Institute of Evolutionary Physiology and Biochemistry

Source localization is a technique used to estimate the sources of brain activity based on signals recorded from the scalp. Accurate source localization critically depends on the precise spatial digitization of sensor locations. In this protocol, we present a practical and reliable method for digitizing sensor locations using the Navigated Brain Stimulation (NBS) system. NBS is a component of Transcranial Magnetic Stimulation (TMS) equipment commonly available in TMS laboratories, but rarely utilized for sensor digitization of electroencephalography (EEG) or functional near-infrared spectroscopy (fNIRS) systems. This approach allows researchers to leverage existing infrastructure to significantly improve the spatial accuracy of source modeling, without investing in dedicated digitization equipment. We guide viewers through the full workflow: (1) digitizing EEG electrode locations using default tools of the Nexstim NBS system; (2) exporting coordinate data in compatible formats; (3) integrating this data into EEG preprocessing and source localization pipelines using the MNE-Python package. The protocol also includes tips for aligning digitized data with MRI images and optimizing coregistration accuracy. To illustrate the method's practical utility, we apply it to analyze data from a tactile stimulation experiment. Custom Python scripts for coordinate processing and coregistration are provided to ensure reproducibility and ease of adoption. The results show that incorporating digitized electrode positions remarkably improves the anatomical accuracy and interpretability of cortical source estimates compared to default electrode montages.

Source localization is a technique used to estimate the sources of brain activity based on signals recorded from the scalp. Accurate source localization critically depends on the precise spatial digitization of sensor locations. In this protocol, we present a practical and reliable method for digitizing sensor locations using the Navigated Brain Stimulation (NBS) system. NBS is a component of Transcranial Magnetic Stimulation (TMS) equipment commonly available in TMS laboratories, but rarely utilized for sensor digitization of electroencephalography (EEG) or functional near-infrared spectroscopy (fNIRS) systems. This approach allows researchers to leverage existing infrastructure to significantly improve the spatial accuracy of source modeling, without investing in dedicated digitization equipment. We guide viewers through the full workflow: (1) digitizing EEG electrode locations using default tools of the Nexstim NBS system; (2) exporting coordinate data in compatible formats; (3) integrating this data into EEG preprocessing and source localization pipelines using the MNE-Python package. The protocol also includes tips for aligning digitized data with MRI images and optimizing coregistration accuracy. To illustrate the method's practical utility, we apply it to analyze data from a tactile stimulation experiment. Custom Python scripts for coordinate processing and coregistration are provided to ensure reproducibility and ease of adoption. The results show that incorporating digitized electrode positions remarkably improves the anatomical accuracy and interpretability of cortical source estimates compared to default electrode montages.

Amphibian retinas contain “green” rods, which are rod-shaped photoreceptors with a cone-type visual pigment. These rods are considered a potentially transitional photoreceptor type, but their phototransduction cascade’s molecular composition has remained uncertain. Here, we present a streamlined electrophysiology-molecular workflow that enables the rapid spectral identification, physical capture, and targeted single-cell reverse transcription-polymerase chain reaction (RT-PCR) of individual amphibian photoreceptors. After suction-pipette spectral screening under alternating red and green illumination, electrophysiologically identified cells are isolated and processed directly for reverse transcription and PCR. Coupling real-time functional phenotyping with sensitive molecular profiling provides a practical tool for resolving photoreceptor molecular heterogeneity and investigating evolutionary transitions between rod and cone phenotypes.Key features• New combined workflow: Rapid electrophysiological spectral typing by suction-pipette, followed by single-cell transfer, reverse transcription, and targeted PCR.

Efficient regulation of intracellular Ca2+ is essential for neuronal survival, and the sodium–calcium exchanger (NCX) is a major pathway for Ca2+ extrusion. NCX activity depends critically on the transmembrane Na+ gradient maintained by the Na/K-ATPase (NKA). Although both proteins co-localize in neuronal lipid rafts, direct functional modulation of NCX by NKA has not been quantitatively demonstrated. Here, we tested the hypothesis that positive modulation of NKA enhances NCX forward-mode transport in cortical neurons. Using whole-cell patch-clamp electrophysiology, we recorded NCX current–voltage (I–V) relationships in rat cortical neurons and applied 0.5 nM ouabain to selectively potentiate NKA. NCX currents were isolated using KB-R7943, and experimentally obtained I–V curves were fitted with a well-established kinetic model to estimate intracellular Na+ concentration. Ouabain produced a marked rightward shift of the NCX I–V curve, increasing forward-mode current at physiological membrane potentials. Modeling indicated a reduction of intracellular Na+ from ~32 mM to ~21 mM, consistent with NKA activation. In neurons loaded with BAPTA, which inhibits NKA, ouabain no longer affected NCX parameters. These findings demonstrate that NKA-NCX interaction modulates NCX transport by shaping the local Na+ gradient and provide a mechanistic explanation for the neuroprotective actions of low-nanomolar ouabain.

The cell surface receptor Down syndrome cell adhesion molecule (Dscam) subjected to mutually exclusive splicing in Arthropoda shows an extraordinary degree of potential immune recognition specificity. We have tested the hypothesis on the existence of similar polymorphism in the homologous receptors from sister-clade protostomian animals, Spiralia, comprising Mollusca and Annelida. Two new members of Dscam family from bivalve mollusks, Mytilus edulis (MeDscam) and Crassostrea gigas (CgDscam) were cloned from ganglia mRNA and their differential expression was estimated in immune cells. The expression of MeDscam and CgDscam in haemocytes is weak, constitutive and was not modulated by immune inducers neither in vivo nor in vitro experiments. Based on the sequencing results, we did not observe diversity in the Dscam transcripts in both ganglia and haemocytes. Our screening of C. gigas genome and genomes of other Spiralia animals, gastropod mollusk Lottia gigantea and polychaete worm Capitella teleta, revealed their Dscam homologues genes do not contain the hypervariable exon arrays and all exons are found at unique positions. This type of gene structure leaves no chance for the generation of multiple splice variants. The immune function of Dscam pattern recognition receptor based on the vast repertoire of isoforms is an evolutionary innovation developed exclusively in Arthropoda lineage and could not be attributed to other non-arthropod Protostomia.

Although cyclic adenosine monophosphate (cAMP) is not the main player in the process of phototransduction, it can modulate it. The effects of cAMP have been widely studied in rods, but its role in the photoreceptors responsible for daytime vision, the cones, has not been thoroughly investigated. The aim of this study was to eхamine the impact of increased intracellular cAMP levels on the photoresponse kinetics and light sensitivity of red-sensitive cones (RCs) in the retinas of adult zebrafish (Danio rerio). Two electrophysiological methods were employed in this study: ex vivo electroretinography with isolating the responses of RCs, and single photoreceptor current recordings performed using a suction pipette. The adenylate cyclase activator forskolin was used to increase intracellular cAMP levels. The investigation of response characteristics revealed that forskolin did not affect the sensitivity of RCs compared to controls. A detailed analysis of the kinetics of the unsaturated response of RCs showed that forskolin, similarly to its effects on amphibian rods and zebrafish blue- and green-sensitive cones, slowed down the process of turning off the phototransduction cascade, while simultaneously increasing the integration time of the response. The activation stages of the cascade remained unaffected by forskolin. At the same time, a closer examination of the rising phase of the photoresponse near its peak revealed that the faster stage of the cascade turning off was further accelerated by the action of forskolin. Collectively, these data indicate that an increase in intracellular cAMP levels can modulate the phototransduction cascade in zebrafish RCs; however, unlike other studied spectral cone types, it does not alter their light sensitivity. The most significant modulation likely occurs in the cascade turning off processes of RCs.

HighlightsThis study provides the first comprehensive analysis of long-term, region-specific changes in the entire glutamatergic system (iGluRs, mGluRs, EAATs) after neonatal febrile seizures.The most severe and persistent molecular alterations occur in the ventral hippocampus and medial prefrontal cortex, key nodes for emotional regulation and cognition.The behavioral outcome is characterized by a hyperanxious phenotype with locomotor and habituation deficits, but with working memory and sociability remaining unaffected.The findings reveal a precise neurobiological mechanism that could underlie the increased risk of anxiety-related disorders following early-life febrile seizures.Febrile seizures (FS) are a common childhood neurological event associated with an increased risk of long-term cognitive and emotional deficits, though the precise mechanisms remain elusive. Using a rat model, we investigated the long-term effects of FS induced on postnatal day 10, assessing outcomes in young adulthood (P45-55). We report region-specific neuronal loss in the hippocampus, more extensive in the ventral segment. Molecular analysis revealed a broad downregulation of genes encoding ionotropic and metabotropic glutamate receptors and excitatory amino acid transporters. These alterations were most severe and persistent in the ventral hippocampus and medial prefrontal cortex. Behaviorally, rats with neonatal FS exhibited a hyperanxious phenotype, characterized by reduced locomotor and exploratory activity and impaired habituation to a novel environment. In contrast, spatial working memory and social behavior remained intact. Our results provide the first comprehensive evidence that neonatal FS trigger long-term, region-specific disruptions of the glutamatergic system within hippocampal–prefrontal circuits. These findings identify vulnerable molecular targets and precise neurobiological mechanisms that may underlie the heightened risk of anxiety-related disorders following early-life FS, suggesting new avenues for therapeutic intervention.

The water channel aquaporin-4 (AQP4) is a key participant in the molecular and cellular mechanisms providing the elimination of various metabolites and amyloid proteins from brain tissue. It is assumed that AQP4 dysfunction may provoke the development of Parkinson’s disease (PD) and lead to its aggravation. The aim of this study was to elucidate whether downregulated AQP4 expression in the substantia nigra pars compacta (SNpc) affects the development of α-synuclein pathology, neurodegeneration, and motor impairment in a rat model of the clinical stage of PD. Experiments were carried out on male Wistar rats aged 6–7 months. To downregulate AQP4 expression in the SNpc, a lentiviral construct containing a nucleotide sequence that encodes a short hairpin RNA (shRNA) interfering with natural AQP4 mRNA and thus silencing the Aqp4 gene (AQP4-LVC) was injected into the SNpc. To reproduce a model of the clinical stage of PD, the proteasome inhibitor lactacystin (LC) was bilaterally injected into the SNpc four weeks following AQP4-LVC administration. The study involved behavioral tests, immunohistochemical labeling, and immunoblotting. AQP4-LVC application caused a 42% decrease in AQP4 protein content in the SNpc after four weeks. The LC model of PD was characterized by the appearance of motor disorders, death of 57% of dopamine (DA)-ergic neurons in the SNpc and 56% of their axons in the striatum, attenuation of compensatory processes aimed at maintaining DA level in the nigrostriatal system, and an increase in the content of a total water-soluble form of α-synuclein, as well as its aggregated and Ser129-phosphorylated forms. Downregulation of AQP4 expression in the SNpc in an LC model of PD exacerbated motor dysfunction and caused the appearance of dystrophy signs, indicating the development of a terminal phase of the clinical stage of PD. The rapid progression of parkinsonian symptoms was associated with enhanced neurodegeneration and the depletion of compensatory processes in the nigrostriatal system, coupled with the accelerated formation of pathological α-synuclein aggregates. The obtained data indicate that AQP4 deficiency in the SNpc accelerates the development of Parkinson’s-like pathology.

Cell lines and primary cultures are widely used to study host-pathogen interactions, reducing the need for challenge trials with live fish, which are costly and raise ethical concerns. We present Atlantic salmon scale explants (SE) as a promising model for bacterial challenges. Comparison with the body map (transcriptomes of seventeen cells and tissues) revealed strong immune activity in SE. SE ranked first in antiviral gene expression and second and third in antibacterial and inflammatory responses, respectively. Additionally, SE shows high expression of skin-specific genes encoding structural proteins of the cytoskeleton and extracellular matrix. Contrast to the epidermis, dermis, and whole skin highlights immune genes, including markers of acute inflammation and stress. Exposure to pathogens (Moritella viscosa and Tenacibaculum finnmarkense) and commensal bacteria consistently upregulated a suite of immune genes in a dose response manner. This panel includes chemokines, cytokines, genes involved in eicosanoid metabolism, and both humoral and cellular effectors. The prevalence of genes related to signaling and communication suggests that salmon scales function as sentinels, mobilizing immune responses upon encountering bacteria. While common immune responses were evident across all trials, scales also revealed differences between bacterial species. Combining commensal bacteria with M. viscosa enhanced responses to pathogens, and differences were observed between M. viscosa and T. finnmarkense. The effects of commensal bacteria varied in magnitude. Exposed cells showed changes in morphology and increased iNOS expression. Future development of this model will focus on time-course experiments and expanding the analytical repertoire to include pathology detection methods.

Tactile imagery (TI) engages somatosensory cortices in both hemispheres, along with widespread brain regions associated with the imagery process itself. Actively simulating touch can influence the processing of actual tactile stimuli, as reflected by modulations in somatosensory evoked potentials (SEPs) components. This study uses high-density electroencephalography (EEG) and sLORETA-based source localization to analyse cortical sources of SEPs components susceptible to active skin sensations imagery. Twenty healthy participants performed TI and tactile attention (TA) tasks. TI enhanced early SEP components (P100), indicating engagement of primary somatosensory cortical networks. This was accompanied with robust μ-rhythm event-related desynchronization (ERD) localized to the postcentral gyrus. While TA also elicited μ-ERD, its cortical distribution was broader, suggesting involvement of more distributed and possibly non-specific attentional mechanisms. Notably, sensor-space analysis revealed an enhanced late frontal P200 peak during TI, potentially indicating increased frontal activation. However, source-space analysis confirmed the absence of frontal pole involvement in SEPs during TI, underscoring the importance of accurate source localization. Thus, TI was found to significantly activate primary somatosensory cortices, influencing early stages of real tactile stimulus processing. Its effects were more spatially focused compared to those induced by TA. These findings suggest that TI could be a prospective approach for sensorimotor rehabilitation. On the other hand, TA could provide an effortless method for modulating sensorimotor rhythms in BCI control.