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R Somogyi

Publications and source records attributed to R Somogyi.

At least 37 records · Page 2Linked to original sources
PubMed1998

GABAergic cells and signals in CNS development.

GABA is formed primarily from decarboxylation of glutamate by a family of cytosolic and membrane-bound GAD enzymes. In the adult, GAD-derived GABA sustains the vitality of the central nervous system (CNS), since blockage of GAD rapidly leads to convulsions and death. In plants, cytosolic GAD synthesizes GABA in response to hormones and environmental stress. Since decarboxylation involves protonation, secretion of GABA serves to buffer cytosolic pH in plant cells. Families of GAD and GABAA receptor/Cl- channel transcripts and encoded proteins emerge early and seemingly everywhere during CNS development, with their abundance closely paralleling neurogenesis and peaking before birth. Micromolar GABA acts at receptor/Cl-channels to depolarize progenitor cells in the cortical neuroepithelium; it also elevates their cytosolic Ca2+ (Cac2+) levels. In some way, these effects decrease proliferation. GABA directs the migration of postmitotic neuroblasts at femtomolar concentrations and stimulates their random motility at micromolar concentrations via Ca2+ signaling mechanisms. Activation of GABAA receptors by micromolar GABA may limit motility via membrane depolarization and elevated Cac2+. These results indicate that in vitro GABA can affect embryogenesis of the CNS through effects on cell proliferation and migration. As neurons differentiate postnatally, Cl(-)-dependent depolarization disappears together with GABAergic Cac2+ signals. Physiologically occurring GABAergic signals at Cl-channels exist in tonic and transient forms. Since the former are found on progenitor cells while both are present in postmitotic neurons, mechanisms to generate transients differentiate in the latter. Surprisingly, tonic and transient forms of GABAergic signaling at Cl-channels are rapidly and smoothly interconvertible and seem to be derived from online GABA synthesis in a surface-accessible compartment of the membrane.

Animals↗
PubMed1997

Novel differentially expressed genes induced by kainic acid in hippocampus: putative molecular effectors of plasticity and injury.

Systemic kainic acid administration in rats induces acute limbic status epilepticus and subsequent neuronal degeneration and development of chronic hyperexcitability with similarities to human temporal lobe epilepsy. The mechanisms mediating the responses to kainic acid likely involve transcriptional changes in genes of importance for cellular injury, protection, and plasticity. We have used an arbitrarily primed PCR technique to identify such changes in the rat dentate gyrus. Three previously uncharacterized transcripts were found to be upregulated in the dentate gyrus 4 h following systemic kainic acid. In situ hybridization using riboprobes transcribed from the cloned PCR fragments were used to confirm differential expression specifically in dentate granule neurons following seizure. Basal expression for all three transcripts is widespread throughout the rat brain, with the highest levels seen in the hippocampal pyramidal and granule cell layers. The novel sequences do not match any known full-length cDNAs and may belong to novel gene families. However, they all showed high homology to human partial cDNA sequences (ESTs) that are expressed in brain as well as several other tissues. Two additional transcripts identified in this study corroborate earlier findings on differential expression of heat-shock proteins after seizure. The novel transcripts found in this study may be involved in epileptogenesis and neuronal responses to damage following seizure.

Animals↗
PubMed1997

Anatomical gradients in proliferation and differentiation of embryonic rat CNS accessed by buoyant density fractionation: alpha 3, beta 3 and gamma 2 GABAA receptor subunit co-expression by post-mitotic neocortical neurons correlates directly with cell buoyancy.

Development of the CNS occurs as a complex cascade of pre-programmed events involving distinct phases of cell proliferation and differentiation. Here we show these phases correlate with cells of specific buoyant densities which can be readily accessed by density gradient fractionation. Sprague-Dawley dams were pulse-labelled with bromodeoxyuridine (BrdU) and selected regions of embryonic (E) CNS tissues at E11-22 dissociated with papain into single-cell suspensions. Proliferative cell populations were assessed by anti-BrdU and propidium iodide staining using flow cytometry. Cell differentiation was evaluated using molecular and immunocytochemical probes against mRNAs and antigens differentiating the neuroepithelial, neuronal and glial cell lineages. The results show the emergence of distinctive spatiotemporal changes in BrdU+ populations throughout the CNS during embryonic development, which were followed by corresponding changes in the cellular distributions of antigens distinguishing specific cell types. Fractionation of neocortical cells using discontinuous Percoll gradients revealed that an increasing number of cells increase their buoyancy during corticogenesis. Immunocytochemical and molecular characterization showed that the proliferative and progenitor cell populations are for the most part associated with lower buoyancy or higher specific buoyant densities (> 1.056 g/ml) whereas the post-mitotic, differentiated neurons generally separated into fractions of higher buoyancy or lower specific buoyant densities (< 1.043 g/ml). Immunostaining with antibodies against several GABAA receptor subunits (alpha 3, beta 3, gamma 2) revealed that the highest percent (70-90%) of immunopositive cells could be identified in the most buoyant, differentiating neurons found in the cortical plate/subplate regions, with the lowest percent of the immunopositive cells found in the least buoyant, proliferative and progenitor cell populations originating from the ventricular/subventricular zones. Taken together, these results indicate that buoyant density is a distinguishing characteristic of embryonic CNS cells transforming from primarily proliferative to mainly differentiating, and that fractionation of these cells according to their buoyant densities provides rapid access to the properties of specific cell lineages during the prenatal period of CNS development.

Animals↗
PubMed1997

Neurotrophins stimulate chemotaxis of embryonic cortical neurons.

During mammalian cortical development, neuronal precursors proliferate within ventricular regions then migrate to their target destinations in the cortical plate, where they organize into layers. In the rat, most cortical neuronal migration occurs during the final week of gestation (Bayer et al, 1991; Jacobson, 1991). At this time (E15-E21), reverse transcriptase-polymerase chain reaction demonstrated that cortical homogenates contain mRNA encoding brain derived neurotrophic factor (BDNF) and the catalytic form of its high-affinity receptor, TrkB. Immunocytochemistry and in situ hybridization of sections revealed that the catalytic TrkB receptors predominantly localize to regions containing migratory cells. Many TrkB+ cells exhibited the classic morphology of migrating neurons, suggesting that TrkB ligands play a role in cortical neuronal migration. We analysed whether TrkB ligands influence the motility of embryonic cortical cells (from E15-E21) using a quantitative in vitro chemotaxis assay. High-affinity TrkB ligands (BDNF and NT4/5) stimulated chemotaxis (directed migration) of embryonic neurons at concentrations ranging from 1 to 100 ng/ml. NT-3, a low-affinity TrkB ligand, only stimulated significant migration at high concentrations (> or =100 ng/ml). Peak migration to BDNF was observed at gestational day 18 (E18). BDNF-induced chemotaxis was blocked by either tyrosine kinase inhibitor, K252a, or the Ca2+-chelator, BAPTA-AM, suggesting that BDNF-induces motility via autophosphorylation of TrkB receptor proteins and involves Ca2+-dependent mechanisms. BDNF-stimulation of increased cytosolic Ca2+ was confirmed with optical recordings of E18 cortical cells loaded with Ca2+ indicator dye. Thus, signal transduction through the TrkB receptor complex directs neuronal migration, suggesting that, in vivo, BDNF exerts chemotropic effects that are critical to morphogenesis of the cortex.

Animals↗
PubMed1995

Localization of functional receptor epitopes on the structure of ciliary neurotrophic factor indicates a conserved, function-related epitope topography among helical cytokines.

By rational mutagenesis, receptor-specific functional analysis, and visualization of complex formation in solution, we identified individual amino acid side chains involved specifically in the interaction of ciliary neurotrophic factor (CNTF) with CNTFR alpha and not with the beta-components, gp130 and LIFR. In the crystal structure, the side chains of these residues, which are located in helix A, the AB loop, helix B, and helix D, are surface accessible and are clustered in space, thus constituting an epitope for CNTFR alpha. By the same analysis, a partial epitope for gp130 was also identified on the surface of helix A that faces away from the alpha-epitope. Superposition of the CNTF and growth hormone structures showed that the location of these epitopes on CNTF is analogous to the location of the first and second receptor epitopes on the surface of growth hormone. Further comparison with proposed binding sites for alpha- and beta-receptors on interleukin-6 and leukemia inhibitory factor indicated that this epitope topology is conserved among helical cytokines. In each case, epitope I is utilized by the specificity-conferring component, whereas epitopes II and III are used by accessory components. Thus, in addition to a common fold, helical cytokines share a conserved order of receptor epitopes that is function related.

Animals↗
PubMed1995

Developmental kinetics of GAD family mRNAs parallel neurogenesis in the rat spinal cord.

GABA (gamma-amino butyric acid), a fast-acting synaptic transmitter in the mature CNS, is synthesized from glutamate by GAD (glutamic acid decarboxylase). We have developed an ultrasensitive PCR technique to quantify the expression of GAD-related mRNAs during the development of the rat cervical spinal cord and have localized them using in situ hybridization. GAD65, GAD67, and an alternatively spliced variant of GAD67, EP10, were quantified each day from embryonic (E) day 11 through E21, and at postnatal days 0, 7, 14, and adult. GAD65 and GAD67 mRNAs were detected at E11 and increased exponentially over three orders of magnitude during embryonic development, then declined approximately threefold in the first-2 postnatal weeks. While the exponential growth phase coincided with the progressive appearance of GAD67 in situ signals in both the ventral and dorsal cord, the postnatal decline coincided with the virtual disappearance of expression in the ventral region. EP10 expression was prominent in the embryo, then declined markedly together with the mRNA encoding the neuroepithelial stem cell marker, nestin. The concerted appearance of GAD-related mRNAs paralleled transcripts encoding neuronal markers (light and heavy neurofilaments) and also closely correlated with the expression of GABA, mRNAs encoding GABAA receptor subunits, and depolarizing responses to GABA. We have used the results on GAD-related mRNA expressions to formulate a simple, minimal mathematical model that accounts for their kinetics in terms of positive and negative feedback loops.

Aging↗
PubMed1994

Recombinant human CNTF receptor alpha: production, binding stoichiometry, and characterization of its activity as a diffusible factor.

The primary ligand-binding protein (CNTFR alpha) of the multicomponent receptor for ciliary neurotrophic factor was produced in Escherichia coli. Using novel applications of size-exclusion chromatography and a protein gel-shift assay, we obtained quantitative separation of correctly refolded protein, as well as analytical monitoring of the refolding process and ligand binding. By these and other methods, we determined a 1:1 stoichiometry for the receptor-ligand complex. To investigate the proposed activity and mechanism of soluble CNTFR alpha as a diffusible factor, we studied the response of TF-1 cells which lack CNTFR alpha to various CNTF ligands and the stimulation of this response by sCNTFR alpha. The results show that sCNTFR alpha combines with CNTF and mediates cell survival with the same relative ligand specificity and relative affinity as the cell-surface form. Thus, soluble receptor can reconstitute on a cell surface active complexes that are analogous to the native complexes. Moreover, both the relative ligand potency in the absence of CNTFR alpha and the kinetics of the response to sCNTFR alpha indicate that the other components of the receptor complex contribute little, but measurably, to the specific potency of CNTF.

Animals↗
PubMed1994

Retrograde axonal transport of LIF is increased by peripheral nerve injury: correlation with increased LIF expression in distal nerve.

Leukemia inhibitory factor (LIF) is a cytokine that affects the survival and differentiation of certain neuronal populations in vitro. To identify LIF-responsive neurons in the adult rat, we have demonstrated retrograde axonal transport of 125I-LIF to sensory and motor neurons. The accumulation of 125I-LIF by both cell types was significantly increased by prior sciatic nerve crush. Retrograde transport of 125I-LIF was inhibited by excess unlabeled LIF but not by related cytokines, indicating a specific receptor-mediated mechanism. Northern blot analysis revealed LIF expression in peripheral nerve that was increased in distal segments after axotomy. The correlation between LIF expression and increased retrograde transport following injury suggests that LIF plays a role in peripheral nerve regeneration.

Animals↗
PubMed1994

GABA-induced chemokinesis and NGF-induced chemotaxis of embryonic spinal cord neurons.

During CNS development, neuroblasts proliferate within germinal zones of the neuroepithelium, and then migrate to their final positions. Although many neurons are thought to migrate along processes of radial glial fibers, increasing evidence suggests environmental factors also influence nerve cell movement. Extracellular matrix molecules are thought to be involved in guiding neuronal migration, and molecules such as NGF and GABA exert trophic effects on immature neurons. The nature of the signals that initiate and direct neuroblast migration, however, is unknown. In vitro, NGF and GABA promote neurite outgrowth from cultured cells, and NGF induces axonal chemotaxis (directed migration along a chemical gradient). At earlier developmental stages, these molecules could influence neuroblast movement. Therefore, we investigated whether these molecules induce embryonic neuronal migration. Using an in vitro microchemotaxis assay, we show that rat embryonic spinal cord neurons migrate toward picomolar NGF and femtomolar GABA beginning at embryonic day 13 (E13). Cells exhibit chemotactic responses to NGF while GABA stimulates chemokinesis (increased random movement). GABA effects are mimicked by muscimol and inhibited by bicuculline and picrotoxin, suggesting GABA motility signals are mediated by GABA receptor proteins. Expression of GABA receptors by embryonic cord cells has been previously reported (Mandler et al., 1990; Walton et al., 1993). We used polymerase chain reaction analysis to demonstrate the presence of NGF and trk mRNA in E13 and E14 cord cells, indicating the cells express message for both NGF and high-affinity NGF receptors. Immunohistochemistry of E13 spinal cord sections indicates that NGF and GABA colocalize in fibers close to the target destinations of migrating neurons, suggesting diffusible gradients of these molecules provide chemoattractant signals to migratory cells. Thus, in vitro, neuroblast migration is induced by specific signaling molecules that are present in the developing spinal cord, and may stimulate migration of embryonic neurons prior to synaptogenesis.

Animals↗
PubMed1993

Ontogeny of GABAA receptor subunit mRNAs in rat spinal cord and dorsal root ganglia.

Relatively little is known about the development of GABAA receptor subunits and their gene expression in mammalian spinal cord. The expression of mRNAs encoding 13 GABAA receptor subunits (alpha 1-6, beta 1-3, gamma 1-3, and delta) in embryonic, postnatal, and adult rat spinal cord and dorsal root ganglia (DRG) cells were studied by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Both techniques revealed the presence of all subunit mRNAs originally found in the rat brain, except for alpha 6, which was not detectable, and delta, which was weakly detected only by RT-PCR. Two anatomically distinctive sets of subunit mRNAs were found by in situ hybridization within the ventricular zone (VZ) and mantle zone (MZ). The trio of alpha 4, beta 1, and gamma 1 subunit mRNAs emerged exclusively in neuroepithelial cells at embryonic day 13 (E13) and remained detectable in the VZ until E17. In the MZ, beta 3 subunit mRNA was first detected at E12, while alpha 2, alpha 3, alpha 5, beta 2, gamma 2, and gamma 3 transcripts appeared at E13. Expressions of the subunit mRNAs in the MZ rapidly increased and expanded in a ventrodorsal sequence from motoneurons to dorsal horn neurons before reaching a peak in the late embryonic/early postnatal period. The mRNA expressions declined during postnatal development, by region-selective depletion, with alpha 4, alpha 5, beta 1, beta 2, gamma 1, and gamma 3 subunit mRNAs becoming barely detectable. In contrast, alpha 2, alpha 3, beta 3, and gamma 2 transcripts persisted into adulthood with distinct anatomical distributions. RT-PCR analysis revealed unique developmental patterns in the intensities of PCR products, most of which were in good agreement with developmental changes in the densities of hybridized mRNA signals. However, RT-PCR amplified minute amounts of mRNAs for alpha 1, alpha 4, alpha 5, beta 1, beta 2, gamma 1, gamma 3, and delta subunits in adults, which were not found in film autoradiograms, but could be detected in a few grain-positive cells in emulsion-dipped sections. DRG cells expressed alpha 2, alpha 3, alpha 5, beta 2, beta 3, and gamma 2 subunit mRNAs during embryogenesis but only alpha 2, beta 3, and gamma 2 subunit mRNAs were reliably detected in the adult.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Modulation of cytosolic-[Ca2+] oscillations in hepatocytes results from cross-talk among second messengers. The synergism between the alpha 1-adrenergic response, glucagon and cyclic AMP, and their antagonism by insulin and diacylglycerol manifest themselves in the control of the cytosolic-[Ca2+] oscillations.

Hepatocytes respond to stimulation by glycogenolytic agonists acting via phosphoinositide (PI) breakdown through oscillations of the free cytosolic concentration of Ca2+ ([Ca2+]cyt.). Since the second-messenger repertoire of hepatocytes includes many other factors besides Ca2+, we investigated to what degree the regulation of [Ca2+]cyt. oscillations is integrated into these other signalling systems. [Ca2+]cyt. was recorded in single rat hepatocytes by using the Ca(2+)-indicator fura-2. Parallel stimulation with phenylephrine (an alpha 1-adrenergic agonist of PI breakdown) and glucagon resulted in a synergistic stimulation of [Ca2+]cyt. oscillations. Direct activation of the cyclic-AMP-dependent pathway with several stimuli (forskolin, 8-bromo cyclic AMP, 8-CPT cyclic AMP) mimicked the response to glucagon. In contrast, [Ca2+]cyt. oscillations induced by various combinations of these agonists could be antagonized by the glycogenic hormone insulin. As one of the options in the insulin-signalling network, we tested a diacylglycerol activator of protein kinase C, DiC8. It also acted as an inhibitor of [Ca2+]cyt. oscillations. We investigated how these observations could be reconciled with our previously introduced model of [Ca2+]cyt. oscillations in hepatocytes [Somogyi and Stucki (1991) J. Biol. Chem. 266, 11068-11077]. First of all, the effect of calmodulin inhibitors (calmidazolium and CGS 9343 B), acting at the core of our model on the feedback of Ca2+ on Ins(1,4,5)P3-induced Ca2+ release, was not altered by the new modulators. In addition, all agonists and antagonists could be used interchangeably in combination and introduced no significant change in the oscillatory pattern or spike shape. Since the response was solely limited to frequency modulation, over- or understimulation of the oscillatory system, there is no need to create a new oscillator or to introduce further reaction steps into the core of the model. We conclude that the regulation of [Ca2+]cyt. via the explored second-messenger pathways can be embedded into the oscillatory system as modulation of rate constants already present in this model.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Hormone-induced calcium oscillations in liver cells can be explained by a simple one pool model.

Hormone-induced oscillations of the free intracellular calcium concentration are thought to be relevant for frequency encoding of hormone signals. In liver cells, such Ca2+ oscillations occur in response to stimulation by hormones acting via phosphoinositide breakdown. This observation may be explained by cooperative, positive feedback of Ca2+ on its own release from one inositol 1,4,5-trisphosphate-sensitive pool, obviating oscillations of inositol 1,4,5-trisphosphate. The kinetic rate laws of the associated model have a mathematical structure reminiscent of the Brusselator, a hypothetical chemical model involving a rather improbable trimolecular reaction step, thus giving a realistic biological interpretation to this hallmark of dissipative structures. We propose that calmodulin is involved in mediating this cooperativity and positive feedback, as suggested by the presented experiments. For one, hormone-induced calcium oscillations can be inhibited by the (nonphenothiazine) calmodulin antagonists calmidazolium or CGS 9343 B. Alternatively, in cells overstimulated by hormone, as characterized by a non-oscillatory elevated Ca2+ concentration, these antagonists could again restore sustained calcium oscillations. The experimental observations, including modulation of the oscillations by extracellular calcium, were in qualitative agreement with the predictions of our mathematical model.

Animals↗
PubMed1989

A G-protein mediates secretagogue-induced gap junctional channel closure in pancreatic acinar cells.

Using the double whole-cell patch-clamp technique, we determined that dialysis of cell pairs by GTP[S] potentiated electrical uncoupling induced by extracellular addition of carbamylcholine (CCh). An inhibitor of diglyceride lipase, RHC 80267, further potentiated CCh/GTP[S]-induced junctional channel closure, probably by accumulation of diacylglycerol. Moreover, the protein kinase C inhibitor polymyxin B completely blocked uncoupling elicited by CCh/GTP[S]. These results provide the first evidence suggesting that gap junction channel closure by cholinergic stimulation is mediated by a G-protein, which acts by increasing phosphatidylinositol biphosphate breakdown and protein kinase C activity.

Animals↗
PubMed1989

Inhibition of electrical coupling in pairs of murine pancreatic acinar cells by OAG and isolated protein kinase C.

Gap junctional coupling was studied in pairs of murine pancreatic acinar cells using the double whole-cell patch-clamp technique. During stable electrical coupling, addition of OAG (1-oleoyl-2-acetyl-sn-glycerol) induced a progressive reduction of the junctional conductance to the detectable limit (approximately 3 pS). Prior to complete electrical uncoupling, various discrete single channel conductances between 20 and 100 pS could be observed. Polymyxin B, a potent inhibitor of the protein kinase C (PKC) system, completely suppressed OAG-stimulated electrical uncoupling. Dialysis of cell pairs with solutions containing PKC, isolated from rat brain, also caused electrical uncoupling. The presence of 0.1 mM dibutyryl cyclic AMP and 5 mM ATP in the pipette solution, which serves to stabilize the junctional conductance, did not suppress the effects of OAG or isolated PKC. We conclude that an increase of protein kinase C activity leads to the closure of gap junction channels, presumably via a PKC-dependent phosphorylation of the junctional peptide, and that this mechanism is dominant over cAMP-dependent upregulatory effects in the experimental time range (less than or equal to 1 hr). A correlation of the observed single channel conductances with the appearance of channel subconductance states or various channel populations is discussed.

Animals↗