J Korean Med Sci.  2006 Jun;21(3):518-526. 10.3346/jkms.2006.21.3.518.

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

• Choi JS
• Pham T
• Jang YJ
• Bui BC
• Lee BH
• Joo KM
• Cha CI
• Lee KH

• KMID: 1778438
• DOI: http://doi.org/10.3346/jkms.2006.21.3.518

Abstract

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.

Keyword

Cerebellum; Interneurons; Corticotropin-Releasing Hormone; urocortin; Protective Agents

MeSH Terms

gamma-Aminobutyric Acid/*metabolism
Time Factors
Receptors, Corticotropin-Releasing Hormone/*metabolism
Peptides/chemistry
Neurons/*metabolism
Mice, Inbred C57BL
Mice
Immunohistochemistry
Image Processing, Computer-Assisted
Corticotropin-Releasing Hormone/biosynthesis/*physiology
Cerebellum/*embryology/*metabolism
Cells, Cultured
Cell Survival
Animals

Figure

• 

  Fig. 1 Images of representative cells cultured in BME complete (control) media. The same field is shown as GABA-labeled fluorescent image (left column), CRF-R1-labeled fluorescent image (middle column), and DIC image (right column). Cells are cultured in BME complete (control) media for 10 days, and fixed at 1, 2, 6 or 10 DIV, followed by double-immunolabeling for GABA and CRF-R1. Not all CRF-R1-positive cells are immunopositive for GABA (B, E, H, K, white arrows; C, F, I, L, black arrows). (A-C) 1 DIV, (D-F) 2 DIV, (G-I) 6 DIV, (J-L) 10 DIV. Cell plating density=100×103 cells/cm2. DIV, days in culture. Magnification; ×200.

• 

  Fig. 2 The number of GABA immunopositive neurons in cultures. Primary cerebellar GABAergic neurons were cultured in control (BME complete) media (bright left column) or in the presence of 1 µM astressin (dark right column). The number of GABA-positive neurons per image (180 µm2/sample) was counted, and expressed as mean value±standard deviation of the mean (a minimum of 400 cells per culture age was counted). At 2 DIV in control culture, the number of GABA-immunopositive neurons decreases to 60% of that in 1 DIV (no treatment). At 6 DIV in control culture, the number of surviving GABAergic interneurons further decreses to 45% of that in 2 DIV, or 27% of that in I DIV. At 10 DIV in control culture, the number of GABAergic neurons does not further decrease compared to 6 DIV, and maintains almost the same level as 6 DIV. Treatment with 1 µM astressin alone does not result in any change in the survival of GABAergic neurons, when compared to control at each culture age. Significance was determined using Student's t-test, two-tailed. Cell plating density=100×103 cells/cm2. DIV; days in culture, DT; Days in treatment.

• 

  Fig. 3 Images of representative cells cultured in BME complete (control) media. The same field is shown as GABA-labeled fluorescent image (left column), CRF-R2-labeled fluorescent image (middle column), and DAPI-labeled fluorescent image (right column). Not all CRFR2-positive cells are immunopositive for GABA (B, C, E and F, white arrows) (A-C) 2 DIV, (D-F) 10 DIV. Cell plating density=100×103 cells/cm2. DIV, days in culture. Magnification; ×200.

• 

  Fig. 4 Effect of CRF on GABAergic neurons in culture. Primary cerebellar cultures were treated with 1 µM CRF alone (middle column), or 1 µM astressin in the presence of 1 µM CRF (right column), for 1, 5, or 9 days (DT). The number of GABA-positive neurons per image (180 µm2/sample) was counted, and presented as mean value±standard deviation of the mean (a minimum of 400 cells per culture age was counted). Significance was determined using Studen's t-test, two-tailed. *represents p<0.05, with respect to control at the corresponding age. Cell plating density=100×103 cells/cm2. DIV, days in culture; DT, days in treatment.

• 

  Fig. 5 Effect of urocortin and urocortin II on GABAergic neurons in culture. Primary cerebellar cultures were treated with 1 µM urocortin alone (2nd column), 1 µM astressin in the presence of 1 µM urocortin (3rd column), or 1 µM urocortin II alone (4th column) for 1, 5, or 9 days (DT). The number of GABA-positive neurons per image (180 µm2/sample) was counted, and presented as mean value±standard deviation of the mean (a minimum of 400 cells in each culture condition was counted). Significance was determined using Student's t-test, two-tailed. *represents p<0.05, with respect to control at the corresponding age. Cell plating density=100×103 cells/cm2. DIV, days in culture; DT, days in treatment.

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