1. Aleksandra Nogaj, Kinga Przybylska, Aleksandra Trenk, Anna Guguła and Anna Błasiak –The role of nucleus incertus originating innervation and relaxin-3/RXFP3 signaling in modulating ventral hippocampus activity – anatomical, neurochemical and electrophysiological studies in rats
The stress-sensitive brainstem structure nucleus incertus (NI) is a primary source of neuropeptide relaxin-3 (RLN3) in the brain. NI provides extensive innervation to the ventral hippocampus (vHPC), an area critical for processing emotional information and involved in control of anxiety-related behaviors. The activation of RLN3 receptors (RXFP3) in vHPC increases anxiety and social avoidance. However, the neuronal mechanisms underlying interactions between NI and vHPC remain unclear.
Viral-based neural tract tracing was performed to map NI-originating fibers within the vHPC. Fluorescent multiplex in situ hybridization was used to characterize vHPC neurons expressing RXFP3 mRNA. Finally, we used ex vivo multielectrode array and patch-clamp recordings, to verify the effects of RXFP3 activation on vHPC network activity.
Neural tract-tracing revealed a high density of NI fibers within the polymorphic and molecular layer of the dentate gyrus (DG), the majority being RLN3-positive. That indicates NI as a primary source of RLN3 in the vHPC. Multiplex in situ hybridization showed that all RXFP3-mRNA expressing vHPC DG cells are vGAT1 mRNA-positive, which indicates their GABAergic nature. Ex vivo multielectrode array and patch-clamp recordings demonstrated that RXFP3 activation exert both inhibitory and excitatory effects on vHPC neurons activity.
These results suggest that NI originating RLN3 innervation controls the activity of DG neurons and points to the NI-vHPC axis as an important component of the neural mechanism underlying stress processing. That influence may account for anxiety-related behaviors and social avoidance following chronic RXFP3 activation in the vHPC.
2. Katarzyna Hryniewiecka, Marcin Lipiec, Magdalena Majkowska, Suelen Baggio, Ewa Kublik, Marta Wiśniewska – Validation of a new mouse model for autism spectrum disorder
Tcf7l2 is a high-confidence risk gene for autism spectrum disorder, but its exact role in its pathogenesis is unknown. Tcf7l2 is highly expressed in the thalamus, where it regulates genes governing neuronal excitability. The thalamus, as a relay of sensory signals from periphery, as well as a modulator of activity between associative, motivational, executive and motor cortical regions, is a candidate structure for ASD pathogenesis. To understand the role of thalamus-expressed Tcf7l2 in the information processing and development of ASD, we investigated functional consequences of its deficiency in the brain.
We hypothesize that Tcf7l2 deficiency in thalamic neurons impairs the activity of thalamo-cortical circuits, whose dysfunctions are common in psychiatric disorders such as ASD.
A mouse strain with the postnatal knockout of Tcf7l2 in the thalamus was used for in vivo and in vitro electrophysiological experiments. Neuropixels probes inserted to somatosensory thalamus and cortex of anesthetised mouse were used to measure field evoked potential responses to single and paired whisker stimulations. The strength of the prepulse inhibition effect (PPI, a measure of sensory gating mechanisms) was estimated from their amplitudes. In vitro, a patch-clamp recordings were conducted to investigate the excitability of thalamic neurons in their standard firing modes – tonic, burst and rebound-burst.
Our results indicate that male knockout mice show impairments in the prepulse inhibition mechanism compared to control mice (t(18) = -3.26, p < 0.01). Preliminary patch-clamp results show a decrease in general excitability and a dysfunction in burst mode firing in thalamic neurons.
These results show an impairment in electrophysiological function, which is crucial for signal processing in thalamic neurons. Moreover, as the PPI is frequently decreased in cognitive disorders, including ASD; and ASD symptoms are more severe in males, we can cautiously conclude that indeed thalamic locus of Tcf7l2 disruption may play a role in the development of ASD. Our next aim is to understand molecular mechanisms behind observed deficits and establish their link to behavior.
3. Bernadeta A. Pietrzak 1*, Andrzej Łach 1*, Agnieszka Wnuk 1, Karolina Przepiórska 1, Małgorzata Kajta 1
Non-nuclear estrogen receptors targeting as a novel neuroprotective strategy against amyloid-β-induced neurotoxicity
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by amyloid-β deposition and tau hyperphosphorylation that result in progressive exacerbation of memory loss and cognitive decline. AD is accompanied by estrogen receptor (ER) signaling dysregulation, as evidenced by high incidence of AD among postmenopausal women. ERs are an important system for neuroprotection. However, activation of classical, nuclear ERs which mediate the vast majority of hormonal effects, in addition to neuroprotection may lead to carcinogenesis and cardiovascular problems. On the contrary, selective activation of non-nuclear i.e., membrane-associated ERs (mERα and mERβ) has been shown a safer alternative that does not induce tumorigenesis and thromboembolism. Therefore, in this study, we assessed the neuroprotective capacity of Pathway Preferential Estrogen-1 (PaPE-1), which selectively activates mERα and mERβ against amyloid-β-induced toxicity.
To model Alzheimer’s disease, we utilized mouse primary neocortical cell cultures that were treated with amyloid-β oligomers 1-42 (10 μM). PaPE-1 was applied in the posttreatment paradigm. The mechanisms of PaPE-1-attributed neuroprotection were assessed in respect to apoptosis, cell viability, the degree of neurodegeneration and neurite outgrowth.
We observed that primary neocortical cell cultures treated with amyloid-β showed increased caspase -3, -8, and -9 activities. Since caspases are known not only for their pathological role in cell death but also in neurite outgrowth and dendrite branching, we evaluated the effects of amyloid-β and posttreatment with PaPE-1 on cell viability (Calcein AM), the degree of cell neurodegeneration (Fluoro-Jade C), apoptosis-related chromatin condensation (Hoechst 33342) and neurite outgrowth. According to our results, PaPE-1 attenuated the abovementioned amyloid-β-induced changes, thus supporting the neuroprotective capacity of PaPE-1 against AD-related pathologies.
Our original data provide evidence that selective activation of mERα and mERβ by PaPE-1 is a promising neuroprotective strategy against amyloid-β-induced neurotoxicity. This research was funded by the National Science Centre of Poland, grant number 2020/39/NZ7/00974.
4. Tomasz Kuliński, Johan Davidsson, Mechanics and Maritime Sciences Division – Acute-phase electrocorticography in rats exposed to rotational traumatic brain injury
The aim of this animal pilot study was to test whether rotational traumatic brain injury causes measurable alterations in resting state electrocorticography in the acute postinjury phase and, if so, what alterations may be found. Our primary hypothesis was that power spectral densities between pre- and postinjury states will differ.
5 male Sprague-Dawley rats (including 1 sham) were sacrificed. Animals were anaesthetized with midazolam-medetomidine-fentanyl mixture injection. Rats with exposed crania were glued to a test rig imparting rotational acceleration-deceleration analogous to car crash-related head injury in humans. Brain activity was measured with 3 epidural screw electrodes and a Biopac MP150 system. Recording lasted for at least 10 minutes before trauma and from immediately after injury until up to 3 hours. Data determined as artefact-free had power spectral density (PSD) estimated with Welch’s modified periodogram over the whole of preinjury and postinjury recordings, and separately for the first and last 300 s of the postinjury recording (early and late postinjury phases).
Our preliminary results indicate that rats exposed to rotational brain injury show suppressed spectral power relative to preinjury in the early postinjury phase for frequencies up to 40 Hz. The spectral power partially returns to or even exceeds preinjury values at the late postinjury phase. We also encountered numerous unexpected episodes of signal loss, both in injured and uninjured animals, which occurred suddenly or gradually over the course of minutes.
We have concluded that resting state cortical activity after trauma does show alterations in spectral power, which vary over time. Our two main speculations on the causes of observed signal loss events are that they may be either (1) “electrode pop” artefacts of a physical or chemical nature, or may be related to (2) spreading depression. We argue for this distinction by noting the differences in the time courses of these events.
5. Piotr Buczkowicz – EEG brain activity in ADHD children
Attention deficit hyperactivity disorder (ADHD) is characterised by persistent inattention and/or hyperactivity symptoms interfering with or reducing social, academic or occupational aspects of functioning.  It is estimated that ADHD occurs in 7 to 10 percent of population. [1, 2]
ADHD is recognised by a primary care provider or a mental health professional, based on the interviews with the parents or the child. The interviews are relatively subjective, they often have poor specificity and are inconsistent between i.e. parent and teachers versions. [5, 6] Use of psychometric assessment, like Conners-3 test is often recommended.  However, it might be sometimes insufficient, or even create a risk of gender bias.  These problems implicate a growing need of alternative, objective methods of diagnosis.
Therefore, attempts have been made to find a biomarker for ADHD. In case of structural neuroimaging, they turned out to be mostly unsuccessful, but the literature is inconsistent. [1, 10] Generally there is no biomarkers for ADHD  and yet the closest to that seems to be EEG, especially changes in the theta-beta ratio (TBR).  The latter is being used as FDA-approved Neuropsychiatric EEG-Based Assessment Aid System NEBA.  Although not entirely consistent, research on the EEG characteristics in ADHD is a promising thread that may help to better understand the neural basis and the possibility of more accurate diagnosis in this disorder.
This work aims to present the current scientific findings on the characteristics of EEG recordings in children with ADHD during the resting recording, but also during the cognitive task. At the end, a research plan (with the description of preliminary results, if possible) will be presented, aiming at creating new EEG-ADHD markers in combination with eye-tracking. The hypothetical results of the planned experiment that I will describe could have crucial meaning on understanding of neural basis of ADHD and diagnosis possibilities.
6. Karolina Pendrasik, Dominika Świerczewska, Kacper Mazurkiewicz, Michalina Wiwatowska-Ligęza – Analysis of influence of prenatal exposition to testosterone and serum testosterone level in patients with multiple sclerosis treated with natalizumab and fingolimod.
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS. Exposition to low levels of testosterone (T) during the prenatal period represent risk factors for developing MS. High serum T levels in adult patients can be a protective factor in MS. T plays also a role in formation and repair of myelin in the CNS suggesting its neuroprotective role. In this work we studied a link between T levels and clinical progression of patients treated for relapsing-remitting form of MS (RRMS).
We enrolled 33 patients with RRMS treated with natalizumab and fingolimod. Digit Ratio (DR) was measured. Serum T concentration was measured with electrochemiluminescence. Neurological outcome was determined using EDSS. For assessment of cognitive functions we used SDMT, MoCA, FSMC and MFIS. Since patients suffering from MS frequently cope with depression. We measured its severity with BDI.
We found a correlation in female patients between serum T level and progression in MFIS and a correlation in female patients between DR and FSMC . In male patients we observed an association between serum T level and progression in SDMT and between T and progression in MFIS . There is a relation in all male patients between DR and progression in BDI.
Our observations indicate an existing link between exposition to T and outcome of patients with RRMS. The data may provide useful information for the use of DR and serum T level as a marker for response to different MS therapies.
7. Alicja Olszewska, Maciej Gaca, Dawid Droździel, Agnieszka Widlarz, Aleksandra Herman, Artur Marchewka – Differential timecourses of functional brain reorganisation in novice pianists.
Learning to play the piano is a complex task, integrating multiple sensory modalities and higher-order cognitive functions. Longitudinal neuroimaging studies on adult novice musicians show training-related functional changes, primarily in music perception tasks. However, these findings are not uniform and are obtained at varying time-scales from minutes to weeks to months. It is possible that the observed diversity of results reflects different stages of complex neuroplastic process. Moreover, the reorganisation of brain activity while actually playing an instrument has been rarely studied. Thus, our aim was to investigate the dynamic complexity of functional brain reorganisation within the first half year of musical training.
We scanned twenty-four novice learners (female, 18-23yo) using fMRI while they played increasingly complex musical pieces after 1, 6, 13 and 26 weeks of training.
In line with previous studies, playing music evoked responses mainly in the auditory and left sensorimotor regions and supplementary motor cortex. The training, however, invoked changes in the right sensorimotor cortex, cerebellum, superior parietal cortex, anterior cingulate, anterior insula, putamen and hippocampus, among others. Post-hoc analysis revealed region-specific time-courses for independent auditory, motor and inferior frontal regions of interest.
These results suggest that while the primary sensory, motor and frontal regions are involved in playing music, the training affects higher-order cognitive control and integrative regions, and basal ganglia. When designing longitudinal experiments, caution should be taken not to limit the analysis only to task-related regions. Moreover, training might affect different brain regions in a sequential manner, providing evidence in favour of the dynamic, complex and nonlinear nature of brain plasticity.
8. Anna Lesniewska, Urszula Górska Ph.D., Marek Binder Ph.D., Małgorzata Hołda Ph.D. – 40Hz ASSR is sensitive to the level of arousal and awareness during natural sleep.
When we fall asleep, we seem to not detect most of incoming auditory stimuli, while some of them could still be detected. Auditory steady-state responses (ASSRs) have been proven to be sensitive in fluctuations in arousal – they tend to reduce in the states where conscious processing is markedly diminished. However, it is unclear whether those effects are caused by fluctuations in arousal or consciousness. We attempted to answer this question in our study by using neuroimaging techniques and sleep questionnaires.
ASSRs are oscillatory responses elicited by periodic auditory stimulation Here, a series of two chirp stimuli, varying in duration and frequency, was presented to 22 healthy volunteers. We analysed the intertrial phase clustering (ITPC) response from the nine frontocentral channels on the EEG data for each stimulus. From 14 of the participants, 42 sleep and dream reports were collected.
We found significant (p < 0.05) effects of reduced ITPC values from wakefulness to N2 and N3 NREM conditions. For the wide-band type of chirp stimulation there was also significant effect between wakefulness and REM sleep, while it was not significant when narrow-band type of chirp stimulation was used. Interestingly, the comparison of subjective reports from serial awakenings paradigm reveals significant difference (p<0.05) of ITPC in 37-43 Hz band between no-dreaming vs dreaming reports in which auditory experience was present, but not when it was absent.
These results indicate that low gamma range ASSRs are sensitive to changes in both consciousness (which decreases during non-dreaming unconsciousness) and arousal (which decreases in NREM sleep). The current findings support the idea that ASSRs in the low gamma range can be used to reliably distinguish between levels of consciousness in clinical settings.
9. Marta Chrustowicz, prof. Łukasz Okruszek, mgr Szymon Mąka – Effects of Loneliness on Neurophysiological Correlates of Cognitive Reappraisal Use in Young Adults
According to the Evolutionary Theory of Loneliness, a prolonged feeling of loneliness may increase bottom-up processes associated with orienting towards social stimuli (e.g. hypervigilance to social threats) while decreasing top-down processes necessary for producing adequate emotional response towards it (mentalizing and emotion regulation). The study aimed to examine the impact of loneliness on ability to engage in cognitive reappraisal, in a group of participants from the general population.
151 young adults from the whole spectrum of loneliness were asked to reappraise or watch negative three-second animations depicting two point-light agents that have been taken from Social Perception and Interaction Database (Okruszek & Chrustowicz, 2020), while the electroencephalography (EEG), electrocardiography (ECG) and galvanic skin response (GSR) were recorded.
The study found that altercations were perceived as more arousing and negative than neutral social interactions. Increased early and late Late Positive Potential (LPP) amplitude to altercations compared to neutral interactions were also found. However, the use of reappraisal strategy was found to reduce these effects. Moreover, the effect of condition was observed also in ECG and GSR signals. Interestingly, no correlation between loneliness levels and behavioural and neurophysiological markers observed during the task were found.
To summarize, the behavioral and neurophysiological data suggest that the participants were capable of comprehending the negative situations presented through point-light displays and employing cognitive reappraisal to decrease their level of arousal and negative assessment of the stimuli. Moreover, the participants’ ability to engage in cognitive reappraisal was found to be unaffected by their level of loneliness.
10. Marta Lotka, Anna Ceglarek, Magdalena Fąfrowicz, Koryna Lewandowska, Tadeusz Marek, Jeremi Ochab, Barbara Sikora-Wachowicz – Functional connectivity estimation from fMRI data: correlation matrix estimation method matters.
Accurate estimation of the correlation matrix from functional magnetic resonance (fMRI) data is crucial to functional integration research. The Pearson correlation matrix is commonly used. However, in the fMRI data setting the number of variables – locations from which the signal is sampled – is of the same or greater order as the number of samples. In such a setting the Pearson correlation coefficient becomes very sensitive to any noise present in the data. Moreover, the Pearson correlation coefficient does not account for the non-stationarity of fMRI data. Therefore, it is of interest to explore other methods of correlation matrix estimation and, in particular, the effects of method choice on detection of statistically significant differences between experimental conditions.
Method comparison is conducted using data from an fMRI experiment investigating the effects of diurnal changes of working memory performance. Three methods of correlation matrix estimation are compared: the Pearson correlation coefficient, the detrended cross-correlation coefficient and a coefficient based on the resting BOLD event triggered average (rBeta) method. Each method is also considered after application of Ledoit-Wolf shrinkage – a procedure reducing the effects of noise present in the data.
All correlation matrix estimation methods besides the rBeta-based method detected statistically significant differences between information encoding and retrieval phases of the experiment. However, differences between experimental tasks were generally not stable with respect to the correlation matrix estimation method used.
To ensure meaningful results, it is necessary to further investigate correlation matrix estimation methods in the context of different data analysis pipelines.
11. Ewelina Turczak, Nina Härtwich, Reinhard König, Patrick J. C. May, Cezary Sielużycki – Optimising the connectivity pattern within auditory cortex: An evolutionary approach.
To investigate signal processing in the auditory cortex (AC), May and colleagues created an advanced computational model based on the anatomy of the AC. It has successfully been applied to describe event-related fields (ERFs) acquired with magnetoencephalography (MEG). In the past, the values of the connection weights between the basic computational units of the model were set manually, with the aim of roughly replicating the morphology of real ERFs. To obtain better parameter values, we fitted simulated ERFs to experimental ERFs using an advanced optimisation approach.
To solve this optimisation problem, we applied evolutionary algorithms (EAs). We utilised a simplified AC architecture with only one cortical column as the basic unit in each of the 14 fields of the model. We have also employed the full architecture of the model with 16 columns per field.
Both approaches provided high-quality fits of the synthetic MEG signal to the real MEG waveform. Our results for both architectures revealed an asymmetry between feedforward and feedback connection strengths, with stronger values for feedback connections. This finding deviates from the originally assumed symmetry of respective weight values.
Our results shed new light on the organisation of the human AC, but more MEG data from different subjects must be examined. Due to the computational burden of EAs, this requires finding new ways for reducing the computational complexity. Our work demonstrates the potential of parameter optimisation methods in the context of the improvement of computational models simulating biological systems.
12. Agata Kozioł, David López Pérez, Zuzanna Laudańska, Anna Malinowska-Korczak, Karolina Babis, Oleksandra Mykhailova, Hana D’Souza, Przemysław Tomalski – Motor overflow during reaching in infancy: quantification of limb movement using Inertial Motion Units
Early in life infants exhibit motor overflow, which can be defined as the generation of involuntary movements accompanying purposeful actions (Addamo et al., 2009). Although this phenomenon has already been explored in infancy (e.g., D’Souza et al., 2017), previous studies relied on manual coding and the exact mechanism of motor overflow is still debated. Here we present the results of the first study quantifying motor overflow with high accuracy and precision provided by wearable motion trackers.
In this study we used Inertial Motion Units to quantify motor overflow in 4-month-old infants. The purpose of the research was to investigate the motor activity across the non-acting limbs during goal-directed action. To this end, we used wearable motion trackers to measure infant motor activity during a baby-gym task designed to capture overflow during reaching movements. The analysis was conducted on the subsample of participants (n = 20), who performed at least four reaches during the task.
A series of Granger causality tests revealed differences in activation patterns depending on the type of the reaching movement and the non-acting limb. Importantly, on average, the non-acting arm preceded the activation of the acting arm. In contrast, the acting arm activity was followed by the activation of the legs.
The registered differences in motor activity may be a reflection of distinct purposes of the non-acting limbs in supporting postural stability and efficiency of movement execution. Finally, our findings demonstrate the utility of wearable motion trackers for precise measurement of infant movement dynamics.