

Volume transmission in the brain is mediated by the diffusion of neurotransmitters, modulators and other neuroactive substances in the extracellular space. The effects of nitric oxide synthase inhibition on extracellular space diffusion properties were studied using two different approaches, the histological dextran method and the real-time iontophoretic tetramethylammonium method. The spread of biotinylated dextran (mol. wt 3000) in the extracellular space was measured morphometrically following microinjection into the neostriatum of male rats. Two parameters were used to describe the spread of biotinylated dextran in brain tissue, namely, total volume of spread and the mean grey value. The nonspecific nitric oxide synthase inhibitors NG-nitro-L-arginine methyl ester (10-100 mg/kg) and NG-monomethyl-L-arginine acetate (30-200 mg/kg) decreased the total volume of spread of dextran in a dose-dependent manner. 7-Nitroindazole monosodium salt (50-100 mg/kg), a specific neuronal nitric oxide synthase inhibitor, did not change the total volume of spread of dextran. Using the tetramethylammonium method, the extracellular space diffusion properties can be described by the volume fraction (alpha = extracellular space volume/total tissue volume), tortuosity lambda (lambda2 = free diffusion coefficient/apparent diffusion coefficient in tissue), and non-specific uptake kappa' [Nicholson C. and Syková E. (1998) Trends Neurosci. 21, 207-215]. Nitric oxide synthase inhibition by NG-nitro-L-arginine methyl ester (50 mg/kg) had relatively little effect on volume fraction and tortuosity, and no changes were observed after NG-monomethyl-L-arginine acetate (20 mg/kg) or 7-nitroindazole monosodium salt (100 mg/kg) treatment. A substantial increase was found only in non-specific uptake, by 13% after NG-nitro-L-arginine methyl ester and by 16% after NG-monomethyl-L-arginine acetate, which correlates with the decreased total volume of spread of dextran observed with the dextran method. NG-Nitro-L-arginine methyl ester treatment (100 mg/kg) decreased striatal blood flow and increased mean arterial blood pressure. The changes in dextran spread and non-specific uptake can be explained by an increased capillary clearance following the inhibition of endothelial nitric oxide synthase, as neuronal nitric oxide synthase inhibition had no effect. The observed changes after non-specific nitric oxide synthase inhibition may affect the extracellular space concentration of neurotransmitters and modulators, and influence volume transmission pathways in the central nervous system by increased capillary and/or cellular clearance rather than by changes in extracellular space diffusion.

Diffusion heterogeneity and anisotropy in rat hippocampus.

We provide evidence for anisotropic diffusion in rat corpus callosum and hippocampus. The preferential diffusion pathway in corpus callosum is along the myelinated axon fibres; in the hippocampus diffusion is easier along the transversal axis (x) than along the sagittal (y) or vertical (z) axes. In all areas studied, i.e. in the cortex, corpus callosum and hippocampus, the mean ECS volume fraction alpha (alpha = ECS volume/total tissue volume) ranged between 0.20 and 0.22 and mean non-specific uptake k' was between 4.0 and 5.9 x 10(-3) s-1. Diffusional anisotropy in the hippocampus may be of importance for extrasynaptic transmission and in the 'cross-talk' between synapses.

Diffusion constraints and neuron-glia interaction during aging.

Changes in brain extracellular space (ECS) volume, composition, and geometry are a consequence of neuronal activity, of glial K+, pH, and amino acid homeostasis, and of changes in glial cell morphology, proliferation, and function. They occur as a result of repetitive neuronal activity, seizures, anoxia, injury, inflammation, and many other pathological states in the CNS, and may significantly affect signal transmission in the CNS. Activity-related or CNS damage-related cellular swelling is compensated for by ECS volume shrinkage and, as a consequence, by a decrease in the apparent diffusion coefficients (ADCs) of neuroactive substances diffusing in the ECS. Changes in cellular morphology, such as occur during aging, could also result in changes of ECS volume and geometry. We provide evidence for limited diffusion in rat cortex, corpus callosum, and hippocampus in the aging brain that correlates with changes in glial volume and the extracellular matrix. In all structures, the mean ECS volume fraction alpha (alpha = ECS volume/total tissue volume) and nonspecific uptake k' are significantly lower in aged rats (26-32 months old) than in young adult brain. Compared to young adult brain, in the aged brain we found an increase in GFAP staining and hypertrophied astrocytes with thicker processes which, in the hippocampus, lost their radial organization. The tortuosity (lambda = square root of D/ADC) was lower in the cortex and CA3 region. Immunohistochemical staining for fibronectin and chondroitin sulfate proteoglycans revealed a substantial decrease that could account for a decrease in diffusion barriers. Diffusion parameters alpha, lambda, and k' in the aging brain after cardiac arrest changed substantially faster than in the young adult brain, although the final values were not significantly different. This suggests that the smaller extracellular space during aging results in a greater susceptibility of the aging brain to anoxia/ischemia, apparently due to a faster extracellular acidosis and accumulation of K+ and toxic substances, for example, glutamate. We conclude that during aging the movement of substances is more hindered in the narrower clefts. This is partly compensated for by a decrease in the diffusion barriers that may be formed by macromolecules of the extracellular matrix. Diffusion parameters can affect the efficacy of synaptic as well as extrasynaptic transmission by a greater accumulation of substances, because they diffuse away from a source more slowly, or induce damage to nerve cells if these substances reach toxic concentrations. Diffusion parameters are also of importance in the "crosstalk" between synapses, which has been hypothesized to be of importance during LTP and LTD. We can, therefore, assume that the observed changes in ECS diffusion parameters during aging can contribute to functional deficits and memory loss.