Feeding specific glutamate surge in the rat lateral hypothalamus revealed by low-flow push-pull perfusion.

Substantial evidence implicates the lateral hypothalamus (LH) in the control of ingestive behavior and previous studies have found that glutamate release within the LH increases during meals. It is not known, however, whether this effect is selective for feeding, or whether similar changes are also seen during drinking. In this work, we examined this question using low-flow push-pull perfusion which allows sampling from small tissue volumes. Presentation of highly palatable solid or liquid foods to food-deprived rats resulted in an immediate increase in glutamate output of more than 200% over baseline. The response was maximal immediately after food presentation. In contrast, significant changes in glutamate output were not seen when water was presented to water-deprived animals, despite the occurrence of vigorous drinking. These findings confirm reports of feeding related glutamate release in the LH and demonstrate that this effect is specific to feeding, rather than being a general concomitant of all ingestive behaviors. The push-pull technique described here may allow the relevant region of the LH to be identified with greater precision than other methods.

Demonstration and use of nanoliter sampling of in vivo rat vitreous and vitreoretinal interface.

PURPOSE: An understanding of the chemical microenvironments at different locations on the retina can provide unique insights into retinal neurochemistry and pathology. The anatomical shape and the small volumes available from a spatially restricted volume greatly complicate these types of measurements. The aim of this study was to demonstrate an in vivo sampling system to probe different regions of the rat retina. METHODS: A low-flow push-pull perfusion probe was developed with concentric fused-silica capillaries. It was designed to fit through a 29-gauge needle for placement in the vitreous and at the vitreoretinal interface of the rat eye. Physiological saline was perfused and withdrawn through outer and inner capillaries, respectively, at flow rates between 10 and 50 nl/min. Samples of 500 nl were collected for amino acid analysis by capillary electrophoresis. Perfusion of a potent and selective inhibitor of the excitatory amino acid transporters was performed through the probe with the tip located 1-2 mm away from the optic nerve head. RESULTS: Ten amino acids were quantified from the perfusates of vitreous and the vitreoretinal interface. Sampling through time showed the use of this system to monitor retinal changes in these amino acids. The infusion of a transport protein antagonist shows a statistically significant increase in the glutamate concentration in collected samples when the probe tip is placed peripheral to but not over the optic nerve head. CONCLUSIONS: We demonstrate a new method for following neurochemical changes at the retina with spatial resolution. This in vivo method is widely applicable to the site-specific study of states of normal and dysfunctional retinal neurochemistry.

Determination of amino acids in rat vitreous perfusates by capillary electrophoresis.

In vivo determinations of amino acids are important for improving our understanding of physiological states of biological tissue function and dysfunction. However, the chemically complex matrix of different biological fluids complicates the assay of this important class of molecules. We introduce a method for characterizing the amino acid composition of submicroliter volumes of vitreous humor perfusates. Low-flow push-pull perfusion sampling is compatible with collecting small volume samples in a complicated matrix that are potentially difficult to separate. An efficient, sensitive, and rapid analysis of amino acids from in vivo perfusates of the vitreous is presented with 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde (CBQCA) derivatitation and capillary electrophoresis (CE) separation with laser-induced fluorescence detection (LIF). Derivatization with CBQCA for up to 2 h provided high sensitivity and low detection limits at the nM level. Seventeen amino acids including D-serine (D-Ser) and D-aspartate (D-Asp) were resolved in less than 10 min. Importantly, D-Ser is separated from its enantiomeric pair. Characterization of vitreal amino acids with this assay technique will be useful for understanding ocular diseases and physiological mechanisms in vision.