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.

The assembly of a new growth cone is a prerequisite for axon regeneration after injury. Creation of a new growth cone involves multiple processes, including calcium signalling, restructuring of the cytoskeleton, transport of materials, local translation of messenger RNAs and the insertion of new membrane and cell surface molecules. In axons that have an intrinsic ability to regenerate, these processes are executed in a timely fashion. However, in axons that lack regenerative capacity, such as those of the mammalian CNS, several of the steps that are required for regeneration fail, and these axons do not begin the growth process. Identification of the points of failure can suggest targets for promoting regeneration.

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.

SUMMARY1. Propagation of action potentials at high frequency was studied in a branching axon of the lobster by means of simultaneous intracellular recording both before and after the branch point.2. Although the branching axon studied has a geometrical ratio close to one (perfect impedance matching) conduction across the branch point failed at stimulation frequencies above 30 Hz.3. The block of conduction after high frequency stimulation occurred at the branch point per se. The parent axon and daughter branches continued to conduct action potentials. 4. Conduction block after high frequency stimulation appeared first in the thicker daughter branch and only later in the thin branch.5. With high frequency stimulation there was a 10-15 % reduction in amplitude of the action potential in the parent axon, a corresponding decrease in the rate of rise ofthe action potential, a 25-30 % decrease in conduction velocity, marked increase in threshold and prolongation of the refractory period. In addition the membrane was depolarized by 1-3 mV.6. Measurements of the membrane current using the patch clamp technique showed a large decrease in the phase of inward current associated with the action potential, before the branching point.7. The small membrane depolarization seen after high frequency stimulation is not the sole cause of the conduction block. Imposed prolonged membrane depolarization (8 mV for 120 sec) was insufficient to produce conduction block.8. In vivo chronic extracellular recordings from the main nerve bundle (which contains the parent axon) and the large daughter branch revealed that: (a) the duration and frequency of trains of action potentials along the axons exceeded those used in the isolated nerve experiments and (b) conduction failure in the large daughter branch could be induced in the whole animal by electrical stimulation of the main branch as in the isolated preparation.9. Possible mechanisms underlying block of conduction after high frequency stimulation in a branching axon are discussed.

The emergence of a neuronal growth cone from a transected axon is a necessary step in the sequence of events that leads to successful regeneration. Yet, the molecular mechanisms underlying its formation after axotomy are unknown. In this study, we show by real time imaging of the free intracellular Ca2+ concentration, of proteolytic activity, and of growth cone formation that the activation of localized and transient Ca2+-dependent proteolysis is a necessary step in the cascade of events that leads to growth cone formation. Inhibition of this proteolytic activity by calpeptin, a calpain inhibitor, abolishes growth cone formation. We suggest that calpain plays a central role in the reorganization of the axon's cytoskeleton during its transition from a stable differentiated structure into a dynamically extending growth cone.

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