Combined in vivo recording of neural signals and iontophoretic injection of pathway tracers using a hollow silicon microelectrode

Highlights

• Spatially controlled neuronal labeling is performed with a hollow silicon microelectrode.

• Combination of iontophoretic injection with electrophysiology using a single micromachined device is demonstrated in acute animal experiment.

• Mapping the projectome of locally labeled neuronal population is demonstrated as possible application of microiontophoresis function of a silicon neural probe.

Abstract

This paper presents the results of in vivo local release of a neuronal tracer, biotinylated dextran amine (BDA) in the rat somatosensory cortex using monolithically integrated microfluidic channel of a silicon neural microelectrode. The tracer injection is controlled by iontophoresis using Pt electrodes in the vicinity of the outlet of the microfluidic channel. Using 3–5 μA, 5–7 s on/off cycle and 15–20 min total injection time the localized injection resulted in clear anterograde and retrograde BDA labeling both within the cortex and in subcortical structures. Anterograde and retrograde labeling revealed the fine details of neuronal processes including dendritic spines and axon terminal-like endings. Injection sites appeared clear lacking any strong diffuse background labeling. Electrophysiological recording performed with the same microdevice immediately after the iontophoresis indicated normal cortical functioning. The results prove that the combination of in vivo multichannel neural recording and controlled tracer injection using a single implanted microdevice is feasible, and therefore it can be a powerful tool for studying the connectome of the brain.

Introduction

Thorough investigation of the brain’s wiring diagram is essential for understanding neural information processing at both local and global scales [1]. Exploring the structure and function of the brain’s connectome is in fact one of the major scope of recent neuroscience research. However, new methods and technologies need to be developed to bridge brain structure and function [2]. Complementing neuronal recording with pathway tracing is still an indispensable tool to achieve such goals specifically at the microcircuit (single neurons) and mesoscale (neuronal populations) levels of the neural network. However, electrophysiology and neuronal tract tracing are usually applied separately even if combined in the same experiment. Typically if recording of neural activity is also needed, an additional electrode is positioned into the investigated brain area close to the micropipette or glass capillary used for neuronal labeling [3]. Similarly, the sequential application of electrode recording and tracer injection within the same experiment has several shortcomings including precision issues (targeting the same tissue volume), increased tissue damage by multiple penetrations, long preparation of electrode configurations. There are several drawbacks of such an approach including the precise alignment of the different electrodes/syringes, crowded instrumentation and the invasive manner of the surgery. Therefore a device integrating both microelectric recording and tract tracing capabilities is highly demanded to enhance efficiency and precision of such a multidisciplinary approach. Also, using integrated devices simplifies the experimental procedures and would perhaps increase the popularity of experiments combining physiological and anatomical approaches. Such progress could ultimately provide significant contribution in understanding the connectome [4].

In vivo intra- and juxtacellular recordings are usually made by electrodes filled with substances as e.g., biocytin, allowing the subsequent anatomical reconstruction of the neurons. These are important tools in exploring the neuronal microcircuitry. However, recent development in the field of multielectrode recording (MUR) provides new opportunities in examining mesoscale, population level functional and structural (spatial patterns) phenomena. Also, understanding the neural basis of MUR related phenomena requires knowledge about the underlying neuronal connectivity. Anterograde and retrograde tracing techniques are fundamental means of connectional neuroanatomy [5]. There are numerous substances used for tracing with varying chemical properties [6]. Biotinylated dextran amine (BDA) is one of the most popular tracers because of its sensitivity and the ease of use for different purposes including fluorescence and electron microscopy. BDA is widely used for labeling the origin as well as the termination of neural connections [4]. High molecular weight BDA (<10 kDa) yields higher anterograde sensitivity and provides detailed labeling of axons and terminals (target regions), while low molecular weight BDA (>3 kDa) yields higher sensitivity and provides detailed retrograde labeling of neuronal cell bodies (origin of pathways) [4]. However, it should be noted that in some amount BDAs of different molecular weights are transported in both directions and that the preference of direction can be controlled for some extent with the experimental procedures applied (method of injection, combined neurochemical lesion with NMDA, survival time etc.)[7].

BDA has been used to trace connections both within the cerebral cortex and between cortical and subcortical structures of different species. BDA labeling revealed projections from numerous regions in the CNS, e.g., from the cortex to the substantia nigra [8], from the brainstem [9], medulla [10], thalamus [7] etc. The two point source diffusion based real time techniques for drug delivery are iontophoresis and pressure ejection, both from a micropipette [11]. BDA can be applied both by pressure and iontophoresis. Iontophoresis is a fundamental technique to eject charged molecules including neuronal tracers in the brain tissue through establishing voltage gradient between the tracer solution suspended in a micropipette and the extracellular space. The advantage of iontophoresis over pressure injection is that ions and not the solution can be released in a modest amount by controlling the iontophoretic current [11]. The polarity of the applied current depends on the charge of the substance to be injected by iontophoresis. If there are no further charged molecules in the solution, then the amount of tracer deposited in the tissue is proportional to the total charge delivered by the current source. This implies that the electric current and injection time are the relevant parameters to be precisely controlled in an experiment. Mostly, pulsed direct current is used to prevent impedance build-up in micropipettes. One of the electrodes is a fine gauge wire which is inserted in the micropipette shaft. For this purpose, gold wire, silver wire, chloridized silver wire or stainless steel wire have been used. The other (reference) electrode is usually connected to some arbitrary site of the animal body, mostly outside of the brain tissue, providing a practically homogenous voltage gradient throughout the corpus of the animal.

In recent years, neural microelectrodes of several functionality have been developed and demonstrated [12], [13], [14]. For convection enhanced drug delivery purposes, a variety of microdevices has been successfully tested in vivo [15], [16], [17], [18], [19]. In our work, we propose an in vivo method of using neural microelectrode with monolithically integrated microfluidic channel to control tracer delivery in the close vicinity of the recording sites by iontophoresis. Additionally, we also prove that recording functionality is still facilitates the monitoring of local field potentials, single and multiple unit activity. Detailed description of the device fabrication and application for convection enhanced delivery of drugs through the blood brain barrier can be found in our previous work [15].