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At least 163 records · Page 9Linked to original sources

Telencephalon-specific Rb knockouts reveal enhanced neurogenesis, survival and abnormal cortical development.

Correct cell cycle regulation and terminal mitosis are critical for nervous system development. The retinoblastoma (Rb) protein is a key regulator of these processes, as Rb-/- embryos die by E15.5, exhibiting gross hematopoietic and neurological defects. The extensive apoptosis in Rb-/- embryos has been attributed to aberrant S phase entry resulting in conflicting growth control signals in differentiating cells. To assess the role of Rb in cortical development in the absence of other embryonic defects, we examined mice with telencephalon-specific Rb deletions. Animals carrying a floxed Rb allele were interbred with mice in which cre was knocked into the Foxg1 locus. Unlike germline knockouts, mice specifically deleted for Rb in the developing telencephalon survived until birth. In these mutants, Rb-/- progenitor cells divided ectopically, but were able to survive and differentiate. Mutant brains exhibited enhanced cellularity due to increased proliferation of neuroblasts. These studies demonstrate that: (i) cell cycle deregulation during differentiation does not necessitate apoptosis; (ii) Rb-deficient mutants exhibit enhanced neuroblast proliferation; and (iii) terminal mitosis may not be required to initiate differentiation.

Alleles↗

Temporal and spatial preferences of c-fos mRNA expression in the rat brain following cortical lesion.

The expression of the proto-oncogene c-fos is increased in neuronal cells by a number of stimuli and the usefulness of this gene as a marker of neuronal activity has been demonstrated. The temporal and spatial expression of c-fos mRNA following the induction of a unilateral cortical lesion have been investigated in the rat brain by Northern blot analysis and in situ hybridization histochemistry. It was observed that the lesion evoked a rapid increase (20-fold) in the content of c-fos mRNA in the ipsilateral cortex, whereas in the contralateral cortex c-fos mRNA expression was more modest (7-fold). In the whole hippocampus a large and very rapid increase (17-fold) of c-fos mRNA expression was detected. The effect of a cortical lesion on Ca2+ uptake and membrane potential was also investigated. Using synaptosomes as a model system, we have provided evidence that Ca2+ entry via membrane depolarization increases in coordination with c-fos gene expression in neuronal cells. The principal conclusions from this study are that cortical lesions induce transient expression of the c-fos gene in specific neuronal cells of the rat brain.

Animals↗

[Harms technique of C1-C2 fixation with polyaxial screws and rods].

PURPOSE OF THE STUDY: The Harms technique of stabilizing C1-C2 by fixation with polyaxial screws and rods is a further option for atlantoaxial fixation from the dorsal approach. Harms and Melcher published this method in 2001, but the operation had first been performed by Harms in August 1997. The aim of this study is to evaluate the first results and try to assign the Harms C1-C2 fixation an appropriate standing in the in broad range of options for stabilization of the atlantoaxial complex. MATERIAL: Between December 2002 and January 2004 we carried out the Harms fixation of C1-C2 on 22 patients admitted to the Department of Spine Surgery, Motol University Hospital, 2nd Medical Faculty in Prague. Out of these, 18 patients were included in this study, 10 men and 8 women between 23 and 84 years of age (average, 55.4 years) followed-up longer than 6 months. In 14 patients we used the Harms technique as a permanent fixation of C1-C2 in order to achieve atlantoaxial arthrodesis and, in four patients, we applied it only for a period of 4 to 6 months without the use of bone grafts or their substitutions. We employed the permanent fixation to treat the following conditions: fracture of the atlas in three patients, type IIA comminuted fracture of the dens base in three patients, fracture of C2 categorized as "other" in two patients, atlantoaxial vertical instability in one patient with rheumatoid arthritis, malunion of the fractured dens in one patient, and complicated trauma to C1-C2 in four patients. The temporary fixation was used for type III displaced fractures of the dens in two and fixed atlantoaxial rotatory dislocations also in two cases. Only one patient showed signs of Frankel C neurological deficit on admission, the rest were without neurological findings. METHODS: All screws were inserted under an image intensifier always in lateral projection. First we retracted the greater occipital nerve in a caudal direction towards C2 with a fine raspatory and, using an awl, marked the entry point in the C1 lateral mass; a pilot hole, reaching through the anterior cortical bone, was made with a 2.5 mm drill. It followed a straight or slightly convergent trajectory in an anterior-posterior direction and parallel to the plane of the C1 posterior arch in the sagittal direction. Individual anatomical variations in the atlantoaxial complex of every patient were respected. The hole was tapped through the entire vertebral body, with the exception of osteoporous bone in which only the posterior cortical bone was treated with a screw tap. At this stage profuse bleeding usually arose from dissection around the epidural venous plexus along the C1-C2 joint. This was effectively controlled by a quick insertion of a screw and compression of the venous plexus with the screw head. To control bleeding by bipolar electrocautery is difficult and is always associated with a risk of nerve injury. Screws 3.5 mm thick, with polyaxial heads, were inserted bicortically into the lateral mass of C1. Subsequently, the intervertebral C2-C3 joint was localized and its medial border in the spinal canal was palpated. The entry point for placement of a C2 pedicle screw was marked with an awl at the point of intersection at a distance of 2 mm from the medial border and 5 mm from the caudal border of the C2 articular process. Under an X-ray intensifier in lateral projection, a hole was drilled approximately parallel to the screws inserted in C1, i. e., at an angle of 20 to 30 degrees cranially, up to and through the anterior cortical bone. In the transversal plane, the screws were situated in a convergent direction at an angle of 20 to 25 degrees. After all screws had been inserted, we reduced the antlantoaxial complex in the correct anatomical position by manipulating the patient's head or by directly adjusting the screws. Connecting 3.0-mm rods were then applied and fastened by cap nuts or inner nuts according to the instrumentation used. RESULTS: Operative time ranged from 35 to 155 min, with an average of 81 min. Intra-operative blood loss ranged from 50 to 1500 ml, with an average of 560 ml. The X-ray intensifier was used for a period of 0.4 to 2.6 min, with an average of 0.9 min. A total of 36 screws were inserted in the atlas; their length ranged from 16 to 34 mm (average, 30.6 mm). All screws were positioned correctly in the C1 lateral mass; two screws did not reach up to the anterior cortical bone and one protruded over it, but without causing clinical problems. Thirty-six screws were inserted in the axis. Their length ranged from 28 to 36 mm (average, 31.7) mm). Twenty-seven screws were correctly applied through the isthmus into the C2 anterior cortical bone, three were too short to reach it and five were placed too close to the vertebral artery canal. Of these, two protruded into the artery canal, but without clinical consequences. One screw inserted too medially passed into the spinal canal, but this also was without clinical response. Of the 36 screws inserted in C2, three (8.3 %) were malpositioned. Bony fusion at C1-C2 was the goal of this operation in 14 patients. At 6 weeks post-operatively, it was achieved in two patients, at 12 weeks in 12 patients and at 6 months in all 14 patients. The C1-C2 segment was stable at 12 weeks in all 18 treated patients. Four patients reported restriction of motion in rotation by 10 to 25 % after removal of the instrumentation. DISCUSSION: Operative time, longer at the beginning than with the Magerl technique, gradually shortened to between 45 and 60 min. Similar trends were seen when intra-operative blood loss and X-ray exposure were evaluated. Using the Harms and Melcher procedure we saved the greater suboccipital nerve. In contrast to these authors, however, we did not resect the atlantoaxial joint. Solid fusion was achieved in all our patients. Of the total of 72 screws inserted, only three (4.2 %) were assessed as malpositioned; however, when related to the 36 screws inserted in C2, this was 8.3 %, which indicates that insertion of screws in C2 was more difficult. We did not observe any clinical consequences in any of these cases. CONCLUSIONS: The Harms fixation of C1-C2 is a very effective technique for stabilizing the atlantoaxial complex. It enables us to provide temporary fixation without damage to atlantoaxial joints and to reduce the vertebrae after the screws and rods had been inserted, which is unique. These advantages compensate for a higher cost of the implant.

Adult↗

Co-ordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system.

Transcellular Ca2+ transport in the distal nephron involves passive Ca2+ influx at the apical membrane, diffusion through the cytosol and active extrusion across the opposing basolateral membrane. The molecular identity of the apical Ca2+ entry step is still elusive, but its regulatory aspects have been analyzed in the present study. To this end, rabbit connecting and cortical collecting tubular cells were cultured on permeable and transparent supports and the apical Ca2+ influx was deduced from Mn2+ quenching of Ca2+ independent Fura-2 fluorescence, while the intracellular Ca2+ concentration ([Ca2+]i) was measured simultaneously. In parallel experiments, transcellular Ca2+ transport was determined isotopically as 45Ca2+ flux from the apical to basolateral compartment. Decreasing the apical pH from 7.4 to 5.9 inhibited transcellular Ca2+ transport by 53 +/- 1%, whereas apical Ca2+ influx was reduced by 39 +/- 7% and [Ca2+]i decreased by 18 +/- 3%. Reversal of the Na+/Ca2+ exchanger by iso-osmotic replacement of Na+ by N-methyl-D-glucamine in the basolateral compartment resulted in 50 +/- 5% inhibition of Ca2+ transport, 46 +/- 3% reduction of apical Ca2+ influx and 60 +/- 3% increase in [Ca2+]i. In the absence of basolateral Ca2+, however, this manoeuvre decreased [Ca2+]i by 21 +/- 8%, while Ca2+ transport and apical Ca2+ influx were reduced by the same magnitude as in the presence of Ca2+, that is by 53 +/- 6% and 45 +/- 4%, respectively. Stimulation of adenylyl cyclase with forskolin (10(-5) M) increased transcellular Ca2+ transport by 108 +/- 40%, stimulated apical Ca2+ influx by 120 +/- 17% and increased [Ca2+]i by 110 +/- 2%. In conclusion, the apical Ca2+ influx is regulated by apical pH, intracellular cAMP and basolateral Na+/Ca2+ exchanger activity, and is coupled in an 1:1 fashion to the rate of transepithelial Ca2+ transport.

Animals↗

Cortical, hippocampal, and striatal mediation of schedule-induced behaviors.

The sequential occurrence of licking, locomotor activity, entries into the food magazine (panel pressing), and nonreenforced lever pressing engendered by a periodic schedule of food presentation were measured in each 60-s interreenforcement interval in normal and brain-damaged rats. The development of these responses was measured over 20 days in different groups of food-deprived rats that had received aspirations of the hippocampus, small lesions of the cortex overlying the hippocampus (hippocampal-operated control group), decortication, or 6-hydroxy-dopamine lesions of the caudate nucleus or nucleus accumbens. All lesions produced distinctive patterns of change in the measured behaviors, and dissociations as well as similarities in their effects were evident. These results are discussed with respect to dissociations in the motor and motivational effects of the various lesions and in terms of contemporary hypotheses of schedule-induced behavior.

Animals↗

Brain electrobiogenesis protection induced by nimodipine and MK-801 during acute hypoxia in hypertensive rats.

The protective effect of nimodipine, a calcium entry blocker, and MK-801, an excitatory amino acid antagonist, on the cortical electrobiogenesis of hypertensive SHR-Charles-River adult rats during a 90-s session of acute hypoxia, was tested either with a single treatment or with a therapeutic association. Pretreatment with nimodipine alone (0.1 mg kg-1, i.v.) was particularly effective in increasing the rat brain resistence to hypoxia when compared to the controls. MK-801 pretreatment alone (3 mg kg-1, subcutaneous), improved significantly the recovery of brain electrical activity from hypoxia, as it not only shortened the recovery time but also abolished the post-hypoxic burst activity. The combined pretreatment with both drugs, using the same doses, displayed a more powerful brain protective effect on the cortical electrobiogenesis than the single drug pretreatment, suggesting the existence of a synergy between nimodipine and MK-801.

Acute Disease↗

Interindividual variability in the hemispheric organization for speech.

A PET activation study was designed to investigate hemispheric specialization during speech comprehension and production in right- and left-handed subjects. Normalized regional cerebral blood flow (NrCBF) was repeatedly monitored while subjects either listened to factual stories (Story) or covertly generated verbs semantically related to heard nouns (Gener), using silent resting (Rest) as a common control condition. NrCBF variations in each task, as compared to Rest, as well as functional asymmetry indices (FAI = right minus left NrCBF variations), were computed in anatomical regions of interest (AROIs) defined on the single-subject MNI template. FAIs were predominantly leftward in all regions during both tasks, although larger FAIs were observed during Gener. Subjects were declared "typical" for language hemispheric specialization based on the presence of significant leftward asymmetries (FAI < 0) in the pars triangularis and opercularis of the inferior frontal gyrus during Gener, and in the middle and inferior temporal AROIs during Story. Six subjects (including five LH) showed an atypical language representation. Among them, one presented a right hemisphere specialization during both tasks, another a shift in hemispheric specialization from production to comprehension (left during Gener, right during Story). The group of 14 typical subjects showed significant positive correlation between homologous left and right AROIs NrCBF variations in temporal areas during Story, and in temporal and inferior frontal areas during Gener, almost all regions presenting a leftward FAI. Such correlations were also present in deactivated areas with strong leftward asymmetry (supramarginalis gyrus, inferior parietal region). These results suggest that entry into a language task translates into a hemispheric reconfiguration of lateral cortical areas with global NrCBF increase in the dominant hemisphere and decrease in the minor hemisphere. This can be considered as the setting up of a "language mode", under the control of a mechanism that operates at a perisylvian level. On top of this global organization, regional variations carry on the performance of the cognitive operations specific to the language task to be performed. Hemispheric relationships could be different in atypical subjects, with either between task hemispheric regulation differences or differences in regional specialization.

Adult↗

Modes of protein movement that lead to the asymmetric localization of partner of Numb during Drosophila neuroblast division.

Partner of Numb (Pon) colocalizes with the determinant Numb and is required for its proper asymmetric localization in Drosophila. How the asymmetric localization of Pon is accomplished is not well understood. Here, we show that Pon localization takes place at the protein level and that its C-terminal region is necessary and sufficient for asymmetric localization. Fusion of the Pon localization domain with green fluorescent protein (GFP) allowed monitoring of the localization process in living embryos. Upon a neuroblast's entry into mitosis, Pon is recruited from the cytoplasm to the cortex. Cortically recruited Pon can move apically or basally within the two-dimensional confines of the cortex. This movement can occur when myosin motor activity is inhibited. However, the restriction of Pon to the basal cortex requires both actomyosin and Inscuteable.

Animals↗

A centrally acting, anxiolytic angiotensin II AT1 receptor antagonist prevents the isolation stress-induced decrease in cortical CRF1 receptor and benzodiazepine binding.

Long-term pretreatment with an angiotensin II AT1 antagonist blocks angiotensin II effects in brain and peripheral organs and abolishes the sympathoadrenal and hypothalamic-pituitary-adrenal responses to isolation stress. We determined whether AT1 receptors were also important for the stress response of higher regulatory centers. We studied angiotensin II and corticotropin-releasing factor (CRF) receptors and benzodiazepine binding sites in brains of Wistar Hannover rats. Animals were pretreated for 13 days with vehicle or a central and peripheral AT1 antagonist (candesartan, 0.5 mg/kg/day) via osmotic minipumps followed by 24 h of isolation in metabolic cages, or kept grouped throughout the study (grouped controls). In another study, we determined the influence of a similar treatment with candesartan on performance in an elevated plus-maze. AT1 receptor blockade prevented the isolation-induced increase in brain AT1 receptors and decrease in AT2 binding in the locus coeruleus. AT1 receptor antagonism also prevented the increase in tyrosine hydroxylase mRNA in the locus coeruleus. Pretreatment with the AT1 receptor antagonist completely prevented the decrease in cortical CRF1 receptor and benzodiazepine binding produced by isolation stress. In addition, pretreatment with candesartan increased the time spent in and the number of entries to open arms of the elevated plus-maze, measure of decreased anxiety. Our results implicate a modulation of upstream neurotransmission processes regulating cortical CRF1 receptors and the GABA(A) complex as molecular mechanisms responsible for the anti-anxiety effect of centrally acting AT1 receptor antagonists. We propose that AT1 receptor antagonists can be considered as compounds with possible therapeutic anti-stress and anti-anxiety properties.

Amphibian Proteins↗

Expression of mos in astrocytic tumors and its potential role in neoplastic progression.

The c-mos gene and its protein product mos, components of the mitogen-activated protein kinase transduction pathway, are known to be involved in the control of meiosis and mitosis. Apart from a study on lung carcinomas, there is little information about its role in human neoplasia. The aim of this study was to investigate expression of mos in astrocytic tumors and to correlate it with accumulation of p53. We studied expression of mos in 62 cases of supratentorial astrocytic tumor. Intracytoplasmic immunostaining for mos was found in 28 (45%) cases: 3 of 20 (15%) grade 2 astrocytomas, 9 of 20 (45%) grade 3 anaplastic astrocytomas, and 16 of 22 (73%) glioblastomas. Immunopositivity for mos correlated significantly (P < 0.01) with tumor grade but not with p53 expression. In contrast to the findings in relation to lung tumors, immunopositivity for mos in astrocytic tumors did not predict recurrence-free or overall survival time. Cytoplasmic immunostaining was observed in scattered large cortical neurons adjacent to tumors, possibly due to stress-induced abortive entry into the cell cycle. The correlation of mos immunopositivity with tumor grade may reflect the expansion of more malignant mos-positive clones. This study provides evidence that mos may be involved in the neoplastic progression of a proportion of astrocytic tumors.

Adolescent↗

Cortical and subcapsular cataracts: significance of physical forces.

Cortical cataracts usually begin with either sharp limited clear fluid clefts, resulting in opaque spokes, or clear lamellar separations, resulting in cuneiform opacities. They do not commence prior to 45 years of age. From this age on an increase in lens nuclei hardening can be detected. Therefore, during disaccommodation in older lenses, mechanical shear stresses must develop between the soft remaining cortices and the harder nuclei. These shear stresses are significant regarding the different cortical ruptures in predisposed lenses: in a privileged radial direction (according to zonular traction) of the sharp limited cortical spokes, or in parallel microridges at the commencement of lamellar separations, as observed when a rubber surface slides against a harder object. In pure cortical cataracts the ion pump (K+ > Na+) and investigated metabolic parameters remain largely intact. Therefore, it is not surprising that, in contrast to subcapsular cataracts, subcapsular opacities do not occur. Subcapsular cataracts are known to be caused by a variety of factors: aging, diabetes, corticosteroids, iridocyclitis, or X-ray, among many others. In contrast to cortical cataracts, subcapsular cataracts were found to be closely associated with ion pump damage (Na+ > K+) and a variety of metabolic activity alterations. In subcapsular cataracts passive fluids (from the vitreous and camera anterior) enter externally through the lens capsule. This initially forms numerous free clear, secondary grey, subcapsular fluid vacuoles. If the ion pump (metabolic barrier) is more severely damaged fluids may also enter the lens nucleus (secondary grey nuclear cataract), which rarely results in intumescent cataract. In cortical and subcapsular cataracts and lens perforations the main cause of grey opalescence appears to be the result of lens proteins (water-soluble crystalline) coming into direct contact with free fluids (water). In cortical cataracts this happens in the area of sharp limited mechanical cortical ruptures (fluid clefts), and in subcapsular cataracts during passive, external fluid entry, resulting in subcapsular fluid vacuoles and opacities, and also later grey-white nuclear opacities. The importance of water contact with water-soluble lens crystallines in behalf of light scattering and turbidness also has been investigated experimentally.

Cataract↗

Hypertrophy of renal mitochondria.

Compensatory renal hypertrophy leads to an increase in the size and metabolic capacity of renal tubular cells. Increased transport and metabolic activities must be sustained by an augmented rate of energy production, which is largely dependent on mitochondrial processes. Although previous studies have suggested that mitochondria proliferate in the hypertrophying cell, the data to support this have not been convincing. This study was designed to determine whether the mitochondria of the hypertrophied renal proximal tubular cell undergo hypertrophy or proliferation. Flow cytometric analysis of proximal tubular cells obtained from the kidneys of uninephrectomized rabbits revealed an increase in cell size and RNA content compared with control cells but showed no change in DNA content and nuclear size and no evidence of entry into the S/G2/M phases of the cell cycle. Histomorphometric analysis of cortical proximal tubules revealed that although cytoplasmic volume increased, mitochondrial density remained constant, indicating that mitochondrial volume increases in proportion to the increase in cell volume. By day 14, mitochondrial volume had increased 66% above control values. Electron microscopic examination of isolated S2 proximal tubules from 5/6 nephrectomized rabbits with maximal hypertrophy revealed mitochondrial cristae which appeared to be more densely packed than that in normal cells. The size of the functional mitochondrial pool per cell was determined by rhodamine-123 fluorescence. This increased within 24 h of uninephrectomy, peaked at approximately 80% above control levels at 5 days, and remained elevated throughout the 16 days of observation. The initial increase (days 1 and 2) occurred before a measurable increase in mitochondrial volume occurred and presumably reflects an increase in mitochondrial membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Transforming growth factor-beta in human diabetic nephropathy: effects of ACE inhibition.

OBJECTIVE: Studies in rodent models have suggested that reduction in renal transforming growth factor (TGF)-beta1 may underlie the renoprotective effects of the renin-angiotensin system (RAS) blockade. However, the role of the RAS blockade in abrogating TGF-beta in human disease is unknown. Accordingly, we sought to examine TGF-beta gene expression and biological activity in human renal biopsies, before and after ACE inhibition. RESEARCH DESIGN AND METHODS: RNA was extracted from renal biopsies taken from participants in the Diabiopsies study, a randomized controlled 2-year trial of 4 mg/day perindopril versus placebo that reported a reduction in proteinuria and cortical matrix expansion in type 2 diabetic nephropathy. Biopsies taken at study entry and at 2 years were obtained in 12 patients (6 placebo and 6 taking perindopril). TGF-beta1 and its receptor mRNA were quantified by real-time PCR, and its biological activity was assessed by examining the activation of its intracellular signaling pathway (phosphorylated Smad2) and the expression TGF-beta-inducible gene H3 (betaig-H3). RESULTS: At baseline, TGF-beta1 expression was similar in both placebo- and perindopril-treated groups and was unchanged over a 2-year period in biopsies of placebo-treated subjects. In contrast, perindopril treatment led to a substantial diminution in TGF-beta1 mRNA (mean 83% reduction, P < 0.05). Phosphorylated Smad2 immunolabeling and betaig-H3 mRNA were similarly reduced with ACE inhibition (P < 0.05) but unchanged in the placebo group. No differences were noted in the gene expression of TGF-beta receptor II in biopsies of either placebo- or perindopril-treated subjects. CONCLUSIONS: This study demonstrates that over a 2-year period, treatment with perindopril in patients with type 2 diabetes and nephropathy leads to a reduction in both renal TGF-beta1 gene expression and its downstream activation.

Adult↗

Re-entry waves of Leao's spreading depression between neocortex and caudate nucleus.

About 40% of spreading depression (SD) waves elicited from the parieto-occipital cortex of anesthetised rats penetrated through the temporal lobe structures (amygdala) into the caudate nucleus. Almost 70% of these SD waves did not terminate in the caudate but returned to the cortex and spread through it toward the site of SD initiation. Longer cortico-caudate (5.9 +/- 0.1 min) than caudate-cortical (4.7 +/- 0.2 min) conduction times suggest that SD enters and leaves caudate through routes of different length. SD waves elicited by KCl microinjection into the caudate reached frontal and parieto-occipital cortical electrodes with latencies indicating that the transit point is 5 mm closer to the rostral than to the caudal electrode. The region best satisfying this condition corresponds to rostral claustrum. The directionally biased SD conduction through the transit zone provides a re-entry path for cortico-caudate-cortical SD propagation and forms thus a natural reverberator the small dimensions of which preclude generation of repetitive SD waves.

Animals↗

Effects of altered cholinergic function on working and reference memory in the rat.

Many data suggest that the brain's cholinergic neurons participate in the control of memory and it has been suggested that cholinergic systems are involved differentially in working and reference memory. To test this hypothesis the effects on memory of unilateral injections of the neurotoxins, quinolinic acid or kainic acid into the cortically projecting cholinergic cells of the nucleus basalis magnocellularis (nbm) were evaluated. In experiment 1, quinolinate-injected (n = 7) and sham-operated (n = 7) rats were tested in a T-maze alternation task that requires working memory. Lesion rats performed significantly more poorly than shams and subsequent biochemical assays of cortical choline acetyltransferase (CAT) activity revealed significant reductions in the lesion rats. In experiment 2, kainate-injected (n = 9) and sham-operated (n = 8) rats were trained in an eight-arm radial maze with only four arms baited. Lesion rats made significantly more working memory errors (entries into baited arms from which the food had already been collected) than reference memory errors (entries into never baited arms). CAT assays showed that the lesion led to a decrease in cortical CAT with no significant change in hippocampal CAT. The results of these studies support the hypothesis that cholinergic neurons of the basocortical system may be differentially involved in working and reference memory.

Animals↗

Identification of PLCgamma-dependent and -independent events during fertilization of sea urchin eggs.

At fertilization, sea urchin eggs undergo a series of activation events, including a Ca2+ action potential, Ca2+ release from the endoplasmic reticulum, an increase in intracellular pH, sperm pronuclear formation, MAP kinase dephosphorylation, and DNA synthesis. To examine which of these events might be initiated by activation of phospholipase Cgamma (PLCgamma), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCgamma as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCgamma and (1) Ca2+ green dextran to monitor intracellular free Ca2+, (2) BCECF dextran to monitor intracellular pH, (3) Oregon Green dUTP to monitor DNA synthesis, or (4) fluorescein 70-kDa dextran to monitor nuclear envelope formation. Microinjection of the tandem SH2 domains of PLCgamma produced a concentration-dependent inhibition of Ca2+ release and also inhibited cortical granule exocytosis, cytoplasmic alkalinization, MAP kinase dephosphorylation, DNA synthesis, and cleavage after fertilization. However, the Ca2+ action potential, sperm entry, and sperm pronuclear formation were not prevented by injection of the PLCgammaSH2 domain protein. Microinjection of a control protein, the tandem SH2 domains of the phosphatase SHP2, had no effect on Ca2+ release, cortical granule exocytosis, DNA synthesis, or cleavage. Specificity of the inhibitory action of the PLCgammaSH2 domains was further indicated by the finding that microinjection of PLCgammaSH2 domains that had been point mutated at a critical arginine did not inhibit Ca release at fertilization. Additionally, Ca2+ release in response to microinjection of IP3, cholera toxin, cADP ribose, or cGMP was not inhibited by the PLCgammaSH2 fusion protein. These results indicate that PLCgamma plays a key role in several fertilization events in sea urchin eggs, including Ca2+ release and DNA synthesis, but that the action potential, sperm entry, and male pronuclear formation can occur in the absence of PLCgamma activation or Ca2+ increase.

Action Potentials↗

Effects of 6-dimethylaminopurine on microtubules and putative intermediate filaments in sea urchin embryos.

The effects of 6-dimethylaminopurine (6-DMAP) (a putative phosphorylation inhibitor) on the state of assembly of microtubules and intermediate filaments have been studied during the first cell cycle of the sea urchin Strongylocentrotus droebachiensis. Changes in the spatial organization of cytoskeletal structures were studied by indirect immunofluorescence with anti-tubulin and anti-IFa antibodies. The rates and patterns of protein phosphorylation in control and treated eggs were also investigated. The transfer of fertilized eggs to 600 microM 6-DMAP within 4 min following insemination inhibits pronuclear migration and syngamy. This also prevents male pronuclear decondensation, while chromatin condensation and nuclear envelope breakdown do not occur in the female pronucleus. Immunolabeling with anti-tubulin antibodies reveals the presence of cortical microtubules as early as 15 min after fertilization in both control and treated eggs. However, no sperm astral microtubules could be detected in the treated eggs. At later stages, from syngamy (40 min) up to nuclear envelope breakdown (90 min), 6-DMAP affects neither cortical microtubule organization nor the state of chromatin condensation but it precludes nuclear envelope breakdown and entry into mitosis. Treatment of the fertilized eggs after nuclear envelope breakdown induces permanent chromosome decondensation and premature disappearance of the mitotic apparatus. This last event involves disruption of the spatial organization of both microtubules and putative intermediate filaments. Quantitative measurements of protein phosphorylation show that 6-DMAP efficiently and reversibly inhibits 32P incorporation into proteins. Qualitative analysis of the autoradiograms of 32P-labeled proteins separated by SDS-PAGE reveals that a major protein band, migrating with an apparent molecular weight of 31 x 10(3)Mr, is specifically dephosphorylated in eggs treated with 6-DMAP. This study suggests that protein phosphorylation is required for sperm aster microtubule growth and migration, but not for cortical microtubule polymerization. It also strengthens the hypothesis that, in sea urchin eggs, putative intermediate filaments are tightly associated with spindle microtubules. Finally, it confirms that inhibiting protein phosphorylation before nuclear envelope breakdown reversibly prevents the entry into mitosis.

Adenine↗

Effects of Ca2+ channel blockers on cortical hypoperfusion and expression of c-Fos-like immunoreactivity after cortical spreading depression in rats.

1. We examined the effects of two Ca2+ channel blockers, lomerizine (KB-2796) and flunarizine, on the cortical hypoperfusion (measured by hydrogen clearance and laser Doppler flowmetry methods) and cortical c-Fos-like immunoreactivity that follow KCl-induced cortical spreading depression in anaesthetized rats. Cortical spreading depression was induced by application of 1 M KCl for 30 s to the cortical surface, 3.0 mm posterior to the area of cerebral blood flow measurement. 2. In control rats, KB-2796 (0.3 and 1 mg kg-1, i.v.) dose-dependently increased cerebral blood flow significantly at 30 min and 15 min, respectively, after its administration. Flunarizine (1 mg kg-1, i.v.) significantly increased cerebral blood flow 15 min after its administration. In contrast, dimetotiazine (3 mg kg-1, i.v.), a 5-HT2 and histamine H1 antagonist, failed to affect cerebral blood flow significantly. 3. After KCl application to the cortex, cerebral blood flow monitored by the laser Doppler flowmetry method increased transiently, for a few minutes, then fell and remained approximately 20 to 30% below control for at least 60 min. Cerebral blood flow monitored by the hydrogen clearance method was also approximately 20 to 30% below baseline for at least 60 min after KCl application. KB-2796 (0.3 and 1 mg kg-1, i.v.) and flunarizine (1 and 3 mg kg-1, i.v.) administered 5 min before KCl application inhibited the cortical hypoperfusion that followed KCl application, but dimetotiazine (1 and 3 mg kg-1, i.v.) did not. 4. An indicator of neuronal activation, c-Fos-like immunoreactivity, was detected in the ipsilateral, but not in the contralateral frontoparietal cortex 2 h after KCl application. No c-Fos-like immunoreactivity was seen on either side of the brain in the hippocampus, thalamus, striatum or cerebellum. 5. KB-2796 (1 mg kg-1, i.v.) and flunarizine (3 mg kg-1, i.v.), but not dimetotiazine (3 mg kg-1, i.v.), significantly attenuated the expression of c-Fos-like immunoreactivity in the ipsilateral frontoparietal cortex. 6. These findings suggest that the inhibitory effects of KB-2796 and flunarizine on the cortical hypoperfusion and expression of c-Fos-like immunoreactivity induced by spreading depression are mediated via the effects of Ca(2+)-entry blockade, which may include an increase in cerebral blood flow and the prevention of excessive Ca2+ influx into brain cells. KB-2796 and flunarizine may prove useful as inhibitors of cortical spreading depression in migraine.

Animals↗