Development of a Versatile System for Advanced Neuronal Recordings with Multisite Microelectrodes

Deliverables

During the research project we studied the system physiology of the limbic cortex in normal conditions and in a model of neurological disease. The collected data helped us to characterize the network mechanisms in the hippocampus/parahippocampal region, a brain area that plays a crucial role in memory and is precociously damaged in dementia and in temporal lobe epilepsy. We demonstrated that the recording system developed by VSAMUEL, comprehensive of silicon probes, connectors, 64 channel amplifier, data acquisition hardware/software and analysis software can be successfully utilized to record brain activity from the isolated guinea-pig brain maintained in vitro by arterial perfusion. The prevalent activity of our group was aimed at validating the silicon probes manufactured by ACREO. We demonstrated that silicon probes that perform 32 recording sites arranged with different configurations can be utilized (and re-utilized) during acute experiments aimed at recording field potentials and single cell activities in the in vitro guinea-pig brain preparation, in particular in the limbic cortex during normal physiological conditions and during epileptogenesis. We published the results of the study in several scientific publications on peer-reviewed Neuroscience Journals (see list below). The use of the VSAMUEL system allows to record activity from different portions of the brain with a peculiar and original arrangement of the recording sites. Our findings support the use of VSAMUEL recording system in Neuroscience research activities that involve multi-site recordings in different experimental in vitro and in vivo preparations. The potential use of VASMUEL recording system in humans, for instance during pre-surgical studies in patients suffering of pharmacoresistant epilepsies or to perform corticographic recording during brain surgery, could be further investigated.

A new micromachining process for batch fabrication of silicon neural probes has been developed and demonstrated in the design and manufacture of probes with 32- and 64-site recording electrode arrays. It is believed that a key to the understanding of the neural system is to make simultaneous observations of the activity of a large number of cells. Probes that can penetrate neural tissue and insert a large number of recording sites, while tissue damage is kept to a minimum, are thus needed, and microsystem technology (MST) is well suited to this end. In comparison, conventional wire electrode technology is limited to fewer recording sites in a small volume of tissue, has less exact relative positioning and/or will cause more damage to the neural tissue. Although micro-machined neural probes have been shown previously, the most commonly used process is based on a combination of wet silicon etching and a p++ diffused etch stop to create fork-like probe shapes. In contrast, we have developed an all-dry silicon-etch based process where the buried oxide layer of a silicon-on-insulator (SOI) substrate acts as an etch stop for a double-sided deep reactive ion etch (DRIE). We believe that this process has several attractive features when considering process related design limitations, process complexity, uniformity and manufacturability. Double-sided lithography and etching allow a thicker base plate to be part of the design, which facilitates the handling of the otherwise very thin probes after processing. The use of step-and-repeat projection lithography enabled structures with down to 1 um line-width. This gives a better trade-off between electrode-count and tissue damage than most other probes available today. The probe chips are mounted on custom designed flexible printed circuits (FPC). Electrical interconnects are made with ultrasonic wire bonding. The chips and bond wires are protected with an epoxy glob top. The back end of the FPC is designed to mate with a zero insertion force (ZIF) connector. Measurement and testing contribute a significant part to the cost of microsystems. Automated measurement set-ups are difficult to arrange for microsensors, which must interact with the environment during the test. In this project we have also demonstrated a novel 3-point impedance measurement procedure for microelectrodes. The concept is based on the use of a saline gel and enables relatively fast measurement at wafer level. a) Basic neuroscience research: Recordings and correlation analysis of multiple signals are made on animal models (e.g. rat, mouse, and guinea pig) to improve basic knowledge of the function of the brain and neural disorders. This application area also includes research on computer algorithms modelled on the function of the brain. A market estimate, based on the number of relevant presentations during the Society for Neurosciences Annual Meetings or the number of its relevant members, leads to the conclusion, that there are 2000 laboratories in North-America engaged in electrophysiological research and therefore potentially interested in acquiring a multi-site recording system. There is today no known commercial competitor producing this type of fork-like neural probes, although academic groups have provided U.S. neuroscientists with probes on a non-commercial basis. b) Pharmaceutical research and drug development: Part of the development cost of a new drug is spent in order to assess the activity of the new drugs in models. However, in particular neurologic active drugs for humans should not be tested just in cell cultures. Due to the unpredictable influence of the drug on higher-level neuronal networks, they have to be tested electrophysiologically on animals. Being able to perform several dozens instead of a handful of recordings at the same time with the same animal and perfect control of the recording sites to each other would mean a significant improvement in testing performance. Savings on the number of lab animals could be realised, not to mention the invaluable data on inter-neuron correlation. An estimate of this market segment leads to at least double the size of the basic research segment. c) Medical treatment, e.g. targeting of brain structures during brain surgery: d) Interfaces for control of prostheses (brain-machine interfaces, BMIs: This two last application areas may require new probe materials.

Potential Applications Scientific use of biosensors in the form of silicone probes with multi-site electrode arrays in research animals for studies of physiological and patho-physiological mechanisms in the nervous system. The end users of this technique are scientific researchers investigating the function of electrical signalling within the cerebellar cortex and similar neural structures. The market consists of scientific laboratories for basic research and the pharmaceutical industry. Technical Results The aim of the research project used to test and evaluate the VSAMUEL probes for use in the cerebellar cortex in vivo is to perform multiple single unit activity recordings from identified neurons. The collected data has provided increased knowledge on the dynamical activity of neuronal networks during rest and during processing of tactile information from the facial skin and whiskers. This data has also been used to constrain and improve models of functional networks in the cerebellar cortex and to validate computer simulations of cerebellar activity. The VSAMUEL silicone probes were evaluated by mechanical insertion tests in the cerebellar cortex of rat. Probe design parameters such as tip configuration, tip profile, tip strength, and insertion technique and speed were evaluated in terms of damage to the tissue, damage to the probe, mechanical stability of implantation, and recording characteristics. We demonstrated that the probes could be successfully inserted into the cerebellar cortex. Data from these studies were used to improve the probe design further. Using the recording system developed by VSAMUEL or a composite system of the VSAMUEL preamplifier and other equipment we showed that the VSAMUEL probes could utilized (and re-utilized) in acute experiments to record field potentials, multi-unit spike activity, and single-unit spike activity from identified cell types in the cerebellar cortex of the rat. The results were published at scientific conferences and in peer-reviewed neuroscience journals. The main advantage of VSAMUEL probes over other available multi-site electrode arrays is the large number of recording sites in small volume, and the possibility of arranging the layout of recording sites in such way that multiple units can be recorded along several parallel electrode tracks simultaneously. This will allow for collection of activity data from several layers from within functional areas/units of layered neural structures (e.g. cerebral cortex, cerebellum, superior colliculus). Critical Success Factors: The VSAMUEL silicone probes are the state-of-the-art in terms of miniaturized multi-site electrode arrays available to scientist for electrophysiological recordings in research animals. Many important issues in biomedical research relevant to human health and disease depend on experiments where a large number of individual nearby nerve cells can be recorded simultaneously. A functional multi-site electrode with sufficient sensitivity and selectivity would provide tremendous benefits for such experiments and attract great interest from scientists and pharmaceutical companies. Its price/performance ratio is better than other multi-site probes and it has a great commercial potential.

Technical results: In order to interface the small probe chips with the comparatively large pre-amplifier system, an interconnect and packaging scheme had to be developed. Flexible printed circuit (FPC) technology was identified as an attractive solution, allowing for flexible and precise positioning of the recording sites in the neural tissue using micromanipulators. The FPCs were developed in cooperation with HP Etch AB (Järfälla, Sweden). The front end of the FPCs is designed to fit the base plate and the bond pad positions of the silicon probe chips. The back end is tailored to mate with the preamplifiers zero insertion force (ZIF) connector (Molex 52559). The front end is stiffened in order to have a solid support for the probe chip and to be able to connect the FPC to a micromanipulator. The probe chips are glued in place and electrically connected with ultrasonically wire-bonded Au wires. The contacts, and wire bonds are isolated and mechanically protected with an epoxy glob top cover. The FPC interconnects were manufactured in a 32-channel version and a 64-channel version. The 32-channel version has the form of a single straight ribbon (~ 125 mm x 9 mm). The 64-channel version is V-shaped with two legs with 32 channels each. Application areas, end users, and markets. See "Silicon probes for 32 and 64 recording sites 6788"

A 128-channel, very low-noise pre-amplifier system for neurophysiological research applications was developed. The end users of this newly developed system are neuroscientists investigating the nervous system. As an initial step, the interface between the multi-channel silicon probes (Acreo) and the preamplifier input has been defined by a closed cooperation between the project partners Acreo and Thomas RECORDING. In order to obtain effective results, different parameters had to be checked (e.g. connectors structural shape). Finally we agreed to use a zero insertion force (ZIF) flexible printed circuit connector (FPC) for the interface between silicone probes and pre-amplifier system. The 128-channel pre-amplifier system was adapted to the mechanical and electro-technical requirements of the silicone probes. These electrodes will be used to extract the electrical signals from nervous system (e.g. brain, peripheral nerve). At the signal output of the probes we are confronted with small amplitude signals in the micro volt range that have to be amplified with a low-noise pre-amplifier system to get a really good signal-to noise ratio. Therefore it was urgently required to use extremely low noise electronic components. Simultaneously it was required to design a miniaturized system, because usually the space in a recording setup is limited. Based on our philosophy to keep our amplifiers compact, reliable, and better than state-of-the-art the main goal was to optimize the packing density of the electronic parts with a simultaneous reduction of inter-channel cross-talk. To meet all these design requirements we decided to realize the pre-amplifier system with a printed circuit design using surface mounted devices (SMD) technology. The result was a miniaturized, very low noise multi-channel preamplifier with negligible cross-talk specifications. Furthermore we do offer custom-based connections between various microwire-/silicone-electrodes and our pre-amplifier system. The pre-amplifier is a unique amplifier system on the market. In contrast to current pre-amplifier solutions it provides: 1) custom-made multi-channel probe interface. This interface can be easily adapted different multi-channel probes currently available on the market. 2) broad bandwidth design for simultaneous LFP and SUA recordings, 3) Small size, 4) The use of high quality electronic components and the ISO 9001 quality management controlled product development and manufacture guarantees a high product quality and reliability. 5) Based of the integrated design a product maintenance is not required. Therefore this design is superior to current multi-channel amplifiers available on the market. There is no other completely integrated 128 channel recording system available at the moment. Currently neuroscientists have to combine products from different manufacturers to get a multi-channel recording system. The result of this R&D project is a unique high tech product that will offer the possibility to the neuroscience community to get a 128 channel recording system from one hand. The integrated philosophy of the 128 channel recording system avoids interface problems between the different system components (e.g. probes, preamplifier, main amplifier, data acquisition system, software, analysis tools, and so on) as often noticed in combinations of different systems from different manufacturers. Beside marketing this product within a relatively large neuroscience community we plan to offer the 128 channel amplifier and data acquisition system also to other research or industrial areas where it is urgently required to acquire data simultaneously with high sample rates on as much as possible recording channels. This usually is required in test systems for example in the car industry, where data from many sensors have to be acquired. Designed for in vivo multi-channel recording, our multi-channel pre-amplifier in combination with programmable gain main amplifier (PGMA), together with silicone probes and the data acquisition system, offers unsurpassed performance on up to 128 channels at a sampling rate of 50 ks/channel with an amplitude resolution of 16bit. The modularity of the system makes it easily possible to adapt the system to the individual requirements of the researcher. With the current flexibility and performance the system is the worlds first fully integrated 128 channel recording system for neurophysiological applications.

Potential Applications a) Chronic implantation of biosensors in the form of silicone probes with multi-site electrode arrays in research animals for studies of physiological and patho-physiological mechanisms in the nervous system, heart, and skeletal muscle. The end users of this technique are scientific researchers investigating the function of electrical signalling within these systems. The market consists of several thousands of scientific laboratories for basic research and the pharmaceutical industry; b) Chronic implantation of biosensors in the form of silicone probes with multi-site electrode arrays in humans for studies of physiological and patho-physiological mechanisms in the nervous system, heart, and skeletal muscle. The end users are medical doctors using this technique as a diagnostic tool and scientific researchers investigating the function of electrical signalling within these systems. The market consists of hospital clinics within these fields, several thousands of scientific laboratories for basic and clinical research and the pharmaceutical industry. Technical Results: Mechanical insertion tests of silicone probes were performed in the peripheral nerve of rabbit and the cerebellar cortex of rat. Probe design parameters such as tip configuration, tip profile, tip strength, and insertion technique and speed were evaluated in terms of damage to the tissue, damage to the probe, mechanical stability of implantation, and recording characteristics. It was demonstrated that the probes could be successfully inserted into the cerebellar cortex in acute experiments, but that available cables and contacts connecting the probes to recording instruments needed modifications in order to be suitable for chronic implantation in the rat cerebellum. A design study of alternative solutions for connecting silicone probes chronically implanted into the brains of research animals were performed. Based on parameters such as optimal design, the available expertise within the VSAMUEL consortium, available resources, and time to design and produce a prototype, a cable based on a shortened version of the same flexible printed circuit (FPC) technology used for acute recordings was selected for further consideration. This short "chronic" FPC has more available channels for other sensors, and for reference and ground electrodes. It is designed to interface with an 80-channel Hiroshi contact, which is connected to its mate on a small printed circuit board attached to the animal during chronic recordings. A prototype version based on the acute probe was made available to research groups outside the consortium and used successfully in chronic recordings in the hippocampus of rats. Critical Success Factors The VSAMUEL silicone probes are the state-of-the-art in terms of miniaturized multi-site electrode arrays available to scientist for electrophysiological recordings in research animals. Many important issues in biomedical research relevant to human health and disease depend on experiments on freely moving and behaving animals during long periods of time, i.e. chronic recordings. A functional chronic multi-site electrode would provide tremendous benefits for such experiments and attract great interest from scientists and pharmaceutical companies. Its price/performance ratio is better than other multi-site probes and it has a great commercial potential. If the probes could be shown suitable for clinical use in humans the market would increase even further. Potential barriers are linked to the limited experience of using the probes in chronic experiments and untested connector prototypes, which may prove unsuitable.

Micro system technology is well suited to batch-fabricate neural probes with multiple electrodes, intended for recording or stimulation in neural tissue. However, a fundamental problem is that the desired spatial electrode distributions often differ between different neuroscience experiments. Up till now, silicon neural probes have been produced using fixed lithographic mask sets, which is straightforward but inflexible and costly with respect to redesigns for small production volumes. Neural probes that are available today are hence designed according to specifications from specific scientific user groups, or offered as fixed standard designs. A commercial setting, on the other hand, prohibits custom made designs for individual researchers due to the associated costs and lead times. We have developed and demonstrated a method to vary the recording site distribution on neural probes with a mask-less finishing process. The concept is based on the use of direct write laser lithography (DWL) in one mask layer, thus enabling on-demand processing of wafers with semi-custom designs at a reasonable cost and lead time. We use the DWL to define windows in the top isolation layer of the device, thus selecting which electrodes, out of a standardised electrode array, should be active. In addition the active electrode area can be varied. The concept was evaluated using a 64-site neural probe design and manufacturing process. Impedance characterisation was made on active and inactive electrodes and on electrodes with varying active area. The results show ~15 times lower impedance for active compared to inactive electrodes at 1 kHz, which is considered sufficient for signal discrimination. Application areas, end users and markets See "Silicon probes for 32 and 64 recording sites 6788".

A 128-channel, very low-noise main amplifier system for neurophysiological research applications was developed. The end users of this newly developed system are neuroscientists investigating the nervous system. A 128 channel programmable gain main amplifier was developed to amplify and filter the preamplifier output signal and to be interfaced to the 128 channel data acquisition system. The 128 channel main amplifier is a microprocessor controlled programmable gain main amplifier (PGMA) system. Within this project we have developed a new microcontroller board based on the microcontroller ST-10F168 from SGS Thomson. This microcontroller board controls the gain settings of each of the 128 amplifier channels and communicates with the host personal computer via standard serial port (RS-232). This serial communication is used to set the amplifier gain via graphical user interface (GUI) of the data acquisition software. Beside this serial communication hardware we have integrated a user interface in the front panel of the programmable gain main amplifier (PGMA) to offer an additional possibility to for amplifier gain selection on all 128 channel. The use of modern real-time digital signal processing eliminates the need to make the filter parameters of the PGMA adjustable. This surely is a shift from today's practice. It can happen, that a scientist is interested in measuring slow local field potentials (LFP) and in a slightly different experiment on the same specimen, be focused on high-frequency spikes (single-unit activity, SUA). In both cases one will work with the same high-quality broadband PGMA, and digitally filter out the interesting frequency ranges with wavelet transform algorithm of the newly developed data acquisition system (ISIP). The user can even obtain both frequency bands simultaneously in a single measurement. Changing the types and characteristics of the digital filters can be done without danger of distorting or even deteriorating the signals very comfortable via the GUI of the data acquisition software. Thus the PGMA is designed to do this job extremely well. The PGMA amplifies the signals and restricts them to a sharply defined frequency band. To meet the requirements for multi-channel neurophysiological recordings the PGMA was designed to have a broad recording bandwidth of 0.06Hz15kHz, a low-noise amplification and modular amplifier modules. This approach helps the user to keep the experimental setup easy, reliable, and reproducible. For common-mode signal rejection the PGMA is equipped with the differential input on each of the 128 channels. Furthermore it offers a wide range of gains. The PGMA is a unique amplifier system on the market. Additionally to current amplifier solutions it provides: 1) gain setting possibilities via software GUI and hardware user interface, 2) broad bandwidth design for simultaneous LFP and SUA recordings, 3) Small size, 4) The use of high quality electronic components and the ISO 9001 quality management controlled product development and manufacture guarantees a high product quality and reliability. 5) Based of the integrated design a product maintenance is not required. Therefore this design is superior to current multi-channel amplifiers available on the market. There is no other completely integrated 128 channel recording system available at the moment. Currently neuroscientists have to combine products from different manufacturers to get a multi-channel recording system. The result of this R&D project is a unique high tech product that will offer the possibility to the neuroscience community to get a 128 channel recording system from one hand. The integrated philosophy of the 128 channel recording system avoids interface problems between the different system components (e.g. probes, preamplifier, main amplifier, data acquisition system, software, analysis tools, and so on) as often noticed in combinations of different systems from different manufacturers. Beside marketing this product within a relatively large neuroscience community we plan to offer the 128 channel amplifier and data acquisition system also to other research or industrial areas where it is urgently required to acquire data simultaneously with high sample rates on as much as possible recording channels. This usually is required in test systems for example in the car industry, where data from many sensors have to be acquired.

Up till now, silicon neural probes have been produced using fixed lithographic mask sets, which is straightforward but inflexible and costly with respect to redesigns for small production volumes. We demonstrated a method to vary the recording site distribution on neural probes with a mask-less finishing process. The concept is based on the use of direct write laser lithography (DWL) in one mask layer, thus enabling on-demand processing of wafers with semi-custom designs at a reasonable cost and lead time. We use the DWL to define windows in the top isolation layer of the device, thus selecting which electrodes, out of a standardised electrode array, should be active. In addition the active electrode area can be varied. The concept was evaluated using a 64-site neural probe design and manufacturing process. Impedance characterisation is made on active and inactive electrodes and on electrodes with varying active area. The results show ~15 times lower impedance for active compared to inactive electrodes at 1 kHz, which is considered sufficient for signal discrimination.

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