Transient extracellular glutamate events in the basolateral amygdala track reward-seeking actions.

The ability to make rapid, informed decisions about whether or not to engage in a sequence of actions to earn reward is essential for survival. Modeling in rodents has demonstrated a critical role for the basolateral amygdala (BLA) in such reward-seeking actions, but the precise neurochemical underpinnings are not well understood. Taking advantage of recent advancements in biosensor technologies, we made spatially discrete near-real-time extracellular recordings of the major excitatory transmitter, glutamate, in the BLA of rats performing a self-paced lever-pressing sequence task for sucrose reward. This allowed us to detect rapid transient fluctuations in extracellular BLA glutamate time-locked to action performance. These glutamate transients tended to precede lever-pressing actions and were markedly increased in frequency when rats were engaged in such reward-seeking actions. Based on muscimol and tetrodotoxin microinfusions, these glutamate transients appeared to originate from the terminals of neurons with cell bodies in the orbital frontal cortex. Importantly, glutamate transient amplitude and frequency fluctuated with the value of the earned reward and positively predicted lever-pressing rate. Such novel rapid glutamate recordings during instrumental performance identify a role for glutamatergic signaling within the BLA in instrumental reward-seeking actions.

A microfabricated, 3D-sharpened silicon shuttle for insertion of flexible electrode arrays through dura mater into brain.

OBJECTIVE: Electrode arrays for chronic implantation in the brain are a critical technology in both neuroscience and medicine. Recently, flexible, thin-film polymer electrode arrays have shown promise in facilitating stable, single-unit recordings spanning months in rats. While array flexibility enhances integration with neural tissue, it also requires removal of the dura mater, the tough membrane surrounding the brain, and temporary bracing to penetrate the brain parenchyma. Durotomy increases brain swelling, vascular damage, and surgical time. Insertion using a bracing shuttle results in additional vascular damage and brain compression, which increase with device diameter; while a higher-diameter shuttle will have a higher critical load and more likely penetrate dura, it will damage more brain parenchyma and vasculature. One way to penetrate the intact dura and limit tissue compression without increasing shuttle diameter is to reduce the force required for insertion by sharpening the shuttle tip. APPROACH: We describe a novel design and fabrication process to create silicon insertion shuttles that are sharp in three dimensions and can penetrate rat dura, for faster, easier, and less damaging implantation of polymer arrays. Sharpened profiles are obtained by reflowing patterned photoresist, then transferring its sloped profile to silicon with dry etches. MAIN RESULTS: We demonstrate that sharpened shuttles can reliably implant polymer probes through dura to yield high quality single unit and local field potential recordings for at least 95 days. On insertion directly through dura, tissue compression is minimal. SIGNIFICANCE: This is the first demonstration of a rat dural-penetrating array for chronic recording. This device obviates the need for a durotomy, reducing surgical time and risk of damage to the blood-brain barrier. This is an improvement to state-of-the-art flexible polymer electrode arrays that facilitates their implantation, particularly in multi-site recording experiments. This sharpening process can also be integrated into silicon electrode array fabrication.

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays.

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas. The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days. This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

High-Density, Long-Lasting, and Multi-region Electrophysiological Recordings Using Polymer Electrode Arrays.

The brain is a massive neuronal network, organized into anatomically distributed sub-circuits, with functionally relevant activity occurring at timescales ranging from milliseconds to years. Current methods to monitor neural activity, however, lack the necessary conjunction of anatomical spatial coverage, temporal resolution, and long-term stability to measure this distributed activity. Here we introduce a large-scale, multi-site, extracellular recording platform that integrates polymer electrodes with a modular stacking headstage design supporting up to 1,024 recording channels in freely behaving rats. This system can support months-long recordings from hundreds of well-isolated units across multiple brain regions. Moreover, these recordings are stable enough to track large numbers of single units for over a week. This platform enables large-scale electrophysiological interrogation of the fast dynamics and long-timescale evolution of anatomically distributed circuits, and thereby provides a new tool for understanding brain activity.

Silicon Wafer-Based Platinum Microelectrode Array Biosensor for Near Real-Time Measurement of Glutamate in Vivo.

Using Micro-Electro-Mechanical-Systems (MEMS) technologies, we have developed silicon wafer-based platinum microelectrode arrays (MEAs) modified with glutamate oxidase (GluOx) for electroenzymatic detection of glutamate in vivo. These MEAs were designed to have optimal spatial resolution for in vivo recordings. Selective detection of glutamate in the presence of the electroactive interferents, dopamine and ascorbic acid, was attained by deposition of polypyrrole and Nafion. The sensors responded to glutamate with a limit of detection under 1muM and a sub-1-second response time in solution. In addition to extensive in vitro characterization, the utility of these MEA glutamate biosensors was also established in vivo. In the anesthetized rat, these MEA glutamate biosensors were used for detection of cortically-evoked glutamate release in the ventral striatum. The MEA biosensors also were applied to the detection of stress-induced glutamate release in the dorsal striatum of the freely-moving rat.

A Fully Automated Approach to Spike Sorting.

Understanding the detailed dynamics of neuronal networks will require the simultaneous measurement of spike trains from hundreds of neurons (or more). Currently, approaches to extracting spike times and labels from raw data are time consuming, lack standardization, and involve manual intervention, making it difficult to maintain data provenance and assess the quality of scientific results. Here, we describe an automated clustering approach and associated software package that addresses these problems and provides novel cluster quality metrics. We show that our approach has accuracy comparable to or exceeding that achieved using manual or semi-manual techniques with desktop central processing unit (CPU) runtimes faster than acquisition time for up to hundreds of electrodes. Moreover, a single choice of parameters in the algorithm is effective for a variety of electrode geometries and across multiple brain regions. This algorithm has the potential to enable reproducible and automated spike sorting of larger scale recordings than is currently possible.

Improved chronic neural stimulation using high surface area platinum electrodes.

We report a novel nano-cluster platinum (NCPt) film that exhibits enhanced performance as an electrode material for neural stimulation applications. Nano-cluster films were deposited using a custom physical vapor deposition process and patterned on a flexible polyimide microelectrode array using semiconductor processing technology. Electrode performance was characterized in vitro using electrochemical impedance spectroscopy and compared with sputtered thinfilm platinum (TFPt) electrodes. We characterized electrode impedance, charge storage capacity, voltage transient properties, and relative surface area enhancement in vitro. Preliminary lifetime testing of the electrode reveals that the NCPt electrodes degrade more slowly than TFPt electrodes. The combination of material biocompatibility, electrochemical performance, and preliminary lifetime results point to a promising new electrode material for neural interface devices.

Electrochemically deposited iridium oxide reference electrode integrated with an electroenzymatic glutamate sensor on a multi-electrode array microprobe.

An implantable micromachined multi-electrode array (MEA) microprobe modified for utilization as a complete electrochemical biosensor for rapid glutamate detection is described. A post-fabrication method for electrochemical deposition of an iridium oxide (IrOx) film onto a designated microelectrode enabled incorporation of an IrOx reference electrode (RE) on the microprobe. The on-probe IrOx RE provides an alternative to the commonly utilized Ag/AgCl wire RE, which has been shown to be unstable and to cause an inflammatory response in living tissue. The IrOx film electrodeposited onto a platinum site was tested as part of a complete chemical sensing system that included a platinum counter electrode and enzymatic glutamate sensing electrodes all on a single silicon-based MEA platform. The thin film IrOx was mechanically robust enough to endure conditions of repeated heating and wetting during the MEA fabrication process. The pH dependence of the IrOx open circuit potential (OCP) was measured at -77±0.4 mV/pH and remained stable over a two-week period. The on-probe IrOx RE was tested in a two- and three-electrode system with glutamate biosensors. The biosensors were shown to detect a physiologically relevant range of glutamate concentrations and to reject the interferents, dopamine and ascorbic acid. By incorporating all of the electrodes onto a single device, baseline noise was reduced by an average of ∼61% in vitro and ∼71% in vivo with reduced tissue damage, since only a single probe needed to be implanted.

Insertion of flexible neural probes using rigid stiffeners attached with biodissolvable adhesive.

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (>3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.