Autoradiographic Results Research Articles

Increased potency of neuropeptide y to antagonize α2-adrenoceptor function in the nucleus tractus solitarii of the spontaneously hypertensive rat

The regulation by neuropeptide Y of α2-adrenoceptors in the nucleus tractus solitarii was evaluated in the adult normotensive Wistar Kyoto rat and the adult spontaneously hypertensive rat. The microinjection of a submaximal dose of l-noradrenaline (800pmol in 50nl) alone into the nucleus tractus solitarii produced a significant reduction in the mean arterial blood pressure in either strain. The threshold dose (1pmol in 50nl) of neuropeptide Y(1–36) for the vasodepressor response in the Wistar Kyoto rat was five times higher than that (0.2pmol in 50nl) in the spontaneously hypertensive rat. Furthermore, neuropeptide Y(1–36) at 0.2pmol in 50nl could significantly counteract the vasodepressor response to l-noradrenaline (800pmol in 50nl) in the spontaneously hypertensive rat, but not in the Wistar Kyoto rat, in which 1pmol in 50nl of neuropeptide Y(1–36) must be employed to counteract the vasodepressor response to l-noradrenaline (800pmol in 50nl), although the vasodepressor responses are of a similar magnitude. The in situ hybridization and quantitative receptor autoradiographical experiments showed that the α2A-adrenoceptor messenger RNA levels and the Bmax value of the α2-adrenoceptor agonist [3H]p-aminoclonidine binding sites measured in the nucleus tractus solitarii of the spontaneously hypertensive rat were substantially lower than those in the Wistar Kyoto rat. The quantitative receptor autoradiographical results were consistent with the cardiovascular results and showed that in the spontaneously hypertensive rat, neuropeptide Y(1–36) at 1nM led to a significant increase in the Kd value of [3H]p-aminoclonidine binding sites. In the Wistar Kyoto rat, neuropeptide Y(1–36) produced this effect only at 10nM. The present study provides evidence for an increase of the potency of neuropeptide Y(1–36) to antagonistically modulate α2-adrenoceptors in the nucleus tractus solitarii of the spontaneously hypertensive rat. This enhanced antagonistic action may partly be related to a reduction in the number of α2A-adrenoceptors in the nucleus tractus solitarii of the spontaneously hypertensive rat, since a decrease has been observed in the α2A-adrenoceptor messenger RNA levels and the α2-adrenoceptor binding sites in the spontaneously hypertensive rat.This increased potency of neuropeptide Y(1–36) to antagonize α2-adrenoceptor function in the nucleus tractus solitarii of the spontaneously hypertensive rat may contribute to the development of high blood pressure in this hypertensive strain.

Skin reservoir formation and bioavailability of dermally administered chemicals in hairless guinea pigs

There is concern as to whether dermally applied chemicals that remain in the skin after exposure are: bioavailable and should be included as part of the systemic dose; this study was conducted to investigate the temporal relationship between the skin depot and absorbed dose. Single doses of 14C-labelled phenanthrene, benzo[a]pyrene or di(2-ethylhexyl) phthalate were administered dermally to groups of four female, Hartley hairless guinea pigs which were housed individually in metabolism cages to collect urine and faeces for radioassay. The animals were sacrificed at 6 hr, 24 hr, 48 hr, 7 days or 14 days after dosing to harvest skin specimens for the determination of radioactivity by autoradiographic and liquid scintillation methods, and to determine the dose that remained in the body. It was found that for all three compounds the amount of chemical left in the skin decreased over time while the cumulative percent dose, excreted in urine and faeces increased. The autoradiographic results were consistent with those obtained from the liquid scintillation method showing a gradual decrease in radioactivity grain accumulation over the time periods for the three compounds, with the highest grain density observed around hair follicles of the skin. The results of this study indicate that the chemicals left in the skin after surface washing eventually enter the systemic circulation and should be considered as part of the total dose absorbed, and that the hair follicle may play an important role in percutaneous penetration.

GABA and GABAA receptor changes in the substantia nigra of the rat following quinolinic acid lesions in the striatum closely resemble Huntington's disease

GABA and GABAA receptors have been studied in the substantia nigra of the rat following quinolinic acid lesions in the striatum. The regional distribution of GABA and GABAA receptors was investigated using immunohistochemical techniques with monoclonal antibodies to GABA and to the beta 2.3 subtypes of the GABAA receptor complex. The distribution, density and cellular localization of GABAA receptors were studied using quantitative receptor autoradiography and 6-hydroxydopamine-induced degeneration of dopaminergic pars compacta neurons. The subunit configuration of GABAA receptors was investigated using in situ hybridization histochemistry and subunit subtype-specific oligonucleotide probes. The results showed that in the normal substantia nigra GABA and GABAA receptors were mainly localized within the pars reticulata. GABAA receptors were mainly of the BZI variety, had a subunit subtype configuration that included alpha 1 and beta 2.3 subtypes, and showed a rostrocaudal gradient in the density of receptors; the density of receptors in the caudal third was 56% higher than that in the rostral third of the pars reticulata. Following quinolinic acid-induced degeneration of the striatonigral pathway, there was a marked loss of GABA immunoreactivity and a 59% increase in the density of GABAA receptors in the substantia nigra pars reticulata. There was a corresponding regional topography in the pattern of loss of GABA immunoreactivity and in the pattern of increase in GABAA receptors in the pars reticulata; the topography varied with the size and placement of the lesion in the striatum and correlated with the known topographical organization of the striatonigral projection. The quantitative autoradiographic results showed that following quinolinic acid lesions in the striatum: (i) the greatest increase in the density of GABAA receptors occurred in the middle third (91% increase) of the pars reticulata; (ii) the receptors were mainly of the GABAA/BZI variety; and (iii) 6-hydroxydopamine-induced degeneration of the dopaminergic pars compacta neurons did not significantly affect the density of receptors, indicating that the increased receptor binding was mainly localized on non-dopaminergic pars reticulata neurons. The immunohistochemical and in situ hybridization studies showed that, as in the normal substantia nigra, GABAA receptors in the substantia nigra pars reticulata on the lesioned side contained the alpha 1 and beta 2.3 GABAA receptor subtypes; the alpha 1 and beta 2.3 subtypes (but not the alpha 2) were increased after quinolinic acid lesions.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of clozapine and risperidone on 5-HT 2 and D 2-dopamine receptor binding in the post-mortem human brain. An autoradiographic study

Effects of the antipsychotics risperidone and clozapine on 5-HT 2 and D 2-dopamine receptor binding were examined using [ 3H]N-methylspiperone ([ 3H]NMSP) and in vitro receptor autoradiography on human whole hemisphere cryosections. The 5-HT 2 receptor antagonist ketanserin and the D 2-dopamine receptor antagonist raclopride were used as references. [ 3H]NMSP binding was observed in caudate nucleus, putamen, and cerebral cortex indicating binding to D 2-dopamine and 5-HT 2 receptors. Risperidone and clozapine counteracted the binding to both receptor types. This was in contrast to raclopride, which selectively blocked the D 2-dopamine receptors in the basal ganglia, and ketanserin, which selectively blocked the 5-HT 2 receptors in the cerebral cortex. Risperidone (100 nM and 10μ M) blocked up to 90% of [ 3H]NMSP binding to both receptor types, whereas the blocking capacity of clozapine (10 μ M) was lower (approximately 60%). The lack of total blockade of D 2-dopamine receptors is in line with results obtained in with [ 11C]raclopride and positron emission tomography studies of clozapine treated human subjects. However, autoradiographic studies of clozapine competition of [ 3H]raclopride binding show total displacement of the binding at high clozapine concentrations, thus contradicting the PET results with [ 11C]raclopride, as well as the autoradiographic results obtained with [ 3H]NMSP. In conclusion it can be stated that pharmacological concentrations of the two drugs clozapine and risperidone block a large proportion of D 2-dopamine receptors and 5-HT 2 receptors in the human brain. Moreover, the study shows the usefulness of human whole hemisphere autoradiography for the study of interaction of drugs with different central neurotransmitter receptors.

An improved fragile X Southern blot probe without the CGGs eliminates background bands

The largest of the commonly used probes for Southern blot diagnosis of fragile X mental retardation syndrome spans the CGG repeat cluster in the FMR-1 gene. This probe causes appearance of 'common' or 'constant' background bands which occasionally complicate the interpretation of autoradiographic results. By removing a 357 bp Sphl to Nhel fragment containing the CGGs from the probe pE5·1, we constructed a probe which eliminates the background bands yet allows the use of a large probe (4·8kb) to detect changes in the diagnostic 5·2 kb genomic EcoRI band. This CGG-deficient probe has been used in routine diagnostic cases as well as in second round testing of pE5·1-probed cases where enlarged mutant bands are suspected to comigrate with the background bands.

Effects of sterigmatocystin and T-2 toxin on the induction of unscheduled DNA synthesis in primary cultures of human gastric epithelial cells.

Primary cultures of human gastric epithelial cells were tested for induction of unscheduled DNA synthesis (UDS) by sterigmatocystin (ST) and T-2 toxin. Autoradiographic results indicated that ST (10(-6)-10(-4)M) induced UDS in the presence of S9 activation system. The repair rates were 24-91% (net grains > or = 3) and 2-71% (net grains > or = 5). T-2 toxin did not induce UDS in this study.

Tissue-specific nuclear repatterning in plant cells

Tissue-specific nuclear repatternings, consisting of changes in the number and size of the chromocenters, were observed by analyzing, in Feulgen squashes and sections, different tissues of several plant species, particularly of Ionopsidium savianum. Nuclear repatternings occur mainly near the base of the meristems. They are due to associations of chromosomes at their heterochromatic regions. This was confirmed by the results of cytophotometric measurements, showing the same contents of both Feulgen/DNA and heterochromatin in nuclei with a different number of chromocenters. These data also showed that chromosome association does not occur in endoreduplicating or endoreduplicated cells. Autoradiographic results after [3H]thymidine treatments indicated that DNA synthesis does not occur in nuclei with extensive chromosome association. A highly significant, positive correlation was found between the number of chromocenters in each nucleus and the amount of RNA synthesis as indicated by [3H]uridine incorporation. It is suggested that chromosome association plays some role in the regulation of the functional activity of the nucleus and in tissue differentiation.

Endothelin Receptors in the Cornea, Iris and Ciliary Processes. Evidence from Binding, Secondary Messenger and PCR Studies

Endothelin (ET)-1 (10 nM) is about six times more effective than ET-3 in contracting the isolated iris sphincter muscle; the ET-1-induced contraction is insensitive to indomethacin treatment. The effect of ET-2 is intermediatory between ET-1 and ET-3 in contracting the muscle. The relative potency of the ETs to stimulate inositol phosphates (InsPs) in the iris-ciliary processes is ET-1 > ET-2 > ET-3, with ET-1 about six times more potent than ET-3; these effects are also insensitive to indomethacin. Studies utilizing the polymerase chain reaction (PCR) show that ET B receptors are present. Although no evidence could be found for the occurrence of ET A receptors, their presence cannot be excluded. These results suggest that the stimulation of InsPs and contraction of the iris sphincter muscle by ET is mediated by ET B receptors and that products generated via activation of phospholipase A 2 are not indirectly involved in the observed responses. However, another type of ET receptor is indicated by the finding that ET-1 reduces the forskolin-elevated cAMP levels in the iris-ciliary epithelium. Autoradiographic results show that specific [ 125I]ET-1 binding sites are associated with the iris, ciliary processes and the corneal endothelium. As in the iris-ciliary process tissues, ET-1 is the most effective of the three ETs stimulating InsPs in the cornea, although statistically the differences were insignificant. Moreover, ET-1 was found to have no effect on the forskolin-elevated cAMP levels in the cornea. Whether these results reflect true differences between the ET receptors in the iris-ciliary processes and corneal endothelium remains to be established.

Characterization of the 5-hydroxytryptamine receptor modulating the release of 5-[3H]hydroxytryptamine in slices of the human neocortex.

In the rat brain, the presynaptic 5-hydroxytryptamine (5-HT) autoreceptors located on 5-HT terminals correspond to the 5-HT1B subtype. The presence of a 5-HT receptor probably located on 5-HT nerve endings and modulating transmitter release in the human neocortex has been reported, but its detailed pharmacological characterization is not yet available. On the other hand, receptor binding and autoradiographic results indicate that the 5-HT1B receptor subtype is not present in the human brain. We, therefore, studied the modulation of the electrically evoked release of [3H]5-HT by various 5-HT receptor agonists and antagonists in preloaded slices of human neocortex obtained from 18 patients undergoing neurosurgery. The nonselective 5-HT1A/1B/1D receptor agonist 5-carboxamidotryptamine produced a potent inhibition (70% at 0.03 microM) of the electrically evoked release of [3H]5-HT which was blocked by 5-HT receptor antagonists with the following relative order of potency: methiothepin greater than metergoline = methysergide greater than propranolol. The selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin at 0.1 microM did not modify the electrically evoked release of [3H]5-HT. The 5-HT1A/1B receptor agonist RU 24969 was 10 times more potent at inhibiting [3H]5-HT overflow in the rat frontal cortex than in the human neocortex. The potent 5-HT1B receptor antagonist cyanopinodolol did not modify the 5-carboxamidotryptamine-induced inhibition of the electrically evoked release of [3H]5-HT in slices of the human neocortex, but produced by itself a small inhibition of [3H]5-HT overflow.(ABSTRACT TRUNCATED AT 250 WORDS)

Internuclear neurons in the ocular motor system of frogs

Medial and lateral rectus motoneurons of frogs were localized after retrograde labeling with horseradish peroxidase (HRP) injected in the medial rectus muscle or applied on the cut end of the abducens nerve. Coordinates of these cell columns were used as target areas for the injection of small amounts of HRP (20-60 nl) and [3H]leucine (25-40 nl) and as search areas for retrogradely and anterogradely labeled internuclear neurons (INT) in in vivo and in vitro experiments. HRP injection in the medial rectus subdivision of the oculomotor nucleus (n = 6) resulted in retrograde labeling of cell bodies in the contralateral principal abducens nucleus. On the average about 16 cells per animal were found. Somatic diameters were about 13.5 +/- 2.8 microns (n = 32). The number and the size of these abducens internuclear neurons (AbINT) are smaller than those of lateral rectus motoneurons (n = 75; diameter: 19 +/- 3.2 microns). A crossed projection of AbINT to medial rectus motoneurons in the contralateral oculomotor nucleus is further supported by autoradiographic results. Following injection of [3H]leucine into the abducens nucleus, a high density of silver grains was visible within the contralateral oculomotor nucleus, mainly in the caudal part of the oculomotor nucleus, where medial rectus motoneurons are located. Injection of [3H]leucine in vivo (n = 4) and in vitro (n = 3) resulted in a similar high density of silver grains within the contralateral oculomotor nucleus, but the background level of silver grains was significantly higher after in vitro (264 +/- 38/2,500 microns2) than after in vivo injections (195 +/- 17/2,500 microns2). HRP injection in the principal abducens nucleus (n = 9) resulted in retrograde labeling of cell bodies in the medial rectus subdivisions of the bilateral oculomotor nuclei. Ipsilateral projections predominated, with about 10 (+/- 8) labeled cells over contralateral projections (about 3 +/- 2). Average diameters of these oculomotor internuclear neurons (OcINT) were again smaller (10.8 +/- 2 microns; n = 18) than those of medial rectus motoneurons (14.4 +/- 3 microns; n = 52). In addition, retrogradely labeled cells were consistently encountered in the bilateral vestibular nuclei, the cerebellar nuclei, the dorsal brainstem caudal to the abducens nuclei, and ipsilaterally in the pretectum. Most of the vestibular neurons were located in the rostral part of the vestibular nuclear complex. These neurons might constitute part of the three-neuronal arc of the vestibulo-ocular reflex in the frog. Labeled cells in the pretectum were restricted to the ipsilateral posterior thalamic nucleus (P).(ABSTRACT TRUNCATED AT 400 WORDS)

Role of the GABA chloride ion channel component on aggressive behavior in the male crab

Picrotoxin blocking effects on GABA-induced inhibition of lateral merus display in the male crab Carcinus mediterraneus suggest that the chloride ion channel component of the GABA molecule is involved in the control of this defensive behavior. The low GABA doses (3 and 5 μ/g body weight) inhibited lateral merus display as early as 10 min after drug treatment while the administration of higher GABA concentrations (>5 μg/g) prolonged the duration of the behavioral effects. The administration of 1 μg/g picrotoxin to the animals treated with the effective GABA doses (5, 10 and 20 μg/g) restored lateral merus display. The antagonizing activity of picrotoxin on the inhibitory GABA effects, in a temporal manner, demonstrates that GABAergic sites other than peripheral ones are probably participating with the regulation of this agonistic posture. Quantitative autoradiography results revealed interesting receptor levels changes of the specific chloride ion channel antagonist [ 35S] t-butylbicyclophosphorothionate in the different brain areas of the male crab displaying lateral merus with respect to normal nonreactive animals. Elevated receptor binding levels were encountered in the middle and posterior brain area whereas low levels were obtained at the anterior level. Both the behavioral and autoradiographic results suggest that the defensive type of aggression behavior in crustacean may not only be mediated by a peripherally controlled GABA-gated chloride ion flux but also by a central GABAergic mechanism.

Autoradiographic localization of α1‐adrenoceptors in the dog prostate and urethra with 3H‐prazosin

AbstractThe regional distribution of α1‐adrenoceptors in the dog urethra has been studied by quantitative autoradiography using the specific α1‐antagonist 3H‐prazosin as a ligand. The binding of 3H‐prazosin to urethral tissue sections was saturable, reversible, and of high affinity (Kd = 0.7 nM); it occurred at a single population of sites and possessed the pharmacological features of the α1‐adrenoceptor. The binding parameters of 3H‐prazosin on urethral tissue sections were found similar to those obtained on urethral membranes. Autoradiographic results revealed that the α1‐adrenoceptors are heterogeneously distributed along the dog urethral axis. The highest densities of 3H‐prazosin binding sites were found in the preprostatic urethra. Comparison between autoradiographic and histological slides revealed that the 3H‐prazosin binding sites were only localized on the smooth muscle fibres of the dog urethra.

Effects of chronic postnatal opioid receptor blockade by naltrexone upon proliferation capacity in the prenatally X-irradiated brain of the rat

We recently reported that in rats prenatally X-irradiated on gestation day 14 with 1 Gy, postnatal chronic application of the opioid antagonist naltrexone (NX) led to a remarkable growth spurt of the microencephalic brain. In the present study we present histological and autoradiographic results found in the subependymal layer (SEL) of the forebrain lateral ventricles. NX led to an intermittent augmentation of the mitotic index of the X-irradiated brains within a postnatal observation period of 24 weeks. The most conspicuous finding was transient hyperplasia of the SEL at 4–6 weeks of age which occurred in close proximity to an intact ependymal lining. Districts of the lateral ventricles which were denuded from ependyme and where the rest of the ependymal layer (EL) was dislocated peripherally showed upon NX treatment a long-lasting SEL hyperplasia with a tendency towards dysplasia. These results revealed that repair proliferation of embryotoxic X-irradiation is normally under strong control by the opioid system. If that system, which exerts a suppressing effect upon glial growth, is blocked by NX, prominent hyperplastic reactions occur which may be useful for repairing the lesion pattern.

Autoradiographic localization of [3H]RU 486 and [3H]progesterone in the uterus, pituitary and hypothalamus of the rat

Twenty-one castrated oestrogen-primed Wistar rats, which were 2-months-old, were injected via the jugular vein with 100 mu Ci/100 g body weight of [3H]RU 486 or [3H]progesterone. Some of these received unlabelled compounds for competition studies. Samples of reproductive tract, pituitary and hypothalamus were excised after 15 min. The 4-microns frozen sections were processed for thaw-mounted autoradiography. The exposure time of the autoradiogram was approximately 6 months. After the injection of [3H]RU 486 and [3H]progesterone, the nuclear concentration of radioactivity was most distinct in muscular and stromal cells of the uterus, and the epithelial nuclei of lumina and glands showed weak labelling. Nuclear localization was also observed in muscle cells of the vagina, cervix and oviduct. After injection of [3H]progesterone, the radioactivity was found in the nuclei and cytoplasm of anterior pituitary cells and some cells showed a preferential nuclear concentration of radioactivity. The distribution of [3H]RU 486 in the anterior pituitary was more extensive than that of [3H]progesterone. In the hypothalamus, specific localization of [3H]RU 486 and [3H]progesterone existed in neurones accumulated in the preoptic nucleus, preoptic suprachiasmatic nucleus and the periventricular nucleus. No localization was found in the diaphragm. Pretreatment with RU 486, but not with dexamethasone, reduced the nuclear concentration of radioactivity of [3H]progesterone in the vagina, uterus, oviduct, pituitary and hypothalamus. The nuclear concentration of radioactivity after injection of [3H]RU 486 was also decreased by preinjection with progesterone. The autoradiographic results suggest that RU 486 and progesterone competed for the specific binding site (possibly a progesterone receptor) in the target cells at the levels of the uterus, pituitary and hypothalamus in vivo.

Renewal rates of murine T-lymphocyte subsets

The proliferative dynamics of T-cell subsets in lymphoid organs of BALB/c mice have been evaluated by means of [ 3H]thymidine injections for up to 3 days, followed by FACS and autoradiography. In the thymus, cells positive for both CD4 and CD8 antigens were more rapidly renewed than single-positive cells. Cell populations showing strong expression of Thy-1 and weak expression of CD5 antigens were more rapidly renewed than populations with the opposite antigen expressions. The results obtained from the spleen and the lymph nodes indicated that the majority of T cells have life spans exceeding a few days, and comparable cell kinetics were found in the different T-cell subpopulations. Results obtained after injections of hydroxyurea, which causes elimination of cycling cells, paralleled the autoradiographic results.

Autoradiographic localization of [3H]RU 486 and [3H]progesterone in the uterus, pituitary and hypothalamus of the rat

Twenty-one castrated oestrogen-primed Wistar rats, which were 2-months-old, were injected via the jugular vein with 100 microCi/100 g body weight of [3H]RU 486 or [3H]progesterone. Some of these received unlabelled compounds for competition studies. Samples of reproductive tract, pituitary and hypothalamus were excised after 15 min. The 4-micron frozen sections were processed for thaw-mounted autoradiography. The exposure time of the autoradiogram was approximately 6 months. After the injection of [3H]RU 486 and [3H]progesterone, the nuclear concentration of radioactivity was most distinct in muscular and stromal cells of the uterus, and the epithelial nuclei of lumina and glands showed weak labelling. Nuclear localization was also observed in muscle cells of the vagina, cervix and oviduct. After injection of [3H]progesterone, the radioactivity was found in the nuclei and cytoplasm of anterior pituitary cells and some cells showed a preferential nuclear concentration of radioactivity. The distribution of [3H]RU 486 in the anterior pituitary was more extensive than that of [3H]progesterone. In the hypothalamus, specific localization of [3H]RU 486 and [3H]progesterone existed in neurones accumulated in the preoptic nucleus, preoptic suprachiasmatic nucleus and the periventricular nucleus. No localization was found in the diaphragm. Pretreatment with RU 486, but not with dexamethasone, reduced the nuclear concentration of radioactivity of [3H]progesterone in the vagina, uterus, oviduct, pituitary and hypothalamus. The nuclear concentration of radioactivity after injection of [3H]RU 486 was also decreased by preinjection with progesterone. The autoradiographic results suggest that RU 486 and progesterone competed for the specific binding site (possibly a progesterone receptor) in the target cells at the levels of the uterus, pituitary and hypothalamus in vivo.

Thyroxine, triiodothyronine, and reverse triiodothyronine processing in the cerebellum: autoradiographic studies in adult rats.

Well confirmed evidence has demonstrated that the cerebellum is an important target of thyroid hormone action during development. Moreover, the presence of nuclear receptors and strong 5'-deiodinase activity in cerebella of adult rats have suggested that this region may continue to respond to thyroid hormones during maturity. Recent autoradiographic observations have focused attention on the cerebellar granular layer, in that [125I]T3 administered iv to adult rats was found to be selectively and saturably concentrated there. To determine the specificity of iodothyronine localization in the granular layer, we have now compared film autoradiographic observations made after iv [125I]T4 and iv [125I]rT3 with those found after iv [125I]T3. The results demonstrated that, as in the case of the latter hormone, labeling within the cerebellar cortex after iv [125I]T4 was both selective and saturable. Moreover, except for a lag in time to resolution and a longer retention time, the distribution of cerebellar radioactivity after iv labeled T4 was qualitatively similar to that seen after iv [125I]T3. However, the ability of T4 to become differentially concentrated in the granular layer of cerebellum was absolutely dependent on its ability to be converted intracerebrally to T3. Thus, pretreatment with ipodate, which blocks brain 5'-deiodinase activity and, therefore, the intracerebral formation of T3 from T4, completely prevented cerebellar granular layer labeling after iv [125I]T4 even though it did not interfere with differential labeling of this region by iv delivered [125I]T3. In the same experiments, propylthiouracil, a potent peripheral, but not central, 5'-deiodinase inhibitor, had no qualitative effect on the distribution of either T4 or T3 in cerebellum. By contrast with the results obtained after administering labeled T3 or T4, brain labeling after iv delivered [125I]rT3 was found to be no different from that produced by markers of cerebral blood flow, which rapidly enter and leave the brain without becoming incorporated into brain cells. This was so even during treatment with propylthiouracil and ipodate, both of which markedly prolonged the normally brief residence time of this iodothyronine in serum and brain. Overall, the autoradiographic results served to highlight the importance of the morphological approach for investigating thyroid hormone action and metabolism in brain. They demonstrated that only T3, whether entering as such from the circulation or formed in situ from T4 (but neither T4 itself nor iv administered rT3) was strongly, selectively, and saturably concentrated in the cerebellar granular layer of adult rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular substrates for interactions between neurons containing phenylethanolamine N-methyltransferase and GABA in the nuclei of the solitary tracts.

Adrenaline and gamma-aminobutyric acid (GABA) have been implicated in autonomic functions involving the intermediate and caudal portions of the medial nuclei of the solitary tracts (m-NTS). We sought to determine whether there was a cellular basis for direct intracellular or synaptic interactions between these transmitters in neurons in the m-NTS of rat brain by using dual-labeling immunocytochemical methods. Light microscopy revealed immunoautoradiographic labeling for the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) in perikarya and processes in close proximity to cells demonstrating peroxidase reaction product for GABA. Electron microscopy of the intermediate m-NTS at the level of the area postrema further established the localization of immunoautoradiographic and peroxidase labels for PNMT and GABA in common as well as separate perikarya and dendrites. All axon terminals were labeled separately for PNMT and GABA. The PNMT-labeled terminals formed both symmetric and asymmetric synapses, whereas the GABA-labeled terminals formed exclusively symmetric synapses. Twenty-four percent (n = 42) of the PNMT- and 39% (n = 128) of the GABA-labeled terminals formed synaptic junctions on unlabeled soma and dendrites. Occasionally both types of terminals converged on a common unlabeled dendrite and on GABA-labeled dendrites. Only 3% of the PNMT- and 12% of the GABA-containing terminals formed synapses on PNMT-labeled soma and dendrites, whereas 7% of each type formed synapses with GABA-labeled profiles. The remaining labeled terminals lacked synaptic relations within the sections examined. The autoradiographic results were confirmed and extended by means of immunogold labeling for PNMT in combination with peroxidase-antiperoxidase localization of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). GAD-labeled terminals formed symmetric synapses with dendrites that were either unlabeled or contained low levels of PNMT (gold particles) or PNMT and GAD. We conclude that in caudal, more cardiovascular portions of the NTS, adrenaline and GABA may coexist, but they are more commonly detected in separate populations of neurons having receptive sites for both transmitters and innervating certain common target neurons.

Distribution of mu, delta, and kappa opiate receptor types in the forebrain and midbrain of pigeons

Ligands that are highly specific for the mu, delta, and kappa opiate receptor binding sites in mammalian brains have been identified and used to map the distribution of these receptor types in the brains of various mammalian species. In the present study, the selectivity and binding characteristics in the pigeon brain of three such ligands were examined by in vitro receptor binding techniques and found to be similar to those reported in previous studies on mammalian species. These ligands were then used in conjunction with autoradiographic receptor binding techniques to study the distribution of mu, delta, and kappa opiate receptor binding sites in the forebrain and midbrain of pigeons. The autoradiographic results indicated that the three opiate receptor types showed similar but not identical distributions. For example, mu, delta, and kappa receptors were all abundant within several parts of the cortical-equivalent region of the telencephalon, particularly the hyperstriatum ventrale and the medial neostriatum. In contrast, in other parts of the cortical-equivalent region of the avian telencephalon, such as the dorsal archistriatum and caudal neostriatum, only kappa receptors appeared to be abundant. Within the basal ganglia, all three types of opiate receptors were abundant in the striatum and low in the pallidum. Within the diencephalon, kappa and delta binding was high in the dorsal and dorsomedial thalamic nuclei, but the levels of all three receptor types were generally low in the specific sensory relay nuclei of the thalamus. Kappa binding and delta binding were high, but mu was low in the hypothalamus. Within the midbrain, all three receptor types were abundant in both the superficial and deep tectal layers, in periventricular areas, and in the tegmental dopaminergic cell groups. In many cases, the distribution of opiate receptors in the pigeon forebrain generally showed considerable overlap with the distribution of opioid peptide-containing fiber systems (for example, in the striatal portion of the basal ganglia), but there were some clear examples of receptor-ligand mismatch. For example, although all three receptor types are very abundant in the hyperstriatum ventrale, opioid peptide-containing fibers are sparse in this region. Conversely, within the pallidal portion of the basal ganglia, opioid peptide-containing fibers are abundant, but the levels of opiate receptors appear to be considerably lower than would be expected. Thus, receptor-ligand mismatches are not restricted to the mammalian brain, since they are a prominent feature of the organization of the brain opiate systems in pigeons.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative macroautoradiography.

Visualizing the localization of given xenobiotics in the body becomes possible by means of application of macroautoradiography, though there are still many problems, which we have to solve near future, and also there are a few limited evaluation points. In the most important of the problems is whether or not autoradiographic results are quantitatively reliable in a conventional manner. Many reports have been published in this field as described in this article briefly since 1961. However, all of them were limited in a mechanical improvement how to read the shading of blackening on the recorded photo film compared with a density control.In this article, the remains of the problems are pointed out and discussed in detail. First, essential factors are determined for quantitative macroautoradiography.(1) To use radioisotopes with soft β emitters for the tracer of given xenobiotics in major cases. (2) To keep a given thickness of the thin sections through a contact period with the photosensitive material., (3) To elucidate whether or not there is a good and simple correlation between the radioactivity concentration (dpm/μm3 × μm in thickness) of a soft β emitters and relative intensity developed at a localized photosensitive material, and finally, (4) To express a resulting image more finely, comparatively, quickly, and easily with computer assistance.Results are given the most important factor for quantitative macroautoradiography to keep a given thickness of the thin section without any dryness. Dryness caused impossible correction of selfabsorption of soft emitters in the substances. The conventional X-ray films have too many and complex factors to calculate and simplify correctly the radioactivity concentration from the resulted autoradiographic image. And the majority of the factors might be in secret for covering the production pattents. Consequently, the conventional X-ray film is available for highly resolving, more impressible, and more microanalytic autoradiography of soft β emitters but not quantitative macroautoradiography.For this, we recommend “Imaging Plate”. Flexible plastic plate coated with fine photosimulable phospher crystals (BaFBr: Eu2+), which was developed by Miyahara et al, Fuji Photo Fim Co. Results indicated good correlation between the relative intensity of PSL (Intensity × exposure time) and the radioactivity concentration (dpm/μm3 × μm in given thickness) of each tissue, assumed by correcting the selfabsorption of 14C in the thin sections.