Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons induce a functional switch in the growth factor responsiveness of astroglia: Involvement of basic fibroblast growth factor

Roberto Avola, Vittoria Spina-Purrello, Francesco Gallo, Maria C. Morale, Nunzio Marletta, Antonino Costa, Cataldo Tirolo, Nuccio Testa, Salvatore Reale, Bianca Marchetti

Research output: Contribution to journalArticle

Abstract

Recent evidence indicates that astroglial-derived growth factors (GFs) participate in the development of luteinizing hormone-releasing hormone (LHRH) neurons, but it is still unknown whether LHRH neurons may exert a reciprocal modulation of glial cell function. Using immortalized hypothalamic LHRH (GT1-1) neurons in co-culture with glial cells, we have recently shown that basic fibroblast growth factor (bFGF) plays a prominent role in the glial-induced acquisition of the mature LHRH phenotype by GT1-1 cells. We have resorted to this model and combined biochemical and morphological approaches to study whether the response of glial cells to a number of GFs (including bFGF, insulin-like growth factor I, IGF-I, epidermal growth factor, EGF and insulin) expressed during LHRH neuron differentiation, is modulated by co-culture with pure LHRH neurons. Pre-treatment of hypothalamic astrocytes with an inactive ('priming') dose of bFGF for 12 h powerfully increased astroglia proliferative response to IGF-I (10 ng/ml), EGF (10 g/ml) and insulin (10 μg/ml), inducing a 65-100% increase in the [3H]thymidine incorporation compared to untreated cultures. When astroglial cells and developing GT1-1 neurons were co-cultured for 5 days in vitro (DIV), the [3H]thymidine incorporation was significantly higher than in astroglial cells cultured without neurons. Application of the different GFs to the co-culture for either 12 or 24 h further stimulated DNA synthesis to various extent according to the GF applied and the time of application. Localization of the proliferating cells by dual immunohistochemical staining, followed by cell counting and bromodeoxiuridine (BrdU) labeling index calculation, revealed that the incorporation of BrdU was restricted to the nuclei of LHRH-immunopositive neurons. Such changes were accompanied by extensive morphological alterations of astroglial and LHRH fiber networks, whereas neutralization of bFGF activity in GT1-1 neuron-glial co-cultures by a bFGF-antibody, dramatically counteracted the observed effects. The functional switch of astroglia proliferative response to GFs coupled to the potent morphological and functional modifications of developing glia and pure LHRH neurons observed in vitro, support a bidirectional interaction between immortalized LHRH neurons and astroglial cells and identify bFGF as a key player in this crosstalk.

Original languageEnglish
Pages (from-to)743-763
Number of pages21
JournalInternational Journal of Developmental Neuroscience
Volume18
Issue number8
DOIs
Publication statusPublished - 2000

Fingerprint

Hypothalamic Hormones
Fibroblast Growth Factor 2
Gonadotropin-Releasing Hormone
Astrocytes
Intercellular Signaling Peptides and Proteins
Neurons
Neuroglia
Coculture Techniques
Epidermal Growth Factor
Insulin-Like Growth Factor I
Thymidine
Insulin
Cultured Cells

Keywords

  • Astroglia proliferation
  • Basic fibroblast growth factor
  • Bromodeoxiuridine incorporation
  • DNA synthesis
  • Glial fibrillary acidic protein (GFAP)
  • Immortalized LHRH neurons
  • LHRH neuron development
  • LHRH neuron-astroglia interactions

ASJC Scopus subject areas

  • Developmental Biology
  • Developmental Neuroscience

Cite this

@article{3b807bd07e834383bfc54bf3b39a5f78,
title = "Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons induce a functional switch in the growth factor responsiveness of astroglia: Involvement of basic fibroblast growth factor",
abstract = "Recent evidence indicates that astroglial-derived growth factors (GFs) participate in the development of luteinizing hormone-releasing hormone (LHRH) neurons, but it is still unknown whether LHRH neurons may exert a reciprocal modulation of glial cell function. Using immortalized hypothalamic LHRH (GT1-1) neurons in co-culture with glial cells, we have recently shown that basic fibroblast growth factor (bFGF) plays a prominent role in the glial-induced acquisition of the mature LHRH phenotype by GT1-1 cells. We have resorted to this model and combined biochemical and morphological approaches to study whether the response of glial cells to a number of GFs (including bFGF, insulin-like growth factor I, IGF-I, epidermal growth factor, EGF and insulin) expressed during LHRH neuron differentiation, is modulated by co-culture with pure LHRH neurons. Pre-treatment of hypothalamic astrocytes with an inactive ('priming') dose of bFGF for 12 h powerfully increased astroglia proliferative response to IGF-I (10 ng/ml), EGF (10 g/ml) and insulin (10 μg/ml), inducing a 65-100{\%} increase in the [3H]thymidine incorporation compared to untreated cultures. When astroglial cells and developing GT1-1 neurons were co-cultured for 5 days in vitro (DIV), the [3H]thymidine incorporation was significantly higher than in astroglial cells cultured without neurons. Application of the different GFs to the co-culture for either 12 or 24 h further stimulated DNA synthesis to various extent according to the GF applied and the time of application. Localization of the proliferating cells by dual immunohistochemical staining, followed by cell counting and bromodeoxiuridine (BrdU) labeling index calculation, revealed that the incorporation of BrdU was restricted to the nuclei of LHRH-immunopositive neurons. Such changes were accompanied by extensive morphological alterations of astroglial and LHRH fiber networks, whereas neutralization of bFGF activity in GT1-1 neuron-glial co-cultures by a bFGF-antibody, dramatically counteracted the observed effects. The functional switch of astroglia proliferative response to GFs coupled to the potent morphological and functional modifications of developing glia and pure LHRH neurons observed in vitro, support a bidirectional interaction between immortalized LHRH neurons and astroglial cells and identify bFGF as a key player in this crosstalk.",
keywords = "Astroglia proliferation, Basic fibroblast growth factor, Bromodeoxiuridine incorporation, DNA synthesis, Glial fibrillary acidic protein (GFAP), Immortalized LHRH neurons, LHRH neuron development, LHRH neuron-astroglia interactions",
author = "Roberto Avola and Vittoria Spina-Purrello and Francesco Gallo and Morale, {Maria C.} and Nunzio Marletta and Antonino Costa and Cataldo Tirolo and Nuccio Testa and Salvatore Reale and Bianca Marchetti",
year = "2000",
doi = "10.1016/S0736-5748(00)00052-6",
language = "English",
volume = "18",
pages = "743--763",
journal = "International Journal of Developmental Neuroscience",
issn = "0736-5748",
publisher = "Elsevier Limited",
number = "8",

}

TY - JOUR

T1 - Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons induce a functional switch in the growth factor responsiveness of astroglia

T2 - Involvement of basic fibroblast growth factor

AU - Avola, Roberto

AU - Spina-Purrello, Vittoria

AU - Gallo, Francesco

AU - Morale, Maria C.

AU - Marletta, Nunzio

AU - Costa, Antonino

AU - Tirolo, Cataldo

AU - Testa, Nuccio

AU - Reale, Salvatore

AU - Marchetti, Bianca

PY - 2000

Y1 - 2000

N2 - Recent evidence indicates that astroglial-derived growth factors (GFs) participate in the development of luteinizing hormone-releasing hormone (LHRH) neurons, but it is still unknown whether LHRH neurons may exert a reciprocal modulation of glial cell function. Using immortalized hypothalamic LHRH (GT1-1) neurons in co-culture with glial cells, we have recently shown that basic fibroblast growth factor (bFGF) plays a prominent role in the glial-induced acquisition of the mature LHRH phenotype by GT1-1 cells. We have resorted to this model and combined biochemical and morphological approaches to study whether the response of glial cells to a number of GFs (including bFGF, insulin-like growth factor I, IGF-I, epidermal growth factor, EGF and insulin) expressed during LHRH neuron differentiation, is modulated by co-culture with pure LHRH neurons. Pre-treatment of hypothalamic astrocytes with an inactive ('priming') dose of bFGF for 12 h powerfully increased astroglia proliferative response to IGF-I (10 ng/ml), EGF (10 g/ml) and insulin (10 μg/ml), inducing a 65-100% increase in the [3H]thymidine incorporation compared to untreated cultures. When astroglial cells and developing GT1-1 neurons were co-cultured for 5 days in vitro (DIV), the [3H]thymidine incorporation was significantly higher than in astroglial cells cultured without neurons. Application of the different GFs to the co-culture for either 12 or 24 h further stimulated DNA synthesis to various extent according to the GF applied and the time of application. Localization of the proliferating cells by dual immunohistochemical staining, followed by cell counting and bromodeoxiuridine (BrdU) labeling index calculation, revealed that the incorporation of BrdU was restricted to the nuclei of LHRH-immunopositive neurons. Such changes were accompanied by extensive morphological alterations of astroglial and LHRH fiber networks, whereas neutralization of bFGF activity in GT1-1 neuron-glial co-cultures by a bFGF-antibody, dramatically counteracted the observed effects. The functional switch of astroglia proliferative response to GFs coupled to the potent morphological and functional modifications of developing glia and pure LHRH neurons observed in vitro, support a bidirectional interaction between immortalized LHRH neurons and astroglial cells and identify bFGF as a key player in this crosstalk.

AB - Recent evidence indicates that astroglial-derived growth factors (GFs) participate in the development of luteinizing hormone-releasing hormone (LHRH) neurons, but it is still unknown whether LHRH neurons may exert a reciprocal modulation of glial cell function. Using immortalized hypothalamic LHRH (GT1-1) neurons in co-culture with glial cells, we have recently shown that basic fibroblast growth factor (bFGF) plays a prominent role in the glial-induced acquisition of the mature LHRH phenotype by GT1-1 cells. We have resorted to this model and combined biochemical and morphological approaches to study whether the response of glial cells to a number of GFs (including bFGF, insulin-like growth factor I, IGF-I, epidermal growth factor, EGF and insulin) expressed during LHRH neuron differentiation, is modulated by co-culture with pure LHRH neurons. Pre-treatment of hypothalamic astrocytes with an inactive ('priming') dose of bFGF for 12 h powerfully increased astroglia proliferative response to IGF-I (10 ng/ml), EGF (10 g/ml) and insulin (10 μg/ml), inducing a 65-100% increase in the [3H]thymidine incorporation compared to untreated cultures. When astroglial cells and developing GT1-1 neurons were co-cultured for 5 days in vitro (DIV), the [3H]thymidine incorporation was significantly higher than in astroglial cells cultured without neurons. Application of the different GFs to the co-culture for either 12 or 24 h further stimulated DNA synthesis to various extent according to the GF applied and the time of application. Localization of the proliferating cells by dual immunohistochemical staining, followed by cell counting and bromodeoxiuridine (BrdU) labeling index calculation, revealed that the incorporation of BrdU was restricted to the nuclei of LHRH-immunopositive neurons. Such changes were accompanied by extensive morphological alterations of astroglial and LHRH fiber networks, whereas neutralization of bFGF activity in GT1-1 neuron-glial co-cultures by a bFGF-antibody, dramatically counteracted the observed effects. The functional switch of astroglia proliferative response to GFs coupled to the potent morphological and functional modifications of developing glia and pure LHRH neurons observed in vitro, support a bidirectional interaction between immortalized LHRH neurons and astroglial cells and identify bFGF as a key player in this crosstalk.

KW - Astroglia proliferation

KW - Basic fibroblast growth factor

KW - Bromodeoxiuridine incorporation

KW - DNA synthesis

KW - Glial fibrillary acidic protein (GFAP)

KW - Immortalized LHRH neurons

KW - LHRH neuron development

KW - LHRH neuron-astroglia interactions

UR - http://www.scopus.com/inward/record.url?scp=0034522510&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034522510&partnerID=8YFLogxK

U2 - 10.1016/S0736-5748(00)00052-6

DO - 10.1016/S0736-5748(00)00052-6

M3 - Article

C2 - 11154844

AN - SCOPUS:0034522510

VL - 18

SP - 743

EP - 763

JO - International Journal of Developmental Neuroscience

JF - International Journal of Developmental Neuroscience

SN - 0736-5748

IS - 8

ER -