NI Neurofiziologijos laboratorija (11.06)

Retinal ganglion cells detect changes in illuminance. An increase and a decrease in illuminance levels induce ON and OFF responses, respectively. This segregation into two major types of visual responses is carried out through the lateral geniculate nucleus (LGN) to the primary visual cortex area V1. In area V1, these responses are partially merged on single neurons, although domains of ON and OFF responses are still found in layer IV, which receives LGN afferents, and in layers II-III, which have no direct inputs from the LGN. However, it is unclear whether feedback circuits in area V1 can process ON and OFF responses independently. Here, we demonstrate that optogenetic local activation of layer IV–VI pyramidal neurons modulates the receptive field (RF) area of ON and OFF responses independently. Although the RF area was reduced for both ON and OFF responses (77.9% of control, Q1 = 56.0%, Q3 = 93.1%, and 60.7% of control, Q1 = 45.1%, Q3 = 92.2%, respectively; n = 60), there was no correlation between these changes in single neurons (Spearman’s rank correlation coefficient ρ = 0.144, p > 0.27, n = 60). In contrast, the ON and OFF response amplitude changes correlated in single neurons (ρ = 0.486, p < 0.0003, n = 60), and the median amplitude change was similar for both types of responses (79.6% and 84.6%, respectively, for OFF and ON responses). These results can be explained by assuming that modulation of the response amplitude is largely dependent on changes in the membrane properties of a neuron, while the RF area is modulated mainly in feedback loops presynaptically.

Glioblastoma (GBM) is an aggressive brain tumor marked by rapid growth, frequent recurrence, and resistance to treatment. Emerging research shows that GBM cells interact with neural stem cells (NSCs) through extracellular vesicles (EVs). Understanding how GBM-derived EVs influence NSCs is key to revealing tumor–microenvironment dynamics and identifying novel therapeutics. Because calcium signaling governs cell proliferation, migration, and differentiation, changes in intracellular calcium may provide an early indicator of EV-driven NSC reprogramming. This study examined how GBM cell lines from different origins affect NSC behavior, focusing on calcium-signaling changes and validating findings with next-generation sequencing (NGS). Human neural stem cells (NSC H9 line) were treated with EVs isolated from GBM cell lines A172 and U87-MG conditioned media using 12% polyethylene glycol (PEG) precipitation. Cells were exposed to GBM-derived EVs (~1250 EVs per cell) for 17 h for Ca2+ signalling analysis and for 24 h for NGS. For Ca²⁺ imaging, NSCs were loaded with 2 µM Oregon Green-488 BAPTA-1 dye and imaged at 2 Hz for 200 s using a 40× objective. Nifedipine (2 µM) and caffeine (5 mM) were applied sequentially to assess L-type Ca²⁺ channel activity and ryanodine receptor (RyR)-mediated Ca²⁺ release, respectively. The pilot study revealed that GBM EVs influence Ca²⁺ signaling in NSCs, with effects dependent on the cell line. A172 EVs increased Ca²⁺ signal frequency in affected NSCs, which was blocked by nifedipine, suggesting elevated L-type Ca²⁺ channel activity. In U87 EV– treated NSCs, signal amplitude increased with nifedipine and further rose with caffeine, indicating enhanced RyR activity. NGS analysis revealed changes in RyR, SLC8B1 and LETM1 gene expression, suggesting increased Ca²⁺ concentration in mitochondria leading to augmented metabolic activity in mitochondria. Changes in Ca²⁺ signaling induced by EVs point to potential cross-talk between cytosolic and mitochondrial Ca²⁺ pathways. The study shows that A172 EVs enhance Ca²⁺ signaling, potentially promoting NSC proliferation and differentiation, whereas U87-MG EVs strongly elevate RyR levels, increasing NSC sensitivity to environmental cues such as growth factors or inflammatory interleukins. These functional effects are further supported by NGS analysis, which confirms the underlying molecular changes driving these EV-mediated responses.

Regos sistema yra vienas svarbiausių jutimo mechanizmų, leidžiančių gyvūnams laiku atpažinti aplinkos stimulus ir tinkamai į juos reaguoti. Žinduolių regos sistemą sudaro keli smegenų centrai, kurie apdoroja regos informaciją skirtingais lygiais. Žiurkėse viršutiniai kalneliai (toliau VK) atsakingi už greitas orientacines reakcijas į galimą pavojų, tuo tarpu pirminė regos žievė (toliau V1) vykdo sudėtingesnį vaizdo analizavimą, bet tam reikia daugiau laiko. Tačiau nėra aišku, kokią dalį neuronų atsakų į grėsmę keliančius stimulus galima priskirti pačių VK funkcijai, o kokią – jų sąveikai su V1. Šis tyrimas atliktas žiurkėse, nes yra anatomiškai patogu tirti viršutinių kalnelių veiklą bei pirminės regos žievės įtaką šiems atsakams. Trys savaitės prieš eksperimentą į V1 buvo suleistas adenoasocijuotas virusas (AAV), sukeliantis šviesai jautraus ChR2 kanalo ekspresiją piramidiniuose neuronuose. Tai leido selektyviai aktyvuoti šiuos neuronus naudojant 465 nm LED šviesą. Žiurkei buvo pateiktas vizualinis stimulas „looming“ tipo – didėjanti tamsi dėmė šviesiame fone, imituojanti artėjantį objektą. VK neuronų aktyvumas registruotas naudojant daugiakanalę 4×4 elektrodų matricą, leidžiančią stebėti kelių neuronų grupių atsakus vienu metu. Po eksperimentų smegenys fiksuotos, atlikta pjūvių nuskaidrinimo procedūra, fluorescencinė analizė bei šviesos pluošto mikroskopija, siekiant patikslinti registracijos vietas ir V1 aksonų projekcijas į VK. Surinkti duomenys apdoroti: atlikta veikimo potencialų detekcija daugiakanalės matricos pagalba, VK atsakų analizė bei jų klasifikavimas pagal tipą bei statistinis atsako dydis t.y. elipsės formos atsakas į vizualinį stimuliavimą. Šių elipsės formos dydžių vertinimas naudojant ploto formulę: bei Wilcoxon testą palyginti kontrolės ir lazerio stimuliuojamų atsakų plotą. Skirtumas tarp kontrolės „ON“ ir lazerio „ON“ atsakų nėra statistiškai reikšmingas (W = 186, Z = –0.07, p = 0.944, n = 27). Taip pat skirtumas tarp kontrolės būsenos „OFF“ ir lazerio „OFF“ nėra statistiškai reikšmingas (W = 147, Z = –0.72, p = 0.469, n = 26). Optogenetinė V1 stimuliacija nepakeitė VK neuronų populiacijos atsakų, lyginant lazerio „ON“ ir „OFF“ būsenas. Atlikti Wilcoxon testai (visuose palyginimuose p > 0.05) parodė, kad V1 optogenetinė aktyvacija šioje eksperimento paradigmoje nepakeitė VK neuronų populiacijos veiklos.

Background. Tumor-associated microenvironmental signaling plays a critical role in cancer proliferation by affecting adjacent healthy cells. Glioblastomas (GBMs), highly aggressive brain tumors, actively reshape their microenvironment through various mechanisms, notably via extracellular vesicles (EVs), which mediate intercellular communication. These EVs likely influence neural stem cells (NSCs), essential for brain homeostasis, neurogenesis, and repair. GBMs often arise near the subventricular zone (SVZ), rich in NSCs, suggesting potential two-way cellular interactions. Recent findings propose that NSCs may even serve as a cell of origin for GBM, though it remains unclear whether this is due to glioblastoma-induced signaling or intrinsic NSC traits. While SVZNSCs are implicated in GBM progression and recurrence, little is known about their direct response to tumor-derived signaling. This study aimed to explore how GBM cell lines of distinct origin affect NSC behavior, focusing on calcium signaling, a crucial second messenger involved in proliferation, differentiation, and cellular communication. Methods. GBM cell lines A172 and U87-MG were cultured in DMEM high-glucose media with 10% EV-depleted FBS for 72 h. EVs were isolated using 12% PEG precipitation and characterized via nanoparticle tracking analysis and ELISA for CD63, HSP70, and APOA1. NSC H9 cells were cultured in KnockOut DMEM/F-12 with StemPro supplement, bFGF, and EGF (20 ng/ml each), and stimulated with GBM EVs (~1250 EVs/cell) or control EVs from unconditioned media signaling was measured using Oregon Green-488 BAPTA- influx via L-type channels and RyR-mediated release from the endoplasmic reticulum. signaling change in NSC after stimulation with GBM EVs and its dependence on cell line EVs. The A172 EVs significantly increased the number signals in affected NSC by 25% compared to unaffected NSC (p<0.01 Kolmogorov-Smirnov, K-S, test), the difference was absent after application of nifedipine indicating increased levels of L-type Ca2+ channels. In NSC affected by U87 EVs Ca2+ signaling was not changed; however, the amplitude of signals was significantly increased by 29% after nifedipine (p<0.01, K-S test). The following caffeine application increased the amplitude by 16% (p<0.05, K-S test), and the number of signals increased by 126% (p<0.001, K-S test) indicating increased levels of RyR in NSC affected by U87 EVs. signaling is altered by GBM-derived EVs, affected by A172 EVs shows possible facilitation of NSC proliferation and differentiation. On the other hand, strong increase in RyR levels in NSCs due to U87-MG EVs shows potentiation of response of affected cells to possible influence by environmental molecular mediators of proliferation or differentiation, like growth factors, or inflammatory interleukins. Since transient Ca2+ signaling regulates cell differentiation, migration and proliferation, different effects of A172 and U87-MG EVs shows selective shaping of GBM cellular environment.

Astrocyte function is controlled by intracellular Ca2+ signaling. On the other hand, hypoxia influences calcium dynamics and its homeostatic range because of reduction of ATP synthesis, which inhibits ATP dependent processes. By using Ca2+ sensitive fluorescence dye, we studied how metformin changed spontaneous oscillating Ca2+ signals in soma of astrocytes in monocultures, prepared from rat brains. Mild hypoxic conditions (2% O2) applied for 24 h had no effect on astrocyte viability; however, it reduced the relative amplitude of Ca2+ signals, slowed the decay of the signals, and increased the period of spontaneous oscillations. Lower concentrations of metformin, 250 μM or 500 μM, applied before hypoxia reduced this influence by partially restoring the amplitude, fastening the decay, and reducing the period of Ca2+ signaling. In contrast, higher concentration, 1mM of metformin exaggerated the effects of hypoxia by reducing signals, slowing their decay and prolonged the period between signals. Unexpectedly, in astrocytes grown under normoxic conditions, all concentrations of metformin after one hour of application had effects similar to hypoxia for Ca2+ signaling. In conclusion, our data show that mild hypoxia reduces Ca2+signaling in astrocyte cell monocultures, and low concentrations of metformin under mild hypoxic conditions help to rescue the functioning of astrocytes by conditioning the cells to prolonged hypoxic influence.

A slowly moving dark spot imitating the shadow of a hovering bird of prey has been shown to induce freezing in rodents. Such visually triggered behaviours are usually initiated in the superior colliculus (SC); therefore, it is likely that such slowly moving dark spots can produce responses in SC neurons. In SC, two types of visual responses are typically distinguished: ON responses are produced by an increase in image brightness, and OFF responses are produced by a decrease in image brightness. Typically, OFF responses are very brief, lasting only a few hundred milliseconds, and may be poorly suited for the detection of slowly moving dark spots. Here, we report that, in the majority of SC neurons of urethane-anaesthetized rats, in addition to these brief OFF responses, very slow OFF responses lasting over 5s were present; thus, OFF responses that occurred >1s after the stimulus offset were termed 'slow OFF' response, while brief, less than 1s long OFF responses were called 'fast OFF' response. Although the slow OFF responses were of similar amplitude as the fast OFF responses (~5Hz), the optimal size (producing the maximal response) was larger for the slow OFF responses (20° for the slow and 10° for the fast OFF responses). Correlation analysis revealed that both the fast and the slow components of the OFF response contribute to the response to a slowly moving spot. Elimination of visual cortex inputs increased the amplitude and duration of the slow OFF responses, indicating that they originate in the retina. It is concluded that in rodent SC, a new type of OFF response that is well suited for predator detection is present.

Astrocyte function is controlled by intracellular Ca2+ signaling. On the other hand, hypoxia influences calcium dynamics and its homeostatic range because of reduction of ATP synthesis, which inhibits ATP dependent processes. By using Ca2+ sensitive fluorescence dye, we studied how metformin changed spontaneous oscillating Ca2+ signals in soma of astrocytes from 5–7-day-old rats grown under normoxic and mild-hypoxic (2% O2) conditions. Mild hypoxic conditions applied for 24 h did not change astrocyte viability; however, it reduced the relative amplitude of Ca2+ signals, slowed the decay of the signals, and increased the period of spontaneous oscillations. Lower concentrations of metformin, 0,25 or 0,5 mM, applied before hypoxia reduced this detrimental influence by partially restoring the amplitude, fastening the decay, and reducing the period of Ca2+ signaling. In contrast, higher concentration, 1mM, of metformin exaggerated the effects of hypoxia by reducing signals, slowing their decay and prolonged the period between signals. Unexpectedly, in astrocytes grown under normoxic conditions all concentrations of metformin after several hours of application had detrimental effects for Ca2+ signaling. Low concentration of metformin under mild hypoxic conditions helps to rescue the functioning of astrocytes by conditioning the cells to prolonged hypoxic influence.

Background: ON and OFF responses are generated in the visual pathway of all mammals following an increase and a decrease in stimulus brightness respectively. This generation of these two fundamental types of visual responses occurs in the retina and their specificity at synaptic level is at least partially preserved in the superior colliculus and the lateral geniculate nucleus, an intermediate visual pathway stations from the retina to the cortex. It was believed that in the cortex this segregation of ON and OFF pathways is mostly lost. However, recent findings suggest that this is not the case. Our experiments further support the hypothesis that ON and OFF pathway specificity is preserved up to the primary cortex, at least in rodents since pyramidal neurons can control each type of response independently. Moreover, there is a mechanism that maintains a balance between these two types of responses since the average effect of optogenetic stimulation of pyramidal neurons on ON and OFF responses is similar. Methods: Wistar rats were anesthetized by peritoneal injections of urethane (1.5 – 2.0 g/kg), then they were placed in a stereotaxic frame. The recordings were done 4-6 weeks following an injection of a virus containing channelrhodopsin (ChR2) under a short CamKII promoter in the primary visual cortex area V1. ChR2 reacts to blue 465nm light by the opening of ion channels resulting in depolarization and activation of the neuron. A tetrode with an attached optical fiber for a 465nm blue LED stimulation was placed in the virus injected area within the deep cortical layers of the primary cortex (area V1). Bright spots were presented on a dark grey background for visual stimulation. Initially, a single neuron RF area was determined during visual stimulation and then the RF area was evaluated again during optic fiber mediated 465nm LED stimulation of pyramidal neurons (CamKII positive cells). Further analysis of recorded data performed by employing Igor pro, Matlab, KlustKwik software packages. Results: During optogenetic stimulation of pyramidal neurons the RF area of OFF responses was reduced to a median value of 70% of control (p <0.001, n = 24, Wilcoxon signed-rank test, WSR test) while the RF area of ON responses to a median value of 79% of control (p <0.027, n = 24, WSR test). Although there was no significant difference in the extent of area reduction (p >0.3, n = 24, Kruskal-Wallis test), there was no correlation between the effect size on the ON and the OFF response RF area for single neurons (Spearman’s ρ = 0.03, p >0.5). Similarly, the OFF response amplitude was reduced to median value of 84% (p <0.013, n = 24, WSR test) while the ON response amplitude to 82% (p <0.001, n = 22, WSR test). Again. although there was no significant difference in the extent of reduction in the ON and OFF response amplitudes (p >0.7, n = 24 and 22, Kruskal-Wallis test), there was no correlation between the effect size onthe ON and the OFF response amplitude for single neurons (Spearman’s ρ = 0.25, p >0.2). Conclusions: In the primary visual cortex optogenetic activation of pyramidal neurons reduced the RF area and the response amplitude of ON and OFF responses to similar extent. However, there was no correlation of these effects on the ON and the OFF responses at single neuron level. In other words, at single neuron level the OFFresponse control by the pyramidal neurons was independent of the ON response control indicating the presence of synaptic pathways originating in the pyramidal neurons that are specific for ON and OFF responses, confirming ON and OFF pathway segregation in the primary visual cortex. Moreover, the similarity of changes induced upon ON and OFF responses by optogenetic stimulation indicates the presence of a global mechanism that keeps balance between the amplitudes of OFF and ON responses as well as between their areas.

The primary visual cortex or area V1 is the most studied visual area in the brain, and, probably, one of the most studied brain areas in general. There is a good reason for that. In the primary visual cortex, the sensory inputs are well defined, they represent images. Moreover, a good deal is known what image features are mostly represented by V1 neurons. Every V1 neuron responds only to images presented inside the receptive field (RF), a small fraction of the viewing field. Although many stimulus features/parameters may influence responses, the visual response amplitude mainly depends on the stimulus orientation, movement direction and size. In spite of multiple studies addressing the mechanisms of orientation and direction selectivity in the primary visual cortex, one of the main issues, the contribution of two major cell types, the pyramidal neurons and the interneurons, to the selectivity of visual responses in area V1 was unsettled before the era of optogenetics. It is not surprising that, when an over a decade ago the first optogenetic tools suitable to study interneuron function appeared, almost all optogenetic studies on V1 focussed on the issues of orientation and direction selectivity. Surprisingly, other fundamental features of the area V1 neurons, such as signal-to-noise ratio (SNR) and the RF area were hardly investigated. Therefore, my focus was to study these two parameters of visual neurons since they directly determine visual information content of a neuron: the location of the visual stimulus can be pinpointed more accurately if the RF area is reduced, and a visual stimulus can be detected with fewer errors if SNR is increased. It turns out that optogenetic stimulation of either pyramidal neurons or interneurons leads to the same result, an increased SNR, and a reduced RF area of both pyramidal cells and interneurons. Thus, both types of stimulation increase information content of area V1 neurons. Stimulation of interneurons produces an overall inhibitory effect; however, this effect is more pronounce on noise than on the visual responses, therefore the SNR is improved. Similarly, the inhibitory effect on RF flanks is more pronounced than in the RF centre leading to the reduced RF area. Stimulation of pyramidal neurons immediately activates inhibitory interneurons, that in turn suppress their activity, leading to an overall inhibitory effect. Moreover, these two effects can be observed on both ON and OFF types of responses, that are triggered by the bright stimulus onset and offset respectively. Interestingly, the effects on ON and OFF responses are independent, i.e., at a single neuron level the effect size on the ON response does not predict the effect size on the OFF response. Nevertheless, on average, the effect size on both types of responses is similar, suggesting the presence of a global control of the balance between ON and OFF responses. It is concluded that pyramidal neurons are connected with each other by at least two types of negative feedback pathways selective to ON and OFF responses. In addition, there is a global network that balances both the amplitude and the RF area of ON and OFF responses.

Microglia are the main resident immune cells that are among the first responders to hypoxic/ischemic brain damages. Because of their sensitivity to blood flow fluctuations, microglia become activated and undergo morphological changes under hypoxic/ischemic conditions. However, intracellular mechanisms mediating microglial activation under hypoxic conditions are not well understood. When activated, microglia can cause a cascade of inflammatory processes and initiate cytokine release, activate reactive oxygen species production. It is known that intracellular Ca2+ signaling is linked with pathophysiological functions of microglia and its signaling changes, arising in response to brain damage. Previous studies found that calcium signaling is important for microglial immune function— cytokine release, P2X receptor trafficking and diffusion. Imaging of calcium-dependent fluorescence in vivo has demonstrated that microglial cells have spontaneous Ca2+ signaling which is important for microglial function. Spontaneous signals and their rate were changed by lipopolysaccharide and by local neuronal tissue injury. Also, changes in neuronal activity triggered increased microglial process Ca2+ signaling, which was related to process extension. However, there is still little knowledge on how Ca2+ spontaneous signals change in response to hypoxia. Metformin is widely used as anti-hyperglycemic agent for non-insulin-dependent (type 2) diabetes therapy. It has been suggested that metformin decreases hepatic glucose production mostly via inhibition of complex I of the mitochondrialrespiratory chain and activation of AMP-kinase signaling pathway. Recent studies suggest that metformin can also act as a neuroprotective agent during hypoxia/ischemia by suppressing mitochondrial complex I activity in neuronal cultures, by reducing ischemic stroke-induced oxidative stress, inhibiting neuronal apoptosis and suppressing neuroinflammation. Metformin has also been suggested to inhibit mitochondrial permeability transition pore (mPTP) opening and to exert neuroprotective effects by this mechanism. However, the link between mitochondrial dysfunction and inflammatory responses mediated by the activated microglia are not clear yet, and the role of metformin in these processes remains largely unknown. It has been demonstrated that metformin and phenformin at pharmacologically relevant concentrations differently improve Ca2+ homeostasis in hypoxia-affected primary cortical neuronal cell cultures, but little is known about their role in microglial cultures. In this study, we investigated the effect of metformin on spontaneous calcium signals in cultured microglia cells grown under normoxic and mild hypoxic conditions in order to elucidate the mechanism by which ischemichypoxic injury induce spontaneous calcium signaling changes in microglia. By using Ca2+ sensitive fluorescence dye, we studied how inhibition of mitochondrial respiration changed spontaneous Ca2+ signals in soma of microglial cells from 5–7-day-old rats grown under normoxic and mild-hypoxic conditions. In microglia under normoxic conditions, metformin or rotenone elevated the rate and the amplitude of Ca2+ signals 10–15 min after drug application. Addition of cyclosporin A, a blocker of mPTP, antioxidant trolox, or inositol 1,4,5-trisphosphate receptor (IP3R) blocker caffeine in the presence of rotenone reduced the elevated rate and the amplitude of the signals implying sensitivity to reactive oxygen species, and involvement of mitochondrial mPTP together with IP3R. Microglial cells exposed to mild hypoxic conditions for 24 h showed elevated rate and increased amplitude of Ca2+ signals. Application of metformin or rotenone but not phenformin before mild hypoxia reduced this elevated rate. Thus, metformin and rotenone had the opposing fast action in normoxia after 10–15 min and the slow action during 24 h mild-hypoxia implying activation of different signaling pathways. The slow action of metformin through inhibition of complex I could stabilize Ca2+ homeostasis after mild hypoxia and could be important for reduction of ischemia-induced microglial activation.