A CMOS neuroelectronic interface based on two-dimensional transistor arrays with monolithically-integrated circuitry.

The ability to monitor and to elicit neural activity with a high spatiotemporal resolution has grown essential for studying the functionality of neuronal networks. Although a variety of microelectrode arrays (MEAs) has been proposed, very few MEAs are integrated with signal-processing circuitry. As a result, the maximum number of electrodes is limited by routing complexity, and the signal-to-noise ratio is degraded by parasitics and noise interference. This paper presents a single-chip neuroelectronic interface integrating oxide-semiconductor field-effect transistors (OSFETs) with signal-processing circuitry. After the chip was fabricated with the standard complementary-metal-oxide-semiconductor (CMOS) process, polygates of specific transistors were etched at die-level to form OSFETs, while metal layers were retained to connect the OSFETs into two-dimensional arrays. The complete removal of polygates was confirmed by high-resolution image scanners, and the reliability of OSFETs was examined by measuring their electrical characteristics. Through a gate oxide of only 7nm thick, each OSFET can record and stimulate neural activity extracellularly by capacitive coupling. The capability of the full chip in neural recording and stimulation was further experimented using the well-characterised escape circuit of the crayfish. Experimental results indicate that the OSFET-based neuroelectronic interface can be used to study neuronal networks as faithfully as conventional electrophysiological tools. Moreover, the proposed simple, die-level fabrication process of the OSFETs underpins the development of various field-effect biosensors on a large scale with on-chip circuitry.

Static magnetic field expose enhances neurotransmission in crayfish nervous system.

PURPOSE: To investigate the effects of static magnetic field (SMF) exposure on the synaptic transmission in a tail-flip circuit of crayfish. MATERIALS AND METHODS: An O-shaped permanent magnet (35 mT intensity) was placed under the isolated nerve cord of crayfish to provide static magnetic field exposure. Using electrophysiological methods, the excitatory post synaptic potential (EPSP) before and after field exposure in the lateral giant interneuron were measured and compared. RESULTS: The EPSP produced via electrical and chemical synapses in the lateral giant neuron were enhanced after 30 min of SMF exposure (8.08 mT). Perfusion of field-exposed crayfish bath solution or preloading of Ca(2+) chelator and intracellular Ca(2+) release blocker failed to observe the SMF-induced enhancement on EPSP. CONCLUSIONS: Exposure of SMF increases the efficacy of synaptic-transmission in crayfish tail-flip escape circuit and this SMF-induced potentiation is a Ca(2+) dependent phenomenon.

Long-lasting potentiation of excitatory synaptic signaling to the crayfish lateral giant neuron.

The neural circuit that underlies the lateral giant fiber (LG)-mediated reflex escape in crayfish has provided findings relating synaptic change to nonassociative learning such as sensitization and habituation. The LGs receive sensory inputs from the primary sensory afferents and a group of mechanosensory interneurons (MSIs). An increase of excitability by suprathreshold repetitive excitation of this circuit, which is similar to Hebbian long-term potentiation (LTP), has been reported. This potentiation was previously thought to result from the enhancement of transmission at cholinergic synapses between primary afferents and MSIs but not the electrical synapses onto LG. In this study, we found that potentiation of synaptic signaling at the electrical synapse onto LG can also be induced when the synapse was activated with subthreshold repetitive pulses or with a few strong suprathreshold shocks. LG LTP was induced in the preparation which had received pulses at limited frequency range. Although whether this LTP is involved in the learning process of escape behavior in crayfish is not clear, the intensity and amount of sensory stimulation used here mimicked those that could easily be produced by a predator trying to catch a crayfish and could be of adaptive significance in life.

Dual and opposing modulatory effects of serotonin on crayfish lateral giant escape command neurons.

Serotonin modulates afferent synaptic transmission to the lateral giant neurons of crayfish, which are command neurons for escape behavior. Low concentrations, or high concentrations reached gradually, are facilitatory, whereas high concentrations reached rapidly are inhibitory. The modulatory effects rapidly reverse after brief periods of application, whereas longer periods of application are followed by facilitation that persists for hours. These effects of serotonin can be reproduced by models that involve multiple interacting intracellular signaling systems that are each stimulated by serotonin. The dependence of the neuromodulatory effect on dose, rate, and duration of modulator application may be relevant to understanding the effects of natural neuromodulation on behavior and cognition and to the design of drug therapies.

Hierarchical folding of intestinal fatty acid binding protein.

Intestinal fatty acid binding protein (IFABP) is a member of the lipid binding protein family, members of which have a clam shell type of motif formed by two five-stranded beta-sheets. Understanding the folding mechanism of these proteins has been hindered by the presence of an unresolved burst phase. By initiating the reaction with a sub-millisecond mixer and following its progression by Trp fluorescence, we discovered three distinct phases in the folding reaction of the W6Y mutant of IFABP from which we postulate the following sequence of events. The first phase (k(1) > 10 000 s(-1)) involves collapse of the polypeptide chain around a hydrophobic core. During the second phase (k(2) approximately 1500 s(-1)), beta-strands B-G, mostly located on the top half of the clam shell structure, propagate from this hydrophobic core. It is followed by the final phase (k(3) approximately 5 s(-1)) involving the formation of the last three beta-strands on the bottom half of the clam shell and the establishment of the native hydrogen bonding network throughout the protein molecule.

Flavohemoglobin, a globin with a peroxidase-like catalytic site.

Biochemical studies of flavohemoglobin (Hmp) from Escherichia coli suggest that instead of aerobic oxygen delivery, a dioxygenase converts NO to NO3(-) and anaerobically, an NO reductase converts NO to N(2)O. To investigate the structural features underlying the chemical reactivity of Hmp, we have measured the resonance Raman spectra of the ligand-free ferric and ferrous protein and the CO derivatives of the ferrous protein. At neutral pH, the ferric protein has a five-coordinate high-spin heme, similar to peroxidases. In the ferrous protein, a strong iron-histidine stretching mode is present at 244 cm(-1). This frequency is much higher than that of any other globin discovered to date, although it is comparable to those of peroxidases, suggesting that the proximal histidine has imidazolate character. In the CO derivative, an open and a closed conformation were detected. The distal environment of the closed conformation is very polar, where the heme-bound CO strongly interacts with the B10 Tyr and/or the E7 Gln. These data demonstrate that the active site structure of Hmp is very similar to that of peroxidases and is tailored to perform oxygen chemistry.

A cooperative oxygen binding hemoglobin from Mycobacterium tuberculosis. Stabilization of heme ligands by a distal tyrosine residue.

The homodimeric hemoglobin (HbN) from Mycobacterium tuberculosis displays an extremely high oxygen binding affinity and cooperativity. Sequence alignment with other hemoglobins suggests that the proximal F8 ligand is histidine, the distal E7 residue is leucine, and the B10 position is occupied by tyrosine. To determine how these heme pocket residues regulate the ligand binding affinities and physiological functions of HbN, we have measured the resonance Raman spectra of the O(2), CO, and OH(-) derivatives of the wild type protein and the B10 Tyr --> Leu and Phe mutants. Taken together these data demonstrate a unique distal environment in which the heme bound ligands strongly interact with the B10 tyrosine residue. The implications of these data on the physiological functions of HbN and another heme-containing protein, cytochrome c oxidase, are considered.

Submillisecond unfolding kinetics of apomyoglobin and its pH 4 intermediate.

Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except for a possible burst phase in unfolding. Submillisecond mixing experiments confirm the unfolding burst phase and show that its properties are consistent with the recently discovered interconversion between two forms of I, Ia equilibrium Ib. In urea-induced unfolding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib-->Ia. The second question is whether the Ia, Ib intermediates accumulate transiently when the native protein (N) unfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib accumulate when U refolds to N at pH 6.0 and the results fit the linear folding pathway U equilibrium Ia equilibrium Ib equilibrium N. We report here that either or both Ia and Ib accumulate transiently when N unfolds to U at pH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2. 7 and refolding at pH 6.0. In unfolding as in refolding, we do not detect a fast track that bypasses the Ia, Ib intermediates.

A cooperative oxygen-binding hemoglobin from Mycobacterium tuberculosis.

Two putative hemoglobin genes, glbN and glbO, were recently discovered in the complete genome sequence of Mycobacterium tuberculosis H37Rv. Here, we show that the glbN gene encodes a dimeric hemoglobin (HbN) that binds oxygen cooperatively with very high affinity (P(50) = 0.013 mmHg at 20 degrees C) because of a fast combination (25 microM(-1).s(-1)) and a slow dissociation (0.2 s(-1)) rate. Resonance Raman spectroscopy and ligand association/dissociation kinetic measurements, along with mutagenesis studies, reveal that the stabilization of the bound oxygen is achieved through a tyrosine at the B10 position in the distal pocket of the heme with a conformation that is unique among the globins. Physiological studies performed with Mycobacterium bovis bacillus Calmette-Guérin demonstrate that the expression of HbN is greatly enhanced during the stationary phase in aerobic cultures but not under conditions of limited oxygen availability. The results suggest that, physiologically, the primary role of HbN may be to protect the bacilli against reactive nitrogen species produced by the host macrophage.