Understanding how the electrical activity of neurons gives rise to the higher functions of the brain is one of the most important questions in neuroscience. Starting in the 1950s, it has been understood that the chemical synapse is keystone of network activity. Synapses bridge the electrical gap between neurons via chemical neurotransmitters by translating action potentials in presynaptic neurons to postsynaptic potentials. In doing so, neurons’ electrical activities can influence each other and give rise to higher-level network functions. In the field of electrophysiology, however, measurement of these synapses across a neuronal network has been difficult: postsynaptic potentials are small amplitude (~1 mV) and therefore very difficult to measure.
For example, the patch clamp technique has been the workhorse method for synapse measurement due to its high-fidelity and reliable cell interface. For measurement, a pipette with an inserted electrode mechanically punctures the cell membrane to gain direct access to inside of the neuron. Such intracellular access results in high-fidelity recording/stimulation and facilitated the measurement of ion-channel currents, for which it won the Nobel prize in 1991, and the quantization of the postsynaptic potential. Nonetheless, its large size and need to be micromanipulated limits its measurement to less than ~10 cells in parallel, preventing it from measuring large-scale network connections.
To extend intracellular access to the network level, we have integrated nanoscale electrodes with custom designed electronic circuit chips in our recent paper1. The research extends the initial demonstration by Hongkun Park’s group in 2012 that vertical nanoelectrodes are capable of intracellular access to neurons2, Fig. 1a. Importantly, because these electrodes were fabricated using standard top-down microfabrication techniques, they had the potential to scale intracellular access to large arrays of electrodes for network-level investigations. A difficult engineering problem arose to record such electrode arrays, however, as wiring many electrodes to off-chip electronics becomes increasingly difficult as the number of electrodes becomes large.
Fig. 1 | Research progression of the CMOS nanoelectrode array technology. a, The vertical nanoelectrode array demonstrated intracellular access to neurons using top-down fabricated, nanoneedles. Off chip electronics were used to record from a 4×4=16 nanoelectrode array. Copyright 2012 Springer Nature. b, The vertical nanowires were combined with CMOS technology to parallelize the system to 32×32=1,024 nanoelectrodes. Intracellular access was only accomplished with cardiac cells: the permeabilization of neuron membranes was too strong and resulted in cell death. Copyright 2017 Springer Nature. c, A new CMOS nanoelectrode array (left) improved the nano-bio interface and permeabilization process to enable intracellular recording from rat neurons (right). Various nanoelectrode geometries were demonstrated to attain the intracellular access using platinum black. The device contains a 64×64=4,096 nanoelectrode array and records intracellular signals from more than 1,700 rat neurons. Copyright 2019 Springer Nature.
To address this issue, a collaboration with Donhee Ham’s group was formed which specializes in complementary metal-oxide-semiconductor (CMOS) integrated circuit design. The CMOS technology, which is the same technology that manufactures the chips in computers and cell phones, is critical for this equation as it facilitates the highly parallel readout and control of large-scale electrode arrays on its surface using integrated digital and analog circuitry just below. We reported a first generation CMOS nanoelectrode array in 20173 in which we fabricated nanoelectrodes on top of a custom designed CMOS circuit, Fig. 1b, but critically, it was only capable of recording from cardiac cells and was not able to record from neurons despite our best efforts. The largest problem encountered stemmed from the delicate nature of mammalian neurons: to gain intracellular access the cell membrane needed to be permeated (liken to the puncturing procedure of the patch clamp) but in doing so the neuron would typically die due to depolarization.
To overcome this nano-bio interface problem, we have developed a gentler way of gaining intracellular access and a new integrated circuit designed to facilitate its operation1, Fig. 1c. Instead of applying a fast and large amplitude voltage signal for membrane permeabilization, as other works have done4, we apply a small amplitude constant current. The current then gently permeabilizes the membrane while also helping to maintain its membrane potential at a healthy value. Additionally, the use of platinum black electrodes improves electrode-neuron coupling where the nanoscale crevasses and protrusions of the platinum form a tight seal with the cell membrane.
The culmination of these research efforts resulted in a high-yield of neuron coupling: using the 4,096 parallel recording channels we have intracellularly measured more than 1,700 rat neurons simultaneously. Measuring and mapping synaptic connections was then possible at a scale far beyond the patch clamp: we have mapped more than 300 synaptic connections from just a 19 min recording, which has provided a never-before-seen look at the neuronal network. In comparison, the patch clamp can typically measure up to ~2 synaptic connections day.
Looking towards the future, we foresee the high-fidelity intracellular access, which we also demonstrate is capable of both ion-channel current measurement and postsynaptic quantization like the patch clamp, as opening the way for a new breed of neuroelectronic interfaces to be developed. At the front-end, a nano-bio interface with strong intracellular interactions, and at the back end, highly parallel CMOS electronics for recording and stimulation. In the short term, we are adapting the device for high-throughput, high-precision drug screening on synaptic connections for neurological disorders such as schizophrenia, Parkinson’s disease, autism, Alzheimer’s disease, and addiction.