Chlorpromazine treatment induced inhibition of intracellular biochemical activity of mouse brain tissue

Chlorpromazine (CPZ), an antipsychotic drug, was found to inhibit intracellular carboxylesterases (CarbEs). As intracellular target carboxylesterases we used alpha-naphthyl acetate esterase (alpha-NA), naphthol AS-D chloroacetate esterase (AS-D) and alpha-naphthyl butyrate esterase (alpha-NB) in mouse polymorphonuclear neutrophils (PMN), hepatocytes (HC) and neuronal brain cells (NC). The impact of CPZ on the cells ranged from no effect to death, with intermediary effects of decreased CarbEs activities without either morphological changes or structural changes. The results of our study indicate that intracellular CarbEs activity inhibition by CPZ was dose-dependent, though the drug concentration required to bring about 50% inhibition of the initial activity (ID-50) varied between the mouse cell types, under the same experimental conditions. CarbEs activity was decreased or completely inhibited at CPZ concentrations ranging from 0.5 to 5.0 mg/ml (1.4 to 14.08 mmol/l). The impact maximum concentration of CPZ 5 mg/ml (14.08 mmol/l) on mouse brain cells resulted in 46.58% inhibition for AS-D, 54.26% for α-NA and 99.52% for α-NB. Our studies established a clear relationship between the increasing concentrations of CPZ and the extent of inhibition of the intracellular esterases of mice. Correlation of the inhibitory effects in all the cell types was demonstrated. The polymorphonuclear neutrophils - leukocytes were the most sensitive (ID-50 = 0.42 mg CPZ/ml) and the hepatocytes most resistant to the CPZ effect (ID-50 = 2.45 mg CPZ/ml). Since leukocytes are human cells much more readily available than hepatocytes or neuronal cells, we presume that CarbEs in peripheral blood leukocytes could be used as markers for the indication of intracellular biochemical damage of hepatocytes and neuronal brain cells by CPZ.Hlorpromazin (CPZ) je neuroleptik koji se koristi za dugotrajni tretman najtežih psihijatrijskih bolesti. Osnovni mehanizam dejstva CPZ je blokada D2 receptora ali se malo zna o efektima CPZ na intracelularnu biohemijsku aktivnost moždanog tkiva. U ovom radu ispitivan je uticaj CPZ-a na intracelularne karboksilesteraze (EC 3.1.1.1.), kao marker biohemijske aktivnosti. Primenom osetljive in vitro metode - MIMDECI detektovane su rane intracelularne biohemijske promene u morfološki očuvanim ćelijama, indukovane primenom CPZ-a. MIMDECI je tehnologija koja koristi dva markera (za enzimsku aktivnost i morfologiju ćelija) i meri delovanje leka na enzim pre pojave nespecifičnog oštećenja ćelija. Intracelularna biohemijska aktivnost praćena je pomoću alfa-naftil acetat karboksilesteraze, naftol AS-D hloroacetat karboksilesteraze i alfa-naftil butarat karboksilesteraze. Karboksilesteraze su citoplazmatski konstituenti mnogih tipova ćelija, tako da su paralelno praćeni efekti CPZ-a u polimorfonuklearima periferne krvi, hepatocitima i nervnim ćelijama mozga sivog miša, u in vitro uslovima. Naši rezultati ukazuju da CPZ inhibira karboksilesteraze u sva tri tipa ćelija i da stepen inhibicije zavisi od vremena aplikacije i primenjene koncentracije. Intraceiularna kar boksiiesterazna aktivnost je smanjena ili potpuno inhibirana primenom CPZ-a u rastućim koncentracijama od 0.5 - 5.0 mg/ml. Korelacijom inhibitornog efekta u sva tri tipa ćelija ustanovili smo da su polimorfonuklearni leukociti periferne krvi najosetljiviji na dejstvo CPZ-a (ID50 = 0.42 mg/ml) dok su hepatociti najrezistentniji (ID50 = 2.45 mg/ml).nul

Modification of the acetylcholine-induced current of the snail Helix pomatia L. by fast temperature changes

Using the single electrode voltage clamp method, we found that acetylcholine (aCh) induces transient inward dose-dependent current on the membrane of the identified Helix pomatia Br neuron. We analyzed the effects of fast cooling and heating as well as thermal acclimation on the aCh inward current. the experiments were conducted on active and dormant snails acclimated to either 20 or 7°C for at least four weeks. the Hill coefficient remained approximately 1 in all cases, which means that there is a single aCh binding site on the membrane. Fast temperature alternations induce binding affinity changes. in the work presented, we analyzed the effects of cooling on the aCh-induced inward current. the amplitude of aCh-induced inward current was markedly reduced after cooling, and the speed of decay of the aCh response was lower.Upotrebom metode nametnute voltaže ustanovili smo da acetilholin (aCh) izaziva dozno-zavisnu ulaznu struju na membrani identifikovanog Br neurona puža Helix pomatia. Analizirali smo efekte brzog hlađenja i zagrevanja kao i termalne aklimacije na aCh ulaznu struju. eksperimenti su rađeni na puževima aklimiranim na 20 i 7°C u toku četiri nedelje. Hill-ov koeficijent je uvek ostajao oko 1, što znači da postoji samo jedno mesto za vezivanje aCh na membrani neurona. Aklimacija na 7°C nije menjala afinitet za vezivanje aCh za razliku od aklimacije na 20°C gde je ustanovljeno povećanje afiniteta za vezivanje aCh na membrani identifikovanog Br neurona puža Helix pomatia.Projekat ministarstv

Photosensitive neurons in mollusks

In addition to regular photoreceptors, some invertebrates possess simple extra ocular photoreceptors. For example, the central ganglia of mollusks contain photosensitive neurons. These neurons are located on the dorsal surface of the ganglia and based on their electrophysiological properties it has been postulated that they are internal photoreceptors. Besides the eye, transduction of light also occurs in these extra-ocular photoreceptors. In the present work, we analyze the reactivity of these nerve cells to light and describe the underlying mechanism mediating the light-induced response.Uz poznate fotoreceptore neki beskičmenjaci imaju jednostavne ekstraokularne fotoreceptore. Na primer, centralna ganglija mekušaca sadrži fotosenzitivne neurone. Ovi neuroni lokalizovani na dorzalnoj strani ganglije i na osnovu elektrofizioloških karakteristika pretpostavlja se da poseduju unutrašnje fotoreceptore. Međutim, pored oka, transdukcija svetla se odvija i u ovim ekstraokularnim fotoreceptorima. U ovom radu analizirali smo reaktivnost ovih nervnih ćelija na svetlo i objasnili mehanizam koji leži u njegovoj osnovi.nul

Chlorpromazine treatment induced inhibition of intracellular biochemical activity of mouse brain tissue

Chlorpromazine (CPZ), an antipsychotic drug, was found to inhibit intracellular carboxylesterases (CarbEs). As intracellular target carboxylesterases we used alpha-naphthyl acetate esterase (alpha-NA), naphthol AS-D chloroacetate esterase (AS-D) and alpha-naphthyl butyrate esterase (alpha-NB) in mouse polymorphonuclear neutrophils (PMN), hepatocytes (HC) and neuronal brain cells (NC). The impact of CPZ on the cells ranged from no effect to death, with intermediary effects of decreased CarbEs activities without either morphological changes or structural changes. The results of our study indicate that intracellular CarbEs activity inhibition by CPZ was dose-dependent, though the drug concentration required to bring about 50% inhibition of the initial activity (ID-50) varied between the mouse cell types, under the same experimental conditions. CarbEs activity was decreased or completely inhibited at CPZ concentrations ranging from 0.5 to 5.0 mg/ml (1.4 to 14.08 mmol/l). The impact maximum concentration of CPZ 5 mg/ml (14.08 mmol/l) on mouse brain cells resulted in 46.58% inhibition for AS-D, 54.26% for α-NA and 99.52% for α-NB. Our studies established a clear relationship between the increasing concentrations of CPZ and the extent of inhibition of the intracellular esterases of mice. Correlation of the inhibitory effects in all the cell types was demonstrated. The polymorphonuclear neutrophils - leukocytes were the most sensitive (ID-50 = 0.42 mg CPZ/ml) and the hepatocytes most resistant to the CPZ effect (ID-50 = 2.45 mg CPZ/ml). Since leukocytes are human cells much more readily available than hepatocytes or neuronal cells, we presume that CarbEs in peripheral blood leukocytes could be used as markers for the indication of intracellular biochemical damage of hepatocytes and neuronal brain cells by CPZ.Hlorpromazin (CPZ) je neuroleptik koji se koristi za dugotrajni tretman najtežih psihijatrijskih bolesti. Osnovni mehanizam dejstva CPZ je blokada D2 receptora ali se malo zna o efektima CPZ na intracelularnu biohemijsku aktivnost moždanog tkiva. U ovom radu ispitivan je uticaj CPZ-a na intracelularne karboksilesteraze (EC 3.1.1.1.), kao marker biohemijske aktivnosti. Primenom osetljive in vitro metode - MIMDECI detektovane su rane intracelularne biohemijske promene u morfološki očuvanim ćelijama, indukovane primenom CPZ-a. MIMDECI je tehnologija koja koristi dva markera (za enzimsku aktivnost i morfologiju ćelija) i meri delovanje leka na enzim pre pojave nespecifičnog oštećenja ćelija. Intracelularna biohemijska aktivnost praćena je pomoću alfa-naftil acetat karboksilesteraze, naftol AS-D hloroacetat karboksilesteraze i alfa-naftil butarat karboksilesteraze. Karboksilesteraze su citoplazmatski konstituenti mnogih tipova ćelija, tako da su paralelno praćeni efekti CPZ-a u polimorfonuklearima periferne krvi, hepatocitima i nervnim ćelijama mozga sivog miša, u in vitro uslovima. Naši rezultati ukazuju da CPZ inhibira karboksilesteraze u sva tri tipa ćelija i da stepen inhibicije zavisi od vremena aplikacije i primenjene koncentracije. Intraceiularna kar boksiiesterazna aktivnost je smanjena ili potpuno inhibirana primenom CPZ-a u rastućim koncentracijama od 0.5 - 5.0 mg/ml. Korelacijom inhibitornog efekta u sva tri tipa ćelija ustanovili smo da su polimorfonuklearni leukociti periferne krvi najosetljiviji na dejstvo CPZ-a (ID50 = 0.42 mg/ml) dok su hepatociti najrezistentniji (ID50 = 2.45 mg/ml).nul

Effect of glutamate antagonists on nitric oxide production in rat brain following intrahippocampal injection

Stimulation of glutamate receptors induces neuronal nitric oxide (NO) release, which in turn modulates glutamate transmission. The involvement of ionotropic glutamate NMDA and AMPA/kainate receptors in induction of NO production in the rat brain was examined after injection of kainate, a non-NMDA receptor agonist; kainate plus 6-cyano- 7-nitroquinoxaline-2,3-dione (CNQX), a selective AMPA/kainate receptor antagonist; or kainate plus 2-amino-5-phosphonopentanoic acid (APV), a selective NMDA receptor antagonist. Competitive glutamate receptor antagonists were injected with kainate unilaterally into the CA3 region of the rat hippocampus. The accumulation of nitrite, the stable metabolite of NO, was measured by the Griess reaction at different times (5 min, 15 min, 2 h, 48 h, and 7 days) in hippocampus, forebrain cortex, striatum, and cerebellum homogenates. The used glutamate antagonists APV and CNQX both provided sufficient neuroprotection in the sense of reducing nitrite concentrations, but with different mechanisms and time dynamics. Our findings suggest that NMDA and AMPA/kainate receptors are differentially involved in nitric oxide production.Stimulacija glutamatskih receptora dovodi do stvaranja azot oksida (NO) u neuronima mozga što dovodi do modulacije glutamatske neurotransmisije. Ispitivana je uloga glutamatskih NMDA i AMPA/kainatnih receptora u stvaranju NO u mozgu pacova posle intracerebralne aplikacija kainata, agonista AMPA/kainatnih receptora, kainata sa 6-ciano-7- nitrokinoksalin-2,3-dionom (CNQX), selektivnim antagonistom AMPA/kainatnih receptora ili kainata sa 2-amino-5-fosfonopentanoiskom kiselinom (APV) selektivnim antagonistom NMDA receptora. Antagonisti glutamata su aplicirani unilateralno u selektivno osetljiv CA3 region hipokampusa. Stvaranje NO je praćeno preko akumulacije nitrita, stabilnih metabolita NO, Griess-ovom metodom. Merenja su vršena u hipokampusu, korteksu, stiatumu i cerebelumu mozga pacova 5 min, 15 min, 2 h, 48 h i 7 dana nakon aplikacije. U svim praćenim moždanim strukturama neuroprotektivno je delovala primena CNQX i APV u smislu smanjenja produkcije NO, ali sa očiglednom razlikom u mehanizmu dejstva i vremenskoj dinamici. Rezultati našeg istraživanja dokazuju da su glutamatski NMDA i AMPA/kainatni receptori različito uključeni u proces produkcije NO.nul

Modification of the acetylcholine-induced current of the snail Helix pomatia L. by fast temperature changes

Using the single electrode voltage clamp method, we found that acetylcholine (aCh) induces transient inward dose-dependent current on the membrane of the identified Helix pomatia Br neuron. We analyzed the effects of fast cooling and heating as well as thermal acclimation on the aCh inward current. the experiments were conducted on active and dormant snails acclimated to either 20 or 7°C for at least four weeks. the Hill coefficient remained approximately 1 in all cases, which means that there is a single aCh binding site on the membrane. Fast temperature alternations induce binding affinity changes. in the work presented, we analyzed the effects of cooling on the aCh-induced inward current. the amplitude of aCh-induced inward current was markedly reduced after cooling, and the speed of decay of the aCh response was lower.Upotrebom metode nametnute voltaže ustanovili smo da acetilholin (aCh) izaziva dozno-zavisnu ulaznu struju na membrani identifikovanog Br neurona puža Helix pomatia. Analizirali smo efekte brzog hlađenja i zagrevanja kao i termalne aklimacije na aCh ulaznu struju. eksperimenti su rađeni na puževima aklimiranim na 20 i 7°C u toku četiri nedelje. Hill-ov koeficijent je uvek ostajao oko 1, što znači da postoji samo jedno mesto za vezivanje aCh na membrani neurona. Aklimacija na 7°C nije menjala afinitet za vezivanje aCh za razliku od aklimacije na 20°C gde je ustanovljeno povećanje afiniteta za vezivanje aCh na membrani identifikovanog Br neurona puža Helix pomatia.Projekat ministarstv

Photosensitive neurons in mollusks

In addition to regular photoreceptors, some invertebrates possess simple extra ocular photoreceptors. For example, the central ganglia of mollusks contain photosensitive neurons. These neurons are located on the dorsal surface of the ganglia and based on their electrophysiological properties it has been postulated that they are internal photoreceptors. Besides the eye, transduction of light also occurs in these extra-ocular photoreceptors. In the present work, we analyze the reactivity of these nerve cells to light and describe the underlying mechanism mediating the light-induced response.Uz poznate fotoreceptore neki beskičmenjaci imaju jednostavne ekstraokularne fotoreceptore. Na primer, centralna ganglija mekušaca sadrži fotosenzitivne neurone. Ovi neuroni lokalizovani na dorzalnoj strani ganglije i na osnovu elektrofizioloških karakteristika pretpostavlja se da poseduju unutrašnje fotoreceptore. Međutim, pored oka, transdukcija svetla se odvija i u ovim ekstraokularnim fotoreceptorima. U ovom radu analizirali smo reaktivnost ovih nervnih ćelija na svetlo i objasnili mehanizam koji leži u njegovoj osnovi.nul

Effect of glutamate antagonists on nitric oxide production in rat brain following intrahippocampal injection

Stimulation of glutamate receptors induces neuronal nitric oxide (NO) release, which in turn modulates glutamate transmission. The involvement of ionotropic glutamate NMDA and AMPA/kainate receptors in induction of NO production in the rat brain was examined after injection of kainate, a non-NMDA receptor agonist; kainate plus 6-cyano- 7-nitroquinoxaline-2,3-dione (CNQX), a selective AMPA/kainate receptor antagonist; or kainate plus 2-amino-5-phosphonopentanoic acid (APV), a selective NMDA receptor antagonist. Competitive glutamate receptor antagonists were injected with kainate unilaterally into the CA3 region of the rat hippocampus. The accumulation of nitrite, the stable metabolite of NO, was measured by the Griess reaction at different times (5 min, 15 min, 2 h, 48 h, and 7 days) in hippocampus, forebrain cortex, striatum, and cerebellum homogenates. The used glutamate antagonists APV and CNQX both provided sufficient neuroprotection in the sense of reducing nitrite concentrations, but with different mechanisms and time dynamics. Our findings suggest that NMDA and AMPA/kainate receptors are differentially involved in nitric oxide production.Stimulacija glutamatskih receptora dovodi do stvaranja azot oksida (NO) u neuronima mozga što dovodi do modulacije glutamatske neurotransmisije. Ispitivana je uloga glutamatskih NMDA i AMPA/kainatnih receptora u stvaranju NO u mozgu pacova posle intracerebralne aplikacija kainata, agonista AMPA/kainatnih receptora, kainata sa 6-ciano-7- nitrokinoksalin-2,3-dionom (CNQX), selektivnim antagonistom AMPA/kainatnih receptora ili kainata sa 2-amino-5-fosfonopentanoiskom kiselinom (APV) selektivnim antagonistom NMDA receptora. Antagonisti glutamata su aplicirani unilateralno u selektivno osetljiv CA3 region hipokampusa. Stvaranje NO je praćeno preko akumulacije nitrita, stabilnih metabolita NO, Griess-ovom metodom. Merenja su vršena u hipokampusu, korteksu, stiatumu i cerebelumu mozga pacova 5 min, 15 min, 2 h, 48 h i 7 dana nakon aplikacije. U svim praćenim moždanim strukturama neuroprotektivno je delovala primena CNQX i APV u smislu smanjenja produkcije NO, ali sa očiglednom razlikom u mehanizmu dejstva i vremenskoj dinamici. Rezultati našeg istraživanja dokazuju da su glutamatski NMDA i AMPA/kainatni receptori različito uključeni u proces produkcije NO.nul

Combined segmentation and classificationbased approach to automated analysis of biomedical signals obtained from calcium imaging

Automated screening systems in conjunction with machine learning-based methods are becoming an essential part of the healthcare systems for assisting in disease diagnosis. Moreover, manually annotating data and hand-crafting features for training purposes are impractical and time-consuming. We propose a segmentation and classification-based approach for assembling an automated screening system for the analysis of calcium imaging. The method was developed and verified using the effects of disease IgGs (from Amyotrophic Lateral Sclerosis patients) on calcium (Ca2+) homeostasis. From 33 imaging videos we analyzed, 21 belonged to the disease and 12 to the control experimental groups. The method consists of three main steps: projection, segmentation, and classification. The entire Ca2+ time-lapse image recordings (videos) were projected into a single image using different projection methods. Segmentation was performed by using a multi-level thresholding (MLT) step and the Regions of Interest (ROIs) that encompassed cell somas were detected. A mean value of the pixels within these boundaries was collected at each time point to obtain the Ca2+ traces (time-series). Finally, a new matrix called feature image was generated from those traces and used for assessing the classification accuracy of various classifiers (control vs. disease). The mean value of the segmentation F-score for all the data was above 0.80 throughout the tested threshold levels for all projection methods, namely maximum intensity, standard deviation, and standard deviation with linear scaling projection. Although the classification accuracy reached up to 90.14%, interestingly, we observed that achieving better scores in segmentation results did not necessarily correspond to an increase in classification performance. Our method takes the advantage of the multi-level thresholding and of a classification procedure based on the feature images, thus it does not have to rely on hand- crafted training parameters of each event. It thus provides a semi-autonomous tool for assessing segmentation parameters which allows for the best classification accuracy

Effect of 7-nitroindazole on superoxide production and MnSOD activity

in the rat brain following kainаte-induced neurotoxicit