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Scott A. Shippy - One of the best experts on this subject based on the ideXlab platform.
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Development of μ-Low-Flow-Push-Pull Perfusion Probes for Ex Vivo Sampling from Mouse Hippocampal Tissue Slices.
ACS chemical neuroscience, 2017Co-Authors: Marissa R. Cabay, Alyssa Mcray, David E. Featherstone, Scott A. ShippyAbstract:This work demonstrates a reduced tip μ-low-flow-Push-Pull Perfusion technique for ex vivo sampling of the extracellular space of mouse hippocampal brain slices. Concentric fused-silica capillary probes are pulled by an in-house gravity puller with a butane flame producing probe tips averaging an overall outer diameter of 30.3 ± 8 μm. The 10-30 nL/min Perfusion flow rate through the probe generates an average recovery of 90%. Sampling was performed with mouse brain tissue slices to characterize basal neurotransmitter content in this model system. Samples were collected from hippocampal tissue slices at a volume of 200 nL per sample. Sample arginine, histamine, lysine, glycine, glutamate, and aspartate content was quantified by micellar electrokinetic chromatography with LED-induced fluorescence detection. Primary amine content was sampled over several hours to determine evidence for tissue damage and loss of extracellular content from the tissue slice. Overall, all amino acid concentrations trended lower as an effect of time relative to tissue slicing. There were significant concentration decreases seen for histamine, lysine, and aspartate between time points 0-2 and 2-6 h (p < 0.05) relative to tissue slicing. Analysis of averaged sampling experiments does not appear to reveal significant probe-insertion-related amino acid changes. The work presented shows the applicability of an 80% reduction of probe tip size relative to previous designs for the collection of extracellular content from thin tissue slices.
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Development of μ‑Low-Flow-Push–Pull Perfusion Probes for Ex Vivo Sampling from Mouse Hippocampal Tissue Slices
2017Co-Authors: Marissa R. Cabay, Alyssa Mcray, David E. Featherstone, Scott A. ShippyAbstract:This work demonstrates a reduced tip μ-low-flow-push–pull Perfusion technique for ex vivo sampling of the extracellular space of mouse hippocampal brain slices. Concentric fused-silica capillary probes are pulled by an in-house gravity puller with a butane flame producing probe tips averaging an overall outer diameter of 30.3 ± 8 μm. The 10–30 nL/min Perfusion flow rate through the probe generates an average recovery of 90%. Sampling was performed with mouse brain tissue slices to characterize basal neurotransmitter content in this model system. Samples were collected from hippocampal tissue slices at a volume of 200 nL per sample. Sample arginine, histamine, lysine, glycine, glutamate, and aspartate content was quantified by micellar electrokinetic chromatography with LED-induced fluorescence detection. Primary amine content was sampled over several hours to determine evidence for tissue damage and loss of extracellular content from the tissue slice. Overall, all amino acid concentrations trended lower as an effect of time relative to tissue slicing. There were significant concentration decreases seen for histamine, lysine, and aspartate between time points 0–2 and 2–6 h (p < 0.05) relative to tissue slicing. Analysis of averaged sampling experiments does not appear to reveal significant probe-insertion-related amino acid changes. The work presented shows the applicability of an 80% reduction of probe tip size relative to previous designs for the collection of extracellular content from thin tissue slices
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Sample collection and amino acids analysis of extracellular fluid of mouse brain slices with low flow Push-Pull Perfusion
The Analyst, 2015Co-Authors: Geovannie Ojeda-torres, David E. Featherstone, L. Williams, Scott A. ShippyAbstract:Brain tissue slices are a common neuroscience model that allows relatively sophisticated analysis of neuronal networks in a simplified preparation. Most experimental methodology utilizes electrophysiological tools to probe these model systems. The work here demonstrates the adaptation of low-flow Push-Pull Perfusion sampling (LFPS) to a brain slice system. LFPS is used to sample from the hippocampus of mouse brain slices. Perfusate amino acid levels are quantified following sampling with capillary electrophoresis. Glutamate was measured from the CA1 region of the hippocampus in slices taken from a cystine-glutamate transporter deletion mutant, xCT(-/-), and the background strain C57BL/6J. Sampling is performed over up to 6.5 h with standard tissue slice preparation and experimentation methods. Four amino acids were quantified to demonstrate the ability to perform LFPS and show good agreement with published literature. Perfusate glutamate levels are found to be significantly lower with xCT(-/-) slices (1.9(±0.5) μM) relative to controls (4.90(±1.1) μM). But, experiments with control slices show a significant decrease in glutamate over the 6 h sampling period that are not seen with xCT(-/-) slices. Increasing the LFPS sample collection rate during the first 90 min of sampling did not show a sampling artifact in perfusate glutamate content. Sampling immediately following slicing did not show an early increasing glutamate level that would be indicative of a significant contribution from blood or tissue damage. The data presented here show a complementarity to electrophysiological studies of tissue slices. The ability to characterize extracellular fluid chemical content with LFPS in these slices provides an alternative data stream for probing neurochemical signaling networks in brain tissue slices.
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Monitoring of in vivo manipulation of nitric oxide synthases at the rat retina using the Push-Pull Perfusion sampling and capillary electrophoresis.
Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2014Co-Authors: Jeanita S. Pritchett, Scott A. ShippyAbstract:Proteins play a variety of functional roles in tissues that underlie tissue health. The measurement of protein function is important to both understand normal and dysfunctional tissue states. Low-flow Push-Pull Perfusion sampling (LFPS) has been used to collect submicroliter volumes of extracellular fluid which are well suited to capillary electrophoresis for compositional quantitative analysis. In this study, LFPS is used to deliver pharmacological agents to the in vivo retinal tissues at the probe sampling tip during sampling to measure protein function. Two native nitric oxide synthase enzymes were pharmacologically inhibited and the enzyme product NO metabolite, nitrate, was determined with capillary electrophoresis from the perfusates. LFPS delivered inhibitors including the non-selective N(G)-nitro-Larginine methyl ester (L-NAME), the nNOS selective 7-nitroindazole (7-NI), and eNOS N5-(1-imioethyl)-L-ornithine, dihydrochloride (L-NIO) were perfused to the sampling region either directly over a rat retina optic nerve head or 1-mm peripheral to the ONH. At the PONH, 65, 55 and 60% of baseline nitrate levels, respectively, were observed with inhibitor infusion. These are statistically significant (P
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High temporal resolution coupling of low-flow Push-Pull Perfusion to capillary electrophoresis for ascorbate analysis at the rat vitreoretinal interface
The Analyst, 2008Co-Authors: Eric E. Patterson, Jeanita S. Pritchett, Scott A. ShippyAbstract:A system is presented demonstrating the high-temporal resolution coupling of low-flow Push-Pull Perfusion sampling (LFPS) to capillary electrophoresis for the absorbance measurement of ascorbate at the rat vitreoretinal interface. This system holds all separation components at a low pressure as the means for withdrawing sample during LFPS. The system uses a flow-gated interface to directly couple the withdrawal capillary from the LFPS probe to a separation capillary and eliminates the need for any offline sample handling. The temporal resolution of the system was limited by injection time and is less than 16 s. This high temporal resolution was applied to the monitoring of in vivoascorbate levels at the rat vitreoretinal interface. Baseline concentrations of ascorbate were found to be 86 µM ± 18 µM at the vitreoretinal interface. Baseline concentrations matched well with those obtained for the postmortem bulk vitreous analysis. Upon stimulation with 145 mM K+, a maximum increase in baseline values between 32–107% for n = 3 was observed. This system demonstrates the first in vivo temporal study of ascorbate at the rat vitreoretinal interface.
Stephen G. Weber - One of the best experts on this subject based on the ideXlab platform.
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Higher Aminopeptidase Activity Determined by Electroosmotic Push–Pull Perfusion Contributes to Selective Vulnerability of the Hippocampal CA1 Region to Oxygen Glucose Deprivation
ACS chemical neuroscience, 2017Co-Authors: Stephen G. WeberAbstract:It has been known for over a century that the hippocampus, the center for learning and memory in the brain, is selectively vulnerable to ischemic damage, with the CA1 being more vulnerable than the CA3. It is also known that leucine enkephalin, or YGGFL, is neuroprotective. We hypothesized that the extracellular hydrolysis of YGGFL may be greater in the CA1 than the CA3, which would lead to the observed difference in susceptibility to ischemia. In rat organotypic hippocampal slice cultures, we estimated the Michaelis constant and the maximum velocity for membrane-bound aminopeptidase activity in the CA1 and CA3 regions. Using electroosmotic push–pull Perfusion and offline capillary liquid chromatography, we inferred enzyme activity based on the production rate of GGFL, a natural and inactive product of the enzymatic hydrolysis of YGGFL. We found nearly 3-fold higher aminopeptidase activity in the CA1 than the CA3. The aminopeptidase inhibitor bestatin significantly reduced hydrolysis of YGGFL in both regi...
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Numerical Modeling of Electroosmotic Push-Pull Perfusion and Assessment of Its Application to Quantitative Determination of Enzymatic Activity in the Extracellular Space of Mammalian Tissue.
Analytical chemistry, 2017Co-Authors: Stephen G. WeberAbstract:Many sampling methods have been developed to measure the extracellular concentrations of solutes in the extracellular space of mammalian tissue, e.g., brain. However, few have been used to quantitatively study the various processes, such as enzymatic degradation, that determines the fate of these solutes. For a method to be useful in this pursuit, it must be able to (1) perfuse tissue and collect the perfusate for quantitative analysis of the solutes introduced and reaction products produced, (2) control the average residence time of the active solutes, and (3) have the appropriate spatial resolution for the process of interest. Our lab previously developed a Perfusion technique based on electroosmosis (EO), called EO Push-Pull Perfusion (EOPPP), that is in principle suitable to meet these needs. However, much like the case for other sampling methods that came before, there are parameters that are needed for quantitative interpretation of data but that cannot be measured easily (or at all). In this paper, we present a robust finite element model that provides a deep understanding of fluid dynamics and mass transport in the EOPPP method, assesses the general applicability of EOPPP to studying enzyme activity in the ECS, and grants a simple approach to data treatment and interpretation to obtain, for example, Vmax and Km for an enzymatic reaction in the extracellular space of the tissue. This model is a valuable tool in optimizing and planning experiments without the need for costly experiments.
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Higher Aminopeptidase Activity Determined by Electroosmotic Push–Pull Perfusion Contributes to Selective Vulnerability of the Hippocampal CA1 Region to Oxygen Glucose Deprivation
2017Co-Authors: Stephen G. WeberAbstract:It has been known for over a century that the hippocampus, the center for learning and memory in the brain, is selectively vulnerable to ischemic damage, with the CA1 being more vulnerable than the CA3. It is also known that leucine enkephalin, or YGGFL, is neuroprotective. We hypothesized that the extracellular hydrolysis of YGGFL may be greater in the CA1 than the CA3, which would lead to the observed difference in susceptibility to ischemia. In rat organotypic hippocampal slice cultures, we estimated the Michaelis constant and the maximum velocity for membrane-bound aminopeptidase activity in the CA1 and CA3 regions. Using electroosmotic push–pull Perfusion and offline capillary liquid chromatography, we inferred enzyme activity based on the production rate of GGFL, a natural and inactive product of the enzymatic hydrolysis of YGGFL. We found nearly 3-fold higher aminopeptidase activity in the CA1 than the CA3. The aminopeptidase inhibitor bestatin significantly reduced hydrolysis of YGGFL in both regions by increasing apparent Km. Based on propidium iodide cell death measurements 24 h after oxygen–glucose deprivation, we demonstrate that inhibition of aminopeptidase activity using bestatin selectively protected CA1 against delayed cell death due to oxygen–glucose deprivation and that this neuroprotection occurs through enkephalin-dependent pathways
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Numerical Modeling of Electroosmotic Push–Pull Perfusion and Assessment of Its Application to Quantitative Determination of Enzymatic Activity in the Extracellular Space of Mammalian Tissue
2017Co-Authors: Stephen G. WeberAbstract:Many sampling methods have been developed to measure the extracellular concentrations of solutes in the extracellular space of mammalian tissue, e.g., brain. However, few have been used to quantitatively study the various processes, such as enzymatic degradation, that determines the fate of these solutes. For a method to be useful in this pursuit, it must be able to (1) perfuse tissue and collect the perfusate for quantitative analysis of the solutes introduced and reaction products produced, (2) control the average residence time of the active solutes, and (3) have the appropriate spatial resolution for the process of interest. Our lab previously developed a Perfusion technique based on electroosmosis (EO), called EO push–pull Perfusion (EOPPP), that is in principle suitable to meet these needs. However, much like the case for other sampling methods that came before, there are parameters that are needed for quantitative interpretation of data but that cannot be measured easily (or at all). In this paper, we present a robust finite element model that provides a deep understanding of fluid dynamics and mass transport in the EOPPP method, assesses the general applicability of EOPPP to studying enzyme activity in the ECS, and grants a simple approach to data treatment and interpretation to obtain, for example, Vmax and Km for an enzymatic reaction in the extracellular space of the tissue. This model is a valuable tool in optimizing and planning experiments without the need for costly experiments
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Electroosmotic Perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures
Analytical and Bioanalytical Chemistry, 2014Co-Authors: Mats Sandberg, Stephen G. WeberAbstract:This review covers recent advances in sampling fluid from the extracellular space of brain tissue by electroosmosis (EO). Two techniques, EO sampling with a single fused-silica capillary and EO push–pull Perfusion, have been developed. These tools were used to investigate the function of membrane-bound enzymes with outward-facing active sites, or ectoenzymes, in modulating the activity of the neuropeptides leu-enkephalin and galanin in organotypic-hippocampal-slice cultures (OHSCs). In addition, the approach was used to determine the endogenous concentration of a thiol, cysteamine, in OHSCs. We have also investigated the degradation of coenzyme A in the extracellular space. The approach provides information on ectoenzyme activity, including Michaelis constants, in tissue, which, as far as we are aware, has not been done before. On the basis of computational evidence, EO push–pull Perfusion can distinguish ectoenzyme activity with a ~100 μm spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus. Graphical Abstract Schematics for the one-probe electroosmotic sampling (EO sampling, left) and two-probe electroosmotic Push-Pull Perfusion techniques (EOPPP, right). An external electric field drives fluid flow in the direction indicated by the arrows. HBSS - Hanks Balanced Salt Solution.
Kazuo Takebe - One of the best experts on this subject based on the ideXlab platform.
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Effects of Intravenous Administration of Interleukin-1-Beta on the Release of Prostaglandin E2, Corticotropin-Releasing Factor, and Arginine Vasopressin in Several Hypothalamic Areas of Freely Moving Rats: Estimation by Push-Pull Perfusion
Neuroendocrinology, 1994Co-Authors: Hajime Watanobe, Kazuo TakebeAbstract:Utilizing the Push-Pull Perfusion technique, we examined the effect of an intravenous bolus injection of recombinant human interleukin (IL)-1 beta on the release of prostaglandin E2 (PGE2), CRF, and AVP in several hypothalamic areas of freely moving rats, simultaneously monitoring plasma ACTH levels. Perfused hypothalamic areas were the median eminence (ME), the paraventricular nucleus (PVN), and the medial preoptic area (MPOA). During the period 12:00-15:00 h, perfusates and blood samples were collected every 10 min (between 13:00 and 13:40 h) or 20 min (between 12:00 and 13:00 h, and between 13:40 and 15:00 h). IL-1 beta (1.0 micrograms/rat) or vehicle only (in control groups) was injected at 13:00 h. In both the ME and the PVN but not in the MPOA, the outputs of CRF and AVP were significantly stimulated by IL-1 beta, prior to the rise in plasma ACTH. A significant stimulation of PGE2 by IL-1 beta was observed only in the PVN, and its temporal profile was very similar to those of CRF and AVP in the PVN. These results suggest that PGE2 may be a trigger in the PVN for the activation of CRF and AVP neurons, and thereby ACTH secretion, which follows IL-1 beta injection.
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Evidence that neuropeptide Y secretion in the median eminence increases prior to the luteinizing hormone surge in ovariectomized steroid-primed rats: estimation by Push-Pull Perfusion.
Neuroscience letters, 1992Co-Authors: Hajime Watanobe, Kazuo TakebeAbstract:Abstract Utilizing the Push-Pull Perfusion technique, we examined the secretory profiles of neuropeptide Y (NPY) and luteinizing hormone (LH)-releasing hormone (LHRH) in the median eminence (ME) of ovariectomized adult rats which were primed with estrogen and progesterone to provoke LH and prolactin (PRL) surges. The ME was perfused with artificial cerebrospinal fluid between 13.00 and 18.00 h, and perfusates and blood samples were collected every 20 min. NPY and LHRH in the ME started to significantly increase 40 min earlier than the initial significant rise in the plasma LH, and the highest ME levels of the neuropeptides clearly preceded the occurrence of the LH surge. Regarding the PRL surge, however, such temporal relationship was not apparent. These in vivo data appear to support the putative facilitatory role of NPY in the generation of the steroid-induced LH surge. This is the first study to characterize the temporal profile of in vivo release of NPY in rat ME in terms of its relationship to LH and PRL surg
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Evidence that intravenous administration of interleukin-1 stimulates corticotropin releasing hormone secretion in the median eminence of freely moving rats: estimation by Push-Pull Perfusion.
Neuroscience letters, 1991Co-Authors: Hajime Watanobe, Shinsuke Sasaki, Kazuo TakebeAbstract:Abstract Utilizing Push-Pull Perfusion, we examined secretory profiles of corticotropin releasing hormone (CRH) in the median eminence (ME) and of plasma adrenocorticotropin (ACTH) in freely moving male rats after intravenous bolus injection of recombinant human interleukin (IL)-lα (1.0 μg) and 1β (1.0 μg). The ME was perfused with artificial cerebrospinal fluid between 11.00 and 14.00 h, and perfusates and blood samples were collected every 20 min. Administrations at 12.00 h of IL-1α and 1β, but not vehicle only, resulted in significant increases in both the plasma ACTH and ME-CRH. The rise in ME-CRH clearly preceded the enhanced ACTH secretion. These in vivo data strongly suggest that IL-1 stimulates ACTH secretion, at least in part, by triggering hypothalamic CRH release. This is the first to characterize the temporal profile of CRH secretion in the ME after intravenous administration of IL-1 to freely moving rats.
Hajime Watanobe - One of the best experts on this subject based on the ideXlab platform.
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A comparative study of the effects of nitric oxide and carbon monoxide on the in vivo release of gonadotropin-releasing hormone and neuropeptide Y from rat hypothalamus during the estradiol-induced luteinizing hormone surge: estimation by Push-Pull p
Neuroendocrinology, 1999Co-Authors: Akira Kohsaka, Hajime Watanobe, Yoshifumi Kakizaki, Toshihiro SudaAbstract:Recent evidence suggests that nitric oxide (NO), a free radical gas, plays an important role in regulating the function of a variety of neuroendocrine systems. With respect to the hypothalamo-pituitary-gonadal axis, a stimulatory effect of NO on the release of gonadotropin-releasing hormone (GnRH) from rat hypothalamus has been demonstrated in vitro. However, no previous study has reported NO-stimulated secretion of GnRH from in vivo hypothalamus, and also the precise cellular site of action of NO within the GnRH neuronal system remains to be elucidated. In the present study, utilizing the Push-Pull Perfusion technique of rat hypothalamus, we examined the effect of L-arginine (L-Arg), an NO donor, on the release of GnRH, neuropeptide Y and cyclic GMP (c-GMP), which is a pivotal second messenger molecule of the NO system. For comparison, we also examined the effect of carbon monoxide (CO), another putative gaseous neurotransmitter, using hematin, a CO donor. During the period of 11.00-18.00 h, we collected blood and hypothalamic perfusates from ovariectomized adult rats that had been implanted with an estradiol capsule 2 days before. During the entire period of observation, L-Arg (1.0 or 10 mM), hematin (10 or 100 microM) or artificial cerebrospinal fluid alone (as the control) was infused into the medial preoptic area (MPOA) where there are cell bodies of GnRH neurons, or the median eminence-arcuate nucleus complex (ME-ARC) where axon terminals of GnRH neurons are localized. Although 10 mM of L-Arg significantly stimulated GnRH and c-GMP, but not neuropeptide Y, levels in both the MPOA and ME-ARC, GnRH and c-GMP in the ME-ARC were already increased by 1.0 mM of L-Arg. By contrast, both concentrations of hematin were without effect at either site of the hypothalamus. This study is the first to demonstrate that NO is capable of stimulating GnRH release from rat hypothalamus in vivo. Our data also suggests that both cell bodies and axon terminals of GnRH neurons may be sites of action of NO. Our data do not support a previous study by other investigators that reported a stimulatory effect of CO on the GnRH release.
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Effects of Intravenous Administration of Interleukin-1-Beta on the Release of Prostaglandin E2, Corticotropin-Releasing Factor, and Arginine Vasopressin in Several Hypothalamic Areas of Freely Moving Rats: Estimation by Push-Pull Perfusion
Neuroendocrinology, 1994Co-Authors: Hajime Watanobe, Kazuo TakebeAbstract:Utilizing the Push-Pull Perfusion technique, we examined the effect of an intravenous bolus injection of recombinant human interleukin (IL)-1 beta on the release of prostaglandin E2 (PGE2), CRF, and AVP in several hypothalamic areas of freely moving rats, simultaneously monitoring plasma ACTH levels. Perfused hypothalamic areas were the median eminence (ME), the paraventricular nucleus (PVN), and the medial preoptic area (MPOA). During the period 12:00-15:00 h, perfusates and blood samples were collected every 10 min (between 13:00 and 13:40 h) or 20 min (between 12:00 and 13:00 h, and between 13:40 and 15:00 h). IL-1 beta (1.0 micrograms/rat) or vehicle only (in control groups) was injected at 13:00 h. In both the ME and the PVN but not in the MPOA, the outputs of CRF and AVP were significantly stimulated by IL-1 beta, prior to the rise in plasma ACTH. A significant stimulation of PGE2 by IL-1 beta was observed only in the PVN, and its temporal profile was very similar to those of CRF and AVP in the PVN. These results suggest that PGE2 may be a trigger in the PVN for the activation of CRF and AVP neurons, and thereby ACTH secretion, which follows IL-1 beta injection.
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Evidence that neuropeptide Y secretion in the median eminence increases prior to the luteinizing hormone surge in ovariectomized steroid-primed rats: estimation by Push-Pull Perfusion.
Neuroscience letters, 1992Co-Authors: Hajime Watanobe, Kazuo TakebeAbstract:Abstract Utilizing the Push-Pull Perfusion technique, we examined the secretory profiles of neuropeptide Y (NPY) and luteinizing hormone (LH)-releasing hormone (LHRH) in the median eminence (ME) of ovariectomized adult rats which were primed with estrogen and progesterone to provoke LH and prolactin (PRL) surges. The ME was perfused with artificial cerebrospinal fluid between 13.00 and 18.00 h, and perfusates and blood samples were collected every 20 min. NPY and LHRH in the ME started to significantly increase 40 min earlier than the initial significant rise in the plasma LH, and the highest ME levels of the neuropeptides clearly preceded the occurrence of the LH surge. Regarding the PRL surge, however, such temporal relationship was not apparent. These in vivo data appear to support the putative facilitatory role of NPY in the generation of the steroid-induced LH surge. This is the first study to characterize the temporal profile of in vivo release of NPY in rat ME in terms of its relationship to LH and PRL surg
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Evidence that intravenous administration of interleukin-1 stimulates corticotropin releasing hormone secretion in the median eminence of freely moving rats: estimation by Push-Pull Perfusion.
Neuroscience letters, 1991Co-Authors: Hajime Watanobe, Shinsuke Sasaki, Kazuo TakebeAbstract:Abstract Utilizing Push-Pull Perfusion, we examined secretory profiles of corticotropin releasing hormone (CRH) in the median eminence (ME) and of plasma adrenocorticotropin (ACTH) in freely moving male rats after intravenous bolus injection of recombinant human interleukin (IL)-lα (1.0 μg) and 1β (1.0 μg). The ME was perfused with artificial cerebrospinal fluid between 11.00 and 14.00 h, and perfusates and blood samples were collected every 20 min. Administrations at 12.00 h of IL-1α and 1β, but not vehicle only, resulted in significant increases in both the plasma ACTH and ME-CRH. The rise in ME-CRH clearly preceded the enhanced ACTH secretion. These in vivo data strongly suggest that IL-1 stimulates ACTH secretion, at least in part, by triggering hypothalamic CRH release. This is the first to characterize the temporal profile of CRH secretion in the ME after intravenous administration of IL-1 to freely moving rats.
Robert T. Kennedy - One of the best experts on this subject based on the ideXlab platform.
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Chemical Gradients within Brain Extracellular Space Measured using Low Flow Push–Pull Perfusion Sampling in Vivo
2016Co-Authors: Thomas R. Slaney, Kirsten A. Porter-stransky, Omar S. Mabrouk, Brandon J. Aragona, Robert T. KennedyAbstract: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 mm3 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
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experimental evaluation and computational modeling of tissue damage from low flow push pull Perfusion sampling in vivo
Journal of Neuroscience Methods, 2015Co-Authors: David E Cepeda, Leah Hains, Joseph L Bull, Stephen I Lentz, Robert T. KennedyAbstract:Background Neurochemical monitoring via sampling probes is valuable for deciphering neurotransmission in vivo. Microdialysis is commonly used; however, the spatial resolution is poor.
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Chemical gradients within brain extracellular space measured using low flow Push-Pull Perfusion sampling in vivo.
ACS chemical neuroscience, 2012Co-Authors: Thomas R. Slaney, Omar S. Mabrouk, Kirsten A. Porter-stransky, Brandon J. Aragona, Robert T. KennedyAbstract: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.
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Push-Pull Perfusion sampling with segmented flow for high temporal and spatial resolution in vivo chemical monitoring.
Analytical chemistry, 2011Co-Authors: Thomas R. Slaney, Jing Nie, Neil D. Hershey, Prasanna Thwar, Jennifer J. Linderman, Mark A. Burns, Robert T. KennedyAbstract: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.
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Microfluidic chip for low-flow Push-Pull Perfusion sampling in vivo with on-line analysis of amino acids
Analytical chemistry, 2005Co-Authors: Nicholas A. Cellar, Scott T. Burns, Jens Christian Meiners, Hao Chen, Robert T. KennedyAbstract:Multilayer soft lithography was used to prepare a poly(dimethylsiloxane) microfluidic chip that allows for in vivo sampling of amino acid neurotransmitters by low-flow push−pull Perfusion. The chip incorporates a pneumatically actuated peristaltic pump to deliver artificial cerebrospinal fluid to a push−pull Perfusion probe, pull sample from the probe, perform on-line derivatization with o-phthaldialdehyde, and push derivatized amino acids into the flow-gated injector of a high-speed capillary electrophoresis−laser-induced fluorescence instrument. Peristalsis was achieved by sequential actuation of six, 200 μm wide by 15 μm high control valves that drove fluid through three fluidic channels of equal dimensions. Electropherograms with 100 000 theoretical plates were acquired at ∼20-s intervals. Relative standard deviations of peak heights were 4% in vitro, and detection limits for the excitatory amino acids were ∼60 nM. For in vivo measurements, push−pull probes were implanted in the striatum of anesthetiz...
