The use of vital dyes to assess embryonic viability in the hamster, Mesocricetus auratus

Experiments were designed to assess the use of the vital dyes trypan blue and fluorescein diacetate as indicators of the viability of hamster ova and embryos. Exclusion of trypan blue and fluorescence with fluorescein diacetate showed high correlations with uptake of [3H]uridine by ova and further development of embryos in vitro. Ova killed by freezing and thawing incorporated [3H]uridine at background levels. Trypan blue exclusion and fluorescein diacetate uptake were highly correlated with each other (r = 0.99). Trypan blue and fluorescein diacetate serve as excellent indices of viability in ova and early embryos of hamsters.     

The effect of diamphenethide on protein synthesis by the liver fluke, Fasciola hepatica

The effect of the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 μg ml⁻¹) on the uptake and incorporation by adult Fasciola hepatica of radioactively labelled precursors of DNA, RNA and protein synthesis ([³H]thymidine, [³H]uridine and [³H] leucine, respectively) was measured by liquid scintillation counting. Comparison was made between the effects of DAMD and those of specific inhibitors of DNA, RNA and protein synthesis, namely, 5-fluorouracil, cordycepin and cycloheximide, respectively. DAMD caused a significant decrease in the overall uptake and incorporation of [³H]uridine by F. hepatica, decreased the incorporation of [³H] leucine and also caused a significant decrease in the overall protein content of the flukes. The effect of DAMD was similar to that of cycloheximide (1 × 10⁻³M), a potent inhibitor of protein synthesis, which also caused a significant decrease in the incorporation of [³H] leucine by the fluke and a decrease in the overall protein content of the fluke. Cordycepin (100 μg ml⁻¹) caused a significant decrease in the protein content of the fluke, but had no effect on the uptake or incorporation of [³H]uridine. 5-Fluorouracil (1 × 10⁻⁴ ) did not affect the uptake or incorporation of [³H]thymidine, nor did it decrease the protein content of the fluke. The results indicate that DAMD inhibits protein synthesis by F. hepatica, possibly by inhibiting RNA synthesis. The results are also consistent with previous morphological investigations involving DAMD.     

Fluorescein Diacetate Uptake to Determine the Viability of Human Fetal Cerebral Cortical Cells

We investigated the accuracy of fluorescein diacetate uptake as an indicator of the viability of human fetal cerebral cortical cells. Cortical cells from 16-26-week-old normal fetuses were studied. The cortices were dissociated mechanically with normal saline to make a suspension. Fluorescein diacetate uptake and trypan blue exclusion were compared as methods for examining cell viability. Our results show that fluorescein diacetate uptake is a simple and sensitive method for examining human fetal cortical cell viability.     

The uptake of the GABA agonist, [H]isoguvacine, by the isolated retina of the rabbit

Previous autoradiographic studies aimed at showing neurones using GABA as their neurotransmitter have been hampered by the fact that the substance is a ubiquitous metabolite and therefore accumulated by a large variety of cells, including glia. Consequently, GABA uptake markers without this widespread uptake are desirable, and one, [³H]isoguvacine, has shown promising results in autoradiographic experiments. Its uptake has now been further studied with quantitative radiochemical techniques.

The uptake of the drug was slow compared to GABA uptake and reached a tissue/medium ratio of about 3 after 120 min. The uptake could be inhibited by GABA, beta-alanine or ouabain, and by incubating the retinas at 0°C. The uptake kinetics were complex but suggested a high affinity uptake system (K_m about 10⁻⁸ M) and perhaps one or several systems with lower affinities.

The results indicate that [³H]isoguvacine and [³H]GABA are accumulated and retained by the same neurones, which most likely use GABA as their neurotransmitter.     

Biochemical and autoradiographic investigations of the retrograde axonal transport of labeled material following [H]norepinephrine injection in the olfactory bulb

Biochemical and autoradiographic methods were used to investigate the retrograde transport of labeled material after injection of [³H]norepinephrine ([³H]NE) in the olfactory bulb (OB) of rat. Mechanical obstruction of the ventricular recess prevented intraventricular diffusion. At different time intervals following bilateral [³H]NE injections, total radioactivity was measured in the OB, locus caeruleus (LC), raphe dorsalis and periaqueductal gray. Preferential accumulation occurred in the LC, and an approximate rate of retrograde transport of 1–6 mm/h could be calculated. Previous administration of 6-hydroxydopamine in the OB reduced this accumulation by 60%. Sixteen hours after [³H]NE injection, the radioactivity in LC was equally distributed between an ethanol-soluble and -insoluble fraction. A small proportion of the soluble material was recovered as [³H]NE and/or [³H]normetanephrine. Following unilateral injections of [³H]NE, light microscopic autoradiograms demonstrated nerve cell body labeling mainly in the ipsilateral LC and of greater intensity after 16 than 4 and 8 h. These data lead to the conclusion that the movement of radioactive material was indeed representative of retrograde axonal transport rather than of other mechanisms such as diffusion. The observation of a preferential labeling of noradrenergic perikarya in LC supports the hypothesis of a process mediated by specific binding and/or uptake of [³H]NE into noradrenergic axon terminals.     

Selective changes of neurotransmitter receptors in middle-aged gerbil brain

Age-related alterations in major neurotransmitter receptors and voltage dependent calcium channels were analyzed by receptor autoradiography in the gerbil brain. [³H]Quinuclidinyl benzilate (QNB). [³H]cyclohexyladenosine (CHA), [³H]muscimol, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 were used to label muscarinic acetylcholine receptors, adenosine A₁ receptors, γ-aminobutyric acid_A (GABA_A) receptors, (NMDA) receptors, dopamine D₁ receptors, opioid receptors, and voltage dependent calcium channels, respectively. In middle-aged gerbils (16 months old), the hippocampus exhibited a significant elevation in [³H]QNB, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 binding, whereas [³H]CHA and [³H]muscimol binding showed a significant reduction in this area, compared with that of young animals (1 month). On the other hand, the cerebellum showed a significant alteration in [³H]QNB, [³H]CHA, and [³H]naloxone binding and the striatum also exhibited a significant alteration in [³H]SCH 23390 and [³H]CHA binding in middle-aged gerbils. The neocortex showed a significant elevation only in [³H]CHA binding in middle-aged animals. The nucleus accumbens and thalamus also showed a significant alteration only in [³H]muscimol binding. However, the hypothalamus and substantia nigra exhibited no significant alteration in these bindings in middle-aged gerbils. These results demonstrate the age-related alterations of various neurotransmitter receptors and voltage dependent calcium channels in most brain regions. Furthermore, they suggest that the hippocampus is most susceptible to aging processes and is altered at an early stage of senescence.     

A differential interaction of putative μ-selective agonists with opiate binding sites in rat brain

The availability of tritium-labelled sufentanil ([³H]SUF) allowed for a further radioligand analysis of opiate binding sites in rat brain. A comparison of the binding characteristics of [³H]SUF and [³H]dihydromorphine ([³H]DHM) revealed a very similar potency in their mutual displacement by unlabelled analogues. Furthermore, a series of putative μ-opiate agonists displayed equal potencies in displacing either [³H]SUF and [³H]DHM, the only striking exception being the highly μ-selective opioid peptide morphiceptin which was 33 times less potent in inhibiting [³H]SUF as compared to [³H]DHM binding. Additional experiments revealed further pronounced differences in [³H]SUF and [³H]DHM binding characteristics: the total amount of binding sites for [³H]SUF was 4 times higher than that for [³H]DHM and the regional distribution within particular brain areas displayed considerable differences. Furthermore, the binding of [³H]SUF was differentially modulated by sodium and GTP as compared to [³H]DHM binding. These data suggest that in rat brain, [³H]SUF interacts both with μ-opiate sites recognizing [³H]DHM and another type of opiate site, which cannot be equated with any of the, as yet, described δ- or κ-binding sites, and rather, represents a subclass of μ-opiate receptor sites. These experiments, thus, support the notion of subclasses (isoreceptors) for different types of opiate receptors.     

Inhibition of pinocytosis in rat yolk sac by trypan blue.

Day 17.5 yolk sacs from rats injected with partially denatured 125I-labeled bovine serum albumin (I-BSA) were cultured in vitro by a raft technique. The rates of release of [125I]iodotyrosine were similar in control yolk sacs and in yolk sacs from rats preinjected with trypan blue. Day 17.5 rat yolk sacs were also cultured in medium containing I-BSA. Following pinocytic uptake the substrate was degraded intracellularly and [135I]iodotyrosine released into the medium. Trypan blue, when present in the medium in concentrations above 100 mug/ml, inhibited pinocytosis of I-BSA and so decreased the rate of [125I]iodotyrosine production. Trypan blue similarly decreased the rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone. Pinocytic uptake of macromolecules was not decreased in yolk sacs from rats pretreated with trypan blue. The relevance of these results to the mechanism of teratogenic action of trypan blue is discussed. It is proposed that if trypan blue in teratogenic doses similarly inhibits pinocytosis by the yolk sac during the organogenetic period teratogenesis might result from a transient interruption in the flow of metabolites through the yolk sac to the embryo.     

μ-opioid receptor-mediated inhibition of the release of radiolabelled noradrenaline and acetylcholine from rat amygdala slices

Presynaptic modulation by opioids of electrically-evoked neurotransmitter release from superfused rat amygdala slices prelabelled with [³H]noradrenaline (NA) and [¹⁴C]choline was examined. Both [³H]NA and [¹⁴C]acetylcholine release were strongly inhibited by morphine, the mixed δ/μ-receptor agonist [ -Ala², -Leu⁵]enkephalin (DADLE) and the highly selective μ-agonist [ -Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO), whereas the highly selective δ-agonist [ -Pen², -Pen⁵]enkephalin and the κ-agonist bremazocine were without effect. The inhibitory effects were potently antagonized by naloxone but not by the selective δ-receptor antagonist fentanylisothiocyanate. When the selective uptake inhibitor desipramine was used to prevent uptake of [³H]NA into noradrenergic nerve terminals, but sparing the uptake into dopaminergic nerve terminals, the electrically evoked release of tritium was strongly inhibited by bremazocine but not by DADLE or DAMGO.

The data indicate, that in the amygdala transmitter release from dopaminergic nerve fibres is inhibited only via activation of κ-receptors, whereas transmitter release from noradrenergic and cholinergic nerve fibers is subjected to inhibition by opioids via activation of μ-receptors only. Regional differences and similarities of modulation of neurotransmitter release by opioids in the rat brain are briefly discussed.     

A comparison of the different methods available for determining BCG-macrophage interactions in vitro, including a new method of colony counting in broth

Abstract Different methods of determining BCG viability based on colony forming unit (CFU) counting and radio-isotope labelling were comparatively assessed. These included radio-isotope labelling with [³H]uracil, [³H]uridine, [³H]glycerol, and CFU counting, by both agar plate dilution, and microcolony counting in broth. The sensitivity ranges of the different techniques were determined in both macrophage-free and macrophage-treated systems and used to assess the anti-mycobacterial potential of human monocyte-derived macrophages following BCG infection.     

Laminar distribution of noradrenergic markers in rat visual cortex

The laminar distribution of ₁-, ₂- and β-adrenoreceptors was studied in the visual cortex of adult rat together with an investigation of noradrenaline uptake sites. The different layers of the visual cortex were separated by cutting serial cryostat sections and binding studies were performed in slide-mounted tissue sections of 10μm thickness collected from one individual cortical layer. [³H]desipramine binding, assumed to label noradrenaline uptake sites, was found to be highest in layer I by about 37%, whereas binding in the remaining layers was uniformly distributed. The laminar distribution of the ₁- and ₂-adrenoreceptors studied using [³H]prazosin and [³H]clonidine as radioligands, was similar to that of the noradrenaline uptake sites: markedly higher binding was detectable in layer I compared to the remaining layers. In contrast, the density of β-adrenoreceptors, as revealed by [³H]dihydroalprenolol binding, was highest in layers I and IV, followed by layer II/III, (58% of that in layer I and IV). Lowest binding was observed in layers V and VI (36%). The similarity in laminar distributions of -adrenoreceptors and noradrenaline uptake sites suggests a close correlation of receptor localization and fibre termination, whereas the localization of β-adrenoreceptors cannot be easily related to the pattern of noradrenergic fibres and terminals.     

Specificity of Muscarinic Acetylcholine Receptor Regulation by Receptor Activity

Abstract— Regulation of muscarinic acetylcholine receptor concentration by receptor activity in neuron-like NG108-15 hybrid cells is a highly specific process. Receptor levels, monitored by binding of [³H]quinuclidinyl benzilate ([³H]QNB), decreased 50-75% following 24-h incubation of cells with muscarinic agonists, but none of the following cellular processes was altered by this chronic receptor stimulation: (1) glycolytic energy metabolism, measured by [³H]deoxy- d -glucose ([³H]DG) uptake and retention; (2) rate of cell division; (3) transport, measured by [³H]valine and [³H]uridine uptake; (4) RNA biosynthesis, measured by [³H]uridine incorporation; (5) protein biosynthesis, measured by [³H]valine and [³⁵S]methionine incorporation into total protein and into protein fractions obtained by polyacrylamide gel electrophoresis. In contrast, chronic stimulation did cause a threefold decrease in the capacity of carbachol to stimulate phosphatidylinositol (PI) turnover, a receptor-mediated response. In addition to cholinomimetics, the neuroeffector adenosine (1 m m for 24 h) also caused a decrease in [³H]QNB binding levels, but chronic stimulation of α -adrenergic, opiate, prostaglandin E₁, and prostaglandin F_2α receptors found on NG108-15 cells caused no changes. The data indicate that loss of muscarinic receptors caused by receptor stimulation is not a consequence of fundamental changes evoked in overall cellular physiology but reflects a specific regulation of cholinoceptive cell responsiveness.     

Characteristics of nitric oxide-evoked [H]taurine release from cerebral cortical neurons

Pharmacological characteristics of [³H]taurine release evoked by nitric oxide (NO) were investigated using mouse cerebral cortical neurons in primary culture. NO generators such as S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) dose-dependently increased [³H]taurine release from neurons. Such stimulatory effects of NO generators were completely abolished by hemoglobin, a NO radical scavenger, indicating that these [³H]taurine releases might be due to NO liberated from SNAP and SNP. Sodium withdrawal from incubation buffer significantly inhibited the SNAP- and SNP-induced [³H]taurine releases, whereas the removal of calcium showed no alterations in the [³H]taurine release evoked by NO generators. β-Alanine and guanidinoethane sulfonate, inhibitors of carrier-mediated taurine transport system, inhibited the SNAP- and SNP-evoked releases of [³H]taurine in a dose-dependent manner. These results indicate that the NO-evoked [³H]taurine release from cerebral cortical neurons is mediated by the reverse process of sodium-dependent carrier-mediated taurine transport system.     

Synaptic vesicles from hog brain—their isolation and the coupling between synthesis and uptake of γ-aminobutyrate by glutamate decarboxylase

Purified synaptic vesicles were isolated from hog cerebral cortex by a rapid procedure consisting of homogenization of cerebral cortex slices in iso-osmotic sucrose, differential centrifugation and sucrose density-gradient centrifugation. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contained high amounts of γ-aminobutyrate (GABA) and acetylcholine at specific concentrations of 390 nmol/mg protein and 7.2 nmol/mg protein respectively.

Glutamate decarboxylase, the enzyme which catalyses GABA formation, binds to the synaptic vesicles in a calcium-dependent manner. The percentage of glutamate decarboxylase bound to the vesicles increases from about 5% without calcium, reaching a plateau of about 60% at 4 mM Ca²⁺. Magnesium in concentrations 0.2–10 mM has no significant effect on glutamate decarboxylase binding. Also in phospholipid vesicles (small unilamellar phosphatidylserine-phosphatidylcholine. 2:1 liposomes) Ca²⁺, but not Mg²⁺, induced the binding of glutamate decarboxylase, reaching a plateau of 50% at 2 mM Ca²⁺. Both in synaptic vesicles and in phospholipid vesicles the calcium-dependent glutamate decarboxylase binding seems to be specific, and not caused by unspecific association of proteins, since the specific binding (bound enzyme activity/mg bound protein) increases 3-fold from 0 to 4 mM Ca²⁺.

The functional role of this binding was studied in GAD containing vesicles by measuring the relationship between the accumulation of [³H]GABA, newly synthetized from [³H]glutamate, and the uptake of added [¹⁴C]GABA. No significant uptake of [¹⁴C]GABA was found under the experimental conditions used, whereas large amounts of [³H]GABA were found within the vesicles. It appears that the [³H]GABA accumulation process is functionally linked to [³H]GABA synthesis and is mediated by the membrane-bound glutamate decarboxylase. This synthesis-coupled uptake of GABA into synaptic vesicles possibly serves to bring about a plasticity effect in previously stimulated GABAergic nerve endings.     

[H]WAY–100635 for 5–HT1A receptor autoradiography in human brain: a comparison with [H]8–OH–DPAT and demonstration of increased binding in the frontal cortex in schizophrenia

WAY–100635 is the first selective, silent 5–HT_1A (5-hydroxytryptamine_1A, serotonin-1A) receptor antagonist. We have investigated the use of [³H]WAY–100635 as a quantitative autoradiographic ligand in post-mortem human hippocampus, raphe and four cortical regions, and compared it with the 5–HT_1A receptor agonist, [³H]8–OH–DPAT. Saturation studies showed an average Kd for [³H]WAY–100635 binding in hippocampus of 1.1 nM. The regional and laminar distributions of [³H]WAY–100635 binding and [³H]8–OH–DPAT binding were similar. The density of [³H]WAY–100635 binding sites was 60–70% more than that of [³H]8–OH–DPAT in all areas examined except the cingulate gyrus where it was 165% higher. [³H]WAY–100635 binding was robust and was not affected by the post-mortem interval, freezer storage time or brain pH (agonal state). Using [³H]WAY–100635, we confirmed an increase of 5–HT_1A receptor binding sites in the frontal cortex in schizophrenia, previously demonstrated with [³H]8–OH–DPAT. Compared to [³H]8–OH–DPAT, [³H]WAY–100635 has two advantages: it has a higher selectivity and affinity for the 5–HT_1A receptor, and it recognizes 5–HT_1A receptors whether or not they are coupled to a G-protein, whereas [³H]8–OH–DPAT primarily detects coupled receptors. Given these considerations, the [³H]WAY–100635 binding data in schizophrenia clarify two points. First, they indicate that the elevated [³H]8–OH–DPAT binding seen in the same cases is attributable to an increase of 5–HT_1A receptors rather than any other binding site. Second, the enhanced [³H]8–OH–DPAT binding in schizophrenia reflects an increased density of 5–HT_1A receptors, not an increased percentage of 5–HT_1A receptors which are G-protein-coupled. We conclude that [³H]WAY–100635 is a valuable autoradiographic ligand for the qualitative and quantitative study of 5–HT_1A receptors in the human brain.     

Fluoxetine-induced inhibition of synaptosomal [H] 5-HT release: Possible CA-channel inhibition

Fluoxetine, a selective 5-HT uptake inhibitor, inhibited 15 mM K⁺-induced [³H] 5-HT release from rat spinal cord and cortical synaptosomes at concentrations > 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K⁺ used to depolarize the synaptosomes and the concentration of external Ca²⁺. Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [³H] 5-HT release induced by the Ca²⁺-ionophore A 23187 or Ca²⁺-independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K⁺-induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca²⁺ channels and Ca²⁺ entry. Whereas fluoxetine and paroxetine inhibited binding of [³H] nitrendipine to the dihydropyridine-sensitive L-type Ca²⁺ channel, the less selective uptake inhibitors did not alter binding. The dihydropyridine antagonist nimodipine partially blocked fluoxetine-induced inhibition of release. Moreover enhanced K⁺-stimulated release due to the dihydropyridine agonist Bay K 8644 was reversed by fluoxetine. Fluoxetine also inhibited the K⁺-induced increase in intracellular free Ca²⁺ in fura-2 loaded synaptosomes. These data are consistent with the suggestion that fluoxetine inhibits K⁺-induced [³H] 5-HT release by antagonizing voltage-dependent Ca²⁺ entry into nerve terminals.     

The short term effects of 5,7-dihydroxytryptamine on peripheral serotonin stores in Rhodnius prolixus and their long-term recovery

The serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) appears to affect invertebrate systems differently from vertebrate ones. The basis for toxicity in vertebrates appears to involve the intraneuronal actions of monoamine oxidase (MAO) upon the toxin. In insects, MAO is not present in appreciable amounts. In this study, we demonstrate that in vitro 5.7-DHT competitively inhibits the uptake of [³H]serotonin by serotonergic neurohaemal areas. The apparent K_M increases from 4.9 × 10⁻⁷ to 1.7 × 10⁻⁶ M. This neurotoxin also causes a significant release of previously accumulated [³H]serotonin in nominally Ca²⁺-free saline. While 5,7-DHT does not affect the uptake of [³H]tryptophan, it reduces the subsequent synthesis of [³H]serotonin. In vivo, the tissues appear to have recovered 2 weeks after toxin treatment, as determined by immunohistochemistry. At 24 h, 1 week and 2 weeks after injection, the tissues are able to take up and release [³H]serotonin normally. 1 and 2 weeks after injection, insects ingest a normal-sized blood meal, a behaviour acutely disrupted by 5,7-DHT treatment. The results of this and other invertebrate studies suggest that 5,7-DHT does not destroy serotonergic neurons, as it does in vertebrates. 5,7-DHT may be a more useful tool to study the functions of serotonin in invertebrates as one may transiently affect serotonin stores.     

[H]lysergic acid diethylamide (LSD): differential agonist and antagonist binding properties at 5-HT receptor subtypes in rat brain

[³H]Lysergic acid diethylamide (LSD) in the presence of 40 nM ketanserin labeled the 5-HT_1A receptor subtype in rat hippocampal membranes. In the presence of guanosine triphosphate (GTP), the B_max and affinity of [³H]LSD binding to the 5-HT_1A binding site were significantly decreased. [³H]LSD in the presence of 40 nM WB4101 labeled the 5-HT₂ receptor subtype in homogenates of rat frontal cortex. In contrast to the effect on [³H]LSD binding to the 5-HT_1A binding site, GTP produced no significant effect on either the B_max or the K_D of [³H]LSD binding to the 5-HT₂ binding site. Competition of 5-HT for [³H]LSD binding to the 5-HT₂ binding site was best described by a computer-derived model assuming two binding sites. In the presence of GTP, the 5-HT competition curve was shifted significantly to the right with an approx. 3-fold increase in the IC₅₀. These binding characteristics are consistent with [³H]LSD acting as an antagonist at the 5-HT₂ receptor which has multiple affinity states for agonists and is coupled to a guanine nucleotide regulatory subunit. Thus, [³H]LSD has binding characteristics consistent with it acting as an agonist at the 5-HT_1A receptor subtype but as an antagonist at the 5-HT₂ receptor subtype in rat brain.     

Role of antioxidants in the nitric oxide-elicited inhibition of dopamine uptake in cultured mesencephalic neurons. Insights into potential mechanisms of nitric oxide-mediated neurotoxicity

Under aerobic conditions the addition of (C₂N₅)₂N(N[O]NO)⁻ · Na⁺(DEA/NO), S-nitroso-N-macetyl penicillamine and nitric oxide (NO)-saturated buffer, but not S-nitroso- -glutathione, to dopamine solutions resulted in dopamine o-semiquinone formation that was dependent on the formation of a NO/oxygen intermediate. High pressure liquid chromatography (HPLC) electrochemical analysis of dopamine demonstrated that the DEA/NO-induced oxidation of dopamine was abrogated in the presence of the antioxidants, ascorbate and glutathione. NO spontaneously released from DEA/NO decreased [³H]dopamine accumulation in primary cultures of mesencephalic neurons in a dose-dependent fashion. In contrast, [³H]γ-aminobutyric acid uptake by mesencephalic neurons tested under the same conditions was unchanged. When DEA/NO was added to incubation buffer that contained [³H]dopamine and the antioxidant, ascorbate or glutathione, [³H]dopamine uptake was also inhibited. These data excluded that oxidation of extracellular [³H]dopamine by the intermediates of the NO/O₂ reaction could have caused this decrease. Instead, NO may have acted directly on a not yet identified target operative in the regulation of dopamine storage and release. Analysis of the rate constants for the NO reaction with ascorbate, glutathione and dopamine revealed that dopamine quinone formation was delayed by the presence of antioxidants. Since the formation of NO as well as neurotransmitter release are activated during ischemia reperfusion injury, it is possible that prolonged NO exposure could deplete antioxidants and facilitate the oxidation of dopamine and thereby cause neurotoxicity.     

Retrograde axonal transport after radioactive hydroxyindole injections into the olfactory bulb—an autoradiographic study

Light microscope autoradiography was used to study the retrograde transport of labelled material after injection of [³H]serotonin ([³H]5-HT), [³H]5-hydroxytryptophan ([³H]5-HTP) and [¹⁴C]5-hydroxyindoleacetic acid ([¹⁴C]5-HIAA) into the olfactory bulb (OB) of rat. A perikaryal labelling was clearly visualized in the Raphe Dorsalis (RD) and the Raphe Centralis (RC) 24 h after injection of [³H]5-HT (but not after injection of [³H]5-HTP or [¹⁴C]5-HIAA) into the OB of rats without monoamine-oxidase inhibitor (MAOI). In the OB, the labelled cells (mitral, granular, periglomerular and tufted cells) and the varicosities (dispersed in granular, plexiform and glomerular layers) were greater in number and intensity at 8 h than at 24 h after [³H]5-HT (10⁻³ M) injection. Five hours after injection of [¹⁴C]5-HIAA (10⁻³ M) some mitral, granular and tufted cells were labelled in the cytoplasm, nuclei and dendrites. A few varicosities were also observed. In contrast, after [³H]5-HTP injection no clear labelling was visualized in axonal processes. A net autoradiographic reaction was seen, however, in the capillary walls and some granular cells.

After injection of [³H]5-HT at various concentrations (10⁻² M to 10⁻⁵ M) into the OB of rats pretreated with MAOI, a selectivity in the pattern of labelling in the injection site and the afferent cell bodies was found at 10⁻⁴ M and 10⁻⁵ M. At these concentrations, the serotoninergic RD and RC neurons were clearly labelled, but the non-serotoninergic neurons such as those originating in the Locus Coeruleus, prepiriform cortex were devoid of label. In the OB, only varicosities and fiber-like structures were reactive. In the RD cell bodies, the intensity of labelling as well as the number of labelled cells were greater at higher concentrations of injected [³H]5-HT and when rats were pretreated with a MAOI.