Notice of Retraction: Therapeutic Effects of Umbilical Cord Blood Derived Mesenchymal Stem Cell-Conditioned Medium on Pulmonary Arterial Hypertension in Rats

The first author of the article has duely admitted that he is mainly responsible for the misconduct.

Therapeutic Effects of Umbilical Cord Blood Derived Mesenchymal Stem Cell-Conditioned Medium on Pulmonary Arterial Hypertension in Rats

BACKGROUND: Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) may have multiple therapeutic applications for cell based therapy including the treatment of pulmonary artery hypertension (PAH). As low survival rates and potential tumorigenicity of implanted cells could undermine the mesenchymal stem cell (MSC) cell-based therapy, we chose to investigate the use of conditioned medium (CM) from a culture of MSC cells as a feasible alternative. METHODS: CM was prepared by culturing hUCB-MSCs in three-dimensional spheroids. In a rat model of PAH induced by monocrotaline, we infused CM or the control unconditioned culture media via the tail-vein of 6-week-old Sprague-Dawley rats. RESULTS: Compared with the control unconditioned media, CM infusion reduced the ventricular pressure, the right ventricle/(left ventricle+interventricular septum) ratio, and maintained respiratory function in the treated animals. Also, the number of interleukin 1alpha (IL-1alpha), chemokine (C-C motif) ligand 5 (CCL5), and tissue inhibitor of metalloproteinase 1 (TIMP-1)-positive cells increased in lung samples and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL)-positive cells decreased significantly in the CM treated animals. CONCLUSIONS: From our in vivo data in the rat model, the observed decreases in the TUNEL staining suggest a potential therapeutic benefit of the CM in ameliorating PAH-mediated lung tissue damage. Increased IL-1alpha, CCL5, and TIMP-1 levels may play important roles in this regard.

Region-specific changes in the immunoreactivity of Atg9A in the central nervous system of SOD1(G93A) transgenic mice / 대한해부학회지

Autophagy is a eukaryotic self-degradation system that plays a pivotal role in the maintenance of cellular homeostasis. Atg9 is the only transmembrane Atg protein required for autophagosome formation. Although the subcellular localization of the Atg9A has been examined, little is known about its precise cell and tissue distribution. In the present study, we used G93A mutation in superoxide dismutase 1 [SOD1(G93A)] mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of Atg9A immunoreactivity in the central nervous system of these mice. Atg9A-immunoreactivity was detected in the spinal cord, cerebral cortex, hippocampal formation, thalamus and cerebellum of symptomatic SOD1(G93A) transgenic mice. By contrast, no Atg9A-immunoreactivity were observed in any brain and spinal cord region of wtSOD1, pre-symptomatic and early symptomatic mice, and the number and staining intensity of Atg9A-positive cells did not differ in SOD1(G93A) mice between 8 and 13 weeks of age. These results provide evidence that Atg9A-immunoreactivity were found in the central nervous system of SOD1(G93A) transgenic mice after clinical symptoms, suggesting a possible role in the pathologic process of ALS. However, the mechanisms underlying the increased immunoreactivity for Atg9A and the functional implications require elucidation.

Region-specific changes in the immunoreactivity of Atg9A in the central nervous system of SOD1(G93A) transgenic mice / 대한해부학회지

Autophagy is a eukaryotic self-degradation system that plays a pivotal role in the maintenance of cellular homeostasis. Atg9 is the only transmembrane Atg protein required for autophagosome formation. Although the subcellular localization of the Atg9A has been examined, little is known about its precise cell and tissue distribution. In the present study, we used G93A mutation in superoxide dismutase 1 [SOD1(G93A)] mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of Atg9A immunoreactivity in the central nervous system of these mice. Atg9A-immunoreactivity was detected in the spinal cord, cerebral cortex, hippocampal formation, thalamus and cerebellum of symptomatic SOD1(G93A) transgenic mice. By contrast, no Atg9A-immunoreactivity were observed in any brain and spinal cord region of wtSOD1, pre-symptomatic and early symptomatic mice, and the number and staining intensity of Atg9A-positive cells did not differ in SOD1(G93A) mice between 8 and 13 weeks of age. These results provide evidence that Atg9A-immunoreactivity were found in the central nervous system of SOD1(G93A) transgenic mice after clinical symptoms, suggesting a possible role in the pathologic process of ALS. However, the mechanisms underlying the increased immunoreactivity for Atg9A and the functional implications require elucidation.

Immunohistochemical study on the expression of calcium binding proteins (calbindin-D28k, calretinin, and parvalbumin) in the cerebral cortex and in the hippocampal region of nNOS knock-out(-/-) mice / 대한해부학회지

Nitric oxide (NO) modulates the activities of various channels and receptors to participate in the regulation of neuronal intracellular Ca2+ levels. Ca2+ binding protein (CaBP) expression may also be altered by NO. Accordingly, we examined expression changes in calbindin-D28k, calretinin, and parvalbumin in the cerebral cortex and hippocampal region of neuronal NO synthase knockout(-/-) (nNOS-/-) mice using immunohistochemistry. For the first time, we demonstrate that the expression of CaBPs is specifically altered in the cerebral cortex and hippocampal region of nNOS-/- mice and that their expression changed according to neuronal type. As changes in CaBP expression can influence temporal and spatial intracellular Ca2+ levels, it appears that NO may be involved in various functions, such as modulating neuronal Ca2+ homeostasis, regulating synaptic transmission, and neuroprotection, by influencing the expression of CaBPs. Therefore, these results suggest another mechanism by which NO participates in the regulation of neuronal Ca2+ homeostasis. However, the exact mechanisms of this regulation and its functional significance require further investigation.

Immunohistochemical study on the expression of calcium binding proteins (calbindin-D28k, calretinin, and parvalbumin) in the cerebellum of the nNOS knock-out(-/-) mice / 대한해부학회지

Nitric Oxide (NO) actively participates in the regulation of neuronal intracellular Ca2+ levels by modulating the activity of various channels and receptors. To test the possibility that modulation of Ca2+ buffer protein expression level by NO participates in this regulatory effect, we examined expression of calbindin-D28k, calretinin, and parvalbumin in the cerebellum of neuronal NO synthase knock-out (nNOS(-/-)) mice using immunohistochemistry. We observed that in the cerebellar cortex of the nNOS(-/-) mice, expression of calbindin-D28k and parvalbumin were significantly increased while expression of calretinin was significantly decreased. These results suggest another mechanism by which NO can participate in the regulation of Ca2+ homeostasis.

Heat shock protein 70 alters the endosome-lysosomal localization of huntingtin

Huntington's disease is caused by CAG trinucleotide expansions in the gene encoding huntingtin. N- terminal fragments of huntingtin with polyglutamine produce aggregates in the endosome-lysosomal system, where proteolytic fragments of huntingtin is generated. Heat shock protein 70 (HSP70) prevents the formation of protein aggregates, but the effect of HSP70 on the huntingtin in the endosome-lysosomal system is unknown. This study was to determine whether HSP70 alters the distribution of huntingtin in endosome-lysosomal system. HSP70 expressing stable cells (NIH/3T3 or cerebral hybrid cell line A1) were generated, and mutant [(CAG)100] huntingtin was transiently overexpressed. Analysis of subcellular distribution by immnuocytochemistry or proteolysis cleavage by Western blotting was performed. 18 CAG repeat wild type [WT; (CAG)18] huntingtin was used as a control. Cells with huntingtin showed patterns of endosome- lysosomal accumulation, from a 'dispersed vacuole (DV)' type into a coalescent 'perinuclear vacuole (PV)' type over time. In WT huntingtin, HSP70 increased the cells with the PV types that enhanced the proteolytic cleavage of huntingtin. However, HSP70 reduced cells of the DV and PV types expressing mutant huntingtin, that result in less proteolysis than that of control. In addition, intranuclear inclusions were formed only in mutant cells, which was not affected by HSP70. These results suggest that HSP70 alters the distribution of huntingtin in the endosome-lysosomal system, and that this contributes to huntingtin proteolysis.

Immunohistochemical Study on the Distribution of Glycogen Synthase Kinase (GSK) 3beta in the Central Nervous System of SOD1G93A Transgenic Mice / 체질인류학회지

In the present study, we investigated influences of glycogen synthase kinase (GSK) 3beta on the development and/or progression of amyotrophic lateral sclerosis (ALS). We used transgenic mice expressing a human Cu/Zn superoxide dismutase mutant (SOD1G93A) as an in vivo model of ALS and examined expressional changes of GSK3beta immunohistochemically in the spinal cord, brain stem and cerebellum. With these experiments we demonstrate that the neurons in these regions of symptomatic SOD1G93A transgenic mice showed increased GSK3beta immunoreactivities compared with wild-type SOD1 transgenic mice. In contrast to symptomatic SOD1G93A transgenic mice, few GSK3beta immunoreactivity changes were detected in 8w- and 13w-old presymptomatic SOD1G93A transgenic mice. These data suggest the possibility that GSK3 functions as a modulating factor of apoptosis-related alterations in ALS and that GSK3beta exert differential functions in the development and/or progression of ALS. But the exact functional significances of these changes require further elucidation.

Caveolin-1 upregulation in senescent neurons alters amyloid precursor protein processing

Lipid rafts provide a platform for regulating cellular functions and participate in the pathogenesis of several diseases. However, the role of caveolin-1 in this process has not been elucidated definitely in neuron. Thus, this study was performed to examine whether caveolin-1 can regulate amyloid precursor protein (APP) processing in neuronal cells and to identify the molecular mechanisms involved in this regulation. Caveolin-1 is up-regulated in all parts of old rat brain, namely hippocampus, cerebral cortex and in elderly human cerebral cortex. Moreover, detergent-insoluble glycolipid (DIG) fractions indicated that caveolin-1 was co-localized with APP in caveolae-like structures. In DIG fractions, bAPP secretion was up-regulated by caveolin-1 over-expression, which was modulated via protein kinase C (PKC) in neuroblastoma cells. From these results we conclude that caveolin-1 is selectively expressed in senescent neurons and that it induces the processing of APP by beta-secretase via PKC downregulation.

Corticotropin-releasing Factor (CRF) and Urocortin Promote the Survival of Cultured Cerebellar GABAergic Neurons Through the Type 1 CRF Receptor

Corticotropin releasing factor (CRF) is known to be involved in the stress response and in some degenerative brain disorders. In addition, CRF has a role as a neuromodulator in adult cerebellar circuits. Data from developmental studies suggest a putative role for CRF as a trophic factor during cerebellar development. In this study, we investigated the trophic role for CRF family of peptides by culturing cerebellar neurons in the presence of CRF, urocortin or urocortin II. Primary cell cultures of cerebella from embryonic day 18 mice were established, and cells were treated for either 1, 5 or 9 days with Basal Medium Eagles complete medium alone or complete medium with 1 micrometer CRF, urocortin, or urocortin II. The number of GABA-positive neurons in each treatment condition was counted at each culture age for monitoring the changes in neuronal survival. Treatment with 1 micrometer CRF or 1 micrometer urocortin increased the survival of GABAergic neurons at 6 days in vitro and 10 days in vitro, and this survival promoting effect was abolished by treatment with astressin in the presence of those peptides. Based on these data, we suggest that CRF or urocortin has a trophic role promoting the survival of cerebellar GABAergic neurons in cultures.

The Effect of Anti-oxidants on Apoptosis Relating Protein Expressions in Mutant Cu/Zn-superoxide Dismutase Motor Neuron Cells

BACKGROUNDS: Free radical theory showed that aging might be correlating with the accumulation of oxidative damage into biomolecules of animals. Since the process was also observed in some neurodegenerative disease, the study on the subject could improve our understanding of the aging process. In this regard, our study laid focus on the effect of various anti-oxidants on the cells including mutant Cu/Zn-superoxide dismutase, which was reported to be deficient in various diseases or aging processes. METHODS: We tested retinol, Vitamin C, E and coenzyme Q10 on mutant Cu/Zn-superoxide dismutase(A4V) motor neuron cells using immunocytochemical study and accompanying statistical analysis. RESULTS: During our trial on the hydrogen peroxide treatment on A4V mutant cells, Vitamin A and C did not show any defensive effect on oxidative stress induced A4V cells while Vitamin E and coenzyme Q10 exhibited meaningful inhibitory effect against apoptosis relating protein expression. CONCLUSION: In case of oxidative damages induced by Cu/Zn-superoxide dismutase deficiencies, the protective role of some anti-oxidants like Vitamin E or coenzyme Q10 should be positively considered in forthcoming studies.

Immunohistochemical Study on the Distribution of Estrogen Receptor-alpha in the Hippocampus of the Normal Aged Rat

BACKGROUND: In recent years, estrogen has also been shown to modulate the development and function of the brain, bur not exclusively in areas involved with sexual behavior. Among the most novel and fascinating effects of estrogen are those on cognitive function and memory process and their alterations during aging and neurodegenarative disease like Alzheimer. Estrogen receptors distributed not only in the hypothalamus but many different areas, like cerebral cortex, hippocampus, basal forebrain, midbrain, spinal cord, and the diverse action of estrogen is supported by this fact. Numerous studies suggest thai estrogen may be beneficial in preserving cognitive function, but it is not clear yet. PURPOSE: In this study, we perform the immunohistochemical staining in the hippocampus of normal aged rat, and show the distribution of estrogen receptor compared with the neonatal rat. METHODS: we have used antibodies against a estrogen receptor(ER)-alpha to determine their distribution in neonatal and aged SD rat hippocampus. RESULTS: In neonatal rat hippocampus, ER-alpha immunoreactivity was observed in the nucleus of Purkinje cells, whereas in aged rat hippocampus, ER-a immunoreactivity was found mainly in the cytoplasm of Purkinje cells. CONCLUSION: We showed the age related intracellular differential distribution of ER-alpha immunoreactivity in the rat hippocampus. But, further investigations are required to establish whether functional relations like cognitive ability exist with this different intracellular expression of ER-alpha immunoreactivity.

Effect of Familial Amyotrophic Lateral Sclerosis-associated Cu, Zn-superox-ide Dismutase Mutation on Neural Differentiation in Motor Neuronal Cells

BACKGROUND: Mutations in the human Cu, Zn-superoxide dismutase(SOD1) gene have been identified in some cases of familial amyotrophic lateral sclerosis(ALS). The aim of this study is to delineate the effect of the SOD1 mutation on neural differentiation, and to investigate the mechanism of neuronal death. METHODS: We studied motorneuron-neurob-lastoma hybrid cells(VSC 4.1) expressing wild type or mutant SOD1(G93A, A4V) during differentiation by dibutyryl cAMP and aphidicolin. RESULTS: Mutant cells(G93A) revealed a decreased viability compared with the control cells, mainly in the early stage ofdifferentiation. The release of cytochrome c and increased nuclear fragmentation were observed. However, cell death was not protected by nonselective caspase inhibitor(z-VAD-fmk), but by the antioxi-dant( Trolox). CONCLUSIONS: The results suggest that oxidative stress may be the main mechanism of neuronal death, particularly in the early stage of differentiation.

Immunohistochemical studies on the distribution of microcirculation-controlling factors in the cerebral cortex and hippocampal formation of the aged rat / 대한해부학회지

Recent studies have explored certain changes of neurons containing neuropeptides that are involved in the cerebral microcirculation with aging. However, the degree of loss of vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing neurons in the aged CNS has not yet been established with certainty. Nitric oxide (NO) from the neuronal NO synthase (nNOS) appears to play the principal role in the cerebral flow response to functional activation. Several findings suggest that NO production may be decreased in the aged rat. Therefore, changes with aging of VIP-, NPY- and NOS-containing neurons were demonstrated by immunohistochemistry in this study. A major loss of VIP-immunoreactive (IR) neurons in the aged rat brain was observed in the frontal cortex area 3, parietal cortex area 1, hindlimb area, temporal cortex area 1 & 2, monocular part of occipital cortex area 1, occipital cortex area 2, and retrosplenial cortex. The axis of VIP neurons in the aged group showed an irregular orientation tendency, especially in layers II and III. Major loss of NPY-IR neurons in the aged rat brain were observed in the retrosplenial cortex, frontal cortex areas 1 and 2, parietal cortex areas 1 and 2, occipital cortex areas 1 and 2, the temporal cortex, hippocampus proper and cingulate cortex. Loss of NPY-IR neurons was observed mostly in layers V and VI. The number of NOS-IR cells was significantly decreased in the aged rat, but the extent of changes was variable in each area.Morphologically, the number of dendritic branches seemed to be decreased in the aged group and the length of dendrites of VIP-, NPY- and NOS-IR neurons showed a tendency to shorten. These results indicate the involvement of VIP-, NPY- and NOS-IR neurons in the aging process in relation to the increased incidence to cerebrovascular disorders in the elderly, and provide the first morphological evidence for the loss of VIP-, NPY- and NOS-IR neurons in each area of cerebral cortex and the hippocampus of the aged rat by immunohistochemistry.

The Alteration of Avian Retinal Microglia Induced by Optic Nerve Transection / 대한해부학회지

Retina, a part of CNS, has served valuable and accessible tissue for elucidating the cellular properties of neurons and glia due to its similarity to brain. Unlike mammalian counterpart, avian retina is devoid of vessels and astrocytes. However little is known about glial reaction to neuronal injuries in this species. Therefore, this study was performed to investigate the microglial responses in the quail retina following neuronal injuries. The retinae from normal and optic nerve transected adult quails were studied immunohistochemically with anti-QH1, a marker known to be specific for microglia. In the normal retina, QH1-labeled microglial cells displayed typical feature of ramified (resting) form and were localized mainly in the inner plexiform layer. After optic nerve transection (ONT) morphology of microglial cells changed from the ramified to the amoeboid form. This feature of microglial cells maintained throughout the post operational periods until 28 days after ONT. Particularly, at 14 and 21 days after ONT amoeboid microglia displayed cell bodies with stout and bushy processes, suggesting active phagocytosis. The distribution pattern of microglia also changed in accord to ganglion cell degeneration: they gradually moved to layers of ganglion cells and optic nerve fibers where ganglion cell bodies and axons were under degeneration. This change of microglial distribution was most prominent at 14 days of ONT. The result of this study is generally consistent with that reported in mammalian counterpart and this similarity between the avascular avian retina and the vascularized mammalian counterpart suggests that processes of microglial activation, such as migration and phagocytosis, can occur in the vessel-free CNS tissue.

Immunocytochemical study on the distribution of NOS-immunoreactive neurons in the cerebral cortex of aged rats / 대한해부학회지

Nitric oxide (NO) involvement has been demonstrated in mechanisms of synaptic plasticity, particularly in hippocampal long-term potentiation, a mechanism that underlies certain forms of learning and memory. Further, NO has been shown to regulate various neurotransmitters which play an important role in learning and memory. Several findings suggest that NO production may be decreased in the aged rat. Changes in the nNOS-containing neurons with aging were demonstrated by immunocytochemistry and in situ hybridization. NOS-immunoreactive cells in aged rats were present in all cortical areas and the hippocampus, and the pattern of distribution was similar to that of the control group. The number of NOS-immunoreactive cells in the cerebral cortex was significantly decreased in the aged rats, but the extent of changes was variable in each area, and ranged from mild decrease (50%). Severely decreased areas were the cingulate cortex, parietal cortex area 1, temporal cortex area 1, 2, 3, medial part of occipital cortex area 2, monocular and binocular part of occipital cortex area 1, entorhinal cortex, hippocampus proper, dentate gyrus and subiculum. Moderately decreased areas (30~50%) were frontal cortex area 1, 2, 3, parietal cortex area 2, forelimb, hindlimb, lateral part of occipital cortex area 2. Slightly decreased area was insular cortex. Morphologically, the number of dendritic branches seemed to be decreased in aged group and the length of dendrites of NOS-IR neurons showed a tendency to shorten. These results indicate the involvement of neuronal system containing NOS in the aging brain, and provide the first morphological evidence for the loss of NOS neurons in the cerebral cortex of the aged rats by immunocytochemistry. Further multidisciplinary investigations involving normal aging and neurodegenerative disease such as Alzheimer's disease are needed to clarify the importance of nitric oxide changes in the cerebral cortex with aging.

Spatial and temporal expression patterns of apoptosis-related genes in rat spinal cord during normal aging / 대한해부학회지

The pattern of distribution in rat spinal cord and changing pattern during normal aging of c-Fos, Bcl-2, Bax, and p53 expression were investigated by immunohistochemical staining. Male Sprague-Dawley rats at the age of one week, five months, and two years were studied. C-Fos immunoreactivity was observed diffusely in gray matters in neonatal rats, preferentially located in deep dorsal horn and around central canal. Compared with those of neonatal rats, immunoreactive cells decreased prominently in adult rats. In aged rats, these cells were not seen in any segments. In a transverse section, spatial expression of Bcl-2 and Bax proteins showed a diffuse distribution pattern with immunore-activity more prominent in the anterior horn. Continuing expression of these proteins was shown in each age group. In adult rats, Bcl-2 immunoreactivity was decreased drastically compared to that of neonatal rats. The immunoreactivity was higher in aged than in adult rats, but the number of immunoreactive cells was not different between aged and neonatal rats. The number of Bax-immunoreactive cells was greater in adult than in neonatal rats; in aged rats, it was similar with that of adult rats. The spinal cords of neonatal rats were not p53-immunoreactive, though p53-positive cells were detected in all segments of adult spinal cord. P53-positive cells were stained along the cellular margin, with a pale central portion. The pattern of p53 immunoreactivity in adult and aged rats was similar; the number of p53-positive cells, however, was higher in aged rats than in adult. In conclusion, we demonstrated that the expression patterns of c-Fos, Bcl-2, Bax, and p53 proteins in rat spinal cord change during normal aging for the first time.

Carbonic anhydrase II immunostaining in the cerebellum of postnatal mice / 대한해부학회지

The carbonic anhydrase II (CA-II) is specifically expressed in oligodendrocytes, the cells responsible for myelination in the central nervous system. However no direct evidence on relationship between myelin formation and CA-II immunoreactivity has been described. The aims of these studies are to investigate the relationship between CA-II and myelination during cerebellar development of mouse. Myelin staining was found on postnatal (P) 14, and its intensity increased in proportion to developmental age. CA-II positive oligodendrocytes were observed in the white matter of cerebellum on P 14 day. CA-II positive oligoden-drocytes also occured in the granular layer and Purkinje cell layers in the later stage of dvelopment. The parallel development in the CA-II expression and myelination during development suggests that CA-II in oligoendrocyte play a role to myelination.

Immunohistochemical Study on the Nitric Oxide Synthase in the Developing Rat Brain / 대한해부학회지

Nitric oxide(NO) is thought to play an important role in development and plasticity of brain. In this study, we aimed to examine the expression of neuronal NOS and NADPH-diaphorase (NADPH-d) activity in the developing rat brain. The results show that there is a great variation in the time of appearance of the earliest NOS containing cells depending on their location: At the 15th embryonic day weakly stained cells were present in caudate-putamen, and neurons in the sensory trigeminal nucleus and the solitary nucleus displayed an intense staining. The NOS neurons in orbital neocortex, bed nucleus of stria terminalis, paraventricular hypothalamic nucleus, lateral hypothalamic area and mammillary body appeared first at the 18th embryonic day. The supraoptic nucleus and superior and inferior colliculi also weakly labeled at the 18th embryonic day, At the loth embryonic day, positive cells appeared in horizontal limb of diagonal band, anterior olfactory nucleus and parafascicular thalamic nucleus. In the cerebellum, weak NOS staining was present in fibers and cells situated below Purkinje cert layer. The Purkinje cell layer displayed a weak, rather diffuse activity throughout the cerebellum at postnatal day 0. At the 4th postnatal day. the reaction product in the Purkinje cell layer became more distinct. At the 10th postnatal day, the inner part of molecular layer became populated by NOS positive basket cells, and the reaction products on the Purkinje cells began to disappear. The present results showed that NOS in the rat brain is expressed in different populations of neurons at different stages of development. This expression pattern of NOS suggests that NO may play a role in the developmental remodelling of the mammalian brain.

Study on the Expression of Vasoactive intestinal polypeptide, Vasopressin and Oxytocin mRNAs in the Rat Brain using Double in situ Hybridization Technique / 대한해부학회지

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.