Ultrasound-Based Neuromodulation for Cognition Enhancement: Exploring Frequency-Specific Effects on Hippocampal Neurogenesis in Rats

Abstract

Neurogenesis, the process of generating functional neurons from neural stem or progenitor cells, holds significant therapeutic potential for treating neurological disorders, including neurodegenerative diseases and cognitive impairments. Neurostimulation, which involves externally modulating neural activity, has emerged as a promising approach to enhancing neurogenesis. While Deep Brain Stimulation (DBS) has shown efficacy in modulating neural circuits and promoting neurogenesis, its invasive nature poses risks such as infection and bleeding, limiting its broader application. In contrast, ultrasound-based neurostimulation offers a non-invasive alternative with substantial potential. Low-intensity ultrasound has been shown to modulate neural activity, enhance synaptic plasticity, and potentially stimulate neurogenesis. This study aimed to investigate the frequency-specific effects of low-intensity ultrasound on hippocampal neurogenesis and its functional outcomes in Sprague-Dawley rats. A four-week experimental protocol was implemented, during which ultrasound stimulation was applied directly to the hippocampus. Three distinct ultrasound frequencies—500 kHz, 1 MHz, and 5 MHz—were systematically examined to determine optimal conditions for promoting neurogenesis. Neural proliferation was assessed using immunofluorescence, with BrdU (5-bromo-2'-deoxyuridine) labeling proliferating cells and NeuN (Neuronal Nuclei) marking mature neurons. Additionally, the novel object recognition (NOR) test, a hippocampal-dependent cognitive task, was used to evaluate functional outcomes, with behavioral metrics including time spent exploring the novel object and latency to explore recorded post-stimulation. The results demonstrated frequency-dependent effects of ultrasound stimulation on hippocampal neurogenesis at both molecular and behavioral levels. Immunofluorescence analysis revealed a significant increase in BrdU-positive cells at 500 kHz, indicating enhanced neural proliferation at this frequency, whereas higher frequencies (1 MHz and 5 MHz) exhibited reduced neurogenic efficacy. Behavioral assessments using the NOR test further corroborated these findings, as rats exposed to 500 kHz ultrasound showed superior cognitive performance, reflected in increased time spent exploring the novel object and shorter latency times. In contrast, rats in the 1 MHz and 5 MHz groups exhibited diminished exploratory behavior and prolonged latency, aligning with the molecular data and suggesting a direct relationship between enhanced neurogenesis and cognitive improvements at 500 kHz