Scott A. Shippy

Excess salt removal with matrix rinsing: direct peptide profiling of neurons from marine invertebrates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) is a viable technique for the examination of biological environments. Clearly, sample preparation plays a pivotal role in the ability to obtain mass spectra from samples as complex as biological cells. The physiological salt concentrations associated with neurons from marine specimens interfere with MALDI analysis. A unique and simple rinsing procedure allows cellular clusters, individual neurons and connective tissues to be directly assayed for peptides with minimal sample handling. Isolated cells and tissues, including egg-laying hormone-releasing cells, from the central nervous systems of the model marine molluscs Aplysia californica and Pleurobranchaea californica are used to demonstrate the salt removal method. In addition to facilitating sample ionization, the MALDI matrix 2,5-dihydroxybenzoic acid serves to (i) aid in microdissections by stabilizing cell membranes, (ii) deactivate endogenous proteolytic enzymes and (iii) reduce high salt concentrations in order to improve spectral quality. Representative MALDI mass spectra are presented which indicate the presence of several neuroactive peptides previously characterized by conventional biochemical methods. More than ten individual peptides can be detected in a single cell. In spite of the chemically complex sample, the mass spectra are surprisingly free of extraneous peaks. Furthermore, both mass resolution and mass accuracy are similar to those encountered with more common MALDI samples and protocols.

Does amygdaloid corticotropin‐releasing hormone (CRH) mediate anxiety‐like behaviors? Dissociation of anxiogenic effects and CRH release

The brain corticotropin‐releasing hormone (CRH) circuits are activated by stressful stimuli, contributing to behavioral and emotional responses. The present study assessed anxiety‐like responses and in vivo neurochemical alterations at the central nucleus of the amygdala (CeA) evoked by exposure to an unfamiliar (anxiogenic) environment. Also, the impact of anxiolytic treatments and those that affect CRH were assessed in this paradigm. Novel environment (new cage) markedly suppressed ingestion of a palatable snack. This effect was dose‐dependently antagonized by diazepam and was utilized as an index of anxiety in the rodent. Although exposure to a novel environment also stimulated the in vivo release of CRH and glutamate at the CeA, various CRH antagonists (e.g. αh‐CRH, Cα‐MeCRH, CP‐154,526, antisauvagine‐30, preproTRH178‐199) did not attenuate the stressor‐elicited behavioral suppression, although Cα‐MeCRH was found to attenuate the freezing response elicited by contextual stimuli that were associated with previously administered footshock. Moreover, central infusion of CRH failed to suppress snack consumption in the home cage. Although diazepam had potent anxiolytic effects in this paradigm, this treatment did not prevent the stressor‐associated release of CRH and glutamate at the CeA. Thus, while neural circuits involving CRH and/or glutamatergic receptors at the CeA may be activated by an unfamiliar environment, the data challenge the view that activation of these receptors is necessary for the expression of anxiety‐like behavioral responses. Rather than provoking anxiety, these systems might serve to draw attention to events or cues of biological significance, including those posing a threat to survival.

Demonstration of low flow push-pull perfusion.

Methods to follow in vivo chemical composition provide information regarding the processes of intercellular communication. There is a need for methods that provide chemical information from small volumes of the central nervous system (CNS) without sacrificing neurochemical recovery. One method that offers potential for providing such information is push-pull perfusion. In this study a low flow push-pull perfusion system is introduced that provides high (70-80%) in vitro recoveries. A concentric probe design is used with a 27-gauge stainless steel outer cannula for saline infusion and an inner fused silica capillary for fluid withdrawal. Flow rates of 10-50 nl/min were reliably generated and were well matched in vitro. Sampling was performed in the striatum of an anesthetized rat generating a 0.5 microl sample every 12 min. Capillary electrophoresis was used to determine glutamate levels in each sample; the basal level was found to be 1.97+/-0.70 microM. The method described was also demonstrated to deliver L-trans-pyrrolidine-2,4-dicarboxylic acid through the perfusion solution while sampling. Post-sampling histological analysis demonstrates little tissue disturbance to the sampled region. These data provide evidence that low flow push-pull method is a viable alternative for studying neurochemical signaling in the CNS.

Regulation of Synaptic Transmission by Ambient Extracellular Glutamate

Many neuroscientists assume that ambient extracellular glutamate concentrations in the nervous system are biologically negligible under nonpathological conditions. This assumption is false. Hundreds of studies over several decades suggest that ambient extracellular glutamate levels in the intact mammalian brain are ~0.5 to ~5 µM. This has important implications. Glutamate receptors are desensitized by glutamate concentrations significantly lower than needed for receptor activation; 0.5 to 5 µM of glutamate is high enough to cause constitutive desensitization of most glutamate receptors. Therefore, most glutamate receptors in vivo may be constitutively desensitized, and ambient extracellular glutamate and receptor desensitization may be potent but generally unrecognized regulators of synaptic transmission. Unfortunately, the mechanisms regulating ambient extracellular glutamate and glutamate receptor desensitization remain poorly understood and understudied. NEUROSCIENTIST 14(2):171—181, 2008. DOI: 10.1177/1073858407308518

A role for excitatory amino acids in diabetic eye disease

Diabetic retinopathy is a leading cause of vision loss. The primary clinical hallmarks are vascular changes that appear to contribute to the loss of sight. In a number of neurodegenerative disorders there is an appreciation that increased levels of excitatory amino acids are excitotoxic. The primary amino acid responsible appears to be the neurotransmitter glutamate. This review examines the nature of glutamatergic signaling at the retina and the growing evidence from clinical and animal model studies that glutamate may be playing similar excitotoxic roles at the diabetic retina.

Analysis of trace level peptides using capillary electrophoresis with UV laser-induced fluorescence

In order to measure trace level peptides (< 1 μM) in exceedingly small sample sizes (< 100 pl) from neuronal or subneuronal samples, sophisticated separation and detection methods are required. Novel instrumentation is described that employs UV laser excitation of fluorescamine labeled peptides for fluorescence detection in capillary electrophoresis. Small injections of standard analyte mixtures or biological samples are efficiently separated on the basis of their electrophoretic mobilities in an electric field applied across the capillary. For sensitive detection, we use the 354 nm line from a helium-cadmium laser for excitation, a reflecting microscope objective for fluorescence collection, and a photomultiplier tube for photon counting. A large excess of fluorescamine derivatizes amino acids and peptides down to concentrations of 10 nM with a previously unreported but manageable fluorescence background resulting from reagent hydrolysis and impurities. This detection method gives a linear working range of four orders of magnitude and a limit of detection for labeled Aplysia californica egg-laying hormone of 2.3 × 10−9 M. Finally, assays of individual Aplysia c. neurons produce electropherograms unique to the identified neuron type and peaks corresponding to peptide standards.

Hemolymph Amino Acid Analysis of Individual Drosophila Larvae

One of the most widely used transgenic animal models in biology is Drosophila melanogaster, the fruit fly. Chemical information from this exceedingly small organism is usually accomplished by studying populations to attain sample volumes suitable for standard analysis methods. This paper describes a direct sampling technique capable of obtaining 50-300 nL of hemolymph from individual Drosophila larvae. Hemolymph sampling performed under mineral oil and in air at 30 s intervals up to 120 s after piercing larvae revealed that the effect of evaporation on amino acid concentrations is insignificant when the sample was collected within 60 s. Qualitative and quantitative amino acid analyses of obtained hemolymph were carried out in two optimized buffer conditions by capillary electrophoresis with laser-induced fluorescence detection after derivatizing with fluorescamine. Thirteen amino acids were identified from individual hemolymph samples of both wild-type (WT) control and the genderblind (gb) mutant larvae. The levels of glutamine, glutamate, and taurine in the gb hemolymph were significantly lower at 35%, 38%, and 57% of WT levels, respectively. The developed technique that samples only the hemolymph fluid is efficient and enables accurate organism-level chemical information while minimizing errors associated with possible sample contaminations, estimations, and effects of evaporation compared to the traditional hemolymph-sampling techniques.

Amino-acid levels in subretinal and vitreous fluid of patients with retinal detachment

PurposeTo compare the concentration of amino acids in subretinal and vitreous fluid of patients with primary rhegmatogenous retinal detachment to that of control vitreous.MethodsThis prospective, observational study measured amino-acid levels in subretinal fluid of patients undergoing scleral buckle placement (n=20) and vitreous fluid in patients undergoing pars plana vitrectomy (n=5) for primary retinal detachment. Vitreous fluid from patients undergoing vitrectomy for macular hole (n=7) or epiretinal membrane (n=3) served as a control. Subretinal fluid and control vitreous were analysed using high-pressure liquid chromatography. Retinal detachment vitreous was analysed using capillary electrophoresis-laser-induced fluorescence.ResultsMean levels of glutamate (27.0±1.7 μM), aspartate (4.1±4.0 μM), and glycine (44.1±31.0 μM) in subretinal fluid and glutamate (13.4±11.9 μM) in the vitreous were significantly elevated in retinal detachment compared to control vitreous. A significant, positive association was observed between levels of aspartate and glutamate in subretinal fluid (Spearmans correlation coefficient: 0.74, P<0.01). Mean arginine levels did not differ significantly between subretinal fluid and control vitreous. Levels of alanine, tyrosine, valine, isoleucine, leucine, and phenylalanine were significantly lower in subretinal fluid compared to control vitreous (all P<0.01).ConclusionsGlutamate levels in subretinal fluid and vitreous of patients with primary retinal detachment is significantly elevated in comparison to control vitreous. This finding lends further support to the hypothesis that elevated glutamate levels may result from ischaemia of the outer retina secondary to retinal detachment.

Detection of Elevated Signaling Amino Acids in Human Diabetic Vitreous by Rapid Capillary Electrophoresis

Elevated glutamate is implicated in the pathology of PDR. The ability to rapidly assess the glutamate and amino acid content of vitreous provides a more complete picture of the chemical changes occurring at the diabetic retina and may lead to a better understanding of the pathology of PDR. Vitreous humor was collected following vitrectomies of patients with PDR and control conditions of macular hole or epiretinal membrane. A capillary electrophoresis method was developed to quantify glutamate and arginine. The analysis is relatively fast (<6 minutes) and utilizes a poly(ethylene)oxide and sodium dodecylsulfate run buffer. Both amino acid levels show significant increases in PDR patients versus controls and are comparable to other reports. The levels of vitreal glutamate vary inversely with the degree of observed hemorrhage. The results demonstrate a rapid method for assessment of a number of amino acids to characterize the chemical changes at the diabetic retina to better understand tissue changes and potentially identify new treatments.

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.