Developmental regulation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury. I. Rodent cerebral white matter and cortex

The immature brain is highly susceptible to hypoxia/ischemia (H/I), and perinatal H/I brain injury represents a major cause of neurodevelopmental disorders in both preterm and term infants (Volpe, 2001; Ferriero, 2004). In preterm infants, H/I causes primarily white matter injury, termed periventricular leukomalacia (PVL; Banker and Larroche, 1962; Okumura et al., 1997; Volpe, 2001). In contrast, H/I in term newborns causes predominantly gray matter lesions and seizures (Hauser et al., 1993; Maller et al., 1998; Roland et al., 1998; Saliba et al., 1999; Volpe, 2001). Rodent models of perinatal H/I brain injury reflect similar age-dependent regional differences, despite slight age variations in different rat strains. During the first week of life (postnatal day P1–P7), H/I results in selective white matter injury, characterized by loss of premyelinating oligodendrocytes (pre-OLs), followed by hypomyelination (Sheldon et al., 1996; Follett et al., 2000; Cai et al., 2001; Back et al., 2002; Liu et al., 2002). During the second postnatal week (P8–14), H/I causes spontaneous electro-graphic and behavioral seizures (Jensen et al., 1991; Owens et al., 1997), as well as extensive cortical and hippocampal neuronal loss (Towfighi et al., 1997; Chen et al., 1998). H/I causes glutamate accumulation in both gray and white matter structures in the developing rat brain (Benveniste et al., 1984; Andine et al., 1991; Silverstein et al., 1991; Hagberg, 1992; Loeliger et al., 2003), implying a key role for glutamate receptors (GluRs) in the pathophysiology of perinatal H/I brain injury. Glutamate receptor subtypes include the N-methyl-D-aspartate receptors (NMDARs), the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs), and kainate receptors (KARs; Hollmann and Heinemann, 1994; Michaelis, 1998). Whereas neurons and astrocytes express both NMDARs and non-NMDARs (Petralia and Wenthold, 1992; Petralia et al., 1994; Conti et al., 1994; Shelton and McCarthy, 1999; Schipke et al., 2001), OLs express primarily non-NMDARs (Patneau et al., 1994; Gallo et al., 1994; Rosenberg et al., 2003). Compelling evidence for a critical role of GluRs in perinatal H/I injury is provided by experimental therapeutic trials. AMPAR antagonists are highly protective to developing OLs against H/I injury at P7 (Follett et al., 2000, 2004) and are effective in suppressing hypoxia-induced seizures at P10–P12 (Jensen et al., 1995; Koh and Jensen, 2001). Similarly, in P7–P10 rats, NMDAR and AMPAR antagonists have been shown to attenuate H/I-induced neuronal injury (Olney et al., 1989; Hagberg et al., 1994; Chen et al., 1998). AMPAR-mediated signaling and excitotoxicity depend on the functional properties of the receptor complex, such as Ca2+ permeability (Gu et al., 1996; Friedman and Koudinov, 1999; Sanchez et al., 2001, 2005; Jensen et al., 2001; Deng et al., 2003; Follett et al., 2004), which in turn are dictated by subunit composition. AMPARs are heteromeric complexes composed of four subunits (GluR1 through GluR4), and receptors lacking the GluR2 subunit are Ca2+-permeable, whereas those including the GluR2 subunit are impermeable to Ca2+ (Burnashev et al., 1992; Seeburg, 1993; Jonas et al., 1994; Washburn et al., 1997). In the immature rat brain, GluR2 expression is low relative to non-GluR2 subunits (Pellegrini-Giampietro et al., 1991, 1992; Sanchez et al., 2001; Kumar et al., 2002), suggesting that AMPARs with increased Ca2+ permeability are abundantly expressed during this time window. Indeed, functional studies in situ in immature rodent brain have confirmed increased Ca2+ influx through AMPARs expressed on pre-OLs (Fulton et al., 1992; Bergles et al., 2000; Follett et al., 2004) and developing hippocampal and pyramidal neurons (Sanchez et al., 2001; Kumar et al., 2002), supporting a close correlation between GluR2 expression level and receptor function. In this two-part series of studies, we examine evidence for a relationship between differential distribution of GluR2-lacking (Ca2+-permeable) AMPARs and selective white and gray matter vulnerability to H/I in both rodent (part I) and human (Talos et al., 2006). We hypothesize that the GluR2-lacking AMPARs represent a key factor in age-dependent regional susceptibility to H/I, so regional and temporal expression of these receptors would correspond in a cell-specific manner to patterns of susceptibility to H/I. In addition, we hypothesize that subtle differences in age windows of susceptibility between rat strains are due to strain-dependent differences in temporal onset and progression of AMPAR subunits on specific cell types. In part I, we analyzed the developmental profile of each AMPAR subunit in both white matter and cortex from Long Evans (LE) rats during the first 3 postnatal weeks (postnatal days P1–P21) and specifically evaluated the developmental regulation of the GluR2 subunit relative to other AMPAR subunits by immunoblotting and immunofluorescence double labeling. To examine strain-dependent differences in AMPAR subunit expression, a subset of immunofluorescence double-labeling experiments for specific AMPAR subunits was conducted in parallel in both LE and Sprague Dawley (SD) rat pups, ages P1–P14. In part II (Talos et al., 2006), human parietal white matter and cortex from cases ranging between 18 postconceptional weeks (PCW) and 210 PCW (approximately 3.3 years) were similarly evaluated for age-dependent variations in AMPAR subunit expression by immunoblotting and immunofluorescence double labeling.