Autoradiographic studies on the uptake of 3H-noradrenaline and 3H-GABA in cultured rat cerebellum

Summary Autoradiographic studies were made on the uptake of ³H-noradrenaline and ³H-GABA in rat cerebellum grown in tissue culture. GABA was taken up by Purkinje cells and interneurones as well as by glial cells. In contrast, ³H-noradrenaline was only accumulated by nerve fibres but not by neuronal cell bodies and by glial cells.     

Effects of chronic morphine intake on binding of NAD and GABA-benzodiazepine agonists to synaptic membranes

Nine weeks of compulsory morphine drinking decreased the specific binding of³H-muscimol to GABA receptors and¹⁴C-NAD to rat brain synaptic membranes and increased the synaptosomal uptake of¹⁴C-GABA. These effects of morphine on the GABA-benzodiazepine receptor complex were reversed by excessive doses of vitamin B₃. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 3, pp. 295–301, March, 1999     

Ontogeny of 3H-muscimol binding to membranes of chick forebrain

Summary A potent GABA agonist, ³H-muscimol, was used to investigate the development of GABA receptors in left and right hemispheres of chick forebrain from day 12 in ovo to day 21 post-hatch. Total specific ³H-muscimol binding (p mol mg^–1 protein) increases rapidly in ovo, reaching a peak at around day one post-hatch and then showing a slow decline to approximately 50% of the maximal level at day 21 post-hatch. Despite the considerable evidence from previous studies of lateralization of avain brain function, no significant hemispheric differences were found in ³H-muscimol binding (either of p mol hemisphere^–1 or p mol mg protein^–1) at any of the developmental ages examined.     

Evidence for GABAB-receptors on cultured astrocytes of rat CNS: autoradiographic binding studies

Summary The cellular localization of GABA-binding sites was studied in explant cultures of rat cerebellum, brain stem and spinal cord by means of autoradiography. Labelling of GABA_B-sites was done with ³H(-)baclofen or ³H-GABA in presence of unlabelled bicuculline. Binding sites for these radio-ligands were found on many neurones and on a large number of astrocytes. Labelling of glial cells was usually weaker than that of neurones. Combining autoradiography with staining with anti-glial fibrillary acidic protein (GFAP) revealed that the glial cells labelled with ³H-baclofen or ³H-GABA were GFAP-positive. In contrast, when GABA_A-sites were localized using ³H-GABA in presence of unlabelled baclofen, the GABA_A-agonists ³H-muscimol and ³H-THIP, or the antagonist ³H-(+)-bicuculline, binding only occurred to neurones but not to astrocytes. Immunohistochemical investigations with the monoclonal antibody (bd-17) against the GABA_A/benzodiazepine/chloride channel complex revealed that neurones were specifically stained whereas glial cells were immunonegative. From our observations it is suggested that astrocytes possess GABA_B-receptors but there is little evidence for the existence of GABA_A-sites on glial elements.     

Comparison between the renal handling of14C-Na ferrocyanide and3H-methoxy inulin in the rat

Summary ¹⁴C-sodium ferrocyanide and³H inulin were simultaneously microinjected into surface convolutions of proximal tubules or cortical peritubular capillaries of the left kidney. Ureteral urine was fractionally collected from both kidneys after each injection in order to demonstrate the subsequent excretory pattern of each substance. The results of 25 intratubular injections revealed no significant difference between the excretory pattern of either indicator. The mean total recovery of³H inulin was not significantly different from that of¹⁴C-Na ferrocyanide (P for mean difference 0.5 pairedt test). However a comparison between the time of 50% excretion of each substance revealed a slight but significant delay in the excretion of ferrocyanide [0.086±0.03 min (SE)].Each of the five cortical peritubular capillary microinjections gave rise to a similar excretory pattern for both indicators from each kidney; in every case the kinetic excretory pattern of¹⁴C ferrocyanide closely followed that of³H inulin. The mean ratio amount of ferrocyanide excreted/amount of inulin excreted, obtained for the experimental (left) kidney, did not differ significantly from that obtained for the control (right) kidney. (P for mean difference 0.2, pairedt test.)It was concluded that after simultaneous microinjection into proximal tubules or cortical peritubular capillaries no significant difference exists between the renal handling of¹⁴C-Na ferrocyanide and³H-inulin, also no loss of ferrocyanide occured either into or through the cells bordering the tubular lumen.     

Autoradiographic identification of cerebral and cerebellar cortical neurons accumulating labeled gamma-aminobutyric acid (3H-GABA)

Summary Small amounts of labeled gamma-aminobutyric acid (³H-GABA), a putative neurotransmitter, were injected stereotaxically into the cerebral and cerebellar cortices of rats pretreated with aminooxyacetic acid (AOAA), a drug which prevents GABA breakdown. After fixation by aldehyde perfusion the tissue was processed for light and electron microscopic autoradiography.Specific autoradiographic uptake patterns were observed in both cortices. A high activity was found mainly over stellate and basket cells in the cerebellar cortex and over stellate cells in the cerebral cortex. Also probable Golgi cells seem to accumulate ³H-GABA. Pyramidal cells and Purkinje cells, on the other hand, showed a low activity. Thus, uptake and retention of ³H-GABA in the brain areas studied seem to be related to interneurons supposed to have an inhibitory function. This is consistent with the view that GABA serves as the major inhibitory transmitter substance in mammalian cortical areas.     

The relationship between GABA immunoreactivity and labelling by local uptake of [3H]GABA in the striate cortex of monkey

Summary An antiserum to GABA was used in the macaque monkey to determine whether neurons that accumulate exogenously applied [³H]GABA in vivo are also immunoreactive for GABA. Following the injection of [³H]GABA into different laminae of striate cortex in two untreated animals and in one animal treated with amino-oxyacetic acid, selective accumulation of the labelled amino acid was demonstrated in perikarya by autoradiography. Radiographically labelled neurons (n, 519) and their unlabelled neighbours were tested in consecutive 0.5 m thick sections by immunocytochemistry for GABA immunoreacitivity. Injection of [³H]GABA did not increase the number of neurons showing GABA immunoreactivity. On the contrary many of the cells that accumulated [³H]GABA were immunonegative. These neurons were mostly located in layers IVC and VA following [³H]GABA injection into layers II–III, and in layers upper III and II following injection into layers V and VI. A comparison of the position of these neurons with known local projection patterns in the striate cortex of monkey suggests that GABA-immunonegative neurons may nevertheless become labelled by [³H]GABA if most of their local axon terminals fall within the injection site. The interlaminar projection of GABA-immunopositive neurons, which probably contain endogenous GABA, could be deduced from the position of the [³H]GABA injection site that leads to their autoradiographic labelling. Although the present study confirmed our previous results on the interlaminar connections of neurons that accumulate [³H]GABA, it demonstrated that [³H]GABA labelling alone may not be a sufficient criterion to assess the GABAergic nature of neurons in the striate cortex of monkey.     

Electron microscopic radioautographic study on the nucleic acids and protein syntheses in the aging mouse testis

The ultrastructural difference between³H-thymidine labeled and unlabeled germ cells in mouse testis was observed by electron microscopic radio-autography. The RNA and protein syntheses in the seminiferous tubules of aging mice were also studied using³H-uridine and leucine radioautography. At embryonic day 19, the Sertoli cells and myoid cells labeled with³H-thymidine were often observed. The gonocytes labeled with³H-thymidine that were not rich in cell organelles appeared from the early postnatal stage. The silver grains were located mainly over the nuclei of labeled gonocytes, but a few were over the mitochondria. The labeling index of spermatogonia for³H-thymidine rapidly increased from 2 weeks on, and stayed relatively constant until old age. The labeling indices of the other two kinds of cells, i.e., Sertoli and myoid cells, decreased to low levels with aging, while these cells continued RNA and protein syntheses through aging, as detected by the incorporation of³H-leucine and uridine.     

Topography of 3H-DHA, 3H-QNB, 3H-Dopamine, and 3H-DAGO Binding Sites Distribution in the Central Part of the Sinoatrial Node in Rat Heart

The topography of distribution of ³H-dihydroalprenolol, ³H-quinucledinyl benzilate, ³H-dopamine, and ³H-DAGO binding sites in the central part of the sinoatrial node in rat heart was studied by autoradiography after electrophysiological identification of the dominant pacemaker region location. Receptor asymmetry between the lateral and median regions of the central part of the sinoatrial node was shown. The dominant pacemaker region lay in the lateral area of the sinoatrial node; the number of binding sites for all four ligands was minimum in it. The number of binding sites gradually increased in the cranial and caudal directions from the dominant pacemaker region along the sinoatrial node artery (more smoothly in the caudal direction). The relative densities of bindings sites for ³H-dihydroalprenolol and ³H-dopamine were higher in the lateral region compared to the perinodal working myocardium, while the densities for ³H-quinucledinyl benzilate and ³H-DAGO were virtually the same. The distribution of binding sites along the artery in the median region of the sinoatrial node was even for ³H-quinucledinyl benzilate and ³H-DAGO. For ³H-DAGO these parameters were close to those in the perinodal atrial myocardium, for ³H-quinucledinyl benzilate somewhat lower. Curves presenting the distribution of binding site densities for ³H-dihydroalprenolol and ³H-dopamine in the median region of the sinoatrial node were similar, with a pronounced peak in the region contralateral to the dominant pacemaker region, and significantly higher binding parameters compared to those for the perinodal atrial myocardium. The difference consisted in higher density of ³H-dopamine binding sites in the median region of the sinoatrial node in comparison with the lateral region. Binding activity was maximum in the wall of the sinoatrial node artery. The distribution of binding sites for ligands to the main autonomic nervous system neurotransmitters in the rat heart sinoatrial node is heterogeneous. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 10, pp. 472–477, October, 2005     

Autoradiographic localization of binding sites for 3H-serotonin and 3H-ketanserin on neurones and astrocytes of cultured rat brain stem and spinal cord

Summary By means of lightmicroscopic autoradiography we have studied the cellular localization of binding sites for ³H-serotonin and ³H-ketanserin in organotypic cultures of rat brain stem and spinal cord. In both types of cultures, a relatively great number of neurones revealed binding sites for ³H-serotonin which predominantly labels S₁-receptors. ³H-ketanserin, an S₂-antagonist was also bound to many neurones although to a lesser extent than ³H-serotonin. Binding sites for both radio-ligands were also observed on astrocytes. These findings together with electrophysiological investigations indicate that astrocytes possess serotonin receptors.     

The effects of thyroid hormones on3H-ouabain binding site concentration,Na, K-contents and86Rb-efflux in rat skeletal muscle

Using a recently developed method based upon vanadate facilitated³H-ouabain binding, the total concentration of³H-ouabain binding sites was determined in biopsies of rat skeletal muscles containing varying proportions of slow-twitch fibres. In extensor digitorum longus, diaphragm, gastrocnemius and soleus muscles from mature (12-week-old) hyperthyroid rats the values obtained were respectively 2.6, 3.5, 5.1 and 9.8 times higher than those found in the same muscles from hypothyroid animals. This indicates that the effect of thyroid hormones is more pronounced on slow-twitch than on fast-twitch fibres. The changes in³H-ouabain binding site concentration with thyroid status could not be accounted for by differences in affinity or the rate of³H-ouabain binding.In young (4–5 week old) rats, where the K-content and the³H-ouabain binding site concentration in muscle had been reduced by K-depletion, T₃-pretreatment produced an even larger relative increase in the³H-ouabain binding site concentration than in age-matched controls, but no increase in K-content. Therefore, the downregulation of³H-ouabain binding sites seen during K-depletion cannot be attributed to a decreased response to thyroid hormones. In normal rats the marked stimulating effect of thyroid hormone on the synthesis of³H-ouabain binding sites was not associated with any significant change in K-content, but clearly preceded by a significant (P<0.001) rise in the efflux of⁸⁶Rb.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

The pharmacokinetics of (125I) 3-deoxy-3-iodine glucose on experimental tumor modelsin Vivo

The dynamics of¹²⁵I distribution is studied in rats with induced tumors of the prostate and mammary gland for intravenous administration of¹²⁵I-3D-G. It is found that 80% of the activity is eliminated in the first 24 hours. A relatively high level of¹²⁵I accumulation is found in necrotically altered regions of the tumor. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, № 3, pp. 294–295, March, 1994.     

Characterization by Golgi impregnation of neurons that accumulate 3H-GABA in the visual cortex of monkey

Summary ³H-GABA was injected into restricted regions of visual areas 1 and 2 (cortical areas 17 and 18) on the lateral surface of the occipital lobe in monkeys. The injected tissue was processed for Golgi impregnation and gold toning. Sections containing Golgi-impregnated neurons were re-embedded, sectioned at 1 m, and prepared for autoradiography to reveal neurones that had selectively accumulated ³H-GABA.Golgi-impregnated pyramidal, spiny stellate and aspiny nonpyramidal neurons were examined for ³H-GABA accumulation. Out of 47 aspiny nonpyramidal neurons 16 were labelled by ³H-GABA. The other cell types did not accumulate the amino acid. Twelve of the labelled neurons were drawn. Eight were bitufted neurons with their dendrites oriented predominantly radially, three were small multipolar neurons, and one could be reconstructed only partially. One neuron had a locally arborizing axon in layer III.Two bitufted, Golgi-impregnated neurons in layer II and upper III of area 18 were labelled from GABA injection radially beneath in layer VI, providing evidence for earlier suggestions that in the monkey's visual cortex the cells in the upper layers which project radially and accumulate ³H-GABA are aspiny non-pyramidal cells. The results indicate the existence of different types of putative GABA-ergic interneurons.During part of this project P. Somogyi was supported by the Wellcome Trust at the Department of Pharmacology, Oxford University. We are grateful to the Hungarian Academy of Sciences, the International Cultural Institute of Budapest, the Wellcome Trust, the Royal Society, the E.P. Abraham Cephalbsporin Trust, and the Medical Research Council for financial support     

Tritiated thymidine autoradiographic study on postnatal development of epididymal adipose tissue in the normal mouse

Summary Cell proliferation was studied in the developing epididymal adipose tissue of mice by means of³H-thymidine autoradiography. 4 mice in each age group were killed 30 min after a single injection of³H-thymidine given at the 6th, 10th, 16th, 21st, 28th, 35th, 42nd, 49th or 56th postnatal day. The labeling index of the adipose tissue was high for several postnatal days but it was highest on the 6th day. The labeling index decreased thereafter, and cell proliferation appeared to almost cease after 56 days. Another 16 mice were given a single injection of³H-thymidine at 6 days old; pairs of animals were killed at 30 min, 2, 4, 6, 8, 10, 14 and 18 days later. From the 2nd to 6th days of pulse labeling, the number of labeled fat cells increased significantly, whereas the number of labeled poorly differentiated mesenchymal cells decreased reciprocally. The labeling index of fat cells remained unchanged from the 6th to the 18th day. These results suggest that there is a population of fat cell-precursor cells in the mesenchymal tissue of the new born mouse, and that it takes 2–6 days for the precursor cells to become mature fat cells in the epidiymal fatty tissue.     

The age-dependent changes in the number of3H-ouabain binding sites in mammalian skeletal muscle

The influence of age on the binding of³H-ouabain in skeletal muscle has been characterized in rats, mice and guinea pigs. Measurements performed using biopsies and intact fibers obtained from different types of rat muscles showed that from birth to the 4th week of life, the number of³H-ouabain binding sites per unit weight increases up to 5-fold, followed by almost the same relative decrease to a plateau around 250 pmol/g wet wt at an age of 22 weeks. These changes were not associated with any major alterations in apparentK _D (1.7–3.1×10^–7M) dissociation rate or heterogeneity in binding characteristics. Measurements of 3-O-methylfluorescein phosphatase activity, an enzyme activity which is closely correlated to the Na–K-ATPase activity, confirmed the³H-ouabain binding data.In mice, the number of³H-ouabain binding sites showed similar, albeit less pronounced changes with age, a maximum being reached at the 4th week of life. In guinea pigs, the number of³H-ouabain binding sites per unit weight decreased by 60% from birth to maturity.The results indicate that the early development and differentiation of individual skeletal muscles is associated with a marked increase in the number of Na–K-pumps (when expressed as pmol/muscle), until at maturity a plateau is reached. However, when expressed as pmol/g wet wt the increase is followed by a decrease to a plateau. This may in part account for the relatively low digitalis sensitivity seen in infants as compared to newborn and mature individuals.     

Blockade by tetraethylammonium (TEA) and rubidium of potassium exchange in sartorius muscle fibers: Distribution of14C-TEA in muscle

Summary Tetraethylammonium (TEA) causes a blockade of⁴²K-exchange in resting sartorius muscle by a mechanism that differs from that caused by rubidium ions. Whereas the blockade by rubidium of⁴²K-efflux was antagonized by elevation of extracellular potassium, that caused by TEA was antagonized only partially. Rubidium-induced blockade has characteristics of competitive inhibition of⁴²K-exchange while the TEA-induced blockade appears to be non-competitive. Moreover, TEA causes a greater blockade of⁴²K-exchange in muscles bathed in hypertonic solutions than in muscles bathed in isotonic solutions. This finding may be related to the more rapid rate of⁴²K-exchange in muscles bathed in hypertonic solutions. The equilibrium constant for the interaction between TEA and membrane receptors estimated during⁴²K-efflux is approximately 20 mM; the equilibrium constant for rubidium ions is 1.4 mM. The¹⁴C-TEA space in sartorius muscle is about 2-times greater than the¹⁴C-inulin or sodium spaces but somewhat smaller than¹⁴C-urea space. The rates of efflux¹⁴C-TEA,¹⁴C-inulin and¹⁴C-urea are comparable and rapid. Thus, the muscle membrane does not appear to offer a barrier to the exchange of TEA.Supported by research grant NS-07540-05 and NS-09148-02 from the Institute of Neurological Sciences and Stroke, National Institutes of Health, U.S.P.H.S., Bethesda, Maryland.     

Transport properties of the basolateral membrane of the oxyntic cells in frog fundic gastric mucosa

The conductive properties of the basolateral membrane of oxyntic cells (OC) of frog fundic gastric mucosa were investigated by utilizing the microelectrode technique. By examining the response of the basolateral cell membrane potential difference,V _cs, to sudden ion concentration changes in the serosal bath it was concluded that the basolateral membrane of OC has a high Ba²⁺-sensitive K⁺-conductance, and no Cl^–-conductance both in resting (cimetidine) and in stimulated (histamine) state. The response ofV _cs to serosal Cl^–-removal, consisting in a slight hyperpolarization (anomalous Nernst response), could not be explained by possible permeability changes to K⁺ and Na⁺ since the potential response to Cl^– was essentially preserved by blocking K⁺-permeability with Ba²⁺ and replacing all Na⁺ by choline. Conversely, hyperpolarization ofV _cs after Cl^–-free perfusion was abolished by exposure to HCO ₃ ^– -free solution, indicating that HCO ₃ ^– -ions are required at the serosal bath for Cl^– to get his effect. It was investigated wether the effect of Cl^– was due to an electrogenic Na⁺(HCO ₃ ^– ) _n /Cl^– exchange mechanism on the basolateral membrane. Experiments showed that the potential response to HCO ₃ ^– -removal and to Na⁺-removal, consisting in a depolarization ofV _cs, was similar both in presence and in absence of Cl^–. Furosemide (0.5 mmol/l) had no effect on steadyV _cs andV _t. The electrophysiological analysis of the data led to excluding the involvement of Na-Cl, Na-2Cl and NaK-2Cl cotransports, and to including the existence of an electrogenic Na⁺(HCO ₃ ^– ) _n /Cl^– exchange process, while suggests the presence of an electroneutral Cl^–/HCO ₃ ^– exchange mechanism to explain Cl^–-transport across the basolateral membrane of OC.This work was supported by a research grant from Ministero della Pubblica Istruzione, Rome, Italy     

Autoradiographic localization of 3H-gamma-aminobutyric acid in the medial hypothalamus

Summary Tritium labelled gamma-aminobutyric acid (³H-GABA) was infused into the third ventricle of rats with normal or deafferented hypothalamus and the distribution of the label was studied by light and electron microscopic autoradiography.In control as well as deafferented hypothalamus a few neurones accumulated radioactivity, while the majority was unlabelled. Characteristic clusters and rows of silver grains were observed in the neuropil of several regions probably indicating labelled cell processes and terminal axons. Electron microscopy showed that at least some of the clusters were over axon terminals with synaptic vesicles. ³H-GABA accumulated also in the ependyma and glial elements.The results suggest that in the medial hypothalamus there is a preferential uptake of GABA in some neurones and nerve fibers; at least some of these are hypothalamic interneurones. This supports the hypothesis that some hypothalamic neurones and nerve endings may use GABA as a transmitter.     

Intratumoral FOXP3VEGFR2 regulatory T cells are predictive markers for recurrence and survival in patients with colorectal cancer

Previously, we have shown that CD8⁺T/FOXP3⁺ cell ratio but not FOXP3⁺ cell number alone is an independent prognostic factor for colorectal cancer. In the present study, we evaluated whether the number of intratumoral FOXP3⁺VEGFR2⁺ (itFOXP3⁺VEGFR2⁺) T cells alone could be a predictive factor for survival prognosis in patients with colorectal cancer. Distribution of regulatory T cells (Tregs) at tumor sites derived from 88 patients with primary colorectal cancer was fluorescence-immunohistochemically examined. Relatively low number of itFOXP3⁺VEGFR2⁺ cells significantly correlated with poor disease-free survival (DS) and overall survival (OS); multivariate analysis indicated that number of itFOXP3⁺VEGFR2⁺ cells is an independent predictive and prognostic factor of DS and OS while neither intratumoral FOXP3⁺ cell number nor intratumoral FOXP3⁺VEGFR2⁻ cell number alone showed significant correlation with DS or OS. These results suggest that FOXP3⁺VEGFR2⁺ may be a better predictive Treg marker than FOXP3⁺ alone for recurrence and survival in patients with colorectal cancer.