Chemical Gradients within Brain Extracellular Space Measured using Low Flow Push–Pull Perfusion Sampling in Vivo

Abstract

Although populations of neurons are known to vary on the micrometer scale, little is known about whether basal concentrations of neurotransmitters also vary on this scale. We used low-flow push−pull perfusion to test if such chemical gradients exist between several small brain nuclei. A miniaturized polyimide-encased push−pull probe was developed and used to measure basal neurotransmitter spatial gradients within brain of live animals with 0.004 mm 3 resolution. We simultaneously measured dopamine (DA), norepinephrine, serotonin (5-HT), glutamate, γ-aminobutyric acid (GABA), aspartate (Asp), glycine (Gly), acetylcholine (ACh), and several neurotransmitter metabolites. Significant differences in basal concentrations between midbrain regions as little as 200 μm apart were observed. For example, dopamine in the ventral tegmental area (VTA) was 4.8 ± 1.5 nM but in the red nucleus was 0.5 ± 0.2 nM. Regions of high glutamate concentration and variability were found within the VTA of some individuals, suggesting hot spots of glutamatergic activity. Measurements were also made within the nucleus accumbens core and shell. Differences were not observed in dopamine and 5-HT in the core and shell; but their metabolites homovanillic acid (460 ± 60 nM and 130 ± 60 nM respectively) and 5-hydroxyindoleacetic acid (720 ± 200 nM and 220 ± 50 nM respectively) did differ significantly, suggesting differences in dopamine and 5-HT activity in these brain regions. Maintenance of these gradients depends upon a variety of mechanisms. Such gradients likely underlie highly localized effects of drugs and control of behavior that have been found using other techniques.

Brain interstitial fluid glutamine homeostasis is controlled by blood–brain barrier SLC7A5/LAT1 amino acid transporter

“…Therefore, our findings of low hippocampal ISF relative to CSF AA levels are supported by our carefully controlled study protocol (including the in vivo determination of K 0 ). Furthermore, while there are some discrepancies in Gln levels, the direct sampling data available for several AAs 33 are consistent with our results. Finally, although CSF and ISF partially co-mingle, the steep AA concentration gradients observed indicates a degree of independent homeostatic regulation of solutes in each fluid compartment may exist.…”

Experimental evaluation and computational modeling of tissue damage from low-flow push–pull perfusion sampling in vivo

“…The streamlines can provide an estimate of how far into the tissue flow, and therefore sampling, occurs. We estimate this to be about 0.003 mm 3 , in agreement with a previous experimental estimate of 0.004 mm 3 (Slaney, et al 2012). The sampling area of the tip was 0.04 mm 2 in PPP.…”

“…These relatively high flow rates were perceived to cause substantial tissue damage (Redgrave, 1977). More recently, highly miniaturized PPP has been reported and used in brain and other tissues (Kottegoda et al, 2002; Thongkhao - On et al, 2004; Lee et al, 2013; Slaney et al, 2012; Slaney et al, 2011). Low-flow PPP uses relatively narrow bore capillaries as the probe and sampling flow rates of just 10-50 nL/min.…”

“…With the use of smaller bore tubing, the spatial resolution is improved relative to microdialysis or conventional PPP. Indeed, this method has been used to sample from the vitreous humor between lens and retina of the rat eye (Thongkhao - On et al, 2004) and to measure chemical gradients around small brain regions (Slaney et al, 2012). This method has been coupled with segmented flow and microscale analytical techniques to achieve temporal resolution of a few seconds (Slaney et al, 2011).…”

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

“…Using low-flow push-pull perfusion sampling to obtain microelectrode-like spatial resolution, Slaney and colleagues describe heterogeneity in basal glutamate concentration of the midbrain with some areas having low glutamate levels (2 µM) and neighboring ones having higher glutamate levels (24 µM). (Slaney et al, 2012). Moreover, MEA measures of resting glutamate (tonic levels) are at least 40%-50% derived from neuronal sources (Hascup et al, 2010) in contrast with the lack of sodium-and calcium-dependence seen with many microdialysis recordings (Timmerman and Westerink, 1997).…”