All sections were then analyzed with the LSM 510
Meta (Zeiss). Nesti n-ir was observed with the blue FITC
filter; ChAT-ir was viewed with the DAPI filter; biocy-
tin-filled cell was viewed with the Rhodamine Red filter.
Confocal images were scanned, edited, reconstructed
and measured using LSM 510 Meta software (Zeiss).
Analyses of the projections of nestin+ and nestin-
To demonstrate whether the nestin+ and nestin- choliner-
gic neurons projected to hippocampus, retrograde label-
ling combined with double immunostaining protocols
were used. Four rats were instilled with 0.5 μlof3%fast
blue dye into the CA1 area (AP-3.8, L 1.5, H 3.0) o f the
hippocampus. After a 5-d transport period, the experimen-
tal animals were sacrificed, immunofluorescence of nestin
and ChAT were performed with a standard protocols.
Rhodamine Red was the fluorescent label for ChAT-ir, cy2
for nestin-ir neurons . The localization of fast blue in
nerve cell bodies of the MS/DBB area were observed,
counted, and analyzed. The e xperimental controls were
performed as described above.
All statistical analyses were performed with SPSS 11.5 for
Windows. Data were analyzed statistically using either the
student’s t-test, one-way ANOVA (post hoc multiple com-
parison by least-significant difference (LSD ) or Dunnett’s
T3 test), or the K olmogorov-Smirnov test. Significance
level for all measures was set at P < 0 .05. Data are pre-
sented as means ± S.E.M (standard error of the mean). All
figures were prepared using CorelDraw 10.0 (Corel;
Ottawa) and Adobe Photoshop CS (Adobe; San Jose, CA).
Acknowledgements and Funding
We gratefully acknowledge the assistance of Monique David in manuscript
revision, Yongjun Chen, Pu Wang, QunFang Yuan and Yao Xie for their help
in experimental techniques. This work was supported by National Nature
Science Foundation of China (No. 30700436, 31160221), Doctoral Program of
Guangdong Natural Science Foundation (No. S2011040004372). The funders
had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Department of Anatomy and Neurobiology, Zhongshan School of Medicine,
Sun Yat-sen University, Guangzhou, China.
Department of Neurobiology,
Southern Medical University, Guangzhou, China.
JHZ designed and carried out the study, performed the statistical analysis,
and drafted the manuscript. HYG participated in the design of the study,
performed the statistical analysis, and helped to draft the manuscript. ZBY
conceived the study, and participated in its design and coordination, and
helped to draft the manuscript. JTZ carried out the molecular genetic
studies, participated in the sequence alignment. KHG participated in the
immunohistochemistry and neuronal projection study. DPL participated in
the design of the study and performed the statistical analysis. TMG
participated in design and coordination of the study, and helped to draft
the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Received: 26 August 2011 Accepted: 20 December 2011
Published: 20 December 2011
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