Autoradiographic analysis of [3H]dopamine and [3H]dopa uptake in the turtle olfactory bulb

Uptake and retention of exogenous tritiated dopamine and L-dopa was observed within turtle olfactory bulb slices. In the more superficial layers, periglomerular and superficial tufted cells, as well as their processes, and intraglomerular dendrites were recognized as labeled. Within the deeper part of the bulb, some labeled cells between the tanycytes, as well as nerve fibers and terminals within the granule cell layer, are reported. The results confirm the presence of specific intrinsic dopaminergic cells within the reptilian olfactory bulb.     

Time course of postmortem modifications in synaptosomal [3H]dopamine uptake and [3H]GBR 12783 binding to the dopamine uptake complex in the rat striatum

The present study focuses on the changes of two biochemical markers of the striatal dopaminergic innervation evaluated after different postmortem storage periods of rat heads either at room temperature (21 degrees C) or at 4 degrees C: (i) the uptake of [3H]dopamine (DA) into striatal synaptosomes and (ii) the specific binding of [3H]GBR 12783, a selective ligand for the neuronal dopamine uptake sites, to a striatal membrane fraction. The uptake of [3H]DA was completely abolished after 24 and 72 h storage of the tissue at 21 degrees C and 4 degrees C, respectively, whereas in the same conditions, the binding of [3H]GBR 12783 was slightly decreased. The Km and Kd of these two processes were virtually unchanged after the different storage periods considered, whereas the Vmax and Bmax were markedly decreased.     

2-[3H]deoxyglucose uptake patterns in rats exploring a six-arm radial tunnel maze: differences between experienced and nonexperienced rats

In an automated tunnel maze, rats were allowed to explore either a 6-arm radial configuration ("experienced") or an alley maze configuration ("nonexperienced"). The activity of control rats was restricted to the center of the maze. After 8 daily sessions and a 5-day break, catheters were implanted into the jugular vein. Two days later, 2-deoxyglucose was administered before both experimental groups were exposed to the 6-arm radial configuration. Nonexperienced rats differed from experienced rats in terms of efficiency of exploration, but not in locomotor activity. Compared with experienced animals, nonexperienced rats showed an increase in 2-deoxyglucose uptake in prefrontal and cingulate cortices and in mediodorsal and laterodorsal thalamic nuclei. Exposure of rats to familiar and unfamiliar maze patterns resulted in different patterns of brain metabolic activity.     

Localization of the dopamine uptake complex using [3H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine ([3H]BTCP) in rat brain

[³H]N-[1-(2-Benzo(b)thiophenyl)cyclohexyl]piperidine ([³H]BTCP) is a novel phencyclidine derivative with considerable selectivity for dopamine uptake sites. [³H]BTCP was used to label dopamine uptake sites in vitro, in rat brain, and the regions containing these sites were visualized with an autoradiographic technique. The binding was found to be highest in the striatum, where > 90% of binding was specific. Furthermore, 6-hydroxydopamine lesions of the nigrostriatal pathway obliterated striatal [³H]BTCP binding ipsilaterally, whereas ibotenic acid injection into the caudate-putamen failed to significantly reduce [³H]BTCP binding in that structure. These results indicate that [³H]BTCP labels dopamine uptake sites in mammalian brain and that it can be employed for autoradiographic studies of this transport complex.     

Sodium independence of the binding of [3H]GBR 12783 and other dopamine uptake inhibitors to the dopamine uptake complex

When Na+ was the only cation present in the incubation medium used for the determination of the specific binding of [3H]GBR 12783 in rat striatal membranes, the Na+-dependence between 10 and 210 mM Na+ was not observed. In media with low (10 mM) or high (130 mM) Na+ concentration, mazindol and nomifensine competed with [3H]GBR 12783 for its specific binding site with the same affinities. With the exception of amineptine, all the tested catecholamine uptake inhibitors were equally potent to compete with [3H]GBR 12783 when Na+ concentration was decreased from 130 to 10 mM. These data suggest that the media previously used for the binding studies of tritiated inhibitors of dopamine uptake (Tris-ions buffer and Krebs-Ringer medium) contain ions which could exert inhibitory effects on the specific binding at low Na+ concentration.     

Sex differences in long-term gonadectomized rats: monoamine levels and [3H]nitroimipramine binding in brain nuclei

Summary The levels of norepinephrine, dopamine, serotonin and the serotonin metabolite 5-hydroxyindoleacetic acid were measured in microdissected brain nuclei from long-term gonadectomized male and female rats. Females had 50 and 120% higher levels of serotonin in the anterior hypothalamic nucleus (AH) and arcuate-median eminence (AR-ME), respectively. No significant differences in norepinephrine, dopamine or 5-hydroxyindoline acetic acid were found in any of the brain nuclei sampled. The differences in serotonin content do not appear to result from a sexually dimorphic distribution of serotonin nerve terminals since quantitative autoradiography of ³H-nitroimipramine revealed no differences in binding between the sexes in the AH or AR-ME.Supported by USPHS grant HD12011 to V.N. Luine and USPHS postdoctoral grant HD06368 to K.J. Renner     

Astrocyte opioid receptors: activation modifies the noradrenaline-evoked increase in 2-[14C]deoxyglucose incorporation into glycogen

Astrocyte-enriched cultures of the newborn rat cortex accumulated 2-[14C]deoxyglucose (2-DG), a proportion portion of which was incorporated into glycogen. Exposure to exogenous noradrenaline resulted in an increase (30%) in the incorporation of 2-[14C]DG into glycogen in these cultures. The extent to which noradrenaline evoked an increase in 2-[14C]DG labelling of glycogen was modified by both morphine and methionine-enkephalin. Whereas morphine augmented the noradrenaline-induced increase in 2-[14C]DG incorporation into glycogen in these cultures, methionine-enkephalin attenuated this response.     

The effect of chronic ethanol consumption on [C]deoxyglucose uptake in rat brain in vivo

The uptake of [¹⁴C]deoxyglucose by brains of rats that were given alcohol in drinking water for 7 months was investigated. There was a general, approximately 50%, increase in deoxyglucose uptake in brains of ethanol-treated rats with areas of the limbic system being particularly affected.     

A radioautographic study of [3H]L-glutamate and [3H]L-glutamine uptake in the guinea-pig cochlea

Since glutamate has been recently proposed as a possible transmitter of the sensory hair cells in the cochlea, a radioautographic study was performed to look for the in vitro uptake of [³H] l-glutamate and [³H]l-glutamine. Several experimental conditions were applied. The control experimental procedures consisted in an incubation with one of the labelled tracers (10 min), followed by a post-incubation (3 × 10 min) without tracer. In these experiments, either with [³H]l-glutamate or [³H]l-glutamine, the following structures were labelled: inner hair cells, glial cells of the osseous spiral lamina and areas of the inner spiral and tunnel spiral bundles. When these experiments were carried out in absence of Na⁺, these labellings were strongly decreased. When the incubation was not followed by a post-incubation, the results differed depending on the tracer: with [³H]l-glutamate, the glial cells and the areas of inner spiral and tunnel spiral bundles were labelled, whereas with [³H]l-glutamine, mainly the inner hair cells were labelled. An addition of l-methionine-dl-sulfoximine, a glutamine synthetase inhibitor, into the incubation and post-incubation media, produced a decrease of the labelling of the inner hair cells and of the glial cells. An addition of unlabelled glutamine to the post-incubation media decreased the inner hair cell labelling, while a similar addition of unlabelled glutamate did not. In either case, neither the outer hair cells, the second type of sensory cells, nor the spiral ganglion neurons were labelled.These results suggest that in the cochlea, glutamate and glutamine have their metabolisms linked together, as in some parts of the central nervous system. Correlated to biochemical and electro physiological data these results support the hypothesis that glutamate could be the neurotransmitter of the inner hair cells.     

Differential effects of adrenocorticotropin(1-24) on [3H]2-deoxy-D-glucose uptake in cultured cells derived from different brain regions

The effect of adrenocorticotropin(1-24) (ACTH(1-24)) on the uptake of [3H]2-deoxy-D-glucose ([3H]2-DG) was compared in cell cultures derived from two regions (hypothalamus, and extrahypothalamic forebrain) of fetal rat brain. Under control conditions, [3H]2-DG uptake was similar in extrahypothalamic (10.9 +/- 1.1 nmol/mg protein/5 min) and hypothalamic (11.9 +/- 1.3) cells. No significant effect of ACTH (1-24) (10(-7) to 10(-5) M) was found on uptake of [3H]2-DG in extrahypothalamic cells. In contrast, in hypothalamic cells, a potent stimulatory effect (P less than 0.0001) up to 174% over the control value of [3H]2-DG uptake was produced by these concentrations of ACTH(1-24). This study suggests that ACTH may be a stimulator of brain glucose uptake, and that this effect varies in different brain regions.     

Degeneration and graft-induced restoration of dopamine innervation in the weaver mouse neostriatum: a quantitative radioautographic study of [3H]dopamine uptake

Summary A recently introduced quantitative radioautographic technique was used to characterize the striatal dopaminergic deficit in weaver mutant mice and to evaluate the extent of DA reinnervation resulting from cell suspension grafts of fetal ventral mesencephalic tissue. Brain slices from normal mice and unilaterally grafted weaver mice were incubated in [³H]DA, in the presence of desipramine and pargyline, 3–5 months after graft surgery. Semi-thin sections from the fixed and resin-embedded slices were subsequently exposed on tritium sensitive film and afterwards dipped in nuclear emulsion for light microscope radioautography. Alternate slices were embedded in Epon for post-embedding tyrosine hydroxylase (TH) immunocytochemistry. The grain density of the film radioautographs matched well the distribution of TH positive fibers. Both methods revealed an almost complete absence of DA axons in the dorsomedial quadrant of the weaver neostriatum and an increasing density of DA innervation towards the ventrolateral areas. In the light microscope radioautographs, only the ventral striatum (i.e. nucleus accumbens and olfactory tubercle) and a narrow ventral and periventricular zone of the caudate-putamen were covered by silver grain clusters typical of DA varicosity labeling. Such labeled varicosities were nevertheless found in reduced numbers in the lateral portion of both nucleus accumbens and the olfactory tubercle. The remaining neostriatum was overlaid by diffuse silver grains, suggesting a deficient DA uptake and storage mechanism in the residual DA fibers in this region. Immunocytochemistry using antibodies specific for DA or TH provided further evidence that the residual DA innervation in the weaver neostriatum was biochemically defective. Weaver mice with grafts of ventral mesencephalic tissue in the right neostriatum showed an amphetamine-induced rotational bias to the contralateral side, which was not seen in the sham-operated animals. In contrast to the intrinsic weaver neostriatal DA innervation, DA fibers of graft origin exhibited the normal, clustered type of varicosity labeling. The computerized image analysis of silver grain density in film radioautographs was calibrated by counting these labeled varicosities in selected areas of light microscope radioautographs from the same sections. Results showed a mean DA reinnervation of neostriatal tissue surrounding the graft of about 20%, in some cases up to 80%, of the density seen in wild type mice, with a gradual decrease with distance up to 1–1.4 mm from the graft. The ventral parts of the neostriatum, which contained higher numbers of residual intrinsic DA fibers, were much more sparsely reinnervated than the dorsal and dorsomedial areas. These data show that a quantitatively significant DA reinnervation of the weaver neostriatum can be provided by fetal mesencephalic cell suspension grafts and that these DA fibers become functional, at least with respect to their DA uptake and storage mechanisms, in a neostriatal environment where intrinsic weaver DA axons are strongly deficient. However, observations in long-term weaver mice (9 months after transplantation) suggested that the graft-derived DA fiber outgrowth was reduced with time in the affected striatum, in spite of good survival of the grafted neurones.     

Localization of the dopamine uptake complex using [H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine ([H]BTCP) in rat brain

[³H]N-[1-(2-Benzo(b)thiophenyl)cyclohexyl]piperidine ([³H]BTCP) is a novel phencyclidine derivative with considerable selectivity for dopamine uptake sites. [³H]BTCP was used to label dopamine uptake sites in vitro, in rat brain, and the regions containing these sites were visualized with an autoradiographic technique. The binding was found to be highest in the striatum, where > 90% of binding was specific. Furthermore, 6-hydroxydopamine lesions of the nigrostriatal pathway obliterated striatal [³H]BTCP binding ipsilaterally, whereas ibotenic acid injection into the caudate-putamen failed to significantly reduce [³H]BTCP binding in that structure. These results indicate that [³H]BTCP labels dopamine uptake sites in mammalian brain and that it can be employed for autoradiographic studies of this transport complex.     

In vivo labelling of the mouse dopamine uptake complex with the phencyclidine derivative [3H]BTCP

[3H]BTCP ([3H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine), a phencyclidine (PCP) derivative which binds with a high affinity to the dopamine (DA) uptake complex in vitro, has been tested for in vivo binding to mouse brain. Using [3H]BTCP as a tracer (5 microCi, i.v.) we found the striatum as the region which accumulated the largest amount of radioactivity (58 dpm/mg tissue). In other brain regions the radioactive level (about 20 dpm/mg tissue) was close to the non-specific binding determined by an injection of unlabeled BTCP (40 mg/kg, s.c.) 2 h prior to the [3H]BTCP injection. In the striatum [3H]BTCP binding was inhibited in a dose-dependent manner by unlabeled BTCP (ID50 = 6.34 mg/kg) and nomifensine (ID50 = 11.06 mg/kg). It was unaffected by the DA receptor antagonist haloperidol and by PCP or its analog TCP at doses of 10 mg/kg. These results suggest that [3H]BTCP binds to the dopamine uptake complex in the mouse brain in vivo. Thus, although PCP has no effect on [3H]BTCP binding in these experimental conditions, this in vivo binding model will be useful for the determination of the precise interaction of PCP and its derivatives with the striatal dopamine uptake complex in vivo independently of their interaction with the N-methyl-D-aspartate receptor-channel complex.     

Low uptake of [3H]2-deoxy-D-glucose by cultured rat Schwann cells

[3H]2-Deoxy-D-glucose (2-DG) was used to investigate the glucose uptake in cultured rat Schwann cells from postnatal Sprague-Dawley rat sciatic nerves. The glucose uptake of Schwann cells slightly increased in a time- and dose-dependent manner. However, the maximal uptake level was much lower than that of ethylnitrosourea (ENU)-induced transformed rat schwannoma-like cells and fibroblasts. By autoradiography of the cultured system, we were able to visualize the accumulation of [3H]2-DG grains in the schwannoma-like cells and fibroblasts, but not in Schwann cells.     

[3H]thymidine uptake in cells of rat condylar cartilage

Weanling rats were injected intraperitoneally with [³H]thymidine and sacrificed from 5 min to 20 days later. Their mandibular condylar cartilages were examined histologically, by thin-layer autoradiography, and by using liquid scintillation and microscopic counting methods. Labeled DNA appeared in some of the chondrocytes of the resting zone as early as 10 min postinjection, and reached the proliferative zone by 24 hr and the hypertrophic zone by 4 days. The labeling pattern in the last zone was more disperse, being oriented toward the periphery of the cells as they became hypertrophic. The maximum number of labeled chondrocytes was reached by 2 hr postinjection. These amounted to approximately 11% of the total chondrocyte population, the majority of which were located in the resting zone (73%). It is concluded that, over this period, the mitotic index for these cells is 50–60 per thousand resulting in approximately 100 labeled chondrocytes. In addition, some of the chondroclasts at the erosion front contained labeled DNA as early as 5 min after [³H]thymidine administration. By 10 min, 65% of these cells exhibited one or more labeled nuclei, and the ratio of labeled cells remained high through 20 days. Chondroclasts were seen to contain a diffuse label within their cytoplasm after 5 days. This label was similar to that seen in hypertrophic chondrocytes that had reached the erosion front by that time. Clearly, chondroclasts exhibit nuclear division and do not form from fusion of hypertrophic chondrocytes, although which specific mononuclear cells may act as chondroclast progenitors is not clear. In addition, these multinucleate resorbing cells are capable of ingesting or phagocytizing nuclear remnants from hypertrophic chondrocytes at the eroding face of cartilage.     

Age-correlated loss of dopamine uptake sites labeled with [H]GBR-12935 in human putamen

The effects of age (19–100 years) upon dopamine uptake sites labeled with [³H]GBR-12935 in human postmortem putamen from 20 individuals were studied. There was a 70% decrease in binding density (B_max) over the adult age range. No significant changes in binding affinity (K_d) were detected, the mean K_d being 1.0 ± 0.2 NM (mean ± S.E.M.). Nor were there any changes in binding related to the postmortem delay. Based on the findings that [³H]GBR-12935 labels the uptake site for dopamine, it is suggested that the age-related loss of [³H]GBR-12935 binding in human putamen reflects a degeneration of dopamine neurites.     

In vivo binding of [3H]GBR 12783, a selective dopamine uptake inhibitor, in mouse striatum

[3H]GBR 12783 (1,2-(diphenylmethoxy)ethyl-4-(3-phenyl-2-propenyl)-piperazine), a specific dopamine uptake inhibitor, was tested for in vivo central binding in mice. The difference between the striatal and cerebellar levels of radioactivity was maximal 1 hour after the i.v. injection of a tracer dose of [3H]GBR 12783. The additional accumulation of radioactivity in striatum, relatively to cerebellum, was dose-dependently decreased by dopamine uptake inhibitors. It was unaffected by high doses of dopamine receptor agonists or antagonists and of serotonin or norepinephrine uptake blockers. The intrastriatal injection of 6-hydroxydopamine (6-OHDA) resulted in an almost similar decrease in both the synaptosomal [3H]dopamine uptake and the in vivo [3H]GBR 12783 binding. These data suggest that [3H]GBR 12783 injected i.v. labels the dopamine transport complex in the striatum and thus can be used for the in vivo assessment of the density of dopaminergic nerve endings in brain areas.     

Phagocytosis of Mycoplasma pneumoniae and Acholeplasma laidlawii measured as inhibition of [3H]uridine uptake by macrophages

Many studies of the interaction between phagocytes and mycoplasmas have given controversial results. This is probably due both to the small size of the microorganisms and their ability to attach to the cell membrane, making it difficult to distinguish between adsorption and ingestion. To overcome these difficulties we took advantage of a phenomenon we noted occuring concomitantly with phase-contrast microscope-monitored phagocytosis of heat-killed C. albicans, i.e., a reduction of [³H]uridine uptake by macrophages from culture medium. This approach allowed us to measure the ability of mouse peritoneal macrophages and the macrophage-like P 388 D 1 continuouscell line to phagocytose Mycoplasma pneumoniae and Acholeplasma laidlawii. Live, UV-killed and specific antiserum-opsonized mycoplasmas were tested. A. laidlawii was ingested under all the conditions mentioned above, while live M. pneumoniae was not phagocytosed unless UV-killed. Phagocytosis of UV-killed M. pneumoniae was directly verified by transmission electron microscopy studies. Data obtained with opsonized M. pneumoniae indicated no ingestion by mouse peritoneal macrophages and incomplete phagocytosis with P388 D 1 macrophages, suggesting that different responses by different types of phagocytes can be observed. In spite of a lack of information concerning the biological meaning of the inhibition of macrophage RNA metabolism during phagocytosis, our data suggest that this phenomenon may be used to study the phagocytosis of microorganisms which are difficult to visualize.