Borna Disease Virus Persistent Infection Activates Mitogen-activated Protein Kinase and Blocks Neuronal Differentiation of PC12 Cells

Abstract

Persistence of Borna disease virus (BDV) in the central nervous system causes damage to specific neuronal populations. BDV is noncytopathic, and the mechanisms underlying neuronal pathology are not well understood. One hypothesis is that infection affects the response of neurons to factors that are crucial for their proliferation, differentiation, or survival. To test this hypothesis, we analyzed the response of PC12 cells persistently infected with BDV to the neurotrophin nerve growth factor (NGF). PC12 is a neural crest-derived cell line that exhibits features of neuronal differentiation in response to NGF. We report that persistence of BDV led to a progressive change of phenotype of PC12 cells and blocked neurite outgrowth in response to NGF. Infection down-regulated the expression of synaptophysin and growth-associated protein-43, two molecules involved in neuronal plasticity, as well as the expression of the chromaffin-specific gene tyrosine hydroxylase. We showed that the block in response to NGF was due in part to the down-regulation of NGF receptors. Moreover, although BDV caused constitutive activation of the ERK1/2 pathway, activated ERKs were not translocated to the nucleus efficiently. These observations may account for the absence of neuronal differentiation of persistently infected PC12 cells treated with NGF. Persistence of Borna disease virus (BDV) in the central nervous system causes damage to specific neuronal populations. BDV is noncytopathic, and the mechanisms underlying neuronal pathology are not well understood. One hypothesis is that infection affects the response of neurons to factors that are crucial for their proliferation, differentiation, or survival. To test this hypothesis, we analyzed the response of PC12 cells persistently infected with BDV to the neurotrophin nerve growth factor (NGF). PC12 is a neural crest-derived cell line that exhibits features of neuronal differentiation in response to NGF. We report that persistence of BDV led to a progressive change of phenotype of PC12 cells and blocked neurite outgrowth in response to NGF. Infection down-regulated the expression of synaptophysin and growth-associated protein-43, two molecules involved in neuronal plasticity, as well as the expression of the chromaffin-specific gene tyrosine hydroxylase. We showed that the block in response to NGF was due in part to the down-regulation of NGF receptors. Moreover, although BDV caused constitutive activation of the ERK1/2 pathway, activated ERKs were not translocated to the nucleus efficiently. These observations may account for the absence of neuronal differentiation of persistently infected PC12 cells treated with NGF. Borna disease virus nerve growth factor nucleoprotein growth-associated protein-43 Tris-buffered saline glyceraldehyde-3-phosphate dehydrogenase mitogen-activated protein kinase extracellular signal-regulated kinase MAPK kinase cAMP response element-binding protein microtubule-associated protein-2 tyrosine hydroxylase epidermal growth factor Borna disease virus(BDV)1 is a nonsegmented, negative stranded RNA virus (1de la Torre J.C. J. Virol. 1994; 68: 7669-7675Crossref PubMed Google Scholar, 2Schneemann A. Schneider P.A. Lamb R.A. Lipkin W.I. Virology. 1995; 210: 1-8Crossref PubMed Scopus (173) Google Scholar), belonging to theBornaviridae family in the Mononegavirales order. BDV causes central nervous system diseases characterized by behavioral abnormalities in a wide variety of animals (3Gosztonyi G. Ludwig H. Koprowski H. Lipkin I. Borna Disease. Springer-Verlag, Berlin, Germany1995: 39-73Google Scholar). There is evidence that BDV infects humans, although its role in neuropsychiatric disorders remains a matter of debate (4Kishi M. Nakaya T. Nakamura Y. Zhong Q. Ikeda K. Senjo M. Kakinuma M. Kato S. Ikuta K. FEBS Lett. 1995; 364: 293-297Crossref PubMed Scopus (111) Google Scholar, 5Bode L. Zimmermann W. Ferszt R. Steinbach F. Ludwig H. Nat. Med. 1995; 1: 232-236Crossref PubMed Scopus (223) Google Scholar, 6de la Torre J.C. Gonzalez-Dunia D. Cubitt B. Mallory M. Mueller-Lantzsch N. Grässer F.A. Hansen L.A. Masliah E. Virology. 1996; 223: 272-282Crossref PubMed Scopus (118) Google Scholar, 7Lipkin W.I. Schneemann A. Solbrig M.V. Trends Microbiol. 1995; 3: 64-69Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 8Planz O. Rentzsch C. Batra A. Winkler T. Büttner M. Rziha H.J. Stitz L. J. Virol. 1999; 73: 6251-6256Crossref PubMed Google Scholar, 9Nakamura Y. Takahashi H. Shoya Y. Nakaya T. Watanabe M. Tomonaga K. Iwahashi K. Ameno K. Momiyama N. Taniyama H. Sata T. Kurata T. de la Torre J.C. Ikuta K. J. Virol. 2000; 74: 4601-4611Crossref PubMed Scopus (109) Google Scholar). When inoculated in adult immunocompetent Lewis rats, BDV causes behavioral disturbances associated with massive brain inflammation and extensive neuronal damage (10Narayan O. Herzog S. Frese K. Scheefers H. Rott R. Science. 1983; 220: 1401-1403Crossref PubMed Scopus (199) Google Scholar, 11Stitz L. Dietzschold B. Carbone K.M. Koprowski H. Lipkin I. Borna Disease. Springer-Verlag, Berlin, Germany1995: 75-92Google Scholar). Neonatal infection also results in a lifelong persistent infection associated with a variety of behavioral abnormalities but without generalized meningitis or encephalitis (10Narayan O. Herzog S. Frese K. Scheefers H. Rott R. Science. 1983; 220: 1401-1403Crossref PubMed Scopus (199) Google Scholar,12Carbone K.M. Park S.W. Rubin S.A. Waltrip R.W., II Vogelsang G.B. J. Virol. 1991; 65: 6154-6164Crossref PubMed Google Scholar, 13Bautista J.R. Schwartz G.J. de la Torre J.C. Moran T.H. Carbone K.M. Brain Res. Bull. 1994; 34: 31-36Crossref PubMed Scopus (96) Google Scholar, 14Bautista J.R. Rubin S.A. Moran T.H. Schwartz G.J. Carbone K.M. Dev. Brain Res. 1995; 90: 45-53Crossref PubMed Scopus (85) Google Scholar, 15Dittrich W. Bode L. Ludwig H. Kao M. Scheider K. Biol. Psychiat. 1989; 20: 818-828Abstract Full Text PDF Scopus (124) Google Scholar). Therefore, this second model allows studying the consequences of persistent BDV infection on neuronal functions in the absence of inflammation. Many lines of evidence suggest that BDV persistence per secan cause damage to specific neuronal populations. For example, infection of adults with BDV disrupts cortical cholinergic innervation prior to encephalitis (16Gies U. Bilzer T. Stitz L. Staiger J.F. Brain Pathol. 1998; 8: 39-48Crossref PubMed Scopus (31) Google Scholar), whereas neonatal infection causes alterations in postnatal development of the cerebellum and hippocampus as well as synaptic pathology (13Bautista J.R. Schwartz G.J. de la Torre J.C. Moran T.H. Carbone K.M. Brain Res. Bull. 1994; 34: 31-36Crossref PubMed Scopus (96) Google Scholar, 14Bautista J.R. Rubin S.A. Moran T.H. Schwartz G.J. Carbone K.M. Dev. Brain Res. 1995; 90: 45-53Crossref PubMed Scopus (85) Google Scholar, 17Eisenman L.M. Brothers R. Tran M.H. Kean R.B. Dickson G.M. Dietzschold B. Hooper D.C. J. Neurovirol. 1999; 5: 181-189Crossref PubMed Scopus (63) Google Scholar, 18Gonzalez-Dunia D. Watanabe M. Syan S. Mallory M. Masliah E. de la Torre J.C. J. Virol. 2000; 74: 3441-3448Crossref PubMed Scopus (60) Google Scholar). In particular, neonatal infection causes Purkinje cell death, dentate gyrus granule neuron degeneration, and a progressive loss of cortical neurons (14Bautista J.R. Rubin S.A. Moran T.H. Schwartz G.J. Carbone K.M. Dev. Brain Res. 1995; 90: 45-53Crossref PubMed Scopus (85) Google Scholar,17Eisenman L.M. Brothers R. Tran M.H. Kean R.B. Dickson G.M. Dietzschold B. Hooper D.C. J. Neurovirol. 1999; 5: 181-189Crossref PubMed Scopus (63) Google Scholar, 18Gonzalez-Dunia D. Watanabe M. Syan S. Mallory M. Masliah E. de la Torre J.C. J. Virol. 2000; 74: 3441-3448Crossref PubMed Scopus (60) Google Scholar, 19Hornig M. Weissenbock H. Horscroft N. Lipkin W.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12102-12107Crossref PubMed Scopus (228) Google Scholar). BDV is a noncytopathic virus, and the mechanism whereby it causes neuronal pathology is still not understood. Neonatal infection leads to variations in the level of proinflammatory cytokines and to changes in monoamine tissue content as well as in the expression of molecules regulating central nervous system plasticity (18Gonzalez-Dunia D. Watanabe M. Syan S. Mallory M. Masliah E. de la Torre J.C. J. Virol. 2000; 74: 3441-3448Crossref PubMed Scopus (60) Google Scholar, 19Hornig M. Weissenbock H. Horscroft N. Lipkin W.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12102-12107Crossref PubMed Scopus (228) Google Scholar, 20Sauder C. de la Torre J.C. J. Neuroimmunol. 1999; 96: 29-45Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 21Pletnikov M.V. Rubin S.A. Schwartz G.J. Carbone K.M. Moran T.H. Brain Res. Dev. Brain Res. 2000; 119: 179-185Crossref PubMed Scopus (58) Google Scholar, 22Weissenbock H. Hornig M. Hickey W.F. Lipkin W.I. Brain Pathol. 2000; 10: 260-272Crossref PubMed Scopus (81) Google Scholar). Although these changes might be responsible for part of the pathology, they, however, appear quite late after infection (i.e. at least 3 weeks post-inoculation). A recent study showing that cerebellar damage is observed only if infection occurs before postnatal day 15 suggests that BDV also interferes with early events of postnatal brain development (23Rubin S.A. Bautista J.R. Moran T.H. Schwartz G.J. Carbone K.M. Brain Res. Dev. Brain Res. 1999; 112: 237-244Crossref PubMed Scopus (39) Google Scholar). Therefore, we hypothesized that infection may affect the response to neurotrophic factors that play key roles in early differentiation and survival of neurons. In particular, we decided to investigate whether BDV infection interferes with the response to nerve growth factor (NGF), the prototypic member of the neurotrophin family (24Levi-Montalcini R. Skaper S.D. Dal Toso R. Petrelli L. Leon A. Trends Neurosci. 1996; 19: 514-520Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). As a first step to test this hypothesis, we studied the response to NGF of PC12 cells infected by BDV. The PC12 cell line is a neural crest-derived adrenal chromaffin cell line obtained from a rat pheochromocytoma. PC12 cells exhibit several features of neuronal differentiation following treatment with NGF. They extend neurites, become post-mitotic, and resemble sympathetic neurons (25Greene L.A. Tischler A.S. Adv. Cell. Neurobiol. 1982; 3: 373-414Crossref Google Scholar). Binding of NGF to its receptor, a tyrosine kinase, triggers a cascade of protein phosphorylation events, leading to the activation of several genes. This signal transduction cascade and the genes involved have been studied extensively (26Sassone-Corsi P. Der C.J. Verma I.M. Cell. Biol. 1989; PubMed Scopus Google Scholar, R. J. 1995; PubMed Scopus Google Scholar). we that persistent infection of PC12 cells with BDV leads to changes in cell and expression of genes in synaptic Moreover, infection causes a block in neurite outgrowth and several changes in the NGF signal transduction PC12 cells were at in with II with and were or a cell were at a to in tissue or to or with PC12 cells were infected with BDV as (13Bautista J.R. Schwartz G.J. de la Torre J.C. Moran T.H. Carbone K.M. Brain Res. Bull. 1994; 34: 31-36Crossref PubMed Scopus (96) Google and 3 after the infection to a persistently infected line cells were the as the and PC12 cells were on in for and in for They were treated with NGF for the before RNA was by by with is to and The were for at in and of and were with at the were to the were by in before The following were from the BDV nucleoprotein rat and tyrosine hydroxylase B. A. F. N. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google as well as from rat and from C. and M. The were with obtained with a to the rat glyceraldehyde-3-phosphate dehydrogenase were in a C. M. J. J. Neurosci. 1998; PubMed Google and a was by for at and protein was by a protein or on the were by before The was blocked for at in Tris-buffered saline with of or the was at with the in with the were for with or by a extensive in and was by with signal and to When the and the of a the were first with the specific for the the was by in blocked as and with the of the were on a and and We the following a the of mitogen-activated protein kinase and a and and protein the of and a the of and a the BDV la Torre J.C. Gonzalez-Dunia D. Cubitt B. Mallory M. Mueller-Lantzsch N. Grässer F.A. Hansen L.A. Masliah E. Virology. 1996; 223: 272-282Crossref PubMed Scopus (118) Google Scholar). on were with in saline by and for as D. Cubitt B. Grässer F.A. de la Torre J.C. J. Virol. PubMed Google Scholar). after with and for at cells were with in saline cells were with and were extensively as for in and with The were to synaptophysin microtubule-associated protein and NGF We also a to the protein as well as the to the of and to BDV When and were cells were with a were and PC12 cells were infected with BDV as and analyzed at after infection after infected cells were from PC12 of the cells were as by of the nucleoprotein and cell to cells in infected cells and and their to the to on the the changes the cell not neurite outgrowth in PC12 cells persistently infected with BDV PC12 or cells after were on for 3 in the or absence of NGF. The cells were and with We also the response of infected PC12 cells to NGF. NGF the of PC12 cells and neurite As in cells treated with NGF to and to PC12 cells in and to NGF The infection not cell as by cells not BDV and infection not cell The of cells was that of cells 3 by We whether cells still neuronal were on treated with and for with PC12 cells of and The expression of in cells in the cells In the expression of a was in cells synaptophysin expression was as cells were of synaptophysin expression with the alterations to BDV persistence cells still synaptophysin and to NGF. These cells in not infected in the of this study were on persistently infected cells that of the infection causes a progressive down-regulation of synaptophysin PC12 cells and cells infected with BDV and and and are PC12 and cells were treated with NGF for and were cells were for synaptophysin and BDV The of cells is due to the that these cells not become in response to NGF. cells with the BDV exhibit a of the nucleus on not with a the progressive of synaptophysin in infected synaptophysin is not in for a cell that is not infected We the level of for synaptophysin in PC12 and with the level of the P. Neurobiol. 1991; 5: PubMed Scopus Google Scholar). We also the expression of the tyrosine hydroxylase a gene by NGF in PC12 cells L.A. Cell. Biol. PubMed Scopus (223) Google Scholar). is that the step in the leading to is in neurons as well as in neural crest-derived chromaffin were also for the gene and for As in PC12 cells constitutive of and to NGF was by the la Torre J.C. Mallory M. M. L. G. Masliah E. Virology. 1996; 220: PubMed Scopus Google in RNA was in PC12 and expression following NGF In for and were or observed by in cells and were not in response to NGF whereas for As we only in BDV persistence in PC12 cells the expression of genes for molecules involved in synaptic plasticity as well as in We whether the absence of response of cells to NGF be due to a down-regulation of the expression of the NGF receptors. NGF are M.V. Trends Neurosci. 1995; Full Text PDF PubMed Scopus Google Scholar). The neurotrophin is a that also the and NGF also and the tyrosine kinase and it been that and may be for the of the and for the M.V. Trends Neurosci. 1995; Full Text PDF PubMed Scopus Google Scholar, P.A. 1994; Full Text PDF PubMed Scopus Google Scholar). We analyzed the expression of and In PC12 we observed a in the level of and molecules following NGF In in were for and were the of for We studied the expression of by The was after NGF treatment in PC12 whereas expression was in PC12 In the cells of with of the whereas that were of not appear to with nucleoprotein expression was the of by in the PC12 The block in neuronal differentiation observed in be in part by the down-regulation NGF results from and showed that cells still of least for and not in response to NGF. Moreover, of expression with a showed that infected cells still of not Therefore, we decided to the of BDV infection on the NGF signal transduction cascade R. J. 1995; PubMed Scopus Google Binding of NGF to the triggers that the signal to the of the and the the activated protein and and their the ERKs a of the factor that to response in several R. J. R. Cell. 73: Full Text PDF PubMed Scopus Google Scholar). In the ERKs the kinase J. Proc. Natl. Acad. Sci. U. S. A. 90: PubMed Scopus Google Scholar), that in as and the of early genes as by to cAMP response element-binding M. G. E. M. S. P. R. 1995; Google Scholar, A. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). The of specific for activated MAPK that only to study in the of BDV infection on the NGF signal transduction events after of PC12 and cells were at after and of by were analyzed by for the expression of the activated In the were after of the activated of the kinase and with that of the kinase and This was at least and the results were to in B. We observed expression of activated or in PC12 cells prior to NGF and only phosphorylation at this and were and This was by activation of and of the cascade and by of the analyzed by were observed in PC12 cells persistently infected with BDV we observed that and ERK1/2 were in the absence of NGF. This was if cells were for of be that activated of and were observed in PC12 cells before NGF after the of protein on the cells to NGF treatment with in the of activated and to with of the PC12 This suggests that the is still in cells and is of the MAPK this activation was in PC12 cells and to activation of the was leading to only level of was constitutive phosphorylation of in and treatment with NGF caused phosphorylation of this The that the MAPK is activated in cells is in with the that these cells not C.J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). we also observed that activation of the cascade was in this activation the of activated we hypothesized that BDV infection may with the of activated To this PC12 and cells were on treated with and analyzed for the of the activated ERK1/2 by and The results are of a of cells analyzed for and the was several with the results of was of activated ERK1/2 in PC12 cells prior to NGF As early as after NGF activated ERK1/2 was and was in the nucleus and in the of PC12 The of cells were for activated ERKs by after In of the signal was the signal and by the signal was In activated ERKs expression was prior to NGF were from cell to and the of cells ERKs NGF were to observed before NGF. the of activated ERKs was from that in and also after with a not that was and that was activated translocated to the nucleus of We whether these changes were in infected PC12 As in the of activated was in the infected cells in the early after Moreover, infected cells were to neurite Therefore, the changes early after infection and in with the virus, to the cell been that the observed in infected at with BDV are to alterations of the of the hippocampus and two brain that to after M. Weissenbock H. Horscroft N. Lipkin W.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12102-12107Crossref PubMed Scopus (228) Google Scholar, M.V. Rubin S.A. Schwartz G.J. Moran T.H. Carbone K.M. 1999; PubMed Scopus Google Scholar). is well that as NGF have a on the development of the central nervous system (24Levi-Montalcini R. Skaper S.D. Dal Toso R. Petrelli L. Leon A. Trends Neurosci. 1996; 19: 514-520Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). The role of in neuronal differentiation and from neuronal cell death, with recent results that BDV neonatal persistent infection causes alterations of synaptic plasticity and neuronal cell by (18Gonzalez-Dunia D. Watanabe M. Syan S. Mallory M. Masliah E. de la Torre J.C. J. Virol. 2000; 74: 3441-3448Crossref PubMed Scopus (60) Google Scholar, 19Hornig M. Weissenbock H. Horscroft N. Lipkin W.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12102-12107Crossref PubMed Scopus (228) Google Scholar, 22Weissenbock H. Hornig M. Hickey W.F. Lipkin W.I. Brain Pathol. 2000; 10: 260-272Crossref PubMed Scopus (81) Google Scholar), led to the of BDV on NGF BDV is noncytopathic, it and at in as well as in PC12 cells in Therefore, this cell line is model to study the of infection on neuronal gene expression and on the to neurotrophic we system to the mechanisms whereby persistent BDV infection leads to neuronal gene expression and We observed that the persistence of BDV in PC12 cells was by a progressive change of Although PC12 cells with to NGF R.A. J. Cell. 1995; PubMed Scopus Google Scholar, R. A. D. R. J. Cell. 1998; PubMed Scopus Google Scholar), we that the phenotype was due to and of PC12 cells we observed a change of phenotype in PC12 cells the and for the of and a change of phenotype was obtained in several Moreover, of the PC12 exhibit the of gene expression and tyrosine kinase observed in at early infected cells were from cells and of This infection of a cell with from of PC12 it is that persistence in PC12 caused a progressive change of BDV infection not change the level of expression of neuronal in PC12 as or The expression of in cells may be due to the change of and caused by BDV. it may be due to the activation of activated ERKs with M. Biol. Cell. 1996; PubMed Scopus Google Scholar). In the expression of neuronal as synaptophysin and was in This is with results showing synaptophysin and protein expression in the central nervous system of infected (18Gonzalez-Dunia D. Watanabe M. Syan S. Mallory M. Masliah E. de la Torre J.C. J. Virol. 2000; 74: 3441-3448Crossref PubMed Scopus (60) Google Scholar). The expression of was also in these results suggest that BDV infection a of genes to NGF without NGF neuronal gene PC12 cells to extend in response to NGF. We showed that the block of NGF was due in part to a down-regulation of NGF We the of BDV infection on tyrosine kinase and early gene that the MAPK also be In we several be as and ERK1/2 were activated and to NGF factor activation the and phosphorylation of the of the cascade was and led to or The MAPK cascade is in the response of cells to several growth factors and In the of PC12 the have been the response to NGF and to epidermal growth factor Although NGF causes neurite and of a treatment leads to by the receptor, is also a tyrosine and early transduction with a activation of The key and in the of ERK1/2 activation that activation of the proliferation, whereas activation is for differentiation of PC12 cells C.J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). we were to that BDV infection caused a activation of was not by activation of early genes and neuronal A of events that the phosphorylation of the phosphorylation of and that the ERKs to a signal in PC12 In particular, the expression of to the was in that ERK1/2 might not to the The level of activation in infected cells be due to phosphorylation by P. FEBS Lett. 2000; PubMed Scopus Google Scholar). The of ERKs to the nucleus of infected cells was by of activated ERKs and by For however, we observed a constitutive activation to the and ERKs as well as response after treatment with NGF. This may be due to the that this is in neuronal to the and to the nucleus before activation P. C. M. R. J. J. Neurosci. 1998; PubMed Google Scholar). may also be due to phosphorylation of I. D. Cell. Biol. 1998; PubMed Scopus (63) Google Scholar). The mechanism leading to activation of ERK1/2 in cells is not We were to ERK1/2 activation following treatment of PC12 cells with from cells not This suggests that factors by the infected cells are not responsible for leading to ERK1/2 A is that BDV affects the of the phosphorylation or in the For example, BDV infection the of of the of the to the nonsegmented, RNA BDV a that is by the Although phosphorylation of the BDV not appear to be to the ERK1/2 M. B. A. Lipkin W.I. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), it with events involved in of ERK1/2 is by a MAPK is in the nucleus H. Cell. Full Text PDF PubMed Scopus Google Scholar). This the activated ERKs after their their to the is that leads to a progressive of activated in BDV of remains to be it in the of RNA and is of the BDV as the the and the protein have been well studied T.H. T. M. M. Virology. 1999; PubMed Scopus Google Scholar, T. R. T. J. J. Virol. 2000; PubMed Scopus Google Scholar, M. M. L. J. Lipkin W.I. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). factors associated with these in the of have not been One that BDV factors to and from their the of as the activated To this is the first of a persistent infection with neuronal differentiation and to been that protein causes a block of NGF differentiation, in this to of with in cell 8: Google Scholar, K. W. 1: Google Scholar). it been that also the of and PC12 cells to NGF A. D. G. C. L. G. de J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In the central nervous the and are of neuronal survival and synaptic plasticity S. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). are in this may to in the of early postnatal We for for and for of and for and for the of of the in this We are also to and for the