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

Abstract

SHORT ABSTRACT This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS. LONG 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.

Time-Resolved Integrative Optical Imaging of Diffusion during Spreading Depression

“…In the RTI method (10), a small extracellular probe, in our case the cation TMA (MW 74), was released from an iontophoretic microelectrode and detected by an ion-selective microelectrode positioned 120 µm away. We followed the protocol described in detail in (38) to perform these measurements in diluted agarose gel or in the brain slices, and to analyze the diffusion curves. Diffusion measurements in diluted agarose gel provided the transport number n t of the iontophoretic microelectrode, i.e., the fraction of iontophoretic current carrying the TMA cations, and the free diffusion coefficient D. The values of n t and D were utilized when analyzing the measurements in brain to yield the ECS volume fraction α, the effective diffusion coefficient D , and the nonspecific clearance κ.…”

Time-Resolved Integrative Optical Imaging of Diffusion during Spreading Depression

“…In the RTI method (10), a small extracellular probe-in our case, the cation TMA (MW 74)-was released from an iontophoretic microelectrode and detected by an ion-selective microelectrode positioned 120 mm away. We followed the protocol described in detail in (39) to perform these measurements in diluted agarose gel or in the brain slices, and to analyze the diffusion curves. Diffusion measurements in diluted agarose gel provided the transport number n t of the iontophoretic microelectrode, i.e., the fraction of iontophoretic current carrying the TMA cations, and the free diffusion coefficient D. The values of n t and D were utilized when analyzing the measurements in brain to yield the ECS volume fraction a, the effective diffusion coefficient D + , and the nonspecific clearance k.…”

Current Techniques for Investigating the Brain Extracellular Space

“…The RTI microelectrode capillary and the respective recording microelectrode can be positioned in the tissue independently or integrated in a single probe. The source/detector pair is calibrated for a given diffusion distance in aqueous agar prior to use, which represents a scenario of unhindered, homogeneous diffusion (Odackal et al, 2017). The technique is readily applied in live tissue, where it yields diffusion curve data ( Figure 1B; Nicholson and Phillips, 1981).…”

“…Regarding temporal resolution, the electrical current pulses used for iontophoretic application of TMA + are typically 1 min duration 100 nA square pulses (Odackal et al, 2017), and subsequent diffusional clearance of TMA + from the area following the pulse takes a few minutes, thus enabling repeated sampling with an interval of 5 min. The technique was from the beginning introduced in an in vivo setting (Nicholson and Phillips, 1981), though a drawback is that in vivo RTI experiments are, to our knowledge, invariably performed in anesthetized animals.…”