Affinity labelling of cholinergic receptors
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The superior colliculus (SC) plays a critical role in orienting movements, in part by integrating modulatory influences on the sensorimotor transformations it performs. Many species exhibit a robust brain stem cholinergic projection to the intermediate and deep layers of the SC arising mainly from the pedunculopontine tegmental nucleus (PPTg), which may serve to modulate SC function. However, the physiological effects of this input have not been examined in vivo, preventing an understanding of its functional role. Given the data from slice experiments, cholinergic input may have a net excitatory effect on the SC. Alternatively, the input could have mixed effects, via activation of inhibitory neurons within or upstream of the SC. Distinguishing between these possibilities requires in vivo experiments in which endogenous cholinergic input is directly manipulated. Here we used anatomical and optogenetic techniques to identify and selectively activate brain stem cholinergic terminals entering the intermediate and deep layers of the awake mouse SC and recorded SC neuronal responses. We first quantified the pattern of the cholinergic input to the mouse SC, finding that it was predominantly localized to the intermediate and deep layers. We then found that optogenetic stimulation of cholinergic terminals in the SC significantly increased the activity of a subpopulation of SC neurons. Interestingly, cholinergic input had a broad range of effects on the magnitude and timing of SC responses, perhaps reflecting both monosynaptic and polysynaptic innervation. These findings begin to elucidate the functional role of this cholinergic projection in modulating the processing underlying sensorimotor transformations in the SC.
Superior colliculus
Pedunculopontine Tegmental Nucleus
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Amino acids such as L-tyrosine, L-histidine, and tryptophan, which bear an aromatic ring in the molecule, could successfully be labelled by radioactive iodine through arylthallium ditrifluoroacetate intermediate. Generally, the labelling reaction could proceed in a short labelling time(ca, 20 minutes) and resulted in a high labelling yields and purity of the labelled product. This procedure has, therefore, been proved to be effective as the labelling method of short labelling time and high specific activity. Labelling proteins such as oval albumin and human albumin could also be achieved in net labelling yield by thallating them at the low temperature , whereas the labelled products were mainly composed of various denatured products by thallating them at the high temperature, though the radioactivity was highly retained in the labelled products. Uracil and hippuric acid could also be labelled in a short labelling time though their thallation required a prolonged heating procedure. It was proved that this procedure may be effective to label these compounds by short lived radioisotopes. The labelling yields were, however, lower than 30%.
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A number of different thiol protective groups have been synthesized and attached to mercaptoacetyltriglycine (MAG3) ligand. The newly made MAG3 analogues were labelled with 99mTc by direct labelling under alkaline condition and by stannous tartrate exchange labelling method. In the latter method, the amount of the ligand, reaction temperature and pH varied and their effects on the labelling efficiencies were studied. Radiochemical purities of 51% to 70%, 58% to 75% and 46% to 81% respectively, were obtained by radio-HPLC analysis for the studied MAG3 precursors when, 0.1 mg, 0.4 mg and 1.6 mg of the ligand was used and labelling was performed at both low temperature (70°C) and pH (pH 3). All the studied ligands were efficiently labelled with 99mTc (up to 99%) when heated for 10 min at pH 9 and 100°C. The labelling efficiency obtained by the direct labelling method for MAG3 analogues varied from 32% to 94% and was in all cases lower than after the exchange labelling at pH 9 and 100°C. It was observed that the radiochemical purities can be improved significantly by heating the “direct labelling mixture” at elevated temperature. © 1997 John Wiley & Sons, Ltd.
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Superior colliculus (SC) is a midbrain structure that integrates sensory inputs and generates motor commands to initiate innate motor behaviors. Its retinorecipient superficial layers (sSC) receive dense cholinergic projections from the parabigeminal nucleus (PBN). Our previous in vitro study revealed that acetylcholine induces fast inward current followed by prominent GABAergic inhibition within the sSC circuits (Endo T, Yanagawa Y, Obata K, Isa T. J Neurophysiol 94: 3893-3902, 2005). Acetylcholine-mediated facilitation of GABAergic inhibition may play an important role in visual signal processing in the sSC; however, both the anatomical and physiological properties of cholinergic inputs from PBN have not been studied in detail in vivo. In this study, we specifically visualized and optogenetically manipulated the cholinergic neurons in the PBN after focal injections of Cre-dependent viral vectors in mice that express Cre in cholinergic neurons. We revealed that the cholinergic projections terminated densely in the medial part of the mouse sSC. This suggests that the cholinergic inputs mediate visual processing in the upper visual field, which would be critical for predator detection. We further analyzed the physiological roles of the cholinergic inputs by recording looming-evoked visual responses from sSC neurons during optogenetic activation or inactivation of PBN cholinergic neurons in anesthetized mice. We found that optogenetic manipulations in either direction induced response suppression in most neurons, whereas response facilitation was observed in a few neurons after the optogenetic activation. These results support a circuit model that suggests that the PBN cholinergic inputs enhance functions of the sSC in detecting visual targets by facilitating the center excitation-surround inhibition.NEW & NOTEWORTHY The modulatory role of the cholinergic inputs from the parabigeminal nucleus in the visual responses in the superficial superior colliculus (sSC) remains unknown. Here we report that the cholinergic projections terminate densely in the medial sSC and optogenetic manipulations of the cholinergic inputs affect the looming-evoked response and enhance surround inhibition in the sSC. Our data suggest that cholinergic inputs to the sSC contribute to the visual detection of predators.
Superior colliculus
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A number of different thiol protective groups have been synthesized and attached to mercaptoacetyltriglycine (MAG3) ligand. The newly made MAG3 analogues were labelled with 99mTc by direct labelling under alkaline condition and by stannous tartrate exchange labelling method. In the latter method, the amount of the ligand, reaction temperature and pH varied and their effects on the labelling efficiencies were studied. Radiochemical purities of 51% to 70%, 58% to 75% and 46% to 81% respectively, were obtained by radio-HPLC analysis for the studied MAG3 precursors when, 0.1 mg, 0.4 mg and 1.6 mg of the ligand was used and labelling was performed at both low temperature (70°C) and pH (pH 3). All the studied ligands were efficiently labelled with 99mTc (up to 99%) when heated for 10 min at pH 9 and 100°C. The labelling efficiency obtained by the direct labelling method for MAG3 analogues varied from 32% to 94% and was in all cases lower than after the exchange labelling at pH 9 and 100°C. It was observed that the radiochemical purities can be improved significantly by heating the “direct labelling mixture” at elevated temperature. © 1997 John Wiley & Sons, Ltd.
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HLA-B-associated transcript 3 (BAT3) was originally identified as one of the genes located within human major histocompatibility complex. It encodes a large proline-rich protein with unknown function. In this study, we found that a fragment of the BAT3 gene product interacts with a candidate tumor suppressor, DAN, in the yeast-based two-hybrid system. We cloned the full-length rat BAT3 cDNA from a fibroblast 3Y1 cDNA library. Our sequence analysis has demonstrated that rat BAT3 cDNA is 3617 nucleotides in length and encodes a full-length BAT3 (1098 amino acids) with an estimated molecular mass of 114,801 daltons, which displays an 87.4% identity with human BAT3. The deletion experiment revealed that the N-terminal region (amino acid residues 1-80) of DAN was required for the interaction with BAT3. Green fluorescent protein-tagged BAT3 was largely localized in the cytoplasm of COS cells. Northern hybridization showed that BAT3 mRNA was expressed in all the adult rat tissues examined but predominantly in testis. In addition, the level of BAT3 mRNA expression was more downregulated in some of the transformed cells, including v-mos- and v-Ha-ras-transformed 3Y1 cells, than in the parental cells.
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