ABSTRACT
Objective To investigate the effect of extracellular signal-regulated kinases(ERK) signal transduction in the process of NSCs differentiating into neurons in the fimbria-transected hippocampi's extracts. Methods Twelve Sprague-Dawley rats'right fimbrias were transected. The extracts were gained from the fimbria-transected hippocampi at the 14th day normal rat, and the extracts supernatant fluid was collected after centrifugal process, then the protein concentration in the extracts was determined. In the serum-free medium,NSCs from the fetal hippocampus were planted on 24 well culture plate, then were divided into three group and eight wells for each group as follows: the transected group contained the extracts of the fimbria-transected hippocampi;the normal group contained the extracts of the normal hippocampi;the pure control group have no extracts. After cultured for 14 days,the cells were detected by using MAP-2 and p-ERK immunofluorescence. Result The number, area, perimeter of MAP-2 positive neurons were all declined in transected group, the normal group and the control group orderly. Statistic results showed significant difference between every two groups. The number of MAP-2/p-ERK double-positive neurons were decreased in transected group, the normal group and the control group orderly, but the percentage of double-labeled neurons in total MAP-2 positive neurons were increased in turn. In these two aspect, there were also significant difference between every two group. And most of the MAP-2/p-ERK double-positive neurons were immature. Conclusion The extracts of the fimbria-transected hippocampi had obvious effects on promoting NSCs differentiating into neurons and speeding up the maturation of neurons than those of the normal hippocampi. The morphological results showed that ERK signal transduction might be related to the differentiation of NSCs into neurons.
ABSTRACT
The technique of in situ hybridization using synthetic oligonucleotides labelled by non-radioactive method was developed to localize vasoactive intestinal polypeptide, arginine-vasopressin and oxytocin mRNAs in the rat brain. Also double in situ hybridization technique where combination of non-radioactive and radioactive probes were applied was developed to localize 2 neuropeptide mRNAs in single tissue section. The results were as follows; In non-radioactive in situ hybridization methods using digoxigenin-labelled oligonucleotide probe, alkaline-phosphates method using NBT and BCIP as substrates gave the best result that specific hybridization signals were observed. In radioactive in situ hybridization methods using 35S-labelled oligonucleotide probe, specific hybridization signals were observed in both nuclear track emulsion and X-ray film autoradiography. In double in situ hybridization methods using combination of 35S-labelled and digoxigenin-labelled oligonucleotide probes, specific hybridization signals were observed in the group where K5 emulsion was applied as nuclear track emulsion. The technique of in situ hybridization using digoxigenin-labelled oligonucleotide applied in this study will be useful as alternative for radioactive in situ hybridization technique. Moreover, combination of non-radioactive and radioactive labelled probes in double in situ hybridization technique will be a useful tool for the simultaneous localization of various mRNAs in single section for the study of various neurotransmitters, neuropeptides, receptors and signal transduction molecules.
Subject(s)
Animals , Rats , Aging , Autoradiography , Brain , Corpus Callosum , Digoxigenin , In Situ Hybridization , Magnetic Resonance Imaging , Mesencephalon , Neuropeptides , Neurotransmitter Agents , Oligonucleotide Probes , Oligonucleotides , Oxytocin , Pons , RNA, Messenger , Signal Transduction , Vasoactive Intestinal Peptide , Vasopressins , X-Ray FilmABSTRACT
In the present study, comparison have been made between two kinds of double labelling methods; single side labelling and both sides labelling. To make the different groups be comparable, all sections were continuously cut from the same specimen and all labelling processes were carried out at the same ti- me. The groups were divided as follows: A, one side anti-?-amylase labelling and one side antitrypsin labelling; B, single side labelling of anti-?-amylase and antitrypsin; C, one incubation with anti-?-amylase, followed by two incu- bations with protein A-gold (PAG) complexes of varied size, C1: 7nm and 20nm, C2: 10nm and 20nm, C3: 20nm and 7nm; D, single side labelling of anti- ?-amylase and an unrelated antiserum (antichathepsin D), applying free protein A between two labellings; E, as C, but with free protein A between two PAG incubations; F, as control. Group A and B showed that the two labelling me- thods had almost the same sensitivity.Group C indicated that the interaction of single side labelling were resulted from the combination of the second PAG with the free Fc region of the first antiserum. To decrease the interaction, it was necessary for the second PAG to be much larger than the first one.Group D de- monstrated that the interaction between the second antiserum and the first PAG was very feeble. Group E proved that free protein A could completely prevent the interaction of single side labelling method. The both sides labelling method avoids interaction, but mistakes resulting from the ultrastructural differences on two sides of the sections may happen. Which method to be selected is dependent upon what to be labelled.
ABSTRACT
Immunoreactivity of neurotensin(NT) and substance P (SP) were studied by means of PAP method with simultaneous immunocytochemical double staining on the same sections using diaminobenzidine (DAB) for SP and benzidinc dihydrochloride(BDHC) for NT as the chromogens for light and electron microscopy in dorsal horn of spinal cord of rat treated with colchicinc. In our hands the reaction products of DAB and BDHC is quite discernible, under LM the former is brown and later is blue; under EM DAB reaction product is homogeneous and diffuse electron opaque while BDHC reaction product is patch electron opaque, it is a new but simpler approach to demonstrate two antigens simultaneously in the same ultrathin section. It would be conformable further to the studies of morphological and functional relationships between different kinds of neurons. The results showed that NT-like immunoreactive (NT-LI) perikarya were mainly located in lamina Ⅱi and out layer of lamina Ⅲ and NT-LI terminals were mainly located in laminae Ⅰ-Ⅲ. Under EM the NT-LI axon terminals may synapse with the unlabeled axon or unlabeled dendrites. SP-like immunoreactive (SP-LI) perikarya were mainly located in lamina Ⅱ. The density of SP-LI terminals were higher in laminae Ⅰ-Ⅱ, while axo (SP)-axonic (SP), axo (SP)-somatic (SP) synapses were identified with EM. In double labelling sections, furthermore under EM, it was found that NT-LI axon terminal (BDHC patch like electron opaque reaction products)can synapse or contact with SP-LI axon (DAB diffuse electron opaque reaction products). Our results suggested that NT-and SP-containing neurons and terminals in dorsal horn might participate in regulating process of primary sensory transmission.
ABSTRACT
The present investigation was performed in fifteen adult rats using four fluorescent tracers, including 4'-6-diamidino-2-phenylindol. 2 HC1 (DAPI), nuclear yellow (NY), propidium iodide (PI), and fast blue (FB), which were divided into four sets: PI-DAPI, PI-NY, DAPI-NY, and FB-NY. In each set two fluorescent tracers were used separately to label gastric corpus and pylorus antrum. In the medulla oblongata, the labelled cells in the dorsal motor nucleus of vagus (DmnX) were observed in every animal, but those in the nucleus ambiguus (nA) were only seen. in 11 cases and in the nucleus tractus solitarius (nTs) in 5 cases. In addition, labelled cells were also found in laminae V of the posterior grey columns of the thoracic (T6-9) spinal cords, spinal ganglia and sympathetic trunks. The results of counting and analysing the labelled cells of DmnX of both sides of 75 sections in four cases are as follows: 1233 cells on the left, including 1143 labelled cells of gastric corpus (92.7%), 63 labelled cells of pylorus antrum (5.1%), 27 double labelled cells (2.2%); 1007 cells on the right, including 930 labelled cells of gastric corpus (92.3%), 41 labelled cells of pylorus antrum (4.1%), and 36 double labelled cells (3.7%). The results indicate that PI is not fitted for 'long distance labelling in peripheral nervous system; the density of vagus innervation of gastric corpus is larger than that of pylorus antrum; a part of the nerve endings of vagus postganglionic neurons are distributed both in gastric corpus and in pylorus antrum; a part of postganglionic neurons of sympathetic fibres are located at the sympathetic trunk.
ABSTRACT
The central nucleus of the amygdala(Ce) is heterogeneous in cyto- and chemoarchitecture, and contains over a dozen neuropeptides. Our recent works suggested that there were reciprocal connections between the Ce and the oval nucleus (Or) of the bed nuclei of the stria terminalis (BST). The present experiment was designed to study the immunocytochemical properties of the Ce neurons projecting to the Ov by the fluoro-gold(FG) retrograde tract-tracing method combined with indirect immunofluorescence staining in male Sprague-Dawley rats.It was found that the neurons in the Ce were labeled densely following injection of FG into the Ov, and some FG labeled neurons were restained by indirect immunofluorescence procedures using antisera to corticotropin-releasing factor (CRF), neurotensin (NT), M-enkephalin(M-ENK) or cholecystokinin(CCK). The findings suggest that the CRF-, NT-, M-ENK- and CCK-containig neurons in the Ce project to the Ov.
ABSTRACT
The two-way projection of the intrinsic cardiac neurons in the rat has been studied by means of CB-HRP retrograde tracing and double-labelling with fluorescent tracers. CB-HRP was injected into the right stellate ganglian of the 20 rats. CB-HRP positive neurons were found in the intrinsic cardiac ganglia, moreover, most of the labelled cells localized in the ganglia which were nearby the epicardium of posterior wall of the atria and the root of large vessles. Bb and PI were injected the right stellate ganglia and the wall of the cardiac ventricle of 30 rats respectively, PI-Bb double labelling neurons were found in the posterior wall of the atria for the first time. The present study demonstrated that a few of intrinsi ccardiac neurons give off two long processes, one of which projects to the ventricle, and the other one extends to the stellate ganglion. They may be sensory and may participate the composition of vegetative nervous reflex pathway of heart outside CNS.
ABSTRACT
In our recent work it was found that corticotropin-releasing factor (CRF) and neurotensin(NT) were coexistent in the neurons of the oval nucleus (Ov) of the bed nuclei of the stria terminalis (BST) and there were reciprocal connections between the Ov and the lateral hypothalamic area(LHA).In the present experiments fluoro-gold (FG) retrograde tract-tracing technique combined with indirect immunofluorescence staining was used to investigate the chemical properties of the Ov neurons projecting to the LHA in the male Sprague-Dawley rats. It was found that after injecting of FG into the posterior LHA the retrogradely labeled neurons on the Ov were positively stained with antisera to CRF or NT. The present study proves that a part of CRF- and NT-containing neurons in the Ov project to the posterior LHA.