Symposium SM06 : The Future of Neuroengineering---Relevant In Vivo Technology

The human medial temporal lobe contains neurons that respond to the semantic contents of a presented stimulus. These "concept cells" may respond to very different pictures of a given person and even to their written or spoken name. Their response latency is far longer than necessary for object recognition, and they are found in brain regions that are crucial for declarative memory formation. It has thus been hypothesized that they may represent the semantic "building blocks" of episodic memories.
In this talk I will present data from single unit recordings in the hippocampus, entorhinal cortex, parahippocampal cortex, and amygdala during paradigms involving perception as well as encoding and consolidation of episodic memories in order to characterize the role of concept cells in these cognitive functions.

In order to understand how behavioral and network level functions emerge from the cooperation of neuronal populations, a representative fraction of neurons must be simultaneously monitored at the single-cell level. Furthermore, in order to properly command these networks, interventions must also be temporally and spatially precisely targeted. Solutions realizing cell-type or neurotransmitter specificity regarding either the input or the output relations may add a further complementary dimension to the studies.
We developed large-scale electrophysiological recording methods that can monitor the activity of thousands of neurons simultaneously. Due to on-head analog signal multiplexing, the recorded signals are instantaneously available for driving e.g. feedback circuits, while it still provides submillisecond resolution and does not constrain the behavior of the freely moving rodents. We combine our recordings with precise optogenetic and transcranially delivered electrical manipulation approaches, which can give birth to on demand, closed-loop therapeutic interventions for many drug resistant neuropsychiatric disorders.
I will briefly introduce our recent results we gained to understand the development of hypersynchronous events in the hippocampus. Our translational work employing closed-loop transcranial electrical stimulation on human patients will be also highlighted in my talk. The goal of these efforts is to establish an acute intervention approach promptly disrupting pathologic network oscillations such as epileptic seizures. Finally, I will shortly present our attempts in developing a new generation of chemosensitive electrodes to report the concentration changes of various neuromodulators in vivo, with a comparable resolution to their secondary electrical aspects.

As our understanding of the brain’s physiology and pathology progresses, increasingly sophisticated materials and technologies are required to advance discoveries in systems neuroscience and develop more effective diagnostics and treatments for neuropsychiatric disease. Localizing brain signals may assist with tissue resection and intervention strategies in patients with such diseases. Precise localization requires large and continuous coverage of cortical areas with high-density recording from populations of neurons while minimizing invasiveness and adverse events. We describe a large-scale, high-density, organic electronic–based, conformable neural interface device (NeuroGrid) with embedded integrated circuitry capable of simultaneously recording local field potentials (LFPs) and action potentials from the cortical surface. We demonstrate the feasibility and safety recording with such devices in anesthetized and awake subjects. Highly localized and traveling physiological and pathological LFP patterns were recorded, and correlated neural firing provided evidence about their local generation. Application of NeuroGrid technology to disorders such as epilepsy may improve diagnostic precision and therapeutic outcomes while reducing complications associated with invasive electrodes conventionally used to acquire high-resolution and spiking data.

Classical inorganic electronic devices are commonly used in medical practice to diagnose and treat neurological disorders. As our knowledge about the organization and function of healthy and pathological neuronal networks of the human brain is improving, there is an increasing need to develop less invasive, biocompatible stimulating and recording devices with better spatiotemporal resolution.
Organic electronic devices provide us unique solutions for these problems, due to their special characteristics. Their mixed conductivity, mechanical flexibility, biocompatibility, and unique capability for drug delivery enable them to be promising candidates for advanced therapeutic applications.
In my talk I would like to highlight how animal models in experimental neuroscience can help to develop and test these organic materials to invent more effective approaches in the diagnosis and the treatment of human brain diseases.

We have assembled miniaturized, brain-implantable/bio-inpsired electronics on biocompatible photocurable polysiloxane stiffer. The electronic devices and sensors show transferrable, bright, and thin micro GaN LEDs, neurotransmitter, and neural gases like NO and CO detection sensors.
In the usual optogenetic technique, the enabled modes of use are impossible to realize using standard approaches that rely on rigid, long, glass fiber optics coupled to external, bulky light sources. Our systems exploit ultrathin, flexible substrates populated with microscale inorganic light emitting diodes (LEDs) together with electrophysiological and temperature sensors, all mounted on removable plastic needles that facilitate insertion into the tissue. Detailed experimental and theoretical studies of the operation, ranging from heat flow aspects to inflammation assessments and comparison to conventional devices, illustrate the unique features of this technology. Also we exploit neural gases NO and CO change with a chronic epilepsy mouse model using injectable microscale neural gas sensors. We believe that these multifunctional, injectable electronics could be beneficial in diverse brain diseases and will open many challenging applications in areas of implantable diagnostics and therapeutics.

One of the most important scientific and technological frontiers of our time lies in the interface between electronics and the human brain. Interfacing the most advanced human engineering endeavor with nature’s most refined creation promises to help elucidate aspects of the brain’s working mechanism and deliver new tools for diagnosis and treatment of a host of pathologies including epilepsy and Parkinson’s disease. Current solutions, however, are limited by the materials that are brought in contact with the tissue and transduce signals across the biotic/abiotic interface. Recent advances in organic electronics have made available materials with a unique combination of attractive properties, including mechanical flexibility, mixed ionic/electronic conduction, enhanced biocompatibility, and capability for drug delivery. I will present examples of novel devices for recording and stimulation of brain activity that go beyond the current state-of-the-art in terms of performance, compatibility with the brain, and form factor. I will show that organic electronic materials offer tremendous opportunities to design devices that improve our understanding of brain physiology and pathology, and can be used to deliver new therapies.

There is currently a surge in the development of neurotechnologies to aid in understanding brain function and in enabling new neuroprosthesis devices. Accompanying these advances is an increased awareness of the tissue and vascular strain and damage as well as the biofouling response to penetrating intraparenchymal/depth electrodes. Surface electrocorticography (ECoG) is viewed as a less invasive alternative that is not challenged by the cellular encapsulation and low spatial coverage of depth electrodes and is being investigated for brain-machine interfaces and in encoding cognitive functions. This paper will focus on the clinical translation of ECoG microelectrode arrays. First, we summarize their performance metrics for high fidelity recording and stimulation with in-vivo data that benchmarks different electrode materials across a variety of species. Second, we discuss the size dependency of recording single units from the cortical surface using these different microelectrode materials. Finally, we provide an update on the utility of the technology for recording in the clinic.

The development of organic bioelectronics has been intensified during last decade, in part sparked by several unique demonstrations of using the technology to bridge the signaling gap between biology and technology. Typically, thin film processing protocols, originally developed for large area electronics, have been explored to realize organic bioelectronic sensors and actuators. In order to distribute and reach target sites, such as in the nervous system for recording and/or stimulation, typically areal 2D device systems are not suitable.
Here, we report organic bioelectronics produced into fiber and capillary systems. The resulting technology defines a 1D filamentous organic bioelectronics platform, which makes penetration and definition of bioelectronics inside the biological system possible, at the same time limiting any damage caused to the nervous system, tissues and organs. Long range fibers and capillaries, extending over inches, have been developed with diameters ranging from 60 to 200 microns including semiconducting and electronic/ionic conductors to define device functionality for sensors and delivery devices. Different bioelectronic devices and distributed systems have been manufactured and applied to both animal and plant tissues to enable recording and stimulation of signaling and physiology at high spatiotemporal resolution with minimal bioelectronic “side effects”.

Brain-computer interfaces and neural prostheses based on the detection of low frequency neural signal are a rapidly growing research and commercial field expected to bring new hope for patients suffering from neural disorders or loss of motor functions. Several technologies are currently competing to be the first reaching the market; however, none of them fulfil all the requirements of an ideal interface with nerve tissue neurons. Thanks to its biocompatibility, low dimensionality, mechanical flexibility and electronic properties, graphene is one of the most promising material candidates for neural interfacing.
In this contribution, we will demonstrate the integration of arrays of solution-gated graphene field-effect transistors onto flexible polyimide neural implants. The devices were placed at the surface and inside the visual and auditory cortex of rats in order to record the activity of the neural network under light and sound stimulation during acute experiments. Similarly, by placing the implants on the cornea of rats, we were able to record the induced activity of the retina under light stimulation. Before each recording, the transistors were fully characterized in-vivo using custom-designed electronics to assess their functionalities. Hence, by varying the location in the brain and the type of neural recording, we prove that our graphene-based technology offers very good performance, in particular high flexibility and good signal-to-noise ratio, for the detection of low frequency neural signals.

The human brain contains about 86 billion neurons of various types, which are the basic functional units of neural circuits that process information. The mainstay of neurophysiology research has been eavesdropping on more and more of these neurons, in order to understand the way they transform their inputs into their outputs, in other words, the computation they perform. For this task, it is crucial to develop the right probes that maximize the information we can gain from neurons while minimize the damage they may introduce.
Our overarching goal is to understand the neural mechanisms underlying cognition, focusing on learning, memory, attention and decision making. I will showcase rodent experiments to study neurons that contribute to associative learning using electrophysiological recordings and optogenetic stimulation in awake behaving transgenic mice. Next, I will demonstrate how recording techniques can be introduced to human patients to gather first hand data on the internal workings of the human brain in health and disease in order to understand how normal cognition works as well as the ways it may go awry.

Given that organ function is regulated by neurological signals, various diseases can potentially be treated via bioelectronic devices that modulate these signals. However, state-of-the-art micro-cuffs based on rigid pad, wire, and pin electrodes do not yet provide fiber type selectivity or precision below the length scale of the electrode, which is a major limitation for achieving targeted stimulation of peripheral nerves that innervate critical organs. Thus, a technique for intrafascicular, molecular-guided formation of fiber type-selective polymer electrodes could potentially enable novel bioelectronic therapies for organ health. Here, we describe the in situ deposition of conjugated polymers for direct, local interfacing with peripheral nerves from custom designed electrodes created by 3D printing. In vitro experiments using hydrogel nerve mimics and explanted peripheral nerve tissue showed that polythiophene-based polymer electrodes could be polymerized in situ by application of cuff voltages above the polymerization threshold of the monomer species. In vivo studies showed precise control of the deposition by changing the size, shape, and location of the working and counter electrodes. 3D printing was leveraged to create micro-cuffs with programmable symmetric and asymmetric pad and wire electrode configurations to explore the potential for omni-directional guidance of the in situ deposited polymer, as well as to customize the mechanical and anatomical matching of printed cuffs and small diameter nerves. The experimentally observed conjugated polymer electrode polymerization trajectories showed agreement with finite element simulations of electric field and current density distributions in the 3D printed micro-cuffs. This work shows the potential to achieve fiber type-selective electrodes for peripheral nerve stimulation via directed in situ polymerization of conducting polymers.

Micro-Electrode-Arrays (MEAs) based systems coupled to neuronal populations constitute a well-established experimental in vitro and in vivo neuro-electronic platforms to study fundamental mechanisms of brain (dys)functions and brain interfaces. 2D neuronal networks coupled to electronic devices have been widely used as a model for understanding basic neurophysiological mechanisms, for in vitro neuropharmacology and as neurotoxicity assays. Upon use of co-culturing techniques and genetic manipulation, these 2D models have been also used to investigate neural diseases via electrophysiological and optical (e.g., optogenetics) means. However, the inherent limitations of existing (in vitro and in vivo) animal models pushed the development of alternative in vitro models, able to better replicate the complex structure and function of a tissue-organ system.
Here we present an array of Organic Charge Modulated FETs (OCMFETs) called MOAs (Micro OCMFET Arrays) coupled with excitable cells for electrophysiological and metabolic monitoring. The neuro-electronic interface is validated with in vitro neuronal networks towards the development of brain-on-a-chip model systems and to understand limits of applicability in in vivo conditions. The microsystem has been fabricated on highly flexible and compliant plastic films, in order to allow the developed system to be employed for in vivo applications such as electrocorticography (ECoG) or intra-cortical brain interfaces.
The developed microtransducer arrays showed optimal biocompatibility, no need for an external reference electrode, good signal-to-noise ratio and good stability. The same OCMFET is able to measure metabolic activity (i.e., through local pH variations; quasi-static measurements) and electrophysiological activity (i.e., through induced charge variations onto the sensing area by ions displacement across the plasma membrane). Moreover, the transduction principle is addressed and compared with the sensing capabilities of microtransducers fabricated with standard MEA and CMOS technologies. Finally, engineered 3D networks, to be coupled to MOA microsystems, are presented together with future applications for the development of disposable and low-cost brain-on-a-chips (i.e., precision-personalized medicine). Comparisons with standard MEA and CMOS technologies and possible applications for in vivo neuro-electronic interfaces are presented and discussed.

Point-of-care (POC) biosensors are integrated diagnostic systems employed for the detection of clinically relevant analytes in biological fluids such as blood, urine and saliva. These devices offer the advantage to provide rapid results directly where the information is needed (e.g. patient’s home, doctor’s office or emergency room), thus facilitating an earlier diagnosis and a prompt patient’s treatment. Various technologies have been proposed for the realization of POC biosensors including label-free techniques based on optical, mechanical and electrochemical transducers. However, reliable, quantitative and ultrasensitive devices have been not yet commercialized. Electronic biosensors based on organic thin-film transistors (OTFTs) [1] are a promising choice for the development of the next generation of POC devices. These biosensors can be combined with integrated electrical circuits, microfluidic systems and wireless technologies. Furthermore, they offer high sensitivity, biocompatibility and possibility to produce all-printed low-cost biosensors in flexible and disposable formats. Among them, electrolyte-gated (EG)-OTFTs [2] have been identified as ideal candidates for biosensors development as they operate at low voltages directly in aqueous buffer solutions. Using these configurations ultrasensitive label-free immunosensors for the detection of C-reactive protein (CRP), a specific biomarker of inflammatory and infection diseases, at the femtomolar concentration level have been developed.[3] The devices are also able to perform chiral differential detection of odorant molecules. The specific features of the proposed EGOTFT biosensors as well as their analytical performances will be discussed.


References

[1] L. Torsi, M. Magliulo, K. Manoli, G. Palazzo, Chemical Society Review, 42, (2013) 8612-862.
[2] K. Manoli et al., Angewandte Chemie International Edition, 54, (2015), 12562-12576.
[3] M.Y. Mulla, E. Tuccori, M. Magliulo, G. Lattanzi, G. Palazzo, K. Persaud and L. Torsi, Nature Communications, 6, (2015), 6010.

Current activities in brain neuroscience research focus on cognitive processes, mapping of neuronal activity in ever-larger segments, in-silico simulations and experiments. Given the number of neurons and the complex neural interconnection web that exist in the human brain, it is unlikely that an artificial brain of significant size can be achieved using approaches based on semiconductor devices.

Can we actually build a section of an artificial brain that has the “look-and-feel” of the biological organ and act like it? Can an artificial brain be implanted in the body and connected to the rest of the “system”? This is clearly a grand challenge to duplicate the enormous web of functions in the most complex organ developed by nature.

We will first define and discuss main aspects that need to be demonstrated materials properties, electrical properties, energy consumption, interconnections, functionality, testability and integration. Then we introduce a possible concept for a “real artificial” brain that has some tentative answers for each set of requirements. This “semi-soft” approach it combines some elements that are organic, but not necessarily biological, with cellular components.

Our approach starts with the observation of similarities between biological neuronal arrays and polymer fibers in membranes formed by electrospinning. Typical fiber diameter range (from nm to µm) and length (from sub-mm to meters) is consistent to that of axons. The number of fiber cross-connects in a typical membrane are ~ 10¹¹-10¹²/cm³, similar to brain synapse density.

The presentation will review the work to date of several groups. At Cincinnati, we have fabricated polymer-based carbon nanofiber (CNF) electrospun membranes with electrical resistivity range of ~0.1 to 1000 W-cm. This range covers typical axon resistance of ~100 W-cm. In addition, we have fabricated core-sheath fibers by coaxial electrospinning that contain an inner conducting medium shielded by an insulating cover, similar to myelin sheath covered axons. Among other reports, Nielsen/Aarhus University has demonstrated that coaxial fiber membranes enhanced the growth of neuronal cells, indicating not just biocompatibility but ability to sustain long term viability. Jakobson and Ottosun (Lund) have demonstrated a unique electrospun fiber membrane with an uncompressed 3D structure that mimics the extracellular matrix of real brain tissue and can serve as scaffold for neuronal cell growth. Lee/Postech has demonstrated that artificial organic synapses fabricated on core-sheath electrospun nanofibers can operate with femtojoule energy consumption.

There is a whole universe of issues associated with the possible integration of an artificial brain section into a living brain in order to repair malfunctioning aspects. Nonetheless, the potential benefit of this concept is of such magnitude that this long and arduous journey is worth embarking on.

Long-term in vivo multimodal studies that combine optogenetics and electrophysiology are essential for investigating the functional connectivity of local neuronal circuits. Graphene holds unique advantages for implantable neural systems by combining properties like optical transparency, flexibility, high conductivity, and biocompatibility. Here we report a graphene-based compact closed-loop optogenetics system with the capability of simultaneous recording and processing of neural data in real time to guide selective control of neural activity through optogenetic stimulation. We report a fabrication process for high-yield large area fabrication of low impedance transparent graphene microelectrode arrays on clear flexible substrates. We extensively investigate light-induced artifacts for graphene microelectrodes in comparison to conventional metal-based microelectrodes through experiments and modeling. We develop an equivalent circuit model which successfully explains the recorded waveforms for metal electrodes under various light intensity and duration conditions. Unlike conventional metal-based microelectrodes, which suffer from huge light-induced artifacts, transparent graphene microelectrodes completely eliminate the artifact problem and thus allow the design of compact closed-loop optogenetics systems. We design and build a compact battery-powered system incorporating transparent graphene microelectrode arrays, fiber-coupled μLEDs and a custom circuit board that integrates different modules. Finally, we successfully demonstrate closed-loop operation based on electrical sensing with transparent graphene microelectrodes and optogenetic stimulation with μLEDs without any crosstalk between the two modalities. This compact system offers fast control of specific neural populations, which can significantly facilitate in vivo studies on neuronal dynamics, neural plasticity, and identification of how specific neuron type contributes to the local neural circuits.

Several psychiatric and neurological disorders - ranging from Alzheimer's and Parkinson’s diseases to schizophrenia and deep depressed mood - are attributable to alterations in the structure and function of the synapse, engendered by neurotransmitter imbalances. For this reason, a deeply understanding of the basic neurotransmission mechanism and its involvement in the pathogenesis is acquiring an ever-increasing scientific significance. The broad impact of this basically fundamental understanding is evident since it will lead to the possibility to treat neurological diseases, now representing a dramatic social and economic burdens worldwide.
In our previous investigations, we validated the ability of glassy carbon (GC) microelectrodes in detecting Dopamine release and recording neural activity in vivo in the Striatum and Caudomedial Neostriatum auditory area of European Starling songbirds. The GC microelectrode arrays were made of 4 recording-detection sites with an area of 1500 µm² and a vertical distance of 220 µm.
In this study, we are presenting an improved version of the GC microelectrode arrays with an higher spatial resolution (8 microelectrodes with 110µm vertical space) and with different recording-detection site areas ranging from 500 µm² to 2000 µm². Such device allowed to evaluate how the electrode size influences (i) the Dopamine (DA) sensing capability in terms of lower detection limit, selectivity and sensitivity (ii) the neural signal recording quality. Indeed, the electrode impedance is one of main factor that determines the signal to noise ratio as well as the spike sorting performance.
The new GC microelectrode arrays were tested in vivo in the Striatum and Caudomedial Neostriatum auditory area of European Starling songbirds for the validation of (i) detection of spontaneous and electrically stimulated DA through fast scan cyclic voltammetry and (ii) neural signal recording performance. Preliminary interpretations of the in-vivo results are reported.
This kind of evaluation leads to the identification to the optimum parameters in order to implement a multiplatform able to simultaneously record neural activity and detect neurotransmitters at the synaptic site, essential to obtain a deeper comprehension of the neurotransmission mechanisms.

We present a novel neural probe which allows not only intracortical electrical signal recording and neurotransmitter detection, but also is capable of simultaneously recording ECoG signals. The neural probe introduced here leverages the versatility of microfabrication process and our recent modular pattern transfer method to make probes that consist of a combination of glassy carbon (GC) surface and penetrating electrodes that are supported on a common polymeric substrate. This probe is hoped to offer a versatile platform that allows for the investigation of the correlation between neurotransmitter concentration, neuronal spiking activity, and also potentially ECoG signals.
For validation, both in vitro and in vivo characterizations of the devices have been performed. The electrochemical behavior of GC microelectrodes has been studied in a 0.9% NaCl aqueous solution through electrochemical impedance spectroscopy - to evaluate the magnitude and phase impedance giving insights of charge transport dynamics - and through cyclic voltammetry and power pulse technique - to quantify their capacitive charging and injection limit. The capability of the GC electrodes to detect neurotransmitters has been in vitro evaluated in PBS (Phosphate Buffer Solution) through Fast Scan Cyclic Voltammetry (FSCV), ramping the potential from -0.4V to 1.3V (Vs Ag/AgCl) and back at 400 V/s with a frequency of 10Hz. The GC electrodes were demonstrated to be capable of detecting Dopamine and Serotonin with very low detection limits and linear trends from 10 nM to 1 μM concentrations. The in vivo characterization has been accomplished by acute experiments, where the device was implanted in rats, and neural signal recordings, both simultaneous intracortical spikes activities at different locations and surface evocated potentials were documented.
In conclusion, the presented devices, giving the possibility to simultaneusly elaborate multiple informations from neural networks, offer significant advantages in a wide range of clinical applications for both the Central and Peripheral Nervous Systems.