Emerging trends in in vivo neurochemical monitoring by microdialysis

Abstract

Mapping chemical dynamics in the brain of live subjects is a challenging but highly rewarding goal because it allows neurotransmitter fluctuations to be related to behavior, drug effects, and disease states. A popular method for such measurements is microdialysis sampling coupled to analytical measurements. This method has become well-established for monitoring low molecular weight neurotransmitters, metabolites, and drugs, especially in pharmacological and pharmacokinetic studies. Recent technological developments which improve the temporal and spatial resolution of the methods will enable it to be used for studying behavior and small brain nuclei. Better assays allow monitoring more neurotransmitters simultaneously. Extension to analysis of aggregating proteins like amyloid β is proving extremely useful for uncovering the roles of these molecules and how they contribute to neurodegenerative diseases.

Figure 1

Illustration of using antibodies to enhance recovery in microdialysis. In normal microdialysis, analytes (represented by shapes) cross the membrane according to their concentration gradient. In “antibody-enhanced” microdialysis, antibody added to the perfusion fluid (“y”-shapes) results in low concentration of free analyte in the dialysate. This maintains a high concentration gradient of free analytes resulting in higher recovered total concentration. (Adapted from [23].)

Figure 2

In vitro validation of method to measure free amyloid β (Aβ) in the extracellular space of a living brain by microdialysis. (A) Diagram of exchangeable Aβ. Triangles represent potential Aβ binding molecules, e.g., apoE, clusterin, and α2M. Only highlighted Aβ molecules are of the appropriate size to pass through a 35 kDA MWCO membrane on the microdialysis probe. (B) Interpolated zero flow method to quantify the pool of measurable Aβ_1-x and Aβ40 within samples of human CSF (n = 4). At 2.2 µl/min, the percentage recovery of eAβ was 9.74 ± 1.53% (mean ± SEM). C, In vitro percentage recoveries for each Aβ species using the interpolated zero flow method. Each recovery point contains error bars and are overlapping for each species (n = 4). In vitro recovery of each Aβ species by microdialysis is the same. (D) The concentration of eAβ and total soluble Aβ are highly correlated within a sample of human CSF (Pearson’s r = 0.9487; p < 0.0001; n = 9). The mean concentrations of soluble Aβ 1-x and e Aβ 1-x were 30.47 ± 4.23 ng/ml (mean_SEM) and 196.2 ± 28.65pg/ml, respectively. (E,F) Human CSF immunoprecipitated (IP’d) for Aβ has undetectable levels of total soluble Aβ and eAβ. Human CSF spiked with an amount of exogenous Aβ40 peptide expected to double Aβ concentration resulted in a 2.1-fold increase in total soluble Aβ and a 1.9-fold increase in eAβ. (Figure and legend adapted from [27].)

Figure 3

Illustration of segmented flow sampling and coupling to mass spectrometry. (A) Dialysate is pumped into a cross fluidic structure mounted on the subject’s head. In the cross, fluorinated oil and internal standard solution are pumped into separate arms. Resulting segmented flow emerges from the fourth arm and is pumped to the electrospray source of the mass spectrometer (MS). Inset shows a photo where aqueous solution of green dye was sampled and segmented within the cross by perfluorodecalin. HV stands for high voltage. (B) Recording of response to in vivo microinjection of neostigmine obtained using system in A with probe inserted in the striatum of a rat. Figure shows a sample trace for simultaneous detection of acetylcholine (ACh), internal standard, d4-acetylcholine (d4-ACh), and (C) neostigmine (Neo). Each compound is recorded at a specific m/z by the mass spectrometer. Arrow indicates beginning of each microinjection. Insert is a magnified view of the 1^st microinjection for neostigmine detection showing the detection of individual droplets and rapid response time possible. (Adapted from [42]).

Figure 4

Comparison of the size of different sampling probes. Top probe is a conventional microdialysis sampling probe from a commercial vendor. White material is the sampling membrane. “Mini” is a small microdialysis probe that can be fabricated manually. The sampling area is the clear area (about 0.5 mm long by 0.2 mm diameter) at the tip. “Push-pull” is two fused silica capillaries, coated in polyimide, fused together with epoxy. Sampling occurs just at the tip. In practice, a rigid sheath if often added to support the fused silica to near the tip. “Microfab push-pull” a Si-based probe made by lithography and bulk etching techniques. The probe is 85 um wide by 70 thick along the shank and tip. Sampling occurs from a 20 um diameter orifice at the tip (not visible in the photo).