Biomedical subjects

D J Price

Publications and source records attributed to D J Price.

At least 37 records · Page 2Linked to original sources

PubMed2000

A role for Pax6 in the normal development of dorsal thalamus and its cortical connections.

The transcription factor Pax6 is widely expressed throughout the developing nervous system, including most alar regions of the newly formed murine diencephalon. Later in embryogenesis its diencephalic expression becomes more restricted. It persists in the developing anterior thalamus (conventionally termed "ventral" thalamus) and pretectum but is downregulated in the body of the posterior (dorsal) thalamus. At the time of this downregulation, the dorsal thalamus forms its major axonal efferent pathway via the ventral telencephalon to the cerebral cortex. This pathway is absent in mice lacking functional Pax6 (small eye homozygotes: Sey/Sey). We tested whether the mechanism underlying this defect includes abnormalities of the dorsal thalamus itself. We exploited a new transgenic mouse ubiquitously expressing green fluorescent protein tagged with tau, in which axonal tracts are clearly visible, and co-cultured dorsal thalamic explants from Pax6(+/+ )or Pax6(Sey/Sey )embryos carrying the transgene with wild-type tissues from other regions of the forebrain. Whereas Pax6(+/+ )thalamic explants produced strong innervation of wild-type ventral telencephalic explants in a pattern that mimicked the thalamocortical tract in vivo, Pax6(Sey)(/Sey) explants did not, indicating a defect in the ability of mutant dorsal thalamic cells to respond to signals normally present in ventral telencephalon. Pax6(Sey)(/Sey) embryos also showed early alterations in the expression of regulatory genes in the region destined to become dorsal thalamus. Whereas in normal mice Nkx2.2 and Lim1/Lhx1 are expressed ventral to this region, in the mutants their expression domains are throughout it, suggesting that a primary action of Pax6 is to generate correct dorsoventral patterning in the diencephalon. Our results suggest that normal thalamocortical development requires the actions of Pax6 within the dorsal thalamus itself.

Alternative Splicing↗

PubMed2000

Severe left main stem stenosis in a cardiac transplant recipient: successful treatment by coronary stenting.

A 58-year-old male underwent orthotopic cardiac transplantation for ischemic cardiomyopathy. At routine coronary angiography 2 years later, he was found to have severe concentric stenosis of the left main coronary artery but was asymptomatic. Revascularization was recommended on prognostic grounds and after discussion with his cardiac transplant surgeon, percutaneous coronary intervention with elective stenting was offered. This was performed successfully with a single stent and a good angiographic result was maintained 6 and 18 months later.

Blood Vessel Prosthesis Implantation↗

PubMed1999

Basic fibroblast growth factor promotes subplate cell survival in explant cultures of embryonic mouse cortex.

Subplate neurons form a transient layer immediately below the embryonic cortex and die early in postnatal life. It has been suggested that trophic factors, perhaps coming from cortical afferents, maintain the initial survival of these cells. Later withdrawal of these factors may cause subplate cell death. We tested whether basic fibroblast growth factor (bFGF) has survival-promoting effects on subplate cells in organotypic cultures from the late embryonic mouse cortex. We found that the survival of subplate cells was promoted by adding bFGF to the cultures. By contrast, there was no effect of bFGF on the survival of overlying cortical neurons. These results indicate that bFGF may have a role in the regulation of subplate cell survival and death in vivo.

Animals↗

PubMed1999

Serotonin receptor activation enhances neurite outgrowth of thalamic neurones in rodents.

Serotonin (5-HT) has been shown to influence the development of the rodent barrel field by affecting the patterning of thalamic axons in the somatic sensory cortex. To determine whether this is a direct effect on thalamocortical neurones, we analyzed primary thalamic cultures taken from E15 mouse embryos. We show that 5-HT enhances neurite outgrowth of thalamic neurones. The sodium channel blocker, TTX, blocks these effects, whereas the selective 5-HT1B agonist CGS-12066A maleate reproduced 5-HT's effect. Using PCR and immunocytochemistry, we found that 5-HT1B receptors are already expressed by thalamic neurones at E15, and that this expression is maintained in vitro. These results suggest that 5-HT-1B receptor activation directly affects the growth of thalamocortical axons.

Animals↗

PubMed1999

CHK down-regulates SCF/KL-activated Lyn kinase activity in Mo7e megakaryocytic cells.

The Csk Homologous Kinase (CHK) has been shown to have an enzymatic activity similar to the tyrosine kinase Csk in that it down-regulates Src family kinase activity by causing phosphorylation of the Src C-terminal tyrosine residue. In megakaryocytic Mo7e cells, CHK associates with a specific phosphotyrosine juxtamembrane sequence of the SCF/KL-activated c-Kit receptor. Here, we show that in Mo7e cells, the major Src family kinase activity is p53/56(Lyn). Studies using immobilized c-Kit phosphopeptides show that Lyn is able to specifically associate with the tyrosine-phosphorylated juxtamembrane 568Y*VY*IDPT sequence of c-Kit which has previously been shown to associate with CHK. In cells over-expressing CHK by means of a recombinant vaccinia virus, we observed an elimination of the SCF/KL-stimulated Lyn kinase peak of activity observed at 2-5 minutes in cells infected with the helper T7-expressing vaccinia virus by itself. Examination of total tyrosine phosphorylation by Western blotting showed that over-expression of CHK resulted in a reduction in the levels of tyrosine phosphorylations in the range of 50-60 kDa, but had no apparent effect on c-Kit autophosphorylation. Taken together, these findings show that CHK is able to down-regulate SCF/KL-stimulated Lyn activity in megakaryocytes.

Cell Line↗

PubMed1999

Effects of the thalamus on the development of cerebral cortical efferents in vitro.

The cerebral cortex is a multilayered tissue, with each layer differing in its cellular composition and connections. Axons from deep layer neurons project subcortically, many to the thalamus, whereas superficial layer neurons target other cortical areas. The mechanisms that regulate the development of this pattern of connections are not fully understood. Our experiments examined the potential of the thalamus to attract and/or select neurites from appropriate cortical layers. First, we cocultured murine cortical slices in close proximity to thalamic explants in collagen gels. The amount of neurite outgrowth from deep layer cells was enhanced by, but not attracted to, the thalamic explants. Second, we cocultured cortical slices in contact with thalamic or cortical explants to test for laminar specificity of connections. Specificity was apparent after culture for about a week, in that deep cortical layers contained the highest proportions of corticothalamic cells and superficial cortical layers contained the highest proportions of corticocortical cells. After shorter culture of only a few days, however, specificity was not apparent and there were larger numbers of corticothalamic projections from the superficial layers than after a week. To study how the early nonspecific pattern of corticothalamic connections was transformed into the more specific pattern, we labeled corticothalamic cells early, after 2 days, but let the cultures survive for 8 days. On day 8, the nonspecific pattern of early-labeled cells was still seen. We conclude that although the thalamus does not block the initial entry of inappropriate axons from the superficial layers, many of these axons are subsequently lost. This suggests that contact-mediated interactions between cortical axons and the thalamus allow cortical efferents from appropriate layers to be distinguished from those arising in inappropriate layers. This may contribute to the development of layer-specific cortical connections in vivo.

Animals↗

PubMed1999

Evidence that the lateral geniculate nucleus regulates the normal development of visual corticocortical projections in the cat.

The aim of this work was to examine the influence of subcortical afferents on the development of corticocortical projections in the cat's visual cortex. In the adult, corticocortical axons project with precision to link retinotopically corresponding points in visual areas 17 and 18. In the newborn kitten, an excess of corticocortical connections is generated, leading to a degree of imprecision in the early pathways. During the first postnatal month, the loss of some of these early connections lowers their densities and increases the accuracy with which they project. These processes occur in an environment already influenced by afferents from the lateral geniculate nucleus and we tested the extent to which these existing inputs are required for corticocortical development. We lesioned the lateral geniculate nucleus with ibotenic acid in newborn kittens and studied connections from area 17 to area 18, and vice versa, after 1 month. In lesioned kittens, there were fewer corticocortical projections than normal in these reciprocal pathways and those that were present retained an immature, widespread pattern of projection. These results suggest that geniculate afferents are crucial for generating sufficient numbers of corticocortical projections and for creating the precision in their mapping.

Animals↗

PubMed1999

Regulation of megakaryocytopoiesis and platelet production by tyrosine kinases and tyrosine phosphatases.

Megakaryocytopoiesis is the process by which bone marrow progenitor cells develop into mature megakaryocytes, which in turn produce platelets required for normal hemostasis. The development of this hematopoietic lineage depends on a variety of growth factors and cytokines. Growth factor-dependent tyrosine kinase receptors important in megakaryocytopoiesis include c-Kit, fibroblast growth factor receptor, the RON receptor, and the macrophage colony-stimulating factor receptor. Binding of growth factors to their respective receptors results in receptor dimerization and subsequent autophosphorylation on tyrosine residues. Tyrosine autophosphorylations become sites of association for cytoplasmic signaling molecules via their SH2 domains. Some of these molecules are themselves cytoplasmic tyrosine kinases such as the Src kinases, TEC, and CHK. Others are molecules such as phospholipase C-gamma, phosphoinositol 3-kinase, Shc, GTPase-activating protein, and the SH2-containing tyrosine phosphatases SHP-1 and SHP-2. These molecules generate second messengers, regulate the phosphorylation of other downstream molecules, and also regulate the phosphorylation of the receptor itself. The different cytoplasmic components activate pathways involved in either changes in cell growth or changes in the cytoskeleton that affect maturation of the cell. Cytokine receptors also generate signals involved in growth and differentiation. Some of these second messengers overlap with those of the receptor tyrosine kinases. Others, such as the JAKs/STATs, are involved in transcriptional control and are unique to the signaling mediated by cytokine receptors. We describe the contribution of these different signals to the growth/differentiation processes of megakaryocytes. We also describe the contribution of receptor and nonreceptor tyrosine phosphatases to these processes. Lastly, we have compiled selected methods related to the study of protein phosphorylation in megakaryocytes.

Animals↗

PubMed1999

The production and elimination of supernumerary blast cells in the leech embryo.

Different species of leech vary greatly in body size but all have 32 body segments. It is unclear how the development of this precise number of segments is regulated, although it is known that the teloblasts of the early leech embryo initially produce more than the required numbers of segment founder cells (blast cells). We used fluorescent dextrans to show that the M teloblast of the Helobdella robusta embryo produces a variable number of additional (supernumerary) cells. These cells fail to enter the germinal band (which contains cells of all lineages and gives rise to the adult leech), but detach from its posterior end and disappear. Our observations suggest that some suffer an increase in membrane permeability while others fuse with the M teloblasts, but that they do not undergo apoptosis. The supernumerary cells of different lineages detach from the germinal band at different times, suggesting that detachment is not triggered by a global signal acting simultaneously on all lineages. We tested the hypothesis that the elimination of the supernumerary m blast cells results from a requirement of m blast cells for close interactions with cells of the other lineages for their survival, a condition that would not be achieved by the last-born m blast cells that fail to enter the germinal band. We cultured isolated M teloblasts and found that they do produce blast cells that themselves divide, indicating that cells of the M lineage can survive in the absence of any interactions with cells of the other lineages.

Animals↗

PubMed1999

The transcription factor, Pax6, is required for cell proliferation and differentiation in the developing cerebral cortex.

The cerebral cortex develops from the dorsal telencephalon, at the anterior end of the neural tube. Neurons are generated by cell division at the inner surface of the telencephalic wall (in the ventricular zone) and migrate towards its outer surface, where they complete their differentiation. Recent studies have suggested that the transcription factor Pax6 is important for regulation of cell proliferation, migration and differentiation at various sites in the CNS. This gene is widely expressed from neural plate stage in the developing CNS, including the embryonic cerebral cortex, where it is required for radial glial cell development and neuronal migration. We report new findings indicating that, in the absence of Pax6, proliferative rates in the early embryonic cortex are increased and the differentiation of many cortical cells is defective. A major question concerns the degree to which cortical defects in the absence of Pax6 are a direct consequence of losing the gene function from defective cells themselves, rather than being secondary to abnormalities in other cells. Cortical defects in the absence of Pax6 become much more pronounced later in cortical development, and we propose that many result from a compounding of abnormalities in proliferation and differentiation that first appear at the onset of corticogenesis.

Animals↗

PubMed1998

Plasma membrane transporters of serotonin, dopamine, and norepinephrine mediate serotonin accumulation in atypical locations in the developing brain of monoamine oxidase A knock-outs.

Genetic loss or pharmacological inhibition of monoamine oxidase A (MAOA) in mice leads to a large increase in whole-brain levels of serotonin (5-HT). Excess 5-HT in mouse neonates prevents the normal barrel-like clustering of thalamic axons in the somatosensory cortex. Projection fields of other neuron populations may develop abnormally. In the present study, we have analyzed the localization of 5-HT immunolabeling in the developing brain of MAOA knock-out mice. We show numerous atypical locations of 5-HT during embryonic and postnatal development. Catecholaminergic cells of the substantia nigra, ventral tegmental area, hypothalamus, and locus ceruleus display transient 5-HT immunoreactivity. Pharmacological treatments inhibiting specific monoamine plasma membrane transporters and genetic crosses with mice lacking the dopamine plasma membrane transporter show that the accumulation of 5-HT in these catecholaminergic cells is attributable to 5-HT uptake via the dopamine or the norepinephrine plasma membrane transporter. In the telencephalon, transient 5-HT immunolabeling is observed in neurons in the CA1 and CA3 fields of the hippocampus, the central amygdala, the indusium griseum, and the deep layers of the anterior cingulate and retrosplenial cortices. In the diencephalon, primary sensory nuclei, as well as the mediodorsal, centrolateral, oval paracentral, submedial, posterior, and lateral posterior thalamic nuclei, are transiently 5-HT immunolabeled. The cortical projections of these thalamic nuclei are also labeled. In the brainstem, neurons in the lateral superior olivary nucleus and the anteroventral cochlear nucleus are transiently 5-HT immunolabeled. None of these structures appear to express the monoamine biosynthetic enzyme L-aromatic amino acid decarboxylase. The administration of monoamine plasma membrane transporter inhibitors indicates that the 5-HT immunolabeling in these structures is attributable to an uptake of 5-HT by the 5-HT plasma membrane transporter. This points to neuron populations that form highly precise projection maps that could be affected by 5-HT during specific developmental stages.

Animals↗

PubMed1998

Effects of monoamine oxidase A inhibition on barrel formation in the mouse somatosensory cortex: determination of a sensitive developmental period.

Genetic inactivation of monoamine oxidase A (MAOA) in C3H/HeJ mice causes a complete absence of barrels in the somatosensory cortex, and similar alterations are caused by pharmacological inhibition of MAOA in wild type mice. To determine when and how MAOA inhibition affects the development of the barrel field, the MAOA inhibitor clorgyline was administered to mice of the outbred strain OF1 for various time periods between embryonic day 15 (E15) and postnatal day 7 (P7), and the barrel fields were analyzed with cytochrome oxidase and Nissl stains in P10 and adult mice. High-pressure liquid chromatography measures of brain serotonin (5-HT) showed three- to eightfold increases during the periods of clorgyline administration. Perinatal mortality was increased and weight gain was slowed between P3 and P6. Clorgyline treatments from E15 to P7 or from P0 to P7 disrupted the formation of barrels in the anterior snout representation and in parts of the posteromedial barrel subfield (PMBSF). Treatments from P0 to P4 caused similar although less severe barrel field alterations. Clorgyline treatments only during embryonic life or starting on P4 caused no detectable abnormalities. In cases with barrel field alterations, a rostral-to-caudal gradient of changes was noted: Rostral barrels of the PMBSF were most frequently fused and displayed an increased size tangentially. Thus, MAOA inhibition resulting in increased brain levels of 5-HT affects barrel development during the entire first postnatal week, with a sensitive period between P0 and P4. The rostral-to-caudal gradient of changes in the barrel field parallels known developmental gradients in the sensory periphery and in the maturation thalamocortical afferents. The observed barrel fusions could correspond to a default in the initial segregation of thalamic fibers or to a continued, exuberant growth of these fibers that overrides the tangential domain that is normally devoted to individual whiskers.

Animals↗

PubMed1998

The relationship of vasogenic waves to ICP and cerebral perfusion pressure in head injured patients.

Slow vasogenic waves are characterised by sudden rises and falls in both the mean ICP and its cardiac pulsatile amplitude for periods of up to an hour. On reviewing the on-line computer records of a series of 200 consecutive head injured patients, some 650 waves with increases in mean ICP of at least 15 mm Hg were recognized. The mean ICP, pulsatile amplitude, perfusion pressure, arterial pressure and pulse rates which had been generated every minute were reviewed in each of a 100 randomly selected waves in the hopes of allocating them into defined groups according to precipitating factors. We had expected to find that the majority of waves were precipitated by a preceding fall in cerebral perfusion pressure as a result of a transient arterial hypotension. Only in 19 of the 100 waves did this sequence become apparent and in the remainder, no recognizable precipitating factors were found. In 21 of the records, there was a fall in ICP of at least 5 mm Hg before the onset of the pressure wave.

Blood Pressure↗

PubMed1998

Electrophysiological and neurochemical study of the rat geniculo-cortical pathway. Evidence for glutamatergic neurotransmission.

The projection from the dorsal lateral geniculate nucleus to the primary visual cortex of the rat was studied electrophysiologically. Electrical stimulation of the dorsal lateral geniculate nucleus and the optic tract produced three types of responses on neurons of area 17: excitation followed by inhibition, excitation and inhibition. These results extend and confirm, in adult rats, previous studies done in rat geniculate-visual cortex cocultures preparations in vitro. The role of glutamate in the neurotransmission of the rat geniculo-cortical pathway was also investigated. In a first set of experiments, the effects of kynurenate, an antagonist of glutamate receptors, on visual cortex neurons with a monosynaptic excitatory response to dorsal lateral geniculate nucleus stimulation were studied. Microiontophoresis of kynurenate in area 17 neurons selectively suppressed the excitatory response to dorsal lateral geniculate nucleus and optic tract stimulation. In a second set of experiments, the effects of electrical stimulation of the dorsal lateral geniculate nucleus and the optic tract on the release of amino acids in the rat visual cortex in vivo were studied. Using the push-pull method, we perfused a discrete region of the visual cortex with artificial cerebrospinal fluid (CSF), and the amino acid content of the perfusates was analysed by high performance liquid chromatography (HPLC). Stimulation of either the dorsal lateral geniculate nucleus or the optic tract significantly increased glutamate release in area 17. The rest of the amino acids studied did not show significant changes. The results provide evidence for the participation of glutamate in the neurotransmission of the geniculo-cortical pathway in the rat.

Animals↗

PubMed1997

Direct association of Csk homologous kinase (CHK) with the diphosphorylated site Tyr568/570 of the activated c-KIT in megakaryocytes.

The Csk homologous kinase (CHK), formerly MATK, has previously been shown to bind to activated c-KIT. In this report, we characterize the binding of SH2(CHK) to specific phosphotyrosine sites on the c-KIT protein sequence. Phosphopeptide inhibition of the in vitro interaction of SH2(CHK)-glutathione S-transferase fusion protein/c-KIT from SCF/KL-treated Mo7e megakaryocytic cells indicated that two sites on c-KIT were able to bind SH2(CHK). These sites were the Tyr568/570 diphosphorylated sequence and the monophosphorylated Tyr721 sequence. To confirm this, we precipitated native CHK from cellular extracts using phosphorylated peptides linked to Affi-Gel 15. In addition, purified SH2(CHK)-glutathione S-transferase fusion protein was precipitated with the same peptide beads. All of the peptide bead-binding studies were consistent with the direct binding of SH2(CHK) to phosphorylated Tyr568/570 and Tyr721 sites. Binding of FYN and SHC to the diphosphorylated Tyr568/570 site was observed, while binding of Csk to this site was not observed. The SH2(CHK) binding to the two sites is direct and not through phosphorylated intermediates such as FYN or SHC. Site-directed mutagenesis of the full-length c-KIT cDNA followed by transient transfection indicated that only the Tyr568/570, and not the Tyr721, is able to bind SH2(CHK). This indicates that CHK binds to the same site on c-KIT to which FYN binds, possibly bringing the two into proximity on associated c-KIT subunits and leading to the down-regulation of FYN by CHK.

Animals↗

PubMed1997

Fates of the earliest generated cells in the developing murine neocortex.

In mammalian species studied to date, the first-born neocortical cells normally form two layers, one above and one below the cortical plate, called the marginal zone (future layer 1) and the subplate. In primates and carnivores, many of these first-born cells die early in postnatal life. Whether this also occurs in rodents is highly controversial. In this study, we injected pregnant mice with bromodeoxyuridine on embryonic days (E) 11-14 to label the earliest generated neocortical cells, and examined their fates between birth and postnatal day 21. At birth, most cells born on embryonic day 11 were below the cortical plate, and a smaller proportion were above it. Very few of these cells remained by postnatal day 3 and there were none at any depth in the neocortex at older ages. At birth, the largest proportion of cells born on embryonic days 12 and 13 were in the subplate and smaller proportions were in the cortical plate and marginal zone. At older ages, almost all of these cells had disappeared from the marginal zone and from below the cortical plate, although some were retained in the cortical plate. The density of the remaining E12- and E13-born cells decreased more than could be explained by neocortical expansion alone. As a control, we studied cells born on embryonic day 14. These cells were restricted to the cortical plate at birth. By postnatal day 21, their density had decreased by an amount that could be explained by neocortical expansion alone. We conclude that, as in other species, many of the earliest generated cells of the murine neocortex die.

Age Factors↗

PubMed1997

A role for neurotrophins in the survival of murine embryonic thalamic neurons.

The mechanisms that determine whether developing CNS neurons live or die are poorly understood. We studied the role of the neurotrophins and fibroblast growth factors in the survival of embryonic thalamic neurons in culture. Dissociated embryonic dorsal thalamic neurons cultured at high density in defined serum-free medium survived and grew neurites. As in vivo, they expressed all the neurotrophins, fibroblast growth factor-1 and their high-affinity tyrosine kinase receptors. The survival of these cells was reduced by the addition of the protein kinase inhibitor K252a at concentrations that block neurotrophin receptor activity but not the activity of other tyrosine kinase receptors. In low-density cultures, most dorsal thalamic neurons died, but their survival was increased by co-culture with thalamic explants or with most of the neurotrophins and fibroblast growth factor-1 added singly. These results indicate that thalamic neurons have remarkably promiscuous trophic responses to a battery of neurotrophins and fibroblast growth factors. They suggest that neurotrophins endogenous to the early embryonic thalamus may be required to promote the survival of its neurons.

Animals↗

PubMed1997

Determination of the migratory capacity of embryonic cortical cells lacking the transcription factor Pax-6.

The cerebral cortex forms by the orderly migration and subsequent differentiation of neuronal precursors generated in the proliferative ventricular zone. We studied the role of the transcription factor Pax-6, which is expressed in the ventricular zone, in cortical development. Embryos homozygous for a mutation of Pax-6 (Small eye; Sey) had abnormalities suggesting defective migration of late-born cortical precursors. When late-born Sey/Sey precursors were transplanted into wild-type embryonic rat cortex, they showed similar integrative, migrational and differentiative abilities to those of transplanted wild-type mouse precursors. These results suggest that postmitotic cortical cells do not need Pax-6 to acquire the capacity to migrate and differentiate, but that Pax-6 generates a cortical environment that permits later-born precursors to express their full developmental potential.

Animals↗