Harvard researchers have mapped and catalogued more than 70,000 synaptic connections from about 2,000 rat neurons, using a silicon chip capable of recording small yet telltale synaptic signals from a large number of neurons.
The research, published in Nature Biomedical Engineering , is a major advance in neuronal recording and may help bring scientists a step closer to drawing a detailed synaptic connection map of the brain.
Higher-order brain functions are believed to be derived from the ways brain cells, or neurons, are connected. Neuron-to-neuron contact points are called synapses, and scientists seek to draw synaptic connection maps that show not only which neurons connect to which other neurons, but also how strong each connection is. While electron microscopy has been used with great success to make visual maps of synaptic connections, these images lack information on connection strengths and thus the ultimate function of the neuronal network.
In contrast, a patch-clamp electrode , the gold standard in neuronal recording, can effectively get inside an individual neuron to record a faint synaptic signal with high sensitivity, and thus can find a synaptic connection and tell its strength. Scientists have long tried to apply such high-sensitivity intracellular recording to a large number of neurons in parallel, in order to measure and characterize a large number of synaptic signals and thus draw a map annotated with connection strengths. But they have seldom gotten further than obtaining intracellular access from a handful of neurons at once.
The researchers, led by Donhee Ham , the John A. and Elizabeth S. Armstrong Professor of Engineering and Applied Sciences at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS), developed an array of 4,096 microhole electrodes on a silicon chip, which performed massively parallel intracellular recording of rat neurons cultured on the chip. From these unprecedented recording data that abounded with synaptic signals, they extracted over 70,000 synaptic connections from about 2,000 neurons.
The work builds on the team's 2020 breakthrough device – an array of 4,096 vertical nanoneedle electrodes sticking out of a silicon chip of the same integrated circuit design. On this previous device, a neuron could wrap around a needle to allow intracellular recording, which was parallelized through the large number of electrodes. In the best case, they could extract about 300 synaptic connections from the recording data – still blowing well past what patch-clamp recording can reach.
With the basic premise in hand, the team suspected they could do better. Co-lead authors Jun Wang and Woo-Bin Jung from the Ham group at SEAS led the design and fabrication of the microhole electrode array on the silicon chip, the electrophysiological recording and the data analysis.
They operated the chip to gently open up cells with small current injections through the electrodes in order to parallelize their intracellular recording. Postdoctoral researcher Wang said the microhole design is similar to the patch-clamp electrode, which is essentially an electrode-housing glass pipette with a hole at the end.
"Not only do microhole electrodes better couple to the interiors of neurons than the vertical nanoneedle electrodes, but they are also much easier to fabricate. This accessibility is another important feature of our work," Wang said.
The new design exceeded the team's expectations. On average, more than 3,600 microhole electrodes out of the total 4,096 – that is, 90 percent – were intracellularly coupled to neurons on top. The number of synaptic connections the team extracted from such unprecedented network-wide intracellular recording data bloomed to 70,000 plausible synaptic connections, compared with about 300 with their previous nanoneedle electrode array. The quality of the recording data was also better, which allowed the team to categorize each synaptic connection based on its characteristics and strengths.
"The integrated electronics in the silicon chip plays as equally an important role as the microhole electrode, providing gentle currents in an elaborate way to obtain intracellular access, and recording at the same time the intracellular signals," said Jung, a former postdoctoral researcher and now a faculty member at Pohang University of Science and Technology in South Korea.
"One of the biggest challenges, after we succeeded in the massively parallel intracellular recording, was how to analyze the overwhelming amount of data," Ham said. "We have since come a long way to gain insight into synaptic connections from them. We are now working toward a newer design that can be deployed in a live brain."
Paper co-authors include Rona S. Gertner of the Department of Chemistry and Chemical Biology, and Hongkun Park, the Mark Hyman, Jr. Professor of Chemistry and Professor of Physics.
The research was supported by the Samsung Advanced Institute of Technology of Samsung Electronics.
