James J. Valdes

Acetylcholine receptor fiber-optic evanescent fluorosensor.

An acetylcholine receptor-based optical biosensor was developed, which uses the evanescent wave to excite fluorescein isothiocyanate-labeled alpha-bungarotoxin (FITC-alpha-BGT), that is bound to a receptor protein immobilized on the surface of a quartz fiber. After excitation of the fluorophore just outside the waveguide boundary, the resultant fluorescence was trapped by and propagated back up the fiber. Pure nicotinic acetylcholine receptor (nAChR) protein, isolated from Torpedo electric organ, was immobilized noncovalently on quartz optic fibers and its density was quantitated by 125I-BGT binding. In the absence of nAChR, FITC-alpha-BGT alone bound to the quartz fiber. This nonspecific (i.e., nonreceptor) binding of FITC-alpha-BGT was totally eliminated by the addition of 0.1 mg/ml bovine serum albumin to the phosphate-buffered saline medium. This solution did not interfere with FITC-alpha-BGT binding to the nAChR that was immobilized on the fiber. Thus, only specific binding was observed under these conditions, resulting in a very high signal-to-noise ratio of greater than 99. Specific FITC-alpha-BGT binding to the nAChR protein on the optic fibers was inhibited by agonists (e.g., acetylcholine, nicotine, and carbamylcholine) and antagonists (e.g., pancuronium and d-tubocurarine) of the nAChR and was insensitive to high salt concentrations (e.g., 154 mM NaCl). The binding was most sensitive to the highly nAChR-specific alpha-BGT and alpha-cobratoxin. The optic-fiber sensor in its present form does not distinguish between receptor agonists or antagonist and cannot be regenerated for repeated use.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine Receptor-Based Biosensor

Abstract The acetylcholine (ACh) receptor protein isolated from Torpedo electric organ was incorporated into asolectin liposomes and immobilized noncovalently on the surface of a Planar Interdigitated Capacitive sensor to produce an ACh receptor-based biosensor. Capacitance of the biosensor, which was stable in buffered saline, increased rapidly when ACh was added. The response was dosedependent and specific for ACh, and the biosensor did not respond to six other neurotransmitters. Impure receptor preparations containing ACh esterase produced a smaller signal in response to ACh but showed enhanced response after inhibition of ACh esterase with diisopropylfluorophosphate. The competitive antagonist d-tubocurare, and the noncompetitive antagonist amantadine, inhibited the response of the biosensor to ACh, but a time-dependent recovery occurred upon perfusion with ACh. The irreversible antagonist α-neurotoxin inhibited the response and there was no recovery following perfusion with ACh. Thus, the pharmacolog...

Lateralization of norepinephrine, serotonin and choline uptake into hippocampal synaptosomes of sinistral rats

Abstract Rats exhibit asymmetry of turn preference which may reflect an underlying neurochemical laterality of the hippocampus, an allocortical structure implicated in spatial memory and preference. Rats were tested in a T maze to determine left (LTP) and right (RTP) turn preferences, after which uptake of tritiated serotonin, norepinephrine and choline into synaptosomes prepared from the left and right hippocampi, along with protein concentrations and wet weights, were assessed. The left hippocampi were consistently heavier then the right hyppocampi, but there were no differences in protein concentrations. LTP rats showed greater neurotransmitter uptake into the right, relative to the left, hippocampus. This asymmetry was evident to a lesser, and more variable, extent in RTP rats. These data provide the first preliminary evidence that sinistrality has neurochemical correlates at the allocortical level.

Lateralization of zinc in rat brain and its relationship to a spatial behavior

Essential metals are differentially accumulated within the brain and have been related to normal neurotransmitter metabolism. Hippocampal glutaminergic pathways have the highest zinc levels in the brain, and lesions to these pathways disrupt behaviors with a spatial component. Zinc distribution may thus reflect glutaminergic activity or innervation and may have functional consequences for spatial behavior. The present data support this hypothesis, indicating that the lateral distribution of zinc between the right and left hippocampus is strongly correlated with the spatial preference of the animal (r = + 0.72). Other parameters tested but shown not to be significantly correlated with spatial preference were zinc in corpus striatum and cortex, and copper in hippocampus corpus striatum and cortex.

Tuberohypophyseal and Tuberoinfundibular Dopamine Systems Exhibit Differential Sensitivity to Neonatal Monosodium Glutamate Treatment

The arcuate nucleus (AN) is the presumed origin of the dopaminergic innervation of posterior lobe of the pituitary. Posterior lobe dopamine levels were determined in rats that had been neonatally treated with monosodium glutamate (MSG) to lesion the AN. MSG-induced AN damage was confirmed neurochemically, histologically and immunocytochemically. MSG treatment resulted in a substantial loss of AN neurons and approximately a 50% loss of dopamine uptake capacity (Vmax) in the mediobasal hypothalamus. Posterior pituitary dopamine levels were not significantly altered by MSG-induced AN damage. These results suggest that MSG treatment spares the tuberohypophyseal dopamine system and that the AN may not be the sole origin of the dopaminergic innervation of the posterior pituitary.

Chronic Lead Administration in Neonatal Rats: Electron Microscopy of the Retina

The morphologic effects on the retina resulting from chronic lead exposure were assessed in neonatal rats. Newborn rats nursed from dams were given a low (0.115%) or a high (4.5%) concentration of lead in their diet. At day 21 the pups were weaned to the mothers diet. The retinas of the pups were studied by electron microscopy at various ages up to day 60. High and low lead concentrations produced necrosis of photoreceptor cells and cells of the inner nuclear layer. The high lead concentration, in addition, was associated with swelling of endothelial cells of the retinal vessels and narrowing of the lumen. Increased permeability of the retinal vessels and pigment epithelium to horseradish peroxidase was also observed under the high-dose condition. The authors conclude that lead can produce direct neuronal damage and, at high doses, produces retinal vascular lesions and alteration of the blood-retinal barrier.

High-Affinity Uptake of Hypothalamic Neurotransmitters in Mice Treated Neonatally with Monosodium Glutamate

Monosodium glutamate (MSG) administration to neonatal mice results in destruction of the arcuate nucleus (AN) of the hypothalamus and numerous behavioral, endocrine and neurochemical sequelae. the present study assessed high-affinity neurotransmitter uptake into hypothalamic synaptosomes isolated from adult mice which were treated on postnatal day 4 with either MSG (4 mg/g) or saline. MSG treatment produced a significant reduction in synaptosomal uptake of dopamine (DA), choline (Ch) and GABA when expressed in terms of hypothalamic wet weight. However, MSG treatment resulted in a significant loss (70%) of synaptosomal protein and consequent increases in synaptosomal uptake of these neurochemicals when expressed per unit of synaptosomal protein. The results indicate that MSG treatment produced an overall reduction in net hypothalamic uptake, with surviving neuronal elements exhibiting an increased uptake which may reflect compensatory changes in these nerve terminals. MSG may this disrupt pituitary and intrahypothalamic functions via its effects on neuronal systems of the AN.

Xanthines alter behavior maintained by intracranial electrical stimulation and an operant schedule

Caffeine and related alkylxanthines are widely used for recreation and therapeutic effects. In behavioral studies, both response rate-enhancing and attenuating effects have been described, depending upon the dose and behavioral measure used. Intracranial self-stimulation (ICSS) and differential reinforcement of low rates of responding (DRL) were assessed after rats were administered one of a range of doses of caffeine or aminophylline. These measures were chosen because of their demonstrated sensitivity to psychotropic drugs and the potential for comparing the alkylxanthine data to the extensive literature of amphetamine effects on ICSS and DRL behavior. Caffeine and aminophylline elicited dose-and drug-dependent changes in ICSS responding, and increased response rates and decreased reinforcements on the DRL schedule. These behavioral results are discussed with reference to alkylxanthine interactions with adenosine receptors.

Reusable Fiber Optic Biosensors for Detection of Drugs and Toxicants

Use of fluorescein derivatives of specific analytes, that are bound reversibly by biological sensing elements (e.g. receptors or antibodies), proved to be a successful strategy for fiber optic biosensors. Two biosensors, one using nicotinic acetylcholine receptor (nAChR) and the other using antibodies, detected anaytes in real time and were easy to regenerate for multiple measurements.