At present, the prime methodology for studying neuronal circuit-connectivity, physiology and pathology under in vitro or in vivo conditions is by using substrate-integrated microelectrode arrays. Although this methodology permits simultaneous, cell-non-invasive, long-term recordings of extracellular field potentials generated by action potentials, it is 'blind' to subthreshold synaptic potentials generated by single cells. On the other hand, intracellular recordings of the full electrophysiological repertoire (subthreshold synaptic potentials, membrane oscillations and action potentials) are, at present, obtained only by sharp or patch microelectrodes. These, however, are limited to single cells at a time and for short durations. Recently a number of laboratories began to merge the advantages of extracellular microelectrode arrays and intracellular microelectrodes. This Review describes the novel approaches, identifying their strengths and limitations from the point of view of the end users--with the intention to help steer the bioengineering efforts towards the needs of brain-circuit research.

Current extracellular multisite recordings suffer from low signal-to-noise ratio, limiting the monitoring to action potentials, and preclude detection of subthreshold synaptic potentials. Here we report an approach to induce Aplysia californica neurons to actively engulf protruding microelectrodes, providing 'in-cell recordings' of subthreshold synaptic and action potentials with signal-to-noise ratio that matches that of conventional intracellular recordings. Implementation of this approach may open new vistas in neuroscience and biomedical applications.

Interfacing neurons with micro-and nano-electronic devices has been a subject of intense study over the last decade. One of the major problems in assembling efficient neuroelectronic hybrid systems is the weak electrical coupling between the components. This is mainly attributed to the fundamental property of living cells to form and maintain an extracellular cleft between the plasma membrane and any substrate to which they adhere. This cleft shunts the current generated by propagating action potentials and thus reduces the signal-to-noise ratio. Reducing the cleft thickness, and thereby increasing the seal resistance formed between the neurons and the sensing surface, is thus a challenge and could improve the electrical coupling coefficient. Using electron microscopic analysis and field potential recordings, we examined here the use of gold micro-structures that mimic dendritic spines in their shape and dimensions to improve the adhesion and electrical coupling between neurons and micro-electronic devices. We found that neurons cultured on a gold-spine matrix, functionalized by a cysteine-terminated peptide with a number of RGD repeats, readily engulf the spines, forming tight apposition. The recorded field potentials of cultured Aplysia neurons are significantly larger using gold-spine electrodes in comparison with flat electrodes.

Hai A, Shappir J, Spira ME. Long-term, multisite, parallel, in-cell recording and stimulation by an array of extracellular microelectrodes. J Neurophysiol 104: 559 -568, 2010. First published April 28, 2010 doi:10.1152/jn.00265.2010. Here we report on the development of a novel neuroelectronic interface consisting of an array of noninvasive gold-mushroom-shaped microelectrodes (gMEs) that practically provide intracellular recordings and stimulation of many individual neurons, while the electrodes maintain an extracellular position. The development of this interface allows simultaneous, multisite, longterm recordings of action potentials and subthreshold potentials with matching quality and signal-to-noise ratio of conventional intracellular sharp glass microelectrodes or patch electrodes. We refer to the novel approach as "in-cell recording and stimulation by extracellular electrodes" to differentiate it from the classical intracellular recording and stimulation methods. This novel technique is expected to revolutionize the analysis of neuronal networks in relations to learning, information storage and can be used to develop novel drugs as well as high fidelity neural prosthetics and brain-machine systems.

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We demonstrate a technique for mapping brain activity that combines molecular specificity and spatial coverage using a neurotransmitter sensor detectable by magnetic resonance imaging (MRI). This molecular functional MRI (fMRI) method yielded time-resolved volumetric measurements of dopamine release evoked by reward-related lateral hypothalamic brain stimulation of rats injected with the neurotransmitter sensor. Peak dopamine concentrations and release rates were observed in the anterior nucleus accumbens core. Substantial dopamine transients were also present in more caudal areas. Dopamine-release amplitudes correlated with the rostrocaudal stimulation coordinate, suggesting participation of hypothalamic circuitry in modulating dopamine responses. This work provides a foundation for development and application of quantitative molecular fMRI techniques targeted toward numerous components of neural physiology.

This study demonstrates the use of on-chip gold mushroom-shaped microelectrodes (gMμEs) to generate localized electropores in the plasma membrane of adhering cultured neurons and to electrophysiologically monitor the ensuing membrane repair dynamics. Delivery of an alternating voltage pulse (0.5-1 V, 100 Hz, 300 ms) through an extracellularly positioned micrometer-sized gMμE electroporates the patch of plasma membrane facing the microelectrode. The repair dynamics of the electropores were analyzed by continuous monitoring of the neuron transmembrane potential, input resistance (R(in)) and action potential (AP) amplitude with an intracellular microelectrode and a number of neighbouring extracellular gMμEs. Electroporation by a gMμE is associated with local elevation of the free intracellular calcium concentration ([Ca(2+)](i)) around the gMμE. The membrane repair kinetics proceeds as an exponential process interrupted by abrupt recovery steps. These abrupt events are consistent with the "membrane patch model" of membrane repair in which patches of intracellular membrane fuse with the plasma membrane at the site of injury. Membrane electroporation by a single gMμE generates a neuron-gMμE configuration that permits recordings of attenuated intracellular action potentials. We conclude that the use of on-chip cultured neurons via a gMμE configuration provides a unique neuroelectronic interface that enables the selection of individual cells for electroporation, generates a confined electroporated membrane patch, monitors membrane repair dynamics and records attenuated intracellular action potentials.

Microelectrode arrays increasingly serve to extracellularly record in parallel electrical activity from many excitable cells without inflicting damage to the cells by insertion of microelectrodes. Nevertheless, apart from rare cases they suffer from a low signal to noise ratio. The limiting factor for effective electrical coupling is the low seal resistance formed between the plasma membrane and the electronic device. Using transmission electron microscope analysis we recently reported that cultured Aplysia neurons engulf protruding micron size gold spines forming tight apposition which significantly improves the electrical coupling in comparison with flat electrodes (Hai et al 2009 Spine-shaped gold protrusions improve the adherence and electrical coupling of neurons with the surface of micro-electronic devices J. R. Soc. Interface 6 1153-65). However, the use of a transmission electron microscope to measure the extracellular cleft formed between the plasma membrane and the gold-spine surface may be inaccurate as chemical fixation may generate structural artifacts. Using live confocal microscope imaging we report here that cultured Aplysia neurons engulf protruding spine-shaped gold structures functionalized by an RGD-based peptide and to a significantly lesser extent by poly-l-lysine. The cytoskeletal elements actin and associated protein cortactin are shown to organize around the stalks of the engulfed gold spines in the form of rings. Neurons grown on the gold-spine matrix display varying growth patterns but maintain normal electrophysiological properties and form functioning synapses. It is concluded that the matrices of functionalized gold spines provide an improved substrate for the assembly of neuro-electronic hybrids.