Changes in plastid proteome during chloroplast-to-chromolast transition

Transformacija kloroplasta u kromoplaste važan je biološki proces tijekom zriobe plodova. Događaju se važne strukturne i metaboličke promjene. Spektralna konfokalna mikroskopska analiza karotenoida i klorofila u plastidima izoliranih iz plodova pokazala je da kromoplasti nastaju iz već postojećih kloroplasta te da se nakon kromoplastogeneze proces diferencijacije završava. Snimanje živog tkiva in situ tehnikom real-time pokazalo je da je proces sinkroniziran na nivou jedne stanice, ali ne i na nivou čitavog tkiva. Proteomske analize dale su bolji uvid u metaboličke promjene tijekom kromoplastogeneze. Promjene u metabolizmu sastoje se od smanjenja količine proteina svjetlosnih reakcija, uključujući proteine uključene u fotosintezu, Calvinov ciklus i fotorespiraciju. Smanjuje se i količina proteina uključenih u metabolizam ugljikohidrata. Raste količina proteina stresa, kao i proteina uključenih u biosintezu terpenoida, uključujući karotenoide. Zanimljiva je činjenica velike prisutnosti podjedinica enzima RUBISCO u kromoplastima, iako se enzim prvenstveno nalazi u fotosintetski aktivnim plastidima. Navedenim metaboličkim promjenama prethodi akumulacija acetil-CoA karboksilaze koja je važna za formiranje spremišnog matriksa u kojemu će se akumulirati karotenoidi. Metaboličke promjene praćene su strukturnim promjenama. Poremećena je biogeneza tilakoida i fotosistema, a dolazi i do povećane proizvodnje energije i gubitka sposobnosti diobe plastida.Transition from chloroplasts to chromoplasts is an important biological process in fruit ripening. Many significant structural and metabolic shifts occur. Spectral confocal microscopy analyses of carotenoids and chlorophylls in isolated fruit plastids showed that chromoplasts arise from already existing chloroplasts, and that no further differentiation process occur. Real-time in-situ recording of intact live tissue showed that the process is synchronized within a cell, but with less synchrony within the whole tissue. Proteomic analyses gave better insight into metabolic shifts during chromoplastogenesis. Major shifts included decrease in abundance of proteins involved in light reactions, including photosynthesis, Calvin cycle and photorespiration. Proteins involved in carbohydrate metabolism also decrease in abundance. Proteins whose abundance increases during chromoplastogenesis include stress proteins, and proteins related to terpenoid biosynthesis. One interesting feature is that the large subunit of the RUBISCO enzyme, an enzyme typically abundant in photosynthetic plastids, although continuously decreasing in abundance, is present in fully developed chromoplasts. These metabolic shifts are preceded by the accumulation of acetyl CoA carboxylase, which accounts for the generation of a storage matrix that will accumulate carotenoids. Metabolic shifts are followed by structural changes. The biogenesis of the machinery for building up of thylakoid and photosystems is disrupted, coupled with elevated energy production components and loss of plastid division machinery

Variability in exons of CYP2D6 gene in Croatian Roma populations

Gen CYP2D6 kodira protein koji sudjeluje u metabolizmu 25% često prepisanih lijekova i drugih tvari iz okoliša. Ovaj gen vrlo je polimorfan što rezultira visokom ekspesijskom varijabilnošću među pojedincima i populacijama. U ovome radu su GT-seq metodom sekvencirani egzoni gena CYP2D6 u populacijama Roma iz Baranje, Međimurja i Zagreba s ciljem utvrđivanja intra- i interpopulacijske različitosti temeljem varijabilnosti egzona gena CYP2D6. U usporedbi s referentnom sekvencom u ukupnom istraživanom uzorku uočena su 24 polimorfna lokusa od kojih kod četri nije u skladu s Hardy- Weinbergovom ravnotežom. Ustanovljene su različite učestalosti mutacija između proučavanih populacija, te je utvrđena statistički značajna razlika u učestalostima genotipova između proučavanih populacija. Između parova lokusa u tri proučavane populacije detektirana je značajna neravnoteža vezanosti. Ovo istraživanje je potvrdilo genetske specifičnosti romskih populacija koje su posljedica njihove izoliranosti uzrokovane endogamijom, te još uvijek izraženog učinka utemeljitelja. Utvrđene varijabilnosti u genu CYP2D6 za posljedicu mogu imati učinak na uspješnost farmakoloških terapija.The CYP2D6 gene codes for a protein which takes part in the metabolism of 25% commonly perscribed drugs and other xenobiotics. The CYP2D6 gene is very polimorfic, and its expression varies between individuals and among populations. In this research exons of the CYP2D6 gene were sequenced by the GT-seq method in three Roma populations, from Baranja and Međimurje regions and the city of Zagreb in order to determine intra- and interpopulation variability based on CYP2D6 exons. 24 loci were shown to be polimorfic and 4 of them were shown not to be in the Hardy-Weinberg equilibrium. Different frequencies of mutated allels and genotypes were shown to occur among the analyzed populations. Significant linkage disequilibrium was determined between a few pairs of loci. This research has shown a specific genetic profile of the three isoalted Roma populations. Their genetic specificities reflect the founder effect from their demographic history and the still existing endogamy practice. Detected variabilities in the CYP2D6 gene may have an effect on the efficiency of different pharmacological therapies

Changes in plastid proteome during chloroplast-to-chromolast transition

Transformacija kloroplasta u kromoplaste važan je biološki proces tijekom zriobe plodova. Događaju se važne strukturne i metaboličke promjene. Spektralna konfokalna mikroskopska analiza karotenoida i klorofila u plastidima izoliranih iz plodova pokazala je da kromoplasti nastaju iz već postojećih kloroplasta te da se nakon kromoplastogeneze proces diferencijacije završava. Snimanje živog tkiva in situ tehnikom real-time pokazalo je da je proces sinkroniziran na nivou jedne stanice, ali ne i na nivou čitavog tkiva. Proteomske analize dale su bolji uvid u metaboličke promjene tijekom kromoplastogeneze. Promjene u metabolizmu sastoje se od smanjenja količine proteina svjetlosnih reakcija, uključujući proteine uključene u fotosintezu, Calvinov ciklus i fotorespiraciju. Smanjuje se i količina proteina uključenih u metabolizam ugljikohidrata. Raste količina proteina stresa, kao i proteina uključenih u biosintezu terpenoida, uključujući karotenoide. Zanimljiva je činjenica velike prisutnosti podjedinica enzima RUBISCO u kromoplastima, iako se enzim prvenstveno nalazi u fotosintetski aktivnim plastidima. Navedenim metaboličkim promjenama prethodi akumulacija acetil-CoA karboksilaze koja je važna za formiranje spremišnog matriksa u kojemu će se akumulirati karotenoidi. Metaboličke promjene praćene su strukturnim promjenama. Poremećena je biogeneza tilakoida i fotosistema, a dolazi i do povećane proizvodnje energije i gubitka sposobnosti diobe plastida.Transition from chloroplasts to chromoplasts is an important biological process in fruit ripening. Many significant structural and metabolic shifts occur. Spectral confocal microscopy analyses of carotenoids and chlorophylls in isolated fruit plastids showed that chromoplasts arise from already existing chloroplasts, and that no further differentiation process occur. Real-time in-situ recording of intact live tissue showed that the process is synchronized within a cell, but with less synchrony within the whole tissue. Proteomic analyses gave better insight into metabolic shifts during chromoplastogenesis. Major shifts included decrease in abundance of proteins involved in light reactions, including photosynthesis, Calvin cycle and photorespiration. Proteins involved in carbohydrate metabolism also decrease in abundance. Proteins whose abundance increases during chromoplastogenesis include stress proteins, and proteins related to terpenoid biosynthesis. One interesting feature is that the large subunit of the RUBISCO enzyme, an enzyme typically abundant in photosynthetic plastids, although continuously decreasing in abundance, is present in fully developed chromoplasts. These metabolic shifts are preceded by the accumulation of acetyl CoA carboxylase, which accounts for the generation of a storage matrix that will accumulate carotenoids. Metabolic shifts are followed by structural changes. The biogenesis of the machinery for building up of thylakoid and photosystems is disrupted, coupled with elevated energy production components and loss of plastid division machinery

Plasma Membrane Calcium ATPase-Neuroplastin Complexes Are Selectively Stabilized in GM1-Containing Lipid Rafts

The recent identification of plasma membrane (Ca2+)-ATPase (PMCA)-Neuroplastin (Np) complexes has renewed attention on cell regulation of cytosolic calcium extrusion, which is of particular relevance in neurons. Here, we tested the hypothesis that PMCA-Neuroplastin complexes exist in specific ganglioside-containing rafts, which could affect calcium homeostasis. We analyzed the abundance of all four PMCA paralogs (PMCA1-4) and Neuroplastin isoforms (Np65 and Np55) in lipid rafts and bulk membrane fractions from GM2/GD2 synthase-deficient mouse brains. In these fractions, we found altered distribution of Np65/Np55 and selected PMCA isoforms, namely PMCA1 and 2. Cell surface staining and confocal microscopy identified GM1 as the main complex ganglioside co-localizing with Neuroplastin in cultured hippocampal neurons. Furthermore, blocking GM1 with a specific antibody resulted in delayed calcium restoration of electrically evoked calcium transients in the soma of hippocampal neurons. The content and composition of all ganglioside species were unchanged in Neuroplastin-deficient mouse brains. Therefore, we conclude that altered composition or disorganization of ganglioside-containing rafts results in changed regulation of calcium signals in neurons. We propose that GM1 could be a key sphingolipid for ensuring proper location of the PMCA-Neuroplastin complexes into rafts in order to participate in the regulation of neuronal calcium homeostasis

The effect of ganglioside composition on expression, submembrane localization and activity of Na+, K+-ATPase and plasma membrane Ca2+-ATPase in mouse brain

U mišjim modelima s poremećenom sintezom glikosfingolipida gangliozida dolazi do promjene u lipidnom okolišu membrane potrebnom za pravilno funkcioniranje membranskih proteina. Cilj ovog istraživanja bio je višerazinskim metodološkim pristupom sustavno utvrditi utjecaj promijenjenog sastava gangliozida u mišjem modelu St8sia1 null na gensku i proteinsku ekspresiju, te submembransku lokalizaciju pojedinih izoformi i podjedinica Na+, K+-ATPaze (NKA), Ca2+-ATPaze stanične membrane (PMCA) i neuroplastina (Np), kao i na enzimsku aktivnost NKA i PMCA u različitim regijama mozga. Analizom genske ekspresije metodom lančane reakcije polimerazom u stvarnom vremenu utvrđene su promjene u ekspresiji gena za katalitičke i β- podjedinice NKA, izoforme PMCA između moždane kore, maloga mozga i hipokampusa miševa St8sia1 null u odnosu na kontrolnu skupinu, ukazujući na različitu staničnu potrebu za pojedinim izoformama, dok je u sve tri regije mozga utvrđen porast ekspresije gena za neuroplastin. Westernskom metodom otiska pokazano je da dolazi do promjene u količinskom sadržaju istraživanih proteina u regijama mozga St8sia1 null u vidu porasta količine katalitičkih podjedinica NKA te izoforme PMCA2 u moždanoj kori, PMCA4 u malome mozgu, te PMCA3 i 4 u hipokampusu miševa St8sia1 null ukazujući na različitu staničnu potrebu za pojedinim izoformama različitih kinetičkih svojstava. Analizom submembranske lokalizacije navedenih proteina utvrđen je proces njihove redistribucije u analiziranim regijama mozga miševa St8sia1 null. Analizom enzimskih aktivnosti NKA i PMCA utvrđeno je kako dolazi do pada aktivnosti u moždanoj kori i malome mozgu miševa St8sia1 null uslijed ukupnih biokemijskih promjena uzrokovanim promijenjenim gangliozidnim sastavom membrane. Dodavanjem egzogenih gangliozida b- serije uspostavljen je porast enzimske aktivnosti NKA i PMCA, dajući ovom istraživanju translacijski potencijal. Rezultati istraživanja nedvojbeno pokazuju da promijenjen sastav gangliozida ima dalekosežne posljedice po funkcioniranje ubikvitarnih staničnih membranskih prijenosnika iona.Mice models with altered biosynthesis of glycosphingolipids gangliosides have altered membrane lipid environment necessary for the proper function of membrane proteins. The aim of this thesis was to systematically determine the effect of altered ganglioside composition on gene and protein expression, submembrane localization of specific isoforms and subunits of Na+, K+-ATPase (NKA), plasma membrane Ca2+-ATPase (PMCA) and neuroplastin (Np), and enzyme activity of NKA and PMCA in different brain regions of St8sia1 null mice. Gene expression analysis revealed changes in expression profiles of different PMCA isoforms and NKA subunits in null mice compared to their controls, indicating the different cellular need for specific isoforms. Neuroplastin gene expression was higher in all analysed regions. Higher protein content of catalytic NKA subunits and PMCA2 isoforms in the cortex, PMCA4 in the cerebellum, and of PMCA3 and PMCA4 isoforms in the hippocampus was determined in null mice illustrating the cellular needs for specific subunits with differing kinetic parameters. Altered submembrane localization of investigated proteins shows their redistribution between different membrane subdomains. Described biochemical changes stemming from altered ganglioside composition, cause lower enzyme activities of NKA and PMCA determined in cortices and cerebella of null mice. We were able to rescue NKA and PMCA activity by administration of exogenous b- series gangliosides, giving this research translational potential. The results of this thesis unequivocally show that changes in ganglioside microenvironment have considerable consequences on the function of membrane ion transporters

Variability in exons of CYP2D6 gene in Croatian Roma populations

Gen CYP2D6 kodira protein koji sudjeluje u metabolizmu 25% često prepisanih lijekova i drugih tvari iz okoliša. Ovaj gen vrlo je polimorfan što rezultira visokom ekspesijskom varijabilnošću među pojedincima i populacijama. U ovome radu su GT-seq metodom sekvencirani egzoni gena CYP2D6 u populacijama Roma iz Baranje, Međimurja i Zagreba s ciljem utvrđivanja intra- i interpopulacijske različitosti temeljem varijabilnosti egzona gena CYP2D6. U usporedbi s referentnom sekvencom u ukupnom istraživanom uzorku uočena su 24 polimorfna lokusa od kojih kod četri nije u skladu s Hardy- Weinbergovom ravnotežom. Ustanovljene su različite učestalosti mutacija između proučavanih populacija, te je utvrđena statistički značajna razlika u učestalostima genotipova između proučavanih populacija. Između parova lokusa u tri proučavane populacije detektirana je značajna neravnoteža vezanosti. Ovo istraživanje je potvrdilo genetske specifičnosti romskih populacija koje su posljedica njihove izoliranosti uzrokovane endogamijom, te još uvijek izraženog učinka utemeljitelja. Utvrđene varijabilnosti u genu CYP2D6 za posljedicu mogu imati učinak na uspješnost farmakoloških terapija.The CYP2D6 gene codes for a protein which takes part in the metabolism of 25% commonly perscribed drugs and other xenobiotics. The CYP2D6 gene is very polimorfic, and its expression varies between individuals and among populations. In this research exons of the CYP2D6 gene were sequenced by the GT-seq method in three Roma populations, from Baranja and Međimurje regions and the city of Zagreb in order to determine intra- and interpopulation variability based on CYP2D6 exons. 24 loci were shown to be polimorfic and 4 of them were shown not to be in the Hardy-Weinberg equilibrium. Different frequencies of mutated allels and genotypes were shown to occur among the analyzed populations. Significant linkage disequilibrium was determined between a few pairs of loci. This research has shown a specific genetic profile of the three isoalted Roma populations. Their genetic specificities reflect the founder effect from their demographic history and the still existing endogamy practice. Detected variabilities in the CYP2D6 gene may have an effect on the efficiency of different pharmacological therapies

The effect of ganglioside composition on expression, submembrane localization and activity of Na+, K+-ATPase and plasma membrane Ca2+-ATPase in mouse brain

U mišjim modelima s poremećenom sintezom glikosfingolipida gangliozida dolazi do promjene u lipidnom okolišu membrane potrebnom za pravilno funkcioniranje membranskih proteina. Cilj ovog istraživanja bio je višerazinskim metodološkim pristupom sustavno utvrditi utjecaj promijenjenog sastava gangliozida u mišjem modelu St8sia1 null na gensku i proteinsku ekspresiju, te submembransku lokalizaciju pojedinih izoformi i podjedinica Na+, K+-ATPaze (NKA), Ca2+-ATPaze stanične membrane (PMCA) i neuroplastina (Np), kao i na enzimsku aktivnost NKA i PMCA u različitim regijama mozga. Analizom genske ekspresije metodom lančane reakcije polimerazom u stvarnom vremenu utvrđene su promjene u ekspresiji gena za katalitičke i β- podjedinice NKA, izoforme PMCA između moždane kore, maloga mozga i hipokampusa miševa St8sia1 null u odnosu na kontrolnu skupinu, ukazujući na različitu staničnu potrebu za pojedinim izoformama, dok je u sve tri regije mozga utvrđen porast ekspresije gena za neuroplastin. Westernskom metodom otiska pokazano je da dolazi do promjene u količinskom sadržaju istraživanih proteina u regijama mozga St8sia1 null u vidu porasta količine katalitičkih podjedinica NKA te izoforme PMCA2 u moždanoj kori, PMCA4 u malome mozgu, te PMCA3 i 4 u hipokampusu miševa St8sia1 null ukazujući na različitu staničnu potrebu za pojedinim izoformama različitih kinetičkih svojstava. Analizom submembranske lokalizacije navedenih proteina utvrđen je proces njihove redistribucije u analiziranim regijama mozga miševa St8sia1 null. Analizom enzimskih aktivnosti NKA i PMCA utvrđeno je kako dolazi do pada aktivnosti u moždanoj kori i malome mozgu miševa St8sia1 null uslijed ukupnih biokemijskih promjena uzrokovanim promijenjenim gangliozidnim sastavom membrane. Dodavanjem egzogenih gangliozida b- serije uspostavljen je porast enzimske aktivnosti NKA i PMCA, dajući ovom istraživanju translacijski potencijal. Rezultati istraživanja nedvojbeno pokazuju da promijenjen sastav gangliozida ima dalekosežne posljedice po funkcioniranje ubikvitarnih staničnih membranskih prijenosnika iona.Mice models with altered biosynthesis of glycosphingolipids gangliosides have altered membrane lipid environment necessary for the proper function of membrane proteins. The aim of this thesis was to systematically determine the effect of altered ganglioside composition on gene and protein expression, submembrane localization of specific isoforms and subunits of Na+, K+-ATPase (NKA), plasma membrane Ca2+-ATPase (PMCA) and neuroplastin (Np), and enzyme activity of NKA and PMCA in different brain regions of St8sia1 null mice. Gene expression analysis revealed changes in expression profiles of different PMCA isoforms and NKA subunits in null mice compared to their controls, indicating the different cellular need for specific isoforms. Neuroplastin gene expression was higher in all analysed regions. Higher protein content of catalytic NKA subunits and PMCA2 isoforms in the cortex, PMCA4 in the cerebellum, and of PMCA3 and PMCA4 isoforms in the hippocampus was determined in null mice illustrating the cellular needs for specific subunits with differing kinetic parameters. Altered submembrane localization of investigated proteins shows their redistribution between different membrane subdomains. Described biochemical changes stemming from altered ganglioside composition, cause lower enzyme activities of NKA and PMCA determined in cortices and cerebella of null mice. We were able to rescue NKA and PMCA activity by administration of exogenous b- series gangliosides, giving this research translational potential. The results of this thesis unequivocally show that changes in ganglioside microenvironment have considerable consequences on the function of membrane ion transporters

The effect of ganglioside composition on expression, submembrane localization and activity of Na+, K+-ATPase and plasma membrane Ca2+-ATPase in mouse brain

U mišjim modelima s poremećenom sintezom glikosfingolipida gangliozida dolazi do promjene u lipidnom okolišu membrane potrebnom za pravilno funkcioniranje membranskih proteina. Cilj ovog istraživanja bio je višerazinskim metodološkim pristupom sustavno utvrditi utjecaj promijenjenog sastava gangliozida u mišjem modelu St8sia1 null na gensku i proteinsku ekspresiju, te submembransku lokalizaciju pojedinih izoformi i podjedinica Na+, K+-ATPaze (NKA), Ca2+-ATPaze stanične membrane (PMCA) i neuroplastina (Np), kao i na enzimsku aktivnost NKA i PMCA u različitim regijama mozga. Analizom genske ekspresije metodom lančane reakcije polimerazom u stvarnom vremenu utvrđene su promjene u ekspresiji gena za katalitičke i β- podjedinice NKA, izoforme PMCA između moždane kore, maloga mozga i hipokampusa miševa St8sia1 null u odnosu na kontrolnu skupinu, ukazujući na različitu staničnu potrebu za pojedinim izoformama, dok je u sve tri regije mozga utvrđen porast ekspresije gena za neuroplastin. Westernskom metodom otiska pokazano je da dolazi do promjene u količinskom sadržaju istraživanih proteina u regijama mozga St8sia1 null u vidu porasta količine katalitičkih podjedinica NKA te izoforme PMCA2 u moždanoj kori, PMCA4 u malome mozgu, te PMCA3 i 4 u hipokampusu miševa St8sia1 null ukazujući na različitu staničnu potrebu za pojedinim izoformama različitih kinetičkih svojstava. Analizom submembranske lokalizacije navedenih proteina utvrđen je proces njihove redistribucije u analiziranim regijama mozga miševa St8sia1 null. Analizom enzimskih aktivnosti NKA i PMCA utvrđeno je kako dolazi do pada aktivnosti u moždanoj kori i malome mozgu miševa St8sia1 null uslijed ukupnih biokemijskih promjena uzrokovanim promijenjenim gangliozidnim sastavom membrane. Dodavanjem egzogenih gangliozida b- serije uspostavljen je porast enzimske aktivnosti NKA i PMCA, dajući ovom istraživanju translacijski potencijal. Rezultati istraživanja nedvojbeno pokazuju da promijenjen sastav gangliozida ima dalekosežne posljedice po funkcioniranje ubikvitarnih staničnih membranskih prijenosnika iona.Mice models with altered biosynthesis of glycosphingolipids gangliosides have altered membrane lipid environment necessary for the proper function of membrane proteins. The aim of this thesis was to systematically determine the effect of altered ganglioside composition on gene and protein expression, submembrane localization of specific isoforms and subunits of Na+, K+-ATPase (NKA), plasma membrane Ca2+-ATPase (PMCA) and neuroplastin (Np), and enzyme activity of NKA and PMCA in different brain regions of St8sia1 null mice. Gene expression analysis revealed changes in expression profiles of different PMCA isoforms and NKA subunits in null mice compared to their controls, indicating the different cellular need for specific isoforms. Neuroplastin gene expression was higher in all analysed regions. Higher protein content of catalytic NKA subunits and PMCA2 isoforms in the cortex, PMCA4 in the cerebellum, and of PMCA3 and PMCA4 isoforms in the hippocampus was determined in null mice illustrating the cellular needs for specific subunits with differing kinetic parameters. Altered submembrane localization of investigated proteins shows their redistribution between different membrane subdomains. Described biochemical changes stemming from altered ganglioside composition, cause lower enzyme activities of NKA and PMCA determined in cortices and cerebella of null mice. We were able to rescue NKA and PMCA activity by administration of exogenous b- series gangliosides, giving this research translational potential. The results of this thesis unequivocally show that changes in ganglioside microenvironment have considerable consequences on the function of membrane ion transporters

Start Me Up: How Can Surrounding Gangliosides Affect Sodium-Potassium ATPase Activity and Steer towards Pathological Ion Imbalance in Neurons?

Gangliosides, amphiphilic glycosphingolipids, tend to associate laterally with other membrane constituents and undergo extensive interactions with membrane proteins in cis or trans configurations. Studies of human diseases resulting from mutations in the ganglioside biosynthesis pathway and research on transgenic mice with the same mutations implicate gangliosides in the pathogenesis of epilepsy. Gangliosides are reported to affect the activity of the Na+/K+-ATPase, the ubiquitously expressed plasma membrane pump responsible for the stabilization of the resting membrane potential by hyperpolarization, firing up the action potential and ion homeostasis. Impaired Na+/K+-ATPase activity has also been hypothesized to cause seizures by several mechanisms. In this review we present different epileptic phenotypes that are caused by impaired activity of Na+/K+-ATPase or changed membrane ganglioside composition. We further discuss how gangliosides may influence Na+/K+-ATPase activity by acting as lipid sorting machinery providing the optimal stage for Na+/K+-ATPase function. By establishing a distinct lipid environment, together with other membrane lipids, gangliosides possibly modulate Na+/K+-ATPase activity and aid in “starting up” and “turning off” this vital pump. Therefore, structural changes of neuronal membranes caused by altered ganglioside composition can be a contributing factor leading to aberrant Na+/K+-ATPase activity and ion imbalance priming neurons for pathological firing