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. 2014 Feb 7;9(2):e88294.
doi: 10.1371/journal.pone.0088294. eCollection 2014.

The neurogenic effects of exogenous neuropeptide Y: early molecular events and long-lasting effects in the hippocampus of trimethyltin-treated rats

Valentina Corvino  1 , Elisa Marchese  1 , Maria Vittoria Podda  2 , Wanda Lattanzi  1 , Stefano Giannetti  1 , Valentina Di Maria  1 , Sara Cocco  2 , Claudio Grassi  2 , Fabrizio Michetti  1 , Maria Concetta Geloso  1
Affiliations

The neurogenic effects of exogenous neuropeptide Y: early molecular events and long-lasting effects in the hippocampus of trimethyltin-treated rats

Valentina Corvino et al. PLoS One. .

Abstract

Modulation of endogenous neurogenesis is regarded as a promising challenge in neuroprotection. In the rat model of hippocampal neurodegeneration obtained by Trimethyltin (TMT) administration (8 mg/kg), characterised by selective pyramidal cell loss, enhanced neurogenesis, seizures and cognitive impairment, we previously demonstrated a proliferative role of exogenous neuropeptide Y (NPY), on dentate progenitors in the early phases of neurodegeneration. To investigate the functional integration of newly-born neurons, here we studied in adult rats the long-term effects of intracerebroventricular administration of NPY (2 µg/2 µl, 4 days after TMT-treatment), which plays an adjuvant role in neurodegeneration and epilepsy. Our results indicate that 30 days after NPY administration the number of new neurons was still higher in TMT+NPY-treated rats than in control+saline group. As a functional correlate of the integration of new neurons into the hippocampal network, long-term potentiation recorded in Dentate Gyrus (DG) in the absence of GABAA receptor blockade was higher in the TMT+NPY-treated group than in all other groups. Furthermore, qPCR analysis of Kruppel-like factor 9, a transcription factor essential for late-phase maturation of neurons in the DG, and of the cyclin-dependent kinase 5, critically involved in the maturation and dendrite extension of newly-born neurons, revealed a significant up-regulation of both genes in TMT+NPY-treated rats compared with all other groups. To explore the early molecular events activated by NPY administration, the Sonic Hedgehog (Shh) signalling pathway, which participates in the maintenance of the neurogenic hippocampal niche, was evaluated by qPCR 1, 3 and 5 days after NPY-treatment. An early significant up-regulation of Shh expression was detected in TMT+NPY-treated rats compared with all other groups, associated with a modulation of downstream genes. Our data indicate that the neurogenic effect of NPY administration during TMT-induced neurodegeneration involves early Shh pathway activation and results in a functional integration of newly-generated neurons into the local circuit.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. TMT-induced neuronal loss in the rat hippocampus.
Micrographs of Nissl-stained 40 µm sagittal sections showing the CA3 subfield, as indicated in the image in the box, from dorsal hippocampus of CTRL+saline- (A), CTRL+NPY- (B), TMT+saline- (C) and TMT+NPY- (D) treated rats. Severe pyramidal neuronal loss is evident in both groups of TMT-treated animals (C, D). Scale bar: 160 µm.
Figure 2
Figure 2. BrdU-immunoreactivity in the DG of the different experimental groups.
A. Representative micrographs showing DAB-stained BrdU-positive cells in the DG of CTRL+saline- (a), CTRL+NPY- (b), TMT+saline- (c), TMT+NPY (d) -treated rats: an increased number of BrdU-positive newly-generated cells is evident in the DG of TMT+NPY-treated animals (d), when compared with the other groups (a, b, c). Scale bar: 40 µm. B. Bar graphs showing the number of BrdU-stained nuclei in the DG of the different experimental groups. A significantly higher number of BrdU-positive cells is evident in TMT+NPY-treated animals compared with the CTRL+saline group. The values are given as means ± SD (*p<0.05).
Figure 3
Figure 3. BrdU/NeuN double-stained cells in the DG of the different experimental groups.
A. Representative confocal microscopy micrographs of CTRL+saline- (a), CTRL+NPY- (b), TMT+saline- (c), TMT+NPY (d) -treated rat DG sections double-labelled for NeuN (red) and BrdU (green): a higher number of BrdU/NeuN-positive cells is evident in TMT+NPY-treated rats (d). Scale bar: 60 µm. B. The percentage of BrdU/NeuN double-stained cells is unchanged in the various experimental groups. The values are given as means ± S.D. (*p<0.05).
Figure 4
Figure 4. Long-term potentiation (LTP) at medial perforant pathway (MPP)-dentate granule cell (DGC) synapses.
A. Time course of MPP-DGC LTP induced by high frequency stimulation (HFS) (delivered at time 0) in hippocampal slices from rats belonging to the four experimental groups (CTRL+saline, CTRL+NPY, TMT+saline, TMT+NPY). Recordings were performed in normal aCSF (i.e, in the absence of GABAA receptor blockade) and show enhanced LTP (aCSF-LTP) in slices from the TMT+NPY group compared to all the other experimental groups. Results are expressed as percentages of baseline fEPSP amplitude (100%). B. Bar graphs comparing LTP magnitudes observed during the last 5 min of recording in the different experimental groups. Error bars indicate S.E.M. values. *p<0.05 vs CTRL+saline-treated rats; #p<0.05 vs TMT+NPY group, (Student’s t test); n.s., not significant.
Figure 5
Figure 5. mRNA expression levels of Klf9 and Cdk5 in rat hippocampus in the different experimental groups.
Results of quantitative real time-PCR obtained using the ΔΔCt method for calculation of the relative quantity (RQ) of Cdk5 and Klf9 genes tested 30 days after NPY administration. *p<0.05, calculated on mean ΔCt across biological replicates.
Figure 6
Figure 6. mRNA expression levels of genes related to the Shh pathway in rat hippocampus in the different experimental groups.
Results of quantitative real time-PCR obtained using the ΔΔCt method for the calculation of relative quantity (RQ) of Shh, Ccnd1, Ptch1 and Kif3a genes at the three tested time points. *p<0.05, **p<0.001, calculated on mean ΔCt across biological replicates.
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This work was supported by funds from Universita’ Cattolica del S. Cuore (D3.2 line) to C.G. and F.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.