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.     

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.     

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.     

Autoradiographic localization of binding sites for muscarinic and nicotinic agonists and antagonists on cultured astrocytes

Summary The cellular localization of cholinergic binding sites was studied in organotypic cultures of rat spinal cord and brain stem by means of autoradiography with radiolabelled muscarinic and nicotinic agonists and antagonists. Many astrocytes in both types of cultures showed intense labelling by ³H-acetylcholine in the presence of nicotine and the muscarinic antagonists ³H-pirenzepine, ³H-quinuclidinyl benzilate and ³H-propylbenzilcholine mustard. Incubation of the cultures with ³H-nicotine or ³H-acetylcholine in the presence of atropine also caused labelling of astrocytes. In addition to glial cells, many neurones showed binding of muscarinic and nicotinic agonists and antagonists. From our results it is suggested that astrocytes, besides neurones, possess both muscarinic and nicotinic receptors.     

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.     

Autoradiographic localization of binding sites for vasotocin in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Specific binding sites for vasotocin (VT) were detected in brain and pituitary of a teleost fish, the sea bass, after in vitro incubation of tissue sections with [³H]arginine-vasopressin (AVP) and light microscopic autoradiography. Conditions for the binding assay were optimized and as a result the binding was saturable and specific. In the brain [³H]AVP binding was found to occur in the pars lateralis and the pars ventralis of the ventral telencephalon, in the pars centralis of the dorsal telencephalon, in the hypothalamic region (especially in the nucleus preopticus, in the tuberal hypothalamus and around the posterior recess), in the tectum opticum and in the noncellular layer of the corpus cerebelli. In the pituitary a high density of [³H]AVP binding was observed in the areas of the pars distalis (PD) occupied by ACTH-, TSH- and GH-cells and also in the pars intermedia (PI). The present study presents the first anatomical evidence for the presence of VT specific binding sites in teleost brain and pituitary.     

Autoradiographic and immunohistochemical analysis of3H-melatonin distribution in endocrine and nonendocrine organs

Research Institute of Medical Radiology, Academy of Medical Sciences of the USSR, Obninsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 10, pp. 438–440, October, 1990.     

Autoradiographic localization of binding sites for vasotocin in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Specific binding sites for vasotocin (VT) were detected in brain and pituitary of a teleost fish, the sea bass, after in vitro incubation of tissue sections with [³H]arginine-vasopressin (AVP) and light microscopic autoradiography. Conditions for the binding assay were optimized and as a result the binding was saturable and specific. In the brain [³H]AVP binding was found to occur in the pars lateralis and the pars ventralis of the ventral telencephalon, in the pars centralis of the dorsal telencephalon, in the hypothalamic region (especially in the nucleus preopticus, in the tuberal hypothalamus and around the posterior recess), in the tectum opticum and in the noncellular layer of the corpus cerebelli. In the pituitary a high density of [³H]AVP binding was observed in the areas of the pars distalis (PD) occupied by ACTH-, TSH- and GH-cells and also in the pars intermedia (PI). The present study presents the first anatomical evidence for the presence of VT specific binding sites in teleost brain and pituitary.     

Autoradiographic localization of muscimol and baclofen binding sites in rodent cingulate cortex

Summary The laminar distribution of ³H-muscimol and ³H-baclofen binding was analyzed autoradiographically in areas 29c and 24b of rat cingulate cortex. Muscimol binding was heterogeneous in area 29c with a single peak in layer Ia of 320±26 grains per 2500 m². Binding in deeper layers was between 46% and 71% of that in layer Ia. There was a marked diurnal variation in muscimol binding in area 29c such that binding was elevated by 320% in layer Ia of brains perfused at 2200–0100 versus those perfused at 1100–1400. Muscimol binding in area 24b was uniform across all layers and was higher than that in area 29c except for in layer Ia. Baclofen binding was homogeneous in both areas, but was 120% greater in area 24b than in area 29c, and showed no diurnal variations. To localize muscimol binding sites at the cellular level, two types of lesion experiments were conducted in area 29c. First, ablation of neurons intrinsic to this cortex with the neurotoxin ibotenic acid reduced muscimol binding to homogeneity with a 70% reduction in layer Ia and a 29–42% reduction in deeper layers. Second, knife cuts, which were placed to isolate cingulate cortex from fiber pathways originating extrinsically, increased muscimol binding in all layers except layer Ia. Conversely, knife cuts which isolated superficial from deep layers yielded a marked drop in muscimol binding in all layers. In conclusion, muscimol binding sites are heterogeneously distributed in area 29c with peak binding in layer Ia at night. Since experimental observations suggest that muscimol binding is located on pyramidal cell apical tuft dendrites, it is possible that excitatory thalamic and intrinsic inhibitory input via GABA_A receptors on apical dendrites interact before arriving at the soma.     

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.     

Evidence for electrogenic sodium-bicarbonate cotransport in cultured rat cerebellar astrocytes

We have studied the regulation of intracellular pH (pH_i), and HCO ₃ ⁻ -dependent membrane currents in cultured astrocytes from neonatal rat cerebellum, using the fluorescent pH-sensitive dye 2,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and the whole-cell patch-clamp technique. The steady-state pH_i was 6.96 in both nominally CO₂/HCO ₃ ⁻ -free, HEPES-buffered saline (6.96 ±0.14;n=48) and in a saline containing 5% CO₂/24 mM HCO ₃ ⁻ (6.96±0.18;n=48) (at pH 7.4). Inhibition of the Na⁺/H⁺ exchange by amiloride (2 mM) caused a significant decrease of pH_i in nominally CO₂/HCO ₃ ⁻ -free saline. Addition of CO₂/HCO ₃ ⁻ in the continuous presence of amiloride induced a large and fast intracellular alkalinization. Removal of external Na⁺ also caused a fall of pH_i, and addition of CO₂/HCO ₃ ⁻ in Na⁺-free saline evoked a further fall of pH_i, while the outward current was reduced or even reversed. The stilbene 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS, 0.3 mM) reduced the pH_i recovery from the CO₂/HCO ₃ ⁻ -evoked acidification, and blocked the prominent intracellular acidification upon removal of CO₂/HCO ₃ ⁻ . Removal of external Cl⁻ had little effect on these pH_i changes. Lowering the external pH from 7.4 to 6.6 in CO₂/HCO ₃ ⁻ -containing saline produced a large and rapid intracellular acidification and inward current, which were both greatly reduced by DIDS and in the absence of CO₂/HCO ₃ ⁻ . The results suggest that the CO₂/HCO ₃ ⁻ -dependent current is partly due to a reversible bidirectional, electrogenic Na⁺-HCO ₃ ⁻ cotransporter, which helps to regulate pH_i in these cells. In addition, a prominent Na⁺/H⁺ exchanger contributes to extrude acid equivalents from these astrocytes to maintain the steadystate pH_i.     

Transient expression of somatostatin sst2 receptors in rat cerebellar nuclei during development

Adult rat cerebellar nuclei contain a single population of [125I][Leu8,D-Trp22,Tyr25]somatostatin-28 binding sites characterized as sst1 receptors. In the present study, we have investigated the evolution of somatostatin receptors in rat cerebellar nuclei during development by means of quantitative autoradiography on tissue sections. The binding of [125I][Leu8,D-Trp22,Tyr25]somatostatin-28, observed in the primordium of the medial cerebellar nuclei at embryonic day 17, reached a maximum at postnatal day 7 or 10 in the different nuclei. Thereafter, the density of binding sites gradually decreased to the adult level. Competition studies were performed using the somatostatin analogues CH-288 and MK-678 as specific sst1 and sst2 ligands, respectively. Partial inhibition of the radioligand binding by CH-288 and MK-678 revealed the presence of a predominant population of sst1 from embryonic day 19-28 day postnatal and a minor population of sst2 receptors. The use of [125I]MK-678 as a radioligand confirmed the presence of a transient population of sst2 receptors, suggesting that somatostatin could act on rat cerebellar nuclei via sst1 and/or sst2 receptors during development.     

Expression and subcellular localization of NHE3 in the human gallbladder epithelium

Background: Enhanced gallbladder concentrating function is an important factor for the pathogenesis of cholesterol gallstone disease (CGD), but the mechanism is unknown. Potential candidates for regulation of gallbladder ion absorption are suggested to be Na⁺/H⁺ exchanger isoform 3 (NHE₃). In this study, we investigated the expression and subcellular localization of NHE₃ in both acalculous and calculous human gallbladders. Methods: Adult human gallbladder tissue was obtained from 23 patients (7 men, 16 women) who had undergone cholecystectomy. The patients were divided into two groups: Group A (acalculous group) and Group B (calculous group). Gene expression of NHE₃ was quantitatively estimated by real-time PCR. Protein expression was studied by Western blotting assays. Furthermore, expression of immunoreactive NHE₃ was investigated by immunohistochemistry. Results: There was no significant difference in the NHE₃ mRNA expression between calculous and acalculous human gallbladders. NHE₃ protein expression in gallbladders from patients with cholelithiasis is increased compared to those without gallstones. Immunohistochemistry studies prove that NHE₃ is located both on the apical plasma membrane and in the intracellular pool in human GBECs. Conclusions: NHE₃ may play a role in the pathogenesis of human CGD. Additional studies are required to further delineate the underlying mechanisms.     

Different types of 3H-GABA accumulating neurons in the visual cortex of the rat. Characterization by combined autoradiography and Golgi impregnation

Summary Labelled neurons were identified by autoradiography following tangential intracortical injection of [³H]--aminobutyrate (GABA).The addition of cis-1,3-aminocyclohexane carboxylic acid to the GABA solution prevented perikaryal labelling. Labelled neurons were found in each injected layer and in addition they were always present directly above the injection track. The labelling of neurons in layer II. and upper III. following injections in layers V. and VI. can be explained by retrograde axonal transport and indicates that some GABA-ergic neurons project vertically.Ninety neurons of different types were Golgi impregnated and examined for selective [³H]-GABA uptake. Sixteen of these were labelled. On the basis of dendritic characteristics they were classified as aspiny multipolar neurons with small, medium or large dendritic fields, sparsely spiny multipolar neurons and one neuron was a bipolar cell. Thus Golgi impregnation of their processes reveals that cortical GABA-ergic neurons are a heterogeneous population.A [³H]-GABA accumulating, aspiny neuron with profoundly branching, bushy dendrites and locally arborizing axon in layer VI. was studied in the electron microscope. Its fine structural characteristics were similar to those of other identified non-pyramidal neurons. The existence of several types of cortical GABA-ergic neurons differing in their synaptic connections is discussed.Financially supported by the Hungarian Academy of Sciences, the International Cultural Institute of Budapest, the Wellcome Trust, the Royal Society, and the E. P. Abraham Cephalosporin TrustDuring part of this project P. Somogyi was supported by the Wellcome trust at Dept. of Pharmacology, Oxford University     

Electrophysiological properties of neurones in cultures from postnatal rat dentate gyrus

Electrophysiological properties of neurofilament-positive neurones in dissociated cell cultures were prepared at postnatal days 4–5 from rat dentate gyrus and studied using the whole-cell patch-clamp technique. These cells expressed a fast-inactivating, 0.5 M tetrodotoxin-sensitive Na⁺ current; a high-voltage-activated (HVA) Ca²⁺ current, which was 30 M Cd²⁺- and partially 2 M nicardipine-sensitive; and an inward rectifier current, which was sensitive to extracellularly applied 1 mM Cs⁺. The outward current pattern was composed of a delayed rectifier-like outward current sensitive to 20 mM tetraethylammonium (TEA) and a fast-inactivating, Ca²⁺-dependent outward current. This transient Ca²⁺-dependent K⁺ outward current was identified by a subtraction procedure. K⁺ currents recorded under conditions of blocked Ca²⁺ currents (after rundown of the HVA Ca²⁺ current or blocked by extracellularly applied Cd²⁺) were subtracted from control currents. By comparison with the current pattern of identified dentate granule cells, it is concluded that the investigated cell type originated from interneurones or projection neurones of the dentate hilus.     

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.     

Autoradiographic experiments to examine uptake, anterograde and retrograde transport of tritiated serotonin in the mammalian brain

Summary In an attempt to define the potential application of neurotransmitter-specific transport as a method of tracing fiber connections, we have examined the uptake and subsequent ortho- and retrograde transport of tritiumlabeled serotonin (³H-5HT) in the cerebellum-raphe pallidus system. Injection of various concentrations of ³H-5HT followed by different post-injection survival times revealed different labeling patterns in the injected sites and different patterns of transport. The most striking feature is that nonserotonin neurons as well as serotonin cells were able to take up and transport the tritium label in both ortho- and retrograde fashion. The non-sertonin-specific nature of this uptake and transport is more obvious at higher concentrations of ³H-5HT (more than 9x10^-5 M), with longer survival times and following pretreatment with monoamine oxidase inhibitors. At a concentration of 9x10^-6 M ³H-5HT, only specific uptake seems to take place as evidenced by label in known serotonin cells and fiber systems; however, it was impossible to detect by autoradiography any ortho- or retrograde transport at this low concentration. Non-specific uptake and transport were observed following injection into the vestibular nuclei and oculomotor complex. This suggests that non-specific uptake and the transport of ³H-5HT or metabolites may also occur in other regions of the central nervous system.This work was supported in part by U.S. Public Health Service Grants NS 03659, NS 14740, a Louise Harkness Ingalls Fellowship in Research in Parkinson's Disease, and an Alfred P. Sloan Foundation Fellowship in Neuroscience