Absence of binding of α-bungarotoxin and cobra neurotoxin to central acetylcholine receptors — An autoradiographic study

Radiolabelled α-bungarotoxin and cobra neurotoxin, ejected from micropipettes in the vicinity of Renshaw cells, failed to label these neurones.     

Effect of a spider toxin (JSTX) on excitatory postsynaptic current at neuromuscular synapse of spiny lobster

1. We studied the blocking properties of a spider (Nephila clavata) toxin (JSTX) purified from venom on the spiny lobster neuromuscular junction. 2. When a small amount of JSTX was applied to the neuromuscular junction, the excitatory postsynaptic potential (EPSP) was partially suppressed. The amplitude of EPSPs remained at a steady level for several hours during the washing of the preparation, showing that the action of JSTX is irreversible. 3. We recorded the excitatory postsynaptic current (EPSC) from synaptic site using a macro-patch electrode. The amplitude of EPSC increased linearly with hyperpolarization of the membrane potential in the presence and absence of JSTX. 4. The decay phase time constant of EPSC and spontaneous EPSC was decreased by hyperpolarizing the membrane potential both in the absence and in the presence of JSTX. The relationship between the decay time constant and the membrane potential was not modified by JSTX. 5. It is suggested that JSTX irreversibly blocks EPSC by acting on the site that is apart from the ionic channel of the glutamate receptor molecule.     

An autoradiographic microtechnique to study lymphocyte stimulation in solid cultures.

A microtechnique to study lymphocyte stimulation in solid cultures by autoradiography is described. Using this technique at a concentration of 2 x 10(6) lymphocytes/chamber, it was found that the percentage of proliferating cells was 8.4% with anti-lymphocyte serum, 3.9% with calcium ionophore A23187, 2.5% with phytohemagglutinin P, 5.8% with phytohemagglutinin HA17 and 4.2% with tetradecanoyl-phorbolacetate. Lymphocyte responses were dependent on cell concentration. Lymphocyte responses to phytohemagglutinins P and HA17 were potentiated by incorporation into the cultures of 5% autologous erythrocytes.     

Myogenic cell formation in regenerating rat skeletal muscle injured by mincing II. An autoradiographic study

Myonuclei and satellite cell nuclei were differentially labelled with ³H-thymidine in uninjured skeletal muscle of young rats and then traced autoradiographically at intervals after mincing the radioactive hindlimb muscles to determine the source of regenerating presumptive myoblasts. Labelled nuclei were detected by light microscopic examination of 1-m? thick autoradiographs and identified by electron microscopic examination of an adjacent section. Repeated injections of ³H-thymidine during fetal and neonatal development, followed by a 4- to 5-week maturation period, resulted in labelling of 20% of the myonuclei. Satellite cells were not observed to be labelled in this series. Eight to sixteen hours after mincing, 20% of the pyknotic myonuclei were labelled, whereas none of the regenerating presumptive myoblasts appeared labelled. A single injection of ³H-thymidine administered to 18-day-old rats, followed by sacrifice within ten hours, resulted in labelling of 23% of the satellite cell nuclei. Myonuclei were not observed to be labelled in this series. Eight to sixteen hours after mincing, silver grains were detected over both pyknotic and regenerating cell nuclei. These experiments indicate that many satellite cells survive muscle injury and transplantation to become regenerating myogenic cells at a time when most, if not all, myonuclei are undergoing pyknosis.     

Aldosterone binding in isolated tubules II. An autoradiographic study of concentration dependency in the rabbit nephron

Specific binding of aldosterone along the rabbit nephron was investigated using an autoradiographic method on microdissected tubules within a large range of [3H]aldosterone ([3H]A) concentrations (3 X 10(-10) to 2 X 10(-8) M). Paired incubations with [3H]A in the presence or absence of unlabeled A (100-fold excess) were done to determine the specific binding. In addition, competition studies (10 times excess) with A, dexamethasone (Dex), and estradiol (E) were performed. No specific nuclear binding was detected in the proximal convoluted tubule and pars recta. In the cortical collecting tubule (CCT), a high specific labeling occurred at concentrations of A as low as 3 X 10(-10) M that plateaued at 1.5 X 10(-9) M. Aldosterone was the best competitor. In the bright and granular distal tubule, the binding resembled that of CCT, but Dex was nearly as potent a competitor as A. All along the loop of Henle and in the medullary collecting duct, the binding of A was weak at low concentrations and gradually rose with concentration without reaching a plateau. An almost equivalent displacement was obtained by A and Dex in 10-fold excess. We conclude that aldosterone binds mostly to mineralocorticoid sites in the collecting tubule, whereas the binding appears to be to both mineralo- and glucocorticoid sites in the distal tubule, in the different parts of the loop of Henle, and in the medullary collecting tubule.     

A histochemical study of glutamate receptor in rat brain using biotinyl spider toxin

Joro spider toxin (JSTX), a specific blocker of glutamate receptors, was conjugated with biotin. Using the avidin-biotin complex method, specific binding sites of biotinylated JSTX were demonstrated in the cerebellum and hippocampus of the rat. In the cerebellum, strong binding of biotinyl JSTX was observed on perikarya and dendrites of the Purkinje cells with much less binding in the granular cell layer. In the hippocampus, a dense staining was observed in the pyramidal cell layer, with more heavy binding in CA3 than in other sectors of Ammon's horn. The area of distribution of biotinyl JSTX binding sites corresponded well with that of receptors preferentially activated by quisqualate.     

The distribution of serotonin binding sites in the hippocampal region of the rat brain. An autoradiographic study

The distribution of serotonin binding sites was studied in the rat hippocampal region by using contact-film autoradiography after in vitro incubations of brain sections with 5-[3H]hydroxytryptamine, [3H]spiperone, and [3H]ketanserin, respectively. Biochemical studies of the 5-[3H]hydroxytryptamine binding to sections cut through the hippocampal region showed that at saturating concentrations of 5-[3H]hydroxytryptamine (2-2.5 nM) the specific binding was at least 50% of the total. The 5-[3H]hydroxytryptamine binding sites were found to be heterogeneously distributed within the hippocampal region with the highest densities present in the following parts: layers I and II and layers IV through VI of the entorhinal area, the radial layer of the subiculum and subfield CA1 of the Ammon's horn and the molecular layer of the area dentata. Moderate to low densities of binding was observed in layer III of the entorhinal area, the pre- and parasubiculum, the stratum pyramidale of the Ammon's horn, and the granular cell layer of the area dentata. Removal of the 5-hydroxytryptamine nerve terminals by systemic injections of the 5-hydroxytryptamine neurotoxin parachloroamphetamine resulted in no detectable reductions of 5-[3H]hydroxytryptamine binding in any brain region. Lesions of hippocampal cell bodies by intrahippocampal injections of ibotenic acid prevented the binding of 5-[3H]hydroxytryptamine within the area of the cell loss. Comparisons between the distribution of 5-hydroxytryptamine immunoreactive nerve terminals and the 5-[3H]hydroxytryptamine binding sites showed that in some areas of sparse 5-hydroxytryptamine innervation the 5-[3H]hydroxytryptamine binding was close to background (e.g. the pyramidal cell layer, the stratum lucidum) whereas in areas with little 5-[3H]hydroxytryptamine binding (e.g. layer III of the lateral entorhinal area, the presubiculum) a very dense 5-hydroxytryptamine innervation was found. The hippocampal 5-[3H]hydroxytryptamine binding was displaced neither by ketanserin (1 microM) nor by spiperone (1 microM), two drugs that bind to cortical 5-hydroxytryptamine2 receptors in the rat brain. Furthermore, the pattern of hippocampal [3H]spiperone binding differed considerably from that of 5-[3H]hydroxytryptamine. The [3H]ketanserin binding in the hippocampal region did not exceed background levels, except in the hilus of area dentata in the ventral hippocampus and entorhinal layer VI at the same level, where moderate binding was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

An autoradiographic study of satellite cells in autonomic ganglia

This investigation was undertaken to observe and to correlate the responses of satellite cells in autonomic ganglia to increased neuronal and synaptic activity. By electrically stimulating preganglionic nerve fibers terminating in the superior cervical sympathetic ganglion in 19 adult cats and concurrently injecting thymidine-H₃ into the stimulated animal, it was possible to alter the neuronal and synaptic activity within the superior cervical ganglion and subsequently observe from autoradiograms the cellular absorption of thymidine-H₃. The specific reaction encountered was incorporation of thymidine-H₃ into the nuclei of satellite cells. Absorption of thymidine by satellite cells and incorporation of this compound into their nuclear structure could be accelerated or inhibited by exposing the organism to specific drugs (viz. neostigmine and atropine sulfate) during the stimulatory period. The results of this investigation combined with information gathered from studies in many other scientific areas implicate satellite cell participation in response to increased neuronal metabolism and possibly to increased synaptic activity. Satellite cells apparently proliferate to cope with the demands of increased ganglionic activity elicited by the experimental procedures employed in this study. Although specific reactions of satellite cells other than cellular proliferation were not identified in this study, their probability may provide a basis for further investigation.     

Arginine-vasopressin binding sites in rat brain: a quantitative autoradiographic study

Specific binding sites for arginine-vasopressin (AVP) were detected in rat brain after incubation of tissue sections with [3H]AVP. AVP and two selective AVP antagonists are capable of displacing [3H]AVP with an IC50 in the 10(-8)-10(-7) molar range, while oxytocin and ACTH4-10 were much less effective. The neuroanatomical distribution of [3H]AVP-labeled sites was studied with autoradiography utilizing tritium-sensitive LKB film and computerized densitometry for quantitative analyses of the film images. The highest density of [3H]AVP binding sites was observed in hippocampal regions, the lateral septum, olfactory and amygdaloid nuclei, and the nucleus tractus solitarii (NTS) of the brainstem.     

Inhibitory effect of 2,4-dihydroxyphenylacetylasparagine, a common moiety of spider toxin, on glutamate binding to rat brain synaptic membranes

2,4-Dihydroxyphenylacetylasparagine (2,4-DHPA-ASN), a common moiety of molecules of spider toxins, was shown to inhibit L-[3H]glutamic acid binding to rat brain synaptic membranes in a dose-dependent manner. The inhibitory effect of 2,4-DHPA-ASN was almost the same as that of intact spider toxin isolated from Nephila clavata, but significantly higher than that of 2,4-dihydroxyphenylacetic acid (2,4-DHPA). In addition, neither 2,4-dihydroxybenzoic acid nor the isomers of 2,4-DHPA suppressed the glutamate binding. These results suggested that 2,4-DHPA might be the functional part and asparagine in the molecules of spider toxins seemed to cause increasing affinity toward the recognition site of glutamate binding.     

An autoradiographic study of neurotensin receptors in the human hypothalamus

The aim of the present investigation was to determine a detailed mapping of neurotensin (NT) in the human hypothalamus, the brain region involved in neuroendocrine control. For this, we investigated the presence and the distribution of neurotensin binding sites in the human hypothalamus, using an in vitro quantitative autoradiography technique and the selective radioligand monoiodo-Tyr3-neurotensin (2000 Ci/mM). This study was performed on nine adult human postmortem hypothalami. We first determined the biochemical kinetics of the binding and found that binding affinity constants were of high affinity and do not differ significantly between all cases investigated. Our analysis of the autoradiographic distribution shows that NT binding sites are widely distributed throughout the rostrocaudal extent of the hypothalamus. However, the distribution of NT binding sites is not homogenous and regional variations exist. In general, the highest densities are mainly present in the anterior hypothalamic level, particularly in the preoptic region and the anterior boarding limit (i.e. the diagonal band of Broca). Important NT binding site densities are also present at the mediobasal hypothalamic level, particularly in the paraventricular, parafornical and dorsomedial nuclei. At the posterior level, relatively moderate densities could be observed in the mammillary complex subdivisions, apart from the supramammillary nucleus and the posterior hypothalamic area. In conclusion, the present study demonstrates the occurrence of high concentrations of NT binding sites in various structures in many regions in the human adult hypothalamus, involved in the control of neuroendocrine and/or neurovegetative functions.     

An autoradiographic study of topographical relationships between pallidal and cerebellar projections to the cat thalamus

Summary Injections of ³H-leucine were made in the entopeduncular nucleus or dentate nucleus of the cerebellum in eight cats. The terminal projection zones of both pathways in the thalamus were studied using the sagittal plane and their relationships to one another as well as to cytoarchitectural boundaries of thalamic nuclei were compared. The data indicate that the territories controlled by the two projection systems are almost entirely segregated. The segregation is mainly along the antero-posterior axis as the main pallidal projection zone occupies the medio-ventral VA while the main dentate projection zone lies posterior to it in the VL. Furthermore, the dorsolateral part of the VA not occupied by pallidal projections receives dentate projections. In the VM, both afferent systems terminate in the lateral part of the nucleus with pallidal territory located anteriorly and dentate territory located posteriorly, again without overlap. As the delineations of nuclear subdivisions in the ventral thalamus of the cat have been a subject of some controversy, it is suggested that the boundaries of the VA, VL and VM in the cat thalamus be defined on the basis of basal ganglia and cerebellar projection zones.Abbreviations used in the Text and in Fig. 5 AM anterior medial nucleus - AV anterior ventral nucleus - BC brachium conjunctivum - CA anterior commissure - CC crus cerebri - CP posterior commissure - CD caudate nucleus - CE centrum medianum - CLN central lateral nucleus - DN dentate nucleus - EPN entopeduncular nucleus - FF Forel's field - FN fastigial nucleus - FR fasciculus retroflexus - HL lateral habenular nucleus - HM medial habenular nucleus - INA anterior interposite nucleus - INP posterior interposite nucleus - IC internal capsule - LD lateral dorsal nucleus - LG lateral geniculate body - MD medial dorsal nucleus - MTT mamillothalamic tract - NR red nucleus - OT optic tract - PAC paracentral nucleus - PF parafascicular nucleus - PV pulvinar - RT reticular thalamic nucleus - SM submedian nucleus - SN substantia nigra - SNr substantia nigra pars reticularis - STN subthalamic nucleus - VF ventral posterior nucleus - VA ventral anterior nucleus - VL ventral lateral nucleus - VM ventral medial nucleus - ZI zona incerta Supported in part by a grant from the American Parkinson Disease Association and NIH grant R01NS19280     

Aldosterone binding along the rabbit nephron: an autoradiographic study on isolated tubules

The sites of action of aldosterone (A) along the tubule of rabbit kidney were studied by autoradiographic localization of mineralocorticoid-binding sites on microdissected tubular segments. Kidney pyramids were incubated at 30 degrees C for 1 h in a collagenase solution with [3H]aldosterone at a concentration of 1.5 X 10(-9) M with and without an excess unlabeled A. Tubular segments were then microdissected and transferred onto dry film; fixation and staining were done only after exposure of the film 4 mo later in order to avoid diffusion. Specific nuclear labeling was 19.0 +/- 1.3 silver grains/100 micrometers2 in distal convoluted tubules (n = 28) and 21.0 +/- 1.8 in cortical collecting ducts (n = 18). No difference between these two structures was observed (P greater than 0.1, paired t test, n = 15). No specific binding was found in the proximal tubule (0.5 +/- 0.4, n = 17). In the thick ascending limb of Henle's loop, the labeling was low (3.9 +/- 0.9, n = 16). We conclude that, in the rabbit kidney, nuclear mineralocorticoid-binding sites, presumably receptors, are present in the distal and cortical collecting tubule.     

Neurogenesis in the cerebellum of the rat: An autoradiographic study

Summary Time of origin of various neuronal elements in the cerebellum of rat was established with the aid of tritiated-thymidine-autoradiography. The earliest nerve cells to form were the Purkinje cells, and they came into existence on days 15 and 16 of gestation. Interstitial nerve cells had their genesis on days 15, 16, 17 and 18, and the marginal cells on day 16 of the embryonic development. The Golgi cells were found to come into existence on days 17, 18 and 19 of gestation. On day 21 of gestation a number of small-medium-sized nerve cells, which were smaller than the Golgi cells but larger than the granule cells, were seen to come into existence. Finally, the earliest stock of granule, basket and stellate cells, primarily in the nodulus, flocculus and para-flocculus, were observed to have been formed on the day 21 of embryogenesis.This research was supported by NIH Research Grant No. NS-08817-03. Histological work by Sheila Anderson and photographic work by Donna Whitehurst is gratefully acknowledged.     

An autoradiographic and immunocytochemical study of the neonatal rat pituitary gland

Two-day-old female rats were injected with 5 nmole/kg of 6,7-³H-11β-methoxy-17-ethylestradiol (R 2858 = moxestrol) and killed one hour later. The animals were decapitated and, the pituitary glands were removed, mounted on tissue holders and frozen in liquified propane. The tissue was then processed for autoradiography according to the thaw mount technique. At the end of the exposure time, prior to photographic development, some of the tissue was fixed in 10% formalin and then photographically developed for autoradiography. The fixed tissue was subsequently stained immunocytochemically using antibodies to luteinizing hormone or prolactin. Between 10 and 15% of the cells of the pars distalis concentrated the synthetic estrogen or its metabolite. The immunocytochemical procedure revealed that both LH-gonadotrophs and lactotrophs concentrated the steroid. These studies along with earlier studies suggest that the neonatal rat pituitary contains only a small portion of the adult complement of estrogen receptors and that these receptors are dispersed across a number of cell types.     

An autoradiographic study of long-term follicular development in the cyclic hamster

The time required for ovarian follicular development in the cyclic hamster was determined by marking follicles with [3H]-thymidine on day 1 of the estrous cycle (day of ovulation) and by following their fate over the next three cycles by autoradiography. A model of follicular development was formulated, based on the total number of follicles and the changing percentage of labelled follicles with time. It was estimated that 20 days elapsed before a preantral follicle with two to three layers of granulosa cells (stage 1) was able to ovulate. Follicles spent eight days in stage 1 and an additional eight days in stage 2 (4-5 granulosa layers). Preantral follicles entering stage 3 (6-7 granulosa layers) and 4 (greater than 8 layers) on day 1 of the cycle ovulated four days later. Approximately 30 follicles per ovary entered and left stage 1 each cycle, representing a 50% turnover. Of the follicles leaving stage 1, about 50% were transformed into stage 2 follicles; the remaining ones underwent atresia. Stage 2 follicles also had a 50% turnover each cycle. Half of the stage 2 follicles underwent atresia; the remainder developed into stages 3 and 4 follicles. Fifty percent of the stages 3 and 4 follicles present on day 1 of the cycle ovulated at the next estrus with the remainder undergoing atresia.     

Characterization of binding sites for spider toxin, [3H]NSTX-3, in the rat brain

A group of spider toxins (JSTX, NSTX, argiopin, argiotoxin etc.) share a basic common structure and have been reported to block strongly quisqualate- and kainate-sensitive glutamate responses in vertebrate and invertebrate nervous systems. They are presumed to be potent antagonists of both quisqualate and kainate receptors and may serve as useful tools for characterizing these receptors. We report here the synthesis of tritium-labeled NSTX-3 and the characterization of its binding sites in the rat brain. We found that high- and low-affinity binding sites exist in the cerebellum (Kd = 7.75 and 202 nM, Bmax = 0.37 and 5.54 pmol/mg protein, respectively). Synthetic NSTX analogs strongly inhibited [3H]NSTX-3 binding in the cerebellum (IC50 = 10(-7)-10(-6) M), whereas competitive agonists of glutamate receptors (AMPA, quisqualate, NMDA, kainate, glutamate and aspartate) exhibited weak or no inhibitory effects.