Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

Abstract

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 µm away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

Rapid volume pulsation of the extracellular space coincides with epileptiform activity in mice and depends on the NBCe1 transporter

“…We performed the RTI technique in a standard manner (Odackal et al 2017) on the electrophysiology recording setup used for in vitro PTQ experiments. In short, TMA + was released from an iontophoretic microelectrode and detected by a TMA-sensitive ISM positioned about 100 μm away.…”

“…A bias current of +20 nA was continuously applied to the iontophoretic microelectrode. Records with duration of 150 s were taken per measurement, during which a step current ranging from +30 nA to +100 nA was applied for a period of 50 s. The recorded signal was analysed using custom MATLAB-based software, WALTER (Odackal et al 2017), in order to extract the transport number (n t ) of the given iontophoretic microelectrode. Afterwards, a brain slice was placed into the submersion tissue chamber, and the ISM and iontophoretic microelectrodes were placed 200 μm deep into the visual cortex of the mouse brain slice 98−122 μm apart.…”

“…Double-barrelled ISMs were fabricated according to a standard protocol (Odackal et al 2017). All ISMs were made from double-barrelled theta glass (TG200-4; Warner Instruments Corp., Hamden, CT, USA).…”

“…The signals were routed to a Cyberamp 320 signal conditioner (Axon Instruments Inc.) that provided an additional 10× gain and a 2 Hz low-pass 8-pole Bessel filter. The signals were digitized with an analogue-to-digital converter (model USB-6221 M Series DAQ Device, BNC; National Instruments, Austin, TX, USA) and acquired using the custom program WANDA (Odackal et al 2017) written in MATLAB (MathWorks, Natick, MA, USA, RRID:SCR_001622).…”

“…We found that NBCe1 blockade inhibited both RVP and epileptiform activity. To accomplish goal 4, the real-time iontophoretic (RTI) technique (Nicholson & Phillips, 1981;Odackal et al 2017) was used to quantify the SES in the 4-AP model and show that NBCe1 blockade reverses the SES.…”