1.1. Basic Principles and Advantages of microdialysis

Microdialysis is one of the most widely used methods for monitoring neurochemicals in vivo. The concept of using dialysis to collect analytes from interstitial fluid in the brain was first reported as early as 1966, where Bito et al. inserted dialysis bags into the cortices of dogs to collect amino acids present in brain interstitial fluid (Bito et al., 1966). Since this initial study, the development of dialysis probes for the active perfusion and collection of dialysate has laid the ground work for modern-day microdialysis (Ungerstedt and Pycock, 1974). Microdialysis probes consist of a shaft housing inlet and outlet tubes that deliver fluids to and from a semi-permeable membrane (Fig. 2). The inlet tube is connected to a perfusion system that delivers perfusion fluid of physiological solute concentrations through the probe, commonly artificial cerebrospinal fluid (aCSF) or Ringer’s solution, matching the electrolyte concentration of the brain interstitial fluid (Chen et al., 1997; Zapata et al., 2009). The perfusion fluid then flows through a semi-permeable membrane of defined molecular weight cut-off across which extracellular analytes can diffuse. The dialysate then flows through the outlet tube where fractions are collected for post hoc chemical analysis. Chemical analysis of the dialysate is often done by electrochemical detection, mass spectrometry (MS), high-performance liquid chromatography (HPLC), or enzymatic detection (Jin et al., 2008; Zestos and Kennedy, 2017).

Schematic of microdialysis membrane and workflow. Yellow boxes highlight areas of recent advancements and future directions in probe engineering, sampling, and chemical analysis. HPLC trace adapted from Reinhoud and colleagues (Reinhoud et al., 2013).

A critical advantage of microdialysis over the other techniques discussed in this review is its ability to monitor many different analytes simultaneously with picomolar range sensitivity in vivo (Ballini et al., 2008; Reinhoud et al., 2013; Yang et al., 2013). The collected samples can be analyzed using HPLC or mass spectrometry, and up to 70 different neurochemical compounds can be detected in a single dialysate sample (Wong et al., 2016). As a direct sampling method, it permits the measurement of basal concentrations of brain analytes in addition to dynamic changes in neurochemical levels. The collection of analytes by microdialysis is governed by passive diffusion (Fick’s first law) of extracellular solutes through the dialysis membrane. At standard flow rates (0.3 to 3uL/min), microdialysis probes do not achieve absolute equilibrium with the interstitial fluid. Therefore, 100% recovery of solutes from the brain is rarely achieved, and calibration is needed to relate experimental dialysate concentrations to absolute extracellular concentrations. Many factors contribute to a microdialysis system’s efficiency of recovery (or relative recovery) for a particular analyte of interest, including flow rate, membrane surface area, analyte diffusion coefficients, and diffusion (penetration) distance (Bungay et al., 1990; Chefer et al., 2009). However, improving many of these dialysate collection parameters come with trade-offs in spatiotemporal resolution and invasiveness (brief discussion below). Probe membranes generally have molecular weight cutoffs of 20 to 60 kilodaltons (kDa) (Nandi and Lunte, 2009), making microdialysis a well-established method for monitoring virtually any low molecular weight analyte, such as amino acids or biogenic monoamines in the extracellular space, with high sensitivity.

Microdialysis permits multimodal studies combining neural activity recording and manipulation with sample collection and neurochemical detection. Retrodialysis is used in neuropharmacological studies, where adding a pharmacological compound into the perfusate allows for simultaneous steady-state drug delivery to the tissue and sample collection from the extracellular fluid (Höcht et al., 2007). A single probe can further be used for microdialysis in conjunction with other neural recording techniques such as single cell recording or EEG (Ludvig et al., 1994; Obrenovitch et al., 1993). Quiroz and colleagues demonstrated the utility of a novel optogenetic-microdialysis probe to optically stimulate and measure glutamate and dopamine release in the posteromedial nuclear accumbens shell (Quiroz et al., 2016). Al-Hasani and colleagues have also developed an opto-dialysis probe to measure optically evoked, picomolar release of dynorphin and enkephalins in the nucleus accumbens shell in awake, freely moving mice (Al-Hasani et al., 2018). The work that has been done to allow for multimodal recording of brain activity with these new probes have further modernized the use of microdialysis to answer emerging questions in neuroscience.