Opioid-induced proliferation through the MAPK pathway in cultures of adult hippocampal progenitors

Abstract

Administration of opioid agonists or antagonists has been reported to regulate proliferation or survival of neural progenitors in vivo. Here we report that β-endorphin and selective mu-opioid receptor (MOR) and delta-opioid receptor (DOR) agonists stimulate proliferation of isolated rat adult hippocampal progenitors (AHPs). The AHPs were found to express DORs and MORs, but not kappa-opioid receptors. Incubation with β-endorphin for 48 h increased the number of AHPs found in mitosis, the total DNA content, and the expression of proliferating cell nuclear antigen. This proliferative effect from β-endorphin on AHPs was antagonized by naloxone. The β-endorphin-induced proliferation was mediated through phosphorylation of extracellular signal-regulated kinases 1 and 2 and dependent on phosphatidylinositol 3-kinase and both intra- and extracellular calcium. These data suggest a role for the opioid system in the regulation of proliferation in progenitors from the adult hippocampus.

Introduction

Neurogenesis has been shown to occur in several species, including humans Altman and Das 1965, Nottebohm 1985, Stanfield and Trice 1988, Kempermann et al 1997, Eriksson et al 1998. The birth of new cells in the dentate gyrus of the hippocampus, a brain region important for learning and memory, is decreased by stress (Gould et al., 1998) and age (Cameron and McKay, 1999) and increased by hippocampal-dependent learning (Gould et al., 1999), environmental enrichment (Nilsson et al., 1999), and voluntary exercise van Praag et al 1999, van Praag et al 2000. Chronic exposure to morphine, widely used for treatment of pain, has been shown to result in impaired memory Saha et al 1991, Li et al 2001 and also to decrease neurogenesis and the survival of progenitors in the dentate gyrus of the adult rat hippocampus (Eisch et al., 2000). Several reports suggest a negative effect in vivo of morphine on DNA synthesis in the neonatal and adult brain Kornblum et al 1987, Hauser et al 1994, Eisch et al 2000 and a positive effect of opioid antagonists on this process Zagon and McLaughlin 1986, Schmahl et al 1989, Hauser et al 1989, Seatriz and Hammer 1993. By contrast, in an early in vivo study, DNA synthesis has been seen to increase after acute administration of morphine to adult rats (Miller et al., 1982).

In vitro, both proliferation of cerebellar granule neuron precursors (Hauser et al., 2000) and astrocytes (Hauser et al., 1996) were decreased after exposure to morphine, whereas the opioid antagonist naloxone has been shown to decrease DNA synthesis of O4-immunoreactive oligodendrocyte progenitors in glial cultures (Hauser et al., 1993). The inhibitory effect of naloxone was not found in cultures enriched with oligodendrocyte progenitors and fewer astrocytes. Furthermore, the mu-opioid receptor (MOR) agonist PL017 increased DNA synthesis in immature O4-immunoreactive oligodendrocytes in vitro (Knapp et al., 1998). In this study, opposite effects of opiates on proliferation were seen in the immature O4-immunoreactive oligodendrocytes compared with mature myelin basic protein (MBP)-immunoreactive oligodendrocytes in which proliferation was down-regulated. Furthermore, in cultured astrocytes the increase or decrease in proliferation is depending on extracellular levels of calcium (Hauser et al., 1996). This suggests a complex regulation of proliferation in neural cell types. The response from opioids on proliferation seems to depend on the specific cell type studied and whether the cell is at an immature or a mature stage. Other influencing factors include the type of surrounding cells, the calcium level, what opioid receptor subtypes are expressed on the cells, and possibly several other unknown factors.

All three opioid receptor subtypes, delta-opioid receptors (DORs), MORs, and kappa-opioid receptors (KORs), have been found in the dentate gyrus of the adult rat hippocampus at both the protein and the mRNA levels Mansour et al 1987, McLean et al 1987, Delfs et al 1994. β-Endorphin, an endogenous opioid peptide derived from the proopiomelanocortin gene, is expressed in both the embryonal (Angelogianni et al., 2000) and the adult Barna et al 1997, Johansson et al 1997 rat hippocampus.

To better understand whether endogenous opioids have direct effects on the proliferation of adult neural progenitors, we used adult hippocampal progenitors (AHPs) isolated from rat hippocampus Gage et al 1995, Palmer et al 1999. These cells are capable of generating neurons as well as glia Palmer et al 1997, Takahashi et al 1999, Aberg et al 2001 and have been transplanted back to the adult hippocampus, where they retain the capacity to become new granule cell layer neurons Gage et al 1995, van Praag et al 2002.

In this study we wanted to determine whether any opioid receptor subtype exists on the AHPs and, if so, whether they mediate effects from the endogenous opioid peptide β-endorphin or specific opioid receptor agonists. Total DNA content and the expression of the cell-cycle markers, phosphorylated histone H3 (phospho-histone H3), and proliferating cell nuclear antigen (PCNA), were used to determine opioid regulation of AHP proliferation. Previous studies have shown that stimulation of opioid receptors increases the level of free intracellular calcium ([Ca²⁺]_i) Stiene-Martin et al 1998, Thorlin et al 1998, a signal transducer known to be involved in regulation of proliferation (Hauser et al., 1996). Opioids also increase phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2; also called mitogen-activated protein kinase (MAPK)) Gutstein et al 1997, Kramer and Simon 2000, Belcheva et al 2001. Inhibited phosphorylation of MAPK has previously been shown to block proliferation of neural progenitor cells, suggesting that this pathway is important for proliferation of neural progenitors (Learish et al., 2000). Therefore, we also wanted to study whether β-endorphin regulates phosphorylation of ERK1/2 and the levels of [Ca²⁺]_i in the AHPs.