The TD Drive: A Parametric, Open-Source Implant for Multi-Area Electrophysiological Recordings in Behaving and Sleeping Rats.

Intricate interactions between multiple brain areas underlie most functions attributed to the brain. The process of learning, as well as the formation and consolidation of memories, are two examples that rely heavily on functional connectivity across the brain. In addition, investigating hemispheric similarities and/or differences goes hand in hand with these multi-area interactions. Electrophysiological studies trying to further elucidate these complex processes thus depend on recording brain activity at multiple locations simultaneously and often in a bilateral fashion. Presented here is a 3D-printable implant for rats, named TD Drive, capable of symmetric, bilateral wire electrode recordings, currently in up to ten distributed brain areas simultaneously. The open-source design was created employing parametric design principles, allowing prospective users to easily adapt the drive design to their needs by simply adjusting high-level parameters, such as anterior-posterior and mediolateral coordinates of the recording electrode locations. The implant design was validated in n = 20 Lister Hooded rats that performed different tasks. The implant was compatible with tethered sleep recordings and open field recordings (Object Exploration) as well as wireless recording in a large maze using two different commercial recording systems and headstages. Thus, presented here is the adaptable design and assembly of a new electrophysiological implant, facilitating fast preparation and implantation.

Research progress on insect visual electrophysiological techniques.

Insect visual electrophysiological techniques are important to study the electrical characteristics of photoreceptor cells and visual neurons in insects, including electroretinography (ERG) and microelectrode intracellular recording (MIR). ERG records the changes of voltage or electric current in the retina of insects in response to different light stimuli, which occurs outside the cell. MIR records the changes in individual photoreceptor cells or visual neurons of an insect exposed to different lights, which occurs inside the cell. Insect visual electrophysiological techniques can explore the mechanism of electrophysiological response of insects' vision to light and reveal their sensitive light spectra and photoreceptor types. This review introduced the basic structure and the principle of ERG and MIR, and summarized their applications in insect researches in the past 20 years, which would provide references for elucidating the mechanism of light perception in insects and the use of insect phototropism to control pests.

Recording Cilia Activity in Ctenophores.

Pelagic ctenophores swim in the water with the help of eight rows of long fused cilia. Their entire behavioral repertoire is dependent to a large degree on coordinated cilia activity. Therefore, recording cilia beating is paramount to understanding and registering the behavioral responses and investigating its neural and hormonal control. Here, we present a simple protocol to monitor and quantify cilia activity in semi-intact ctenophore preparations (using Pleurobrachia and Bolinopsis as models), which includes a standard electrophysiological setup for intracellular recording.

Electrophysiology of Ctenophore Smooth Muscle.

Unlike in the Cnidaria, where muscle cells are coupled together into an epithelium, ctenophore muscles are single, elongated, intramesogleal structures resembling vertebrate smooth muscle. Under voltage-clamp, these fibers can be separated into different classes with different sets of membrane ion channels. The ion channel makeup is related to the muscle's anatomical position and specific function. For example, Beroe ovata radial fibers, which are responsible for maintaining the rigidity of the body wall, generate sequences of brief action potentials whereas longitudinal fibers, which are concerned with mouth opening and body flexions, often produce single longer duration action potentials.Beroe muscle contractions depend on the influx of Ca2+. During an action potential the inward current is carried by Ca2+, and the increase in intracellular Ca2+ concentration generated can be monitored in FLUO-3-loaded cells. Confocal microscopy in line scan mode shows that the Ca2+ spreads from the outer membrane into the core of the fiber and is cleared from there relatively slowly. The rise in intracellular Ca2+ is linked to an increase in a Ca2+-activated K+ conductance (KCa), which can also be elicited by iontophoretic Ca2+ injection. Near the cell membrane, Ca2+ clearance monitored using FLUO3, matches the decline in the KCa conductance. For light loads, Ca2+ is cleared rapidly, but this fast system is insufficient when Ca2+ influx is maintained. Action potential frequency may be regulated by the slowly developing KCa conductance.

Integration of in vivo electrophysiology and optogenetics in rodents with PEDOT:PSS neural electrode array.

Here, we present a protocol for the fabrication of transparent implantable electrode arrays for integrating optogenetics and electrophysiology. We describe steps for fabricating microelectrodes using the conductive polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate). We then detail procedures for analyzing performance of the electrodes and recording light-evoked neural activities from the transgenic mouse. This protocol utilizes photolithography rather than conventional electrodeposition. For complete details on the use and execution of this protocol, please refer to Cho et al. (2022).1.

Water-responsive supercontractile polymer films for bioelectronic interfaces.

Connecting different electronic devices is usually straightforward because they have paired, standardized interfaces, in which the shapes and sizes match each other perfectly. Tissue-electronics interfaces, however, cannot be standardized, because tissues are soft1-3 and have arbitrary shapes and sizes4-6. Shape-adaptive wrapping and covering around irregularly sized and shaped objects have been achieved using heat-shrink films because they can contract largely and rapidly when heated7. However, these materials are unsuitable for biological applications because they are usually much harder than tissues and contract at temperatures higher than 90 °C (refs. 8,9). Therefore, it is challenging to prepare stimuli-responsive films with large and rapid contractions for which the stimuli and mechanical properties are compatible with vulnerable tissues and electronic integration processes. Here, inspired by spider silk10-12, we designed water-responsive supercontractile polymer films composed of poly(ethylene oxide) and poly(ethylene glycol)-α-cyclodextrin inclusion complex, which are initially dry, flexible and stable under ambient conditions, contract by more than 50% of their original length within seconds (about 30% per second) after wetting and become soft (about 100 kPa) and stretchable (around 600%) hydrogel thin films thereafter. This supercontraction is attributed to the aligned microporous hierarchical structures of the films, which also facilitate electronic integration. We used this film to fabricate shape-adaptive electrode arrays that simplify the implantation procedure through supercontraction and conformally wrap around nerves, muscles and hearts of different sizes when wetted for in vivo nerve stimulation and electrophysiological signal recording. This study demonstrates that this water-responsive material can play an important part in shaping the next-generation tissue-electronics interfaces as well as broadening the biomedical application of shape-adaptive materials.

Simultaneous electrophysiology and optogenetic perturbation of the same neurons in chronically implanted animals using µLED silicon probes.

Micro-light-emitting-diode (µLED) silicon probes feature independently controllable miniature light-emitting-diodes (LEDs) embedded at several positions in each shank of a multi-shank probe, enabling temporally and spatially precise optogenetic neural circuit interrogation. Here, we present a protocol for performing causal and reproducible neural circuit manipulations in chronically implanted, freely moving animals. We describe steps for introducing optogenetic constructs, preparing and implanting a µLED probe, performing simultaneous in vivo electrophysiology with focal optogenetic perturbation, and recovering a probe following termination of an experiment. For complete details on the use and execution of this protocol, please refer to Watkins de Jong et al. (2023).1.

Validation of transparent and flexible neural implants for simultaneous electrophysiology, functional imaging, and optogenetics.

The combination of electrophysiology and neuroimaging methods allows the simultaneous measurement of electrical activity signals with calcium dynamics from single neurons to neuronal networks across distinct brain regions in vivo. While traditional electrophysiological techniques are limited by photo-induced artefacts and optical occlusion for neuroimaging, different types of transparent neural implants have been proposed to resolve these issues. However, reproducing proposed solutions is often challenging and it remains unclear which approach offers the best properties for long-term chronic multimodal recordings. We therefore created a streamlined fabrication process to produce, and directly compare, two types of transparent surface micro-electrocorticography (µECoG) implants: nano-mesh gold structures (m-µECoGs) versus a combination of solid gold interconnects and PEDOT:PSS-based electrodes (pp-µECoGs). Both implants allowed simultaneous multimodal recordings but pp-µECoGs offered the best overall electrical, electrochemical, and optical properties with negligible photo-induced artefacts to light wavelengths of interest. Showing functional chronic stability for up to four months, pp-µECoGs also allowed the simultaneous functional mapping of electrical and calcium neural signals upon visual and tactile stimuli during widefield imaging. Moreover, recordings during two-photon imaging showed no visible signal attenuation and enabled the correlation of network dynamics across brain regions to individual neurons located directly below the transparent electrical contacts.

A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice.

In vivo electrophysiology provides unparalleled insight into the sub-second-level circuit dynamics of the intact brain and represents a method of particular importance for studying mouse models of human neuropsychiatric disorders. However, such methods often require large cranial implants, which cannot be used in mice at early developmental time points. As such, virtually no studies of in vivo physiology have been performed in freely behaving infant or juvenile mice, despite the fact that a better understanding of neurological development in this critical window would likely provide unique insights into age-dependent developmental disorders such as autism or schizophrenia. Here, a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy are described that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in mice as they age from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond, a time window roughly corresponding to the human ages of 2 years old through to adulthood. The number of recording electrodes and final recording sites can be easily modified and expanded, thus allowing flexible experimental control of the in vivo monitoring of behavior- or disease-relevant brain regions across development.

Investigating Neural Correlates of Behavior Through In Vivo Electrophysiology.

Behavioral neuroscience has long relied on in vivo electrophysiology to provide spatially and temporally precise answers to complex questions about the neural dynamics underlying sensory processing and action execution. Investigating the neural correlates of behavior can be challenging in freely behaving animals, especially when making inferences related to internal states that are temporally or conceptually ambiguous, such as decision-making or motivation. This necessitates careful creation of appropriate and rigorous controls and awareness of the many potential confounds when attributing neural signals to animal behavior. This article discusses fundamental considerations for the optimal design and interpretation of in vivo rodent electrophysiological recording experiments and focuses on the different optimization strategies required when investigating neural encoding of external stimuli versus free behavior. The first protocol offers suggestions specific to intracranial surgical implantation of multielectrode arrays. The second protocol delves into optimization strategies and tips useful for designing and interpreting recording experiments conducted in freely behaving rodents. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Surgical implantation of the multielectrode array Basic Protocol 2: Optimizing experimental design and parameters.

Mosquito (Diptera: Culicidae) Vision and Associated Electrophysiological Techniques.

Mosquitoes are considered the world's deadliest animal because of the pathogens they spread. Additionally, they are an unbearable nuisance in many areas. Visual stimuli play an important role in the mosquito life cycle, helping them find vertebrate hosts, floral nectar, and oviposition sites. Here, we review mosquito vision, including its influences on mosquito behavior, the photoreceptors involved, and mosquito spectral sensitivity, as well as provide an overview of techniques used for the analysis of mosquito vision, including electroretinograms, single-cell recordings, and the use of opsin-deficient mutants. We anticipate that this information will be useful for researchers studying mosquito physiology, evolution, ecology, and management.

Solid-Supported Membrane (SSM)-Based Electrophysiology Assays Using Surface Electrogenic Event Reader Technology (SURFE²R) in Early Drug Discovery.

This article presents detailed descriptions of procedures and troubleshooting tips for solid-supported membrane (SSM)-based electrophysiology assays (SURFE²R) to measure electrogenic solute carrier transporter proteins (SLCs) and assess the effects of compounds that modulate their activity. SURFE²R allows the use of the standard 96-well format, making it an ideal platform for tertiary assays in a drug-discovery campaign. The assays are performed with cell-line-derived membrane fractions or proteoliposomes containing the transporter of interest. Three main protocols are described for the isolation of membrane fractions from cell culture and the generation of proteoliposomes containing the transporter of interest. Additionally, detailed protocols for SURFE²R single concentration and dose-response experiments are included to measure the potencies of test compounds in stimulating or inhibiting transporter function (EC50 or IC50 values, respectively) and kinetic functional assays to calculate apparent affinity (kM ) and maximal velocity (Vmax ) of substrate uptake. © 2023 Sanofi. Current Protocols published by Wiley Periodicals LLC. PROTOCOL GROUP 1: Sample preparation for SSM-based electrophysiology assays Support Protocol 1: Production of cell batches Support Protocol 2: Simple isolation of cell membranes Alternate Protocol 1: Isolation of cell membranes with sucrose gradient pre-purification Support Protocol 3: Production and isolation of liposomes Support Protocol 4: Preparation of sensor with isolated cell membranes Alternate Protocol 2: Preparation of sensor with isolated proteoliposomes PROTOCOL GROUP 2: Determination of assay parameters for SSM-based electrophysiology assay Support Protocol 5: Assay with stable buffer Alternate Protocol 3: Assay with ion gradient Support Protocol 6: Determination of membrane/liposome concentration Support Protocol 7: Determination of substrate dependency kM PROTOCOL GROUP 3: Determination of advanced assay parameters for SSM-based electrophysiology assays Support Protocol 8: Assessment of ion concentration dependency Support Protocol 9: Assessment of pH dependency Support Protocol 10: Assessment of DMSO dependency Support Protocol 11: Assessment of signal stability with multiple activations PROTOCOL GROUP 4: Compound testing through SSM-based electrophysiology assays using SURFE²R apparatus Support Protocol 12: Assessment of signal specificity of a published inhibitor or unknown compound(s) Support Protocol 13: Compound wash-out Support Protocol 14: Statistical analysis.

A mesh microelectrode array for non-invasive electrophysiology within neural organoids.

Organoids are emerging in vitro models of human physiology. Neural models require the evaluation of functional activity of single cells and networks, which is commonly measured by microelectrode arrays. The characteristics of organoids clash with existing in vitro or in vivo microelectrode arrays. With inspiration from implantable mesh electronics and growth of organoids on polymer scaffolds, we fabricated suspended hammock-like mesh microelectrode arrays for neural organoids. We have demonstrated the growth of organoids enveloping these meshes and the culture of organoids on meshes for up to one year. Furthermore, we present proof-of-principle recordings of spontaneous electrical activity across the volume of an organoid. Our concept enables a new class of microelectrode arrays for in vitro models of three-dimensional electrically active tissue.

Experimental Verification for Numerical Simulation of Thalamic Stimulation-Evoked Calcium-Sensitive Fluorescence and Electrophysiology with Self-Assembled Multifunctional Optrode.

Owing to its capacity to eliminate a long-standing methodological limitation, fiber photometry can assist research gaining novel insight into neural systems. Fiber photometry can reveal artifact-free neural activity under deep brain stimulation (DBS). Although evoking neural potential with DBS is an effective method for mediating neural activity and neural function, the relationship between DBS-evoked neural Ca2+ change and DBS-evoked neural electrophysiology remains unknown. Therefore, in this study, a self-assembled optrode was demonstrated as a DBS stimulator and an optical biosensor capable of concurrently recording Ca2+ fluorescence and electrophysiological signals. Before the in vivo experiment, the volume of tissue activated (VTA) was estimated, and the simulated Ca2+ signals were presented using Monte Carlo (MC) simulation to approach the realistic in vivo environment. When VTA and the simulated Ca2+ signals were combined, the distribution of simulated Ca2+ fluorescence signals matched the VTA region. In addition, the in vivo experiment revealed a correlation between the local field potential (LFP) and the Ca2+ fluorescence signal in the evoked region, revealing the relationship between electrophysiology and the performance of neural Ca2+ concentration behavior. Concurrent with the VTA volume, simulated Ca2+ intensity, and the in vivo experiment, these data suggested that the behavior of neural electrophysiology was consistent with the phenomenon of Ca2+ influx to neurons.

Patch Clamp: The First Four Decades of a Technique That Revolutionized Electrophysiology and Beyond.

Forty years ago, the introduction of a new electrophysiological technique, the patch clamp, revolutionized the fields of Cellular Physiology and Biophysics, providing for the first time the possibility of describing the behavior of a single protein, an ion-permeable channel of the cell plasma membrane, in its physiological environment. The new approach was actually much more potent and versatile than initially envisaged, and it has evolved into several different modalities that have radically changed our knowledge of how cells (not only the classical "electrically excitable "ones, such as nerves and muscles) use electrical signaling to modulate and organize their activity. This review aims at telling the history of the background from which the new technique evolved and at analyzing some of its more recent developments.

Robotic multi-probe single-actuator inchworm neural microdrive.

A wide range of techniques in neuroscience involve placing individual probes at precise locations in the brain. However, large-scale measurement and manipulation of the brain using such methods have been severely limited by the inability to miniaturize systems for probe positioning. Here, we present a fundamentally new, remote-controlled micropositioning approach composed of novel phase-change material-filled resistive heater micro-grippers arranged in an inchworm motor configuration. The microscopic dimensions, stability, gentle gripping action, individual electronic control, and high packing density of the grippers allow micrometer-precision independent positioning of many arbitrarily shaped probes using a single piezo actuator. This multi-probe single-actuator design significantly reduces the size and weight and allows for potential automation of microdrives. We demonstrate accurate placement of multiple electrodes into the rat hippocampus in vivo in acute and chronic preparations. Our robotic microdrive technology should therefore enable the scaling up of many types of multi-probe applications in neuroscience and other fields.

Studying Synaptic Connectivity and Strength with Optogenetics and Patch-Clamp Electrophysiology.

Over the last two decades the combination of brain slice patch clamp electrophysiology with optogenetic stimulation has proven to be a powerful approach to analyze the architecture of neural circuits and (experience-dependent) synaptic plasticity in such networks. Using this combination of methods, originally termed channelrhodopsin-assisted circuit mapping (CRACM), a multitude of measures of synaptic functioning can be taken. The current review discusses their rationale, current applications in the field, and their associated caveats. Specifically, the review addresses: (1) How to assess the presence of synaptic connections, both in terms of ionotropic versus metabotropic receptor signaling, and in terms of mono- versus polysynaptic connectivity. (2) How to acquire and interpret measures for synaptic strength and function, like AMPAR/NMDAR, AMPAR rectification, paired-pulse ratio (PPR), coefficient of variance and input-specific quantal sizes. We also address how synaptic modulation by G protein-coupled receptors can be studied with pharmacological approaches and advanced technology. (3) Finally, we elaborate on advances on the use of dual color optogenetics in concurrent investigation of multiple synaptic pathways. Overall, with this review we seek to provide practical insights into the methods used to study neural circuits and synapses, by combining optogenetics and patch-clamp electrophysiology.

Liquid crystal electro-optical transducers for electrophysiology sensing applications.

Objective.Biomedical instrumentation and clinical systems for electrophysiology rely on electrodes and wires for sensing and transmission of bioelectric signals. However, this electronic approach constrains bandwidth, signal conditioning circuit designs, and the number of channels in invasive or miniature devices. This paper demonstrates an alternative approach using light to sense and transmit the electrophysiological signals.Approach.We develop a sensing, passive, fluorophore-free optrode based on the birefringence property of liquid crystals (LCs) operating at the microscale.Main results.We show that these optrodes can have the appropriate linearity (µ± s.d.: 99.4 ± 0.5%,n = 11 devices), relative responsivity (µ± s.d.: 57 ± 12%V-1,n = 5 devices), and bandwidth (µ± s.d.: 11.1 ± 0.7 kHz,n = 7 devices) for transducing electrophysiology signals into the optical domain. We report capture of rabbit cardiac sinoatrial electrograms and stimulus-evoked compound action potentials from the rabbit sciatic nerve. We also demonstrate miniaturisation potential by fabricating multi-optrode arrays, by developing a process that automatically matches each transducer element area with that of its corresponding biological interface.Significance.Our method of employing LCs to convert bioelectric signals into the optical domain will pave the way for the deployment of high-bandwidth optical telecommunications techniques in ultra-miniature clinical diagnostic and research laboratory neural and cardiac interfaces.

An improved platform for cultured neuronal network electrophysiology: multichannel optogenetics integrated with MEAs.

Cultured neuronal networks (CNNs) are powerful tools for studying how neuronal representation and adaptation emerge in networks of controlled populations of neurons. To ensure the interaction of a CNN and an artificial setting, reliable operation in both open and closed loops should be provided. In this study, we integrated optogenetic stimulation with microelectrode array (MEA) recordings using a digital micromirror device and developed an improved research tool with a 64-channel interface for neuronal network control and data acquisition. We determined the ideal stimulation parameters including light intensity, frequency, and duty cycle for our configuration. This resulted in robust and reproducible neuronal responses. We also demonstrated both open and closed loop configurations in the new platform involving multiple bidirectional channels. Unlike previous approaches that combined optogenetic stimulation and MEA recordings, we did not use binary grid patterns, but assigned an adjustable-size, non-binary optical spot to each electrode. This approach allowed simultaneous use of multiple input-output channels and facilitated adaptation of the stimulation parameters. Hence, we advanced a 64-channel interface in that each channel can be controlled individually in both directions simultaneously without any interference or interrupts. The presented setup meets the requirements of research in neuronal plasticity, network encoding and representation, closed-loop control of firing rate and synchronization. Researchers who develop closed-loop control techniques and adaptive stimulation strategies for network activity will benefit much from this novel setup.

Nanoneedle-Electrode Devices for In Vivo Recording of Extracellular Action Potentials.

Microscale needle-like electrode technologies offer in vivo extracellular recording with a high spatiotemporal resolution. Further miniaturization of needles to nanoscale minimizes tissue injuries; however, a reduced electrode area increases electrical impedance that degrades the quality of neuronal signal recording. We overcome this limitation by fabricating a 300 nm tip diameter and 200 µm long needle electrode where the amplitude gain with a high-impedance electrode (>15 MΩ, 1 kHz) was improved from 0.54 (-5.4 dB) to 0.89 (-1.0 dB) by stacking it on an amplifier module of source follower. The nanoelectrode provided the recording of both local field potential (<300 Hz) and action potential (>500 Hz) in the mouse cortex, in contrast to the electrode without the amplifier. These results suggest that microelectrodes can be further minimized by the proposed amplifier configuration for low-invasive recording and electrophysiological studies in submicron areas in tissues, such as dendrites and axons.