Alix Charlotte Thomson

Despite the widespread use of the SH-SY5Y human neuroblastoma cell line in modeling human neurons in vitro, protocols for growth, differentiation and experimentation differ considerably across the literature. Many studies fully differentiate SH-SY5Y cells before experimentation, to investigate plasticity measures in a mature, human neuronal-like cell model. Prior to experimentation, serum is often removed from cell culture media, to arrest the cell growth cycle and synchronize cells. However, the exact effect of this serum removal before experimentation on mature, differentiated SH-SY5Y cells has not yet been described. In studies using differentiated SH-SY5Y cells, any effect of serum removal on plasticity markers may influence results. The aim of the current study was to systematically characterize, in differentiated, neuronal-like SH-SY5Y cells, the potentially confounding effects of complete serum removal in terms of morphological and gene expression markers of plasticity. We measured changes in commonly used morphological markers and in genes related to neuroplasticity and synaptogenesis, particularly in the BDNF-TrkB signaling pathway. We found that complete serum removal from already differentiated SH-SY5Y cells increases neurite length, neurite branching, and the proportion of cells with a primary neurite, as well as proportion of βIII-Tubulin and MAP2 expressing cells. Gene expression results also indicate increased expression of PSD95 and NTRK2 expression 24 h after serum removal. We conclude that serum deprivation in differentiated SH-SY5Y cells affects morphology and gene expression and can potentially confound plasticity-related outcome measures, having significant implications for experimental design in studies using differentiated SH-SY5Y cells as a model of human neurons.

Transcranial Magnetic Stimulation (TMS) is a form of non-invasive brain stimulation, used to alter cortical excitability both in research and clinical applications. The intermittent and continuous Theta Burst Stimulation (iTBS and cTBS) protocols have been shown to induce opposite after-effects on human cortex excitability. Animal studies have implicated synaptic plasticity mechanisms long-term potentiation (LTP, for iTBS) and depression (LTD, for cTBS). However, the neural basis of TMS effects has not yet been studied in human neuronal cells, in particular at the level of gene expression and synaptogenesis. To investigate responses to TBS in living human neurons, we differentiated human SH-SY5Y cells toward a mature neural phenotype, and stimulated them with iTBS, cTBS, or sham (placebo) TBS. Changes in (a) mRNA expression of a set of target genes (previously associated with synaptic plasticity), and (b) morphological parameters of neurite outgrowth following TBS were quantified. We found no general effects of stimulation condition or time on gene expression, though we did observe a significantly enhanced expression of plasticity genes NTRK2 and MAPK9 24 h after iTBS as compared to sham TBS. This specific effect provides unique support for the widely assumed plasticity mechanisms underlying iTBS effects on human cortex excitability. In addition to this protocol-specific increase in plasticity gene expression 24 h after iTBS stimulation, we establish the feasibility of stimulating living human neuron with TBS, and the importance of moving to more complex human in vitro models to understand the underlying plasticity mechanisms of TBS stimulation.

Repetitive Transcranial Magnetic Stimulation (rTMS) is an established neuromodulation technique, using electromagnetic pulses that, depending on the precise parameters, a re assumed to lead to lasting neural excitability changes. rTMS has widespread applications in both research and therapy, where it has been FDA approved and is considered a first-line treatment for depression, according to recent North American and European guidelines. However, these assumed excitability effects are often difficult to replicate, and highly unreliable on the single subject/patient level. Given the increasing application of rTMS, especially in clinical practice, the absence of a method to unequivocally determine effects of rTMS on human neuronal excitability is problematic. We have taken a first step in addressing this bottleneck, by administering excitatory and inhibitory rTMS protocols, iTBS and cTBS, to a human in vitro neuron model; differentiated SH-SY5Y cells. We use live calcium imaging to assess changes in neural activity following stimulation, through quantifying fluorescence response to chemical depolarization. We found that iTBS and cTBS have opposite effects on fluorescence response; with iTBS increasing and cTBS decreasing response to chemical depolarization. Our results are promising, as they provide a clear demonstration of rTMS after-effects in a living human neuron model. We here present an in-vitro live calcium imaging setup that can be further applied to more complex human neuron models, for developing and evaluating subject/patient-specific brain stimulation protocols.

In recent years, the influence of alpha (7–13 Hz) phase on visual processing has received a lot of attention. Magneto‐/encephalography (M/EEG) studies showed that alpha phase indexes visual excitability and task performance. Studies with transcranial alternating current stimulation (tACS) aim to modulate oscillations and causally impact task performance. Here, we applied right occipital tACS (O2 location) to assess the functional role of alpha phase in a series of experiments. We presented visual stimuli at different pre‐determined, experimentally controlled, phases of the entraining tACS signal, hypothesizing that this should result in an oscillatory pattern of visual performance in specifically left hemifield detection tasks. In experiment 1, we applied 10 Hz tACS and used separate psychophysical staircases for six equidistant tACS‐phase conditions, obtaining contrast thresholds for detection of visual gratings in left or right hemifield. In experiments 2 and 3, tACS was at EEG‐based individual peak alpha frequency. In experiment 2, we measured detection rates for gratings with (pseudo‐)fixed contrast. In experiment 3, participants detected brief luminance changes in a custom‐built LED device, at eight equidistant alpha phases. In none of the experiments did the primary outcome measure over phase conditions consistently reflect a one‐cycle sinusoid. However, post hoc analyses of reaction times (RT) suggested that tACS alpha phase did modulate RT for specifically left hemifield targets in both experiments 1 and 2 (not measured in experiment 3). This observation requires future confirmation, but is in line with the idea that alpha phase causally gates visual inputs through cortical excitability modulation.

In recent years the influence of alpha (7-13 Hz) phase on visual processing has received a lot of attention. Magneto-/encephalography (M/EEG) studies showed that alpha phase indexes visual excitability and task performance. If occipital alpha phase is functionally relevant, the phase of occipital alpha-frequency transcranial alternating current stimulation (tACS) could modulate visual processing. Visual stimuli presented at different pre-determined, experimentally controlled, phases of the entraining tACS signal should then result in an oscillatory pattern of visual performance. We studied this in a series of experiments. In experiment one, we applied 10 Hz tACS to right occipital cortex (O2) and used independent psychophysical staircases to obtain contrast thresholds for detection of visual gratings in left or right hemifield, in six equidistant tACS phase conditions. In experiments two and three, tACS was at EEG-based individual peak alpha frequency. In experiment two, we measured detection rates for gratings with (pseudo-)fixed contrast levels. In experiment three, participants detected brief luminance changes in a custom-built LED device, at eight equidistant alpha phases. In none of the experiments did the primary outcome measure over phase conditions consistently reflect a one-cycle sinusoid as predicted. However, post-hoc analyses of reaction times (RT) suggested that tACS alpha phase did modulate RT in both experiments 1 and 2 (not measured in experiment 3). This observation is in line with the idea that alpha phase causally gates visual inputs through cortical excitability modulation.

Cognitive effort and self-control are exhausting. Although evidence is ambiguous, behavioural studies have repeatedly suggested that control-demanding tasks seem to deplete a limited cache of self-regulatory resources leading to performance degradations and fatigue. While resource depletion has indirectly been associated with a decline in right prefrontal cortex capacity, its precise neural underpinnings have not yet been revealed. This study consisted of two independent experiments, which set out to investigate the causal role of the right dorsolateral prefrontal cortex (DLPFC) in a classic dual phase depletion paradigm employing non-invasive brain stimulation. In Experiment 1 we demonstrated a general depletion effect, which was significantly eliminated by anodal transcranial Direct Current Stimulation to the right DLPFC. In Experiment 2, however, we failed to replicate the basic psychological depletion effect within a second independent sample. The dissimilar results are discussed in the context of the current ‘replication crisis’ and suggestions for future studies are offered. While our current results do not allow us to firmly argue for or against the existence of resource depletion, we outline why it is crucial to further clarify which specific external and internal circumstances lead to limited replicability of the described effect. We showcase and discuss the current inter-lab replication problem based on two independent samples tested within one research group (intra-lab).