Optimal excitation and emission wavelengths to analyze amino acids and optimize neurotransmitters quantification using precolumn OPA-derivatization by HPLC.

We describe an analytical methodology to obtain high sensitivity and better resolution through the study of fluorometric excitation (λex) and emission (λem) spectrum wavelengths of OPA-amino acids. The spectrum emission study revealed a maximum signal peak at 450 nm for aspartate and glutamine. For glycine, taurine, and GABA, the maximum signal peak was at 448 and for glutamate at 452 nm. The remaining amino acids analyzed showed a maximum emission around 450 nm. The best signal obtained within the spectrum excitation experiments was using 229- to 450-nm λex-λem. The drawbacks observed at these wavelengths were a baseline drift and negative peaks occurrence. Thus, the excitation wavelength of 240 nm was chosen (240- to 450-nm λex-λem) as a compromise between a very good signal response and a baseline stability to resolve the 18 amino acids studied. Furthermore, this protocol was properly validated. On the other hand, the elution gradient program used for neuroactive amino acids (aspartate, glutamate, glycine, taurine and GABA) showed separation to the baseline, in a 15-min run in all of them. Other amino acids, up to 18, also exhibited a very good separation in a 25-min run. In conclusion, we propose the use of 240- to 450-nm λex-λem wavelengths, in OPA-amino acids analysis, as the most suitable protocol to obtain the best signal response, maintaining an optimum chromatographic resolution.

[Acute tryptophan depletion in eating disorders]. / Depleción aguda de triptófano en trastornos de la conducta alimentaria.

INTRODUCTION: This work describes the rational bases justifying the use of acute tryptophan depletion technique in eating disorders (ED) and the methods and design used in our studies. Tryptophan depletion technique has been described and used in previous studies safely and makes it possible to evaluate the brain serotonin activity. Therefore it is used in the investigation of hypotheses on serotonergic deficiency in eating disorders. Furthermore, and given the relationship of the dysfunctions of serotonin activity with impulsive symptoms, the technique may be useful in biological differentiation of different subtypes, that is restrictive and bulimic, of ED. METHODS: 57 female patients with DSM-IV eating disorders and 20 female controls were investigated with the tryptophan depletion test. A tryptophan-free amino acid solution was administered orally after a two-day low tryptophan diet to patients and controls. Free plasma tryptophan was measured at two and five hours following administration of the drink. Eating and emotional responses were measured with specific scales for five hours following the depletion. A study of the basic characteristics of the personality and impulsivity traits was also done. Relationship of the response to the test with the different clinical subtypes and with the temperamental and impulsive characteristics of the patients was studied. RESULTS: The test was effective in considerably reducing plasma tryptophan in five hours from baseline levels (76%) in the global sample. The test was well tolerated and no severe adverse effects were reported. Two patients withdrew from the test due to gastric intolerance. CONCLUSIONS: The tryptophan depletion test could be of value to study involvement of serotonin deficits in the symptomatology and pathophysiology of eating disorders.

In vitro and in vivo characterization of neural stem cells.

Neural stem cells are defined as clonogenic cells with self-renewal capacity and the ability to generate all neural lineages (multipotentiality). Cells with these characteristics have been isolated from the embryonic and adult central nervous system. Under specific conditions, these cells can differentiate into neurons, glia, and non-neural cell types, or proliferate in long-term cultures as cell clusters termed "neurospheres". These cultures represent a useful model for neurodevelopmental studies and a potential cell source for cell replacement therapy. Because no specific markers are available to unequivocally identify neural stem cells, their functional characteristics (self-renewal and multipotentiality) provide the main features for their identification. Here, we review the experimental and ultrastructural studies aimed at identifying the morphological characteristics and the antigenic markers of neural stem cells for their in vitro and in vivo identification.

Developmental expression of fibroblast growth factor (FGF) receptors in neural stem cell progeny. Modulation of neuronal and glial lineages by basic FGF treatment.

Neural stem cells (NSCs) are self-renewable, multipotential cells capable of differentiating into the three major neural cell types, but the mechanisms which regulate their development are not fully understood. Both basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promote the proliferation of NSCs. However, studies on the role of FGFs in the differentiation of EGF-expanded NSCs are still incomplete. We have studied the expression of distinct FGF receptors (FGFRs) in the progeny of EGF-expanded NSCs isolated from E15 rat striatum. In situ hybridization analysis and immunocytochemistry showed a developmentally related expression pattern and a cell lineage-specific distribution of these receptors. FGFR1 and FGFR2 were identified in many early precursors and in the oligodendrocyte lineage. The latter receptor was also present in a subpopulation of astrocytes. FGFR3 was detected in a restricted population of early precursors, in oligodendroglial progenitors, and in neurons and protoplasmic astrocytes of late-term cultures. Basic FGF treatment of the progeny of NSCs increased the proliferative rate of precursors and the number of oligodendrocytes generated, whereas the number of differentiating neurons was significantly reduced. Together these data provide evidence that FGFs modulate the development of EGF-expanded NSCs, and that this is at least partly determined by a cell lineage-specific expression of multiple FGFRs.

Hyperhomocyst(e)inemia is a risk factor of secondary vascular events in stroke patients.

OBJECTIVE: Moderate hyperhomocyst(e)inemia is an independent risk factor for stroke, but it is unclear whether it also would be a risk factor for secondary vascular events after stroke. METHODS: Longitudinal study of 137 consecutive ischemic stroke patients (age 45-91 years) who were prospectively studied with a standard clinical protocol. Vascular events (stroke recurrence, ischemic heart disease, deep venous thrombosis or peripheral arterial disease) were identified during 2 years of follow-up. Serum homocyst(e)ine was determined 3 months after the stroke. The cumulative proportion of patients with homocyst(e)ine above or below the 75th percentile who survived free of vascular events was determined by Kaplan-Meier analysis. Cox models were used to estimate the relative risk of vascular events after controlling for other confounding factors. RESULTS: Serum homocyst(e)ine was significantly higher in patients with vascular events (26.2 versus 19.4 micromol/l; p = 0.016). The cumulative proportion of patients with vascular events was 46.5% in the group with homocyst(e)ine over the 75th percentile (>30 micromol/l) and 20.2% in the other group (log-rank test 7.5; p = 0.0062). After adjustment for age, sex, high blood pressure, diabetes, heart disease, previous cerebrovascular disease, smoking and serum cholesterol, the relative risk of vascular event for patients above compared with those below the 75th percentile of serum homocyst(e)ine was 2.8 (CI 95% 1.3-6; p = 0.01). CONCLUSION: Hyperhomocyst(e)inemia is a significant risk factor for vascular events after ischemic stroke. This finding is independent of other risk factors such as hypertension, and may have therapeutic relevance in the secondary prevention of vascular diseases in stroke patients.

Nitric oxide induces differentiation in the NB69 human catecholamine-rich cell line.

The nitric oxide (NO) donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), induced differentiation of human neuroblastoma NB69 cells to a dopamine phenotype, as shown by phase-contrast microscopy and tyrosine hydroxylase (TH) immunocytochemistry. NB69 cells were treated with 50 to 750 microM SNAP in serum-free-defined medium for 24 h. SNAP treatment did not increase the number of necrotic or apoptotic cells. However, a decrease in the number of viable cells was observed at 750 microM SNAP. In addition, a decrease in (3)H-thymidine uptake was detected at the highest dose of SNAP. An increase in the antiapoptotic Bcl-2 and Bcl-xL protein levels and a decrease in the proapoptotic Bax and Bcl-xS protein levels were also detected by Western blot analysis after SNAP treatment. At low doses (50-125 microM), SNAP induced an increase in catecholamine levels, (3)H-dopamine uptake, TH activity and monoamine metabolism, while a decrease in all these parameters was observed at high doses (250-750 microM). The TH protein content, analyzed by Western blot, remained unchanged in SNAP-treated cells throughout the range of doses studied, when compared with the control group. SNAP produced a dose-dependent decrease in the glutathione (GSH) content of the culture medium, without altering intracellular GSH. In addition, cGMP levels and nitrite concentration, measured in the supernatant of SNAP-treated cells, increased in a dose-dependent manner, as compared to control levels. The guanylate cyclase inhibitor lH-[1,2, 4]oxadiazolo[4,3a]quinoxaline-l-one (ODQ) did not revert the SNAP-induced effect on (3)H-dopamine uptake to control values. These results suggest that NO, released from SNAP, induces differentiation of NB69 cells and regulates TH protein at the post-transcriptional level through a cGMP-independent mechanism.

Localization of gliosis in the corpus striatum of mice following cortical lesions.

Localized lesions of the medial and lateral frontal cortex were used to study gliosis, neurofilament content and changes in synaptic density in the mouse striatum. Relationships between the sites of cortical lesions and the localization of changes in different regions of the striatum were examined after 3 and 12 weeks. Independent of the location of frontal cortex lesions, glial fibrillary acidic protein (GFAP) immunoreactivity was increased throughout the entire striatum after 3 weeks. Twelve weeks after lesioning, increases in GFAP were confined to the dorsomedial (DM) striatum following medial cortical lesions, and to the dorsolateral (DL) striatum following lateral cortical lesions, suggesting persistent gliosis only in areas of striatal deafferentation. It appears, therefore, that the mechanisms which induce gliosis after short and long time periods are different.

Effects of frontal cortical lesions on mouse striatum: reorganization of cell recognition molecule, glial fiber, and synaptic protein expression in the dorsomedial striatum.

Brain injury induces trophic effects within adjacent tissue through an unknown molecular mechanism. One model of this lesion effect involves the enhanced outgrowth of neuronal processes from transplanted substantia nigra in animals with cerebral cortex lesions. Since cell recognition molecules are involved in the molecular mechanisms of contact between cells and surrounding extracellular matrix components, and are important in plasticity of the nervous system, we investigated changes in L1, N-CAM, and tenascin, as well as synapse-associated proteins and gliosis, in the striatum of mice with cortical lesions. The removal of somato-sensory and motor cortex would be expected to produce changes predominantly in the dorsal striatum. Lesioned mice, however, showed a significant enhancement of both L1 and N-CAM immunostaining intensity only within the most medial-periventricular and dorsomedial parts of the striatum, as compared to the nonlesioned side. Tenascin expression was significantly decreased, but only in the most medial part of the striatum. The changes in intensity of immunostaining with L1, N-CAM, and tenascin did not diminish with time after lesioning. These changes in cell recognition molecule expression indicate a possible molecular basis of lesion-induced plasticity in neuronal circuits within the dorsomedial striatum. These changes were accompanied by decreased synapsin and synaptophysin expression, but without any significant change in neurofilament expression. In contrast, glial fibrillary acidic protein and vimentin immunoreactivities were increased in almost the entire striatum on the lesioned side. Therefore, the areas of changes in cell recognition molecule expression did not simply correlate to the increased astrogliosis or neuronal fiber damage. We postulate that the periventricular dorsomedial striatum is relatively sensitive to disturbances of corticostriatonigral circuits and, simultaneously, this striatal area has a unique ability to support and promote neurite growth.

Weak organic acids induce taurine release through an osmotic-sensitive process in in vivo rat hippocampus.

Isotonic media containing sodium salts from weak organic acids induce cell swelling in several experimental preparations (Grinstein et al., 1984; Jakubovicz et al., 1987). In vivo perfusion of rat dentate gyrus, using a microdialysis probe, with modified Krebs-Ringer bicarbonate solutions in which 50 mM NaCl was isotonically substituted by the sodium salts from organic acids with a pKa value of greater than 2 (acetate, propionate, or pyruvate), induced a reversible increase in the extracellular taurine concentration. By contrast, similar NaCl substitutions with sodium salts from the stronger organic acids isethionate and methane-sulfonate did not change extracellular taurine levels. Extracellular taurine increases evoked by acetate, propionate, or pyruvate were almost completely abolished when the perfusion liquid was made hypertonic by adding sucrose (50 mM). A 30% reduction of the acetate-induced extracellular taurine increase was observed both when amiloride was present or when the [Na+]0 was lowered. Both conditions are known to inhibit Na+/H+ exchange. These results are compatible with the hypothesis that acid load-induced taurine release is stimulated by an osmotic sensitive mechanism, part of which is dependent on activation of the Na+/H+ exchange.

The epileptogenic action of the taurine analogue guanidinoethane sulfonate may be caused by a blockade of GABA receptors.

The aim of this paper is to clarify the mechanism through which the taurine analogue guanidinoethane sulfonate (GES) produces its epileptogenic effects. Experiments were performed in the rat hippocampus in vivo, using a brain dialysis probe also containing a recording electrode. Perfusion of 10 mM GES induced an enhancement of extracellular taurine levels probably as a result of forced efflux through the taurine uptake systems in a heteroexchange process. This taurine increase was highly reversible. GES also induced an increase of neuronal excitability and an impairment of recurrent inhibition as judged by the neuronal pattern discharge of evoked potentials. These results indicate the possible implication of GABA receptors in the epileptogenic effect of GES. Specific binding of [3H]-GABA to P2 fractions was inhibited by both bicuculline methiodide (BMI) and GES with the same potency. Similar results were obtained using cerebral sections. Autoradiographic experiments confirm the binding results. GES and BMI completely displaced [3H]-GABA binding. All these results suggest that the epileptogenic GES action is due to a direct antagonism on GABAA receptors.

Extracellular taurine increase in rat hippocampus evoked by specific glutamate receptor activation is related to the excitatory potency of glutamate agonists.

Taurine increases in brain extracellular space due to glutamate agonists were studied in vivo in the rat hippocampus using a dialysis technique, both in the absence and in the presence of glutamate receptor antagonists. Extracellular taurine levels increased during perfusions of agonists, listed in descending order of potency: kainate (KA), N-methyl-D-aspartate (NMDA), and quisqualate (QA). While taurine increases due to KA or QA perfusions were inhibited by 6,7-dinitro-quinoxaline-2,3-dione (DNQX), those induced by NMDA were abolished in the presence of 3-(carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). These results indicate that increases in extracellular taurine levels evoked by NMDA, KA or QA in the rat hippocampus are caused by activation of their specific receptors. Field potentials, concomitantly recorded, were quickly abolished during NMDA or KA perfusions (0.1 mM), while QA (0.25 mM) induced the appearance of bicuculline-like evoked responses. Since taurine has been proposed as an osmoregulatory substance in the rat brain, and cell swelling is known to be an early component of glutamate agonists neurotoxicity, the increases in extracellular taurine reported here could be due to taurine released through an osmoregulatory process, counteracting the neurotoxic cellular oedema induced by glutamate agonists.

Synaptic transmission at the Schaffer-CA1 synapse is blocked by 6,7-dinitro-quinoxaline-2,3-dione. An in vivo brain dialysis study in the rat.

6,7-Dinitro-quinoxaline-2,3-dione (DNQX, FG 9041), a new non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, has been reported to block non-NMDA receptor-mediated excitatory amino acidic responses in cultured neurons. We have perfused this compound in vivo through a dialysis fiber placed in the CA1 regions of anesthetized rats to test its effects on CA1 field-evoked potentials. Perfusions of 25-100 microM DNQX completely abolished field excitatory postsynaptic potentials (EPSP) and orthodromic population spikes (PS). This effect was dose-dependent and was reversed after washing with fresh Krebs-Ringer-bicarbonate. Antidromic population spikes and fiber volley potentials were unaffected by perfusions of DNQX up to 100 microM. On the contrary, perfusion of 50 microM D-2-amino-5-phosphonovalerate, a specific NMDA receptor antagonist, left unchanged both field EPSP and orthodromic PS. Results demonstrate that low-frequency transmission at the Schaffer collaterals-CA1 synapse is mediated by non-NMDA glutamate receptors.

Sensory modulation of hippocampal transmission. II. Evidence for a cholinergic locus of inhibition in the Schaffer-CA1 synapse.

The present work studied the neurotransmitter mediating the depressive effect of sensory stimulation on the Schaffer-CA1 transmission. Field responses of the CA1 region evoked by ipsilateral CA3 stimuli were recorded in paralyzed, locally anesthetized rats following the same experimental paradigm as in the previous work. The tissue zone under recording was perfused in vivo by an implanted hollow fiber (brain dialysis device) with either Krebs-Ringer bicarbonate (KRB), or KRB with penicillin, atropine, acetylcholine or eserine. Results were the following: (1) atropine increased the field excitatory postsynaptic potential (EPSP) amplitude in a dose-dependent manner and totally abolished the modulatory action of sensory stimulation; (2) both the field EPSP and the modulatory action of sensory stimulation remained unaltered during the blockade of GABAergic activity by penicillin; (3) acetylcholine as well as eserine induced a great diminution of both field EPSP and population spike amplitudes, without altering the effect of sensory stimulation; (4) penicillin and atropine induced multiple population spikes, reversing the effect of sensory stimulation and increasing the cell excitability. These results demonstrate that the sensory modulation of information transfer through the Schaffer-CA1 synapse is mediated by a muscarinic cholinergic mechanism. The dose-dependent increase in the field EPSP by muscarinic blockade is evidence for the existence of a cholinergic presynaptic inhibition on the Schaffer collateral terminals.

Does taurine act as an osmoregulatory substance in the rat brain?

The effects of hypotonic media on extracellular free amino acid levels were studied 'in vivo' in the rat dentate gyrus by means of the brain dialysis technique. Extracellular taurine levels increased specifically during perfusions with Krebs-Ringer bicarbonate in which the NaCl concentration was reduced by 25 or 50 mmol/l (hypotonic solutions). These taurine increases were markedly reduced in the presence of furosemide. With further NaCl reductions the enhanced taurine levels remained stable, whereas other amino acids such as glutamate started in increase in a dose-dependent manner. Isoosmolar replacement of NaCl by sucrose did not affect extracellular amino acid levels. These results indicate the possible involvement of taurine in osmoregulatory processes in the brain.

Low chloride-dependent release of taurine by a furosemide-sensitive process in the in vivo rat hippocampus.

Extracellular amino acid levels and field potentials evoked by perforant pathway stimuli were studied in vivo by means of a dialysis device, perfusing the rat dentate gyrus with low chloride solutions. When balanced with acetate, these perfusions enhanced the granule cell population spike amplitude. A specific extracellular taurine enhancement occurred whenever Cl- was replaced by acetate solution, reaching an increase of 20-fold over the basal taurine levels when 125 mM Cl- was replaced, whereas other amino acids remained unchanged. A considerable degree of Cl- replacement with iodide was needed, however, to obtain significant increases of extracellular taurine. Perfusions with bromide instead of Cl- did not cause any change in levels of extracellular amino acids including taurine. Furosemide, an inhibitor of Cl- transport, greatly reduced the taurine increase evoked by the low extracellular concentration of permeant anions. This drug also inhibited the taurine release induced by perfusion with 9 mM K+. These findings indicate that the extracellular increase of taurine, evoked by low permeant anion concentrations, may result from the taurine release through a furosemide-sensitive process.

Effects of dihydrokainic acid on extracellular amino acids and neuronal excitability in the in vivo rat hippocampus.

The effect of inhibition of the high-affinity uptake of glutamate on the extracellular concentration of amino acids and on neuronal excitability was studied in vivo in the hippocampus of the rat. The dentate gyrus or CA1 field were perfused through a dialytrode with Krebs-Ringer-bicarbonate or dihydrokainic acid solutions. The spontaneous electrical activity and evoked field potentials were recorded concomitantly at dendritic or somatic levels. The results showed that with dihydrokainic acid: the extracellular concentrations of both glutamate and taurine were markedly increased in both areas of the hippocampus, the response of taurine being greater in CA1, while other amino acids were unaffected; in the dentate gyrus, the field excitatory postsynaptic potential was decreased while the population spikes were augmented, indicating an increased excitability of the neuronal population. In CA1, both the excitatory postsynaptic potential and spikes were reduced in amplitude. These results indicate that changes in the extracellular concentration of endogenous glutamate influences excitability of the tissue and that inhibition of the uptake processes for putative amino acid neurotransmitters increases the postsynaptic action of synaptically-released endogenous amino acids.

In vivo determination of extracellular concentration of amino acids in the rat hippocampus. A method based on brain dialysis and computerized analysis.

Extracellular (EC) concentrations of amino acids were determined in the rat dentate gyrus by means of non-linear regression analysis of 'in vivo' brain dialysis data, considering a simple model of diffusion through a dialysis membrane. The apparent diffusion constants (K) of several amino acids were also calculated in the 'in vivo' situation. While putative amino acid neurotransmitters (glutamate, aspartate and gamma-aminobutyric acid (GABA) were present in the EC fluid at the low micromolar range (0.8-2.9 microM), glutamine was by far the most prominent (193.4 microM). The values of intra/extracellular concentration ratios formed 3 groups: high (greater than 2000) for putative neurotransmitters; low (less than 100) for serine, glutamine, arginine and alpha-alanine; and intermediate (about 400) for taurine. The 'in vivo' calculated K values proved useful for estimation of both basal and changing EC concentrations of amino acids in relatively brief perfusions. These data were evaluated in terms of the functional significance of absolute EC concentrations and tissue-EC fluid ratios. Present findings indicate the simultaneous existence of both an inhibitory and an excitatory tonus as well as the utility of high intra/extracellular concentration ratios in determination of the possible neurotransmitter role of specific amino acids.

Variation of potassium ion concentrations in the rat hippocampus specifically affects extracellular taurine levels.

The effects of different K+ concentrations (3-100 mM) on both the extracellular amino acid levels and field potentials, evoked by perforant pathway stimulation, were studied 'in vivo' in the rat dentate gyrus by means of a brain dialysis device, formed by a hollow fiber plus a stainless-steel electrode. Perfusion with low K+ concentrations (3-12 mM; Krebs-Ringer bicarbonate) specifically enhanced the dialysate levels of taurine and concomitantly increased the population spike amplitude. High K+ concentrations in perfusate (greater than 25 mM) did not further increase the levels of taurine but enhanced both glutamate and gamma-aminobutyric acid levels, whereas the population spike diminished drastically. The absence of calcium ions in the perfusion liquid increased both basal and K+-enhanced taurine levels. The specific enhancement of extracellular taurine by physiological K+ concentrations may represent an autoregulative mechanism of nervous tissue excitability.