Understanding the intricate workings of neural circuits is paramount to deciphering the functional connections within the brain and unraveling the mechanisms underlying neurological disorders. While in vitro models of neural circuits have advanced significantly, the technology to precisely monitor and manipulate neural activity within three-dimensional (3D) models remains underdeveloped. Existing 3D microelectrode arrays (MEAs) lack the combined capabilities of stimulating nearby neurons and simultaneously monitoring the temporal evolution of network formation in real-time. This limitation hinders comprehensive investigations into the dynamics of complex neural networks. This study addresses this gap by introducing a novel 3D high-density multifunctional MEA that integrates optical stimulation and drug delivery. This innovative technology allows for precise measurements of synaptic latencies and other crucial parameters within engineered 3D neural tissues, paving the way for a deeper understanding of complex neural circuit dynamics.

Numerous studies have highlighted the limitations of two-dimensional (2D) cell culture in accurately representing the complex architecture and physiological functions of the brain. The need for 3D in vitro models that better mimic the in vivo environment has fueled the development of various 3D neural cultures and microelectrode array technologies. However, these existing systems often fall short in providing the crucial capabilities needed for comprehensive investigation. Many lack the combination of high-density recording, precise localized stimulation, and controlled drug delivery, limiting the scope of experiments that can be performed. This paper builds upon previous research on 2D MEAs and 3D neural culture methods, but critically integrates these elements into a single, sophisticated system, thus providing a significant advancement in the field.

The researchers designed and fabricated a 3D high-density multifunctional MEA by stacking three separately fabricated 2D MEAs. Each 2D MEA consisted of multiple shanks, each containing recording microelectrodes. One shank was designed as a multifunctional shank, incorporating microfluidic channels for drug delivery and a thinned optical fiber for optical stimulation. The 3D structure was achieved through precise bonding of the 2D MEAs. The resulting MEA had a high electrode density (~33 sites·mm⁻³), significantly higher than previously reported 3D MEAs. The multifunctional shanks facilitated precise local stimulation and drug delivery, while the high density of recording electrodes enabled the monitoring of extensive regions of neural tissue. The researchers carefully evaluated the MEA's key functionalities. Electrical impedance measurements confirmed the low impedance (~0.015 ± 0.004 MΩ at 1 kHz) of the Pt-black-electroplated electrodes, significantly enhancing signal-to-noise ratios. Flow rate measurements through the microfluidic channels demonstrated precise control of drug delivery. Optical power measurements and Monte Carlo simulations confirmed the efficacy of the optical stimulation, demonstrating sufficient irradiance to activate channelrhodopsin-2 (ChR2). A miniaturized cubicle was designed for in vitro experiments, incorporating the 3D MEA, a custom microdrive for precise positioning, a PDMS culture chamber, and an acrylic enclosure to maintain a stable environment. The researchers used two 3D neural network models: a single-group model and a compartmentalized two-group model, to investigate neural circuit dynamics. Primary rat cortical neurons were cultured in collagen scaffolds within the chambers. The crucial timing of MEA insertion before collagen gelation was established to ensure proper scaffold integration. Optogenetic techniques were used to study synaptic connectivity by stimulating ChR2-expressing neurons. Chemical modulation was performed by locally delivering synaptic blockers through the microfluidic channels. Electrophysiological data were analyzed using spike-sorting algorithms, synchronization scores, and network analysis methods to characterize spontaneous and optically evoked activity, burst activity, and network connectivity.

The developed 3D high-density multifunctional MEA successfully enabled the investigation of neural circuit dynamics in engineered 3D neural tissues. In the single-group model, spontaneous neural activity increased with culture time, showing an increase in firing rates, active electrodes, and burst activity. Network analysis revealed the formation and maturation of functional connections among neurons. Optogenetic stimulation at DIV 6 resulted in activation only near the stimulation site, whereas at DIV 14 widespread activation occurred throughout the network, indicating the formation of extensive synaptic connections. Chemical silencing via CNQX/AP5 injection locally blocked synaptic transmissions and network synchronization, confirming the role of excitatory synapses in the observed network activity. Following the washout, neural activity and synchronization recovered. In the two-group model, optical stimulation at early time points (DIV 6) activated neurons primarily around the stimulation site, with increasing spread of activation to the second somatic region from DIV 7-9. By DIV 10, significant increases in firing rate and synchronized activities were observed in both regions, demonstrating functional connectivity. The synaptic latency between the two groups was measured at DIV 14, indicating signal propagation with latencies ranging from 2 to 8 ms. The synaptic transmission velocities were faster in the longitudinal direction (449 ± 106 mm-s⁻¹) compared to the transverse direction (202.6 ± 69.8 mm-s⁻¹). This difference was attributed to the lower density of synapses in the neurite region along the longitudinal path. The system's applicability was validated using human spinal cord organoids, where neural activity and synchronization were successfully measured and chemically modulated using TTX.

This study successfully demonstrates a significant technological advancement in the field of in vitro neural circuit research. The developed 3D multifunctional MEA provides unparalleled capabilities for investigating complex neural network dynamics. The integration of high-density recording, precise optical stimulation, and localized drug delivery provides a powerful toolkit for studying the formation, maturation, and modulation of functional neural networks. The results from both the single-group and two-group models provide valuable insights into the temporal evolution of synaptic connections and network organization in 3D neural tissues. The ability to measure synaptic latency in a 3D setting is a particular strength, offering direct evidence of functional connectivity. The successful application to human spinal cord organoids further highlights the versatility of this system and its potential for diverse research applications.

This study successfully developed a 3D high-density multifunctional MEA system for investigating neural circuit dynamics. The system's capabilities for high-density recording, optical stimulation, and drug delivery were demonstrated, along with its applicability to both engineered neural tissues and human-derived organoids. Future work could focus on enhancing the MEA's capabilities by increasing the number of electrodes, integrating light source arrays, and incorporating advanced microfabrication techniques. Expanding the system's capabilities to include other neuromodulatory techniques would further broaden the range of applications in neuroscience research.

While the study successfully demonstrates the capabilities of the 3D multifunctional MEA, some limitations should be acknowledged. The current design uses a single multifunctional shank for optical and chemical stimulation. Expanding to multiple functional shanks for independent stimulation and drug delivery in different locations within the 3D tissue would enhance the system's capabilities. The current system's scalability for even larger and more complex neural circuit models may require further technological development to enhance functionality without compromising ease of use. The study primarily used rat cortical neurons; further validation with other neuronal types and species is recommended.