, Oct. 2001, 53 (5) : 391 395 391 Acta Physiologica Sinica 52HT 3,, (, 250012) : 6 WGA2HRP C5, 52HT FITC, 52HT, 52HT HRP252HT, HRP ( ) 52HT : 52HT ; 52HT : 52HT ; ; ; 52HT ; WGA2HRP : Q473 ; R33213 Projections of 52HTergic fibers to the spinal phrenic nucleus and medullary phrenic premotor neurons in the cat SONG Gang 3, LI Qin, SHAO Feng2Zhi ( Department of Physiology, Medical College, Shandong University, Jinan 250012) Abstract : Experiments were performed on 6 adult cats. Brainstem neurons that project to the phrenic nu2 cleus were retrogradely labeled by microinjection of WGA2HRP into the spinal phrenic nucleus1 The brainstem sections were double processed for HRP histochemical staining with TMB2DAB method and 52HT immunohisto2 chemical staining with anti252ht primary antibody and FITC conjugated immunofluorescent secondary antibody. HRP252HT double2labeled neurons were observed mainly in the nucleus raphe pallidus. A few double2labeled neurons were also observed in nucleus raphe obscurus and nucleus raphe magnus. 52HT immunopositive termi2 nal fibers were observed in the vicinity of HRP single2labeled neurons (phrenic premotor neurons) located in the nucleus ambiguus and ventrolateral solitary tract nucleus. The results indicate that the 52HTergic neurons in the nucleus raphe pallidus send axonal projections to the phrenic nucleus, and that the brainstem phrenic premotor neurons also receive 52HTergic axonal projections. Key words : 52HTergic neuron ; raphe nucleus ; phrenic nucleus ; anti252ht antibody ; WGA2HRP, 52HT,,, 52HT, [1 ] [8,9, ] [2, 3 52HT ] 52HT 52HT? 52HT Lalley [4 ] Aoki [5 ]? [6, 7 ], Received 2001201215 Accepted 2001203204 This work was supported by grants from the National Natural Science Foundation of China (No139970278) and Ministry of Education1 3 Corresponding author. Tel : 053122942037 ; E2mail : gangsg @sdu1edu1cn
392 Acta Physiol. Sin. 53 1 111 HRP 6 ( 118 215 kg) (Nembutal TM, 30 mg/ kg, ip), SN23, 20 24 h PBS, VECTASHIELD C4 6, ( 115 215 mm, 315 415 mm ), 5 % WGA2HRP 30 min, TMB ( 15 m) 113 52HT HRP 52HT : 52 HT (DiaSorin), 1 1 000, 20 72 h ; : FITC IgG (Vector), 1 100 2, 15 30 nl, 211 HRP 112 HRP 72 h :, HRP (Nembutal TM, 50 mg/ kg, ip),, 2 (3 ) 24 h C1, (colchicine) 300 g : 1 000 ml 0105 mol/ L PBS (37 ), 1000 ml 3 % 201125 % 215 % 011 mol/ L HRP,, PBS (4 ), 20 % PBS, 4 30 40 m : HRP 5 d (40 m) HRP KF, (15 [10 TMB ] 30 m) : 0105 % DAB20101 % H 2 O 2 20102 % CoAc TBS HRP, 1. HRP 52HT (Berman ) Fig1 1. Distribution patterns of HRP labeled neurons and 52HT immunopositive neurons at different levels of Berman atlas in the brainstem1 7, facial nucleus ; AMB, nucleus ambiguus ; KF, K lliker2fuse nucleus ; LC, locus coeruleus ; NRM, nucleus raphe magnus ; NRP, nucleus raphe pallidus ; P, pyramidal tract ; STN, solitary tract nucleus.
5 : 52HT 393 2. 52HT Fig1 2. Microphotographs showing the 52HT immunopositive neurons and fibers. Aa. The 52HT immunopositive neurons observed in the rostral part of nucleus raphe pallidus. The arrow indicates a HRP labeled 52HT immunopositive neurons ( HRP252HT double2 labeled neuron). Ab. The HRP reaction products in the same HRP252HT double2labeled neuron can be clearly seen under the light field microscope1 Ba. A 52HT immunopositive neuron (arrow) observed in the caudal part of nucleus raphe pallidus. Bb. Under light field microscope, this neuron is identified as a HRP labeled neuron (arrow). Ca. 52HT immunopositive terminal fibers (ar2 row) observed in the area of nucleus ambiguus. At the same site, a HRP labeled neuron (right upper corner) can be observed un2 der light field microscope. Cb. 52HT immunopositive terminal fibers (arrow) observed in the ventrolateral part of solitary tract nu2 cleus1 Arrow head indicates HRP labeled neurons1 Da1 52HT immunopositive terminal fibers observed in the rostral part of nucleus ambiguus (corresponding to pre2b tzinger complex, square area) 1 Db. Photograph of the square area at higher magnification1 Scale bars, 30 m1
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