Push–Pull Perfusion Sampling with Segmented Flow for High Temporal and Spatial Resolution in Vivo Chemical Monitoring

Abstract

Low-flow push-pull perfusion is a sampling method that yields better spatial resolution than competitive methods like microdialysis. Because of the low flow rates used (50 nL/min) it is challenging to use this technique at high temporal resolution which requires methods of collecting, manipulating, and analyzing nanoliter samples. High temporal resolution also requires control of Taylor dispersion during sampling. To meet these challenges, push-pull perfusion was coupled with segmented flow to achieve in vivo sampling at 7 s temporal resolution at 50 nL/min flow rates. By further miniaturizing the probe inlet, sampling with 200 ms resolution at 30 nL/min (pull only) was demonstrated in vitro. Using this method, L-glutamate was monitored in the striatum of anesthetized rats. Up to 500 samples of 6 nL each were collected at 7 s intervals, segmented by an immiscible oil and stored in a capillary tube. The samples were assayed offline for L-glutamate at a rate of 15 samples/min by pumping them into a reagent addition tee fabricated from Teflon where reagents were added for a fluorescent enzyme assay. Fluorescence of the resulting plugs was monitored downstream. Microinjection of 70 mM potassium in physiological buffered saline evoked L-glutamate concentration transients that had an average maxima of 4.5 ± 1.1 μM (n = 6 animals, 3–4 injections each) and rise times of 22 ± 2 s. These results demonstrate that low-flow push-pull perfusion with segmented flow can be used for high temporal resolution chemical monitoring and in complex biological environments.

A novel method for simultaneous glutamate and extracellular activity measurement in brain slices with high temporal resolution

“…A sophisticated procedure using enzymatic assay and pushpull perfusion sampling with segmented flow was tested to determine Glu concentration in a very small samples getting a high temporal resolution in comparison with the classical microdialysis procedure. The concentration of Glu was measured with the Amplex Red method [31]. Also used in the approach presented here, although, it was not necessary to collect samples since the monitoring was carried out continuously.…”

Using copper ions to amplify ROS-mediated fluorescence for continuous online monitoring of extracellular glucose in living rat brain

“…In the case of acute brain injury, the response of a rapid release of massive intracellular K þ ions into brain ECF (Katayama et al, 1990;Reinert et al, 2000) has a profound influence on the dynamic actions of neurotransmitters (Zheng et al, 2000;Wang et al, 2008Wang et al, , 2010Slaney et al, 2011), the membrane-associated transport system, the metabolic activities, and the synaptic functions of neurons (Kimelberg et al, 1979;Marion, 1998). Because the brain extracellular glucose dominates the Na þ /K þ homeostasis and its associated biochemical interactions, the ability to describe the dynamic variations in the concentration of glucose would enhance the study of these essential functions in living rat brains.…”

REAL-TIME IN VIVO NEUROTRANSMITTER MEASUREMENTS USING ENZYME-BASED CERAMIC MICROELECTRODE ARRAYS: WHAT WE HAVE LEARNED ABOUT GLUTAMATE SIGNALING

“…Recently, using MEA recordings we have shown that tonic (resting) and phasic (transient release) glutamate signals are dissociable events. For example, (Day et al, 2006;Quintero et al, 2007;Rutherford et al, 2007;Hascup et al, 2008;Thomas et al, 2009;Hascup et al, 2010;Hinzman et al, 2010;Stephens et al, 2010;Hascup et al, 2011b;Hascup et al, 2011a;Mattinson et al, 2011;Stephens et al, 2011;Hascup et al, 2012;Hinzman et al, 2012;Matveeva et al, 2012a;Matveeva et al, 2012b;Onifer et al, 2012;Thomas et al, 2012;Nevalainen et al, 2013); Microdialysis: (Ueda and Tsuru, 1995;Yang et al, 1995;Battaglia et al, 1997;Biggs et al, 1997;Timmerman et al, 1999;Reinstrup et al, 2000;Schulz et al, 2000;Giovannini et al, 2001;Segovia et al, 2001;Galvan et al, 2003;Cavus et al, 2005;Clinckers et al, 2005;Calcagno et al, 2006;Segovia et al, 2006;Ballini et al, 2008;Hernandez et al, 2008;Lupinsky et al, 2010;Slaney et al, 2011;Li et al, 2012;Vasylieva et al, 2013). initial MEA studies conducted in rats under anesthesia at various ages revealed that tonic levels of glutamate are often ...…”