New Insights into Functional Connectivity and Neuronal Activation

Functional Connectivity and Brain Activation: New Clinical Insights in Neuro-Rehab Therapy. Transcranial Pulse Stimulation (TPS) continues to advance as a promising non-invasive neuromodulation technique in neuroscience. With growing evidence of therapeutic benefits in Alzheimer’s disease — including improvements in memory, mood, and cognition — research is increasingly focused on uncovering the underlying mechanisms of TPS. This is the second publication of the research group in Zurich from their study investigating TPS in mouse models. Building upon earlier work that identified TPS-induced vascular changes, the current research provides critical insights into the neuronal activity and functional connectivity network effects of TPS. Using advanced imaging and histological techniques, the authors show that TPS modulates brain activity and initiates coordinated activation across memory, emotion, and motor-related networks of the brain. These findings deepen the mechanistic understanding of TPS and highlight its potential as a targeted neuromodulation approach. Methods. To explore how TPS affects brain activity and connectivity, the study was conducted on two groups of mice: genetically healthy controls and commonly used mouse models of Alzheimer’s disease. TPS was applied using the NEUROLITH® system, delivering pulses at clinically established energy levels. Each animal received two rounds of stimulation, spaced 15 minutes apart, with each round consisting of three bursts delivered in a defined low–low–high energy sequence (0.05–0.05–0.25 mJ/mm²). The low energy level of 0.05 mJ/mm 2 is roughly equivalent to the energy of 0.25 mJ/mm 2 used in the clinical setting, taking into account the different thicknesses of the skull between humans and mice, whereas the highest allowable energy (0.25 mJ/mm 2 ) was applied to examine the vessel response and potential adverse effects and to explore whether stronger neuromodulatory responses could be elicited within the established safety range. The effects were analysed both during stimulation (calcium imaging) and after stimulation, with fMRI performed immediately post-TPS and histological analysis conducted later. To assess neuronal activation and network dynamics, the researchers used a multimodal approach:. In vivo epifluorescent imaging for calcium response was performed to visualise neuronal activity in real-time during TPS., Immunohistochemistry for c-Fos expression served as a molecular marker for neuron activation., Resting-state functional MRI (rs-fMRI) was used to detect changes in brain-wide functional connectivity before and after stimulation. This combination of techniques enabled the team to observe both local cellular responses and broader changes in brain network communication. Results. TPS induced a clear and energy-dependent neuronal activation, as demonstrated by:. Robust calcium influx during stimulation, visualised via in-vivo fluorescent calcium imaging, indicating immediate neuronal excitation in response to each pulse., Significant increase in c-Fos expression in the dentate gyrus of the hippocampus, observed after stimulation, confirming delayed activation of memory-relevant neurons on a molecular level. These observations suggest that TPS likely engages rapid electrophysiological mechanisms, such as the activation of mechanosensitive ion channels, leading to potassium influx and neuronal depolarisation — a hypothesis supported by the temporal dynamics and energy dependence of the responses. These effects were consistent across Alzheimer model mice and healthy control mice, indicating a general mechanism of action. The stimulation also led to a rapid and transient reorganisation of functional connectivity, observed through resting-state fMRI. Notable increases in network activity were detected in:. Limbic regions (hippocampus, amygdala, entorhinal cortex), Hypothalamic subregions, especially the anterior and ventromedial hypothalamus, Subcortical structures, including the basal ganglia and midbrain. These regions are involved in memory, emotional regulation, and motor control. The similar connectivity patterns seen in both Alzheimer and control mice suggest that TPS engages core neural networks rather than acting disease-specifically. Conclusion. Findings from animal studies cannot be directly translated to clinical outcomes. However, the results support the hypothesis that mechanical stimulation via TPS can safely influence brain activity by engaging neuronal and network processes without inducing thermal or cavitational effects. These insights contribute to a deeper understanding of TPS mechanisms and provide a solid basis for advancing its refinement as a neuromodulatory approach in the context of neurodegenerative disease research.